ANTI-CXCR4 ANTIBODIES, THEIR USE, ANTIBODY-DRUG CONJUGATE AND PHARMACEUTICAL COMPOSITION

专利摘要:
anti-cxcr4 antibodies, their production method, their use, antibody-drug conjugate, pharmaceutical composition, isolated polynucleotide, vector and cell The present invention relates to antibodies and related molecules that bind to the chemokine receptor 4 (cxcr4). the invention further provides antibody-drug conjugates comprising such antibodies, antibody encoding nucleic acids and methods of obtaining such antibodies. the invention further relates to therapeutic methods for using such antibodies and anti-cxcr4 antibody-drug conjugates for the treatment of a disorder associated with cxcr4 function or expression (e.g. cancer) such as colon cancer, rcc, esophagus, gastric, head and neck, lung, ovary, pancreatic or hematologic cancers.
公开号:BR112016002008B1
申请号:R112016002008-1
申请日:2014-07-28
公开日:2021-06-22
发明作者:Wei-Hsien Ho；Shu-Hui Liu；Flavia Mercer Pernasetti
申请人:Pfizer Inc.；
IPC主号:

专利说明:

[0001] The present invention relates to antibodies and related molecules that bind to chemokine receptor 4 (CXCR4). The present invention also relates to molecules comprising, or alternatively consisting of whole antibodies, antibody fragments, or variants thereof. The present invention also relates to encoding amino acid and nucleic acid sequences for such antibodies. The present invention also relates to antibody conjugates (e.g., antibody drug conjugates) comprising the anti-CXCR4 antibodies, compositions comprising the CXCR4 antibodies, and methods of using the anti-CXCR4 antibodies, and their conjugates to treat conditions associated with CXCR4 expression (eg, cancer). The invention also comprises the use of said antibodies, their antigen-binding fragments, or antibody drug conjugates and corresponding processes, for detection and diagnosis of pathological disorders associated with the expression of CXCR4. In certain respects, the disorders are oncogenic disorders associated with an increased expression of CXCR4 relative to normal pathology or any other pathology connected with overexpression of CXCR4. In other respects, the disorders are inflammatory or immunological disorders, allergic disorders, infections (HIV infection, etc.), autoimmune disorders (eg, rheumatoid arthritis), fibrosis disorders (eg, pulmonary), and cardiovascular disorders . The invention finally comprises products and/or compositions or kits comprising at least such antibody or antibody drug conjugates for the prognosis or diagnosis or therapy monitoring of such disorders. BACKGROUND OF THE INVENTION
[0002] Chemokines are small secreted peptides that control the migration of leukocytes along the chemical gradient of ligands, known as the chemokine gradient, especially during immunological reactions (Zlotnik et al, 2000, Immunity, 12: 121 to 127). They are classified into four classes according to the location of Cys residues at the N-terminus. The CXC class consists of chemokines with a pair of Cys separated by a single residue. The most important elements of this class are interleukin-8 (IL-8, CXCL8), stromal factor-1 derivative (SDF-I, CXCL12), gamma-interferon 10 inductive proteins (IP-10, CXCLlO), factor platelet-4 (PF-4, CXCL4), neutrophil activating protein 2 (NAP-2, CXCL7) and melanoma growth-stimulating activity (MGSA, CXCL1). The CC class of chemokines has two adjacent Cys at the N-terminus and includes macrophage inflammatory protein 1 (MIP-lα, CCL3; MIP-ljSa, CCL4), adjusted to the activation of normal expressed and secreted T (RANTES, CCL5), monocyte chemoattractant protein -1 (MCP-I, CCL2). The CX3C class of chemokines contains two Cys separated by three residues at the N-terminus and are represented by fractalkine/neurotactin (CX3CL1). Class C chemokines contain a single N-terminal Cys and are represented by lymphotactin/ATAC/SCM (CL1). Chemokine receptors are grouped according to their chemokine binding selectivity. For example, CXCR4 binds to SDF-I and CXCR5 binds to chemokines that attract B cells (BCAl). The interaction of CXCR4 and SDF-1 plays an important role in multiple phases of the tumor formation process, including tumor growth, invasion, angiogenesis and metastasis.
[0003] CXCR4 is a receptor linked to a 7G transmembrane protein (GPCR) (Herzog et al., DNA Cell Biol. 12: 465 (1993); Rimland et al, Mol. Pharmacol. 40: 869 (1991) ; WO03014153, WO02061087). Many medically significant biological processes are mediated by signal transduction reactions involving G proteins (Lefkowitz, Nature, 351: 353 to 354, (1991)). G-coupled protein receptors (GPCRs) are integral membrane proteins containing 7 putative transmembrane domains (TMs). These proteins mediate signals into the cell through the activation of heterotrimeric G proteins which in turn activate various executing proteins, ultimately resulting in a psychological response, homeostasis and inflammation.
[0004] CXCR4 plays a role in embryogenesis shown to function as a coreceptor for T lymphotrophic HIV-I isolates (Feng, Y. et al, Science 272: 872 (1996)). CXCR4 also plays a pleiotropic role in human cancer. Its expression is genetically regulated in many types of tumors, including breast, lung, colon, pancreas, brain, prostate, ovarian, as well as hematopoietic cancers. Some reports in the literature suggest that SDF-1 may act through CXCR4 as a growth and/or survival factor for some tumors. CXCR4 is expressed in stem cells or tumor-initiating subpopulations of many tumors, and may mediate the ability of these cells to support the recurrence and metastatic development of cancers. Additionally, CXCR4 is expressed in endothelial precursor cells (EPCs), and its activity is required for incorporation of EPCs into functional vessels during angiogenesis. This can make a significant contribution to vascularization and tumor survival. CXCR4 signaling can also lead to the induction of pro-angiogenic cytokines (eg, VEGF), as well as integrins, adhesion molecules, and matrix-degrading enzymes that can mediate invasion by tumor cells. In addition, CXCR4 expression is detected in tumor-infiltrating lymphocytes and fibroblasts, as well as tumor-associated macrophages. These cells tend to suppress immune recognition and attack in the tumor, and rebuild the tumor microenvironment to encourage tumor growth and metastasis.
[0005] The multiple roles of CXCR4 in tumor growth, and metastasis, and its wide expression in many common tumor types, make this receptor an attractive target for therapeutic intervention using inhibitory agents. While peptides and small molecule inhibitors of CXCR4 and anti-CXCR antibodies have been identified or registered in the clinic, their usefulness has been limited by pharmacokinetic and toxicological properties. An agent, such as an antibody or antibody drug conjugate, that is selective, has a long half-life, improved efficacy, and safety profile would be a desirable agent for use in the treatment of cancers.
Although there are several agents under development that target CXCR4, there is a need for additional therapeutic agents that target CXCR4 (such as antibodies and antibody drug conjugates) that have improved efficacy and safety profile. And that they are suitable for use with human patients. The antibodies and antibody drug conjugates of the present invention are therapeutically useful anti-CXCR4 antibodies that possess a number of desirable properties such as reducing tumor formation, tumor growth, angiogenesis and metastasis. Additionally, antibodies and antibody drug conjugates of the present invention induce apoptosis of tumor cells. SUMMARY OF THE INVENTION
[0007] The present invention provides isolated antibodies, antigen binding fragments and their derivatives and antibody drug conjugates that bind to chemokine receptors 4 (CXCR4). The invention includes the amino acid sequences of the heavy and light chains of antibodies and their corresponding nucleic acid sequences.
[0008] In one aspect, the present invention includes the complementary determining region (CDR) sequences of antibodies to obtain binding molecules comprising one or more CDR regions, or derived CDR regions, which retain the CXCR4 binding capacity of the molecule from which the CDR(s) was (were) obtained.
[0009] In another aspect, the invention provides an antibody drug conjugate comprising the anti-CXCR4 antibodies described herein.
[0010] In another aspect, the invention comprises the use of anti-CXCR4 antibodies, their antigen binding fragments, and antibody drug conjugates and the corresponding processes, to detect and diagnose disorders associated with the expression or function of CXCR4 . In one aspect, the disorders are cancer disorders associated with an increased expression of CXCR4 and, relative to normal or any other pathology connected with overexpression of CXCR4.
[0011] In another aspect, the invention comprises products and/or compositions or kits comprising at least such an antibody, its antigen binding fragments, or antibody drug conjugates for the prognosis or diagnosis or monitoring of the therapy of certain cancers .
[0012] In one aspect, the invention provides an isolated antibody, or antigen-binding fragment thereof, that binds chemokine receptor 4 (CXCR4) and comprises: a) a variable heavy chain (VH) region comprising (i) ) a VH CDR1 selected from the group consisting of SEQ ID NOS: 107, 113, 114, 108, 109, 115, 116, 117, 121 and 122; (ii) a VH CDR2 selected from the group consisting of SEQ ID NOS: 162, 128, 110, 111, 118, 119, 154, 123, 158, 124, 159, 125, 160, 126, 161, 127, 163, 164 , 165, 166, 167, 168, 155, 129, 156, and 130, and, (iii) a VH CDR3 selected from the group consisting of SEQ ID NOS: 112, and 120; and/or; b) a variable light chain region (VL) comprising (i) a VL CDR1 selected from the group consisting of SEQ ID NOS: 144, 131, 135, 138, 141, 142, 143, 146, 147, 148, 149, 150 , and 151; (ii) a VL CDR2 selected from the group consisting of 145, 132, 136, and 152; and (iii) a VL CDR3 selected from the group consisting of SEQ ID NOS 139, 133, 137, 140, and 153.
[0013] In another aspect, the invention provides an isolated antibody, or antigen-binding fragment thereof, which binds chemokine receptor 4 (CXCR4) and comprises: a) a variable heavy chain (VH) region comprising determinant regions complements selected from the group consisting of (i) a VH CDR1 comprising the sequence set as SEQ ID NOS: 107, 113, 114, 108, 109, 115, 116, 117, 121 or 122; (ii) a VH CDR2 comprising the sequence adjusted as SEQ ID NOS: 162, 128, 110, 111, 118, 119, 154, 123, 158, 124, 159, 125, 160, 126, 161, 127, 163, 164 , 165, 166, 167, 168, 155, 129, 156, or 130; and (iii) a VH CDR3 comprising the sequence adjusted as SEQ ID NOS: 112, or 120; and/or b) a variable light chain region (VL) comprising complementary determining regions selected from the group consisting of (i) a VL CDR1 comprising the sequence set as SEQ ID NOS: 144, 131, 135, 138, 141, 142, 143, 146, 147, 148, 149, 150, or 151; (ii) a VL CDR2 comprising the sequence set as SEQ ID NOS 145, 132, 136, or 152; and (iii) a VL CDR3 comprising the sequence set as SEQ ID NOS 139, 133, 137, 140, or 153.
[0014] In another aspect, the invention provides an isolated antibody, or antigen-binding fragment thereof, which binds to chemokine receptor 4(CXCR4) and comprises: a) a light chain variable region (VL) comprising ( i) VL CDR1 comprising the sequence X1SX2X3SLFNSX4X5RKNYLX6 where X1 is R or K; X2 is S or A; X3 is W, N or Q; X4 is H or R; X5 is T or F; and/or X6 is A, L, N, or M (SEQ ID NO:151); (ii) a VL CDR2 comprising the sequence WASARX1S where X1 is G or E (SEQ ID NOS: 152), and (iii) VL CDR3 comprising the sequence KQSFX1LRT where X1 is N or R (SEQ ID NO: 153); and/or b) a variable heavy chain (VH) region comprising (i) VH CDR1 comprising the sequence set as SEQ ID NOS: 107, 108, 109, 113, or 114; (ii) a VH CDR2 comprising the sequence set as SEQ ID NO: 157 and (iii) a VH CDR3 comprising the sequence set as SEQ ID NO: 112.
[0015] In one aspect, the invention provides an isolated antibody, or antigen-binding fragment thereof, which binds to CXCR4 and comprises: a variable heavy chain region (VH) comprising the sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVR-QAPGKGLEWVX1FIRHKYX7LYSSQ3FTXNTXVQVTVXVTLXVDl SEQ ID NO: 106), where X1 is G or S; X2 is V or A; X3 is G, F, K, V, T, L or I; X4 is E or Y; X5 is T or R; X6 is W or S; X7 is D or V; X8 is K, T or R; X9 is D or N; X10 is T or S; X11 is R or K; X12 is A or T; and/or X13 is K or R.
[0016] In one aspect, the invention provides an isolated antibody or antigen-binding fragment thereof that binds to chemokine receptor 4 (CXCR4) is selected from the group consisting of: a) a variable light chain region (VL) having a VH CDR1 of SEQ ID NO: 107, 113 or 114; a VH CDR2 of SEQ ID NO: 162 or 128 and a VH CDR3 of SEQ ID NO: 112 and a variable light chain region (VL) having a VL CDR1 of SEQ ID NO: 144; a VL CDR2 of SEQ ID NO: 145 and a VL CDR3 of SEQ ID NO: 139; b) a VH region having a VH CDR1 of SEQ ID NO: 115, 116, 117, 121 or 122; a VH CDR2 of SEQ ID NO: 118 or 119 and a VH CDR3 of SEQ ID NO: 120 and a VL region having a VL CDR1 of SEQ ID NO: 135; a VL CDR2 of SEQ ID NO: 136 and a VL CDR3 of SEQ ID NO: 137; c) a VH region having a VH CDR1 of SEQ ID NO: 107, 108, 109, 113 or 114; a VH CDR2 of SEQ ID NO: 110 or 111 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 131; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 133; d) a VH region having a VH CDR1 of SEQ ID NO: 107, 108, 109, 113 or 114; a VH CDR2 of SEQ ID NO: 154 or 123 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 139; and e) a VH region having a VH CDR1 of SEQ ID NO: 107, 108, 109, 113 or 114; a VH CDR2 of SEQ ID NO: 157 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 151; a VL CDR2 of SEQ ID NO: 152 and a VL CDR3 of SEQ ID NO: 153.
[0017] In one aspect, the invention provides an isolated antibody or antigen binding fragment thereof is selected from the group consisting of: a variable heavy chain (VH) region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 162 and a VH CDR3 of SEQ ID NO: 112 and a variable light chain region (VL) having a VL CDR1 of SEQ ID NO: 144; a VL CDR2 of SEQ ID NO: 145 and a VL CDR3 of SEQ ID NO: 139; b) a VH region having a VH CDR1 of SEQ ID NO: 115; a VH CDR2 of SEQ ID NO: 118 and a VH CDR3 of SEQ ID NO: 120 and a VL region having a VL CDR1 of SEQ ID NO: 135; a VL CDR2 of SEQ ID NO: 136 and a VL CDR3 of SEQ ID NO: 137; c) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 110 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 131; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 133; d) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 154 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 139; and e) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 157 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 151; a VL CDR2 of SEQ ID NO: 152 and a VL CDR3 of SEQ ID NO: 153.
[0018] In one aspect, the invention provides an isolated antibody or antigen binding fragment thereof is selected from the group consisting of: a) a variable heavy chain (VH) region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 158 and a VH CDR3 of SEQ ID NO: 112 and a variable light chain region (VL) having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 139; b) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 158 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; c) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 158 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 150; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; d) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 158 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 141; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; e) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 158 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 144; a VL CDR2 of SEQ ID NO: 145 and a VL CDR3 of SEQ ID NO: 140; f) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 158 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 147; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; g) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 159 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; h) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 160 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; i) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 161 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; j) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 162 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; k) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 163 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; l) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 164 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; m) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 165 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; o) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 166 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; p) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 167 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; q) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 168 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; r) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 168 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; s) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 163 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 144; a VL CDR2 of SEQ ID NO: 145 and a VL CDR3 of SEQ ID NO: 140; t) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 158 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 144; a VL CDR2 of SEQ ID NO: 145 and a VL CDR3 of SEQ ID NO: 139; u) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 162 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 144; a VL CDR2 of SEQ ID NO: 145 and a VL CDR3 of SEQ ID NO: 139; v) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 163 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 144; a VL CDR2 of SEQ ID NO: 145 and a VL CDR3 of SEQ ID NO: 139; and w) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 162 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 144; a VL CDR2 of SEQ ID NO: 145 and a VL CDR3 of SEQ ID NO: 140. In particular aspects of the invention, the antibody or antigen binding fragment thereof comprises: a variable heavy chain (VH) region comprising three CDRs set as SEQ ID NOS: 107, 162 and 112. In some aspect, the antibody or antigen binding fragment thereof comprises: a variable light chain (VL) region comprising three CDRs set as SEQ ID NOS: 144, 145 and 139. In some aspects, the antibody or antigen binding fragment thereof comprises: a variable heavy chain (VH) region comprising three CDRs set as SEQ ID NOS: 107, 162 and 112; and a variable light chain region (VL) comprising three CDRs set as SEQ ID NOS: 144, 145 and 139.
In some aspects of the invention, the antibody or antigen binding fragment thereof comprises: a variable heavy chain (VH) region comprising VH CDR1, VH CDR2 and VH CDR3 from a VH region of SEQ ID NO: 33. In other aspects, the antibody or antigen binding fragment thereof comprises: a variable light chain (VL) region comprising VL CDR1, VL CDR2 and VL CDR3 from a VL region of SEQ ID NO: 73.
In some aspects of the invention, the isolated antibody, or antigen binding fragment thereof comprises: a variable heavy chain (VH) region comprising VH CDR1, VH CDR2 and VH CDR3 from a VH region of SEQ ID NO : 33; and a variable light chain (VL) region comprising VL CDR1, VL CDR2 and VL CDR3 from a VL region of SEQ ID NO: 73.
In some aspects of the invention, the isolated antibody or antigen binding fragment thereof is selected from the group consisting of: a) an antibody or antigen binding fragment thereof comprising a VH region of SEQ ID NO: 33 and a region VL of SEQ ID NO: 73; b) an antibody or antigen binding fragment thereof comprising a VH region of SEQ ID NO: 13 and a VL region of SEQ ID NO: 15; c) an antibody or antigen-binding fragment thereof comprising a VH region of SEQ ID NO:5 and a VL region of SEQ ID NO:7; d) an antibody or antigen binding fragment thereof comprising a VH region of SEQ ID NO:21 and a VL region of SEQ ID NO:47; and e) an antibody or antigen binding fragment thereof comprising a VH region of SEQ ID NO: 106 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132; and a VL CDR3 of SEQ ID NO: 139.
[0023] In some aspects of the invention, the isolated antibody or antigen fragment thereof comprises a variable heavy chain region having an amino acid sequence that is at least 95% identical to SEQ ID NO: 33 and a variable light chain having a sequence of amino acid that is at least 95% identical to SEQ ID NO: 73.
In particular aspects of the invention, the antibody or antigen binding fragment thereof comprises: a) a variable heavy chain (VH) region of SEQ ID NO: 33; and/or b) a variable light chain (VL) region of SEQ ID NO: 73.
In some aspects of the invention, the antibody or antigen binding fragment comprises a variable heavy chain (VH) region produced by the expression vector with ATCC Accession No. PTA-121353. In some aspects of the invention, the antibody or antigen binding fragment comprises a light chain variable region (VL) produced by the expression vector with ATCC Accession No. PTA-121354.
[0026] In some aspects of the invention, the isolated antibody or antigen-binding fragment thereof is selected from the group consisting of:
a) a variable heavy chain (VH) region having a variable heavy chain (VH) having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 158 and a VH CDR3 of SEQ ID NO: 112 and a variable light chain region (VL) having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 139;
b) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 158 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; c) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 158 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 150; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; d) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 158 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 141; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; e) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 158 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 144; a VL CDR2 of SEQ ID NO: 145 and a VL CDR3 of SEQ ID NO: 140; f) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 158 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 147; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; g) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 159 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; h) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 160 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; i) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 161 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; j) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 162 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; k) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 163 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; l) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 164 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; m) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 165 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; n) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 166 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; o) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 167 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; p) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 168 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; q) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 168 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 138; a VL CDR2 of SEQ ID NO: 132 and a VL CDR3 of SEQ ID NO: 140; r) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 163 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 144; a VL CDR2 of SEQ ID NO: 145 and a VL CDR3 of SEQ ID NO: 140; s) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 158 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 144; a VL CDR2 of SEQ ID NO: 145 and a VL CDR3 of SEQ ID NO: 139; t) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 162 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 144; a VL CDR2 of SEQ ID NO: 145 and a VL CDR3 of SEQ ID NO: 139; u) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 163 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 144; a VL CDR2 of SEQ ID NO: 145 and a VL CDR3 of SEQ ID NO: 139; and v) a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 162 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 144; a VL CDR2 of SEQ ID NO: 145 and a VL CDR3 of SEQ ID NO: 140.
[0029] In some aspects of the invention, an isolated antibody or antigen-binding fragment thereof, which binds CXCR4, competes to bind to CXCR4 with and/or binds to the same epitope of CXCR4 as an antibody or its binding fragment. antigen described here.
In some aspects of the invention, the isolated antibody or antigen binding fragment thereof comprises a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 162 and a VH CDR3 of SEQ ID NO: 112 and a VL region having a VL CDR1 of SEQ ID NO: 144; a VL CDR2 of SEQ ID NO: 145 and a VL CDR3 of SEQ ID NO: 139.
In some aspects of the invention, the antibody or antigen-binding fragment thereof is a humanized, chimeric, humanized CDR, or a recombinant human antibody. In other aspects of the invention, the antibody or antigen-binding fragment thereof is not caninized or felinized.
[0032] In some aspects of the invention, the antibody or antigen-binding fragment thereof comprises a tag containing modified acyl glutamine donor at a specific site.
In some aspects of the invention, drug conjugates of anti-CXCR4 antibodies are provided. The antibody drug conjugate of the invention is generally of the formula: Ab-(TLD), where: Ab is an antibody or antigen-binding fragment thereof that binds to the chemokine receptor 4 (CXC4), T is a label containing acyl donor which may optionally be included, is a linker and D is a drug.
In particular aspects, the antibody drug conjugate of the invention is selected from the group consisting of: a) an antibody or antigen binding fragment thereof comprising a VH region comprising CDRs of SEQ ID NOS: 107, 162 and 112 and a VL region comprising CDRs of SEQ ID NOS: 144, 145 and 139; b) an antibody or antigen binding fragment thereof comprising a VH region comprising CDRs of SEQ ID NOS: 115, 118 and 120 and a VL region comprising CDRs of SEQ ID NOS: 135, 136 and 137; c) an antibody or antigen binding fragment thereof comprising a VH region comprising CDRs of SEQ ID NOS: 107, 110 and 112 and a VL region comprising CDRs of SEQ ID NOS: 131, 132 and 133; d) an antibody or antigen binding fragment thereof comprising a VH region comprising CDRs of SEQ ID NOS: 107, 154 and 112 and a VL region comprising CDRs of SEQ ID NOS: 138, 132 and 139; e) an antibody or antigen binding fragment thereof comprising a VH region comprising CDRs of SEQ ID NOS: 107, 157 and 112 and a VL region comprising CDRs of SEQ ID NOS: 151, 152 and 153; f) an antibody or antigen binding fragment thereof comprising a VH region of SEQ ID NO:33 and a VL region of SEQ ID NO:73; g) an antibody or antigen binding fragment thereof comprising a VH region of SEQ ID NO: 13 and a VL region of SEQ ID NO: 15; h) an antibody or antigen binding fragment thereof comprising a VH region of SEQ ID NO:5 and a VL region of SEQ ID NO:7; and i) an antibody or antigen binding fragment thereof comprising a VH region of SEQ ID NO:21 and a VL region of SEQ ID NO:47.
In some antibody drug conjugates of the invention, the Ab is a grafted, humanized, chimeric CDR, or a recombinant human antibody or antigen-binding fragment thereof. In some antibody drug conjugates of the invention, the T is selected from the group consisting of SEQ ID NOS: 171, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 172, 173, 102, 103 and LLQ. In some antibody drug conjugates of the invention, L is selected from the group consisting of acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), amino PEG6-propionyl, maleimidocapronic-valine-citrulline- p-aminobenzyloxycarbonyl (vc) and maleimidocaproyl (mc). In some antibody drug conjugates of the invention, D is selected from the group consisting of a cytotoxic agent, an immunomodulatory agent, an imaging agent, a therapeutic protein, a biopolymer, and an oligonucleotide.
Any antibody drug conjugate of the invention can be prepared with a drug that is a cytotoxic agent. The cytotoxic agent may be an anthracycline, an auristatin, a camptothecin, a combretastein, a dolastatin, a duocarmycin, an enediin, a geldanamycin, an indoline-benzodiazepine dimer, a maytansine, a puromycin, a pyrrolo-benzodiazepine dimer, a taxane, an alkaloid vinca, a tubulisin, a hemiasterlin, a spliceostatin, a pladienolide, and calicheamicin. Any antibody drug conjugate of the invention can be prepared with a drug that is auristatin. In one aspect of the invention, the auristatin may be 0101 (2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy -2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl -1-oxoheptan-4-yl]-N-methyl-L-valinamide), MMAD (Monomethyl Auristatin D or monomethyl dolastatin 10) and 8261 (2-Methylalanyl-N-[(3R,4S,5S)-1 -{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine-1- yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide).
In particular aspects, the antibody drug conjugate of the invention is selected from the group consisting of: a) Ab-LLQGA (SEQ ID NO: 91)-(acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys- VC-PABC))-0101; Ab-LLQGA (SEQ ID NO: 91)-(AcLys-VC-PABC)-MMAD; c) Ab-LLQX1X2X3X4X5 (SEQ ID NO: 102)-(AcLys-VC-PABC)-0101; d) Ab-LLQX1X2X3X4X5 (SEQ ID NO: 102)-(AcLys-VC-PABC)-MMAD; e) Ab-GGLLQGA (SEQ ID NO: 92)-(AcLys-VC-PABC)-0101; and f) Ab-GGLLQGA (SEQ ID NO: 92)-(AcLys-VC-PABC)-MMAD.
In some aspects of the invention, a CXCR4 antibody drug conjugate comprises any of the antibodies or antigen binding fragments thereof described herein. In a particular aspect of the invention, the drug conjugate of the invention comprises an antibody wherein the antibody comprises a VH region comprising CDRs of SEQ ID NOS: 107, 162 and 112 and a VL region comprising CDRs of SEQ ID NOS: 144, 145 and 139.
[0039] Pharmaceutical compositions comprising CXCR4 antibody, its antigen binding fragment, or an antibody drug conjugate described herein and a pharmaceutically acceptable carrier are also provided.
In some aspects of the invention, isolated polynucleotides comprising a nucleotide sequence encoding any of the anti-CXCR4 antibodies or antigen binding fragments thereof described herein are provided. In some aspects, host cells that combined produce the antibody or antigen-binding fragment thereof described herein are provided.
[0041] In other aspects, methods of treating a disorder associated with CXCR4 function or expression in a person are provided comprising administering to the person an effective amount of the pharmaceutical composition of the present invention. In particular aspects of the invention, the disorder is cancer.
[0042] In other aspects, methods are provided for decreasing the metastasis of CXCR4-expressed cancer cells in a person, comprising administering to the person in need thereof an effective amount of the pharmaceutical composition described herein. In other aspects, methods are provided which induce tumor regression in a person having a CXCR4 expressed tumor, comprising administering to the person in need thereof an effective amount of the pharmaceutical composition described herein.
[0043] These and other aspects of the invention will be appreciated by a review of the application as a whole. BRIEF DESCRIPTION OF THE DRAWINGS
[0044] Figure 1 shows the alignment of the variable Heavy Chain (VH) h3G10.
[0045] Figure 2 shows the alignment of the variable Light Chain (VL) h3G10.
Figure 3A shows the cross-reactivity of anti-human CXCR4 Ab h3G10.1.91.A58B to cynomolgus CXCR4 by flow cytometry in a dilution series (0.007 - 267 nM) in HPB-ALL (human leukemia T cells ) and transfected CHO cells.
Figure 3B shows the cross-reactivity of anti-human CXCR4 CXCR4 Ab h3G10.1.91.A58B to cynomolgus CXCR4 by flow cytometry in a dilution series (0.007 - 267 nM) in Raji (NHL; Human Non-Hodgkin's Lymphoma ) and HSC-F (cynomologist T cell culture).
Figure 4 shows the ADCC(A-C) and CDC(D-F) activities of anti-human CXCR4 antibodies in cultures of human cells displaying CXCR4.
Figure 4A shows the activities of anti-human CXCR4 ADCC antibodies of 12A11, 6B6 and 3G10 on Ramos cells (NHL).
Figure 4B shows the comparison of ADCC activity of anti-human CXCR4 antibodies on MOLT-4 (acute T-lymphoblastic leukemia) cells.
[0051] Figure 4C shows the ADCC activities of mouse (m3G10-) and humanized (h3G10-) anti-human CXCR4 antibodies on MOLT-4 (acute T-lymphoblastic leukemia) cells.
[0052] Figure 4D shows the CDC activities of CXCR4 anti-human 12A11, 6B6 and 3G10 antibodies in Ramos cells (NHL).
[0053] Figure 4E shows the comparison of CDC activity of anti-human CXCR4 antibodies m3G10-hIgG1 and m3G10-hIgG4 in Daudi cells (NHL).
[0054] Figure 4F shows the CDC activity of mouse anti-human (m3G10-) and humanized (h3G10-) CXCR4 antibodies in Daudi cells (NHL).
[0055] Figure 5 shows the inhibition of calcium flux by the anti-human CXCR4 antibodies 3G10, 6B6 and 12A11.
[0056] Figure 6A shows that anti-human CXCR4 antibodies 3G10, 6B6 and 12A11 are able to induce cell death in Ramos cells in a dose-dependent manner.
[0057] Figure 6B shows that the ability of the 3G10's anti-human CXCR4 antibody to induce cell death is dependent on bivalence.
[0058] Figure 7 shows that the anti-human CXCR4 antibodies 3G10, 6B6 and 12A11 significantly inhibit tumor growth in a Non-Hodgkin's Lymphoma (NHL) model (Ramos) compared to the isotype control antibody.
Figure 8A shows that the anti-human CXCR4 antibodies 3G10, 6B6 and 12A11 have a significant effect compared to the isotype control antibody on the tumor burden of animals systemically implanted with Raji-LUC cells.
[0060] Figure 8B shows that treatment with anti-human CXCR4 antibodies 3G10, 6B6 and 12A11 had comparable activity and significant tumor growth inhibition (TGI) relative to the isotype control antibody in this model.
[0061] Figure 8C is a survival curve showing the significant effect of anti-human CXCR4 antibodies 3G10, 6B6 and 12A11 compared to the isotype control antibody on the survival of animals systemically implanted with Raji-LUC cells.
Figure 9A shows the effect of CXCR4 Ab 6B6 on tumor burden in an acute myeloid leukemia (AML) model (MV4-11-LUC). The representative bioluminescence image of 5 animals/treatment group from Day 20 to Day 41 is shown.
Figure 9B shows the significant effect of anti-CXCR4 antibody 6B6 compared to isotype control antibody on the survival of animals systemically implanted with MV4-11-LUC AML cells.
Figure 9C shows the significant reduction in human AML tumor burden in Peripheral Blood (PB) of animals treated with anti-CXCR4 antibody 6B6 compared to animals treated with isotype control on Day 35 of the study.
[0065] Figure 10A shows the representative image of luciferase in mice with Chronic Systemic Lymphocyte Leukemia Tumors treated with the antibody CXCR4 3G10.
[0066] Figure 10B is a survival curve showing the significant effect of the anti-human 3G10 CXCR4 antibody compared to the isotype control antibody on the survival of animals systemically implanted with Chronic Lymphocyte Leukemia cells.
[0067] Figure 11 shows humanized anti-human CXCR4 antibodies significantly inhibited tumor growth in a Non-Hodgkin's Lymphoma (NHL) model (Ramos) compared to the isotype control antibody.
[0068] Figure 12A shows the effect of CXCR4 Ab h3G10.1.91.A58B on tumor burden in a multiple myeloma (MM) model (OPM-2-LUC). The representative bioluminescence image of 5 animals/treatment group from Day 8 to Day 30 is shown.
[0069] Figure 12B shows the quantification of bioluminescence (Luciferase activity) in the Systemic Multiple Myeloma (MM) Model of Mice treated with the anti-human CXCR4 antibody h3G10.1.91.A58B, compared to animals treated with the isotype control antibody and Melphalan.
[0070] Figure 12C is a survival curve showing the significant effect of a CXCR4 h3G10.1.91.A58B antibody compared to the isotype control antibody and to Melfalan in mice systemically implanted with OPM-2-Luc MM cells.
Figure 13A shows the significant antitumor effect of CXCR4 ADCs 3G10-TG6-vc0101 and 6B6-TG6-vc0101 in the Ramos tumor model (NHL).
Figure 13B shows the significant antitumor effect of CXCR4 ADC 3G10-TG6-vc0101 in the HPB-ALL (T-ALL) tumor model. DESCRIPTION DETAILED INVENTION
The invention described herein provides antibodies and antibody conjugates (eg, antibody drug conjugates) that bind to CXCR4 (eg, human CXCR4). The invention also provides polynucleotides encoding such antibodies, compositions comprising such antibodies, and methods of producing and using such antibodies. In certain aspects, the antibodies of that disclosure are derived from particular heavy and light chain germline sequences and/or comprise particular structural features such as CDR regions comprising particular amino acid sequences. This description provides isolated antibodies, methods of producing such antibodies, their antigen binding fragments, antibody drug conjugates, and bispecific molecules of this description. This description also refers to methods of using the antibodies, such as detecting CXCR4, modulating CXCR4 activity, and/or targeting CXCR4 expressing cells for destruction (eg, ADCC, CDC, toxin), in disorders associated with the function or expression of CXCR4 such as cancer. The invention also provides methods for preventive and/or therapeutic treatment of a disorder associated with CXCR4 function or expression in a person, such as cancer (e.g. solid tumor cancers or hematologic cancers). Finally, the invention comprises compositions comprising such antibodies in conjugation (e.g. antibody drug conjugates) or in combination with other anti-cancer compounds such as antibodies, toxins, cytotoxics/cytostatics, and the use thereof for prevention and/or treatment of disorders associated with CXCR4 function or expression such as cancer. GENERAL TECHNIQUES
[0074] The practices of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the scope of the art. Such techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al, 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, edition, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, edition, 1998) Academic Press; Animal Cell Culture (R.I. Freshney, edition, 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, editions, 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, editions); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Callos, editions, 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al, editions, 1987); PCR: The Polymerase Chain Reaction, (Mullis et al, editions, 1994); Current Protocols in Immunology (J.E. Coligan et al, editions, 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, editions, Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); and, The Antibodies (M. Zanetti and J.D. Capra, editions, Harwood Academic Publishers, 1995). DEFINITIONS AND ABBREVIATIONS
The terms "Chemokine (C-X-C motif) receptor 4" and "CXCR4" as used herein refer to a G protein-coupled 7-transmembrane domain chemokine receptor that is normally embedded in the membrane of a cell. Terms also include variants, isoforms, homologs, orthologs, and paralogs. Human CXCR4 nucleic acid and polypeptide sequences are described as GenBank Accession NOS. NM-003467 and NP-003458, respectively. Another description of human CXCR4 can be found in Federsppiel, B. et al Genomics 16(3): 707 to 712 (1993); Herzog, H. et al DNA Cell Biol. 12(6): 465 to 471 (1993); Jazin, E.E. et al Regul. Pept 47(3): 247 to 258 (1993); Nomura, H.E et al Int. Immunol. 5(10): 1,239 to 1,249 (1993); Loetscher, M. et al J. Biol. Chem. 269(1): 232 to 237 (1994); Moriuchi, M. et al J. Immunol. 159(9): 4,322 to 4,329 (1997); Caruz, A. et al FEBS Lett. 426(2): 271 to 278 (1998); and Wegner, S.A. et al J. Biol. Chem. 273(8): 4,754 to 4,760 (1998).
[0076] CXCR4 is also known in the art, for example, as LESTR, CD 184, CD184 antigen, CXC chemokine receptor type 4, CXCR-4, CXCL-12, CXCR-R4, D2S201E, FB22, fusion, Fusin, HM89 , HSY3RR, LAP3, LCR1, leukocyte-derived transmembrane domain 7 receptor, NPY3R receptor, NPYR, NPYRL, NPYY3R, SDF-1, or stromal cell-derived factor-1 receptor.
For purposes of the present invention, the term "CXCR4 antigen" encompasses any CXCR4, including human CXCR4, other mammalian CXCR4 (such as mouse CXCR4, rat CXCR4, canine CXCR4, feline CXCR4, or CXCR4 from primates) as well as different forms of CXCR4 (eg, glycosylated CXCR4).
The terms "antibody" and "Ab" as used herein refer to an immunoglobulin molecule capable of recognizing and binding to a target specific antigen, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc. , through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" can encompass any type of antibody, including, but not limited to monoclonal antibodies, polyclonal antibodies, and antigen-binding fragments of intact antibodies that retain the ability to specifically bind a given antigen (by example, CXCR4), bispecific antibodies, heteroconjugate antibodies, their mutants, fusion proteins having an antibody, single chain antibodies (ScFv) and single domain (eg shark and camelids), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology 23(9): 1126 to 1136), humanized antibodies, chimeric antibodies, and any other modified configuration of the immuno-molecule molecule. globulin that includes an antigen recognition site of the required specificity, including antibody glycosylation variants, antibody amino acid sequence variants, and antibodies covalently modified. Antibodies can be murine, rat, human, or of any other origin (including chimeric or humanized antibodies). In some aspects of the invention, the antibody, or antigen binding fragment thereof for use in the methods of the invention is a chimeric, humanized or recombinant human antibody, or CXCR4 binding fragment thereof.
[0079] There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these can also be divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant regions that correspond to different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.
Native or naturally occurring antibodies are typically heterotetrameric glycoproteins of about 150,000 daltons composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has an end in a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain, and the variable domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. It is believed that particular amino acid residues form an interface between the variable domains of the light and heavy chains. The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies.
The terms "antigen-binding fragment", "antigen-binding portion", and "antibody portion" as used herein refer to one or more fragments of an intact antibody that retain the ability to bind an given antigen (eg, CXCR4). The antigen binding functions of an antibody can be performed by fragments of an intact antibody. Examples of antigen binding fragments include Fab; Fab'; F(ab')2; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a single domain antibody (dAb) fragment (Ward et al, Nature 341: 544 to 546, 1989), an isolated complementarity determining region (CDR), a nanobody, and a variable heavy chain region containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded for separate genes, they can be joined, using recombinant methods, by a synthetic linker that allows them to be made as a single protein chain in which the VL and VH regions join to form monovalent molecules (known as single-chain Fv (scFv), see, for example, Bird et al, Science 242: 423 to 426 (1988), and Huston et al, PNAS 85: 5,879 to 5,883 (1988)). Such single chain antibodies can also be encompassed within the term "antigen binding portion" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are classified to be used in the same manner as intact antibodies.
[0082] The term "CDR" as used herein refers to a region in the variable domain of an antibody that confers its binding specificity. There are 3 CDRs in which each heavy and light chain of an antibody. Amino acid residues within the variable region that make up the CDRs can be identified using methods known in the art including, but not limited to, Kabat (Kabat et al, 1992, Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, NIH, Washington DC), Chothia (Chothia et al, 1989, Nature 342: 877 to 883), the accumulation of Kabat and Chothia, definition of AbM (which is a compromise between Kabat and Chothia is derived using Oxford Molecular's AbM antibody modeling software (now Accelrys®), contact definition (MacCallum et al, 1996, J. Mol. Biol., 262: 732 to 745), and/or adaptive definitions (Makabe et al, 2008, Journal of Biological Chemistry, 283: 1156 to 1166.) As used herein, a CDR may refer to CDRs defined by any approach known in the art, including combinations of approaches.The methods used herein may use CDRs defined according to any of these approaches.
The term "Fc region" as used herein refers to a C-terminal region of an immunoglobulin heavy chain. The "Fc region" can be a native Fc region sequence or an Fc region variant. Although the edges of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined to expand from an amino acid residue at position Cys226, or from Pro230, to its carboxy terminus. . Residue numbering in the Fc region is that of the EU index as in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
The term "Fc region" as used herein refers to a C-terminal region of an immunoglobulin heavy chain. The "Fc region" can be a native Fc region sequence or an Fc region variant. Although the edges of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined to expand from an amino acid residue at position Cys226, or from Pro230, to its carboxy terminus. . Residue numbering in the Fc region is that of the EU index as in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
The term "isolated antibody" as used herein refers to an antibody that is substantially free of other antibodies having different antigenic specificities (for example, an isolated antibody that specifically binds to CXCR4 is substantially free of antibodies that bind to antigens other than CXCR4). An isolated antibody that specifically binds to CXCR4 may, however, be cross-reactive to other antigens, such as CXCR4 molecules from other species. Furthermore, an isolated antibody can be substantially free of other cellular materials and/or chemicals.
The terms "humanized antibody" and "CDR grafted antibody" as used herein refer to non-human (e.g., murine) forms of antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab , Fab', F(ab')2 or other antibody antigen binding subsequences) which contain a minimal sequence derived from non-human immunoglobulins. Preferably, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR from a non-human species (donor antibody) such as a mouse, rat, or rabbit that have the desired specificity, affinity, and capacity.
In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are discovered neither in the recipient antibody nor in the imported CDR nor in the framework sequences, but are included to further refine and optimize the performance of the antibodies. In general, the humanized antibody will comprise substantially all or at least one, and typically two, variable domains in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a consensus sequence. of human immunoglobulin. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Antibodies having modified Fc regions as described in WO 99/58572 are preferred. Other forms of humanized antibodies have one or more CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, or CDR H3) that are altered from the original antibody, which are also called one or more "derived from " one or more CDRs from the original antibody.
Humanization can essentially be performed following the method of Winter and partners (Jones et al Nature 321: 522 to 525 (1986); Riechmann et al. Nature 332: 323 to 327 (1988); Verhoeyen et al Science 239: 1534 to 1536 (1988)), by substituting CDRs or CDR sequences from rodents or mutant rodents with the corresponding sequences from a human antibody. See also US Patents 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205; incorporated herein by reference. In some cases, residues within the framework regions of one or more human immunoglobulin variable regions are replaced by corresponding non-human residues (see, for example, US Patents 5,585,089; 5,693,761; 5,693,762; and 6,180. 370). Furthermore, humanized antibodies may comprise residues that are not discovered in the recipient antibody or in the donor antibody. These modifications are made to also refine the performance of the antibodies (eg, obtain the desired affinity). In general, the humanized antibody will comprise substantially all or at least one, and typically two, variable domains, in which all or substantially all hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all framework regions those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details see Jones et al. Nature 331: 522 to 525 (1986); Riechmann et al Nature 332: 323 to 329 (1988); and Provision Curr. Op. Structure Biol. 2: 593 to 596 (1992); incorporated herein for reference. Accordingly, such "humanized" antibodies can include antibodies where substantially less than an intact human variable domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework residues are replaced by residues from analogous sites in rodent antibodies. See, for example, US Patents 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205. See also US Patent No. 6,180,370, and International Publication No. WO 01/27160, where humanized antibodies and techniques for producing humanized antibodies having improved affinity for a predetermined antigen are described.
[0089] The term "human antibody" as used herein refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and/or which has been made using any of the techniques for producing human antibodies. known to those skilled in the art or described herein. Such a definition of a human antibody includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. An example of this is an antibody comprising murine light chain and human heavy chain polypeptides. Human antibodies can be produced using various techniques known in the art. In one embodiment, the human antibody is selected from a phage library, which phage library expresses human antibodies (Vaughan et al, Nature Biotechnology, 14: 309 to 314, (1996); Sheets et al, Proc. Natl. Acad. Sci. (USA) 95: 6,157 to 6162, (1998); Hoogenboom and Winter, J. Mol. Biol., 227: 381, (1991); Marks et al, J. Mol. Biol., 222 :581, (1991)). Human antibodies can also be produced by immunizing animals into which human immunoglobulin has been transgenically introduced in place of endogenous sites, for example, mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in US Patents 5,545,807; 5,545,806; 5,569,825; 5,625.126; 5,633,425; and 5,661.016. Alternatively, the human antibody can be prepared by immortalizing human B lymphocytes which produce an antibody directed against a target antigen (such B lymphocytes may be recovered by cloning a single or individual cell from cDNA, or may have been immunized in vitro). See, for example, Cole et al Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, (1985); Boerner et al, J. Immunol., 147 (1): 86 to 95, (1991); and US Patent 5,750,373.
The term "chimeric antibody" as used herein refers to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the region sequences are derived variables are derived from a mouse antibody and constant region sequences are derived from a human antibody. For veterinary applications, the constant domains of the chimeric antibody can be derived from those other species, such as a cat or a dog.
The terms "canine antibodies" and "feline antibodies" as used herein refer to chimeric antibodies useful as therapeutics in canines and felines, respectively. In both cases, an antigen binding domain from a donor antibody from the heterologous species is combined with a non-antigen binding domain from a recipient antibody from the same species.
The terms "preferably bound" or "specifically bound" as used herein when used in the context of binding an antibody to a target (e.g., CXCR4 protein) is a term well understood in the art. Methods to determine such specific or preferential binding are also known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, with longer duration and/or with greater affinity with a particular cell or substance than with alternative cells or substances. An antibody "specifically binds" or "binds preferentially" to a target if it binds with greater affinity, avidity, more readily, and/or longer duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to a CXCR4 epitope is an antibody that binds to that epitope with greater affinity, avidity, more readily and/or longer than it binds to another CXCR4 epitope or non-epitope. CXCR4.
[0093] The term "binding affinity" and "KD" as used herein is intended to refer to an equilibrium constant dissociation of a particular antibody-antigen interaction. The KD is the ratio of the dissociation rate, also called the "rate constant for association" or "kd" to the rate of association, or "rate constant for dissociation" or "ka". Thus, KD is equal to kd/ka and is expressed as a molar concentration (M). It follows that the smaller the KD, the stronger the binding affinity. Therefore, a KD of 1 µM indicates weak binding affinity compared to a KD of 1 nM. KD values for antibodies can be determined using methods well established in the art. One method of determining the KD of an antibody is using surface plasmon resonance, typically using a biosensor system such as a BIACORE® system.
The term "compete" as used herein in relation to an antibody means that a first antibody binds to an epitope sufficiently similar to binding a second antibody such that the result of binding the first antibody with its epitope. cognate po is detectably decreased in the presence of the second antibody compared to binding of the first antibody in the absence of the second antibody. Antibodies that compete for epitope binding with an antibody of the invention are encompassed by the present invention. Those of skill would appreciate, based on the teachings provided herein, that such competing antibodies may be useful for the methods described herein.
[0095] The term "drug" as used herein refers to any substance that has biological or detectable activity. The term drug should encompass cytotoxic agents, therapeutic agents, immunomodulating agents, detectable labels, imaging agents, binding agents, prodrugs (which are metabolized to an active agent in vivo), growth factors, hormones, cytokines, anti- hormones, xanthines, interleukins, interferons and cytotoxic drugs. Drugs can be small molecules, polypeptides, oligonucleotides or biopolymers. The drug can be conjugated to an antibody of the invention via a linker to form an antibody-drug conjugate.
[0096] The term "causing function" as used herein refers to biological activities attributable to the Fc region of an antibody. Examples of the causative functions of an antibody include, but are not limited to cytotoxicity with antibody-dependent cell-mediated immunity (ADCC), Fc receptor binding, complement dependent cytotoxicity (CDC), phagocytosis, C1q binding, impaired regulation of Fc receptors. cell surface (eg, B cell receptor; BCR). See, for example, Patent No. 6,737,056. Such causative functions generally require that the Fc region be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays known in the art to assess such antibody causative functions. An exemplary measure of causative function is via the Fcy3 and/or C1q binding.
The term "epitope" as used herein includes any protein determinant capable of binding to an immunoglobulin or T-cell receptor or of interacting with a molecule. Epitopic determinants generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally have specific three dimensional structural characteristics as well as specific charge characteristics. An epitope can be "linear" or "adaptive". In a linear epitope, all points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the main amino acid sequence of the protein. In an adaptive epitope, points of interaction occur through amino acid residues in the protein that are separated from each other. Once a desired epitope or antigen is determined, it is possible to generate antibodies to that epitope, for example, using the techniques described in the present invention. Alternatively, during the discovery process, the generation and characterization of antibodies can elucidate information about desirable epitopes. From this information, it is then possible to competitively classify antibodies for binding to the same epitope. One approach to achieving this is to conduct competition studies to discover antibodies that competitively bind to each other, that is, the antibodies compete to bind to the epitope. A high throughput process for "discarding" antibodies on the basis of their competition is described in International Patent Application No. WO 03/48731. As used herein, the term "discard" refers to a method of grouping antibodies based on their antigen-binding characteristics.
The terms "polynucleotide", "nucleic acid/nucleotide" and "oligonucleotide" as used herein refer to polymeric forms of nucleotides of any length, or deoxyribonucleotides or ribonucleotides, their analogs, or any substrate that may be incorporated into a strand by DNA or RNA polymerase. "Polynucleotides" can have any three-dimensional structure, and can perform any function, known or unknown. The following examples are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, messenger RNA (mRNA), carrier RNA, RNA in ribosomes, ribozymes, DNA, cDNA, genomic DNA, recombinant polynucleotides, polynucleotides branches, plasmids, vectors, DNA isolated from any sequence, RNA isolated from any sequence, nucleic acid probes and primers. Polynucleotides can be naturally occurring, synthetic, recombinant, or any combination thereof. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogues. If present, the modification to the nucleotide structure can be transmitted either before or after strand assembly. The nucleotide sequence can be interrupted by non-nucleotide components. A polynucleotide can also be modified after polymerization, such as by conjugation with a component identification. Other types of modifications include, for example, "capsules", replacement of one or more of the naturally occurring nucleotides by an analogue, internucleotide modifications such as, for example, those with uncharged bonds (eg, methyl phosphanates, phosphotriesters, phosphoamidates, internucleotide modifications such as, for example, those with uncharged bonds (e.g., methyl phosphanates, phosphoamidates, carbamates, etc.) and with charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.). ), those containing pendant moieties, such as, for example, proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (eg, acridine, psoralen , etc.), those containing chelators (eg metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified bonds (eg alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Furthermore, any of the hydroxyl groups commonly present on sugars can be substituted, for example, by phosphanate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional bonds to additional nucleotides, or can be attached to solid supports. The 5' and 3' OH terminals can be phosphorylated or substituted by amines or moieties of organic covering groups of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups. Polynucleotides may also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido -ribose, carbocyclic sugar analogues, alpha- or beta-anomeric sugars, epimeric sugars such as arabinose, xyloses or lixoses, pyranose sugars, furanose sugars, sedoeptuloses, acyclic analogues and abasic nucleoside analogues such as methyl ribosides. One or more phosphodiester linkages can be replaced with alternative linking groups. Such alternative linking groups include, but are not limited to configurations where the phosphate is replaced by P(O)S("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate") , P(O)R, P(O)OR', CO or CH2 ("formacetal"), in which each R or R' is independently H substituted or unsubstituted (1-20 C) alkyl optionally containing an ether linkage ( -O-), aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all bonds in a polynucleotide need to be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.
The terms "polypeptide", "oligopeptide", "peptide" and "protein" as used herein refer to chains of amino acids of any length, preferably relatively short (eg, amino acids 10-100). The chain can be straight or branched, it can comprise modified amino acids, and/or it can be interrupted by non-amino acids. The terms also encompass an amino acid chain that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a component identification. Also included in the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that polypeptides can occur as single chains or associated chains.
[00100] Amino acids may be referred to here either by their commonly known three letter symbols or by the one letter symbols recommended by IUPAC-IUB Biochemical.
The terms "amino acid" and "natural amino acid" as used herein refer to arginine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, glycine, alanine, histidine, serine, proline, glutamic acid, aspartic acid, threonine , cystine, methionine, leucine, asparagine, isoleucine and valine.
The term "derivative amino acid" as used herein refers to an amino acid that has substitutions or modifications by covalently bonding a matrix amino acid, such as, for example, by alkylation, glycosylation, acetylation, phosphorylation, etc. In addition, included within the definition of "derivatives" are, for example, one or more analogs of an amino acid with substituted bonds, as well as other modifications known in the art.
[00103] The term "vector" as used herein refers to a construct capable of delivering, and preferably expressing, one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, naked DNA or RNA expression vectors, plasmids, cosmids or phagotyping vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors involved in liposomes, and certain eukaryotic cells, such as producer cells.
[00104] The term "percent sequence identity" as used herein refers to the degree of similarity between two sequences. In the context of nucleic acid sequences, it means residues in two sequences that are the same when aligned for maximum correspondence. The length of the sequence identity comparison can be over the range of at least about nine nucleotides, generally at least about 18 nucleotides, more generally at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 28 nucleotides about 32 nucleotides, and preferably at least 36, 48 or more nucleotides. There are a number of different algorithms known in the art that can be used to measure nucleotide sequence identity. For example, polynucleotide sequences can be compared using FASTA, Gap, or Bestfit, which are programs from Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin. FASTA, which includes, for example, the FASTA2 and FASTA3 programs, provide alignments and percent sequence identity of the regions of best overlap between the query and search sequences. (Pearson, Methods Enzymol. 183: 63 to 98 (1990); Pearson, Methods MoI. Biol. 132: 185 to 219 (2000); Pearson, Methods Enzymol. 266: 227 to 258 (1996); Pearson, J. MoI Biol 276: 71 to 84 (1998); incorporated herein by reference). Unless otherwise specified, default parameters are used for a particular program or algorithm. For example, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word length of 6 and the NOPAM factor for the score matrix) or using the Gap with its default parameters as provided in GCG Version 6.1, incorporated herein by reference.
[00105] Reference to a nucleotide sequence encompasses its complement unless otherwise specified. Thus, reference to a nucleic acid having a particular sequence is to be understood as encompassing its complementary streak, with its complementary sequence.
[00106] In the context of amino acid sequences, it means that two amino acid sequences, when optimally aligned, such as by the GAP or BESTFIT programs using the standard gap weights as provided by the programs, divide at least 70%, 75% or 80% of the sequence identity, preferably at least 90% or 95% sequence identity, and more preferably at least 97%, 98% or 99% sequence identity. In some substantially similar amino acid sequences, residue positions that are not identical differ by conservative amino acid substitutions.
[00107] Substantially similar polypeptides also include conservatively substituted variants in which one or more residues have been conservatively substituted by a functionally similar residue. Examples of conservative substitutions include replacing a non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine with another; the replacement of a polar (hydrophilic) residue by another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine; replacing a basic residue such as lysine, arginine or histidine with another; or replacing one acidic residue such as aspartic acid or glutamic acid with another.
[00108] Another indication that two proteins are substantially identical is that they share an overall three-dimensional structure, or are biologically functional equivalents.
[00109] The term "cytotoxic activity" as used herein refers to a cell killing effect, a cytostatic effect, or an anti-proliferative effect of an antibody, an ADC, or an intracellular metabolite of said ADC. Cytotoxic activity can be expressed as the IC50 value, which is the concentration (molar or mass) per unit volume at which half of the cells survive.
[00110] The term "contact" as used herein refers to bringing an antibody or its antigen-binding portion of the present invention and a CXCR4 target, or its epitope, together in such a way that the antibody can affect the biological activity of CXCR4 . Such "contact" can be consummated "in vitro," that is, in a test tube, Petri dish or similar. In a test tube, contact may involve only an antibody or its antigen-binding portion and CXCR4 or its epitope, or it may involve whole cells. Cells can also be maintained or grown in cell culture dishes and contacted with antibodies or their antigen-binding portions in that environment. In this context, the ability of a particular antibody or its antigen-binding portion to affect the CXCR4-related disorder, i.e., the IC50 of the antibody, can be determined before using the antibody in vivo with more complex living organisms. For cells outside the organism, multiple methods exist, and are well known to those skilled in the art, to contact CXCR4 with the antibodies or antigen binding fragments thereof. In another setting, a significant amount of effectiveness is generated by the causative function. Immune causative functions that have been shown to contribute to antibody-mediated cytotoxicity include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement dependent cytotoxicity (CDC).
The term "tag containing acyl glutamine donor" and "glutamine" as used herein refer to a polypeptide or protein containing one or more Gln residues that act as an acceptor of amine transglutaminase. See, for example, WO2012059882.
[00112] The term "pharmaceutically acceptable carrier" and "pharmaceutically acceptable excipient" as used herein refers to any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the immune system of the individual. Examples include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Standard pharmaceutical carriers include, but are not limited to phosphate buffered saline, water, emulsions such as oil/water emulsion, and various types of wetting agents. Preferred diluents for worse aerosol or parenteral administration are phosphate buffered saline (PBS) or normal saline (0.9%). Compositions comprising such vehicles are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, edition, Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 21st Edition Mack Publishing, 2005). Preferably, the vehicle is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal (e.g., injection or infusion) administration. Depending on the route of administration, the active compound (eg monoclonal antibody, its antigen-binding fragment, antibody drug conjugate) can be coated with a material to protect the compound from the action of acids and other natural conditions that can deactivate the compound.
[00113] The present invention also provides for compositions comprising, or alternatively consisting of one, two, three, four, five, ten, fifteen, twenty or more antibodies of the present invention (including molecules comprising, or alternatively consisting of antibody fragments or its variants). A composition of the invention may comprise, or alternatively consist of, one, two, three, four, five, ten, fifteen, twenty or more amino acid sequences of one or more antibodies or fragments of variants thereof. Alternatively, the composition of the invention may comprise, or alternatively consist of, nucleic acid molecules encompassing one or more antibodies of the invention.
[00114] The term "CXCR4-associated disorder" is any condition where the pathology is due, at least in part, to increased or inappropriate expression of CXCR4 or to inappropriate function of CXCR4. Examples of such disorders include, but are not limited to cancer associated with increased CXCR4 expression over normal, inflammatory and immunological disorders, allergic disorders, infections (HIV infection, etc.), autoimmune disorders (eg, rheumatoid arthritis) , fibrosis disorders (eg pulmonary) and cardiovascular disorders.
[00115] The term "cancer" refers to the physiological condition or disorder in mammals that is typically characterized by irregular cell growth. A blood cancer refers to a blood cancer including but not limited to leukemia, lymphoma and myeloma among others. A solid cancer refers to a cancer of tissue other than blood including but not limited to bladder cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, esophageal cancer, stomach cancer, glioblastoma, head and skin cancers. neck, kidney cancer, lung cancer, mouth cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer and liver carcinomas, etc.
[00116] The term "patient" as used herein refers to an individual suffering from a disorder associated with CXCR4. Patents may include, but are not limited to, humans, non-human primates (eg, monkeys), rats, mice, guinea pigs, pigs, goats, cows, horses, dogs, cats, birds, and poultry. In preferred embodiments, the patient who has been administered an antibody or antibody drug conjugate is a human.
The terms "effective amount" or "effective dosage" as used herein refer to an amount of a drug, compound or pharmaceutical composition necessary to achieve one or more beneficial or desired therapeutic results. For prophylactic use, beneficial or desired outcomes include eliminating or reducing risk, lessening the severity, or delaying the onset of the disorder, including biochemical, histological, and/or behavioral symptoms of the disorder, its complications, and intermediate pathological phenotypes that present during development of the disorder. For therapeutic use, beneficial or desired outcomes include clinical outcomes such as reduced incidence or amelioration of one or more symptoms of the disorder, ability to decrease the dose of other drugs needed to treat the disorder, increase the effect of another drug used to treat the disorder, and/or slow the progression of the disorder.
[00118] For purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to one or more of the following: reduce the proliferation of (or destroy) neoplastic or cancer cells in a CXCR4-associated disorder, reduce or inhibit neoplastic cell metastasis in a CXCR4-associated disorder, shrink or decrease the size of the CXCR4 expressed tumor, remission from a CXCR4-associated disorder, increase the life expectancy of an individual affected by a CXCR4-associated disorder, decrease symptoms resulting from a CXCR4-associated disorder, increasing the quality of life of those suffering from a CXCR4-associated disorder, ability to decrease the dosage of other drugs needed to treat a CXCR4-associated disorder without detrimental effect, delay progression of CXCR4-associated disorder, cure a CXCR4-associated disorder, and/or prolong the survival of patients who have an assic disorder. oted to CXCR4.
[00119] Unless defined otherwise, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those having knowledge of the art. For example, the terms "comprises", "comprising", "containing" and "having" and the like may have the meaning ascribed to them in US Patent law and may mean "includes", "including" etc.; "consisting essentially of" or "consists essentially" in the same form have the meaning ascribed in US Patent law and the term is open-ended, allowing for the presence of more than what is reported hitherto as basic or novel features of what is reported it is not changed by the presence of more than what is reported, but excludes prior art configurations.
[00120] In addition, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
[00121] Reference to "about" a value or parameter here includes (and describes) settings that are targeted to that value or parameter per se. For example, a description referring to "about X" includes the description of "X". Numeric ranges are inclusive of the numbers that define the range. Where aspects of the configurations of the invention are described in terms of a Markush group or other grouping of alternatives, the present invention encompasses not only the entire group listed as a whole, but each group member individually and all possible subgroups of the main group, but also the main group with the absence of one or more of the group members. The present invention also contemplates the explicit exclusion of one or more of any of the members of the group in the claimed invention.
[00122] Throughout this report and claims, the word "comprise", or variations such as "comprises" or "comprising" shall be understood to imply the inclusion of a particular whole number or group of whole numbers, but not the exclusion of any other whole number. or group of whole numbers. Unless otherwise required by context, singular terms must include pluralities and plural terms must include the singular.
[00123] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one having knowledge of the art to which this invention belongs. In case of conflict, the present specification including definitions will control. Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. The materials, methods, and examples are illustrative only and are not intended to be limiting. ANY ANTI-CXCR4 ANTIBODIES AND METHODS FOR THEIR PRODUCTION
The present invention provides anti-CXCR4 antibodies or antigen binding fragments that bind to human CXCR4. In that section of the specification, functions and structural characteristics of exemplary anti-CXCR4 antibodies, or antigen-binding fragments of the description are described in detail. It should be understood that antibodies or antigen binding fragments of the description may be described based on any one or more (2, 3, 4, 5, 6, 7, 8, 9, etc.) of the structures and/or functional characteristics described here. Throughout this portion of the description, when a functional or structural feature is described in relation to the antibodies of the description, it is to be understood that, except where the context clearly indicates otherwise, such structural or functional features may similarly be used to describe a fragment of description antigen binding.
[00125] The nucleotide sequence of the human CXCR4 cDNA and the predicted amino acid sequence of the human CXCR4 protein are shown as SEQ ID NOS: 104 and 105, respectively (Table 1). The human CXCR4 gene, which is approximately 1679 nucleotides in length, encompasses the full-length protein having a molecular weight of approximately 38.7 kD and which is approximately 352 amino acid residues in length. Further description of human CXCR4 nucleic acid and polypeptide sequences can be found in GenBank Accession NOS. NM-003467 and NP-003458, respectively; as well as in Federsppiel, B. et al Genomics 16(3): 707 to 712 (1993); Herzog, H. et al DNA Cell Biol. 12(6): 465 to 471 (1993); Jazin, EE et al Regul. Pept 47(3): 247 to 258 (1993); Nomura, H. et al Int. Immunol. 5(10):1,239 to 1,249 (1993); Loetscher, M. et al J. Biol. Chem. 269(1): 232 to 237 (1994); Moriuchi, M. et al J. Immunol. 159(9): 4,322 to 4,329 (1997); Caruz, A. et al FEBS Lett. 426(2): 271 to 278 (1998); and Wegner, SA et al J. Biol. Chem. 273(8): 4,754 to 4,760 (1998). TABLE 1




The human CXCR4 sequence as used herein may differ from the human CXCR4 of SEQ ID NO: 105 in having, for example, conserved mutations or mutations in non-conserved regions and the CXCR4 has substantially the same biological function as the human CXCR4 of SEQ ID NO: 105. For example, the biological function of human CXCR4 is to have an epitope in the extracellular domain of CXCR4 that is bound by an antibody of momentary description or the biological function of human CXCR4 is chemokine binding or involvement in the metastatic process.
[00127] A particular human CXCR4 sequence will generally be at least 90% identical in amino acid sequence to the human CXCR4 of SEQ ID NO: 105 and certain amino acid residues that identify the amino acid sequence as being human when compared to the amino acid sequences CXCR4 from other species (eg murine). In certain instances, a human CXCR4 may be at least 95%, or even at least 96", 97%, 98% or 99% identical in amino acid sequence to the CXCR4 of SEQ ID NO: 105. In some aspects of the invention, a The human CXCR4 sequence will exhibit no more than 10 amino acid differences from the CXCR4 of SEQ ID NO: 105. In certain aspects of the invention, the human CXCR4 may exhibit no more than 5, or even no more than 4, 3, 2, or 1 differences. amino acid of CXCR4 SEQ ID NO: 105. Percent identity can be determined as described herein.
Antibodies, their antigen binding fragments, and antibody drug conjugates of the invention are characterized by any one or more of the following characteristics: (a) binding to CXCR4; (b) down-regulation or down-regulation of CXCR4 protein expression; (c) treat, prevent, ameliorate one or more symptoms of the disorder associated with CXCR4 function or expression in an individual (eg, cancer such as NHL, AML, MM, CLL, T-ALL, gastric, head and neck , lung, ovary or pancreatic). (d) decrease or inhibit tumor growth or progression in an individual (who has a tumor expressing CXCR4); (e) decrease or inhibit the n=metastasis of cancer cells expressing CXCR4 in an individual (who has one or more cancer cells expressing CXCR4); (f) induce regression (e.g., long-term regression) of a tumor expressing CXCR4 (g) exert cytotoxic activity on cells expressing CXCR4; (h) disable or downgrade the CXCR4 path; and (i) block the interaction of CXCR4 with CXCR4 extracellular binding partners. For example, the antibody, its antigen binding fragment, and the antibody drug conjugate of the invention block the function of SDF-1.
Antibodies useful in the present invention may encompass monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusion proteins comprising an antibody moiety (eg, an antibody domain), humanized antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including antibody glycosylation variants, antibody amino acid sequence variants, and covalently modified antibodies. Antibodies can be murine, rat, human, or of any other origin (including chimeric or humanized antibodies).
[00130] In some aspects of the invention, the CXCR4 antibody as described herein as described herein is a monoclonal antibody. For example, the anti-CXCR4 antibody is a humanized monoclonal antibody or a chimeric monoclonal antibody.
In some aspects of the invention, antibodies of the present invention cross-react with CXCR4 from species other than human, such as CXCR4 from mouse, rat, canine, feline, or primate, as well as different forms of CXCR4 (for example, glycosylated CXCR4). In some aspects of the invention, antibodies specific for human CXCR4 may be completely specific for human CXCR4 and may not exhibit species or other types of cross-reactivity.
In some aspects of the invention, the CXCR4 antibody as described herein is a canine antibody, or a feline antibody. Antibodies according to the present invention that have been caninized or felineized are capable of binding to at least one canine CXCR4 protein or feline CXCR4 protein. In one aspect, a monoclonal antibody of the present invention binds to a canine CXCR4 protein or a feline CXCR4 protein and prevents its binding to a stromal cell-derived factor-1 (SDDF-1).
In some aspects of the invention, the antibody comprises a modified constant region, such as, for example, without limitation, a constant region that has increased potential to elicit an immune response. For example, the constant region can be modified to have increased affinity to an Fc gamma receptor such as, for example, FCYRI, FCYRIIA or FCYIII.
In some aspects of the invention, the antibody comprises a modified constant region, such as a constant region that has increased affinity for a human Fc gamma receptor, that is immunologically inert, i.e., having a reduced potential to elicit a immune response. In some aspects of the invention, the constant region is modified as described in Eur. J. Immunol., 29: 2613 to 2624, 1999; PCT application in PCT/GB99/01441; and/or UK Patent Application 98099518. The Fc may be human IgG1, human IgG2, human IgG3, or human IgG4. The Fc may be human IgG2 containing the mutation A330P331 to S330S331 (IgG2Δa), in which the amino acid residues are numbered relative to the wild-type IgG2 sequence. Eur. J. Immunol., 29: 2,613 to 2,624, 1999. In some aspects of the invention, the antibody comprises an IgG4 constant region comprising the following mutations ( Armouet al, Molecular Immunology 40 585 to 593, 2003): E233F234L235 to P233V234A235 (IgG4Δc), in which the numbering is in relation to wild-type IgG4. In still other aspects of the invention, the Fc is human IgG4 E233F234L235 to P233V234A235 with G236 deletion (IgG4Δb). In another aspect of the invention, the Fc is any human IgG4 Fc (IgG4, IgG4Δb or IgG4Δc) containing hinge stabilizing mutation S228 to P228 (Aalberse et al, Immunology 105, 9 to 19, 2002). In a particular aspect of the invention, the Fc can be aglycosylated Fc.
In some aspects of the invention, the constant region is aglycosylated by mutating oligosaccharide binding residues (such as Asn297) and/or flanking residues that are part of the glycosylation recognition sequence in the constant region. In some aspects of the invention, the constant region is enzymatically aglycosylated by N-linked glycosylation. The constant region can be enzymatically aglycosylated by N-linked glycosylation or by expression in a glycosylation-deficient host cell.
One way to determine the binding affinity of antibodies to CXCR4 is to measure the binding affinity of monofunctional Fab fragments of the antibody. To obtain monofunctional Fab fragments, an antibody (eg, IgG) can be cleaved with papain or expressed recombinantly. The affinity of a CXCR4 Fab fragment of an antibody can be determined by surface plasmon resonance (Biacore™3000™ surface plasmon resonance (SPR) system, Biacore™, INC, Piscataway NJ) equipped with sensor chips. Pre-immobilized streptavidin (AS) or anti-mouse Fc or anti-human Fc using continuous HBS-EP buffer (0.01M HEPES, pH 7.4, 0.15 NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20 ). Biotinylated WGA can be coated onto the SA chip to facilitate the capture of lipoparticles containing CXCR4 proteins. A dilution series (3X dilution factor, 5-membered, with a top concentration of 10 or 30 nM) of Fab was injected from low to high concentration (with an association time of 3 minutes for each concentration) to perform the analysis. kinetics of data using a "kinetic titration" methodology as described in Karlsson, et al. (Karlsson, R., Katsamba, PS, Nordin, H., Pol, E. & Myszka, DG Analyzing a kinetic titration series using affinity biosensors Anal Biochem 349, 136-147 (2006) For some rounds of analysis, buffer was injected onto captured particles rather than Fab to provide empty cycles for double referencing purposes (double referencing was performed as per described in Myszka et al., (Myszka, DG Improving biosensor analysis. J. Mol. Recognit. 12, 279-284 (1999).
[00137] Fab protein concentrations are determined by ELISA and/or SDS-PAGE electrophoresis using a Fab of known concentration (as determined by amino acid analysis) as a standard. Kinetic association (kon) and kinetic dissociation (koff) rates are obtained simultaneously by fitting the data globally to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson , B. (1994). Methods Enzymology 6.99 to 110) using the BIAevaluation program. Equilibrium dissociation constant values are calculated as koff/kon. This protocol is suitable for use in determining the binding affinity of an antibody to any CXCR4, including human CXCR4, other mammalian CXCR4 (such as mouse CXCR4, rat CXCR4, canine CXCR4, or primate CXCR4), as well as different forms of CXCR4 (for example, glycosylated CXCR4). The binding affinity of an antibody is generally measured at 25°C, but it can also be measured at 37°C.
[00138] In some aspects of the invention, an antibody or antigen-binding fragment thereof, which binds to CXCR4, has an MFI of less than 10,000 µg/ml. In particular aspects of the invention, an antibody or antigen-binding fragment thereof, which binds to CXCR4, has an MFI of less than 6,000 µg/ml. In particular aspects of the invention, an antibody or antigen-binding fragment thereof, which binds to CXCR4, has an MFI of less than 5,000 µg/ml. In particular aspects of the invention, an antibody or antigen-binding fragment thereof, which binds to CXCR4, has an MFI in the range of 2000 µg/ml to 5000 µg/ml.
[00139] As used herein, "MFI" refers to mean fluorescence intensity or median fluorescence intensity.
[00140] In some aspects of the invention, an antibody or antigen binding fragment thereof, which binds to CXCR4 has an EC50 of less than 3.00E-09 M. In some aspects of the invention, an antibody or its binding fragment of antigen, which binds to CXCR4 has an EC50 in a range of about 1.00E-09M to about 3.00E-09M. For example, an antibody or its antigen-binding fragment, which binds binds to CXCR4 has an EC50 of 2.00E-09M.
[00141] In some aspects of the invention, an antibody or its antigen-binding fragment binds to CXCR4, inhibits the induced calcium flux of SDF-1. For example, an antibody or antigen-binding fragment thereof inhibits alpha-induced calcium flux of SDF-1, with IC50s for inhibition within the range of about 1 nM to 50 nM. In one aspect, an antibody or antigen-binding fragment thereof inhibits alpha-induced calcium flux from SDF-1, with IC50s for inhibition of less than 30 nM. In one aspect, an antibody or antigen-binding fragment thereof inhibits alpha-induced calcium flux from SDF-1, with IC50s for inhibition of less than 2 nM.
[00142] In some aspects of the invention, an antibody or antigen-binding fragment thereof significantly reduces the number of AML cells in AML cell models. For example, the number of human cell numbers was significantly decreased in animals treated with the 6B6 anti-CXCR4 antibody. Furthermore, the survival of animals treated with the anti-CXCR4 antibodies of the present invention was significantly increased.
[00143] In some aspects, antibodies of the present invention have increased survival time and decreased tumor burden in a mouse non-Hodgkin's systemic lymphoma (NHL) and acute myeloid leukemia (AML) cell models. In one aspect, antibodies of the present invention increased survival in a mouse systemic chronic lymphoid leukemia (CLL) tumor model. In one aspect, antibodies of the present invention inhibited NHL tumor growth in vivo. In some aspects, antibodies of the present invention increased survival time and decreased tumor burden in a mouse systemic multiple myeloma (MM) model.
[00144] Antibodies of the invention can be produced using techniques well known in the art, for example recombinant technologies, phage display technologies, synthetic technologies or combinations of such technologies or other technologies readily known in the art (see, for example, Jayasena, SD, Clin. Chem., 45: 1628 to 1650 (1999) and Fellouse, FA, et al, J. MoI. Biol., 373(4): 924 to 940 (2007)).
The anti-CXCR4 antibodies or antigen binding fragments thereof as described herein can be made by any method known in the art. For example, for the production of hybridoma cell lines, the path and schedule of host animal immunization is generally maintained with established and conventional techniques for antibody simulation and production, as also described herein. General techniques for producing human and mouse antibodies are known in the art and/or are described herein.
[00146] It is contemplated that any mammal including humans or antibody-producing cells thereto can be engineered to serve as the basis for the production of mammalian cell lines, including humans and hybridomas. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantarly, and/or intradermally with an amount of antigen, including as described herein.
[00147] Hybridomas can be prepared from lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C., Nature 256: 495 to 497 (1975) or as modified by Buck, DW, et al., In Vitro, 18: 377 to 381 (1982). Available myeloma lines, including but not limited to X63-Ag8.653 and those from the Salk Institute, Cell Distribution Center, San Diego, Calif., USA, can be used in hybridization. Generally, the technique involves fusing myeloma cells and lymphoid cells using a fusogen such as polyethylene glycol, or by meioelectrics well known to those skilled in the art. After fusion, cells are separated from the fusion medium and grown in a selective growth medium, such as a hypoxanthine-aminopterin-thymidine (HAT) medium, to eliminate unhybridized stem cells. Any of the media described herein, supplemented with or without serum, can be used to culture hybridomas that secrete monoclonal antibodies. As another alternative to the cell fusion technique, EBV immortalized B cells can be used to produce CXCR4 monoclonal antibodies of the invention. Hybridomas are expanded and subcloned, if desired, and supernatants are examined for anti-antigen activity by conventional immunoassay procedures (eg, radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).
[00148] Hybridomas that can be used as a source of antibodies encompass all derivatives, progeny cells of matrix hybridomas that produce monoclonal antibodies specific for CXCR4, or a portion of them.
[00149] Hybridomas that produce such antibodies can be developed in vitro or in vivo using known procedures. Monoclonal antibodies can be isolated from culture media or body fluids by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography. And ultrafiltration if desired. Unwanted activity, if present, can be removed, for example, by running the preparation over sorbents made of antigen attached to a solid phase and eluting or releasing the desired antibodies to the antigen. Immunization of a host animal with a human CXCR4, or a fragment containing the target amino acid sequence conjugated to a protein that is antigenic in the species to be immunized, e.g. keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, inhibitors of soybean trypsin using a bifunctional or derivatizing agent, for example, maleimido-benzoyl sulfosuccinimide ester (conjugation via cysteine residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R1N =C=NR, where R and R1 are different alkyl groups, can produce a population of antibodies (eg, monoclonal antibodies).
If desired, the anti-CXCR4 antibody (monoclonal or polyclonal) of interest can be sequenced and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequence encompassing the antibody of interest can be maintained in vector in a host cell and the host cell can then be expanded and frozen for future use. Production of recombinant monoclonal antibodies in cell culture can be accomplished by cloning antibody genes from B cells by means known in the art. See, for example, Tiller et al, J. Immunol. Methods 329, 112 (2008); US Patent 7,314,622.
[00151] In an alternative, the polynucleotide sequence can be used for genetic manipulation to "humanize" the antibody or to improve the affinity, or other characteristics of the antibody. For example, the constant region can be designed to look more closely like human constant regions to avoid immune responses if the antibody is used in clinical trials and treatments in humans. It may be desirable to genetically engineer the antibody sequence to obtain greater affinity for CXCR4 and/or greater efficacy in inhibiting CXCR4.
[00152] There are four general steps to humanizing a monoclonal antibody. They are: (1) determining the nucleotide and projected amino acid sequence of the light and heavy variable domains of the starting antibody; (2) design the humanized antibody, that is, decide which region of the antibody framework to use during the humanization process; (3) current humanization methodologies/techniques; and (4) transfection and expression of the humanized antibody. See, for example, Patent Nos. 4,816,567; 5,807,715; 5,866.692; 6,331,415; 5,530.101; 5,693,761; 5,693,762; 5,585,089; and 6,180,370.
A number of "humanized" antibody molecules comprising an antigen binding site derived from a non-human immunoglobulin have been described, including chimeric antibodies having rodent or modified rodent V regions and their associated CDRs fused to constant regions human beings. See, for example, Winter et al Nature 349: 293 to 299 (1991), Lobuglio et al. Proc. Nat. Sci. USA 86: 4,220 to 4,224 (1989), Shaw et al. J Immunol. 138: 4534 to 4538 (1987), and Brown et al. Cancer Res. 47: 3577 to 3583 (1987) Other references describe rodent CDRs grafted onto a human scaffold region (FR) prior to fusion with a constant region of suitable human antibody. See, for example, Riechmann et al. Nature 332: 323 to 327 (1988), Verhoeyen et al. Science 239: 1534 to 1536 (1988), and Jones et al. Nature 321: 522 to 525 ( 1986). Another reference describes rodent CDRs supported by the recombinantly designed rodent framework region. See, for example, European Patent Publication No. 0519596. These "humanized" molecules are designed to minimize undesirable immune responses to anti-antibody molecules. -rodent humans that limit the duration and effectiveness of therapeutic applications of these human recipient halves. For example, the antibody constant region can be designed so that it is immunologically inert (eg, does not trigger comp lysis). elementary). See, for example, PCT Publication in PCT/GB99/01441; UK Patent Application 9809951.8. Other humanizing antibody methods that can also be used are described by Daugherty et al., Nucl. Acids Res. 19: 2,471 to 2,476, 1991, and in US Patents 6,180,377; 6,054,297; 5,997,867; 5,866,692; 6,210,671; and 6,350,861; and in PCT Publication No. WO 01/27160.
The general principles relating to humanized antibodies discussed above are also applicable to customizing antibodies for use, for example, in dogs, cats, primates, horses, and bovines. Furthermore, one or more aspects of humanizing an antibody described herein can be combined, for example, with CDR grafting, framework mutation and CDR mutation.
[00155] In some aspects of the invention, fully human antibodies can be obtained using commercially available mice that have been designed to express specific human immunoglobulin proteins. Transgenic animals that are designed to produce a more desirable (eg, fully human antibodies) or more robust immune response can also be used to generate humanized or human antibodies. Methods for obtaining human antibodies from transgenic mice are described by Green et al, Nature Genet. 7: 13 (1994), Lonberg et al, Nature 368: 856 (1994), and Taylor et al, Int. Immun. 6:519 (1994). A non-limiting example of such a System is the XenoMouse® {e.g., Green et al., J. Immunol. Methods 231: 11 to 23 (1999), incorporated herein by reference) by Abgenix (Fremont, CA). IN XenoMouse® and similar animals, mouse antibody gems have been inactivated and replaced by functional human antibody genes, while the rest of the mouse's immune system remains intact.
[00156] The XenoMouse® was transformed with germline-configured YACs (yeast artificial chromosomes) that contained portions of the human IgH and Igkappa sites, including most of the variable region sequences, along with accessory genes and regulatory sequences. The human variable region repertoire can be used to generate antibody-producing B cells, which can be processed into hybridomas by known techniques. A XenoMouse® immunized with a target antigen will produce human antibodies by the normal immune response, which can be harvested or produced by standard techniques described above. A variety of XenoMouse® strains are available, each of which is capable of producing a different class of antibody. Transgenically, human antibodies produced have been shown to have therapeutic potential while retaining the pharmacokinetic properties of normal human antibodies (Green et al, J. Immunol. Methods 231: 11 to 23 (1999)). The expert will verify that the claimed compositions and methods are not limited to the use of the XenoMouse® system but can use any transgenic animal that has been genetically engineered to produce human antibodies.
In some aspects of the invention, an antibody can be prepared using an antibody that has one or more of the VH or VL sequences described herein as starting material to produce a modified antibody, which modified antibody may have altered properties from the starting antibody. An antibody can be produced by modifying one or more residues within one or both of the variable regions (VH and/or VL), for example, within one or more CDR regions and/or within one or more framework regions. . Additionally or alternatively, an antibody can be produced by modifying residues within constant region(s), for example, to alter the antibody's effect function(s). In certain aspects, a CDR graft can be used to produce variable regions of antibodies. Antibodies interact with target antigens predominantly through amino acid residues that are located in the six regions that determine the complementarity of heavy and light chains (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse between individual antibodies than in sequences outside the CDRs. As CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific antibodies that occur naturally by constructing expression vectors that include CCDR sequences from the specific naturally occurring antibody grafted onto the framework sequences from a different antibody with different properties (see, for example, Riechmann, L et al., Nature 332 323 to 327 (1995), Jones, P et al., Nature 32J. 522 to 525 (1986), Queen, C et al, Proc Natl Acad See US 86 10029-10033 (1989), US Patents 5,225,539; 5,530,101; 5,585,089; 5,693,762; and 6,180 .370).
[00158] Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the human germline sequence database "Vbase" (available on the internet at www mrc-cpe cam ac uk/vbase ) as well as in Kabat, E A , et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication NO 91-3242, Tomlinson, IM, et al. (1992) "The Repertoire of Human Germline VH Sequences Reveals about Fifty Groups of VH Segments with Different Hypervariable Loops" Cox, J P L et al, J MoI. Biol TTL 776-798 (1994) "A Directory of Human Germ Line Vn Segments Reveals a Strong Bias in their Usage" Ew. J. Immunol 2A 827-836, the contents of each of which are expressly incorporated herein by reference. As another example, germline DNA sequences for human heavy and light variable chain region genes can be found in the Genbank database. For example, the following heavy chain germline sequences found in mouse HCo7 HuMAb are available in Genbank accession attachment numbers 1 -69 (NG_0010109, NT_024637 and BC070333), 3-33 (NG_0010109 and NT_024637) and 3-7 (NG_0010109 and NT_024637). As another example, the following heavy chain germline sequences found in the mouse HCoI 2 HuMAb are available from Genbank accession attachment numbers 1 -69 (NG_0010109, NT_024637 and BCO7O333), 5-51 (NG_0010109 and NT_024637), 4 -34 (NG_0010109 and NT_024637), 3-30 3 (CAJ556644) and 3-23 (AJ406678). Yet another source of human heavy and light chain germline sequences is the human immunoglobulin gene database available from the IMGT (http://imgt.cines.fr).g.
Preferred framework sequences for use in the antibodies of this disclosure are those which are structurally similar to the framework sequences used by the selected antibodies of this disclosure. The VH sequences CDR1, CDR2, and CDR3, and the VL sequences CDR1, CDR2, and CDR3 can be grafted into framework regions that have a sequence identical to that found in the germline immunoglobulin gene from which the framework sequence is derived. , or the CDR sequences can be grafted into framework regions that contain one or more mutations compared to the germline sequences. Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "de-immunization" and is described in greater detail in Patent Publication No. US20030153043.
[00160] In addition to or alternatively to modifications made within the framework or CDR regions, antibodies of this description may be designed to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life , complement fixation, Fc receptor binding and/or antigen-dependent cellular cytotoxicity. Additionally, an antibody of this disclosure may be chemically modified (for example, one or more chemical moieties may be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these modalities is described in additional detail below. The numbering of residues in the Fc region is that of the EU index of Kabat and/or Chothia. For example, it has been found that in certain cases it is beneficial to mutate residues within framework regions to maintain or enhance the antigen-binding ability of the antibody (see, for example, US Patent Nos. 5,530,101; 5,585,089; 5,693,762). In some aspects of the invention, the hinge region of CH1 is modified so that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of CH1 is altered to, for example, facilitate assembly of light and heavy chains or to increase or decrease antibody stability. In some aspects of the invention, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc hinge fragment such that the antibody has impaired Staphylococcyl A (SpA) protein binding relative to domain SpA binding of native Fc articulation. This approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et al.
In some aspects of the invention, the antibody is modified to increase its biological half-life. Several approaches are possible. For example, one or more of the following mutations can be introduced T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375. Alternatively, to increase the biological half-life, the antibody can be altered within the CH1 or CL region to contain a wild-type receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described. in US Patent Nos. 5,869,046 and 6,121,022 by Presta et al.
In some aspects of the invention, antibodies can be made recombinantly and expressed using any method known in the art. In another alternative, antibodies can be made recombinantly by phage display technology. See, for example, U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., Annu. Rev. Immunol. 12:433-455, 1994). Alternatively, phage display technology (McCafferty et al., Nature 348:552 to 553 (1990)) can be used to produce human antibodies and antibody fragments in vitro from variable immunoglobulin (V) gene repertoires. domain from non-immunized donors. According to this set of procedures, antibody V domain genes are cloned in-frame into a larger or smaller coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the particle. of phage. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in the selection of the gene encoding the antibody that exhibits those properties. In this way, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats; for review, see, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564 to 571 (1993). Several sources of V gene segments can be used for phage display. Clackson et al., Nature 352:624 to 628 (1991), isolated a diverse set of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse set of antigens (including autoantigens) can be isolated following essentially the sets of procedures described by Mark et al., J. Mol. Biol . 222:581 to 597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993). In a natural immune response, antibody genes accumulate mutations at a high rate (somatic hypermutation). Some of the changes introduced will confer higher affinity, and B cells that exhibit high affinity surface immunoglobulin are preferentially replicated and differentiated during subsequent antigen challenge. This natural process can be mimicked using the set of procedures known as "chain shuffling." (Marks et al., Bio/Technol. 10:779 to 783 (1992)). In that method, the affinity of "primary" human antibodies obtained by phage display can be improved by sequentially replacing the heavy and light chain V region genes with repertoires of naturally occurring variants (repertoires) of V domain genes obtained from from unimmunized donors. This set of procedures allows the production of antibodies and antibody fragments with affinities in the pM-nM range. A strategy to make very large phage antibody repertoires (also known as "the mother of all libraries") has been described by Waterhouse et al., Nucl. Acids Res. 21:2265 to 2266 (1993). Gene shuffling can also be used to derive human antibodies from rodent antibodies, where the human antibody has similar affinities and specificities to the starting rodent antibody. According to this method, which is also referred to as "epitope imprinting", the rodent antibody heavy or light chain V domain gene obtained by set of phage display procedures is replaced by a repertoire of V domain genes human, which creates rodent-human chimeras. Antigen selection results in an isolation of human variable regions capable of restoring a functional antigen-binding site, ie, the epitope governs (imprints) the choice of partner. When the process is repeated in order to replace the remaining rodent V domain, a human antibody is obtained (see PCT Publication No. WO 93/06213). Unlike traditional humanization of rodent antibodies by CDR grafting, this set of procedures provides fully human antibodies, which have no structure or CDR residues of rodent origin.
Antibodies can be made recombinantly by first isolating the antibodies and antibody by producing cells from host animals, obtaining the gene sequence and using the gene sequence to recombinantly express the antibody in host cells (for example, CHO cells). Another method that can be employed is to express the antibody sequence in plants (eg, tobacco) or transgenic milk. Methods to express antibodies recombinantly in plants or milk have been disclosed. See, for example, Peeters, et al., Vaccine 19:2756, (2001); Lonberg, N. and D. Huszar Int. Rev. Immunol 13:65 (1995); and Pollock, et al., J Immunol Methods 231:147 (1999). Methods for making antibody derivatives, e.g., humanized, single-chain, etc. are known in the art.
[00164] Sets of immunoassay classification procedures and flow cytometry such as fluorescence activated cell classification (FACS) can also be employed to isolate antibodies that are specific for CXCR4.
Antibodies, as described herein, can be linked to many different carriers. Carriers can be active and/or inert. Examples of well known carriers include polypropylene, polystyrene, polyethylene, dextran, nylon, amylases, glass, natural and modified celluloses, polyacrylamides, agaroses and magnetite. The nature of the vehicle can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable vehicles for binding antibodies, or will be able to verify the same, using routine experimentation. In some aspects of the invention, the vehicle comprises a moiety that targets the myocardium.
[00166] The DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (for example, with the use of oligonucleotide probes that have the ability to specifically bind to genes encoding the heavy and light chains of antibodies monoclonals). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors (such as expression vectors disclosed in PCT Publication No. WO 87/04462), which are then transfected into host cells such as E. coli cells, simian COS cells, monkey cells. Chinese Hamster Ovary (CHO) or myeloma cells that do not otherwise produce the immunoglobulin protein, to obtain monoclonal antibody synthesis in the recombinant host cells. See, for example, PCT Publication No. WO 87/04462. DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant regions in place of homologous murine sequences, Morrison et al., Proc. Nat. Sci. 81:6851, 1984, or by covalently joining the entire immunoglobulin coding sequence or part of the coding sequence for a non-immunoglobulin polypeptide. In that manner, "chimeric" or "hybrid" antibodies are prepared so that they have the binding specificity of a CXCR4 monoclonal antibody herein.
[00167] Methods to determine the binding specificity of an anti-CXCR4 antibody are well known to those of skill in the art. General methods are provided, for example, by Mole, "Epitope Mapping," in METHODS IN MOLECULAR BIOLOGY, VOLUME 10: IMMUNOCHEMICAL PROTOCOLS, Manson (ed.), pages 105 to 116 (The Humana Press, Inc. 1992).
[00168] Anti-CXCR4 antibodies or antigen-binding fragments thereof, as described herein, can be identified or distinguished using methods known in the art, by which reduced CXCR4 expression levels are detected and/ or measure. In some aspects of the invention, an anti-CXCR4 antibody, or antigen binding fragment thereof, is identified by incubating a candidate agent with CXCR4 and monitoring binding and/or attended reduction of CXCR4 expression levels. The binding assay can be performed with purified CXCR4 polypeptide(s), or with cells that naturally express, or are transfected to express, CXCR4 polypeptide(s). In one aspect, the binding assay is a competitive binding assay, in which the ability of a candidate antibody to compete with a known anti-CXCR4 antibody, or antigen binding fragment thereof, for CXCR4 binding is evaluated. The assay can be performed in a variety of formats, including the ELISA format.
[00169] Following initial identification, the activity of a candidate anti-CXCR4 antibody, or antigen-binding fragment thereof, can be further confirmed and refined by bioassays known to test targeted biological activities. Alternatively, bioassays can be used to rank candidates directly. Some of the methods to identify and distinguish anti-CXCR4 antibodies, or antigen binding fragments thereof, are described in detail in the Examples.
[00170] Anti-CXCR4 antibodies, or antigen binding fragments thereof, can be distinguished using methods well known in the art. For example, one method is to identify the epitope to which they bind or "epitope mapping." There are many methods known in the art for mapping and distinguishing the location of epitopes on proteins, including resolving the crystal structure of an antibody-antigen complex, competition assays, gene fragment expression assays, and peptide-based assays synthetic, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999. In a further example, epitope mapping can be used to determine the sequence to which an anti-CXCR4 antibody, or antigen-binding fragment thereof, binds. Epitope mapping is commercially available from various sources, eg Pepscan Systems (Edelhertweg 15, 8219 PH Lelystad, Netherlands). The epitope can be a linear epitope, that is, contained in a single range of amino acids, or a conformational epitope formed by a three-dimensional interaction of amino acids that may not necessarily be contained in a single range. Peptides of varying lengths (eg, at least 4 to 6 amino acids) can be isolated or synthesized (eg, recombinantly) and used for binding assays with an anti-CXCR4 antibody, or antigen binding fragments thereof. . In another example, the epitope to which the anti-CXCR4 antibody, or antigen-binding fragments thereof, binds can be determined in a systematic classification using overlapping peptides derived from the CXCR4 sequence and determining binding by the antibody anti-CXCR4, or antigen-binding fragments thereof. According to gene fragment expression assays, the open reading frame encoding CXCR4 is fragmented randomly or by specific genetic constructs and the reactivity of the expressed fragments of CXCR4 with the antibody to be tested is determined. Gene fragments can, for example, be produced by PCR and then transcribed and translated into protein in vitro, in the presence of radioactive amino acids. Antibody binding to radioactively labeled CXCR4 fragments is then determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in single binding assays. In a further example, mutagenesis of an antigen-binding domain, domain switching experiments, and alanine scanning mutagenesis can be performed to identify required, sufficient, and/or necessary residues for epitope binding. For example, domain switching experiments can be performed using a mutant CXCR4 in which several CXCR4 protein fragments have been replaced (swapped) with CXCR4 sequences from other species (eg mouse), or a closely related protein , but antigenically distinct (eg, CXCR4). By assessing antibody binding to mutant CXCR4, the importance of the particular CXCR4 fragment to antibody binding can be assessed.
Yet another method that can be used to distinguish an anti-CXCR4 antibody is to use competition assays with other antibodies known to bind the same antigen, i.e., multiple fragments in CXCR4, to determine whether the anti- CXCR4 competes with and/or binds to the same epitope as other antibodies. Competition trials are well known to those skilled in the art.
In some aspects of the invention, an isolated antibody or antigen-binding fragment thereof, which binds CXCR4, competes to bind to CXCR4 with and/or binds to the same epitope of CXCR4. Among antibodies that compete for binding to CXCR4 and/or bind to the same epitope of CXCR4 include antibodies that comprise at least one heavy chain variable region (VH) which comprises (i) a VH CDR1 selected from the group consisting of SEQ ID NOs :107, 113, 114, 108, 109, 115, 116, 117, 121 and 122; (ii) a VH CDR2 selected from the group consisting of SEQ ID NOs: 162, 128, 110, 111, 118, 119, 154, 123, 158, 124, 159, 125, 160, 126, 161, 127, 163, 164, 165, 166, 167, 168, 155, 129, 156, and 130, and (iii) a VH CDR3 selected from the group consisting of SEQ ID NOs: 112; and 120; and/or; at least one light chain variable region (VL) region comprising (i) a VL CDR1 selected from the group consisting of SEQ ID NOs: 144, 131, 135, 138, 141, 142, 143, 146, 147, 148, 149, 150 and 151; (ii) a VL CDR2 selected from the group consisting of 145, 132, 136 and 152; and (iii) a VL CDR3 selected from the group consisting of SEQ ID NO: 139, 133, 137, 140 and 153
In some aspects of the invention, an antibody competes to bind to CXCR4 with an antibody or antigen binding fragment thereof, which comprises a heavy chain variable region (VH) comprising three CDRs as SEQ ID NOs: 107, 162 and 112.
In some aspects of the invention, an antibody competes to bind to CXCR4 with an antibody or antigen binding fragment thereof, which comprises a light chain variable region (VL) comprising three CDRs as SEQ ID NOs: 144, 145 and 139.
In some aspects of the invention, an antibody competes to bind to CXCR4 with an antibody or antigen binding fragment thereof, comprising a) a heavy chain variable region (VH) comprising three CDRs as SEQ ID NOs: 107, 162 and 112; and b) a variable light chain region (VL) comprising three CDRs as SEQ ID NOs: 144, 145 and 139.
In some aspects of the invention, an antibody competes to bind to CXCR4 with an antibody or antigen binding fragment thereof, which comprises a) a heavy chain variable region (VH) which comprises VH CDR1, VH CDR2 and VH CDR3 from a VH region of SEQ ID NO: 33; and/or b) a variable light chain region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 from a VL region of SEQ ID NO: 73.
In some aspects of the invention, an antibody competes to bind to CXCR4 with an antibody or antigen binding fragment thereof, which comprises a) a heavy chain variable region (VH) of SEQ ID NO: 33; and/or b) a light chain variable region (VL) of SEQ ID NO: 73.
[00178] An expression vector can be used to direct the expression of an anti-CXCR4 antibody, or antigen binding fragments thereof. A person skilled in the art is familiar with administering expression vectors to obtain expression of an exogenous protein in vivo. See, for example, U.S. Patents No. 6,436,908; 6,413,942 and 6,376,471. Expression vector administration includes local or systemic administration, including injection, oral administration, particle gun or catheter administration, and topical administration. In another aspect of the invention, the expression vector is administered directly to the sympathetic trunk or ganglion, or into a coronary artery, atrium, ventricle or pericardium.
[00179] Targeted delivery of therapeutic compositions that contain an expression vector or subgenomic polynucleotides can also be used. Sets of receptor-mediated DNA delivery procedures are described in, for example, Findeis et al., Trends Biotechnol. 11:202 (1993); Chiou et al., Gene Therapeutics: Methods and Applications of Direct Gene Transfer, J.A. Wolff, ed., 1994; Wu et al., J. Biol. Chem., 263:621 (1988); Wu et al., J. Biol. Chem., 269:542 (1994); Zenke et al., Proc. Natl. Academic Sci. USA, 87:3655 (1990); and Wu et al., J. Biol. Chem., 266:338 (1991). Therapeutic compositions containing a polynucleotide are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. Concentration ranges from about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg and about 20 μg to about 100 μg of DNA can also be used. during a gene therapy protocol. Therapeutic polynucleotides and polypeptides can be delivered using gene delivery vehicles. The gene delivery vehicle can be of viral or non-viral origin (see, generally, Jolly et al., Cancer Gene Therapy, 1:51 (1994); Kimura, Human Gene Therapy, 5:845 (1994); Connelly et al. al., Human Gene Therapy, 1:185 (1995); and Kaplitt, Nature Genetics, 6:148 (1994)). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence can be either constitutive or regulated.
[00180] Virus-based vectors for delivering a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary virus-based carriers include, but are not limited to, recombinant retroviruses (see, for example, PCT Publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93 /10218; WO 91/02805; US Patent Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EP Patent No. 0 345 242), alphavirus-based vectors (e.g. Sindbis virus vectors, virus Semliki forest (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associated virus (AAV) vectors (see, for example, PCT Publication Nos. WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95 /00655). Administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Ther., 1992, 3:147 can also be employed.
[00181] Non-viral delivery vehicles and methods can also be employed, including, but not limited to, polycationic condensed DNA linked or unlinked to independently killed adenovirus (see, e.g., Curiel et al., Hum. Gene Ther. , 3:147 (1992)); linker-ligated DNA (see, for example, Wu, J. et al., Biol. Chem., 264:16985 (1989)); cells from eukaryotic cell delivery vehicles (see, for example, US Patent No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic charge neutralization or fusion with cell membranes. Naked DNA can also be employed. Exemplary naked DNA introduction methods are described in PCT Publication No. WO 90/11092 and U.S. Patent No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Patent No. 5,422,120; PCT Publication Nos. WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additional approaches are described in Philip et al., Mol. Cell Biol., 14:2411 (1994) and in Woffendin et al., Proc. Natl. Academic Sci., 91:1581 (1994).
In some aspects of the invention, the invention encompasses compositions, including pharmaceutical compositions, which comprise antibodies described herein or made by the methods and having the characteristics described herein. As used herein, compositions comprise one or more antibodies that bind to CXCR4, and/or one or more polynucleotides that comprise sequences that encode one or more such antibodies. Such compositions may further comprise suitable excipients, such as pharmaceutically acceptable excipients including buffers, which are well known in the art.
Accordingly, the invention provides any of the following, or compositions (including pharmaceutical compositions) which comprise any of the following sequences: TABLE 2















[00184] In Table 2, the underlined sequences are CDR sequences, according to Kabat, and in bold, according to Chothia.
The invention also provides CDR portions of antibodies to CXCR4 (including Chothia, Kabat CDRs and CDR contact regions). The determination of CDR regions is satisfactorily within the skill of the art. It is understood that in some aspects of the invention, CDRs may be a combination of the Kabat and Chothia CDRs (also called "combined CRs" or "extended CDRs"). In some modes, the CDRs are Kabat CDRs. In other modalities, the CDRs are the Chothia CDRs. In other words, in modalities with more than one CDR, the CDRs can be any one of Kabat, Chothia, combination CDRs or combinations thereof. Tables 3 and 4 provide examples of the CDR sequences provided here. TABLE 3



TABLE 4



In one aspect, the present invention provides an antibody that binds to CXCR4 and/or competes with the antibody as described herein, such as m6B6, h6B6, m12A11, h12A11, m3G10, h3G10, h3G10.A57, h3G10.B44, h3G10.1.7, h3G10.1.60, h3G10.2.5, h3G10.1.91, h3G10.2.37, h3G10.2.45, h3G10.2.42, h3G10.1.33, h3G10.3.25, h3G10, h3G10.2.72, h3G10.A11A, h3G10.A18A, h3G10.A19A, h3G10.A58A, h3G10.A65A, and h3G10.B12A, h3G10.B13A, h3G10.B18A, h3G10.A11B, h3G10.A18B, h3G10.A19B, h3G10.A58B, h3G10.h3G10. .B13B, h3G10.B18B, h3G10.2.25, h3G10.A59, h3G10.A62 or h3G10.L94D.
In another aspect, the antibody competes for binding of CXCR4 with antibody m6B6, h6B6, m12A11, h12A11, m3G10, h3G10, h3G10.A57, h3G10.B44, h3G10.1.7, h3G10.1.60, h3G10.2.5, h3G10 .1.91, h3G10.2.37, h3G10.2.45, h3G10.2.42, h3G10.1.33, h3G10.3.25, h3G10, h3G10.2.72, h3G10.A11A, h3G10.A18A, h3G10.A19A, h3G10.A58A, h3G10.A65 h3G10.B12A, h3G10.B13A, h3G10.B18A, h3G10.A11B, h3G10.A18B, h3G10.A19B, h3G10.A58B, h3G10.A65B, h3G10.B12B, h3G10.B13B, h3G2. A59, h3G10.A62 or h3G10.L94D.
In some aspects of the invention, the antibody competes for binding of CXCR4 with an antibody of the present invention and has a monovalent antibody binding affinity (KD) of about any one or less than about any one of 6, 5nM, 6.0nM, 5.5nM, 5.0nM, 4.5nM, 4.0nM, 3.5nM, 3.0nM, 2.5nM, 2.0nM, 1.5nM, 1.0 nM, 0.5 nM or 0.25 nM as measured by surface plasmon resonance. In some aspects of the invention, the antibody competes for binding of CXCR4 with antibody h3G10 and has a monovalent antibody binding affinity (KD) of about any one or less than any one of 30 nM, 25 nM, 22 nM, 20 nM, 15nM or 10nM.
[00189] In some aspects, the invention also provides CDR portions of antibodies to anti-CXCR4 antibodies based on CDR contact regions. CDR contact regions are regions of an antibody that confer antibody specificity for an antigen. In general, CDR contact regions include residue positions in the CDRs and Vernier zones that are constricted in order to maintain proper loop structure for the antibody to bind a specific antigen. Ber, for example, Makabe et al., J. Biol. Chem., 283:1156 to 1166, 2007. The determination of CDR contact regions is satisfactorily within the skill of the art.
[00190] In some aspects of the invention, an isolated antibody, or antigen-binding fragment thereof, that binds to a chemokine receptor 4 (CXCR4) comprises: a heavy chain variable region (VH) comprising (i) a VH CDR1 selected from the group consisting of SEQ ID NOs:107, 113, 114, 108, 109, 115, 116, 117, 121 and 122; (ii) a VH CDR2 selected from the group consisting of SEQ ID NOs: 162, 128, 110, 111, 118, 119, 154, 123, 158, 124, 159, 125, 160, 126, 161, 127, 163, 164, 165, 166, 167, 168, 155, 129 156, and 130, and (iii) a VH CDR3 selected from the group consisting of SEQ ID NOs: 112; and 120; and/or; b) a light chain variable region (VL) comprising (i) a VL CDR1 selected from the group consisting of SEQ ID NOs: 144, 131, 135, 138, 141, 142, 143, 146, 147, 148, 149 , 150 and 151; (ii) a VL CDR2 selected from the group consisting of 145, 132, 136 and 152; and (iii) a VL CDR3 selected from the group consisting of SEQ ID NO: 139, 133, 137, 140 and 153
[00191] In one aspect, an antibody or antigen-binding fragment thereof that binds to CXCR4 comprises heavy chain (VH) variable CDR regions that comprise an amino acid sequence that is at least 90%, 92%, 94 %, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs: 107, 113, 114, 108, 109, 115, 116, 117, 121, 122; 162, 128, 110, 111, 118, 119, 154, 123, 158, 124, 159, 125, 160, 126, 161, 127, 163, 164, 165, 166, 167, 168, 155, 129, 156, 130, 112, and 120. In one aspect, an antibody or antigen-binding fragment thereof that binds to CXCR4 comprises heavy chain variable CDR regions (VL) that comprise an amino acid sequence that is at least 90% , 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs: 144, 131, 135, 138, 141, 142, 143, 146, 147, 148, 149, 150 , 151, 145, 132, 136, and 152, 139, 133, 137, 140 and 153.
[00192] In some aspects of the invention, an isolated antibody, or antigen-binding fragment thereof, that binds to CXCR4 comprises: a) a heavy chain variable region (VH) comprising complementary determining regions selected from the group consisting of (i) a VH CDR1 comprising the sequence as SEQ ID NOs:107, 113, 114, 108, 109, 115, 116, 117, 121 AND 122; (ii) a VH CDR2 comprising the sequence as SEQ ID NOs: 162, 128, 110, 111, 118, 119, 154, 123, 158, 124, 159, 125, 160, 126, 161, 127, 163, 164 , 165, 166, 167, 168, 155, 129, 156, E 130 and; (iii) a VH CDR3 comprising the sequence as SEQ ID NOs: 112; or 120; and/or b) a light chain variable region (VL) which will comprise complementary determining regions selected from the group consisting of (i) a VL CDR1 which comprises the sequence as SEQ ID NOs: 144, 131, 135, 138, 141, 142 , 143, 146, 147, 148, 149, 150, or 151; (ii) the VL CDR2 comprising the sequence as SEQ ID NOs 145, 132, 136, or 152; and (iii) a VL CDR3 comprising the sequence as SEQ ID NOs: 139, 133, 137, 140 or 153. In some aspects of the invention, an isolated antibody, or antigen binding fragment thereof, that binds to CXCR4 , comprises: a) a variable light chain region (VL) comprising (i) VL CDR1 comprising the sequence X1SX2X3SLFNSX4X5RKNYLX6 wherein X1 is R or K; X2 is S or A; X3 is W, N or Q; X4 is H or R; X5 is T or F; and/or X6 is A, L, N or M (SEQ ID NOs:151); (ii) a VL CDR2 comprising the sequence WASARX1S where X1 is G or E (SEQ ID NOs: 152), and (iii) VL CDR3 comprising the sequence KQSFX1LRT where X1 is N or R (SEQ ID NO: 153 ); and/or b) a heavy chain variable region (VH) which comprises (i) VH CDR1 which comprises the sequence as SEQ ID NOs:107, 108, 109, 113 or 114; (ii) a VH CDR2 comprising the sequence as SEQ ID NO: 157 and (iii) a VH CDR3 comprising the sequence as SEQ ID NO: 112.
[00193] In another aspect, the invention provides an isolated antibody, or antigen-binding fragment thereof, that binds to chemokine receptor 4 (CXCR4) and comprises: a heavy chain variable region (VH) sequence which comprises: EVQLVES- GGGLVQPGGSLRLSCAASGFTFSDYYMSWVR- QAPGKGLEWVX1FIRHKX2NX3X4TX5EYSTX6X7X8GRFTISRDX9SKNX1 0LYLQMNSLX11X12EDTAVYYCAX13 IDDLFAY SS1 is GQG in which XWGTLV X2 is V or A; X3 is G, F, K, V, T, L or I; X4 is E or Y; X5 is T or R; X6 is W or S; X7 is D or V; X8 is K, T or R; X9 is D or N; X10 is T or S; X11 is R or K; X12 is A or T; and X13 is K or R.
[00194] In some aspects of the invention, an isolated antibody or antigen-binding fragment thereof that binds to chemokine receptor 4 (CXCR4) is selected from the group consisting of: a) a heavy chain variable region (VH) that has a VH CDR1 of SEQ ID NO: 107, 113 or 114; a VH CDR2 of SEQ ID NO:162 or 128 and a VH CDR3 of SEQ ID NO:112 and a variable light chain region (VL) having a VL CDR1 of SEQ ID NO:144; a VL CDR2 of SEQ ID NO:145 and a VL CDR3 of SEQ ID NO:139; b) a VH region having a VH CDR1 of SEQ ID NO:115, 116, 117, 121 or 122; a VH CDR2 of SEQ ID NO:118 or 119 and a VH CDR3 of SEQ ID NO:120 and a VL region having a VL CDR1 of SEQ ID NO:135; a VL CDR2 of SEQ ID NO:136 and a VL CDR3 of SEQ ID NO:137; c) a VH region having a VH CDR1 of SEQ ID NO:107, 108, 109, 113 or 114; a VH CDR2 of SEQ ID NO:110 or 111 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:131; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:133; d) a VH region having a VH CDR1 of SEQ ID NO:107, 108, 109, 113 or 114; a VH CDR2 of SEQ ID NO:154 or 123 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:139; and e) a VH region having a VH CDR1 of SEQ ID NO:107, 108, 109, 113 or 114; a VH CDR2 of SEQ ID NO:157 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:151; a VL CDR2 of SEQ ID NO:152 and a VL CDR3 of SEQ ID NO:153.
[00195] In some aspects of the invention, an isolated antibody or antigen-binding fragment thereof that binds to chemokine receptor 4 (CXCR4) is selected from the group consisting of: a) a heavy chain variable region (VH) which has a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:162 and a VH CDR3 of SEQ ID NO:112 and a variable light chain region (VL) having a VL CDR1 of SEQ ID NO:144; a VL CDR2 of SEQ ID NO:145 and a VL CDR3 of SEQ ID NO:139; b) a VH region having a VH CDR1 of SEQ ID NO:115; a VH CDR2 of SEQ ID NO:118 and a VH CDR3 of SEQ ID NO:120 and a VL region having a VL CDR1 of SEQ ID NO:135; a VL CDR2 of SEQ ID NO:136 and a VL CDR3 of SEQ ID NO:137; c) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:110 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:131; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:133; d) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:154 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:139; and e) a VH region that has a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:157 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:151; a VL CDR2 of SEQ ID NO:152 and a VL CDR3 of SEQ ID NO:153.
[00196] In some aspects of the invention, an isolated antibody or antigen-binding fragment thereof that binds to chemokine receptor 4 (CXCR4) is selected from the group consisting of: a) a heavy chain variable region (VH) which has a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:158 and a VH CDR3 of SEQ ID NO:112 and a variable light chain region (VL) having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:139; b) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:158 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; c) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:158 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:150; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; d) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:158 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:141; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; e) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:158 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:144; a VL CDR2 of SEQ ID NO:145 and a VL CDR3 of SEQ ID NO:140; f) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:158 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:147; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; g) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:159 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; h) a VH region that has a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:160 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; i) a VH region that has a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:161 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; j) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:162 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; k) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:163 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; l) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:164 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; m) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:165 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; o) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:166 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; p) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:167 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; q) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:168 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; r) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:168 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; s) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:163 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:144; a VL CDR2 of SEQ ID NO:145 and a VL CDR3 of SEQ ID NO:140; t) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:158 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:144; a VL CDR2 of SEQ ID NO:145 and a VL CDR3 of SEQ ID NO:139; u) a VH region that has a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:162 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:144; a VL CDR2 of SEQ ID NO:145 and a VL CDR3 of SEQ ID NO:139; v) a VH region that has a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:163 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:144; a VL CDR2 of SEQ ID NO:145 and a VL CDR3 of SEQ ID NO:139; and w) a VH region that has a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:162 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:144; a VL CDR2 of SEQ ID NO:145 and a VL CDR3 of SEQ ID NO:140.
In some aspects of the invention, an isolated antibody, or an antigen binding fragment thereof, comprises: a heavy chain variable region (VH) comprising three CDRs as SEQ ID NOs: 107, 162 and 112. some aspects of the invention, an isolated antibody, or an antigen binding fragment thereof, comprises: a light chain variable region (VL) which comprises three CDRs as SEQ ID NOs: 144, 145 and 139. In some aspects of the invention, an isolated antibody, or an antigen binding fragment thereof, comprises: a heavy chain variable region (VH) comprising three CDRs as SEQ ID NOs: 107, 162 and 112; and b) a variable light chain region (VL) comprising three CDRs as SEQ ID NOs: 144, 145 and 139.
In some aspects of the invention, an isolated antibody or antigen binding fragment thereof comprises: a heavy chain variable region (VH) comprising VH CDR1, VH CDR2 and VH CDR3 from a VH region of SEQ ID NO: 33. In some aspects of the invention, an isolated antibody or antigen binding fragment thereof comprises: a variable light chain (VL) region comprising VL CDR1, VL CDR2 and VL CDR3 from one region VL of SEQ ID NO: 73. In still other aspects of the invention, an isolated antibody or antigen binding fragment thereof comprises: a) a heavy chain variable region (VH) comprising VH CDR1, VH CDR2 and VH CDR3 from a VH region of SEQ ID NO: 33; and b) a variable light chain region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 from a VL region of SEQ ID NO: 73.
[00199] In some aspects of the invention, an isolated antibody or antigen binding fragment thereof is selected from the group consisting of: a) an antibody or antigen binding fragment thereof which comprises a VH region of SEQ ID NO:33 and a VL region of SEQ ID NO:73; b) an antibody or antigen binding fragment thereof which comprises a VH region of SEQ ID NO:13 and a VL region of SEQ ID NO:15; c) an antibody or antigen binding fragment thereof which comprises a VH region of SEQ ID NO:5 and a VL region of SEQ ID NO:7; d) an antibody or antigen binding fragment thereof which comprises a VH region of SEQ ID NO:21 and a VL region of SEQ ID NO:47; and e) an antibody or antigen binding fragment thereof which comprises a VH region of SEQ ID NO:106 and a VL region which has a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:139.
In one aspect of the invention, an isolated antibody, or an antigen binding fragment thereof, comprises: a) a heavy chain variable region (VH) of SEQ ID NO: 33; and/or b) a light chain variable region (VL) of SEQ ID NO: 73.
In one aspect, the present invention provides an antibody, or an antigen binding fragment thereof, that binds to CXCR4, wherein the antibody comprises a heavy chain variable region (VH) comprising a sequence shown in SEQ ID NOs: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 85, 87 or 106; and/or a light chain variable region (VL) comprising a sequence shown in SEQ ID NOs: 3, 7, 11, 15, 19-47, 49, 51, 53, 55, 57, 59, 61, 63, 65 67, 69, 71, 73, 75, 77, 79, 81, 83, or 169. In one aspect, an antibody or antigen-binding fragment thereof that binds to CXCR4 comprises a heavy chain variable CDR region ( VH) which comprises an amino acid sequence that is at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs: 1, 5, 9, 13, 17 , 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 85, 87 or 106. In one aspect, an antibody or antigen-binding fragment thereof. binds to CXCR4 comprises a light chain variable region (VL) that comprises an amino acid sequence that is at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the SEQ ID NOs: 3, 7, 11, 15, 19-47, 49, 51, 53, 55, 57, 59, 61, 63, 65 67, 69, 71, 73, 75, 77, 79, 81, 83 or 169 .
[00202] In some aspects of the invention, there is provided the isolated antibody or antigen-binding fragment thereof, according to claim 6, selected from the group consisting of: a) an antibody or antigen-binding fragment thereof as with - comprises a VH region of SEQ ID NO:33 and a VL region of SEQ ID NO:73; b) an antibody or antigen binding fragment thereof which comprises a VH region of SEQ ID NO:13 and the VL region of SEQ ID NO:15; c) an antibody or antigen binding fragment thereof which comprises a VH region of SEQ ID NO:5 and the VL region of SEQ ID NO:7; d) an antibody or antigen binding fragment thereof which comprises a VH region of SEQ ID NO:21 and the VL region of SEQ ID NO:47; and e) an antibody or antigen binding fragment thereof which comprises a VH region of SEQ ID NO:106 and a VL region which has a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132; and a VL CDR3 of SEQ ID NO:139.
In some aspects of the invention, the antibody or antigen binding fragment thereof comprises a) a variable heavy chain (VH) region of SEQ ID NO: 33; and/or b) a variable light chain (VL) region of SEQ ID NO: 73. In particular aspects, the antibody or antigen binding fragment thereof comprises a variable heavy chain (VH) region produced by the expression vector with ATCC Access No. PTA-121353. In other aspects, the antibody or antigen binding fragment comprises a variable light chain (VL) region produced by the expression vector with ATCC Accession No. PTA-121354.
[00204] In some aspects of the invention, the antibody or antigen-binding fragment thereof that binds to chemokine receptor 4 (CXCR4) is selected from the group consisting of: a) a variable heavy chain region (VH) which has a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:158 and a VH CDR3 of SEQ ID NO:112 and a variable light chain region (VL) having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:139; b) a VH region that has a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:158 and a VH CDR3 of SEQ ID NO:112 and a region of VL having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; c) a VH region that has a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:158 and a VH CDR3 of SEQ ID NO:112 and a region of VL having a VL CDR1 of SEQ ID NO:150; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; d) a VH region that has a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:158 and a VH CDR3 of SEQ ID NO:112 and a region of VL having a VL CDR1 of SEQ ID NO:141; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; e) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:158 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:144; a VL CDR2 of SEQ ID NO:145 and a VL CDR3 of SEQ ID NO:140; f) the VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:158 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:147; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; g) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:159 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; h) a VH region that has a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:160 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; i) a VH region that has a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:161 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; j) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:162 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; k) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:163 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; l) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:164 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; m) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:165 and a VH CDR3 of SEQ ID NO:112 and a VL region having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; n) a VH region that has a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:166 and a VH CDR3 of SEQ ID NO:112 and a region of VL having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; o) a VH region that has a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:167 and a VH CDR3 of SEQ ID NO:112 and a region of VL having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; p) a VH region having a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:168 and a VH CDR3 of SEQ ID NO:112 and a region of VL having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; q) a VH region that has a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:168 and a VH CDR3 of SEQ ID NO:112 and a region of VL having a VL CDR1 of SEQ ID NO:138; a VL CDR2 of SEQ ID NO:132 and a VL CDR3 of SEQ ID NO:140; r) a VH region that has a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:163 and a VH CDR3 of SEQ ID NO:112 and a region of VL that has a VL CDR1 of SEQ ID NO:144; a VL CDR2 of SEQ ID NO:145 and a VL CDR3 of SEQ ID NO:140; s) a VH region that has a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:158 and a VH CDR3 of SEQ ID NO:112 and a region of VL having a VL CDR1 of SEQ ID NO:144; a VL CDR2 of SEQ ID NO:145 and a VL CDR3 of SEQ ID NO:139; t) a VH region that has a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:162 and a VH CDR3 of SEQ ID NO:112 and a region of VL having a VL CDR1 of SEQ ID NO:144; a VL CDR2 of SEQ ID NO:145 and a VL CDR3 of SEQ ID NO:139; u) a VH region that has a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO:163 and a VH CDR3 of SEQ ID NO:112 and a region of VL that has a VL CDR1 of SEQ ID NO:144; a VL CDR2 of SEQ ID NO:145 and a VL CDR3 of SEQ ID NO:139; and v) a VH region that has a VH CDR1 of SEQ ID NO:107; a VH CDR2 of SEQ ID NO:162 and a VH CDR3 of SEQ ID NO:112 and a region of VL having a VL CDR1 of SEQ ID NO:144; a VL CDR2 of SEQ ID NO:145 and a VL CDR3 of SEQ ID NO:140.
In particular aspects of the invention, the antibody or antigen binding fragment thereof comprises a VH region having a VH CDR1 of SEQ ID NO: 107; a VH CDR2 of SEQ ID NO: 162 and a VH CDR3 of SEQ ID NO: 112 and a region of VL that has a VL CDR1 of SEQ ID NO: 144; a VL CDR2 of SEQ ID NO: 145 and a VL CDR3 of SEQ ID NO:139.
The binding affinity (KD) of anti-CXXR4 antibody or antigen-binding fragments of, as described herein, to CXCR4 (such as human CXCR4) is about 0.002 to about 200 nM . In some aspects of the invention, the binding affinity is any one of about 200 nM, about 100 nM, about 50 nM, about 45 nM, about 40 nM, about 35 nM, about 30 nM, about about 25 nM, about 20 nM, about 15 nM, about 10 nM, about 8 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5.5 nM, about about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 500 pM, about 100 pM, about 60 pM, about 50 pM, about 20 pM, about of 15 pM, about 10 pM, about 5 pM, or about 2 pM. In some aspects of the invention, the binding affinity is less than any one of about 250 nM, about 200 nM, about 100 nM, about 50 nM, about 30 nM, about 20 nM, about 10 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5 nM, about 4.5 nM, about 4 nM, about 3.5 nM, about 3 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about 1 nM, about 500 pM, about 100 pM, about 50 pM, about 20 pM, about 10 pM, about 5 pM or about 2 pM.
In some aspects of the invention, the binding affinity (e.g., monovalent antibody binding) of antibodies as described herein is about 35 nM or less as measured by surface plasmon resonance. In some aspects of the invention, the binding affinity (e.g., monovalent antibody binding) of antibodies as described herein is about 6.5 nM or less as measured by surface plasmon resonance.
The invention also provides methods for producing any such antibody. Antibodies of this invention can be produced by procedures known in the art. Polypeptides can be produced by proteolytic degradation or other degradation of antibodies, by recombinant methods (i.e., single or fusion polypeptides) as described above, or by chemical synthesis. Antibody polypeptides, especially shorter polypeptides of about 50 amino acids, are conveniently produced by chemical synthesis. Chemical synthesis methods are known in the art and are commercially available. For example, an antibody could be produced by an automated polypeptide synthesizer employing the solid phase method. See also, U.S. Patent Documents 5,807,715; 4,816,567 and 6,331,415.
In another aspect of the invention, antibodies can be produced recombinantly using procedures that are well known in the art. In another aspect, a polynucleotide comprises a sequence encoding antibody heavy chain and/or light chain variable regions m6B6, h6B6, m12A11, h12A11, m3G10, h3G10(VH), h3G10.A57, h3G10. B44, h3G10.1.7, h3G10.1.60, h3G10.2.5, h3G10.1.91, h3G10.2.37, h3G10.2.45, h3G10.2.42, h3G10.1.33, h3G10.3.25, h3G10.2.54, h3G10.A59, h3G10.A62, h3G10 (VL), h3G10.2.72, h3G10.A11A, h3G10.A18A, h3G10.A19A, h3G10.A58A, h3G10.A65A and h3G10.B12A, h3G10.B13A, h3G10.B18A, h3G10.G10.A11.B .A19B, h3G10.A58B, h3G10.A65B, h3G10.B12B, h3G10.B13B, h3G10.B18B, h3G10.2.25 or h3G10.L94D. The sequence encoding the antibody of interest can be maintained in a vector in a host cell and the host cell can then be expanded and frozen for future use. Vectors (which includes expression vectors) and host cells are further described herein.
The invention also encompasses scFv antibodies of this invention. Unique variable chain region fragments are produced by linking variable light and/or heavy chain regions through the use of a short link peptide (Bird et al., Science 242:423 to 426, 1988). An example of a linker peptide is (GGGGS)3 (SEQ ID NO: 89) which bridges approximately 3.5 nm bridges between the carboxy terminals of a variable region and the one designed and used (Bird et al., 1988, supra ). Linkers should be flexible, short polypeptides and preferably have less than about 20 amino acid residues. In turn, the linkers can be modified for additional functions, such as drug binding or solid supports binding. Single-chain variants can be produced recombinantly or synthetically. For synthetic scFv production, an automated synthesizer can be used. For recombinant production of scFv, a suitable plasmid containing polynucleotide encoding scFv can be introduced into a suitable eukaryotic host cell, such as yeast, plant, insect or mammalian or prokaryotic cell, amino terminal of the other va region. -riable. Linkers from other sequences were such as E. coli. Polynucleotides encoding the scFv of interest can be produced by routine manipulations such as polynucleotide ligation. The resulting scFv can be isolated using standard protein purification techniques known in the art.
Other forms of single chain antibodies such as dia-bodies or minibodies are encompassed as well. Diabodies are bivalent, bispecific antibodies in which the variable heavy chain (VH) and variable light chain (VL) domains are expressed in a single polypeptide chain, but through the use of a linker that is too short to allow pairing between the two domains on the same chain and thus forces the domains to pair with complementary domains from another chain and create two antigen-binding sites (see, for example, Holliger, P., et al., Proc. Natl. Acad Sci. USA 90: 6444 to 6448 (1993); Poljak, RJ, et al., Structure 2:1121 to 1123 (1994)). Minibody includes the VL and VH domains of a native antibody fused to the hinge region and CH3 domain of the immunoglobulin molecule. See, for example, U.S. Patent Document 5,837,821.
[00212] A bispecific antibody is an antibody that can simultaneously bind two different targets. Bispecific antibodies (bsAb) and bispecific antibody fragments (bsFab) can have at least one arm that binds, for example, to a tumor-associated antigen and at least one other arm that binds to a targetable conjugate that bears a therapeutic or diagnostic agent. A variety of bispecific fusion proteins can be produced using molecular engineering.
For example, bispecific antibodies, monoclonal antibodies that have binding specificities for at least two different antigens, can be prepared using the antibodies disclosed herein. Methods for producing bispecific antibodies are known in the art (see, for example, Suresh et al., Methods in Enzymology 121:210 (1986)). Traditionally, recombinant production of bispecific antibodies has been based on the co-expression of two immunoglobulin light chain-heavy chain pairs, the two heavy chains having different specificities (Millstein and Cuello, Nature 305, 537 to 539 (1983)) .
According to an approach to producing bispecific antibodies, variable antibody domains with the desired binding specificities (antibody-antigen binding sites) are fused to immunoglobulin constant region sequences. The fusion is preferably with an immunoglobulin heavy chain constant region which comprises at least part of the hinge, CH2 and CH3 regions. It is preferable to have the first heavy chain constant region (CH1) with the site necessary for light chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and are co-transfected into a suitable host organism. This provides great flexibility to adjust the mutual proportions of the three polypeptide fragments in modalities when unequal ratios of the three polypeptide chains used in the construction provide optimal yields. However, it is possible to insert the coding sequences for two or all three polypeptide chains into an expression vector when expression of at least two polypeptide chains at equal ratios results in large yields or when the ratios are not significant.
In some aspects of the invention, bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity on one arm and a hybrid immunoglobulin light chain-heavy chain pair (which provides a second specificity link) on the other arm. This asymmetric structure, with an immunoglobulin light chain in only one half of the bispecific molecule, facilitates separation of the desired bispecific compound from unwanted immunoglobulin chain combinations. This approach is described in WO 1994/004690.
[00216] In another aspect, bispecific antibodies are composed of amino acid modification in the first hinge region in one arm and the substituted/returned amino acid in the first hinge region has an opposite charge to the corresponding amino acid in the second hinge region in another arm . This approach is described in Document WO 2011/143545.
[00217] In another aspect, bispecific antibodies can be generated through the use of a glutamine-containing peptide tag designed for the antibody directed to an epitope (eg, CXCR4) on one arm and another peptide tag (by example, a peptide tag containing Lys or an endogenous reactive Lys) designed for a second antibody directed to a second epitope on another arm in the presence of transglutaminase. This approach is described in PCT Publication WO 2012/059882.
Heteroconjugate antibodies comprising two covalently joined antibodies are also within the scope of the invention. Such antibodies have been used to target immune system cells into unwanted cells (U.S. Patent Document 4,676,980) and to treat HIV infection (PCT Publication No. WO 1991/000360). Heteroconjugate antibodies can be produced using any convenient cross-linking methods. Suitable crosslinking agents and techniques are well known in the art and are described in U.S. Patent Document 4,676,980.
[00219] Chimeric or hybrid antibodies can also be prepared in vitro through the use of known methods of synthetic protein chemistry, which include those involving cross-linking agents. For example, immunotoxins can be constructed through the use of a disulfide exchange reaction or through the formation of a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.
[00220] The invention encompasses modifications in the antibodies and polypeptides of the variants of the invention shown in Table 2, which include functionally equivalent antibodies that do not significantly affect the properties thereof and variants that have improved or decreased activity and/or affinity . For example, the amino acid sequence can be mutated to obtain an antibody with the desired binding affinity for CXCR4. Modification of polypeptides is routine practice in the art and need not be described in detail herein. Examples of modified polypeptides include polypeptides with conservative sequence modifications. As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions that do not deleteriously significantly change the functional activity or that mature (intensify) the affinity of the polypeptide with its ligand or the use of chemical analogues.
[00221] Modifications can be introduced into an antibody of this description by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which the amino acid residue is replaced by an amino acid residue that has a similar side chain. Families of amino acid residues that have similar side chains have been defined in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine , glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues within the CDR regions of an antibody of this description can be replaced by other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e., functions set out above) through the use of the functional assays described in this document.
Amino acid sequence insertions include amino- and/or carboxy terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single amino acid residues or multiples. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to an epitope tag. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody of an enzyme or polypeptide that increases the antibody's half-life in the bloodstream.
[00223] Substitution variants have at least one amino acid residue in the antibody molecule removed and a different residue inserted in its place. Sites of greatest interest for substitutional mutagenesis include hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in Table 5 under the heading of "conservative substitutions". If such substitutions result in a change in biological activity, then more substantial changes, termed "exemplary substitutions" in Table 5 or as further described below in reference to amino acid classes can be introduced and the products examined. TABLE 5 AMINO ACID SUBSTITUTIONS

[00224] Substantial modifications in the biological properties of the antibody are achieved by selecting substitutions that differ significantly in their effect on maintaining (a) the polypeptide backbone structure in the area of substitution, for example, as a conformation in sheet or helical, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring amino acid residues are divided into groups based on common side chain properties:
(1) Non-polar: Norleucine, Met, Ala, Val, Leu, Ile;
(2) Uncharged Polar: Cys, Ser, Thr, Asn, Gln;
[00227] (3) Acid (negatively charged): Asp, Glu;
[00228] (4) Basic (positively charged): Lys, Arg;
[00229] (5) Residues that influence chain orientation: Gly, Pro; and
[00230] (6) Aromatic: Trp, Tyr, Phe, His.
[00231] Non-conservative substitutions are made by switching a member of one of these classes to another class.
Any cysteine residue not involved in maintaining the proper conformation of the antibody can be substituted as well, usually with serine, to enhance the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the antibody to enhance the stability of the antibody, particularly when the antibody is an antibody fragment such as an Fv fragment.
Amino acid modifications can range from changing or modifying one or more amino acids to the complete reprojection of a region, such as the variable region. Changes in the variable region can alter binding affinity and/or specificity. In some aspects of the invention, no more than one to five conservative amino acid substitutions are made within a CDR domain. In other aspects of the invention, no more than one to three conservative amino acid substitutions are made within a CDR domain.
Modifications also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation with different sugars, acetylation and phosphorylation. Antibodies are glycosylated at conserved positions in the constant regions thereof (Jefferis and Lund, Chem. Immunol. 65:111 to 128 (1997); Wright and Morrison, Tib-TECH 15:26 to 32 (1997)). The oligosaccharide side chains of immunoglobulins affect protein function (Boyd et al., Mol. Immunol. 32:1311 to 1318 (1996); Wittwe and Howard, Biochem. 29:4175 to 4180 (1990)) and intramolecular interaction between portions of the glycoprotein, which can affect the conformation and three-dimensional surface presented of the glycoprotein (Jefferis and Lund, supra; Wyss and Wagner, Current Opin. Biotech. 7:409 to 416 (1996)). Oligosaccharides can also serve to target a given glycoprotein on certain molecules based on specific recognition structures. Antibody glycosylation has also been reported to affect antibody-dependent cellular cytotoxicity (ADCC). In particular, CHO cells with tetracycline-regulated expression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase catalyzing formation of GlcNAc in bisection have been reported with enhanced ADCC activity (Umana et al., Mature Biotech. 17:176 to 180 (1999)).
[00235] Antibody glycosylation is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the side chain of asparagine. Therefore, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
The addition of glycosylation sites to the antibody is conveniently achieved by altering the amino acid sequence so that it contains one or more of the tripeptide sequences described above (for N-linked glycosylation sites). The alteration can also be made by adding or substituting one or more serine or threonine residues to the original antibody sequence (for O-linked glycosylation sites).
[00237] The glycosylation pattern of antibodies can also be altered without altering the underlying nucleotide sequence. Glycosylation is largely dependent on the host cell used to express the antibody. Since the cell type used for expression of recombinant glycoproteins, eg antibodies, as potential therapeutics is rarely the native cell, variations in the glycosylation pattern of antibodies can be expected (see eg Hse et al. , J. Biol. Chem. 272:9062 to 9070 (1997)).
[00238] In addition to the choice of host cells, factors that affect glycosylation during recombinant antibody production include growth mode, medium formulation, culture density, oxygenation, pH, purification schemes and the like. Several methods have been proposed to alter the glycosylation pattern achieved in a particular host organism which includes introducing or overexpressing certain enzymes involved in the production of oligosaccharides (Patent Documents Nos. 5,047,335; 5,510,261 and 5,278,299). Glycosylation or certain types of glycosylation can be enzymatically removed from the glycoprotein, for example, through the use of endoglycosidase H (Endo H), N-glycosidase F, endoglycosidase F1, endoglycosidase F2, endoglycosidase F3. Additionally, the recombinant host cell can be genetically engineered to be defective when processing certain types of polysaccharides. These and similar techniques are well known in the art.
[00239] Another modification of the antibodies herein that is contemplated by this description is pegylation. An antibody can be pegylated, for example, to increase the biological (eg, serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivatives of PEG, under conditions in which one or more PEG groups becomes attached. to the antibody or antibody fragment. Preferably, pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derive other proteins, such as mono(Cl-ClO)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain aspects of the invention, the antibody to be pegylated is an antibody-glycosylated. Methods for pegylating proteins are known in the art and can be applied to the antibodies of that description (See, for example, EP 0154316 by Nishimura et al. and EP0401384 by Ishikawa et al).
The present invention is not limited to traditional antibodies and includes the use of antibody fragments and antibody mimetics. Wide varieties of antibody fragment and antibody mimicry technologies have been developed and are well known in the art. While numerous of these technologies, such as domain antibodies, Nanobodies, and Unibodies, Unibodies make use of fragments of, or other modifications to traditional antibody structures, there are also alternative technologies, such as afibodies, DARPins, Avimers, Anticalins, and Versabodies that employ binding structures which, despite mimicking the traditional antibody, are generated from and function through distinct mechanisms.
Domain antibodies (dAbs) are the smallest functional binding units of antibodies that correspond to the variable regions of the heavy (VH) or light (VL) chains of human antibodies. Domantis has developed a series of large, highly functional libraries of human VH and VL dAbs (over ten billion different sequences in each library) and uses these libraries to select dAbs that are specific for therapeutic targets. In contrast to many conventional antibodies, Domain Antibodies are highly expressed in bacterial, yeast and mammalian cell systems. Additional details of domain antibodies and methods of producing the same can be obtained by reference to Patent Documents Nos. 6,291,158, 6,582,915, 6,593,081, 6,172,197 and 6,696,245, Patent Documents No. 2004/ 0110941, European Patent Documents No. 1433846 and European Patents 0368684 & 0616640, WO05/035572, WO04/101790, WO04/081026, WO04/058821, WO04/003019 and WO03/002609, each of which is incorporated herein by way of reference in its entirety.
[00242] Nanobodies are antibody-derived proteins that contain the unique structural and functional properties of naturally occurring heavy chain antibodies. These heavy chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3). The cloned and isolated VHH domain is a perfectly stable polypeptide that has the complete antigen-binding capability of the original heavy chain antibody. Nanobodies have a high homology to the VH domains of human antibodies and can be further humanized without any loss of activity. Furthermore, nanobodies combine the advantages of conventional antibodies with important features of small molecule drugs such as conventional antibodies. Nanobodies show high target specificity, high affinity for their targets and low inherent toxicity. Nanobodies are encoded by unique genes and are efficiently produced in most prokaryotic and eukaryotic hosts, e.g., E. coli (see, for example, Patent Document No. 6,765,087, which is incorporated herein to reference title in its entirety), molds (eg Aspergillus or Tπchoderma) and yeast (eg Saccharomyces, Kluiveromices, Hansenula or Pichia) (see, for example, Patent Document No. 6,838,254, which is incorporated herein by reference in its entirety).
[00243] In some aspects, the present invention provides the use of Unibodies. Unibodies are another antibody fragment technology based on the removal of the hinge region of IgG4 antibodies. Deletion of the hinge region results in a molecule that is essentially half the size of traditional IgG4 antibodies and has a univalent binding region instead of the bivalent binding region of IgG4 antibodies. It is also well known that IgG4 antibodies are inert and therefore do not interact with the immune system, which can be advantageous for the treatment of diseases in which an immune response is not desired and this advantage is passed on to unibodies . For example, unibodies can function to inhibit or silence, but not kill, the cells to which they are attached. Additionally, the unibody that binds to cancer cells does not stimulate them to proliferate. In addition, because unibodies are approximately half the size of traditional IgG4 antibodies, they may show better distribution across larger solid tumors with potentially advantageously effective unibodies cleared from the body at a rate similar to full-length IgG4 antibodies and have the ability to bind with a similar affinity for their antigens as whole antibodies. Additional details of unibodies can be obtained by reference to PCT Publication WO2007/059782, which is incorporated herein by reference in its entirety.
[00244] In some aspects, the present invention encompasses afibody molecules. Afibody molecules are a new class of affinity proteins based on a 58 amino acid residue protein domain derived from one of the IgG binding domains of staphylococcal protein A. This triple helix set domain was used as a a framework for the construction of combinatorial phagemid libraries, from which aphibody variants targeting the desired molecules can be selected through the use of phage display technology (Nord K, Gunnettsson E, Ringdahl J, Stahl S, Uhlen M, Nygren PA, Binding proteins selected from combinatorial libraries of an α-helical bacterial receptor domain, Nat Biotechnol 15 772-7 (1997), Ronmark J, Gronlund H, Uhlen M, Nygren PA, Human immunoglobulin A (IgA)-specific ligands from combinatorial engineering of protein A, Eur J Biochem., 269 264755 (2002)). The simple and robust structure of afibody molecules in combination with their low molecular weight (6 kDa) makes them suitable for a wide variety of applications, eg as detection reagents (Ronmark J, Hansson M, Nguyen T , et al, Construction and characterization of Affibody-Fc chimeras produced in Escherichia coli, J Immunol Methods, 261 199 to 211 (2002)) and to inhibit receptor interactions (Sandstorm K, Xu Z, Forsberg G, Nygren PA, Inhibition of the CD28-CD80 co-stimulation signal by a CD28-binding Affibody ligand developed by combinatorial engineering, protein Eng., 16 691 to 697 (2003)). Additional details of aphibodies and methods of producing them may be obtained by reference to U.S. Patent Document 5,831,012 which is incorporated herein by reference in its entirety.
[00245] Another antibody mimicry technology useful in the context of the present invention is Avimers. Avimers are evolved from a large family of human extracellular receptor domains by in vitro exon shuffling and phage display that generate multi-domain proteins with binding and inhibitory properties. Binding multiple independent binding domains has been shown to create avidity and results in enhanced affinity and specificity compared to conventional single epitope binding proteins. Other potential advantages include simple and efficient production of specific multi-target molecules in Escherichia coli, enhanced thermostability and resistance to Avimers proteases with subnanomolar affinities have been obtained against a variety of targets. Additional information regarding Avimers can be found in Patent Documents US2006/0286603, 2006/0234299, 2006/02231 14, 2006/0177831, 2006/0008844, 2005/0221384, 2005/0164301, 2005/0089932, 2005/ 0053973, 2005/0048512, 2004/0175756, all of which are incorporated herein by reference in their entirety.
[00246] Versabodies are another antibody mimetic technology that could be used in the context of the present invention. Versabodies are small 3 to 5 kDa proteins with > 15% cysteines, which form a high-density disulfide scaffold, which replaces the hydrophobic core that typical proteins have. Replacing a large number of hydrophobic amino acids, which comprises the hydrophobic core, with a small number of disulfides results in a protein that is smaller, more hydrophilic (less aggregation and non-specific binding), more protease and heat resistance, and has a lower density of T-cell epitopes, as the residues that contribute most to MHC presentation are hydrophobic. All of these properties are well known to affect immunogenicity and together they are expected to cause a large decrease in immunogenicity. Additional information regarding Versabodies can be found in U.S Patent Document 2007/0191272 which is incorporated herein by reference in its entirety.
[00247] The detailed description of antibody fragment and antibody mimetic technologies provided above is not intended to be a comprehensive list of all technologies that could be used in the context of this specification. For example, and also without being by way of limitation, a variety of technologies could be used in the context of the present invention additionally which include technologies based on alternative polypeptides, such as complementary determining region fusions as described in Qui et al., Nature Biotechnology, 25(8) 921 to 929 (2007), which is incorporated herein by reference in its entirety, as well as acid-based technologies such as the RNA aptamer technologies described in the Patent Documents US 5,789,157; 5,864,026; 5,712,375; 5,763,566; 6,013,443; 6,376,474; 6,613,526; 6,114,120; 6,261,774 and 6,387,620, all of which are incorporated herein by reference in their entirety.
[00248] Other methods of modification include through the use of coupling techniques known in the subject art, which include, but are not limited to, enzymatic means, oxidative substitution and chelation. Modifications can be used, for example, to attach labels to immunoassays. Modified polypeptides are produced using established procedures in the art and can be examined using standard assays known in the art, some of which are described below and in the Examples.
[00249] In some aspects of the invention, the antibody comprises a modified constant region, such as a constant region that has increased affinity for a human Fc gamma receptor, is immunologically inert or partially inert, e.g., does not trigger mediated by complement, does not stimulate antibody-dependent cell-mediated cytotoxicity (ADCC) or does not activate macrophages or has reduced activities (compared to the unmodified antibody) in any of the following: trigger complement-mediated lysis, stimulate dependent cell-mediated cytotoxicity antibody (ADCC) or activate microglia. Modifications other than constant region can be used to achieve optimal level and/or combination of effector functions. See, for example, Morgan et al., Immunology 86:319 to 324 (1995); Lund et al., J. Immunology 57:4963-9 157:4963-4969 (1996); Idusogie et al., J. Immunology 164:4178-4184, (2000); Tao et al., J. Immunology 143:2595-2601 (1989) and Jefferis et al., Immunological Reviews 163:59-76 (1998). In some aspects of the invention, the constant region is modified as described in Eur. J. Immunol., 29:2613-2624 (1999); PCT Publication No. WO1999/058572. In other aspects of the invention, the antibody comprises a human heavy chain IgG2 constant region which comprises the following mutations: A330P331 to S330S331 (amino acid numbering with reference to wild type IgG2 sequence). Eur. J. Immunol., 29:2613-2624 (1999). In still other aspects of the invention, the constant region is aglycosylated for N-linked glycosylation. In some aspects of the invention, the constant region is aglycosylated for N-linked glycosylation by mutating the glycosylated amino acid residue or flanking residues that are part of the N-glycosylation recognition sequence in the constant region. For example, N-glycosylation site N297 can be mutated to A, Q, K, or H. See, Tao et al., J. Immunology 143:25952601 (1989) and Jefferis et al., Immunological Reviews 163:59- 76 (1998). In some aspects of the invention, the constant region is aglycosylated for N-linked glycosylation. The constant region can be aglycosylated for N-linked glycosylation enzymatically (such as by removing carbohydrate by PNGase enzyme) or by expression in a host cell different in glycosylation.
[00250] Other antibody modifications include antibodies that have been modified as described in PCT Publication No. WO 99/58572. Such antibodies comprise, in addition to a binding domain directed towards the target molecule, an effector binding domain which has an amino acid sequence substantially homologous to all or part of a constant region of a human immunoglobulin heavy chain. These antibodies are capable of binding the target molecule without triggering significant complement-dependent lysis or cell-mediated destruction of the target. In some aspects of the invention, the effector domain is capable of binding FcRn and/or FcYRIIb. These are typically based on chimeric domains derived from two or more human immunoglobulin heavy chain CH2 domains. Antibodies modified in this way are particularly suitable for use in chronic antibody therapy to avoid inflammatory and other adverse reactions in conventional antibody therapy.
The invention includes antibodies with mature affinity. For example, antibodies with enhanced affinity can be produced by the procedure known in the art (Marks et al., Bio/Technology, 10:779 to 783 (1992); Barbas et al., Proc Nat. Acad. Sci, USA 91:3809 to 3813 (1994); Schier et al., Gene, 169:147 to 155 (1995); Yelton et al., J. Immunol., 155:194 to 2004 (1995); Jackson et al., J. Immunol. , 154(7):3310-9 (1995), Hawkins et al., J. Mol. Biol., 226:889 to 896 (1992) and PCT Publication No. WO2004/058184.
[00252] The following methods can be used to adjust the affinity of an antibody and to characterize a CDR. One way of characterizing an antibody's CDR and/or altering (such as enhancing) the binding affinity of a polypeptide, such as an antibody, is called "library scanning mutagenesis". In general, library scan mutagenesis works as follows. One or more amino acid positions in the CDR are replaced by two or more (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acids through the use of art-recognized methods. This generates small libraries of clones (in some aspects of the invention, one for each amino acid position that is analyzed), each with a complexity of two or more members (if two or more amino acids are substituted at each position). In general, the library also includes a clone comprising the native (unsubstituted) amino acid. A small number of clones, for example about 20 to 80 clones (depending on library complexity), from each library are screened for binding affinity to the target polypeptide (or other binding target) and binding candidates. increased, same binding, or none are identified. Methods for determining binding affinity are well known in the art. Binding affinity can be determined using Biacore™ surface plasmon resonance analysis, which detects differences in binding affinity of about 2-fold or more. Biacore™ is particularly useful when the starting antibody already binds with relatively high affinity, for example, a KD of about 10 nM or less. Examining through the use of Biacore™ surface plasmon resonance is described in the Examples in this document.
[00253] In another aspect, the invention relates to the antibody or antigen-binding fragment thereof, which binds CXCR4, the antibody or antigen-binding fragment thereof having an EC50 of less than 100 nM. In yet another aspect, the invention relates to the antibody or antigen-binding fragment thereof, which binds CXCR4, wherein the antibody or antigen-binding fragment thereof has an EC50 of less than 50 nM. In yet another aspect, the invention relates to the antibody or antigen-binding fragment thereof, which binds CXCR4, wherein the antibody or antigen-binding fragment thereof has an EC50 of less than 1 nM. The term "EC50" means the concentration of the test antibody, antigen-binding fragment thereof or antibody-drug conjugate that is required for 50% inhibition over the maximum effect of the same in vitro. The term "IC50" is defined as "the 50% inhibitory concentration"
Binding affinity can be determined through the use of Kinexa Biocensor, scintillation proximity assays, ELISA, ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence transfer and/or yeast display. Binding affinity can also be examined through the use of a suitable bioassay.
[00255] In some aspects of the invention, each amino acid position in a CDR is replaced (in some embodiments, one at a time) with all 20 naturally occurring amino acids through the use of art-recognized mutagenesis methods (some of which are described in this document). This generates small libraries of clones (in some aspects of the invention, one for each amino acid position that is analyzed), each with a complexity of 20 members (if all 20 amino acids are replaced at each position).
[00256] In some aspects of the invention, the library to be examined comprises substitutions at two or more positions, which may be in the same CDR or in two or more CDRs. Therefore, the library can comprise substitutions at two or more positions in a CDR. The library may comprise substitution at two or more positions in two or more CDRs. The library may comprise substitution at 3, 4, 5 or more positions, said positions found in two, three, four, five or six CDRs. Substitution can be prepared through the use of low redundancy codons. See, for example, Table 2 of Balint et al., Gene 137(1):109 to 118 (1993).
[00257] The CDR can be CDRH3 and/or CDRL3. The CDR can be one or more of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and/or CDRH3. The CDR can be a Kabat CDR, a Chothia CDR or an extended CDR.
[00258] Candidates with enhanced binding can be sequenced, which identifies a CDR substitution mutant that results in improved affinity (also called an "enhanced" substitution). Candidates that bind can also be sequenced and identify a CDR substitution that retains the binding.
[00259] Multiple examination cycles may be conducted. For example, candidates (each with an amino acid substitution at one or more positions of one or more CDRs) with enhanced binding are also useful for designing a second library that contains at least the original and substituted amino acid at each enhanced CDR position (ie. ie, amino acid position in the CDR at which a substitution mutant showed enhanced binding). The preparation, examination and selection of this library are discussed further below.
[00260] Scan library mutagenesis also provides a means of characterizing a CDR, as the frequency of clones with enhanced binding, same binding or no binding also provides information regarding the importance of each amino acid position for the stability of the antibody-antigen complex. For example, if a CDR position retains binding when changed to all 20 amino acids, that position is identified as a position that is unlikely to be required for antigen binding. Conversely, if a CDR position retains binding in only a small percentage of substitutions, that position is identified as a position that is important for CDR function. Therefore, library scanning mutagenesis methods generate information regarding positions in CDRs that can be changed to many different amino acids (which includes all 20 amino acids) and positions in CDRs that cannot be changed or which can be changed. changed to only a few amino acids.
[00261] Candidates with enhanced affinity can be combined into a second library, which includes the enhanced amino acid, the original amino acid at that position, and may additionally include other substitutions at that position, depending on the complexity of the library that is desired or allowed through the use of selection method or desired exam. Additionally, if desired, the adjacent amino acid position can be randomized to at least two or more amino acids. Randomization of adjacent amino acids may allow additional conformational flexibility in the mutant CDR, which may, in turn, allow or facilitate the introduction of a large number of enhancement mutations. The library may also comprise substitution in positions that do not show improved affinity in the first exam cycle.
[00262] The second library is screened or screened for library members with enhanced and/or altered binding affinity through the use of any method known in the art, which includes screening through the use of Biacore™ surface plasmon resonance analysis and selection by using any method known in the art for selection, which includes phage display, yeast display and ribosome display.
The invention also encompasses fusion proteins comprising one or more fragments or regions of the antibodies of this invention. In some aspects of the invention, a fusion polypeptide is provided which comprises at least 10 contiguous amino acids from the VH region chain region shown in SEQ ID NOs: 1, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 85, 87 and 106; and/or at least 10 amino acids from the VL region region shown in SEQ ID NOs: 3, 7, 11, 15, 19, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83 and 169. In various aspects of the invention, a fusion polypeptide is provided which comprises at least about 10, at least about 15, at least about 20 , at least about 25, or at least about 30 contiguous amino acids from the variable light chain region, and/or at least about 10, at least about 15, at least about 20, at least about 25, or at least about of 30 contiguous amino acids from the variable heavy chain region. In another aspect, the fusion polypeptide comprises a variable light chain region and/or a variable heavy chain region, as shown in any of the sequence pairs in Table 2. For example, SEQ ID NOs:3 and 5; 3 and 9; 7 and 5; 11 and 13; 15 and 13; 19 and 17; 69 and 31; 49 and 37 and 33 and 73. In some aspects, the fusion polypeptide comprises one or more CDR(s). In still other aspects, the fusion polypeptide comprises CDR H3 (VH CDR3) and/or CDR L3 (VL CDR3). For purposes of this invention, a fusion protein contains one or more antibodies and another amino acid sequence to which it does not bind in the native molecule, for example, a heterologous sequence or a homologous sequence from another region. Exemplary heterologous sequences include, but are not limited to a "tag" such as a FLAG tag or a 6His tag. Tags are well known in the art.
[00264] A fusion polypeptide can be created by methods known in the art, for example, synthetically or recombinantly. Typically, the fusion proteins of this invention are produced by making and expressing a polynucleotide encoding them through the use of the recombinant methods described herein, although they may also be prepared by other means known in the art, which include , for example, chemical synthesis.
This invention also provides compositions comprising antibodies conjugated (e.g., linked) to an agent that facilitates coupling to a solid support (such as biotin or avidin). For the sake of simplicity, reference will be made generally to antibodies with the understanding that such methods apply to any of the anti-CXXR4 antibody or antigen binding fragments thereof, modalities described herein. Conjugation generally refers to linking these components as described herein. Binding (which is usually affixing these components in close association at least for administration) can be achieved in a number of ways. For example, a direct reaction between an agent and an antibody is possible when each has a substituent capable of reacting with the other. For example, a nucleophilic group, such as an amino or sulfhydryl group, may have the ability to react with a carbonyl-containing group, such as an anhydride or an acid halide, or with an alkyl group that contains a good leaving group (by example, a halide) in the other.
[00266] The invention also provides isolated polynucleotides encoding the antibodies of the invention and vectors and host cells comprising the polynucleotide.
[00267] Thus, the invention provides polynucleotides (or com-positions, which includes pharmaceutical compositions) comprising polynucleotides encoding any of the following: m6B6, h6B6, m12A11, h12A11, m3G10, h3G10, h3G10.A57, h3G10.B44 , h3G10.1.7, h3G10.1.60, h3G10.2.5, h3G10.1.91, h3G10.2.37, h3G10.2.45, h3G10.2.42, h3G10.1.33, h3G10.3.25, h3G10, h3G10.2.72, h3G10.A11A, h3G10.A , h3G10.A19A, h3G10.A58A, h3G10.A65A and h3G10.B12A, h3G10.B13A, h3G10.B18A, h3G10.A11B, h3G10.A18B, h3G10.A19B, h3G10.A58B, h3G12.h3G10. .B13B, h3G10.B18B, h3G10.2.25, h3G10.A59, h3G10.A62, h3G10.L94D or any fragment or part thereof which has the ability to bind CXCR4.
[00268] In some aspects, the invention provides polynucleotides that encode any of the antibodies (which includes antibody fragments) and polypeptides described herein, such as antibodies and polypeptides that have impaired or enhanced function. Polynucleotides can be produced and expressed by the procedure known in the art.
[00269] In other aspects, the invention provides compositions (such as pharmaceutical compositions) that comprise any of the polynucleotides of the invention. In some aspects, the composition comprises an expression vector that comprises a polynucleotide that encodes any of the antibodies described herein. For example, the composition comprises one of or both polynucleotides shown in SEQ ID NO: 2 and 4 or SEQ ID NO: 6 and 8 or SEQ ID NO: 86 and 68.
[00270] Expression vectors and administration of polynucleotide compositions are further described herein.
[00271] In other aspects, the invention provides a method of producing any of the polynucleotides described herein.
[00272] Polynucleotides complementary to any such sequences are also encompassed by the present invention. Polynucleotides can be single-stranded (coding or antisense) or double-stranded and can be DNA molecules (genomic, cDNA or synthetic) or RNA. RNA molecules include HnRNA molecules that contain introns and correspond to a DNA molecule in a one-to-one way and mRNA molecules that do not contain introns. Additional coding or non-coding sequences can, but need not, be present within a polynucleotide of the present invention and a polynucleotide can, but need not be linked to other molecules and/or support materials.
Polynucleotides may comprise a native sequence (i.e., an endogenous sequence encoding an antibody or a portion thereof) or may comprise a variant of such a sequence. Polynucleotide variants contain one or more substitutions, additions, deletions and/or insertions such that the immunoreactivity of the encoded polypeptide is not diminished relative to a native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide can generally be assessed as described herein. The variants preferably exhibit at least about 70% identity, more preferably, at least about 80% identity, even more preferably, at least about 90% identity, and most preferably, at least about 95%. % identity with a polynucleotide sequence encoding a native antibody or a portion thereof.
[00274] Two polynucleotide or polypeptide sequences are said to be "identical" if the nucleotide or amino acid sequence in the two sequences is the same when aligned for maximum correspondence as described below. Du-sequence comparisons are typically performed by comparing sequences in a comparison window to identify and compare local regions of sequence similarity. A "comparison window" as used herein, refers to a segment of at least about 20 contiguous positions, generally 30 to about 75, or 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are ideally aligned.
[00275] Optimal alignment of sequences for comparison can be conducted through the use of the Megalign program in the Lasergene bioinformatics software package (DNASTAR, Inc., Madison, WI), through the use of standard parameters. This program incorporates several alignment schemes described in the following references: Dayhoff, M.O., 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345 to 358; Hein J., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M., CABIOS 5:151 to 153 (1989); Myers, E.W. and Muller W., CABIOS 4:11 to 17 (1988); Robinson, E.D., Comb. Theor. 11:105 (1971); Santou, N., Nes, M., Mol. Biol. Evolution 4:406-425 (1987); Sneath, P.H.A. E Sokal, R.R., 1973, Numerical Taxonomy and the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, D.J., Proc. Natl. Academic Sci. USA 80:726 to 730 (1983).
[00276] Preferably, the "percentage of sequence identity" is determined by comparing two ideally aligned sequences in a comparison window of at least 20 positions, with the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (ie, spans) of 20 percent or less, generally 5 to 15 percent or 10 to 12 percent, as compared to reference sequences (which do not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which identical nucleic acid bases or amino acid residues occur in both sequences to produce the number of corresponding positions, by dividing the number of corresponding positions by the total number of positions in the sequence of reference (that is, the window size) and multiply the result by 100 to produce the sequence identity percentage.
Variants may also, or alternatively, be substantially homologous to a native gene or a portion or complement thereof. Such polynucleotide variants are able to hybridize under moderately stringent conditions to a naturally occurring DNA sequence that encodes a native antibody (or a complementary sequence).
Suitable "moderately stringent conditions" include prewash in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridize at 50°C to 65°C, 5X SSC, overnight; then a double wash at 65°C for 20 minutes with each 2X, 0.5X and 0.2X SSC with 0.1% SDS.
[00279] As used herein, "highly stringent conditions" or "high stringency conditions" are those that: (1) employ low ionic strength and high temperature to wash, for example, 0.015M sodium chloride/0, 0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent such as formamide, e.g. 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/ 50 mM sodium phosphate buffer pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employs 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate , 5 x Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by a high stringency wash consisting of 0.1 x SSC containing EDTA at 55°C. The knowledgeable person will recognize how to adjust temperature, ionic strength, etc. as needed to accommodate factors such as probe length and the like.
It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. Even so, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Furthermore, gene alleles comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are altered as a result of one or more mutations, such as nucleotide deletions, additions and/or substitutions. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles can be identified through the use of standard techniques (such as hybridization, amplification and/or database sequence comparison).
[00281] The polynucleotides of this invention can be obtained through the use of chemical synthesis, recombinant methods or PCR. Chemical polynucleotide synthesis methods are well known in the art and need not be described in detail herein. A person skilled in the art can use the sequences provided herein and a commercial DNA synthesizer to produce a desired DNA sequence.
[00282] To prepare polynucleotides through the use of recombinant methods, a polynucleotide comprising a desired sequence can be inserted into a suitable vector and the vector, in turn, can be introduced into a suitable host cell for replication and amplification, as discussed additionally in this document. Polynucleotides can be inserted into host cells by any means known in the art. Cells are transformed by introducing an exogenous polynucleotide by direct absorption, endocytosis, transfection, F-conjugation or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as an unintegrated vector (such as a plasmid) or integrated into the host cell genome. The polynucleotide amplified in this way can be isolated from the host cell by methods well known in the art. See, for example, Sambrook et al., 1989.
[00283] Alternatively, PCR allows the reproduction of DNA sequences. PCR technology is well known in the art and is described in U.S. Patent Documents 4,683,195, 4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase Chain Re-action, Mullis et al. eds., Birkauswer Press, Boston, 1994.
[00284] RNA can be obtained by using the isolated DNA in an appropriate vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using methods well known to those skilled in the art, as set out in Sambrook et al., 1989, supra, for example.
[00285] Suitable cloning vectors can be constructed according to standard techniques or can be selected from a large number of cloning vectors available in the art. Although the cloning vector selected may vary with the host cell being used, useful cloning vectors will generally have the ability to self-replicate, may have a unique target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones that contain the vector. Suitable examples include bacterial plasmids and viruses, e.g. pUC18, pUC19, Bluescript (e.g. pBS SK+) and derivatives thereof, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs and such shuttle vectors as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene and Invitrogen.
[00286] Expression vectors in general are replicable polynucleotide constructs that contain a polynucleotide according to the invention. It is implied that an expression vector must be replicable in host cells as episomes or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to plasmids, viral vectors, which include adenoviruses, adeno-associated viruses, retroviruses, cosmids and expression vector(s) disclosed in PCT Publication No. WO 87/04462. Vector components may generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcriptional control elements (such as promoters, enhancers and terminator). For expression (i.e., translation), one or more translational control elements are also generally required, such as ribosome binding sites, translation initiation sites, and stop codons.
[00287] Vectors containing the polynucleotides of interest can be introduced into the host cell by a number of appropriate means, which include electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; microprojectile bombardment; lipofection and infection (eg where the vector is an infectious agent such as vaccinia virus). The choice of introducing vectors or polynucleotides will often depend on the host cell.
[00288] The invention also provides host cells that comprise any of the polynucleotides described in this document. Any host cells with the ability to overexpress heterologous DNAs can be used for the purpose of isolating the genes encoding the antibody, polypeptide or protein of interest. Non-limiting examples of mammalian host cells include, but are not limited to COS, HeLa and CHO cells. See also PCT Publication No. WO 87/04462. Suitable mammalian host cells include prokaryotes (such as E. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe; or K. lactis). Preferably, host cells express the cDNAs at a level about 5 times higher, more preferably 10 times higher, even more preferably 20 times higher than the corresponding endogenous antibody or protein of interest, if required. -sit, in the host cells. Examining host cells for specific binding to a CXCR4 or a CXCR4 domain (eg, domains 1 to 4) is affected by an immunoassay or FACS. A cell that overexpresses the antibody or protein of interest can be identified. ANTIBODY-DRUG CONJUGATES ANTI-CXXR4
[00289] The present invention provides drug-antibody of the formula Ab-(T-LD), whereby a) Ab is an antibody or antigen-binding fragment thereof, which binds to CXCR4, b) T is a tag of glutamine-containing acyl donor which may optionally be included; c) L is a linker; and d) D is a drug. Methods of making and manufacturing such antibody-drug conjugates and their use in clinical applications are also provided. "Antibody-drug conjugate" or "ADC" refers to antibodies, or antigen-binding fragments thereof, which include antibody derivatives that bind to CXCR4 and are conjugated to a drug.
[00290] In some aspects of the invention, Ab is selected from the group consisting of: a) an antibody or antigen-binding fragment thereof comprising a region of VH comprising CDRs of SEQ ID NOs: 107, 162 and 112 and a region of VL comprising CDRs of SEQ ID NOs: 144, 145 and 139; b) an antibody or antigen binding fragment thereof comprising a VH region comprising CDRs of SEQ ID NOs: 115, 118 and 120 and a VL region comprising CDRs of SEQ ID NOs:135, 136 and 137; c) an antibody or antigen binding fragment thereof comprising a VH region comprising CDRs of SEQ ID NOs: 107, 110 and 112 and a VL region comprising CDRs of SEQ ID NOs:131, 132 and 133; d) an antibody or antigen binding fragment thereof comprising a VH region comprising CDRs of SEQ ID NOs: 107, 154 and 112 and a VL region comprising CDRs of SEQ ID NOs:138, 132 and 139; e) an antibody or antigen binding fragment thereof which comprises a VH region which comprises CDRs of SEQ ID NOs: 107, 157 and 112 and a VL region which comprises CDRs of SEQ ID NOs:151, 152 and 153; f) an antibody or antigen binding fragment thereof which comprises a VH region of SEQ ID NO:33 and a VL region of SEQ ID NO:73; g) an antibody or antigen binding fragment thereof which comprises a VH region of SEQ ID NO:13 and a VL region of SEQ ID NO:15; h) an antibody or antigen binding fragment thereof which comprises a VH region of SEQ ID NO:5 and a VL region of SEQ ID NO:7; and i) an antibody or antigen binding fragment thereof which comprises a VH region of SEQ ID NO:21 and a VL region of SEQ ID NO:47.
[00291] Methods for conjugating cytotoxic agent or other therapeutic agents to antibodies have been described in several publications. For example, chemical modification can be made to antibodies either by lysine side chain amines or through cysteine sulfhydryl groups activated by the reduction of interchain disulfide bonds for the conjugation reaction to occur. See, for example, Tanaka et al., FEBS Letters 579:2092 to 2096, (2005) and Gentle et al., Bioconjug. Chem. 15:658 to 663, (2004). Reactive cysteine residues engineered into specific antibody sites for specific drug conjugation with defined stoichiometry have also been described. See, for example, Junutula et al., Nature Biotechnology, 26:925-932, (2008). Conjugation through the use of an acyl donor tag containing glutamine and/or an endogenous glutamine made reactive (ie, the ability to form a covalent bond as an acyl donor) by designing the polypeptide in the presence of transglutaminase and an amine (for example, a cytotoxic agent that comprises or attaches to a reactive amide) is also described in International Patent Document Serial No. PCT/IB2011/054899 (WO2012/059882), Strop et al., Chem. Biol. 20(2):161 to 167 (2013) and Farias et al., Bioconjug. Chem. 25(2): 245 to 250 (2014).
[00292] An example of a suitable conjugation procedure is based on the conjugation of hydrazides and other nucleophiles to al-deides generated by the oxidation of naturally occurring carbohydrates in antibodies. Hydrazone-containing conjugates can be produced with introduced carbonyl groups which provide the desired drug release properties. Conjugates can also be made with a linker that has a disulfide at one end and an alkyl chain in the middle and a hydrazine derivative at the other end. Anthracyclines are an example of cytotoxins that can be conjugated to antibodies through the use of this technology.
[00293] In other aspects of the invention, the anti-CXXR4 antibody or antigen binding fragments thereof or the conjugate as described herein comprises a tag containing donor acyl glutamine (T) designed at a specific site on the antibody ( for example, a carboxy terminus, an amino terminus, or elsewhere in the anti-CXXR4 antibody. In some aspects of the invention, the tag comprises an amino acid glutamine (Q) or amino acid sequence LLQGG (SEQ ID NO:171), GGLLQGG (SEQ ID NO:90), LLQGA (SEQ ID NO:91), GGLLQGA (SEQ ID NO: 92), LLQ, LLQGPGK (SEQ ID NO: 93), LLQGPG (SEQ ID NO: 94), LLQGPA (SEQ ID NO: 95), LLQGP (SEQ ID NO: 96), LLQP (SEQ ID NO: 97), LLQPGK (SEQ ID NO: 98), LLQGAPGK (SEQ ID NO: 99), LLQGAPG (SEQ ID NO: 100), LLQGAP (SEQ ID NO: 101), GGLLQGPP (SEQ ID NO: 172), LLQGPP ( SEQ ID NO: 173), LLQX1X2X3X4X5, where X1 is G or P, where X2 is A, G, P, or absent, where X3 is A, G, K, P or absent, where X4 is K, G or absent and where X5 is K or absent (SEQ ID NO: 102) or LLQX1X2X3X4X5 where X1 is any naturally occurring amino acid and where X2, X3, X4, and X5 are any naturally occurring amino acids or absent (SEQ ID NO: 103. In a particular aspect of the invention, T is selected from the group consisting of any one of SEQ ID NOs: 91, 92 and 102.
[00294] In other aspects of the invention, an isolated antibody is provided which comprises an acyl donor tag containing glutamine and an amino acid modification at position 222, 340 or 370 of the antibody (EU numbering scheme), wherein the modification is an amino acid deletion, insertion, substitution, mutation, or any combination thereof. For example, the amino acid modifications are K222R, K340R and K370R.
[00295] In some aspect of the invention, an antibody conjugate or antigen-binding fragment is conjugated to a drug, with the drug being selected from the group consisting of a cytotoxic agent, an immunomodulatory agent, an agent of imaging, a therapeutic agent (eg, protein therapeutic), a biopolymer and an oligonucleotide.
In some aspects of the invention, a drug may be linked or conjugated to the anti-CXXR4 antibody or antigen-binding fragment thereof as described herein for targeted site delivery of the drug to tumors (e.g., CXCR4 which express tumor). In particular aspects of the invention, the drug is a cytotoxic agent. Examples of an agent include, but are not limited to, an anthracycline, an auristatin, a camptothecin, a combretastapine, a dolastatin, a duocarmycin, an enediin, a geldanamycin, an indoline-benzodiazepine dimer, a maytansine, a puromycin, a dimer of pyrrolobenzodiazepine, a taxane, a vinca alkaloid, a tubulisin, a hemiasterlin, a spliceostatin, a pladienolide, calicheamicin and stereoisomers, isoesters, analogues and derivatives thereof. In some aspects of the invention, anthracyclines are derived from the bacterium Strepomyces and have been used to treat a wide range of cancers, such as leukemias, lymphomas, breast, uterine, ovarian and lung cancers. Exemplary anthracyclines include, but are not limited to, daunorubicin, doxorubicin (i.e., adriamycin), epirubicin, idarubicin, valrubicin, and mitoxantrone.
[00297] Dolastatins and the peptide analogues of them and derivatives, auristatins, are highly potent antimitotic agents that have been shown to have anticancer and antifungal activity. See, for example, U.S. Patent Document 5,663,149 and Pettit et al., Antimicrob. Chemother Agents. 42:2961 to 2965 (1998). Exemplary dolastatins and auristatins include, but are not limited to, dolastatin 10, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), MMAD (Auristatin Monomethyl D or dolastatin monomethyl 10), MMAF (Auristatin Monomethyl F or N -methylvaline-dolaisoleucine-dolaproine-phenylalanine), MMAE (Auristatin Monomethyl E or N-methylvaline-valine-dolaisoleucine-dolaproine-norephedrine), 5-benzoylvaleric acid ester-AE (AEVB) and other new auristatins (such as those described in US Publication Document No. 20130129753).
[00298] In particular aspects of the invention, auristatin is selected from the group consisting of 0101 (2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)- 2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino} propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide), MMAD (Auristatin Monomethyl D or dolastatin monomethyl 10 and 8261(2-Methylalanyl-N-[ (3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl - 3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide).
[00299] In some aspects of the present invention, auristatin is 0101 (2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)- 1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}- 5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide) which has the following structure:

[00300] In other aspects, auristatin is 3377 (N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S) -1-carboxyl-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl }-N-methyl-L-valinamide) which has the following structure:

[00301] In other aspects of the invention, auristatin is 0131 (2-methyl-L-proly-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3 -{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan -4-yl]-N-methyl-L-valinamide) which has the following structure:

[00302] In some aspects, auristatin is 0121(2-methyl-L-proly-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{ [(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl - 1-oxoheptan-4-yl]-N-methyl-L-valinamide) which has the following structure:

[00303] In other aspects, auristatin is 8261, (8261 2-Methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[( 1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]- N-methyl-L-valinamide), which has the following structure:

[00304] Camptothecin is a cytotoxic quinoline alkaloid that inhibits the enzyme topoisomerase I. Examples of camptothecin and derivatives thereof include, but are not limited to, topotecan and irinotecan and the metabolites thereof, such as SN-38.
[00305] Combretastatins are natural phenols with vascular disrupting properties in tumors. Exemplary combretastatins and derivatives thereof include, but are not limited to, combretastatin A-4 (CA-4) and ombrabulin.
[00306] Duocarmycins are DNA alkylating agents with cytotoxic potency. See Boger and Johnson, PNAS 92:3642 to 3649 (1995). Exemplary duocarmycins include, but are not limited to, duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, and CC-1065. Enedyins are a class of bacterial antitumor products characterized by either nine- or ten-membered rings or the presence of a conjugated triple-double-triple bond system. Exemplary enediins include, but are not limited to, calicheamicin, esperamycin, and dinemycin.
[00307] Geldanamycins are ansamycin benzoquinone antibiotics that bind to Hsp90 (Heat Shock Protein 90) and have been used in antitumor drug. Exemplary geldanamycins include, but are not limited to, 17-AAG (17-N-Allylamino-17-Demethoxygeldanamycin) and 17-DMAG (17-Dimethylaminoethylamino-17-demethoxygeldanamycin).
[00308] Maytansins or derivatives thereof maytansinoids inhibit cell proliferation by inhibiting the formation of microtubules during mitosis by inhibiting the polymerization of tubulin. See Remillard et al., Science 189:1002 to 1005 (1975). Exemplary maytansines and maytansinoids include, but are not limited to, mertansine (DM1) and its derivatives as well as ansamitocin.
[00309] Pyrrolobenzodiazepine dimers (PBDs) and indoline-benzodiazepine dimers (IGNs) are antitumor agents that contain one or more functional imine groups or equivalents thereof that bind to duplex DNA. PBD and IGN molecules are based on the natural product of athramycin and interact with DNA in a sequence-selective manner, with a preference for purine-guanine-purine sequences. Exemplary PBDs and analogs thereof include, but are not limited to, SJG-136.
[00310] Spliceostatins and pladienolides are antitumor compounds that inhibit division and interact with spliceosome, SF3b. Examples of spliceostatins include, but are not limited to, spliceostatin A, FR901464. Examples of pladienolides include, but are not limited to, Pladienolide B, Pladienolide D and E7107.
[00311] Taxanes are diterpenes that act as antitubulin agents or mitotic inhibitors. Exemplary taxanes include, but are not limited to, paclitaxel (eg, TAXOL®) and docetaxel (TAXOTERE®).
[00312] Vinca alkaloids are also antitubulin agents. Exemplary vinca alkaloids include, but are not limited to, vincristine, vinblastine, vindesine, and vinorelbine.
[00313] In some aspects of the invention, the drug is an immunomodulatory agent. Examples of an immunomodulatory agent include, but are not limited to, ganciclovier, etanercept, tacrolimus, sirolimus, voclosporin, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexrate, glucocorticoid and their analogues, cytokines, stem cell growth factors, lymphotoxins, tumor necrosis factor (TNF), hematopoietic factors, interleukins (eg, interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL- 10, IL-12, IL-18 and IL-21), colony stimulating factors (eg, granulocyte colony stimulating factor (G-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF)), interferons (eg, interferons-α, -β, and -Y), the stem cell growth factor called "factor S 1", erythropoietin and thrombopoietin, or a combination of these.
[00314] In some aspects of the invention, the drug is an imaging agent (for example, a fluorophore or a chelator), such as fluorescein, rhodamine, lanthanide phosphors and derivatives thereof. Examples of fluorophores include, but are not limited to, fluorescein isothiocyanate (FITC) (eg, 5-FITC), fluorescein amidite (FAM) (eg, 5-FAM), eosin, carboxyfluorescein, erythrosine, Alexa Fluor® (for example Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700 or 750), carboxytetramethylrhodamine (TAMRA) (by example 5,-TAMRA), tetramethylrhodamine (TMR) and sulphorodamine (SR) (for example SR101). Examples of chelators include, but are not limited to, 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-acid 1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane, 1-glutaric acid-4,7-acetic acid (NODAGA), diethylenetriaminepentaacetic acid (DTPA) and 1,2-bis(o-aminophenoxy) acid ) ethane-N,N,N',N-tetraacetic)(BAPTA).
[00315] In some aspects of the invention, therapeutic or diagnostic radioisotopes or other identifications (e.g., PET or SPECT tags) may be incorporated into the drug for conjugation to anti-CXCR4 antibodies or antigen-binding fragments as described in this document. Examples of a radioisotope or other identifications include, however, without limitation, H, C, N, C, N, O, S, F, P, P , Sc, Cr, Co, 58 59 62 64 67 67 68 75 76 77 86 89 90 Co, Fe, Cu, Cu, Cu, Ga, Ga, Se, Br, Br, Y, Zr, Y, rj/ rjc rj "7 nn drQ drcdrcdr~7 drr» dddddd 94 95 97 99 103 105 105 107 109 111 111 Tc, Ru, Ru, Tc, Ru, Rh, Ru, Hg, Pd, Ag, In, d OC d OC d O dd Qd d O dd QO d/O d/Q 125 125 126 131 131 133 142 143 I, Te, I, I, In, I, Pr, Pr 153 153 161 165 166 166 167 168 169 177 186 Pb, Sm, Tb, Tm, Dy, H, Tm, Tm, Yb, Lu, Re, d Qo d on d Ck~7 d no d nn ond ono nd d od od od od Q 188 189 197 198 199 201 203 211 212 212 213 Re, Re, Pt, Au, Au, T1, Hg, At, Bi, Pb, Bi,223Ra, 224Ac and 225Ac.
In some aspects of the invention, the drug is a therapeutic protein including, but not limited to, a toxin, a hormone, an enzyme and a growth factor.
Examples of a toxin protein (or polypeptide) include, but are not limited to, diphtheria (eg, diphtheria A chain), Pseudomonas exotoxin and endotoxin, ricin (eg, ricin A chain), abrin (eg, abrin A chain), modecin (eg, modecin A chain), alpha-sarcin, Aleurites fordii proteins, diantin proteins, ribonuclease (RNase), DNase I, staphylococcal enterotoxin-A, protein Phytolaca antiviral, gelonin, diphtherin toxin, American Phytolaca proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcina, crotin, sapaonaria officinalis inhibitor, mitogelin, restrictocin, phenomycin, enomicin, trichothecenes, peptides of cystine inhibitory node (ICK) (e.g., keratotoxins) and conotoxin (e.g., KIIIA or SmIIIa).
[00318] In some aspects of the invention, the drug is a biocompatible polymer. Anti-CXCR4 antibodies or antigen binding fragments as described herein can be conjugated to the biocompatible polymer to increase serum half-life and bioactivity and/or to extend half-lives in vivo. Examples of biocompatible polymers include water-soluble polymer such as polyethylene glycol (PEG) or its derivatives and biocompatible polymers that contain zwitterion (for example, a polymer that contains phosphorylcholine).
[00319] In some aspects of the invention, the drug is an oligonucleotide, such as antisense oligonucleotides.
[00320] In another aspect, the invention provides an antibody conjugate or antigen binding fragment as described herein, wherein the antibody-drug conjugate comprises the formula: antibody-(c)-(linker)-(drug ), where the acyl donor glutamine-containing tag is modified at a specific site on the antibody or antigen-binding fragment (eg, at a carboxy terminus of the heavy or light chain or elsewhere), where the tag is conjugated to a linker (eg, a linker that contains one or more reactive amines (eg, primary amine NH2)) and wherein the linker is conjugated to a cytotoxic agent (eg, MMAD or other auristatins as described herein ). In some aspects of the invention, the antibody-drug conjugate does not comprise the acyl donor glutamine-containing tag.
Examples of a linker that contains one or more reactive amines include, but are not limited to, acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC) or amino PEG6-propionyl. See, for example, document no. WO2012/059882.
[00322] In some aspects of the invention, the linker may be a dipeptide linker, such as a valine-citrulline (val-cit), a fenulalanine-lysine (phe-lys) linker or a maleimido-capronic-valine-linker citrulline-p-aminobenzyloxycarbonyl (vc). In another aspect, the linker can be Sulfosuccinimidyl-4-[Nmaleimidomethyl] cyclohexane-1-carboxylate (smcc). Sulfo-smcc conjugation occurs via a maleimide group that reacts with sulfifriles (thiols, -SH), while its Sulfo-NHS ester is reactive to primary amines (as found in Lysine and at the N-terminus of protein or peptide. of the). Furthermore, the linker can be maleimidocaproila (mc). In some aspects of the invention, the acyl donor glutamine containing tag comprises LLQGG (SEQ ID NO:171), GGLLQGG (SEQ ID NO:90), LLQGA (SEQ ID NO:91), GGLLQGA (SEQ ID NO:92) , LLQ, LLQGPGK (SEQ ID NO: 93), LLQGPG (SEQ ID NO: 94), LLQGPA (SEQ ID NO: 95), LLQGP (SEQ ID NO: 96), LLQP (SEQ ID NO: 97), LLQPGK ( SEQ ID NO: 98), LLQGAPGK (SEQ ID NO: 99), LLQGAPG (SEQ ID NO: 100), LLQGAP (SEQ ID NO: 101), GGLLQGPP (SEQ ID NO: 172), LLQGPP (SEQ ID NO: 173 ), LLQX1X2X3X4X5, where X1 is G or P, where X2 is A, G, P or absent, where X3 is A, G, K, P or absent, where X4 is K, G or absent, and where X5 is K or absent (SEQ ID NO: 102) or LLQX1X2X3X4X5, where X1 is a naturally occurring amino acid and where X2, X3, X4 and X5 are any naturally occurring or absent amino acids (SEQ ID NO: 103) .
In some aspects of the invention, the anti-CXCR4 antibody or conjugate as described herein comprises an amino acid substitution of asparagine (N) for glutamine (Q) or of N for alanine (A) at position 297 of the antibody anti-CXCR4.
In some aspects of the invention, the acyl donor glutamine containing tag comprising, for example, LLQ, SEQ ID NOs: 91, 90, 94, 95, 95, 97, 98, 99, 100, 171,101, 172 or 173 is modified at the C-terminus of the antibody heavy chain, wherein the lysine residue at the C-terminus is deleted. In other aspects, the label containing acyl donor glutamine (e.g., GGLLQGA (SEQ ID NO: 92)) is modified at the C-terminus of the antibody light chain. Examples of the antibody include, but are not limited to, m6B6, h6B6, m12A11, h12A11, m3G10, h3G10, h3G10.A57, h3G10.B44, h3G10.1.7, h3G10.1.60, h3G10.2.5, h3G10.1.91, h3G10. 2.37, h3G10.2.45, h3G10.2.42, h3G10.1.33, h3G10.3.25, h3G10, h3G10.2.72, h3G10.A11A, h3G10.A18A, h3G10.A19A, h3G10.A58A, h3G10.A65A, and h3G10B. .B13A, h3G10.B18A, h3G10.A11B, h3G10.A18B, h3G10.A19B, h3G10.A58B, h3G10.A65B, h3G10.B12B, h3G10.B13B, h3G10.B18B, h3G3, h10.10.25. or h3G10.L94D.
[00325] In other aspects of the invention, the conjugate comprises the formula: antibody-(label containing acyl donor glutamine)-(L)-(cytotoxic agent). In some aspects of the invention, the conjugate is a) Ab-LLQGA (SEQ ID NO: 91)-(acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC))-0101; b) Ab-LLQGA (SEQ ID NO: 91)-(AcLys-VC-PABC)-MMAD; c) Ab-LLQX1X2X3X4X5 (SEQ ID NO: 102)-(AcLys-VC-PABC)-0101; d) Ab-LLQX1X2X3X4X5 (SEQ ID NO: 102)-(AcLys-VC-PABC)-MMAD; e) Ab-GGLLQGA (SEQ ID NO: 92)-(AcLys-VC-PABC)-0101; and f) Ab-GGLLQGA (SEQ ID NO: 92)-(AcLys-VC-PABC)-MMAD.
Examples of the antibody include, but are not limited to, m6B6, h6B6, m12A11, h12A11, m3G10, h3G10, h3G10.A57, h3G10.B44, h3G10.1.7, h3G10.1.60, h3G10.2.5, h3G10.1.91 , h3G10.2.37, h3G10.2.45, h3G10.2.42, h3G10.1.33, h3G10.3.25, h3G10, h3G10.2.72, h3G10.A11A, h3G10.A18A, h3G10.A19A, h3G10.A58A, h3G10.A65A, and h3G10.A65A. B12A, h3G10.B13A, h3G10.B18A, h3G10.A11B, h3G10.A18B, h3G10.A19B, h3G10.A58B, h3G10.A65B, h3G10.B12B, h3G10.B13B, h3G10.B10.A19. h3G10.A59, h3G10.A62 or h3G10.L94D.
In some aspects of the invention, Ab is an antibody or antigen binding fragment thereof which comprises a VH region comprising CDRs of SEQ ID NOs: 107, 162 and 112 and a VL region comprising CDRs of SEQ ID NOs : 144, 145 and 139.
In some aspects of the invention, the anti-CXCR4 antibody-drug conjugate induces tumor regression. METHOD FOR USING ANTI-CXCR4 ANTIBODIES, ANTIGEN-BINDING FRAGMENTS THEREOF OR ANTIBODY-DRUG CONJUGATES THEREOF
The antibodies and antibody-drug conjugates of the present invention are useful in various applications including, but not limited to, therapeutic treatment methods and diagnostic treatment methods.
In some aspects of the invention, when administered to a patient, the antibody or conjugate and pharmaceutically acceptable carriers are sterile. Water is an exemplary vehicle when the compound or conjugate is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. The present compositions may also, if desired, contain minor amounts of wetting or emulsifying agents or pH buffering agents.
[00331] The present compositions may take the form of solutions, pellets, powders, extended release formulations or any other form suitable for use. Other examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.
In some aspects, the antibody of the invention and/or the antibody-drug conjugate thereof are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to animals, particularly humans. Typically, vehicles or vehicles for intravenous administration are sterile isotonic aqueous buffer solutions. When necessary, the compositions can also include a solubilizing agent. Compositions for intravenous administration may optionally comprise a local anesthetic such as lignocaine to lessen pain at the injection site. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the amount of active agent. When a compound of the invention and/or antibody-drug conjugate thereof is to be administered by infusion, it may be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the compound of the invention and/or antibody-drug conjugate thereof is to be administered by injection, an ampoule of sterile water for injection or saline solution may be provided so that the ingredients can be mixed prior to administration.
The composition can include various materials that modify the physical form of a solid or liquid dosage unit. For example, the composition can include materials that form a coating shell around the active ingredients. The materials forming the coating shell are typically inert and can be selected from, for example, sugar, shellac and other enteric coating agents. Alternatively, the active ingredients can be enclosed in a gelatin capsule.
[00334] In solid or liquid form, the present compositions may include a pharmacological agent used in the treatment of cancer. In one aspect, the invention provides a method of treating a condition associated with CXCR4 expression in an individual. In some aspects of the invention, the method of treating a disorder associated with CXCR4 function or expression in a subject comprises administering to the subject in need thereof an effective amount of a composition (e.g. pharmaceutical composition) comprising the anti-CXCR4 antibodies , antigen binding fragments thereof or the anti-CXCR4 antibody-drug conjugates as described herein. Disorders associated with CXCR4 expression include, but are not limited to, abnormal CXCR4 expression, altered or aberrant CXCR4 expression, CXCR4 overexpression, and a proliferative disorder (eg, cancer).
Accordingly, in some aspects provided is a method of treating cancer in an individual which comprises administering to the individual in need thereof an effective amount of a composition comprising an anti-CXCR4 antibody, antigen binding fragments thereof or conjugate anti-CXCR4 antibody-drug as described herein. As used herein, cancers include, but are not limited to bladder, breast, cervical, choriocarcinoma, colon, esophageal, gastric, glioblastoma, head and neck, kidney, lung, oral, ovarian, pancreatic cancer, prostate, skin and hematological. In some aspects of the invention, there is provided a method of decreasing tumor progression or growth in an individual having a tumor that expresses CXCR4 which comprises administering to the individual in need thereof an effective amount of a composition comprising an anti-antibody antibody. -CXCR4 or antigen binding fragments thereof or an anti-CXCR4 antibody-drug conjugate as described herein. In other aspects, a method of decreasing metastasis of cancer cells expressing CXCR4 in an individual is provided which comprises administering to the individual in need thereof an effective amount of a composition comprising an antibody, antigen binding fragments thereof or an anti-CXCR4 antibody-drug conjugate as described herein. In other aspects, a method of inducing regression of a CXCR4 expressing tumor in an individual is provided which comprises administering to the individual in need thereof an effective amount of a composition comprising an anti-CXCR4 antibody or antigen binding fragments of the yourself or an anti-CXCR4 antibody-drug conjugate as described herein.
[00336] In some aspects, the invention provides an isolated antibody, an antigen-binding fragment or an antibody-drug conjugate of any of the antibodies as described herein for use in the detection, diagnosis and treatment of pathological disorders associated with the CXCR4 function or expression. In one aspect, the disorders are oncogenic disorders associated with increased over-expression of CXCR4 over normal or any other pathology connected with overexpression of CXCR4.
[00337] In some aspects of the invention, a method of treating a cancer in an individual is provided which comprises administering to the individual in need thereof an effective amount of a composition comprising the heavy chain variable region (VH) comprising (i ) a VH CDR1 selected from the group consisting of SEQ ID NOs: 107, 113, 114, 108, 109, 115, 116, 117, 121 and 122; (ii) a VH CDR2 selected from the group consisting of SEQ ID NOs: 162, 128, 110, 111, 118, 119, 154, 123, 158, 124, 159, 125, 160, 126, 161, 127, 163, 164, 165, 166, 167, 168, 155, 129, 156, and 130 and (iii) a VH CDR3 selected from the group consisting of SEQ ID NOs: 112; and 120; and/or; b) a light chain variable region (VL) comprising (i) a VL CDR1 selected from the group consisting of SEQ ID NOs: 144, 131, 135, 138, 141, 142, 143, 146, 147, 148 , 149, 150 and 151; (ii) a VL CDR2 selected from the group consisting of 145, 132, 136 and 152; and (iii) a VL CDR3 selected from the group consisting of SEQ ID NO: 139, 133, 137, 140 and 153.
[00338] In some aspects of the invention, a method of treating a cancer in an individual is provided which comprises administering to the individual in need thereof an effective amount of a composition comprising an antibody or antigen binding fragment thereof, wherein the antibody comprises: a heavy chain variable region (VH) comprising three CDRs defined as SEQ ID NOs: 107, 162 and 112. In some aspects of the invention, a method of treating a cancer in a subject comprising administering to the individual in need thereof an effective amount of a composition comprising an antibody or antigen binding fragment thereof, wherein the antibody comprises: a light chain variable region (VL) comprising three CDRs defined as SEQ ID NOs : 144, 145 and 139.
[00339] In some aspects of the invention, a method of treating a cancer in an individual is provided which comprises administering to the individual in need thereof an effective amount of a composition comprising an antibody or antigen binding fragment thereof as defined in any one of the preceding claims, wherein the antibody comprises: the heavy chain variable region (VH) which comprises three CDRs defined as SEQ ID NOs: 107, 162 and 112; and a light chain variable region (VL) comprising three CDRs defined as SEQ ID NOs: 144, 145 and 139.
[00340] In some aspects, the present invention provides a method of treating a cancer in an individual which comprises administering to the individual in need thereof an effective amount of a composition comprising an isolated antibody or an antigen binding fragment thereof which binds to CXCR4 and comprises: a heavy chain variable region (VH) comprising VH CDR1, VH CDR2 and VH CDR3 from a VH region of SEQ ID NO: 33; and a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 from a VL region of SEQ ID NO: 73.
In still other aspects, the present invention provides a method of treating a cancer in a subject comprising administering to the subject in need thereof an effective amount of a composition comprising an antibody or antigen binding fragment thereof. as defined in any one of the preceding claims, wherein the antibody comprises: a) a heavy chain variable region (VH) of SEQ ID NO: 33; and b) a light chain variable region (VL) of SEQ ID NO: 73.
[00342] In some aspects, the invention provides a use of an isolated antibody, an antigen-binding fragment or an antibody-drug conjugate of any of the antibodies as described herein in the manufacture of a remedy to treat a disorder associated with CXCR4 function or expression. In one aspect, the disorders are oncogenic disorders associated with increased over-expression of CXCR4 over normal or any other condition connected with overexpression of CXCR4.
In other aspects of the invention, an anti-CXCR4 antibody-drug conjugate includes an antibody or antigen-binding fragment thereof, which has at least one heavy chain variable region and at least one light chain variable region, in that the at least one heavy chain variable region includes three CDRs defined as SEQ ID NOs: 107, 113, 114, 162, 128, and 112. In some aspects of the invention, an antibody-anti-CXCR4 drug conjugate includes an antibody or antigen binding fragment thereof, which has at least one heavy chain variable region and at least one light chain variable region, wherein the at least one light chain variable region includes three CDRs defined as SEQ ID NOs: 144, 145 and 139.
[00344] In some aspects of the invention, an antibody-drug conjugate, which binds to CXCR4, includes an antibody or antigen-binding fragment thereof that has a heavy chain variable region defined as any one of SEQ ID NOs: 33, 5, 9, 13, 17, 21, 23, 25, 27, 29, 31, 35, 37, 39, 41, 43, 45, 85 or 87 and/or a light chain variable region defined as any one among the SEQ ID NOs: 73, 3, 7, 11, 15, 19, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 75, 77, 79, 81, 83, or 169. For example, an anti-CXCR4 antibody-drug conjugate of the invention may include an antibody or antigen-binding fragment thereof that has a heavy chain variable region that has an amino acid sequence that is at least 90 % identical to SEQ ID NO:33 and a light chain variable region that has an amino acid sequence that is at least 90% identical to SEQ ID NO:73; or an antibody or antigen binding fragment thereof which has a heavy chain variable region defined as SEQ ID NO: 33 and a light chain variable region which has an amino acid sequence defined as SEQ ID NO: 73.
In particular aspects of the invention, the antibody is not caninized or felinated.
Antibodies may also be modified, for example, in the variable domains of heavy and/or light chains, for example, to alter a binding property of the antibody. For example, a mutation can be made in one or more of the CDR regions to increase or decrease the KD of an anti-CXCR4 antibody, to increase or decrease the koff, or to alter the antibody's binding specificity. Techniques in site-directed mutagenesis are well known in the art. See, for example, Sambrook et al. and Ausubel et al., supra.
[00347] In some aspects of the invention, a method for detecting, diagnosing and/or monitoring a disorder associated with the function or expression of CXCR4 is provided. For example, anti-CXCR4 antibodies as described herein can be identified with a detectable moiety such as an imaging agent and an enzyme-substrate identification. Antibodies as described herein can also be used for in vivo diagnostic assays, such as in vivo imaging (e.g. PET or SPECT) or a staining reagent.
[00348] In one aspect of the invention, a method is provided for detecting, diagnosing and/or monitoring a disorder associated with the function or expression of CXCR4 comprising the steps of: (i) obtaining a biological sample from a patient suspected of having a disorder associated with CXCR4 function or expression; (ii) contacting the sample to be tested with the antibody or an antigen-binding portion thereof under conditions that allow the formation of a complex between the antibody and the protein-antigen CXCR4 (iii) detecting said antibody-complex protein antigen, wherein the presence of said antibody-protein antigen complex is indicative that said patient has a disorder associated with CXCR4 function or expression. In a particular aspect of the invention, the method further comprises administering to the patient a therapeutically effective amount of the isolated antibody, antigen-binding fragment or antibody-drug conjugate of any of the antibodies as described herein.
In some aspects of the invention, an ADC can be used to target compounds (eg, therapeutic agents, identifications, cytotoxins, radiotoxins, immunosuppressants, etc.) to cells that have CXCR4 cell surface receptors by binding such compounds to the antibody or a fragment thereof. Thus, in some aspects of the invention, methods are provided to localize ex vivo or in vivo cells expressing CXCR4 (e.g., detectable identification such as radioisotope, fluorescent compound as enzyme. Alternatively, ADCs can be used to kill cells which have CXCR4 cell surface receptors targeting cytotoxins or radiotoxins to CXCR4.
In some aspects of the invention, the methods described herein further comprise a step to treat an individual with an additional form of therapy. In some aspects, the additional form of therapy is additional anti-cancer therapy including, but not limited to, chemotherapy, radiation, surgery, hormonal therapy and/or additional immunotherapy.
In some aspects of the invention, the additional form of therapy comprises administering one or more therapeutic agents in addition to an anti-CXCR4 antibody, antigen binding fragments thereof or an anti-CXCR4 antibody-drug conjugate as described in this document. Therapeutic agents include, but are not limited to, a second antibody (e.g., an anti-VEGF antibody, an anti-HER2 antibody, anti-CD25 antibody, and/or an anti-CD20 antibody), an angiogenesis inhibitor, an agent cytotoxic, an anti-inflammatory agent (for example, paclitaxel, sweet taxel, cisplatin, doxorubicin, prednisone, mitomycin, progesterone, tamoxifen or fluorouracil). In one aspect, the additional forms of therapy may be administered simultaneously or sequentially or concomitantly with an anti-CXCR4 antibody, antigen binding fragments thereof, or an anti-CXCR4 antibody-drug conjugate as described herein.
The anti-CXCR4 antibody, antigen-binding fragments thereof, or the anti-CXCR4 antibody-drug conjugate of the present invention can be administered to a subject by any suitable route. It should be understood by those skilled in the art that the examples described herein are not intended to be limiting, but to be illustrative of the available techniques. Accordingly, in some aspects of the invention, the anti-CXCR4 antibody, antigen binding fragments thereof or the anti-CXCR4 antibody-drug conjugate is administered to a subject according to known methods, such as intravenous administration, for example , as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, intracranial, transdermal, subcutaneous, intra-articular, sublingual, intrasynovial, through insufflation, intrathecal, oral, inhalation or topical routes. Administration can be systemic, for example intravenous or localized administration. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers are useful for administration. Liquid formulations can be directly nebulized and lyophilized powder can be nebulized after reconstitution. Alternatively, the anti-CXCR4 antibody, antigen binding fragments thereof or the anti-CXCR4 antibody-drug conjugate can be sprayed into an aerosol with the use of a fluorocarbon formulation and a metered dose inhaler or inhaled as a lyophilized powder and crushed. In some aspects of the invention, the anti-CXCR4 antibody, antigen binding fragments thereof, or the anti-CXCR4 antibody-drug conjugate may be administered via inhalation, as described herein. In other aspects, the anti-CXCR4 antibody, antigen-binding fragments thereof or the anti-CXCR4 antibody-drug conjugates of the present invention can be combined with pharmaceutically acceptable carriers such as saline, Ringer's solution, solution of dextrose and the like. The particular dosage regimen, i.e., dose, timing and repetition, will depend on the particular individual and that individual's medical history.
In one aspect, the anti-CXCR4 antibody, antigen binding fragments thereof, or the anti-CXCR4 antibody-drug conjugate is administered via site-specific or targeted local delivery techniques. Examples of targeted or site-specific local delivery techniques include various implantable depot sources of anti-CXCR4 antibody, antigen-binding fragments of the same or anti-CXCR4 antibody-drug conjugate, or local delivery catheters such as catheters infusion, indwelling catheters or needle catheters, synthetic grafts, adventitious wraps, shunts and stents or other implantable devices, site-specific vehicles, direct injection or direct application. See, for example, PCT Publication No. WO 00/53211 and U.S. Patent No. 5,981,568.
Various formulations of anti-CXCR4 antibody, antigen binding fragments thereof or anti-CXCR4 antibody-drug conjugate can be used for administration. In some aspects of the invention, the anti-CXCR4 antibody, (antigen binding fragments thereof or the anti-CXCR4 antibody-drug conjugate) and a pharmaceutically acceptable excipient may be in various formulations. Some formulations of the present invention comprise pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients are known in the art and are relatively inert substances that facilitate the administration of a pharmacologically effective substance. For example, an excipient can provide form or consistency or act as a diluent. Suitable excipients include, but are not limited to, stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers and skin penetration enhancers. Excipients as well as formulations for parenteral and non-parenteral drug delivery are defined in Remington, The Science and Practice of Pharmacy 20th Edition. Mack Publishing, 2000.
Generally, for the administration of an anti-CXCR4 antibody, antigen binding fragments thereof and/or anti-CXCR4 antibody-drug conjugate, an initial candidate dosage may be about 2 mg/kg. For the purpose of the present invention, a typical daily dosage might range from about anywhere from 3 μg/kg to 30 μg/kg to 300 μg/kg to 3 mg/kg, to 30 mg/kg, to 100 mg/ kg or more depending on the factors mentioned above. For example, a dosage of about 1 mg/kg, about 2.5 mg/kg, about 5 mg/kg, about 10 mg/kg and about 25 mg/kg can be used. For repeated administrations over several days or more, depending on the condition, treatment is sustained until a desired suppression of symptoms occurs or until sufficient therapeutic levels are reached, for example, to inhibit or delay tumor growth/progression or metastasis of the cancer cells. An exemplary dosing regimen comprises administering an initial dose of about 2 mg/kg, followed by a weekly maintenance dose of about 1 mg/kg of the anti-CXCR4 antibody, antigen binding fragments thereof or the anti-antibody conjugate. -CXCR4-drug, or followed by a maintenance dose of about 1 mg/kg every two weeks. Another exemplary dosing regimen comprises administering increasing doses (eg, starting dose of 1 mg/kg and gradually increasing to one or more larger doses each week or over a longer period of time). Other dosing regimens may also be useful, depending on the pattern of pharmacokinetic decay the practitioner wishes to achieve. For example, in some respects dosing from one to four times a week is contemplated. In other respects, dosing once a month or once every two months or every three months is contemplated. The progress of this therapy is easily monitored by conventional techniques and trials. The dosing regimen (including the anti-CXCR4 antibody, antigen-binding fragments thereof, or the anti-CXCR4 antibody-drug conjugate used) may vary over time.
For the purpose of the present invention, the appropriate dosage of an anti-CXCR4 antibody, antigen binding fragments thereof or an anti-CXCR4 antibody conjugate will depend on the anti-CXCR4 antibody, antigen binding fragments of the the same or the CXCR4 antibody-drug conjugate (or compositions thereof) employed, the type and severity of the symptoms to be treated, whether the agent is administered for therapeutic purposes, prior therapy, the patient's medical history and response to the agent , the rate of release from the patient to the administered agent and the discretion of the responsible physician. Typically, the physician will administer an anti-CXCR4 antibody, antigen-binding fragments thereof, or an anti-CXCR4 antibody-drug conjugate until a dosage is reached that achieves the desired result. The dose and/or frequency may vary over the course of treatment. Empirical considerations, such as halfway, will generally contribute to the determination of dosage. For example, antibodies that are compatible with the human immune system, such as humanized antibodies or fully human antibodies, can be used to prolong the antibody's half-life and to prevent the antibody from being attacked by the host's immune system. The frequency of administration can be determined and adjusted over the course of therapy and is generally, but not necessarily, based on treatment and/or suppression and/or improvement and/or delay of symptoms, eg, inhibition or delay of tumor growth. , etc. Alternatively, sustained-release formulations of anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates may be appropriate. Various formulations and devices to achieve extended release are known in the art.
In some aspects of the invention, dosages for anti-CXCR4 antibody, antigen-binding fragments thereof or anti-CXCR4 antibody-drug conjugate can be determined empirically in individuals who have received one or more administrations of the anti-CXCR4 antibody , antigen-binding fragments of the same or its anti-CXCR4 antibody-drug conjugate. Subjects receive incremental dosages of an anti-CXCR4 antibody, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugate. To assess effectiveness, an indicator of disease can be followed.
[00358] Administration of an anti-CXCR4 antibody, antigen binding fragments thereof or anti-CXCR4 antibody-drug conjugate in accordance with the method in the present invention may be continuous or intermittent, depending, for example, on the physiological condition of the recipient if the purpose of administration is therapeutic or prophylactic and other factors known to knowledgeable practitioners. Administration of an anti-CXCR4 antibody, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugate may be essentially continuous over a preselected period of time or may be in a series of spaced doses.
In some aspects of the invention, more than one anti-CXCR4 antibody, antigen-binding fragment thereof or anti-CXCR4 antibody-drug conjugate may be present. At least one, at least two, at least three, at least four, at least five different or more anti-CXCR4 antibodies, antigen binding fragments thereof or anti-CXCR4 antibody-drug conjugates may be present. In general, those anti-CXCR4 antibodies, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugates may have complementary activities that do not adversely affect each other. For example, one or more of the following anti-CXCR4 antibodies can be used: a first anti-CXCR4 antibody targeting an epitope on CXCR4 and a second anti-CXCR4 antibody targeting a different epitope on CXCR4.
Therapeutic formulations of anti-CXCR4 antibody, antigen-binding fragments thereof or anti-CXCR4 antibody-drug conjugate used in accordance with the present invention are prepared for storage by mixing an antibody having a grade of purity with optional pharmaceutically acceptable vehicles, excipients or stabilizers (Remington, The Science and Practice of Pharmacy 21st Edition. Mack Publishing, 2005), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed and may comprise buffers such as phosphate, citrate and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenolic, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m -cresol); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming contraions such as sodium; metal complexes (for example Zn-protein complexes); and/or nonionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).
Liposomes containing the anti-CXCR4 antibody, antigen binding fragments thereof or anti-CXCR4 antibody-drug conjugate are prepared by methods known in the art, such as described in Epstein, et al., Proc. Natl. Academic Sci. USA 82:3,688 (1985); O'Hare et al., Proc. Natl Acad. Sci. USA 77:4,030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with improved circulation time are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be generated by the reversed-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derived phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
[00362] The active ingredients can be captured in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (eg liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington, The Science and Practice of Pharmacy 21st Edition. Mack Publishing, 2005.
[00363] Extended release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, such matrices being in the form of shaped articles, for example, films or microcapsules. Examples of extended release matrices include polyesters, hydrogels (eg, poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (US Patent No. 3,773,919), L-glutamic acid copolymers, and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) , sucrose acetate isobutyrate and poly-D-(-)-3-hydroxybutyric acid.
[00364] The formulations to be used for in vivo administration must be sterile. This is readily achieved, for example, by filtration through sterile filtration membranes. Anti-CXCR4 antibody, antigen-binding fragments thereof or anti-CXCR4 antibody-therapeutic drug conjugate compositions are generally placed in a container that has a sterile access port, e.g., an intravenous solution bag or vial that has a stop pierceable by a hypodermic injection needle.
The compositions according to the present invention may be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions or suppositories for oral, parenteral or rectal administration or administration by inhalation or insufflation.
[00366] To prepare solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutical carrier, for example conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, stearate of magnesium, dicalcium phosphate or gums, and other pharmaceutical diluents, for example, water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention or a non-toxic pharmaceutically acceptable salt thereof. By referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. The solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.01 to about 500 mg of the active ingredient of the present invention. Tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form that takes advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the second being in the form of an envelope over the first. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and allows the inner component to pass intact through the duodenum or be released in a delayed manner. A variety of materials can be used in such enteric layers or coatings, such materials including various polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
[00367] Suitable surface active agents include particularly non-ionic agents such as polyoxyethylene sorbitan (eg TweenTM 20, 40, 60, 80 or 85) and other sorbitans (eg SpanTM 20, 40, 60 , 80 or 85). Compositions with a surface active agent will conveniently comprise between 0.05 and 5% surface active agent and may be between 0.1 and 2.5%. It will be appreciated that other ingredients can be added, for example, mannitol or other pharmaceutically acceptable carriers, if necessary.
Suitable emulsions can be prepared using commercially available fat emulsions such as IntralipidTM, LiposynTM, InfonutrolTM, LipofundinTM and LipiphysanTM. The active ingredient can be either dissolved in a premixed emulsion composition or alternatively it can be dissolved in an oil (eg soybean oil, saffron oil, cottonseed oil, sesame oil, oil of corn or almond oil) and an emulsion formed by mixing with a phospholipid (eg, egg phospholipids, soy phospholipids, or soy lecithin) and water. It will be appreciated that other ingredients may be added, for example glycerol or glucose, to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, for example between 5 and 20%. The fat emulsion may comprise fat droplets between 0.1 and 1.0 µm, particularly 0.1 and 0.5 µm and have a pH in the range of 5.5 to 8.0.
[00369] Emulsion compositions can be those prepared by mixing an anti-CXCR4 antibody, antigen-binding fragments thereof or anti-CXCR4 antibody-drug conjugate with IntralipidTM or its components (soy oil, phospholipids of egg, glycerol and water).
Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof and powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set out above. In some aspects of the invention, the compositions are administered via the nasal or oral airway for local or systemic effect. Compositions in pharmaceutically acceptable, preferably sterile, solvents can be nebulized using gases. Nebulized solutions can be breathed directly from the nebulizing device or the nebulizing device can be attached to a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension or powder compositions can be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner. COMPOSITIONS
Compositions used in the methods of the invention comprise an effective amount of an anti-CXCR4 antibody, antigen binding fragments thereof or anti-CXCR4 antibody-drug conjugate as described herein. Examples of such compositions, as well as how to formulate, are also described in a previous section and below. In some aspects of the invention, the composition comprises one or more anti-CXCR4 antibodies, antigen binding fragments thereof or anti-CXCR4 antibody-drug conjugates. For example, the anti-CXCR4 antibody recognizes human CXCR4. In some aspects, the CXCR4 antibody is a human antibody, a grafted CDR, a humanized antibody, or a chimeric antibody. In other aspects, the anti-CXCR4 antibody comprises a constant region that has the ability to activate a desired immune response, such as antibody-mediated lysis or ADCC. In still other aspects, the anti-CXCR4 antibody comprises a constant region that does not activate an unwanted or undesirable immune response, such as antibody-mediated lysis or ADCC. In some aspects of the invention, the anti-CXCR4 antibody comprises one or more CDR(s) of an anti-CXCR4 antibody or an anti-CXCR4 antibody or antigen binding fragments thereof as described herein (such as a , two, three, four, five or, in some modalities, all six CDRs).
[00372] It is understood that compositions may comprise more than one anti-CXCR4 antibody, antigen binding fragments thereof or anti-CXCR4 antibody-drug conjugate (e.g. a mixture of anti-CXCR4 antibodies that recognize different epitopes of CXCR4). Other exemplary compositions comprise more than one anti-CXCR4 antibody, antigen binding fragments of the same or anti-CXCR4 antibody-drug conjugate that recognizes the same epitope(s) or different species of anti-CXCR4 antibody , antigen-binding fragments of the same or anti-CXCR4 antibody-drug conjugate that bind to different epitopes of CXCR4 (e.g., human CXCR4).
The composition used in the present invention may further comprise pharmaceutically acceptable carriers, excipients or stabilizers (Remington: The Science and Practice of Pharmacy 21st Edition, 2005, Lippincott Williams and Wilkins, Ed. KE Hoover), in the form of formulations lyophilized or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at dosages and concentrations and may comprise buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenolic, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (for example Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein. KITS
[00374] The invention also provides kits for use in the present methods. Kits of the invention include one or more containers comprising an anti-CXCR4 antibody, antigen-binding fragments thereof or anti-CXCR4 antibody-drug conjugate as described herein and instructions for use in accordance with any of the above. methods of the invention described herein. Generally, these instructions comprise a description of the administration of anti-CXCR4 antibody, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugate for the therapeutic treatments described above.
[00375] Instructions regarding the use of anti-CXCR4 antibody, antigen-binding fragments thereof, or anti-CXCR4 antibody-drug conjugate, as described herein, generally include information regarding dosing, dosing schedule and route of administration for the intended treatment. Containers can be unit doses, batch packs (eg, multiple dose packs) or sub-unit doses. Instructions provided in kits of the invention are typically instructions written on an identification or package insert (eg, a sheet of paper included in the kit), but machine-readable instructions (eg, instructions loaded on a magnetic or optical storage disk) are also acceptable.
[00376] The kits of this invention are in suitable packaging. Suitable packaging includes, but is not limited to, jars, bottles, jars, flexible packaging (eg, sealed plastic or Mylar pouches) and the like. Packaging for use in combination with a specific device such as an inhaler, nasal delivery device (for example an atomizer) or an infusion device such as a minipump are also contemplated. The kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial that has a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (for example, the container may be an intravenous solution bag or a vial that has a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-CXCR4 antibody. The container can further comprise a second pharmaceutically active agent.
[00377] Kits can optionally provide components such as buffers and interpretive information. Typically, the kit comprises a container and an identification or package insert(s) on or associated with the container. MUTATIONS AND MODIFICATIONS
[00378] To express the anti-CXCR4 antibodies or antigen binding fragments thereof of the present invention, DNA fragments encoding the VH and VL regions can first be obtained using any of the methods described above. Various modifications, for example, mutations, substitutions, deletions and/or additions can also be introduced into the DNA sequences using standard methods known to those skilled in the art. For example, mutagenesis can be performed using standard methods such as PCR-mediated mutagenesis, in which mutated nucleotides are incorporated into PCR primers so that the PCR product contains the desired mutations or mutagenesis directed to site.
One type of substitution, for example, that can be made is to change one or more cysteines in the antibody, which may be chemically reactive, to another residue, such as, without limitation, alanine or serine. For example, there may be a substitution of a non-canonical cysteine. The substitution can be made in a CDR or framework region of a variable domain or in the constant region of an antibody. In some aspects of the invention, cysteine is canonical.
Antibodies can also be modified, for example, in the variable domains of heavy and/or light chains, for example, to alter a binding property of the antibody. For example, a mutation can be made in one or more of the CDR regions to increase or decrease the antibody's KD for CXCR4, to increase or decrease the koff, or to alter the antibody's binding specificity. Techniques in site-directed mutagenesis are well known in the art. See, for example, Sambrook et al. and Ausubel et al., supra.
A modification or mutation can also be made in a framework region or constant region to increase the half-life of an anti-CXCR4 antibody. See, for example, PCT Publication No. WO 00/09560. A mutation in a framework region or constant region can also be made to alter the immunogenicity of the antibody to provide a site for covalent or non-covalent binding to another molecule or to alter such properties as complement fixation, FcR binding and antibody-dependent cell-mediated cytotoxicity. According to the invention, a single antibody may have mutations in any one or more of the variable domain CDRs or framework regions or in the constant region.
[00382] In a process known as "germline", certain amino acids in the VH and VL sequences can be mutated to match those found naturally in the VH and VL germline sequences. In particular, the amino acid sequences of the framework regions in the VH and VL sequences can be mutated to match the germline sequences to reduce the risk of immunogenicity when the antibody is administered. Germline DNA sequences for human VH and VL genes are known in the art (see, for example, the human germline database "Vbase"; see also Kabat, EA, et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 913242; Tomlinson et al., J. Mol. Biol. 227:776 to 798 (1992); and Cox et al., Eur. J. Immunol 24:827 to 836 (1994).
[00383] Another type of amino acid substitution that can be made is to remove potential proteolytic sites on the antibody. Such sites can occur in a CDR or framework region of a variable domain or in the constant region of an antibody. Replacing cysteine residues and removing proteolytic sites can decrease the risk of heterogeneity in the antibody product and thus increase its homogeneity. Another type of amino acid substitution is to eliminate asparagine-glycine pairs, which form potential deamidation sites, by changing one or both residues. In another example, the C-terminal lysine of the weigh chain of an anti-CXCR4 antibody of the invention can be cleaved. In various aspects of the invention, the heavy and light chains of anti-CXCR4 antibodies can optionally include a signal sequence.
[00384] Once the DNA fragments encoding the VH and VL segments of the present invention are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into genes length antibody chain, in Fab fragment genes or in an scFv gene. In such manipulations, a DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked", as used in this context, is intended to mean that two DNA fragments are joined so that the amino acid sequences encoded by the two DNA fragments remain in frame.
Isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the DNA encoding VH to another DNA molecule encoding heavy chain constant regions (CH1, CH2 and CH3 ). Human heavy chain constant region gene sequences are known in the art (see, for example, Kabat, EA, et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments spanning these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably it is an IgG1 or IgG2 constant region. The IgG constant region sequence can be any one of several alleles or allotypes known to occur among different individuals, such as Gm(1), Gm(2), Gm(3) and Gm(17). These allotypes represent the naturally occurring amino acid substitution in the IgG1 constant regions. For a Fab fragment heavy chain gene, the VH encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region. The heavy chain CH1 constant region can be derived from any of the heavy chain genes.
The isolated DNA encoding the VL region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by covalently linking the DNA encoding the VL to another DNA molecule encoding the light chain constant region, CL. Human light chain constant region gene sequences are known in the art (see, for example, Kabat, EA, et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments spanning these regions can be obtained by standard PCR amplification. The light chain constant region can be either a kappa or a lambda constant region. The kappa constant region can be any one of several alleles known to occur among different individuals, such as Inv(1), Inv(2) and Inv(3). The lambda constant region can be derived from any of the three lambda genes.
[00387] To create a scFv gene, the DNA fragments encoding VH and VL are operatively linked to another fragment encoding a flexible linker, for example, encoding the amino acid sequence (Gly4 -Ser)3, (SEQ ID NO :80) so that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see, for example, Bird et al., Science 242:423a 426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5,879 to 5,883 (1988); McCafferty et al., Nature 348:552 to 554 (1990). monovalent, if only a single VH and VL is used, bivalent, if two VH and VL are used, or polyvalent, if more than two VH and VL are used. Bispecific or polyvalent antibodies can be generated that bind to CXCR4 and to another molecule.
In some aspects of the invention, an immunoadhesin or antibody fusion can be made that comprises all or a portion of an anti-CXCR4 antibody of the invention linked to another polypeptide. In other aspects, only the variable domains of the anti-CXCR4 antibody are linked to the polypeptide. In some aspects of the invention, the VH domain of an anti-CXCR4 antibody is linked to a first polypeptide, whereas the VL domain of an anti-CXCR4 antibody is linked to a second polypeptide that associates with the first polypeptide of way that the VH and VL domains can interact with each other to form an antigen-binding site. In other aspects, the VH domain is separated from the VL domain by a linker so that the VH and VL domains can interact with each other. The VH-linker-VL antibody is then linked to the polypeptide of interest. Additionally, fusion antibodies can be created in which two (or more) single-chain antibodies are linked together. This is useful if you want to create a bivalent or polyvalent antibody on a single polypeptide chain or if you want to create a bispecific antibody.
In some aspects of the invention, other modified antibodies can be prepared using nucleic acid molecules encoding the anti-CXCR4 antibody. For example, "Kappa bodi es"(Ill et al., Protein Eng. 10:949 to 957 (1997)), "Minibodies"(Martin et al., EMBO J., 13:5303 to 5.309 (1994)), "Diabodies" (Holliger et al., Proc. Natl. Acad. Sci. USA 90:6,444 to 6,448 (1993)) or "Janusins" (Traunecker et al., EMBO J. 10:3,655 to 3,659 (1991) and Traunecker et al., Int. J. Cancer (Suppl.) 7:51 to 52 (1992)) can be prepared using standard molecular biological techniques following the teachings of the descriptive report.
Bispecific antibodies or antigen binding fragments can be produced by a variety of methods including hybridoma fusion or binding of Fab' fragments. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315 to 321 (1990), Kostelny et al., J. Immunol. 148:1,547 to 1,553 (1992). Additionally, bispecific antibodies can be formed as "diabodies" or "Janusins". In some aspects of the invention, the bispecific antibody binds to two different epitopes of CXCR4. In some aspects, the modified antibodies described above are prepared using one or more of the variable domains or CDR regions of the anti-CXCR4 antibodies provided herein.
[00391] In one aspect, the present invention comprises the use of multispecific antibodies. A multispecific antibody is an antibody that can simultaneously bind to at least two targets that are of different structure, for example, two different antigens, two different epitopes on the same antigen, or a hapten and/or an antigen or epitope. Multispecific and multivalent antibodies are constructs that have more than one binding site, and the binding sites are of different specificity.
[00392] Representative materials of the present invention were deposited in the American Type Culture Collection (ATCC) on June 19, 2014. A vector having ATCC accession number PTA-121353 is a polynucleotide encoding a variable region of ca- humanized anti-CXCR4 antibody heavy chain, and the vector having ATCC accession number PTA-121354 is a polynucleotide encoding a humanized anti-CXCR4 antibody light chain variable region. The deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and Regulations thereunder (Budapest Treaty). This ensures maintenance of a viable deposit culture for 30 years from the date of deposit. The deposit will be made available by the ATCC under the terms of the Budapest Treaty and subject to an agreement between Pfizer, Inc. and ATCC, which guarantees permanent and unrestricted availability of the progeny of the deposit culture to the public upon grant of the relevant US patent or upon open to the public any US or foreign patent application, whichever comes first, and ensure the availability of the progeny to anyone determined by the US Patent and Trademark Commissioner to be entitled thereto under 35 USC Section 122 and the rules of the Commissioner hereunder (including 37 CFR Section 1.14 with particular reference to 886 OG 638).
[00393] The assignee of this application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials must be promptly replaced upon notification by others of the same. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.
The invention also relates to the use of such anti-CXCR4 antibodies, for example, full length antibodies, antigen binding fragments thereof, or antibody-anti-CXCR4 drug conjugates and pharmaceutical compositions comprising the antibodies of CXCR4 receptor, eg, full length antibodies or antigen-binding fragments thereof, in the treatment of diseases and conditions associated with CXCR4 modulation, such as bone marrow transplantation, chemosensitization, cancer, metastatic disease (eg cancer) , autoimmune disease (eg, rheumatoid arthritis), fibrosis disease (eg, pulmonary), AIDS infection, cardiovascular disease, uveitis, inflammatory diseases, celiac disease, HIV infection, and stem cell-based regenerative medicine. CANCER
The CXCR4 receptor is overexpressed in a large number of cancers, including but not limited to breast (Muller, A. et al. Nature 410:50 to 56 (2001)); ovarian (Scotton, C. et al. Br. J. Cancer 85:891 to 897 (2001); from prostate (Taichman, RS et al. Cancer Res. 62:1,832 to 1,837 (2002); from non-small cell lung (Spano JP et al. Ann. Oncol. 15:613 to 617 (2004)); pancreatic (Koshiba, T. et al. Clin. Cancer Res. 6:3,530 to 3,535 (2000)); thyroid (Hwang , JH et al. J. Clin. Endocrinol. Metab. 88:408 to 416 (2003)); nasopharyngeal carcinoma (Wang, N. et al. J. Transl. Med. 3:26 to 33 (2005)); melanoma (Scala, S. et al. Clin. Cancer Res. 11:1,835 to 1,841 (2005)); renal cell carcinoma (Staller, P. et al. Nature 425:307 to 311 (2003)); lymphoma (Bertolini, F. et al. Cancer Res. 62:3,530 to 3,535 (2002)); neuroblastoma (Geminder, H. et al. J. Immunol. 167:4,747 to 4,757 (2001)); glioblastoma (Rempel, SA et al. Clin Cancer Res. 6:102 to 111 (2000)); rhabdomyosarcoma (Libura, J. et al. Blood 100:2,597 to 2606 (2002)); colorectal ( Zeelenberg, IS et al. Cancer Res. 63:3833 to 3839 (2003)); renal (Schrader, AJ et al. Br. J. C. ancer 86:1,250 to 1,256 (2002)); osteosarcoma (Laverdiere, C. et al. Clin. Cancer Res. 11:2,561 to 2,567 (2005)); acute lymphoid leukemia (Crazzolara, R. et al. Br. J. Haematol. 115:545 to 553 (2001)); and acute myeloid leukemia (Rombouts, E.J.C. et al. Blood 104:550 to 557 (2004)).
[00396] In view of the foregoing, the anti-CXCR4 antibody, antigen binding fragments thereof, or anti-CXCR4 antibody-drug conjugate of this description can be used in the treatment of cancers, including, but not limited to, breast, ovarian, prostate, non-small cell lung, pancreatic, thyroid, nasopharyngeal carcinoma, melanoma, renal cell carcinoma, lymphoma, neuroblastoma, glioblastoma, rhabdomyosarcoma, colorectal, renal, os-theosarcoma, acute lymphoid leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), multiple myeloma (MM), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, mantle cell lymphoma (MCL), follicular lymphoma, Waldenstrom's macroglobulinemia (WM) and B-cell lymphoma and diffuse large B-cell lymphoma (DLBCL). The antibody can be used alone or in combination with other cancer treatments, such as surgery and/or radiation, and/or with other anti-neoplastic agents, such as the anti-neoplastic agents discussed and set out above, including chemotherapy drugs and other anti-cancer antibodies. antitumor antigens, such as those that bind CD20, Her2, PSMA, Campath-1, EGFR and the like.
In some aspects, the anti-CXCR4 antibodies of the present invention can be used in combination with anti CD22 or anti CD33 antibodies, antibody-drug conjugates or compositions comprising such antibodies. For example, the anti-CXCR4 antibodies of the present invention can be combined with inotuzumab ozogamycin or gentuzumab ozogamycin (Mylotarg®).
[00398] "Combination therapy" or administration "in combination with" one or more additional therapeutic agents includes simultaneous, concomitant or consecutive administration in any order. The administration of the constituents of the combined preparations of the present invention can be carried out simultaneously, separately or sequentially.
[00399] According to the present invention, a method for the treatment of cancers is provided which comprises the simultaneous, concomitant or consecutive administration of anti-CXCR4 antibodies of the present invention and inotuzumab ozogamycin. For example, anti-CXCR4 antibodies can be administered before or after or simultaneously with inotuzumab ozogamycin. Additionally, the present invention provides herein a method for treating cancers, such as AML, which comprises the simultaneous, concomitant or consecutive administration of anti-CXCR4 antibodies of the present invention and Mylotarg. For example, anti-CXCR4 antibodies can be administered before or after or simultaneously with Mylotarg.
The anti-CXCR4 antibody, or a fragment thereof, can be conjugated to a therapeutic moiety and/or diagnostic agent, such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin. Such conjugates are referred to herein as antibody-drug conjugates. Antibody-drug conjugates can include one or more cytotoxins.
In some aspects of the invention, the treatment comprises administering an anti-CXCR4 antibody, antigen-binding fragment thereof, or antibody-anti-CXCR4 drug conjugate of the present invention with one or more bioactive agents selected from antibodies, growth factors, hormones, cytokines, anti-hormones, xanthines, interleukins, interferons and cytotoxic drugs. In particular aspects of the invention, the bioactive agent is an antibody, and is directed against a cell surface antigen expressed in B cell malignancies.
In some aspects of the invention, antibody directed against cell surface antigens expressed on B cell malignancies is selected from a group consisting of anti-CD19, anti-CD20 and anti-CD33 antibodies. Such antibodies include the anti-CD20 antibody, rituximab (Rituxan™).
In some aspects of the invention, bioactive agents are cytokines or growth factors and include, but are not limited to, interleukin 2 (IL-2), TNF, CSF, GM-CSF and G-CSF. In particular aspects of the invention, bioactive agents are hormones and include estrogens, androgens, progestins and corticosteroids.
[00404] In some aspects of the invention, the drug is a drug selected from doxorubicin, daunorubicin, idarubicin, aclarubicin, zorubicin, mitoxantrone, epirubicin, carubicin, bendamustine, bevacizumab, bortesomib, lenalidomide, melicogaril, nonorhin valrubicin, cytarabine, gemcitabine, trifluridine, ancitabine, enocitabine, azacitidine, doxifluridine, pentostatin, broxuridine, capecitabine, cladribine, decitabine, floxuridine, fludarabine, gougerotin, puromycin, tegafur, adriazofurin cyclophosphamide, dacarbazine, vinblastine, vincristine, mitoxantrone, bleomycin, mechlorethamine, prednisone, procarbazine methotrexate, flurouracils, etoposide, taxol, taxol analogues and mitomycin.
In some aspects of the invention, the therapeutically effective dose of the anti-CXCR4 antibody, a fragment thereof or the anti-CXCR4 antibody-drug conjugate of the present invention is administered together with one or more combinations of tyrosine inhibitors kinase. Tyrosine kinase inhibitors include both protein and non-protein moieties. A tyrosine kinase inhibitor can be, for example, an antibody, a receptor ligand or a small molecule inhibitor. Examples of suitable tyrosine kinase inhibitors for use in the methods of the present invention include, but are not limited to, gefitinib, sunitinib, erlotinib, lapatinib, canertinib, semaxinib, vatalanib, sorafenib, imatinib, dasatinib, leflunomide, vandetanib, derivatives thereof, analogues thereof and combinations thereof. Additional tyrosine kinase inhibitors suitable for use in the present invention are as described, for example, in US Patent Nos. 5,618,829; 5,639,757; 5,728,868; 5,804,396; 6,100,254; 6,127,374; 6,245,759; 6,306,874; 6,313,138; 6,316,444. 6,329,380; 6,344,459; 6,420,382; 6,479,512; 6,498,165; 6,544,988; 6,562,818; 6,586,423; 6,586,424; 6,740,665; 6,794,393; 6,875,767. 6,927,293; and 6,958,340.
In some aspects of the invention, the therapeutically effective dose of the anti-CXCR4 antibody, a fragment thereof, or the anti-CXCR4 antibody-drug conjugate of the present invention is administered together with one or more drug combinations as a part of a treatment regimen, in which the combination of cytotoxic agents is selected from: CHOPP (cyclophosphamide, doxorubicin, vincristine, prednisone and procarbazine); CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone); COP (cyclophosphamide, vincristine and prednisone); CAP-BOP (cyclophosphamide, doxorubicin, procarbazine, bleomycin, vincristine and prednisone); m-BACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone and leucovorin); ProMACE-MOPP (prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide, leucovorin, mecloethamine, vincristine, prednisone and procarbazine); ProMACE-CytaBOM (prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide, leucovorin, cytarabine, bleomycin and vincristine); MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin and leucovorin); MOPP (mecloethamine, vincristine, prednisone and procarbazine); ABVD (adriamycin/doxorubicin, bleomycin, vinblastine and dacarbazine); MOPP (mecloethamine, vincristine, prednisone and procarbazine) alternating with ABV (adriamycin/doxorubicin, bleomycin and vinblastine); MOPP (mecloethamine, vincristine, prednisone and procarbazine) alternating with ABVD (adriamycin/doxorubicin, bleomycin, vinblastine and dacarbazine); ChIVPP (chlorambucil, vinblastine, procarbazine and prednisone); IMVP-16 (ifosfamide, methotrexate and etoposide); MIME (methyl-gag, ifosfamide, methotrexate and etoposide); DHAP (dexamethasone, high-dose cytaribine, and cisplatin); ESHAP (etoposide, methylpredisolone, high dose cytaribine and cisplatin); CEPP(B) (cyclophosphamide, etoposide, procarbazine, prednisone and bleomycin); CAMP (lomustine, mitoxantrone, cytarabine and prednisone); CVP-1 (cyclophosphamide, vincristine and prednisone), ESHOP (etoposide, methylpredisolone, high dose cytarabine, vincristine and cisplatin); EPOCH (etoposide, vincristine and doxorubicin for 96 hours with bolus doses of cyclophosphamide and oral prednisone), ICE (ifosfamide, cyclophosphamide and etoposide), CEPP(B) (cyclophosphamide, etoposide, procarbazine, prednisone and bleomycin), CHOP-B. (cyclophosphamide, doxorubicin, vincristine, prednisone and bleomycin), CEPP-B (cyclophosphamide, etoposide, procarbazine and bleomycin) and P/DOCE (epirubicin or doxorubicin, vincristine, cyclophosphamide and prednisone).
[00407] In some aspects of the invention, the drug may be administered simultaneously, sequentially or concomitantly with the anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate as described herein. . For example, anti-CXCR4 antibody, antigen binding fragment thereof or anti-CXCR4 antibody-drug conjugate can be administered prior to administration of one or more combinations of cytotoxic agents as a part of a treatment regimen. In some aspects of the invention, therapeutically effective doses of anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate are administered subsequent to administration of one or more combinations of cytotoxic agents as part of a treatment regimen.
In some aspects of the invention, the anti-CXCR4 antibody, antigen-binding fragment thereof or anti-CXCR4 antibody-drug conjugate are subsequently administered together with one or more combinations of cytotoxic agents as part of a treatment regimen.
[00409] The anti-CXCR4 antibody, antigen-binding fragment thereof or anti-CXCR4 antibody-drug conjugate of the present invention may also be administered in conjunction with non-drug treatment such as surgery, radiotherapy, chemotherapy, immunotherapy and diet/exercise regimens. The other therapy may be administered before, concurrently with or after treatment with the anti-CXCR4 antibody, an antigen binding fragment of the same or anti-CXCR4 antibody-drug conjugate of the present invention. There may also be a delay of several hours, days and, in some cases, weeks between the administration of the different treatments, such that the anti-CXCR4 antibody, antigen-binding fragment of the same or anti-CXCR4 antibody-drug conjugate of the present invention can be administered before or after the other treatment. THERAPEUTIC USE OF CXCR4
In accordance with various aspects of the invention, anti-CXCR4 antibodies, antigen-binding fragments thereof or anti-CXCR4-drug antibody can be used to treat or produce drugs to treat a variety of disorders, including various cancers, inflammatory disorders, allergic disorders, infections (HIV infection, etc.), autoimmune disorders (eg, rheumatoid arthritis), fibrosis disorder (eg, pulmonary) and cardiovascular disorders. Cancer disorders include solid tumor cancers (eg, gastric, head of neck, lung, ovarian, and pancreatic cancers) and hematologic cancers (eg, myelodysplastic syndromes, myeloproliferative disorders, and acute leukemias). Examples of hematopoietic disorders include non-B lineage derivatives such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), non-B cell acute lymphoid leukemia (ALL), myelodysplastic disorders, myeloproliferative disorders, polycythemias, thrombocythemias or lymphoproliferations Atypical non-B cell-derived disorders. Examples of a B-cell-derived or B-cell lineage disorder include chronic lymphoid leukemia (CLL), B lymphocyte lineage leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), B-cell pro-lymphoid leukemia , precursor B lymphoblastic leukemia, hairy cell leukemia or plasma cell disorders, for example amyloidosis or Waldenstrom's macroglobulinemia. HEMATOLOGICAL DISORDERS
[00411] Hematological disorders comprise diseases of the blood and all its constituents as well as diseases of organs and tissues involved in the generation or degradation of all blood constituents. In some aspects of the invention, the hematological disorder includes, but is not limited to, acute lymphoid leukemia (ALL), acute myeloid leukemia (AML), chronic lymphoid leukemia (CLL), small lymphocytic lymphoma (SLL), multiple myeloma (MM) , non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, mantle cell lymphoma (MCL), follicular lymphoma, Waldenstrom's macroglobulinemia (WM), B-cell lymphoma, and diffuse large B-cell lymphoma (DLBCL). NHL can include Indolent Non-Hodgkin's Lymphoma (iNHL) or Aggressive Non-Hodgkin's Lymphoma (aNHL). In certain respects, individuals are recidivists or refractory to another treatment. In certain respects, individuals are relapsed or refractory to at least two or more other treatments. In some aspects of the invention, individuals are relapsed or refractory to at least three or more other treatments. In some aspects of the invention, individuals are relapsed or refractory to at least five or more other treatments.
[00412] The present invention contemplates the use of anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate comprising anti-CXCR antibodies as the main therapeutic composition for the treatment of bioshematologic disorders. Such a composition may contain polyclonal anti-CXCR4 or monoclonal anti-CXCR4 antibodies. Additionally, a therapeutic composition of the present invention may contain a mixture of monoclonal anti-CXCR4 antibodies targeting different non-blocking CXCR4 epitopes or mixture of anti-CXCR4 antibody-drug conjugates or mixture of anti-CXCR4 and anti-antibody conjugates Monoclonal CXCR4 and drug. CHRONIC LYMPHOID LEUKEMIA
[00413] CLL cells express high levels of CXCR4. (Burger JA, Burger M, Kipps TJ. Chronic lymphocytic leukemia B cells express functional CXCR4 chemokine receptors that mediate spontaneous migration beneath bone marrow stromal cells. 33, 3658 to 3667 (1999). CXCR4, antigen-binding fragment thereof, or antibody-anti-CXCR4 drug conjugate which comprises an anti-CXCR antibody as the primary therapeutic composition for the treatment of B-cell chronic lymphoid leukemia (CLL). -Polyclonal or anti-CXCR4 monoclonal antibodies or a mixture of polyclonal or anti-CXCR4 monoclonal antibodies Additionally, a therapeutic composition of the present invention may contain a mixture of monoclonal anti-CXCR4 antibodies directed to different CXCR4 epitopes of non-blocking or mixture of anti-CXCR4 antibody-drug conjugates or mixture of monoclonal anti-CXCR4 and anti-CXCR4 antibody and drug conjugates OTHER B-CELL LYMPHOMAS
CXCR4 expression has been demonstrated in B-cell non-Hodgkin's lymphoma (NHL) (Burger et. al., Chronic lymphocytic leukemia B-cell expression functional CXCR4 chemokine receptors that mediate spontaneous migration beneath bone marrow stromal cells. Blood. 94: 3,658 to 3,667 (1999)) and T cells (Trentin L, Agostini C, Facco M, et al. The chemokine receptor CXCR4 is expressed on malignant B cells and mediates chemotaxis. J Clin Invest. 104:115 to 121 (1999)) . Malignant B cells from patients with B-NHL express functional CXCR4 receptors. The distinct pattern of chemokine receptor expression is believed to be involved in lymphoma cell trafficking and addressing and may allow for the distinction of different subsets of NHL. (Jones D, Benjamin RJ, Shahsafaei A, Dorfman DM. The chemokine receptor CXCR4 is expressed in a subset of B-cell lymphomas and is a marker of B-cell chronic lymphoid leukemia. Blood. 95:627–632 ( 2000)). In an animal model, mice were stimulated with T-cell hybridoma cells that were modified to retain CXCR4 within the cytoplasm. In another mouse model of human high-grade NHL, neutralization of CXCR4 by monoclonal antibodies inhibited the circulation of NHL cells and enhanced survival. (Bertolini F, Dell'Agnola C, Mancuso P, et al. CXCR4 neutralization, a novel therapeutic approach for non-Hodgkin's lymphoma. Cancer Res. 62:3.106 to 3.112 (2002)), therefore, suggested the neutralization of CXCR4 as a innovative therapeutic approach in NHL. The present invention contemplates the use of an anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate comprising anti-CXCR4 antibodies as the main therapeutic composition for the treatment of chronic B cell lymphomas. Such a composition may contain polyclonal anti-CXCR4 or monoclonal anti-CXCR4 antibodies. Additionally, a therapeutic composition of the present invention may contain a mixture of monoclonal anti-CXCR4 antibodies targeting different non-blocking CXCR4 epitopes or mixture of anti-CXCR4 antibody-drug conjugates or mixture of anti-CXCR4 and anti-CXCR4 monoclonal antibody conjugates and drug. CXCR4 IN MULTIPLE MYELOMA
[00415] Multiple myeloma (MM) is a large incurable neoplasm of plasma cells. The chemokine receptor CXCR4 is expressed by most MM cells in patients. It promotes the migration and targeting of myeloma cells to the bone marrow (BM) compartment, supports tumor cell survival, and protects myeloma cells against chemotherapy-induced apoptosis. Correspondingly, the anti-CXCR4 antibody, antigen-binding fragment thereof or antibody-anti-CXCR4 drug conjugate which comprises an anti-CXCR antibody of the present invention that inhibits CXCR4 activity (e.g., antagonistic antibodies) can be used to treat haematological disorders such as multiple myeloma. Additionally, a therapeutic composition of the present invention may contain a mixture of monoclonal anti-CXCR4 antibodies directed to different non-blocking CXCR4 epitopes or mixture of anti-CXCR4 antibody-drug conjugates or mixture of anti-CXCR4 and anti-antibody conjugates Monoclonal CXCR4 and drug CXCR4 IN ACUTE LEUKEMIA
[00416] Due to the fact that CXCR4 plays an essential role in the retention of hematopoietic progenitors in the marrow, several groups have examined the role played by CXCR4 in progenitor cell leukemias. Precursor B-cell acute lymphoid leukemia (ALL) expresses functional CXCR4 receptors (Bradstock KF, Makrynikola V, Bianchi A, Shen W, Hewson J, Gottlieb DJ. Effects of the chemokine stromal cell-derived factor-1 on the migration and localization of precursor-B acute lymphoblastic leukemia cells within bone marrow stromal layers. Leukemia. 14:882 to 888 (2000)) that participate in marrow targeting of leukemia cells in immunodeficient mice combined with severe non-obese diabetes (NOD/SCID) (Shen W, Bendall LJ, Gottlieb DJ, Bradstock KF. The chemokine receptor CXCR4 enhances integrin-mediated in vitro adhe-sion and facilitates engraftment of leukemic precursor-B cells in the bone marrow. Exp Hematol. 29:1,439 to 1,447 ( 2001).) In a refined animal model, Sipkins et al (Sipkins DA, Wei X, Wu JW, et al. In vivo imaging of specialized bone marrow endothelial microdomains for tumor engraftment. Nature. 435: 969 to 973 (2005) ) provided evidence that the CXC Functional R4 is required for addressing ALL cells to the marrow microenvironment. The present invention contemplates the use of an anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate comprising anti-CXCR antibodies as the main therapeutic composition for the treatment of acute lymphoid leukemia (ALL) . Such a composition may contain polyclonal anti-CXCR4 or monoclonal anti-CXCR4 antibodies.
Additionally, a therapeutic composition of the present invention may contain a mixture of monoclonal anti-CXCR4 antibodies directed to different non-blocking CXCR4 epitopes or mixture of anti-CXCR4 antibody-drug conjugates or mixture of anti-CXCR4 antibody conjugates and monoclonal anti-CXCR4 and drug. CXCR4 IN ACUTE MYELOID LEUKEMIA (AML)
[00418] Despite an overall sensitivity to chemotherapy, long-term disease-free survival in AML remains low due to the fact that most patients relapse from minimal residual disease (MRD). CXCR4 appears to be the central regulator of survival signals that explain anticancer drug resistance. This concept is supported by the conclusion that high-level expression of CXCR4 by leukemia cells is an adverse prognostic indicator in AML. Spoo AC, Wierda WG, Burger JA. The CXCR4 score: a new prognostic marker in acute myelogenous leukemia [abstract]. Blood. 104:304a (2004). The present invention contemplates the use of an anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate comprising anti-CXCR antibodies as the primary therapeutic composition for the treatment of acute myeloid leukemia (AML) ._Such a composition may contain polyclonal anti-CXCR4 or monoclonal anti-CXCR4 antibodies.
Additionally, a therapeutic composition of the present invention may contain a mixture of monoclonal anti-CXCR4 antibodies directed to different non-blocking CXCR4 epitopes or mixture of anti-CXCR4 antibody-drug conjugates or mixture of anti-CXCR4 antibody conjugates and monoclonal anti-CXCR4 and drug CXCR4 IN NON-HEMATOPOIETIC CANCER
[00420] One of the most intriguing and perhaps most important roles that chemokines and chemokine receptors have is to regulate the metastasis of solid tumors. CXCR4 is one of the best-studied chemokine receptors, which selectively binds to stromal cell-derived chemokine CXC factor 1 (SDF-1), also known as CXCL12 (Fredriksson et. al., Mol Pharmacol. 63:1.256 to 1,272 (2003)). To date, CXCR4 has been shown to be overexpressed in more than 20 human malignancies, including breast cancer, prostate cancer, kidney cancer, colon cancer, thyroid cancer, and pancreatic cancer (Müller et al., Nature 410: 50 to 6 (2001); Akashi et al. Cancer Sci. 99(3):539 to 542 (2008); Maréchal et al. Br J Cancer, 100, 1444 to 1451 (2009); Wang et al., Clin Exp Metastasis , 26, 1049 to 1054.(2009); He X et al., Pathol Res Pract, 206, 712 to 715. (2010)).
[00421] The present invention contemplates the use of an anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate comprising anti-CXCR antibodies as the main therapeutic composition for the treatment of non-hematopoietic cancers. Such a composition may contain polyclonal anti-CXCR4 or monoclonal anti-CXCR4 antibodies. Additionally, a therapeutic composition of the present invention may contain a mixture of monoclonal anti-CXCR4 antibodies targeting different non-blocking CXCR4 epitopes or mixture of antibody-anti-CXCR4 drug conjugates or mixture of antibody-anti-CXCR4 drug conjugates and anti-CXCR4 monoclonals. CXCR4 IN BREAST CANCER
[00422] High-level expression of CXCR4 in neoplastic cells is associated with relatively poor overall survival in breast cancer patients. (Li YM, Pan Y, Wei Y, et al. Up-regulation of CXCR4 is essential for HER2-mediated tumor metastasis. Cancer Cell. 6:459 to 469 (2004)). High-level expression of HER2/neu, which is seen in about 30% of all breast cancers, is also associated with a relatively poor prognosis. Li et. al. recently demonstrated that HER2/neu enhances CXCR4 expression and function by inhibiting CXCR4 degradation. (Li YM, Pan Y, Wei Y, et al. Up-regulation of CXCR4 is essential for HER2-mediated tumor metastasis. Cancer Cell. 6:459 to 469 (2004)). Therefore, the present invention contemplates the use of an anti-CXCR4 antibody, antigen binding fragment thereof, or anti-CXCR4 antibody-drug conjugate which comprises anti-CXCR antibodies as the main therapeutic composition for the treatment of breast cancer. Such a composition may contain polyclonal anti-CXCR4 or monoclonal anti-CXCR4 antibodies. Additionally, a therapeutic composition of the present invention may contain a mixture of monoclonal anti-CXCR4 antibodies targeting different non-blocking CXCR4 epitopes or mixture of anti-CXCR4 antibody-drug conjugates or mixture of anti-CXCR4 and anti-CXCR4 antibody-drug conjugates CXCR4 monoclonal CXCR4 IN LUNG CANCER
[00423] Small cell lung cancer (SCLC) is an aggressive and rapidly metastasizing neoplasm with a high propensity for marrow involvement. Even with chemotherapy and radiotherapy treatment in combination, the 5-year survival is only about 5% due to the rapid development of drug resistance. In SCLC cells, activation of CXCR4 induces migratory and invasive responses and adhesion to marrow stromal cells in a CXCR4 and integrin-dependent manner. CXCR4 can target the distinct metastatic pattern seen in patients with SCLC. Thus, the present invention contemplates the use of an anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate which comprises anti-CXCR antibodies as the main therapeutic composition for the treatment of lung cancer. Such a composition may contain polyclonal anti-CXCR4 or monoclonal anti-CXCR4 antibodies. Additionally, a therapeutic composition of the present invention may contain a mixture of monoclonal anti-CXCR4 antibodies targeting different non-blocking CXCR4 epitopes or mixture of antibody-anti-CXCR4 drug conjugates or mixture of antibody-anti-CXCR4 drug conjugates and anti-CXCR4 monoclonals. CXCR4 IN RENAL CELL CARCINOMA (RCC)
[00424] Recently, the role of CXCR4 in mRCC has been fully elucidated. The results demonstrated that high expression of CXCR4 was strongly associated with poor survival of patients with mRCC (Wang et al., Clin Exp Metastasis, 26, 1049 to 1054(2009); Zhao et al., Mol Biol Rep, 38 , 1,039 to 1,045 (2011)). Furthermore, the results in a murine model show that the metastatic capacity of RCC cells expressing CXCR4 strongly correlated with the level of CXCR4 protein in cancer cells and the expression of SDF-1α in target organs. (Motzer et. al. The New England Journal of Medicine, volume 335, number 12, page 865 to 875 (1996)). Correspondingly, CXCR4 may be an interesting therapeutic agent in a multimodal therapy of clear cell renal carcinoma.
[00425] The present invention contemplates the use of an anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate comprising anti-CXCR antibodies as the main therapeutic composition for the treatment of cell carcinoma kidneys (RCC). Such a composition may contain polyclonal anti-CXCR4 or monoclonal anti-CXCR4 antibodies.
Additionally, a therapeutic composition of the present invention may contain a mixture of monoclonal anti-CXCR4 antibodies directed to different non-blocking CXCR4 epitopes or mixture of anti-CXCR4 antibody-drug conjugates or mixture of anti-CXCR4 antibody-drug conjugates. -CXCR4 and anti-CXCR4 monoclonals. CXCR4 IN NON-ONCOLOGICAL INDICATIONS
[00427] Chemokine receptors are expressed in several specific cells and at a specific time. They are widely associated with the control of inflammatory and immune responses through a mechanism by which their effector cells accumulate in a location where the chemokine is produced. For example, SDF-1 has been shown to specifically inhibit T-cell (X4)-targeted HIV infection in vitro (Bleul et al. Nature, 382:829 to 833 (1996), Oberlin et al. Nature, 833 to 835 (1996)). It can be considered that SDF-1 binds to CXCR4 before HIV, thus taking a framework to infect an HIV cell, resulting in inhibition of HIV infection. Furthermore, an inhibitor of HIV infection has been shown to be a CXCR4 antagonist (Nat. Med., 4, 72 (1998)).
Correspondingly, the present invention contemplates the use of anti-CXCR4 antibody, antigen-binding fragment thereof, or anti-CXCR4 antibody-drug conjugate anti-CXCR antibodies as a therapeutic composition for the treatment of inflammatory or autoimmune diseases, allergic diseases, infections (HIV infection, etc.), diseases associated with HIV infection (acquired immunodeficiency syndrome, etc.). Such a composition may contain polyclonal anti-CXCR4 or monoclonal anti-CXCR4 antibodies.
Additionally, a therapeutic composition of the present invention may contain a mixture of monoclonal anti-CXCR4 antibodies targeting different non-blocking CXCR4 epitopes or mixture of anti-CXCR4 antibody-drug conjugates or mixture of anti-CXCR4 antibody-drug conjugates. -CXCR4 and monoclonal anti-CXCR4. WHIM SYNDROME
[00430] WHIM syndrome is a congenital immunodeficiency disorder characterized by the main clinical manifestations: warts, hypogammaglobulinemia, recurrent bacterial infections and myelocatexia [McDermott, DH et al. Blood 118 (18): 4,957 to 4,962 (2011); Mcermott DH et al. J. Cell. Mol. Med. 15(10): 2,071 to 2,081 (2011)]. Myelocatexia can be further characterized as an unusual hematologic disorder in which mature neutrophils lack bone marrow salts and the abundance or function of B and T cells is deficient (Zueler WW et al. N. Engl. J. Med. 270:699 to 704 (1964)). Hernandez PA et al first described that mutations in CXCR4 are associated with the WHIM syndrome, with CXCR4R334X being the most common and best-studied variant (Hernandez PA et al. Nature Genetics 34: 70 to 74 (2003)). CXCR4R334X , as a gain-of-function mutation, exhibits enhanced signaling capacity for the endogenous ligand CXCL12. Therefore, controlling the increased signaling capacity of CXCR4R334X can be used to treat WHIM syndrome.
In some aspects of the invention, the present invention contemplates the use of an antibody-drug conjugate comprising anti-CXCR4 antibodies as the main therapeutic composition for the treatment of WHIM syndrome, wherein said composition may contain anti- Polyclonal CXCR4 or monoclonal anti-CXCR4. In addition, a therapeutic composition of the present invention may contain a mixture of monoclonal anti-CXCR4 antibodies targeting different non-blocking CXCR4 epitopes or mixture of anti-CXCR4 antibody-drug conjugates or mixture of anti-CXCR4 and anti antibody-drug conjugates -CXCR4 monoclonal In addition, in some aspects, the therapeutic composition disclosed by the present invention can be administered alone or in combination with some current treatments for WHIM syndrome, such as G-CSF (filgrastim (Neupogen; Amgen Inc.)), immunoglobulin intravenous injection and an AMD3100 small molecule CXCR4 antagonist (plerixafor, trade name Mozobil (Genyzme Corporation)).
[00432] The following examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will be made apparent to those skilled in the art from the foregoing description and are within the scope of the appended claims. EXAMPLE 1 ANTIBODY BINDING AFFINITY DETERMINATION FOR CHIMERIC ANTI-CXCR4 MOUSE ANTIBODIES
The binding of mouse anti-CXCR4 chimeric antibodies 12A11, 6B6 and 3G10 (expressed as the hIgG1 subtype) was evaluated in human Ramos Non-Hodgkin's Lymphoma (NHL) cells expressing CXCR4 by flow cytometry. 100,000 cells were incubated with anti-CXCR4 antibodies serially diluted in 100 µl binding buffer (PBS + 0.5% BSA), followed by incubation with Dylight488-conjugated anti-human Fc secondary antibody from Jackson Immunoresearch Laboratories. The % was derived by normalizing the MFI values of each dilution to the maximum value. EC50 was calculated by PRISM software. TABLE 6
EXAMPLE 2 CONNECTION OF HUMANIZED FAB 3G10 TO HPB-ALL CELLS EXPRESSING CXCR4
Binding of humanized 3G10 Fabs was assessed in CXCR4 expressing HPB-ALL cells by flow cytometry. 150,000 cells were incubated with various h3G10 Fabs at 2.5 or 0.25 μg/ml in 100 μl binding buffer (PBS + 0.5% BSA), followed by incubation with secondary antibody specific for anti-human Fab of goat coupled with APC from R&D systems. TABLE 7
EXAMPLE 3 CXCR4 ANTIBODY LINK: CELL LINK AND AB CXCR4 FABS AFFINITY MEASUREMENT
Binding of CXCR4 Fabs to cells expressing CXCR4 was measured by flow cytometry in HPB-ALL (human T cell leukemia) cells. 150,000 HPB-ALL cells were resuspended in 100 μl of FACS buffer (IxPBS + 0.5% BSA) in 96-well plates. Anti-CXCR4 Fabs were added to each well at a final concentration of 0.25 μg/ml and incubated at 4 degrees for 30 min. After removal of primary antibodies, cells were washed twice with FACS buffer and then resuspended in FACS buffer and 2 µl (3 µg) of secondary Ab (Alexa Fluor 647-conjugated goat anti-human IgG, F(ab) specific '2)2, Jackson ImmunoResearch Laboratories, West Grove PA) were then added to each well. Plates were incubated at 4 degrees for another 30 min. Fluorescence signals were acquired by the LSRII cell analyzer (BD Biosciences, San Jose CA). The MFI (Average Fluorescence Intensity) of each sample is shown in the table.
The binding affinity of each Fab was determined using human CXCR4-enriched lipoparticles (LEV101, Integral Molecular, Philadelphia, PA). Biotinylated WGA (Sigma Aldrich, St. Louis MO) can be coated onto the SA chip to facilitate the capture of lipoparticles containing CXCR4 proteins. A dilution series (3X dilution factor, 5-membered, with a top concentration of 10 or 30 nM) of Fab was injected from low to high concentration (with an association time of 3 minutes for each concentration) to perform the analysis kinetics of the data using a "kinetic titration" methodology as described in Karlsson, et al. (Karlsson, R., Katsamba, PS, Nordin, H., Pol, E. & Myszka, DG Analyzing a kinetic titration series using affinity biosensors. Anal. Biochem. 349, 136 to 147 (2006). For some cycles of analysis , buffer was injected over the captured particles in place of Fab to provide blank cycles for double reference purposes (double reference was performed as described in Myszka et al. Improving biosensor analysis. J. Mol. Recognit. 12, 279-284 (1999)). TABLE 8
EXAMPLE 4 CONNECTION OF AB OF CXCR4 TO CXCR4 OF CYNO AND HUMAN MONKEY
[00437] The anti-human CXCR4 Ab h3G10.1.91.A58B was tested for its cross-reactivity to cynomolgus CXCR4 by flow cytometry in a series of dilutions (0.007 to 267 nM) in 1) HPB-ALL (cell leukemia) cells human T) and CHO transfected with Cyno-CXCR4 and 2) Raji (Non-Hodgkin's Lymphoma) and HSC-F (Cynomolgus T cell line). Binding was detected by secondary Ab (Alexa Fluor 647-conjugated goat anti-human IgG, specific Fcgamma, Jackson ImmunoResearch Laboratories, West Grove PA) and purchased with the LSRFortessa cell analyzer (BD Biosciences, San Jose CA). Curve fit calculation and EC50 were performed with Prism software (GraphPad Software, La Jolla CA). Tables 9A and 9B and Figures Figures 3A and 3B. TABLE 9A
TABLE 9B
EXAMPLE 5 EFFECTOR FUNCTION OF CXCR4 ANTIBODIES
[00438] Recombinant therapeutic antibodies rely on two types of functionalities to achieve clinical efficacy: target-specific binding by the Fab domain (antigen binding fragment) and immune-mediated effector functions — such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)—through interaction of the antibody's Fc domain with Fc receptors on various cell types. In ADCC, the Fc region of the antibody binds to Fc receptors (FCYRS) on the surface of immune effector cells, such as natural killers and macrophages, leading to phagocytosis or lysis of target cells. In CDC, antibodies kill target cells by targeting the complement cascade on the cell surface. The Fc portion of a therapeutic antibody may therefore play an important role in its mechanism of action through its influence on ADCC or CDC.
[00439] Each human IgG subclass (IgG1, IgG2, IgG3 and IgG4) exhibits a distinct effector function profile, which is dictated by differential binding to each of the FCYRS and complement complex proteins. Although both IgG1 and IgG3 bind relatively tightly, IgG2 and IgG4 have much lower affinity for Fc receptors and do not produce high levels of ADCC. IgG1 also has high affinity for complex complement proteins, with much less CDC produced by IgG2, IgG3 and IgG4.
The ADCC (antibody dependent cytotoxicity) activity of anti-CXCR4 antibodies was determined with the cytoTox 96 non-reactive cytotoxicity assay kit (Promega, Madison WI). Human tumor cells expressing CXCR4 were seeded on 10,000 cells the day before the assay. Donor PBMC cells were isolated by Ficoll gradient and cultured overnight at 37°C in x-vivo medium. The next day, 10 or 20 µg/ml of antibody was added to the wells with or without the addition of 1,000,000 PBMC cells (E:T=100:1) in RPMI+5% FBS. The plates were then incubated at 37 °C for 4 hours. At the 3.5 hour time point, 20 µl of the lysis solution was added to the target cell wells alone. After rotating the plate at 8000 rpm for 3 min, 50 µl of supernatants were transferred to another plate. 50 μl of substrate was then added to each well and the plate was incubated at room temperature for 30 min in the dark. The reaction was stopped by adding 50 μl of stop solution to each well. The plates were then read at 490 nm with a spectrometer (Molecular Devices). The percentages (%) of specific lysis were calculated with the following formula: % specific lysis = (treatment LDH release - target cell spontaneous LDH release - effector cell spontaneous LDH release) / (maximum LDH release from target cell - spontaneous LDH release from target cell) x 100
The CDC (complement dependent cytotoxicity) activity of anti-CXCR4 antibodies was determined with the cytoTox 96 non-reactive cytotoxicity assay kit (Promega, Madison WI). Human tumor cells expressing CXCR4 were seeded on 10,000 cells the day before the assay. The next day, 5 to 20μg/ml of each Ab was added to the cells alone or with 2.5%, 10% or 20% donor AB complement (from Innovative Biotech or Sigma). Plates were developed and specific lysis analyzed similarly to the ADCC assay.
[00442] The ADCC assay was performed on Ramos (NHL) or MOLT-4 (human T cell leukemia) cells expressing CXCR4 using 20 μg/ml (Figure 4A) or 10 μg/ml (Figure 4C) of antibody plus 100:1 (Effector:Target) ratio of normal donor PBMC cells. After 4 hours of incubation, ADCC activity of anti-human CXCR4 mouse Abs 12A11, 6B6 and 3G10 produced as chimeric hIgG1 in Ramos (NHL) cells (Figure 4A) or humanized 3G10 antibodies in MOLT-4 cells (Figure 4C). Approximately 80% cell lysis was seen from m3G10-hIgG1 treatment, compared to approximately 20% cell lysis from the m3G10-hIgG4 subtype in MOLT-4 cells (Figure 4B).
[00443] The CDC activity was observed from the treatment of 20 μg/ml of mouse 12A11-, 6B6- and 3G10-hIgG1 chimeric antibodies in Ramos cells expressing CXCR4 (Figure 4D) and with 5 μg/ml of humanized antibodies in Daudi cells (NHL) (Figure 4F). A maximum of 20 to 30% specific lysis activity was detected with 5 μg/ml of anti-CXCR4 antibody 3G10 in IgG1 main chain, similarly to Rituxan positive control antibody in Daudi cells, whereas 3G10 in hIgG4 format did not showed CDC activity (Figure 4E) EXAMPLE 6 INHIBITION OF SDF-1-INDUCED CALCIUM FLOW BY ANTI-CXCR4 ANTIBODIES
[00444] Upon the binding of SDF-1 (ligand) to the CXCR4 receptor, a calcium flux reaction is activated. As evidence for the functional antagonistic activity of CXCR4 Abs, the ability of anti-human CXCR4 antibodies to inhibit SDF-1 induced calcium flux, human T cell leukemia (Jurkat) cells were used in conjunction with the kit. Fluo-NW Calcium calcium assay (Molecular Probes/life technologies). Cells were grown to subconfluence in RPMI 1640 medium containing 10% fetal bovine serum, 1% glutamine at 37°C in a CO2 incubator. On the day of the assay, cells were plated in a 384-well, clear black bottom plate at 70,000 cells per well in 25 µl of assay buffer. Kit assay dye was then added to the plates, 25 µl/well for 110 min at room temperature, protected from light. A 1:3 serial dilution of 11 points was prepared in Fluo-NW Ca Assay Kit Buffer for each anti-human CXCR4 antibody, resulting in a concentration range of 500 nM to 8 pM. Cells were incubated with antibodies at room temperature for 20 minutes. Cells were then stimulated with SDF-1 alpha (Invitrogen) to a final concentration of 8 nM. Calcium flux was then measured for 95 seconds using a FLIPR Tetra (Molecular Devices). The positive control consisted of cells in the presence of SDF-1 alpha and no antibody treatment. Baseline was measured from cells lacking SDF-1 alpha and no antibody treatment. Induction with SDF-1 alpha of calcium was measured by fluorescence signal development over time. Data were exported and plotted using GraphPad Prism software and a non-linear curve fit with sigmoid dose response formula was used to calculate EC50 values. The resulting inhibition of calcium flux by anti-human CXCR4 antibodies is shown in Figure 5. Antibodies 3G10, 6B6 and 12A11 inhibited SDF-1 alpha induced calcium flux, with IC50s for inhibition of 1.555 nM, 1.418 nM and 27.07 nM, respectively (Table 10). IC50s for humanized CXCR4 antibodies evaluated in the Functional Calcium Flux assay are summarized in Table 11 (mean n=3 independent experiments/antibody). In summary, the potential of humanized CXCR4 antibodies is similar to that of the chimeric m3G10 antibody in this assay. TABLE 10: CALCIUM FLOW ACTIVITY OF CHIMERIC CXCR4 ANTIBODIES
TABLE 11: CALCIUM FLOW ACTIVITY OF HUMANIZED CXCR4 ANTIBODIES
EXAMPLE 7 ACTIVITY OF CXCR4 ANTIBODIES IN A CYCLIC AMP CELL (CAMP) BASED ON FUNCTIONAL ASSAY
By inhibiting CXCL12 (binding) to the CXCR4 receptor, it is known that cAMP is inhibited. As evidence for antagonist activity of CXCR4 Abs, a cAMP assay was performed using human CXCR4-transfected CHO-K1 cells purchased from DiscoveRx, Fremont, CA. The cAMP Hunter eXpress GPCR Assay Kit (DiscoveRx) was used to perform the assay. In the absence of CXCR4 antibodies, treatment with the CXCR4 ligand, CXCL12, inhibited cAMP production. Upon treatment with CXCR4 antibodies, this inhibition was abrogated, and cAMP production increased. The EC50 for this response is shown in Table 12. In summary, all CXCR4 antibodies tested had similar potential activity in this assay, in the range of 24.3 to 45.6 nM. This study confirmed that mouse 3G10 (chimeric) and humanized 3G10 antibodies can compete and block CXCR4 ligand activity in the cAMP functional assay. TABLE 12: EC50S ABS ANTI-CXCR4 ABS SUMMARIES IN CAMP TEST
EXAMPLE 8 INDUCTION OF CELL DEATH BY ANTI-CXCR4 ANTIBODIES
Anti-CXCR4 antibodies were examined for their ability to induce cell death in the Ramos Non-Hodgkin Lymphoma cell line. Cells were grown to subconfluence in RPMI 1640 medium containing 10% fetal bovine serum and 2 nM glutamine at 37°C in a CO2 incubator. Cells were seeded in growth medium in a 48-well plate at 2.5 x 106 cells/ml. Anti-CXCR4 antibodies, diluted in PBS at the indicated concentrations, were added to the cells and incubated for 24 h at 37°C. Cell death was measured by Annexin V-PE and propidium iodide (PI) fluorescent signal measured by LSRII Flow Cytometer (BD Biosciences). Total cell death was determined by adding the Annexin V+/PI+ population (late) with the Annexin V+/PI- population (early).
The results in Figure 6A demonstrate that the anti-CXCR4 antibodies 6B6 and 3G10 are able to induce cell death in Ramos cells in a dose-dependent manner. At the highest concentration tested, 100 nM, 3G10 and 6B6 resulted in >70% cell death. This effect was greater than positive control Staurosporine (STS), a known potent cell death inducer (approximately 60%). Untreated cells showed approximately 30% cell death in this assay. The 12A11 anti-CXCR4 antibody was not able to induce cell death under the conditions tested. Similar results were observed when testing the activity of these antibodies in non-Hodgkin Raji lymphoma cells.
[00448] In a similar study, single-chain (Fab) and bivalent F(ab)2' were generated from the 3G10 antibody. Fab is a single-chain (monovalent) antibody and contains the immunoglobulin variable regions that are part of the antigen-binding site and the first immunoglobulin constant region. This fragment was obtained by digestion of the 3G10 antibody with the proteolytic enzyme papain. F(ab')2 was generated by pepsin digestion to remove most of the Fc region while leaving part of the hinge region intact. The F(ab')2 fragments had two antigen-binding Fab moieties linked together by disulfide bonds and therefore are bivalent. Fab and F(ab')2 were tested side by side with the full length 3G10 antibody for their ability to induce cell death of Ramos cells. The results shown in Figure 6B indicate that the ability to induce cell death is dependent on bivalence, with the intact antibody (3G10) and F(ab')2 antibodies capable of inducing cell death, whereas the 3G10 Fab had very limited effect on cell death. EXAMPLE 9 INHIBITION OF SOLID NON-HODGKIN LYMPHOMA TUMOR GROWTH IN VIVO BY ANTI-CXCR4 ANTIBODIES
[00449] SDF-1/CXCR4 signaling is believed to play an important role in multiple stages of tumor development, including tumor growth, angiogenesis, invasion and metastasis. To assess the ability of anti-human CXCR4 Abs to inhibit tumor growth, a tumor xenograft model using 4-6 week old female CB17 SCID Beige mice (Jackson Laboratories) and implanted Ramos Non-Hodgkin Lymphoma cells subcutaneously was used. Cells were grown at 37°C in a 5% CO2 incubator in RPMI 1640 medium containing 10% fetal bovine serum. In this study, 5 x 106 cells were implanted in the right rear flank region of each mouse and allowed to grow as solid tumors to an average size volume of approximately 175 mm3, calculated by the formula (volume=length x width2)/2. The mice were then randomized into 4 different treatment groups, n=10 animals per group. Groups of mice were treated with intravenous (i.v.) injection of the antibodies in a sterile PBS solution: (i) Isotype Control Ab (15 mg/kg); (ii) 3G10 (15 mg/kg); (iii) 6B6 (15mg/kg); and (iv) 12A11 (15 mg/kg). Animals were dosed with antibodies once a week for 3 weeks for a total of 3 doses. Tumor volumes were measured for caliper one to three times a week for the duration of the study. The results of the experiment are shown in Figure 7. The results indicate that all 3 anti-CXCR4 antibodies tested significantly inhibited tumor growth relative to the isotype control antibody. The results indicate that anti-CXCR4 antibodies have the ability to inhibit the growth of an established solid tumor in vivo. This tumor growth inhibitory effect was maintained for a long period of time (42 days) after the antibody dosage was discontinued. The activity of all three anti-CXCR4 antibodies tested was comparable in this solid tumor model. Figure 7. EXAMPLE 10 INCREASED SURVIVAL TIME AND DECREASED TUMOR LOAD BY ANTI-CXCR4 ANTIBODIES IN A MOUSE SYSTEMIC NON-HODGKIN LYMPHOMA CELL MODEL
[00450] The antitumor activity of anti-CXCR4 antibodies was further investigated in a hematologic lymphoma model using Non-Hodgkin Raji Lymphoma cells. The Raji cells used in this study were transfected with the luciferase (LUC) gene to allow "in-life" monitoring of tumor burden over time. Cells were grown at 37°C in a 5% CO2 incubator in RPMI 1640 medium containing 10% fetal bovine serum. The model was established by injecting 1 x 106 Raji-LUC cells into 6-8 week old female SCID Beige mice (from the Charles River Laboratories) via the tail vein. On Day 0 (day of implant) Raji-LUC cells lodged in the lungs, indicating accurate i.v. injection of tumor cells in all animals. One day after cell implantation, mice were assigned to treatment groups (10 animals per group) based on the presence of lung bioluminescence. Mice were treated with Isotype Control antibody, 3G10, 6B6 and 12A11, at 10 mg/kg in a sterile PBS solution once a week intraperitoneally (ip) until Day 67. Tumor burden was monitored by bioluminescence imaging using the Xenogen IVIS 200 imaging system. Mice were imaged every 5 days in a ventral position, delivered IP at 15 mg/ml, 200 μl injection, and whole-body bioluminescence was de- completed using Xenogen Living Image. When the mice began to show hind limb paralysis or reached other human endpoints, they were euthanized. Both tumor burden (by bioluminescence) and survival were assessed as end points.
[00451] Figures 8A and B show the bioluminescence results of this study. The decrease in tumor burden is shown by a decrease in the level of bioluminescence over time. Figure 8A shows the representative image of 4 to 5 animals from each group on Days 0 (Implant Baseline), 15 and 25 of the study. All three anti-CXCR4 antibodies significantly decreased tumor burden as measured by luminescence level. Figure 8B shows that treatment with 3G10, 6B6, and 12A11 antibodies had comparable TGI activity to the isotype control antibody in this model.
The survival curve shown in Figure 8C demonstrates a very significant effect of the anti-CXCR4 antibodies 3G10, 6B6 and 12A11 compared to the isotype control antibody on the survival of animals implanted with the Raji-LUC cells systemically. While animals treated with isotype control showed a median survival of 18 days, animals treated with anti-CXCR4 antibodies did not achieve median survival before the end of the study. The effect of all 3 anti-CXCR4 antibodies tested was comparable with no statistical difference between them. The survival of animals treated with anti-CXCR4 antibody was prolonged well beyond Day 67, when the last dose of antibody was administered. EXAMPLE 11 INCREASED SURVIVAL TIME AND DECREASED TUMOR LOAD BY ANTI-CXCR4 ANTIBODIES IN AN ACUTE MYELOID MICE LEUKEMIA (AML) CELL MODEL OF MOUSE
[00453] The anti-CXCR4 antibody 6B6 was tested for its ability to increase survival and reduce tumor burden of NSG mice using an intravenous disseminated/systemic AML model. The human AML cancer line MV4-11 was transduced with the luciferase gene (MV4-11-LUC) to allow "in-life" monitoring of tumor burden over time. Cells were grown at 37°C in a 5% CO2 incubator in IMDM medium containing 10% fetal bovine serum with 1 μg/ml puromycin for selection of luciferase expression. Female NSG mice 4 to 6 weeks old (from Jackson Laboratories) were injected intravenously with AML MV4-11-LUC cells (1 x 106/animal).
[00454] On Day 13 after cell implantation, mice were randomized into three treatment groups (10 animals per group) based on total body bioluminescence intensity. Treatment with weekly subcutaneous (sc) antibody at 10 mg/kg in a sterile PBS solution was initiated on Day 13 (isotype control and anti-CXCR4 6B6 antibody) and on Day 20 (anti-CXCR4 6B6 antibody) as indicated in the figure (Day 13; Day 20). Tumor burden was assessed using the Xenogen IVIS 200 imaging system. Mice were imaged every 7 days in a ventral position and whole-body bioluminescence was determined using the Xenogen Living Image software. When mice began to show hind limb paralysis or reached other human endpoints, they were euthanized. Both tumor burden (by bioluminescence) and survival were assessed as end points. The number of circulating human AML cells in the peripheral blood (PB) of mice (n=10 per group) was monitored in blood samples collected on Day 35 of the study. AML markers CD45 and CD33 were used to determine the percentage of circulating human AML cells by flow cytometry staining with anti-human CD45 and anti-human CD33 antibodies (from BD Biosciences)
Figure 9A shows representative bioluminescence image of 5 animals/treatment group for this study (Day 20 to Day 41). The decrease in tumor burden is shown by a decrease in the level of bioluminescence over time. Inhibition of tumor burden with the anti-CXCR4 antibody 6B6 with early onset (Day 13) was more pronounced than when it was started one week later (Day 20). The anti-CXCR4 antibody 6B6 significantly decreased tumor burden in both treatment groups compared to the isotype control antibody, indicating that the anti-CXCR4 antibody is effective in inhibiting tumor burden in a staged disseminated mouse model of human AML.
The survival curve shown in Figure 9B demonstrates a significant effect of the anti-CXCR4 6B6 antibody compared to the isotype control antibody on the survival of animals systemically implanted with MV4-11-LUC AML cells. While animals treated with isotype control showed a median survival of 40 days, animals treated with the anti-CXCR4 antibody 6B6 on Day 13 and Day 20 had median survivals of 53 and 61 days, respectively. These differences were statistically significant (p<0.05) by the Mantel-Cox test.
[00457] Figure 9C shows the significant reduction in AML cell numbers in animals treated with the anti-CXCR4 antibody 6B6 compared to animals treated with isotype control on Day 35 of the study. EXAMPLE 12 ANTI-CXCR4 ANTIBODY ENHANCED SURVIVAL IN A MOUSE SYSTEMIC CHRONIC LYMPHOID LEUKEMIA TUMOR MODEL
[00458] The antitumor activity of CXCR4 antibody was further investigated in a model of disseminated intravenous chronic lymphoid leukemia (CLL). The human tumor cell line JVM-13 stably transfected to express the luciferase gene was grown at 37°C in a 5% CO2 incubator in RPMI 1640 medium containing 10% fetal bovine serum and 0.25 mg/ml Puromycin . The model was established by injecting 1 x 106 JVM-13-Luc cells into 6-8 week old SCID Beige female mice through the tail vein. 21 days after cell implantation, mice were assigned to treatment groups (10 animals per group) based on bioluminescence readout (BLI). Mice were treated with Isotype Control antibody and 3G10 and were dosed at 10 mg/kg in a sterile PBS solution once a week by the subcutaneous (s.c.) route. Rituximab, an anti-CD20 Ab, approved for the treatment of patients with CLL, was used as a positive control. Rituximab was dosed at 10 mg/kg in a sterile PBS solution once a week, sc Tumor burden was assessed using the Xenogen IVIS 200 imaging system. Mice were imaged every 7 days in a ventral position and whole body bioluminescence was determined using Xenogen Living Image software. When mice begin to show hind limb paralysis or reach other human endpoints, they undergo rameuthanasia. Both tumor burden (by bioluminescence) and survival were assessed as end points.
[00459] Figure 10A shows the image of representative luciferase activity for mice in each treatment group on Days 28, 35, 42, 49 and 56 of the study. Treatment with 3G10 significantly decreased the JVM-13-Luc tumor burden in large bones, as measured by luminescence level over time, compared to the isotype control antibody. Rituximab treatment also significantly reduced tumor burden compared to the isotype control antibody.
[00460] Figure 10B shows a Kaplan-Meyer survival curve for this study. A significant increase in survival was observed for 3G10 and Rituximab antibodies compared to isotype control Ab (p<0.0001). While animals treated with isotype control antibody showed median survival of 32.5 days, animals treated with Rituxan showed median survival of 45 days and animals treated with 3G10 did not achieve median survival before Day 60. The results of this study indicate that the anti-CXCR4 antibody is effective in inhibiting tumor burden in a mouse model disseminated in stages of human CLL. EXAMPLE 13: INHIBITION OF NON-HODGKIN BRANCH LYMPHOMA TUMOR GROWTH IN VIVO BY HUMANIZED ANTI-CXCR4 ANTIBODIES
To assess the ability of humanized CXCR4 Abs to inhibit tumor growth, a tumor xenograft model using 4-6 week old female CB17.cg-Prkdc SCID Beige mice (Charles River) and Ramos cells of Non-Hodgkin's Lymphoma implanted subcutaneously was used. Cells were grown at 37°C in a 5% CO2 incubator in RPMI 1640 medium containing 10% fetal bovine serum. In this study, 5 x 106 cells were implanted in the right rear flank region of each mouse and allowed to grow as solid tumors to an average size volume of approximately 350 mm3, calculated by the formula (volume=length x width2)/2. The mice were then randomized into 7 different treatment groups, n=10 animals per group. Groups of mice were treated subcutaneously (sc) with 10 mg/kg of each antibody in a sterile PBS solution: (Isotype Control antibody; m3G10; h3G10.A57.WT; h3G10.2.37.2.72; h3G10.A57.A58; h3G10.1.91.A58A; h3G10.1.91.A58B. Animals were dosed with antibodies once weekly for 2 weeks for a total of 2 doses. Tumor volumes were measured for caliber twice per week for the duration of the study. The results of the experiment are shown in Figure 11. The results indicate that all anti-CXCR4 antibodies tested significantly inhibited tumor growth relative to the isotype control antibody. that the tested humanized CXCR4 antibodies had similar efficacy to that of the chimeric m3G10 antibody.The tumor growth inhibitory effect was maintained for several days after antibody dosing was stopped (Day 7). EXAMPLE 14 INCREASED SURVIVAL TIME AND DECREASED TUMOR LOAD BY HUMANIZED ANTI-CXCR4 ANTIBODY IN A MOUSE SYSTEMIC MULTIPLE MYELOMA (MM) MODEL
The humanized anti-CXCR4 antibody h3G10.1.91.A58B was tested for its ability to increase survival and reduce the tumor burden of NSG mice using an intravenous disseminated/systemic MM model. The human MM cancer line OPM-2 was transduced with the luciferase gene (OPM-2-LUC) to allow "in-life" monitoring of tumor burden over time. Cells were cultured at 37°C in a 5% CO2 incubator in RPMI 1640 medium containing 10% fetal bovine serum with 0.5μg of puromycin for selection of luciferase expression. Female NSG mice 4 to 6 weeks old were injected intravenously with OPM-2-Luc cells (5 x 106/animal).
[00463] On Day 8 after cell implantation, mice were randomized into three treatment groups (10 animals per group) based on total body bioluminescence intensity. Weekly subcutaneous (s.c.) antibody treatment at 10 mg/kg in a sterile PBS solution was initiated and carried out for 5 weeks. Melfalan, one of the drugs approved for the treatment of patients with multiple myeloma, was dosed at 1 mg/kg twice/week and carried out for 3 weeks. Tumor burden was assessed using the Xenogen IVIS 200 imaging system. Mice were imaged every 7 days in a ventral position and whole-body bioluminescence was determined using the Xenogen Living Image software. When the mice began to show hind limb paralysis or reached other human endpoints, they were euthanized. Both tumor burden (by bioluminescence) and survival were assessed as end points.
[00464] Figures 12A and B show the results of bioluminescence (Luciferase activity) in this study. The decrease in tumor burden is shown by a decrease in the level of bioluminescence over time. Figure 12A shows the representative bioluminescence image of 5 animals/treatment group for this study. Figure 12B shows that treatment with the CXCR4 antibody h3G10.1.91.A58B significantly inhibited tumor growth compared to the isotype control antibody over time (p<0.0001 on Day 30), indicating that the antibody anti-CXCR4 is effective in inhibiting tumor burden in a staged disseminated xenograft model of human multiple myeloma. The survival curve for this study is shown in Figure 12C and demonstrates a significant effect of the anti-CXCR4 antibody 6h3G10.1.91.A58B compared to the isotype control antibody on the survival of animals systemically implanted with MM cells OPM-2-Luc . While animals treated with isotype control and Melphalan showed median survivals of 33.5 and 36 days, respectively, animals treated with CXCR4 antibody h3G10.1.91.A58B had indefinite median survival, with no death observed until Day 50 of the study. Table 13. These differences were statistically significant (p<0.0001) by the Mantel-Cox test. TABLE 13
EXAMPLE 15 ANTI-CXCR4-ADCS CYTOTOXICITY IN CXCR4-POSITIVE CELLS
Anti-CXCR4 antibodies (eg 3G10) were expressed as human IgG1 subtypes and modified with trans-glutaminase tag containing glutamine ("Q") TG6 (SEQ ID NO: 91 (LLQGA)) and conjugated to AcLys-vc-PABC-MMAD (Acetyl-Lysine-Valine-Citrulline-p-aminobenzyloxycarbonyl-MMAD) and AcLys-vc-PABC-0101 ((2-methylalanyl-N-[(3R,4S,5S)-3-methoxy -1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol- 2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide). antibody heavy chain. The conjugation of the anti-CXCR4 antibody to the cytotoxic agent MMAD or 0101 was then achieved through a transamidation reaction catalyzed by microbial transglutaminase between the anti-CXCR4 antibody that carries the tag containing glutamine at the specific site ( eg, carboxyl terminus of the antibody heavy chain) and an amine-containing derivative of the payload (eg, MMAD or 0101). In some cases, the wild-type amino acid lysine at the carboxy terminus (position 447 in accordance with the EU numbering scheme) has been deleted and replaced by the Q tag. In other cases, the wild-type amino acid lysine at position 222 (in accordance with the EU numbering scheme) was replaced by the amino acid arginine ("K222R"). Substitution of K222R provided the significant effect of resulting in more homogeneous antibody and payload conjugate and/or better intermolecular crosslinking between antibody and payload. In the transamidation reaction, the glutamine in the glutamine-containing tag acted as an acyl donor and the amine containing compound acted as an acyl acceptor (amine donor). Purified anti-CXCR4 antibody was incubated with excess acyl acceptor in the presence of Streptoverticillium mobaraense transglutaminase (ACTIVA™, Ajinomoto, Japan) in 150 to 900 mM NaCl, and 25 mM MES, HEPES [4-acid (2-hydroxyethyl)-1-piperazineethanesulfonic acid] or Tris HCl buffer in a pH range from 6.2 to 8.8. Reaction conditions were adjusted for the individual acyl acceptor derivatives. After incubation at room temperature for 2.5 hours, the antibody-drug conjugate was purified on MabSelect resin (GE Healthcare, Waukesha, WI) using standard affinity chromatography methods known to those skilled in the art, such as chromatography Healthcare's business affinity agreement.
[00466] Cells expressing CXCR4 were then seeded in light-bottomed plates with white walls at 4,000 to 8,000 cells/well for 24 hours before treatment. Cells were then treated with antibody-drug conjugates serially diluted 4 times in triplicate. Cell viability was determined by CellTiter-Glo® 96 Luminescent Cell Viability Assay (Promega, Madison WI) 96 hours after treatment. Relative cell viability was determined as percentage of untreated control. The IC50 was then calculated. Anti-CXCR4 antibodies conjugated to MMAD or 0101 via transglutaminase tag exert potent cell killing activity on cells expressing CXCR4. TABLE 14: CYTOTOXICITY OF CXCR4 ADCS IN CELLS EXPRESSING CXCR4
EXAMPLE 16 IN VIVO ANTITUMOR ACTIVITY OF CXCR4-ADCS
The in vivo antitumor efficacy of CXCR4 ADCs was evaluated in Ramos (NHL) and HPB-ALL (T-ALL) xenograft models. 4- to 4-week-old female CB17 SCID mice (Jackson Laboratories) were implanted subcutaneously with 5 x 106 Ramos (NHL) as described in Example 9 up to a mean size volume of approximately 500 mm3, calculated by the formula (volume =length x width2)/2. 10 x 106 HPB-ALL cells were implanted into 4-6 week old female CB17 SCID mice until the mean tumor size also reached approximately 500 mm3. In the Ramos model, groups of mice were treated with intravenous (iv) injection of a single dose of negative control ADC (negative control-TG6-vc0101) at 6.0 mg/kg or CXCR4 ADCs (3G10-TG6-vc0101 and 6B6-TG6-vc0101) at 1.5, 3.0 or 6.0 mg/kg. Tumor volumes were measured for caliber once a week for the duration of the study. The results of the experiment are shown in Figure 13A. The results indicate that both CXCR4 ADCs induced tumor regression at doses >3 mg/kg. Control ADC had no effect on tumor growth. In the HPB-ALL model, 1.5 mg/kg of a single dose injection of 3G10-TG6-vc0101 is sufficient to induce tumor regression (Figure 13B).

权利要求:
Claims (17)
[0001]
1. Isolated humanized antibody, or an antigen-binding fragment thereof, characterized by the fact that it binds to chemokine receptor 4 (CXCR4) and comprises: a) a variable heavy chain (VH) region that has a VH CDR1 of SEQ ID NOs:107, 113 or 114; a VH CDR2 of SEQ ID NO: 162 or 128, and a VH CDR3 of SEQ ID NOs: 112; and b) a variable light chain region (VL) having a VL CDR1 of SEQ ID NOs: 144; a VL CDR2 of SEQ ID NO: 145; and a VL CDR3 of SEQ ID NO: 139.
[0002]
2. Isolated humanized antibody, or an antigen-binding fragment thereof, according to claim 1, characterized in that the antibody comprises: a) a variable heavy chain (VH) region that has a VH CDR1 of SEQ ID NOs:107; a VH CDR2 of SEQ ID NO: 162, and a VH CDR3 of SEQ ID NOs: 112; and b) a variable light chain region (VL) having a VL CDR1 of SEQ ID NOs: 144; a VL CDR2 of SEQ ID NO: 145; and a VL CDR3 of SEQ ID NO: 139.
[0003]
3. Isolated humanized antibody, or an antigen-binding fragment thereof, according to claim 1 or 2, characterized in that the antibody comprises a variable heavy chain (VH) region comprising VH CDR1, VH CDR2 and VH CDR3 from a VH region of SEQ ID NO:33; and a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 from a VL region of SEQ ID NO: 73.
[0004]
4. Isolated humanized antibody or antigen-binding fragment thereof, according to claim 3, characterized in that it consists of an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) of SEQ ID NO: 33; and/or a variable light chain (VL) region of SEQ ID NO: 73.
[0005]
5. Isolated humanized antibody or antigen-binding fragment, according to claim 4, characterized in that the antibody comprises a variable heavy chain (VH) region produced by the expression vector with the ATCC Accession No. PTA-121353 or a variable light chain (VL) region produced by the expression vector with ATCC Accession No. PTA-121354.
[0006]
6. Isolated humanized antibody or antigen-binding fragment, according to any one of claims 1 to 5, characterized in that the antibody is humanized, chimeric, CDR-grafted.
[0007]
7. Isolated humanized antibody or antigen-binding fragment, according to any one of claims 1 to 6, characterized in that the antibody comprises an acyl donor marker containing glutamine projected to a specific site
[0008]
8. Antibody-drug conjugate, characterized in that it has the formula: Ab-(TLD), in which: (a) Ab is an antibody or antigen-binding fragment thereof, as defined in any one of claims 1 to 7 , which binds to chemokine receptor 4 (CXC4); (b) T is a glutamine-containing acyl donor tag which may optionally be included; (c) L is a linker; and (d) D is a drug.
[0009]
9. Antibody-drug conjugate, according to claim 8, characterized in that Ab is selected from the group consisting of: a) an antibody or antigen-binding fragment thereof comprising a VH region comprising CDRs of SEQ ID NOs: 107, 162 and 112 and a VL region comprising CDRs of SEQ ID NOs: 144, 145 and 139; or b) an antibody or antigen binding fragment thereof which comprises a VH region of SEQ ID NO:33 and a VL region of SEQ ID NO:73.
[0010]
10. Antibody-drug conjugate, according to claim 8 or 9, characterized in that (a) Ab is a humanized, chimeric, CDR-grafted or recombinant human antibody or antigen-binding fragment thereof; (b) T is selected from the group consisting of SEQ ID NOs: 171, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 172, 173, 102, 103 and LLQ ; or (c) L is selected from the group consisting of acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), amino PEG6-propionyl, maleimidocapronico-valine-citrulline-p-aminobenzyloxycarbonyl (vc) and maleimidocaproyl (mc); or (d) the antibody or antigen-binding fragment is conjugated to a drug, wherein D is selected from the group consisting of a cytotoxic agent, an immunomodulating agent, an imaging agent, a therapeutic protein, a biopolymer, and an oligonucleotide .
[0011]
11. Antibody-drug conjugate, according to claim 10, characterized in that the cytotoxic agent is selected from the group consisting of an anthracycline, an auristatin, a camptothecin, a combretastain, a dolastatin, a duocarmycin , an enedine, a geldanamycin, an indoline-benzodiazepine dimer, a maytansin, a puromycin, a pyrrolobenzodiazepine dimer, a taxane, a vinca alkaloid, a tubulisin, a hemiasterlin, a spliceostatin, a pladienolid, and calicheamicin.
[0012]
12. Antibody-drug conjugate according to any one of claims 8 to 11, characterized in that it is selected from the group consisting of a) Ab-LLQGA (SEQ ID NO: 91) - (acetyl-lysine-valine- citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC)) - 0101; b) Ab-LLQGA (SEQ ID NO: 91)-(AcLis-VC-PABC)-MMAD; c) Ab-LLQX1X2X3X4X5 (SEQ ID NO: 102)-(AcLis-VC-PABC)-0101; d) Ab-LLQX1X2X3X4X5 (SEQ ID NO: 102)-(AcLys-VC-PABC)-MMAD; e) Ab-GGLLQGA (SEQ ID NO: 92)-(AcLis-VC-PABC)-0101; and f) Ab-GGLLQGA (SEQ ID NO: 92)-(AcLis-VC-PABC)-MMAD.
[0013]
Antibody-drug conjugate according to claim 12, characterized in that Ab is an antibody or antigen-binding fragment thereof comprising a VH region comprising CDRs of SEQ ID NOs: 107, 162 and 112 and a VL region comprising CDRs of SEQ ID NOs: 144, 145 and 139.
[0014]
14. Pharmaceutical composition, characterized in that it comprises the antibody or antigen-binding fragment thereof, as defined in any one of claims 1 to 7, or the conjugate, as defined in any one of claims 8 to 13, and a pharmaceutically acceptable vehicle.
[0015]
15. Isolated polynucleotide, characterized in that it comprises a nucleotide sequence encoding the antibody or antigen-binding fragment thereof, as defined in any one of claims 1 to 7.
[0016]
16. Vector, characterized in that it comprises the polynucleotide, as defined in claim 15.
[0017]
17. Use of an isolated antibody or antigen-binding fragment thereof, as defined in any one of claims 1 to 7, of the antibody-drug conjugate, as defined in any one of claims 8 to 13, characterized in that it is in the manufacture of a composition for the treatment of cancer.

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SG11201600171SA|2016-02-26|

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US20190092867A1|2019-03-28|

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法律状态:
2018-03-06| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: C07K 16/28 (2006.01), A61K 47/00 (2006.01), A61K 3 |

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2019-12-17| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2020-06-23| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-11-10| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-02-09| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-06-22| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 28/07/2014, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201361861706P| true| 2013-08-02|2013-08-02|

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US61/861,706|2013-08-02|

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PCT/IB2014/063483|WO2015015401A2|2013-08-02|2014-07-28|Anti-cxcr4 antibodies and antibody-drug conjugates|BR122016023010A| BR122016023010A2|2013-08-02|2014-07-28|anti-cxcr4 antibodies and antibody-drug conjugates, use and methods of producing them, pharmaceutical composition, isolated polynucleotide, vector and isolated host cell|