ror1 antibody immunoconjugates

专利摘要:
The present invention relates to immunoconjugates comprising an anti-ROR1 antibody or an antigen fragment fragment and a drug moiety. These immunoconjugates are useful in the treatment of cancers of ROR1 expression.
公开号:BR112019027448A2
申请号:R112019027448-0
申请日:2018-06-22
公开日:2020-07-07
发明作者:Brian Lannutti；Katti Jessen；Thanh-Trang Vo；Jeffry Dean Watkins
申请人:VelosBio Inc.；
IPC主号:

专利说明:

[001] [001] This application claims priority for US Patent Applications 62 / 524,382, 62 / 524,386 and 62 / 524,388, all of which were filed on June 23, 2017. Descriptions of these priority applications are incorporated by reference here in its entirety. SEQUENCE LISTING
[002] [002] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference in its entirety. The ASCII copy, created on June 21, 2018, is called 024651_WO002_SL.txt and is 58,100 bytes in size. BACKGROUND OF THE INVENTION
[003] [003] Cancer is the second leading cause of human death, close to coronary artery disease. Receptor tyrosine kinases (RTKs) play a key role in oncogenic transformation, growth and metastasis. RTKs regulate cell differentiation, proliferation, migration, angiogenesis and survival. The orphan receptor tyrosine kinase receptor 1 (ROR1) is an evolutionarily conserved type I membrane protein that belongs to the ROR subfamily and has extracellular domains that contain domains similar to immunoglobulin (Ig), Frizzled, and Kringle. ROR1-deficient mice exhibit a variety of phenotypic defects in the skeletal and urogenital systems, as well as postnatal growth retardation. ROR1 is expressed during embryogenesis and for a variety of different cancers, but not in normal postpartum tissues, and can be considered an oncoembryonic surface antigen. Functional data suggest that ROR1 may function in non-canonical WNT signaling to promote the survival of malignant cells.
[004] [004] The expression and activation of ROR1 seems to be correlated with characteristics of tumor aggressiveness in models of chronic lymphocytic leukemia (CLL), breast cancer, lung cancer, gastric cancer and melanoma (Li et al., PLoS One 5 (7): e11859 (2010); Gentile et al., Cancer Res. 71 (8): 3132-41 (2011); Zhang et al., PLoS One 7 (3): e31127 (2012); Yamaguchi et al. , Cancer Cell. 21 (3): 348-61 (2012); Daneshmanesh et al., Leukemia 26 (6): 1348-55 (2012); Daneshmanesh et al., Leuk Lymphoma 54 (4): 843- 50 (2013); O'Connell et al., Cancer Discov. 3 (12): 1378-93 (2013); Hojjat-Farsangi et al., PLoS One 8 (4): e61167 (2013); Hojjat-Farsangi et al., PLoS One 8 (10): e78339 (2013); Ida et al., Cancer Sci. 107 (2): 155-61 (2016); and Janovska et al., Clin Cancer Res. 22 (2 ): 459-69 (2016)). High levels of ROR1 expression in patients and cell lines are associated with genes involved in the epithelial-mesenchymal transition (EMT) (Cui et al., Cancer Res. 73 (12): 3649-60 (2013)). In patients with CLL, high levels of ROR1 expression are associated with lower treatment-free survival and overall survival (OS) (Cui et al., Blood 128 (25): 2931-2940 (2016)). Similarly, in patients with ovarian cancer, high expression of ROR1 is associated with poor clinical results (Zhang et al., Sci Rep. 4: 5811 (2014)).
[005] [005] In view of the role of ROR1 in cancer, there is a need for new and improved therapies that target ROR1 positive cancer cells. SUMMARY OF THE INVENTION
[006] [006] An immunoconjugate having the formula of Ab - ((L) m– (D)) n is provided here, where: Ab is an antibody or antigen-binding fragment that specifically binds to the receptor human receptor tyrosine kinase 1 orphan (ROR1); L is a linker, and m is 0 or 1; D is a portion of cytotoxic drug; and n is an integer from 1 to 10.
[007] [007] The cytotoxic drug moiety can be selected from the group consisting of, for example, an anti-tubulin agent, a DNA alkylating agent, a DNA cross-linking agent, a DNA intercalating agent and an RNA polymerase II inhibitor. In some embodiments, the cytotoxic drug portion is selected from the group consisting of monomethyl auristatin E (MMAE), azonafide, α-amanitin, duocarmycin TM, pyrrolobenzodiazepine (PBD), PNU-159682 and pharmaceutically acceptable salts, esters and analogs thereof. .
[008] [008] The ligand in the immunoconjugate may comprise a cleavable moiety. It can be cleaved within a target cell. Alternatively, the linker is not cleavable. The linker can be branched or unbranched. In some embodiments, the binder comprises one or more selected portions of valine-citrulline (VC), valine-alanine (VA), para-aminobenzyloxycarbonyl (PAB), polyethylene glycol (PEG), diaminopropionic acid (DPR), Phe- C4, C2-Gly3, C6 alkyl, dimethylethylamine (DMEA) and ethylene diamine (EDA). In certain embodiments, the ligand is covalently attached to the antibody or antigen-binding fragment in a succinimide, carbonyl or cyclooctene or triazole group of the ligand.
[009] [009] In certain embodiments, the antibody or fragment in the immunoconjugate is covalently linked to the ligand by reaction with a portion selected from the group consisting of 6-maleimidocaproyl (MC) -VC-PAB; 6-MC-C6; 6-MC-PEG4-VC-PAB-DMEA; 6- MC-PEG4-VA; 6-MC-DPR-VC-PAB; 6-MC-Phe-C4-VC-PAB; 6-MC-Phe-C4-VC-PAB-DMEA; 6-MC-C2-Gly3-EDA; dibenzylcyclooctin (DBCO) - (PEG2-VC-PAB) 2; DBCO-PEG4-VC-PAB-DMEA; and N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate-VC-PAB. When used herein, VC represents a valine-citrulline dipeptide; VA represented
[0010] [0010] An immunoconjugate having the formula of Ab - ((L) m– (D)) n is also provided here, where: Ab is an antibody or an antigen-binding fragment of the same that specifically binds to the human receptor tyrosine kinase 1 orphan receptor (ROR1); L is a cleavable linker and m is 0 or 1; D is an auristatin (for example, MMAE); and n is an integer from 1 to 10.
[0011] [0011] In an immunoconjugate of the present description, the linker may comprise, for example, a heterocycle or carbonyl covalently linked to the antibody or antigen binding fragment, a spacer group covalently linked to the heterocycle or carbonyl and an ester, thioester, amide, carbonate, thiocarbonate or carbamate covalently linked to the cytotoxic drug moiety. In some modalities, the spacer group comprises an amino acid group, a polyamino acid or benzyl amino or a combination thereof. In some embodiments, the ligand in an immunoconjugate of the present description forms a covalent bond with a cysteine or lysine residue in the antibody or fragment.
[0012] [0012] The Ab component (antibody or fragment thereof) of an immunoconjugate of the present description can bind to the same epitope of ROR1 as an antibody comprising the heavy and light chain amino acid sequences of SEQ ID NOs: 3 and 4 , respectively. The antibody or fragment can comprise the heavy chain complementarity determining region (CDR) 1-3 (HCDR1-3) in SEQ ID NO: 3 and the light chain CDR1-3 (LCDR1-3) in SEQ ID NO : 4. In some embodiments, the antibody or fragment comprises the amino acid sequences of SEQ ID NOs: 7-9 and the light chain of the antibody comprises the amino acid sequences of SEQ ID NOs: 10-12. The antibody or fragment can be humanized. The antibody or fragment can have one or more of the following properties: a) it facilitates the internalization of ROR1 in a human cell; b) binds to human ROR1 with a KD of less than 100 nM (for example, less than 50, 40, 30, 20 or 10 nM); and c) inhibits the growth of human ROR1 + cancer cells in vitro with an EC50 of 500 nM or less (for example, 400 nM or less, 300 nM or less, 200 nM or less, 200 nM or less or 100 nM or less ).
[0013] [0013] In some embodiments, the heavy chain variable domain (VH) and the light chain variable domain (VL) of the antibody in the immunoconjugate comprise the amino acid sequences of: a) SEQ ID NOs: 5 and 6, respectively ; b) SEQ ID NOs: 5 and 50, respectively, c) SEQ ID NOs: 48 and 6, respectively; or d) SEQ ID NOs: 48 and 50, respectively. The antibody can comprise a human IgG1 constant region and optionally also a human cadeia light chain constant region. In other embodiments, the antibody heavy chain and light chain comprise the amino acid sequences of: a) SEQ ID NOs: 3 and 4, respectively; b) SEQ ID NOs: 3 and 49, respectively; c) SEQ ID NOs: 47 and 4, respectively; or d) SEQ ID NOs: 47 and 49, respectively.
[0014] [0014] In some embodiments, the Ab component of the immunoconjugate is a Fab, F (ab) 2 or scFv, for example, a Fab, F (ab) 2 or scFv.
[0015] [0015] Specific embodiments of the present description include an immunoconjugate comprising an antibody conjugated to a cytotoxic drug moiety, wherein the VH and VL of the antibody comprise the amino acid sequences of SEQ ID NOs: 5 and 6, respectively. mind. Examples of such an immunoconjugate are shown in Tables 2 and 3 below and include Antibody-Drug Sets (ADC) -A,
[0016] [0016] In the immunoconjugate of the present description, the number of the drug portion per antibody or fragment, or the ratio of the cytotoxic drug portion to the antibody or fragment (DAR), can be from 1 to 10, for example, 1 to 7 , 1 to 6, 1 to 5, 2 to 7, 2 to 6 or 2 to 5.
[0017] [0017] Pharmaceutical compositions comprising an immunoconjugate of the present description and a pharmaceutically acceptable excipient are also provided herein. The pharmaceutical compositions may further comprise an additional therapeutic agent selected from the group consisting of a Bruton tyrosine kinase inhibitor (BTK), an inhibitor of B cell lymphoma 2 (Bcl-2), a mammalian target of the rapamycin inhibitor (mTOR) and a phosphoino-3-kinase (PI3K) inhibitor. For example, the additional therapeutic agent is selected from ibrutinib, acalabrutinib, venetoclax, everolimus, sapanisertib and idelalisib.
[0018] [0018] A therapy or method for treating cancer in a patient in need thereof is also provided herein, comprising administering to the patient a therapeutically effective amount of an immunoconjugate of the present invention. The cancer can be homogeneous or heterogeneous for the expression of ROR1 and can be, for example, leukemia, lymphoma or solid tumor. In some modalities, the cancer is chronic lymphocytic leukemia (CLL), T cell leukemia (TCL), liner cell lymphoma (MCL), diffuse large B cell lymphoma (DLBCL), Burkitt's lymphoma, myeloma multiple (MM), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL) or a non-Hodgkin lymphoma (NHL) that underwent Richter transformation. In some fashion-
[0019] [0019] The therapy or treatment method of the present description may further comprise administering to the patient an additional anti-cancer therapeutic agent, which can be, for example, a Bruton tyrosine kinase (BTK) inhibitor, a lymphoma inhibitor 2 of B cells (Bcl-2), a mammalian target of the rapamycin inhibitor (mTOR) and an inhibitor of phosphoinositide 3-kinase (PI3K). In some modalities, the additional therapeutic agent is selected from ibrutinib, acalabrutinib, venetoclax, everolimus, sapanisertib and idelalisib.
[0020] [0020] In certain modalities of the present method of therapy or treatment, the cancer is CLL, MCL or an NHL that has undergone Richter's transformation.
[0021] [0021] Immunoconjugates and pharmaceutical compositions described herein are also provided herein for use in the treatment of cancer in the therapy or treatment methods described herein. For example, an immunoconjugate having the formula of Ab - ((L) m– (D)) n for use in the treatment of cancer in a patient in need is provided here, where: Ab is an antibody or fragment of binding to the same antigen that specifically binds to the orphan receptor tyrosine kinase human receptor 1 (ROR1); L is a linker and m is 0 or 1; D is a portion of cytotoxic drug; and n is an integer from 1 to 10. Exemplary modalities of the immunoconjugate and treatment are described above and will be described later.
[0022] [0022] Provided here is also the use of an immunoconjugate here for the manufacture of a medicine for use in the treatment of cancer in a patient in need of it. For example, provided here is the use of an immunoconjugate having the formula of Ab-
[0023] [0023] The present description also provides a method for manufacturing an immunoconjugate, comprising: providing an antibody or antigen-binding fragment that specifically binds to the orphan receptor human tyrosine kinase type 1 (ROR1); conjugating to the antibody a portion of cytotoxic drug selected from the group consisting of an anti-tubulin agent, a DNA alkylating agent, a DNA cross-linking agent, a DNA intercalating agent and an RNA polymerase II inhibitor; wherein the heavy chain of the antibody comprises the amino acid sequences of SEQ ID NOs: 7-9 and the light chain of the antibody comprises the amino acid sequences of SEQ ID NOs: 10-12. Exemplary modalities of the immunoconjugate are described above and will be described later.
[0024] [0024] Manufacturer articles are also provided here as kits, comprising an immunoconjugate of the present description. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] [0025] FIG. 1 is a schematic diagram illustrating a non-limiting example of an immunoconjugate of the present description.
[0026] [0026] FIGS. 2A and 2B are graphs illustrating the binding of various concentrations of Ab1 and ADC-A to ROR1 Jeko-1 (2A) and MDA-MB-231 (2B) positive cells. EC50 values for Ab1 and ADC-A are shown below each graph. The similarity between the EC50 values for Ab1 and unconjugated ADC-A demonstrates that the
[0027] [0027] FIGS. 3A and 3B are graphs illustrating the binding of Ab1, 4A5, ADC-A and ADC-T (3A) and Ab1, ADC-A, D10 and ADC-S (3B) to Jeko-1 cells. EC50 values for antibodies and immunoconjugates are shown below each graph. The similarity between the EC50 values of unconjugated antibodies and the corresponding ADC constructs demonstrates that the drug conjugation had minimal impact on the binding of antibodies to the target cells. The difference in EC50 values between Ab1 / ADC-A and D10 / ADC-S reflects the greater affinity of Ab1 for ROR1 compared to D10.
[0028] [0028] FIGS. 4A and 4B are graphs illustrating the internalization of Ab1, ADC-A and ADC-B in Jeko-1 cells (4A) and the internalization of Ab1 and ADC-A in MDA-MB-231 (4B) cells. The addition of ligand and payload to Ab1 did not negatively affect its binding or internalization, as demonstrated with ADC-A and AB.
[0029] [0029] FIG. 5 is a graph illustrating the internalization rate of Ab1 in MDA-MB-231 cells. The graph shows an initially rapid rate and then a slower rate of cell surface receptor clearance.
[0030] [0030] FIG. 6 is a graph illustrating the expression of the cell surface of ROR1 during the internalization of Ab1 in Jeko-1 cells. While Ab1 is rapidly internalized, the quantification of cell surface ROR1 shows a small decrease in the first 10 minutes, with subsequent measurements indicating the restoration of ROR1 surface expression to initial or slightly higher levels.
[0031] [0031] FIGS. 7A-C are graphs illustrating the expression of ROR1 cell surface during Ab1 internalization in Jeko-1 cells (7A), MDA-MB-468 cells (7B) and MDA-MB-231 cells (7C).
[0032] [0032] FIGS. 8A-8I are representative IC50 plots showing binding to ROR1 by immunoconjugates of the present description, as well as unconjugated MMAE, in cancer cell lines TMD-8 (8A), HBL-1 (8B), DOHH2 (8C) , MDA-MB-468 (8D), Bt549 (8E), TOV112D (8F), JHOM1 (8G), SKOvr3 (8H) and Mino (8I).
[0033] [0033] FIG. 9 is a graph illustrating the inhibition of cell proliferation by 3, 10 or 30 µg / mL of ADC-A in Jeko-1 cells, with or without pretreatment with 100 µg / mL of Ab1. ADC-A inhibited cell proliferation in a dose-dependent manner. Pre-incubation of cells with Ab1 reduced this activity, demonstrating that ADC-A inhibitory activity in cell proliferation was mediated by the binding of ADC-A to ROR1.
[0034] [0034] FIG. 10 is a graph illustrating the dose-dependent inhibition of leukemic cell tumor burden in a mouse model with chronic lymphocytic leukemia TCL1-ROR1 after vehicle treatment, 10 mg / kg of Ab1 or 1 mg / kg, 2 mg / kg or 5 mg / kg of ADC-A.
[0035] [0035] FIG. 11 is a graph illustrating tumor growth inhibition in an MCL xenograft model after vehicle treatment, 5 mg / kg ADC-A or ADC-Q intravenously (IV) every four days (Q4D), 10 mg / kg of Ab1 IV once a week (QW) or 20 mg / kg of ibrutinib per os (PO) every day (QD). ADC-A treatment caused tumor regression.
[0036] [0036] FIG. 12 is a pair of graphs showing the expression of ROR1 (left panel) and inhibition of tumor growth in a DLBCL-GCB xenograft model after treatment with control, 10 mg / kg of QW Ab1, 50 mg / kg of QD venetoclax , Ab1 + venetoclax or 5 mg / kg of ADC-A QW (right panel). Treatment with ADC-A resulted in complete tumor regression in all treated animals. Ab1 alone, venetoclax alone and a combination of Ab1 and veneto-
[0037] [0037] FIG. 13 is a set of graphs showing the expression of ROR1 (left panel) and inhibition of tumor growth after treatment with vehicle, 2.5 or 5 mg / kg of ADC-A or 10 mg / kg of Ab1 (right panel) in a mouse model of Richter transformation xenograft resistant to chemotherapy. Although only 20-30% of intratumor cells are positive for ROR1, complete and prolonged tumor regressions were observed with 5 mg / kg ADC-A.
[0038] [0038] FIG. 14 is a graph illustrating tumor growth inhibition in vehicle treatment, 1 or 5 mg / kg of ADC-A IV QW, 1 or 5 mg / kg of ADC-B IV QW or 10 mg / kg of Ab1 IV QW in a mouse model of breast fatty pad xenograft with triple negative breast cancer (TNBC) MDA-MB- 231.
[0039] [0039] FIG. 15 is a graph illustrating the inhibition of tumor growth after treatment with vehicle, 1 or 5 mg / kg of ADC-A IV Q4D or 10 mg / kg of Ab1 IV QW in a mouse model of human TNBC xenograft BR5011. Although only 58% of intratumor cells were positive for ROR1, complete and prolonged regressions were observed with 5 mg / kg of ADC-A, where the tumor regression was maintained for at least 28 days after the last disease.
[0040] [0040] FIG. 16 is a graph illustrating the inhibition of tumor growth after treatment with vehicle, 1 or 5 mg / kg of ADC-A IV Q4D or 10 mg / kg of Ab1 IV QW in a xenograph model of human TNBC BR5015 (low expression of ROR1). Tumor regression was observed, although only 58% of intratumor cells were positive for ROR1.
[0041] [0041] FIG. 17 is a graph illustrating the inhibition of tumor growth in a mouse model of human lining cell lymphoma xenograft Jeko-1. The mice were treated with a vehicle; 1 mg / kg of ADC-N, ADC-P or ADC-R; or 5 mg / kg of ADC-A, ADC-L, ADC-M, ADC-S or ADC-T. Vehicle and ADC constructions were administered IV Q4D. Significant tumor regression was observed in animals treated with ADC-A, ADC-L, ADC-M and ADC-S, while inhibition of tumor growth was observed in animals treated with ADC-N, ADC-P, ADC-R and ADC - T.
[0042] [0042] FIGS.18A and 18B are graphs illustrating the combination index of treatment with ADC-A and BTK inhibitors ibrutinib (18A) or ACP-196 / acalabrutinib (18B) in various cell lines. ADC-A also had a synergistic effect with ibrutinib and ACP-196 / acalabrutinibone in inhibiting cell proliferation.
[0043] [0043] FIGS. 19A and 19B are graphs illustrating the inhibition of Jeko-1 cell proliferation after treatment with ADC-A, ibrutinib ("Ib") or a combination of ADC-A and ibrutinib (19A); or ADC-A, ACP-196 / acalabrutinib ("ACP196" or "196") or a combination of ADC-A and ACP-196 / acalabrutinib (19B).
[0044] [0044] FIGS. 20A-C are graphs illustrating the combination index of treatment with ADC-A and Bcl-2 ABT-199 / venetoclax inhibitor ("ABT199") in various cell lines (20A), or of ADC-A with inhibitor of Bcl-2 Bcl-2i-1 or Bcl-2i-2 in Jeko-1 cells (20B) or Mino cells (20C). ADC-A exhibited a synergistic effect with ABT-199 in also inhibiting the proliferation of MCL and DLBCL cells. ADC-A also exhibited a synergistic effect with other Bcl-2 inhibitors (Bcl-2i-1 and Bcl-2i-2) on the inhibition of Jeko-1 cell proliferation, and exhibited an additive effect with both inhibitors in inhibiting the proliferation of Mino cells.
[0045] [0045] FIG. 21 is a graph illustrating the inhibition of Jeko-1 cell proliferation after treatment with ADC-A, ABT-199 or a combination of ADC-A and ABT-199.
[0046] [0046] FIG. 22 is a graph illustrating the combination index of treatment with ADC-A and mTOR1 / 2 inhibitor INK128 / sapanisertib ("INK128") in various cell lines. ADC-A exhibited a synergistic effect with INK128 on the inhibition of MCL and DLBCL cell proliferation as well.
[0047] [0047] FIG. 23 is a graph illustrating the inhibition of Jeko-1 cell proliferation after treatment with ADC-A, INK128 or a combination of ADC-A and INK128.
[0048] [0048] FIG. 24 is a graph illustrating the combination index of treatment with ADC-A and PI3K inhibitor CAL-101 / idelalisib ("CAL101") in various cell lines. ADC-A exhibited a synergistic effect with CAL101 on inhibiting the proliferation of MCL and DLBCL cells.
[0049] [0049] FIGS. 25A and 25B are graphs illustrating the inhibition of cell proliferation after treatment with ADC-A, PI3K inhibitor CAL-101 / idelalisib ("CAL101" or "101") or a combination of ADC-A and CAL101, in cell line of DLBCL-ABC TMD-8 (25A) or in the DLBCL-GCB DOHH2 cell line (25B). DETAILED DESCRIPTION OF THE INVENTION
[0050] The present invention provides immunoconjugates of the formula Ab - ((L) m– (D)) n, where Ab is an antibody or antigen-binding fragment thereof that specifically binds to the ROR1 protein; L is a linker; D is a portion of a drug that has therapeutic activity in cancer; m is 0 or 1; and n is an integer from 1 to 10. In the formula, the dash "-" denotes a covalent or non-covalent bond. The antibody or fragment includes, however, is not limited to an antibody or antibody fragment that competes with the D10 or Ab1 antibody for binding to human ROR1 or binds to the same epitope as D10 or Ab1. The drug portion includes, however, is not limited to another antibody or antigen-binding fragment, a polypeptide.
[0051] [0051] An "antibody-drug conjugate" or "ADC" or "immunoconjugate" refers to an antibody molecule, or an antigen-binding fragment thereof, that is covalent or not covalently, with or without a linker, to one or more biologically active molecule (s). The present immunoconjugates comprise antibodies or fragments thereof that are specific for human ROR1 and can therefore serve as excellent targeting moieties for releasing conjugated payloads to ROR1 positive cells. In some embodiments, an ROR1 immunoconjugate provided here has an equilibrium dissociation constant (KD) of about 1 μM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM or 0.001 nM or less (for example, 10-8 M or less, from 10-8 M to 10-13 M or 10-9 M to 10-13 M) for human ROR1. KD can be measured by any suitable assay, such as surface plasmon resonance assays (for example, using a BIACORE®-2000 or BIACORE®-3000). In certain embodiments, the KD of an immunoconjugate of the invention is less than the KD for the D10 antibody. In certain embodiments, the KD of an immunoconjugate of the invention for human ROR1 is less than about 50, 40, 30, 20 or 10 nM (e.g., 40 nM). In some embodiments, a ROR1 immunoconjugate provided here inhibits the growth of human ROR1 + cancer cells in vitro with an EC50 of about 500, 400, 350, 300 or 250 nM or less (for example, 300 nM or less). When used herein, an antibody is said to specifically bind to an antigen when it binds to the antigen with a KD of 100 nM or less, such as less than 10 nM or less (for example, 1-5 nM), as determined for example, superficial plasmonium resonance or Bio-Layer Interferometry.
[0052] [0052] In certain embodiments, the immunoconjugate provided here is internalized by a ROR1 positive cell mainly via the lysosome / endosome pathway. In particular modalities, internalization is independent of the level of expression of ROR1 on the cell surface.
[0053] [0053] Modalities of the antibody or fragment thereof, the ligand and the drug portion used in the immunoconjugates are described in more detail below.
[0054] [0054] The term "antibody" is used here in the broadest sense and includes polyclonal and monoclonal antibodies, such as whole antibodies and their functional fragments (antigen binding). The term encompasses genetically modified and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugate antibodies, multispecific antibodies (for example, bispecific), diabody, tribodies and tetrabodies, tandem di scFv and tandem tri-scFv. Unless otherwise indicated, the term encompasses intact or life-size antibodies, including antibodies of any class or subclass (for example, IgG and its subclasses, such as IgG1, IgG2, IgG3 and IgG4; IgM; IgE; IgA; and IgD), as well as antibody fragments.
[0055] An antibody can include a heavy chain (or a sequence of polypeptide derived therefrom) and a light chain (or a sequence of polypeptide derived therefrom). The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy chain and the light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four regions of conserved structure and three regions determining complementarity. A single VH or VL domain can sometimes be sufficient to confer all or most of an antibody's antigen-binding specificity. In addition, antibodies that bind to a specific antigen can be isolated using a VH or VL domain of an antibody that binds to the antigen to screen a library of complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).
[0056] [0056] The terms "complementarity determining region" and "CDR", which are synonymous with "hypervariable region" or "HVR", refer to sub-regions within the variable domains of the antibody, which confer specificity and / or affinity of the antibody to its antigen. In general, there are three CDRs in each heavy chain variable domain (HCDR1, HCDR2 and HCDR3) and three CDRs in each light chain variable domain (LCDR1, LCDR2 and LCDR3). "Framework regions" ("FRs") refers to the non-CDR portions of the variable domains. In general, there are four FRs in each natural-sized heavy chain variable domain and four FRs in each natural-sized light chain variable domain. The precise limits of the amino acid sequence of a given CDR or FR can be easily determined using any of several known schemes, including those described by Kabat et al., 5th Ed., Public health Service, National Institutes of Health, Bethesda, MD. (1991) ("Kabat" numbering scheme); Al-Lazikani et al., JMB 273, 927-948 (1997) ("Chothia" numbering scheme); MacCallum et al., J.
[0057] [0057] The limits of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on sequence alignments, while the Chothia scheme is based on structural information. The numbering for both Kabat and Chothia schemes is based on the most common sequence lengths of the antibody region, with inserts modified by insertion letters, for example, "30a". The two schemes place certain insertions and deletions ("indels") in different positions, resulting in differential numbering. The contact scheme is based on the analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. Unless otherwise indicated, the CDRs of the antibodies referred to herein can be identified according to any of the methods of Kabat, Chothia, IMGT and contact.
[0058] [0058] An antigen-binding fragment of a life-sized antibody can be used in the production of an immunoconjugate of the present invention. Examples of antibody fragments include, however, are not limited to, Fv, Fab, Fab ', Fab'-SH, F (ab') 2; recombinant IgG fragments (rIgG); diabodies; linear antibodies; single chain antibody molecules (for example, scFv or sFv); single domain antibodies (for example, sdAb, sdFv, nanobodies); and multispecific antibodies formed from antibody fragments. In certain embodiments, the fragments are single chain antibody fragments comprising a variable heavy chain region and / or a variable light chain region, such as scFvs.
[0059] [0059] An immunoconjugate of the invention comprises an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, for example, human ROR1. The antibody or fragment binds to an extracellular portion of the ROR1 protein, such as an epitope in one or more of the immunoglobulin (Ig), Frizzled and Kringle-like domains of the ROR1 protein. In certain embodiments, the ROR1 binding antibody or fragment binds to an ROR1 amino acid sequence shown in SEQ ID NO: 1 or 2 (not including terminal cysteine, which is added for the convenience of conjugation) and it can be internalized by a ROR1 + cell; examples of such an antibody are murine antibodies D10 and 99961. See, Pats. U.S. 9,217,040 and 9,758,591, the descriptions of which are incorporated by reference here in their entirety. In certain embodiments, the antibody or fragment competes with D10 or 99961 for binding to human ROR1. The amino acid sequences of exemplary anti-ROR1 antibodies used in the immunoconjugates of the invention are shown in Table 1 below, where Ab1-Ab4 are humanized variants of antibody 99961. Table 1 SEQ ID NOs of Exemplary Anti-ROR1 Antibodies Ab HCDR1 HCDR2 HCDR3 VH HC LCDR1 LCDR2 LCDR3 VL LC 99961 7 8 9 45 - 10 11 12 46 - Ab1 7 8 9 5 3 10 11 12 6 4 Ab2 7 8 9 5 3 10 11 12 50 49 Ab3 7 8 9 48 47 10 11 12 6 4 Ab4 7 8 9 48 47 10 11 12 50 49 D10 27 28 29 25 - 30 31 32 26 -
[0060] [0060] In some embodiments, the antibody or antibody fragment in the immunoconjugate specifically binds to human ROR1, and its heavy and light chains, respectively, comprise: a) the amino acid sequences of CDR1-3 (HCDR1-3 ) heavy chain in SEQ ID NO: 3 and the amino acid sequences of light chain CDR1-3 (LCDR1-3) in SEQ ID NO: 4;
[0061] [0061] In some embodiments, the antibody or fragment is humanized or chimeric with human constant regions. In other embodiments, the antibody or fragment may comprise a human IgG1, IgG2, IgG3 or IgG4 constant region and optionally a human constante constant region.
[0062] [0062] In certain embodiments, the immunoconjugate of the invention comprises an anti-ROR1 antibody, or an antigen binding fragment, wherein the antibody comprises: a) a variable heavy chain (VH) domain or region comprising an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% (for example, at least 90%) identical to SEQ ID NO: 5 and a light chain variable domain (VL) comprising an amino acid sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% (for example, at least 90%) identical to SEQ ID NO: 6; b) a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 5 and 6, respectively; c) a heavy chain (HC) comprising an amino acid sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98% or 99% (for example, at least 90%) identical to SEQ ID NO: 3 and a light chain (LC) comprising an amino acid sequence 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98% or 99% (for example, at least 90%) identical to SEQ ID NO: 4; or d) an HC and an LC comprising the amino acid sequences of SEQ ID NOs: 3 and 4, respectively.
[0063] [0063] In certain embodiments, the VH and VL of the antibody comprised
[0064] [0064] In some embodiments, the antibody or fragment comprises a human IgG1, IgG2, IgG3 or IgG4 constant region and, optionally, a human constante constant region.
[0065] [0065] In certain embodiments, the HC and LC of the antibody respectively comprise the amino acid sequences of: a) SEQ ID NOs: 3 and 49; b) SEQ ID NOs: 47 and 4; or c) SEQ ID NOs: 47 and 49.
[0066] [0066] In certain embodiments, the immunoconjugate of the invention comprises an antibody or fragment thereof derived from a murine antibody with the amino acid sequences VH and VL of (i) SEQ ID NOs: 25 and 26, respectively; (ii) SEQ ID NOs: 35 and 36, respectively; or (iii) SEQ ID NOs: 45 and 46, respectively. Antibodies derived from these sequences can be, for example, antibodies that have been humanized or joined to a human Fc region (for example, chimeric). For example, the antibody or antigen-binding fragment in the immunoconjugate comprises: a) a VH comprising an amino acid sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98% or 99% identical to SEQ ID NO: 45 and a VL comprising an amino acid sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 46; b) a VH comprising the amino acid sequence of SEQ ID NO: 45 and a VL comprising the amino acid sequence of SEQ ID NO: 46;
[0067] [0067] Exemplary coding sequences for the above mentioned antibodies are shown in Table 12 below. For example, the antibody in the immunoconjugate may comprise: a) a VH encoded by (i) nucleotides 73-420 of SEQ ID NO: 21, or (ii) SEQ ID NO: 23; and a VL encoded by SEQ ID NO: 22 or 24; b) a VH encoded by SEQ ID NO: 52 and a VL encoded by SEQ ID NO: 54; c) a VH encoded by SEQ ID NO: 33 and a VL encoded by SEQ ID NO: 34; d) an HC encoded by nucleotides 73-1,410 of SEQ ID NO: 19 and an LC encoded by nucleotides 73-714 of SEQ ID NO: 20; or e) an HC encoded by SEQ ID NO: 51 and an LC encoded by nucleotides SEQ ID NO: 53.
[0068] [0068] In certain embodiments, the immunoconjugate of the invention comprises an antigen-binding fragment of an anti-ROR1 antibody, wherein the antigen-binding fragment comprises the sequence of any one of SEQ ID NOs: 64-68. In certain modalities, the antigen-binding fragment comprises the VH and VL amino acid sequences of:
[0069] [0069] Percentage sequence identity (%) in relation to a reference polypeptide sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference sequence, after aligning sequences and enter intervals, if necessary, to achieve the maximum percentage of sequence identity. Alignment for purposes of determining the percentage of amino acid sequence identity can be achieved in several ways that are known; for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR). For the purposes here, however, the% amino acid sequence identity values are generated using the ALIGN-2 sequence comparison computer program. The ALIGN-2 sequence comparison computer program was created by Genen- tech, Inc., and the source code was filed with user documentation at the US Copyright Office, Washington DC, 20559, where it is registered. under US Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source code.
[0070] [0070] In situations where ALIGN-2 is used to compare the amino acid sequence, the% amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B is calculated as follows : 100 times the X / Y fraction, where X is the number of amino acid residues registered as identical pairings by the ALIGN-2 sequence alignment program in this A and B program alignment, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the% identity of amino acid sequence from A to B will not be equal to the% identity of amino acid sequence from B to A. Unless specifically stated otherwise, all% of the amino acid sequence identity values used here are obtained as described in the immediately preceding paragraph using the ALIGN computer program. -2.
[0071] [0071] In some embodiments, amino acid sequence variants of the antibodies provided here are contemplated. A variant typically differs from a polypeptide described herein specifically in one or more substitutions, deletions, additions and / or insertions. Such variants can be naturally occurring or can be generated synthetically, for example, by modifying one or more of the polypeptide sequences above the invention and evaluating one or more biological activities of the polypeptide as described herein and / or using any of several known techniques. For example, it may be desirable to improve binding affinity and / or other properties
[0072] [0072] When used herein, the term "substantially identical" refers to two or more sequences having a percentage of sequential units (eg, amino acid residues) that are the same when compared and aligned for maximum correspondence in a comparison window, or in a designated region as measured using comparison algorithms. For example, two or more sequences can be "substantially identical" if the sequential units are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical or about 99% identical in a specified region. Such percentages describe the "percent identity" between two strings.
[0073] [0073] Anti-ROR1 antibodies for use in the immunoconjugates of the present invention can be produced by immunizing an animal with human ROR1 or a human ROR1 protein fragment. Antibodies that bind to the immunization fragment with high affinity (for example, with a KD in the nM range or less) can be screened using routine methods, such as ELISA.
[0074] [0074] If the antibody is a non-human antibody, it can be humanized. A "humanized" antibody is an antibody in which all or substantially all of the CDR amino acid residues are derived from a non-human antibody (for example, mouse or rat) and all or substantially all of the FR amino acid residues are derived from FRs human. A humanized antibody can optionally include at least a portion of a constant region derived from a human antibody. A "humanized form" of a non-human antibody refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity for humans, while maintaining antigen binding specificity and affinity of the parental non-human antibody. In some embodiments, some RF residues in a humanized antibody are replaced by the corresponding residues of the cognate non-human antibody to restore or improve the specificity and / or affinity of binding to the resulting antibody antigen.
[0075] [0075] The antibodies or fragments of ROR1 can be manufactured recombinantly in mammalian host cells containing coding sequences for the antibodies or fragments of ROR1, where the coding sequences are operationally linked to regulatory elements of transcription suitable for expression in host cells. Coding sequences can be introduced into host cells in one or more vectors. Useful mammalian host cells include, inter alia, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293 Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, kidney cells baby hamster (BHK), African green monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, Hep G2) and A549 cells. Cell lines can be selected based on their levels of expression. Other cell lines that can be used include insect cell lines, such as Sf9 or Sf21 cells, and yeast cell lines.
[0076] [0076] In some embodiments, an originating ROR1 antibody may be modified by introducing one or more amino acid substitutions to improve binding to the antibody's antigen, to decrease immunogenicity (eg, de-immunize; see, for example, Jones et al., Methods Mol Biol. 525: 405-23 (2009)) and / or to improve antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).
[0077] [0077] In some modalities, substitutions, insertions or deletions can be made within one or more CDRs, in which the changes do not substantially reduce the binding of the antibody to its antigen. For example, conservative substitutions that do not substantially reduce binding affinity can be made.
[0078] [0078] Changes (for example, substitutions) can be made on CDRs to improve the affinity of the antibody. CDR residues involved in binding to the antigen can be identified using, for example, alanine scanning mutagenesis or computer modeling. HCDR3 and LCDR3, in particular, are generally targeted. A crystal structure of an antigen-antibody complex can also be used to identify points of contact between the antibody and its antigen. Such contact residues and their neighboring residues can be targeted for mutations. Variants can be tracked to determine whether they achieve the desired properties. In vitro affinity maturation (for example, using error-prone PCR, chain rearrangement, CDR randomization or oligonucleotide-directed mutagenesis) can also be used to improve antibody affinity (see, for example, Hoo-
[0079] [0079] Amino acid sequence insertions and deletions made in an antibody or antibody fragment include amino and / or carboxyl terminal fusions ranging in length from one or a few residues to polypeptides containing one hundred or more residues, as well as single or multiple amino acid residues intrassequences and deletions. Examples of terminal inserts include an antibody with an N-terminal methionyl residue. Other variants of insertion of the antibody molecule include the fusion of the N or C terminal of the antibody to an enzyme (for example, for ADEPT) or a polypeptide that increases the serum half-life of the antibody. Examples of variant insertion of antibody molecule intrassequency include an insertion of 3 amino acids in the light chain. Examples of terminal deletions include an antibody with a deletion of 7 or less amino acids at one end of the light chain and the removal of C-terminal lysine in the heavy chain.
[0080] [0080] In some embodiments, the antibodies to ROR1 are changed to increase or decrease its glycosylation (for example, by changing the amino acid sequence so that one or more glycosylation sites are created or removed). A carbohydrate attached to an antibody's Fc region can be changed. Native antibodies to mammalian cells typically comprise a branched, biantenary oligosaccharide linked by an N to Asn297 bond of the CH2 domain of the Fc region (see, for example, Wright et al., TIBTECH 15: 26-32 (1997) ). Asn297 refers to the asparagine residue located around position 297 in the Fc region (EU number of residues in the Fc region; see, for example, Edelman et al. PNAS 63 (1): 78-85 (1969)). However, Asn297 can also be located about ± 3 amino acids upstream or downstream from position 297, that is, between positions 294 and 300, due to small sequence variations in antibodies. The oligosaccharide can be any one of several carbohydrates, for example, mannose, N-acetyl glucosamine (GlcNAc), galactose, sialic acid or fucose linked to a GlcNAc on the stem of the biantenar oligosaccharide structure. Modifications of the oligosaccharide in an antibody can be made, for example, to create antibody variants with certain improved properties. Antibody glycosylation variants can have an improved ADCC and / or CDC function.
[0081] [0081] In some embodiments, antibody variants are provided having a carbohydrate structure that does not have or has a reduced level of fucose linked (directly or indirectly) to an Fc region. For example, the amount of fucose in such an antibody can be 1% to 80%, 1% to 65%, 5% to 65% or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain in Asn297, in relation to the sum of all glycostructures linked to Asn297 (see, for example, PCT Patent Publication WO 2008 / 077546). Such fucosylation variants can have an improved ADCC function (see, for example, Okazaki et al., J. Mol. Biol. 336: 1239-1249 (2004); and Yamane-Ohnuki et al., Bio-tech Bioeng. 87: 614 (2004)). Cell lines (eg knockout cell lines) can be used to produce defucosylated antibodies, for example, protein fucosylation-deficient CHO Lec13 cells and alpha-1,6-fucosyltransferase gene knockout CHO cells ( FUT8) (see, for example, Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); and Kanda et al., Biotechnol. Bioeng. 94 (4): 680-688 (2006)). Other variants of antibody glycosylation, as described in, for example, U.S. Patent 6,602,684) can also be produced for antibodies to ROR1 or antibody fragments for use in the present immunoconjugates.
[0082] [0082] In some embodiments, one or more amino acid modifications can be introduced in the Fc region of an ROR1 antibody to generate an ROR1 antibody with a variant Fc region that imparts new properties to the antibody. A variant Fc region may comprise a human Fc region sequence (for example, a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (for example, a substitution) at one or more amino acid positions . For example, an ROR1 antibody with a variant Fc region may have some, but not all, effector functions, which makes it a desirable candidate for applications in which the half-life of the antibody is important, but some effector functions ( such as complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor binding assays (FcR) can be conducted to ensure that the antibody is not bound to FcR (therefore, probably without ADCC activity), but maintains the ability to bind to FcRn. Non-limiting examples of in vitro assays to evaluate the ADCC activity of a molecule of interest are described in U.S. Patents 5,500,362 and 5,821,337. Alternatively, non-radioactive assay methods can be used (for example, ACTI ™ and CytoTox 96® non-radioactive cytotoxicity assays). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs), monocytes, macrophages and natural killer cells (NK).
[0083] [0083] Antibodies may have increased half-lives and improved binding to the neonatal Fc receptor (FcRn) (see, for example, U.S. Patent Publication 2005/0014934). Such antibodies may comprise an Fc region with one or more substitutions in it that improve the binding of the Fc region to FcRn and include those with substitutions.
[0084] [0084] In some embodiments, it may be desirable to create antibodies modified with cysteine, for example, "thioMAbs", in which one or more residues of an antibody are replaced by cysteine residues. In some embodiments, the substituted residues occur at accessible sites of the antibody. Reactive thiol groups can be positioned at sites for conjugation to other moieties, such as drug moieties or moieties of drug binding, to create an immunoconjugate. Any one or more of the following residues can be replaced by cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) from the Fc region of the heavy chain.
[0085] [0085] An antibody provided herein can also be modified to include non-protein portions. Suitable portions for antibody shedding include, however, are not limited to water-soluble polymers. The term "polymer", when used herein, refers to a molecule composed of repeated subunits; such molecules include, however, are not limited to polypeptides, polynucleotides or polysaccharides or polyalkylene glycols. Non-limiting examples of water-soluble polymers are polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane , ethylene / maleic anhydride copolymer, polyamino acids (homopolymers or randomized copolymers) and dextran or poly (N-vinyl pyrrolid-
[0086] An immunoconjugate of the invention comprises an anti-ROR1 antibody or an antigen-binding fragment of the same conjugate to one or more cytotoxic agents, such as chemotherapeutic agents, growth inhibitory agents, toxins (for example , protein toxins, enzymatically active toxins from bacteria, fungi, plant, or animal origin or fragments thereof) or radioactive isotopes. The cytotoxic agent (s) can be conjugated to the anti-ROR1 antibody or fragment by a linker covalently linked to an amino acid residue of the antibody. Many drugs that can serve as a cytotoxic moiety in an immunoconjugate are independently too toxic to be used in the treatment of cancer and are therefore more effective when targeted specifically at the cancer cell by an antibody or antibody fragment.
[0087] [0087] The term "cytotoxic drug moiety" or "cytotoxic agent" refers to a compound that can cause damage, disturbance or death to a cell. Examples of portions of cytotoxic drugs that can be used as part of an ROR1 immunoconjugate include, but are not limited to: NCA1, auristatin, auristatin E, binding agents to smaller DNA grooves, alkylating agents to smaller grooves of DNA, enedin, lexitropsin, duocarmicin, taxane, puromycin dolastatin, maytansinoid, vinca alkaloid, AFP, MMAF,
[0088] [0088] In some embodiments, a cytotoxic agent suitable for use in the ROR1 immunoconjugate is a) an antitubulin agent (for example, auristatin or do-lastatin, such as auristatin E, monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF ), dimethylvaline-valine-dolaisoleuin-dolaproin-phenylalanine-p-phenylenediamine (AFP), 5-benzoylvaleric acid-auristatin E ester (AEVB), AEB, a maytansinoid, ansamitokinin, mertansin / entansine (DM1) or ravtansin / soravtansin (DM4)), b) a DNA alkylating agent or a minor DNA groove alkylating agent (eg, duocarmicin), c) a DNA crosslinking agent (eg, pyrrole-benzodiazepine (PBD)) , d) a DNA intercalating agent (for example, PNU-159682), e) a minor DNA groove binding agent (for example, CC-1065), or f) an RNA polymerase II inhibitor (for example example, amanitin, such as α-amanitin).
[0089] [0089] When used herein, the term "intercalating agent" refers to a chemical that can be inserted into the intramolecular space of a molecule or the intermolecular space between molecules. For example, a DNA intercalating agent can be a molecule that inserts itself into the stacked bases of the DNA double helix.
[0090] [0090] In some embodiments, the cytotoxic agent used in the ROR1 immunoconjugate is selected from the group consisting of an enedin, a lexitropsin, a duocarmicin, a taxane, a puromycin, a podophyllotoxin, a baccatin derivative, a cryptophysin, a combrestatin, a dolastatin, a maytansinoid, an alkaloid vinca, paclitaxel, docetaxel, ansamitocin, CC-1065, SN-38, topo-tecano, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin , calicheamicin, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, epothilone A, epothilone B, nocodazole, colchicines, colcimide, estramustine, cemadotine, maytansine discodermolide, eleuterobin and netropsin.
[0091] [0091] In some embodiments, the portion of cytotoxic drug in the immunoconjugate is a prodrug.
[0092] [0092] In some embodiments, the median number of the drug portion for the antibody in the immunoconjugate (i.e., drug-to-antibody ratio or DAR) is 1; or at least 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2 , 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5 , 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 6, 7, 8, 9 or 10. Methods specimens for measuring DAR are described in the examples below.
[0093] [0093] In certain embodiments of an immunoconjugate of the invention, the antibody can be conjugated directly to the cytotoxic agent, or it can be conjugated through a ligand. Suitable linkers include, for example, cleavable and non-cleavable linkers. In some embodiments, the linker is a cleavable linker. A cleavable linker refers to a linker that comprises a cleavable portion and is typically susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include, for example, peptide linkers cleavable by an intracellular protease (such as a lysosomal protease or an endosomal protease) and acid cleavable linkers. In exemplary embodiments, the linker can be a dipeptide, such as a valine-citrulline (val-cit or VC) or a phenylalanine-lysine (phe-lys) linker. The linker can also be a tripipeptide or a polypeptide having four or more amino acid residues. Other suitable binders include hydrolyzable binders at a pH below 5.5, such as a hydrazone binder. Additional suitable cleavable linkers include disulfide linkers. In some embodiments, the binder is a non-polymeric binder. In some cases, the linker is either a non-peptide linker or a linker that does not contain an amino acid residue.
[0094] [0094] In some embodiments, the linker includes a C1-C6 alkyl group (for example, a C5, C4, C3, C2 or C1 alkyl group). When used herein, the term "alkyl" refers to a straight or branched hydrocarbon chain radical consisting only of carbon and hydrogen atoms, which does not contain unsaturation. C1-Cx includes C1-C2, C1-C3, ..., C1-Cx, where x is an integer. C1-Cx refers to the number of carbon atoms in the designated group. In some modalities, an alkyl comprises one to eight carbon atoms (C1-8 alkyl). In some embodiments, an alkyl comprises two to six carbon atoms (C2-6 alkyl).
[0095] [0095] When used here, a ligand prior to the chemical reaction to bind the Ab (antibody or fragment) and D (payload) components of the immunoconjugate is also called a "ligand precursor". It will be evident to the person skilled in the ADC technique whether a particular chemical entity described here is a precursor of the ligand based on its reactive capabilities, or a component of the ligand in the final immunoconjugate product.
[0096] [0096] In some embodiments, the bond between the Ab and D components of the immunoconjugate can be formed by reacting the components with a homobifunctional ligand. Exemplary homobifunctional ligands include, but are not limited to, Lomant dithiobis reagent (succinimidylpropionate) DSP, 3′3′-dithiobis (sulfosuccinimidyl propionate) (DTSSP), dissuccinimidyl suberate (DSS), bis (sulfosuccinate) (BS), disuccinimidyl succinate tartrate (DST), disulfosuccinimidyl tartrate (sulfo DST), ethylene glycobis (succinimidyl succinate) (EGS), dissuccinimidyl glutarate (DSG), N, N'-dissuccinimidyl carbonate (DSC ), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3′- dithiobispropionimidate (DTBP), 1,4-di-3 ′ - (2′-pyridildithio) propionamido) butane (DPDPB), bismaleimido-hexane (BMH), compound containing aryl halide (DFDNB), such as, for example, 1,5-difluoro-2,4-
[0097] [0097] In some embodiments, the bond between the Ab and D components of the immunoconjugate can be formed by reacting the components with a heterobifunctional ligand. Exemplary heterobifunctional linkers include, but are not limited to, amine and sulfhydryl reactive crosslinkers, such as N-succinimidyl 3- (2-pyridyldithio) propionate (sPDP), N-succinimidyl 3- (2-pyridyldithio) ) long-chain propionate (LC-sPDP), N-succinimidyl 3- (2-pyridyldithio) water-soluble long-chain propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α- (2-pyridyldithio) ) toluene (sMPT), sulfosuccinimidyl-6- [α-methyl-α- (2-pyridyldithium) toluamido] hexanoate (sulfo-LC-sMPT), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1- carboxylate (SMCC), sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs), m-ma-leimidobenzyl-N- ester hydroxysulfosuccinimide (sulfo-MBs), N-succinimidyl (4-iodoacetyl) aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (sulfo-sIAB), succinimidyl-4- (p-maleimidophenyl) butyrate (sMP -4- (p-maleimidophenyl) butyrate (sulfo- sMPB), N- (-maleimidobutyryloxy) succinimide ester (GMBs), N- (-maleimidobutyryloxy) sulfosuccinimide ester (sulfo-GMBs), succinimidyl 6 - ((io-doacetyl) amino) hexanoate (sIAX), succinimidyl 6- [6 - (((iodoacetyl) amino) hexanoyl) amino] hexanoate (sIAXX), succinimidyl 4 - (((iodoacetyl) amino) methyl) cyclohexane-1-carboxylate (sIAC), succinimidyl 6- (((((4-io-doacetyl) amino) methyl) cyclohexane-1-carbonyl) amino) hexanoate (sIACX),
[0098] [0098] In some modalities, the connection between the components
[0099] [0099] In some embodiments, the ligand / payload conjugation to the antibody or fragment can be formed by reacting with a maleimide group (which can also be referred to as a maleimide spacer). In certain embodiments, the maleimide group is maleimidocaproíla (mc); thus, the ligand / payload is conjugated to the antibody or fragment through the reaction between a residue in the antibody or fragment and the mc group in the ligand precursor. In some modalities, the maleimide group comprises a maleimidomethyl group, such as succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) or sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1 - carboxylate (sulfo-SMCC), as described here.
[00100] [00100] In some modalities, the maleimide group is a self-stabilizing maleimide. In some embodiments, the self-stabilizing maleimide uses diaminopropionic acid (DPR) to incorporate a basic amino group adjacent to the maleimide to provide intramolecular catalysis of the hydrolysis of the thiosuccinimide ring, thereby decreasing the ability of the maleimide to undergo an elimination reaction through of a retro-Michael reaction. In some embodiments, the self-stabilizing macheimide is a maleimide group described in Lyon et al., Nat. Biotechnol. 32 (10): 1059-1062 (2014). In certain modalities,
[00101] [00101] In some embodiments, the linker may include a portion of peptide. In some embodiments, the peptide portion comprises at least 2, 3, 4, 5, 6, 7, 8 or more amino acid residues. In some embodiments, the peptide moiety is cleavable (for example, enzymatically or chemically). In some modalities, the peptide portion is not cleavable. In some embodiments, the peptide portion comprises Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly (SEQ ID NO: 55), Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe -Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 56), or Gly-Phe-Leu-Gly (SEQ ID NO: 57). In certain embodiments, the binder comprises Val-Cit (VC). In certain embodiments, the linker is Val-Cit (VC).
[00102] [00102] In some embodiments, the linker may include a benzoic acid or benzyloxy group, or a derivative thereof. For example, the binder can compromise para-amino-benzoic acid (PABA). In some embodiments, the linker includes a para-amino-benzyloxycarbonyl (PAB) group. In some embodiments, the ligand comprises gamma-amino-butyric acid (GABA).
[00103] [00103] In some embodiments, the bond between the Ab and D components of the immunoconjugate can be formed by reacting the components with a linker comprising a maleimide group, a peptide moiety and / or a benzoic acid (e.g., PABA) or benzyloxycarbonyl group, in any combination. In certain modalities, the maleimide group is maleimidocaproíla (mc). In certain embodiments, the peptide group is Val-Cit (VC). In certain embodiments, the linker comprises a Val-Cit-PABA group. In certain modalities, conjugation of the ligand to the antibody or fragment can be
[00104] [00104] In some modalities, the ligand is an auto-immolating ligand or a self-eliminating ligand (for example, a cyclization self-eliminating ligand). In some embodiments, the linker may be a linker described in U.S. Pat. U.S. 9,089,614 or PCT Publication WO 2015/038426.
[00105] [00105] In some embodiments, the ligand is a ligand of the dendritic type. In certain embodiments, the dendritic ligand comprises a portion of branched multifunctional ligand. The dendritic ligand can have two or more branches. In certain modalities, the dendritic ligand is used to increase the molar ratio of the drug portion to the antibody or fragment. In certain modalities, the dendritic ligand comprises PA-MAM dendrimers.
[00106] [00106] In some embodiments, the ligand is a non-screening ligand or a ligand that does not leave behind a ligand portion (for example, an atom or a ligand group) after cleavage. Examples of non-tracking binders include, however, are not limited to germanium binders, silicon binders, sulfur binders, selenium binders, nitrogen binders, phosphorus binders, boron binders, chrome binders and binders phenylhydrazide. In some embodiments, the linker is an aryl-triazene linker without screening, as described in Hejesen et al., Org Biomol Chem 11 (15): 2493-2497 (2013). In some embodiments, the ligand is an unlabeled ligand described in Blaney et al., Chem. Rev. 102: 2607-2024 (2002). In some modalities, a ligand is a link
[00107] [00107] In some embodiments, the linker comprises a functional group that exercises a steric impediment at the binding site between the linker and a conjugating portion (for example, any of the ADC-A, B, C, E, F and HT described herein ). In some embodiments, the steric impediment is a steric impediment around a disulfide bond. An exemplary linker that exhibits steric impairment can be, for example, a heterobifunctional linker (for example, as described herein). In some embodiments, a linker that exhibits steric impediment comprises SMCC and SPDB.
[00108] [00108] In some embodiments, the ligand is an acid-cleavable ligand. In some embodiments, the acid-cleavable linker comprises a hydrazone bond, which is susceptible to hydrolytic cleavage. In some embodiments, the acid-cleavable linker comprises a thiomaleamic acid linker. In some embodiments, the acid-cleavable ligand is a thiomaleamic acid ligand as described in Castaneda et al., Chem. Common. 49: 8187-8189 (2013).
[00109] [00109] In some embodiments, the linker is a linker described in any of U.S. Patents 6,884,869, 7,498,298, 8,288,352,
[00110] [00110] Ligands suitable for use in the immunoconjugates of the invention may include, for example, ligands that are cleavable intracellularly with high extracellular stability. In certain modalities, the linker comprises a functional group that allows the linker to be attached to any of the antibodies or fragments described herein (for example, a maleimide derivative). In certain modalities
[00111] [00111] In some embodiments, the ligands described herein may be linked to the antibodies or fragments binding the antigen described herein to a naturally occurring amino acid residue, such as a lysine or reduced cysteine. In some embodiments, the linkers can be linked to an unnatural amino acid (eg azidophenylalanine, p-acetylphenylalanine or p-azidomethylphenylalanine) via an alkaline / azide click reaction, carbonyl condensations, additions like Michael, and Mizoroki-Heck replacements. Additional ligand sites can be added genetically and can comprise a polypeptide motif that allows enzymatic addition of the ligand. Such polypeptide motifs may comprise, for example, a glutamine tag (eg, LLQGA (SEQ ID NO: 58)), an aldehyde tag (eg, CxPxR, where x is any amino acid), a sortase motif ( for example, LPxTG (SEQ ID NO: 59, where x is any amino acid), NPQTN (SEQ ID NO: 60)) or a BirA marker (for example, GFEIDKVWYDLDA (SEQ ID NO: 61)). The binding of ligands to a glutamine marker can be achieved using a bacterial transglutaminase. The binding of the ligands to an aldehyde marker is achieved using the formylglycine-generating enzyme (FGE), which oxidizes the cysteine residue of the consensus sequence, creating an aldehyde; this aldehyde can be reacted with an aminooxy group in a binder to form a stable oxime. Binding of ligands to a sortase motif can be achieved using a bacterial transpeptidase that recognizes a C-terminal LPxTG sequence (SEQ ID NO: 59) or NPQTN sequence (SEQ ID NO: 60), cleaves the binding of TG or TN, and facilitates - through a thioacyl-threonine enzyme intermediate - the nucleophilic attack of the alpha-amine protein entering the threonine. The attacking residue can be a glycine dimer or trimer of a linker or added to a linker. The binding of ligands to a sortase motif or to a BirA marker can be achieved using a biotin ligase. In certain embodiments, the binder is not bound to a lysine residue. In certain embodiments, the binder is attached only to a cysteine residue. In certain modalities, the cysteine residue was modified in the antibody by converting one or more non-cysteine residues in the light chain and / or heavy chain to a cysteine. In certain devices, cysteine conversion does not interfere with antigen binding. In certain modalities, selenocysteine is incorporated into the antibody by genetic modification. See, for example, Sochaj et al., Biotechnology Advances 33: 775-784 (2015).
[00112] [00112] In some embodiments, the ligand is conjugated to the antibody or anti-ROR1 fragment by a chemical bonding process. In some embodiments, the linker is conjugated to the anti-ROR1 antibody or fragment by a native link. In some embodiments, the conjugation is as described in Dawson et al., Science 266: 776-779 (1994); Dawson et al., J. Am. Chem. Soc. 119: 4325-4329 (1997); Hackeng et al., PNAS 96: 10068-10073 (1999); or Wu et al., Angew. Chem. Int. Ed. 45: 4116-4125 (2006). In some embodiments, the arrangement is as described in U.S. Patent 8,936,910. In some modalities, the ligand is conjugated to the anti-ROR1 antibody or fragment, specifically at the site or not specifically through native binding chemistry.
[00113] [00113] In some embodiments, the ligand is conjugated to the anti-ROR1 antibody or antigen-binding fragment by a method directed to the site using a "no tracking" coupling technology (Philochem). In some embodiments, the "no tracking" coupling technology uses an N-terminal 1,2-aminothiol group in the binding portion which is then conjugated to the antibody or its fragment containing an aldehyde group. See, for example, Casi et al., JACS 134 (13): 5887-5892 (2012). In some embodiments, the linker is conjugated to the anti-ROR1 antibody or fragment by a method directed to the site using an unnatural amino acid incorporated into the binding portion. In some embodiments, the unnatural amino acid comprises p-acetylphenylalanine (pAcPhe). In some embodiments, the keto group of pAcPhe is selectively coupled to an alkoxyamine-derived conjugation moiety to form an oxime bond. See, for example, Axup et al., PNAS 109 (40): 16101-16106 (2012).
[00114] [00114] In some embodiments, the ligand is conjugated to the anti-ROR1 antibody or antigen-binding fragment by a system conjugated to the anti-ROR1 antibody by a site-directed method, using an enzyme-catalyzed process. In some modalities, the method directed to the site uses SMARTag ™ technology (Redwood). In some embodiments, SMARTag ™ technology comprises the generation of a formylglycine residue (FGly) from cysteine by the formylglycine-generating enzyme (FGE) through an oxidation process under the presence of an aldehyde marker and the subsequent conjugation of FGly to an amino acid molecule functionalized by alkylhydrazine through the hydrazino-Pictet-Spengler bond (HIPS). See, for example, Wu et al., PNAS 106 (9): 3000-3005 (2009) and Agarwal et al., PNAS 110 (1): 46-51 (2013)).
[00115] [00115] In some embodiments, the process catalyzed by enzymes
[00116] [00116] The ligands can be conjugated to the anti-ROR1 antibodies and antigen-binding fragments of the present description in several ways. Generally, a linker and a cytotoxic moiety are synthesized and conjugated before binding to an antibody. A method of binding a binder-drug conjugate to an antibody involves reducing disulfides exposed to solvent with dithiothreitol (DTT) or tris (2-carboxyethyl) phosphine (TCEP), followed by modifying the resulting thiois with portions of binder-drug containing maleimide (e.g., 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-VC-PAB)). A native antibody contains 4 interchain disulfide bonds and 12 intrachain disulfide bonds, as well as unpaired cysteines. Thus, antibodies modified in this way can comprise more than one linker-drug moiety per antibody. In certain embodiments, the immunoconjugates described herein comprise at least 1, 2, 3, 4, 5, 6, 7 or 8, 9 or 10 linker / drug moieties. In certain embodiments, the immunoconjugates described herein comprise 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, ligand / drug moieties , or 1 portion of binder / drug. In cases where the linker is branched and can each connect to multiple drug moieties, the ratio of the drug moiety to the antibody will be greater than the use of an unbranched ligand.
[00117] [00117] In some modalities, the ligand is of the formula:; wherein X is C2-8 alkyl; Y is - (CH2CH2O) qCH2CH2-; W is a unit of amino acid; Zé; n is 0 or 1; p is 0 or 1; q is an integer from 0 to 12; u is an integer from 0 to 5; and v is 0 or 1; where ** indicates the point of attachment to the drug portion (D); and * indicates the point of attachment to the antibody or fragment (Ab).
[00118] [00118] In some modalities, p is 0. In some modalities, p is 1. In some modalities, p is 1 and X is - (CH2) 2-. In some embodiments, p is 1 and X is - (CH2) 3-. In some embodiments, p is 1 and X is - (CH2) 4-. In some modalities, p is 1 and X is - (CH2) 5-. In some modalities, p is 1 and X is - (CH2) 6-. In some modalities, q is an integer from 1 to 12. In some modalities, q is an integer from 4 to 12. In some modalities, q is an integer from 4 to 8. In some modalities, q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In some embodiments, W is an amino acid unit that comprises naturally occurring amino acid residues, as well as minor and minor amino acids. non-naturally occurring amino acid analogues. In some modalities, W is selected from among
[00119] [00119] In some modalities, the ligand is of the formula:; wherein X is C2-8 alkyl; Y is - (CH2CH2O) qCH2CH2-; W is a unit of amino acid; Zé; n is 0 or 1; p is 0 or 1; q is an integer from 0 to 12; u is an integer from 0 to 5; and v is 0 or 1; where ** indicates the point of attachment to the drug portion (D); and * indicates the point of attachment to the antibody or fragment (Ab).
[00120] [00120] In some modalities, n is 0. In some modalities, n is 1. In some modalities, X is - (CH2) 2-. In some modalities, X is - (CH2) 3-. In some modalities, X is - (CH2) 4-. In some modalities, X is - (CH2) 5-. In some modalities, X is - (CH2) 6-. In some modalities, W is an amino acid unit comprising naturally occurring amino acid residues, as well as amino acids.
[00121] [00121] In some modalities, the immunoconjugate optionally also comprises an endosolytic portion. In some cases, the endosolytic portion is a component of the cell compartment release, such as a compound capable of freeing itself from any of the cellular compartments known in the art, such as endosome, lysosome, endoplasmic reticulum (ER), Golgi apparatus, microtubule, peroxisome or other vesicular bodies with the cell. In some cases, the endossomolytic portion comprises an endosolytic polypeptide, an endossomolytic polymer, an endosolytic lipid or a small endossomolytic molecule. In some cases, the endossomolytic portion comprises an endosolytic polypeptide. In other cases, the endossomolytic portion comprises an endosolytic polymer. In some embodiments, an endosolytic polymer described here is an pH-responsive endosolytic polymer. A pH-responsive polymer comprises a polymer that increases in size (swells) or collapses, depending on the pH of the environment. Polyacrylic acid and chitosan are examples of pH-responsive polymers. In some embodiments, an endosolytic portion described herein is a membrane-disruptive polymer. In some cases, the membrane-disruptive polymer comprises a cationic polymer, a neutral or hydrophobic polymer or an anionic polymer. In some embodiments, the membrane-disruptive polymer is a hydrophilic polymer.
[00122] [00122] The term "bond", when used herein, refers to a bond or chemical moiety formed from a chemical reaction between the functional group of a molecular entity and another molecular entity. Such bonds may include, however, are not limited to, covalent and non-covalent bonds, while such chemical moieties may include, however, are not limited to, esters, carbonates, carbamates, imine phosphate esters, hydrazones, acetals, ortho - res, peptide bonds, and oligonucleotide bonds. The hydrolytically stable bond means that the bond is substantially stable in water and does not react with water at useful pH values, including, however, not limited to physiological conditions, for an extended period of time, perhaps even indefinitely. Hydrolytically unstable or degradable bonding means that the bond is degradable in water or aqueous solutions, including, for example, blood. Enzymatically unstable or degradable binding means that the binding can be degraded by one or more enzymes. For example only, PEG and related polymers may include degradable bonds in the polymer backbone or in the linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule. Such degradable bonds include, however, are not limited to ester bonds formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups in a biologically active agent, where such ester groups generally hydrolyze under conditions to release the biologically active agent. Other hydrolytically degradable bonds include, but are not limited to, carbonate bonds; turns on-
[00123] [00123] In some embodiments, the ADC of the present invention has the structure: where Ab is an antibody and D is a drug moiety. In some embodiments, the ADC has the structure: where Ab is an antibody and D is monomethyl auristatin E (MMAE). In some modalities, the ADC has the structure:
[00124] [00124] In some modalities, the ADC has the structure: Ab THE O O H N N N D The H THE
[00125] [00125] In some modalities, the ADC has the structure:
[00126] [00126] In some embodiments, the ADC has the structure: where Ab is an antibody and D is a drug moiety. In some embodiments, the ADC has the structure: where Ab is an antibody and D is mertansin. In some modalities, the ADC has the structure:
[00127] [00127] In some embodiments, the ADC has the structure: where Ab is an antibody and D is a drug portion. In some embodiments, the ADC has the structure: where Ab is an antibody and D is MMAE. In some embodiments, the ADC has the structure: where Ab is an antibody. In some modalities, the ADC has the structure:
[00128] [00128] In some embodiments, the ADC has the structure: where Ab is an antibody and D is a drug moiety. In some embodiments, the ADC has the structure: where Ab is an antibody and D is a duocarmycin. In some modalities, the ADC has the structure:
[00129] [00129] In some embodiments, the ADC has the structure: where Ab is an antibody and D is a portion of drug. In some embodiments, the ADC has the structure: where Ab is an antibody and D is an amanitin, such as α-amanitin. In some embodiments, the ADC has the structure: where Ab is an antibody.
[00130] [00130] In some embodiments, the ADC has the structure: where Ab is an antibody and D is a drug portion. In some modalities, the ADC has the structure:
[00131] [00131] In some embodiments, the ADC has the structure: where Ab is an antibody and D is a drug portion. In some embodiments, the ADC has the structure: where Ab is an antibody and D is PBD. In some embodiments, the ADC has the structure: where Ab is an antibody.
[00132] [00132] Exemplary immunoconjugates of the present invention are shown in the table below:
[00133] [00133] In the table above, the abbreviations are used as follows: maleimide chemistry (MAL); maleimidocaproíla (mc); succinimide (SC) chemistry; succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC); dibenzylcyclooctin (DBCO); diaminopropionic acid (DPR); benzyl (Phe), polyethylene glycol (PEG); valine-citrulline (VC); valine-alanine (VA); para-amino-benzyloxycarbonyl (autoimmune portion) (PAB); dimethylethylamine (DMEA); ethylene diamine (EDA); monomethyl auristatin E (MMAE); N2'-Desacetyl-N2 '- (3-mercaptol-oxopropyl) methylansin (DM1); and pyrrolobenzodiazepine (PBD), [1,2] Diazepino [3,4- e] indole. For ADC-Q and ADC-R, site-specific conjugation of the payload can be done using microbial transglutaminase.
[00134] [00134] The chemical structures of the immunoconjugates in Table 2 are shown in the table below:
[00135] [00135] In some modalities, the immunoconjugates described here
[00136] [00136] Exemplary synthesis methods for the ADCs shown in Table 3 are illustrated in Example 1 below. In ADC-A, C, H to P, S and T, the antibody is covalently linked to the linker / payload fraction via cysteine residues. In ADC-B, E and F, the antibody is covalently linked to the linker / payload portion (or payload for B) via lysine residues. In ADC-Q and R, the antibody is covalently linked to the linker / payload via glutamine residues.
[00138] [00138] The ROR1 immunoconjugates of the invention, such as those made with an antibody that binds an ROR1 epitope shown in SEQ ID NO: 1 or 2 (for example, Ab1, Ab2, Ab3 or Ab4), are effective in the treatment of cancers such as solid tumors that are heterogeneous in the expression of ROR1. Tumors with less than 20% of their cells that express ROR1 can be effectively treated by ROR1 immunoconjugates; for example, tumors can have 20% or more, 30% or more, 40% or more, 50% or more, 60% or more or 70% or more of their cells that express ROR1. Without wishing to be bound by theory, it is considered that the ROR1 immunoconjugates of the invention can cause cell death in ROR1 negative tumor cells through the observed toxicity effect (ie, the payload released from a tumor cell death causes cytotoxicity in a neighboring tumor cell) or improving the immune system's anti-tumor immunity, or both.
[00139] [00139] "Treating", "treating" and "treatment" refer to a method to alleviate or revoke a biological disorder and / or at least one of its associated symptoms. When used herein, "alleviating" a disease, disorder or condition means reducing the severity and / or frequency of occurrence of the symptoms of the disease, disorder or condition. In addition, references here to "treatment" include references to curative, palliative and prophylactic treatment. Cancer treatment includes inhibiting cancer growth (including causing partial or complete cancer regression), inhibiting cancer progression or metastasis, preventing cancer recurrence or residual disease and / or prolonging patient survival .
[00140] [00140] In some modalities, the cancer treatable by the immunoconjugates described here is a cancer that expresses ROR1. The cancer that expresses ROR1 can be determined by any suitable method to determine the expression of genes or proteins, for example, by histology, flow cytometry, RT-PCR or RNA-Seq. The cancer cells used for the determination can be obtained by tumor biopsy or by collecting circulating tumor cells. In certain embodiments, if an antibody-based assay, such as flow cytometry or immunohistochemistry, is used, cancers that express ROR1 are any cancers with cells that show reactivity to the anti-ROR1 antibody greater than the of an isotype control antibody. In certain embodiments, if an RNA-based assay is used, cancers that express ROR1 are those that show a high level of ROR1 RNA compared to a negative control cell or cancer that does not express ROR1.
[00141] [00141] In certain embodiments, antibodies and immunoconjugates are for use in the treatment of hematological malignancies. In a certain embodiment, antibodies and immunoconjugates are for use in the treatment of solid tumors. The cancer to be treated can be selected from, for example, lymphoma, small lymphocytic lymphoma, marginal zone lymphoma, marginal cell B cell lymphoma, Burkitt lymphoma, lining cell lymphoma, diffuse lymphoma large B cells, a non-Hodgkin lymphoma that underwent Richter Transformation, chronic lymphocytic leukemia, T cell leukemia, osteosarcoma, renal cell carcinoma, hepatocellular carcinoma, cannula
[00142] [00142] In certain embodiments, the methods for treating cancer described herein comprise treatment with an immunoconjugate of the invention and treatment with an additional therapeutic agent or biologically active molecule. Examples of biologically active molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, prodrugs, carbohydrates, imaging agents, lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells, antibiotics, fungicides, antiviral agents, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones, growth factors, steroid agents, toxins derived from bacteria and the like. Other examples of biologically active molecules are those listed above, under the heading "Portions of cytotoxic drugs".
[00143] [00143] In certain modalities, the immunoconjugate and the additional therapeutic agent or biologically active molecule are administered at the same time, for example, in the same formulation. In certain embodiments, they are administered separately, in the same or different dosage schedules. In some embodiments, the additional therapeutic agent is an inhibitor of vascular endothelial growth factor (VEGF), an inhibitor of Bruton tyrosine kinase (BTK), an inhibitor of the mammalian target of rapamycin (mTOR), an inhibitor of phosphoinositide 3- kinase (PI3K), a Janus kinase signaling inhibitor / signal transducers and transcription activators (Jak / STAT), an inhibitor of B cell lymphoma 2 (Bcl-2), a spleen tyrosine kinase inhibitor ( SYK), a microtubule inhibitor, an epithelial growth factor (EGFR) receptor, a poly ADP ribose polymerase (PARP) inhibitor, an anaplastic lymphoma kinase (ALK) inhibitor, a repair repair inhibitor DNA, a DNA crosslinker, a DNA crosslinker, a nucleoside analog or an immunomodulatory agent.
[00144] [00144] In some embodiments, the additional therapeutic agent is a) an antibody such as rituximab (anti-CD20) or bevacizumab (anti-VEGF); b) a Bruton tyrosine kinase inhibitor, such as acalabrinibib or ibrutinib; c) an mTOR inhibitor, such as sapanisertib, everolimus or BEZ235; d) a PI3K inhibitor such as idelalisib or buparlisib; e) a Jak / STAT signaling inhibitor such as ruxolitinib; f) a Bcl-2 inhibitor such as ABT-199 / venetoclax, Bcl-2i-1 or Bcl-2i-2; g) a SYK inhibitor such as fostamatinib; h) a microtubule inhibitor such as paclitaxel or vincristine; i) an EGFR inhibitor like erlotinib; j) a PARP inhibitor like olaparib; k) an ALK inhibitor such as crizotinib; l) a DNA repair inhibitor such as carboplatin; m) a DNA crosslinker such as oxaliplatin / cisplatin; n) a nucleoside analog such as gemcitabine; or o) an immunomodulatory drug (IMiD), such as lenalidomide or pomalidomide.
[00145] [00145] In a specific modality, the additional therapeutic agent is venetoclax.
[00146] [00146] In certain embodiments, an immunoconjugate of the invention and an additional therapeutic agent or biologically active molecule are used in combination to treat CLL, MCL or a non-Hodgkin's lymphoma that has undergone Richter's transformation. In particular embodiments, the additional therapeutic agent or biologically active molecule is, for example, ibrutinib, acalabrutinib, venetoclax, Bcl-2i-1, Bcl-2i-2, everolimus, sapanisertib or idelalisib.
[00147] [00147] Additional examples of the additional therapeutic agent are pacritinib, buparlisib, BEZ235, ruxolitinib, fostamatinib, rituximab, lenalidomide, pomalidomide, paclitaxel, vincristine, erlotinib, crizotin-be, carboplatin, oxaliplatin, cisplatin, cisplatin, cisplatin, cisplatin, cisplatin, cisplatin, cisplatin and cisplatin.
[00148] [00148] In certain embodiments, an immunoconjugate of the invention is used in combination with an immune checkpoint modulator that enhances the patient's immune system. For example, the conjugate is used with an immune checkpoint inhibitor, such as an antibody or antibody derivative, an antisense oligonucleotide, a small interfering RNA, an aptamer or a peptide that targets the programmed death ligand 1 (PD-L1, also known as B7-H1, CD274), programmed death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3, TIM3, 2B4, A2aR, B7H1, B 7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (T cell stimulator inducible)), KIR, LAIR1, LIGHT, MARCO (macrophage receptor with collagen structure), PS (phosphatidylserine), OX-40, SLAM, TIGHT, VISTA, VTCN1 or any combination thereof.
[00149] [00149] It is understood that the immunoconjugates of the invention can be used in a treatment method as described herein, can be used in a treatment as described herein and / or can be used in the manufacture of a medicament for a treatment as described herein. The invention also provides kits and articles of manufacture comprising the immunoconjugates of the invention, as described herein.
[00150] [00150] In some embodiments, the immunoconjugate of the invention can be seized in a pharmaceutical composition further comprising one or more pharmaceutically acceptable excipients, vehicles and diluents. For example, the antibodies and immunoconjugates of the invention can be administered suspended in a sterile solution (for example, a solution comprising 0.9% NaCl). In certain embodiments, the solution further comprises one or more of the following: buffers (for example, acetate, citrate, histidine, succinate, phosphate, bicarbonate and hydroxymethylaminomethane (Tris) buffers); surfactants (for example, polysorbate 80 (Tween 80), polysorbate 20 (Tween 20) and poloxamer 188); polyols / disaccharides / polysaccharides (for example, glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose and dextran 40); amino acids (for example, glycine and arginine); antioxidants (eg, ascorbic acid and methionine); and / or chelating agents (for example, EDTA and EGTA). Any combination of these excipients is also considered. In certain embodiments, the immunoconjugates of the invention are transported / stored lyophilized and reconstituted prior to administration. In certain embodiments, lyophilized or immunoconjugate antibody formulations comprise a bulking agent, such as mannitol, sorbitol, sucrose, trehalose and / or dextran
[00151] [00151] The immunoconjugates of the invention, as used herein, encompass pharmaceutically acceptable salts or esters of the conjugates. Pharmaceutically acceptable salts can be formed when an acidic proton in the polypeptides is replaced by a metal ion, for example an alkali metal ion, an alkaline earth ion or an aluminum ion; or coordinates with an organic base. In addition, the salt forms of the immunoconjugates can be prepared using salts of the starting materials or intermediates. The immunoconjugates described herein can be prepared as pharmaceutically acceptable acid addition salts (which are a type of pharmaceutically acceptable salt) by reacting the free base form of the polypeptides described herein with a pharmaceutically acceptable inorganic or organic acid. Alternatively, the immunoconjugates described herein can be prepared as pharmaceutically acceptable base addition salts (which are a pharmaceutically acceptable salt type) by reacting the free acid form of amino acids in polypeptides described herein with a pharmaceutically acceptable inorganic or organic base .
[00152] [00152] Pharmaceutically acceptable salts include, but are not limited to: (1) acid addition salts, formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or formed with organic acids, such as acetic acid, propionic acid, hexanoic acid, cyclopentanopropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid , benzoic acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 2-naphthalenes-
[00153] [00153] In some embodiments, a pharmaceutical composition of the invention includes multiparticulate formulations. In some modalities, the pharmaceutical composition includes formulations of nanoparticles. In some embodiments, the nanoparticles comprise cMAP, cyclodextrin and / or lipids. In some cases, nanoparticles comprise solid lipid nanoparticles, polymeric nanoparticles, self-emulsifying nanoparticles, liposomes, microemulsions and / or micellar solutions. Additional exemplary nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as covalently bonded metal chelates), nanofibers, nanohorns, nano-
[00154] [00154] Any method for administering immunoconjugates accepted in the art can be used. The pharmaceutical compositions of the invention are typically suitable for parenteral administration. When used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical violation of an individual's tissue and administration of the pharmaceutical composition through rupture in the tissue, generally resulting in direct administration in the blood stream , in the muscle or in an internal organ. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a non-surgical wound that penetrates tissues and the like . In particular, parenteral administration is considered to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intratumoral and intrasynovial; renal dialysis techniques. Regional perfusion is also considered. Particular modalities include intravenous and subcutaneous routes.
[00155] [00155] The present invention also provides articles of manufacture,
[00156] [00156] Certain embodiments of the invention are further illustrated below.
[00157] [00157] Unless the context requires otherwise, throughout the specification and claims, the word "understand" and its variations, such as "understands" and "understanding", must be interpreted in an open and inclusive, that is, as "Including, but not limited to". When used herein and in the appended claims, the singular forms "one", "one" and "o (a)" include plural referents, unless the content clearly indicates otherwise. It should also be noted that the term "or" is generally used in its sense, including "and / or", unless the content clearly indicates otherwise. When used herein, the term "about" refers to a numerical range of 10%, 5% or 1%, more or less, of a stated numerical value in the context of the specific use. In addition, the titles provided here are for convenience only and do not interpret the scope or meaning of the claimed modalities.
[00158] [00158] All publications and patents mentioned here are incorporated by reference in their entirety, in order to describe and disclose, for example, the constructions and methodologies described in the publications, which can be used in connection with the inventions currently described. The publications discussed here are provided for publication only prior to the date of submission of this application. Nothing in this document should be construed as an admission that the inventors of the present invention have no right to precede such disclosure by virtue of the previous invention or for any other reason.
[00159] [00159] Unless otherwise defined, all the technical and scientific terms used here have the same meaning as is commonly understood by someone of ordinary experience in the technique to which the inventions described herein belong. Any methods, devices and materials similar or equivalent to those described herein may
[00160] [00160] The following examples illustrate representative embodiments of the present invention and are not intended to limit in any way. Example 1: Synthesis of exemplary immunoconjugates
[00161] [00161] The conjugation of Ab1 with MC-VC-PAB-MMAE (ADC-A) was performed on several scales (2 mg, 30 mg and 350 mg) with similar results. For the smallest scale, 2 mg of Ab1 (10 mg / mL in PBS, pH 6.5) was treated with 2.50 equivalents (eq) of tris (2-carboxyethyl) phosphine (TCEP, 5 mM) in buffer solution conjugation in a water bath at 37 ° C for 2 hours. The PBS buffer contained 15.75 mM Na2HPO4, 34.25 mM NaH2PO4, 2 mM EDTA and 50 mM NaCl, at pH 6.5. Subsequently, the reaction was cooled to 4 ° C. Then, 7 eq of MC-VC-PAB-MMAE in N, N-dimethylacetamide (DMA) were added and the mixture was left at 4 ° C for an additional 1 hour. The buffer was exchanged with 20 mM histidine, pH 5.5 (MMAE buffer) using a spin desalination column (40 kD, 0.5 ml). The number of MMAE drug molecules linked per antibody molecule (DAR) was determined using HIC-HPLC, SEC-HPLC, RP-HPLC and UV / Vis and is summarized in Table 4. Consistent results were obtained at all scales , with DAR ranging from 3.89 to 5.09, on average, depending on the methodology used. Table 4 Characterization of ADC-A Scale (mg) Aggregate (%) Recovery (%) D0 * (%) DAR
[00162] [00162] Other binders and payloads (ADC-C and ADC-H to ADC-P) were conjugated to Ab1 in a similar manner, and the resulting DARs are summarized in Table 5. Table 5 Average DAR for additional ADC constructions DAR Construction Methodology 6.33 UV 6.62 SEC ADC-C 3.97 HIC 4.15 RP 4.24 MS 1.98 UV ADC-H 1.93 SEC ADC-I 4.1 SEC / UV ADC-J 2.1 HIC ADC-K 1.9 PLRP ADC-L 3.9 HIC ADC-M 4.1 HIC ADC-N 4.1, 1.9 PLRP ADC-O 1.8 SEC ADC-P 1.9 PLRP
[00163] [00163] Ab1 in PBS (pH 6.5) was combined with SC-VC-PAB-MMAE (ADC-E) or SC-VC-PAB-DM1 (ADC-F) in DMA and placed on a rotating platform at 10 rpm at 22 ° C for 2-4 hours. For ADC-B, Ab1 in PBS (pH 7.0) was combined with SMCC-DM1 and placed on a rotating platform at 10 rpm at 22 ° C for 2-4 hours. The unreacted payload was removed by buffer exchange, using Amicon ultrafiltration (50 kDa, 0.5 mL). ADC-B was exchanged with 20 mM succinic acid (pH 5.0). DAR values were determined using SEC-HPLC, MS and UV / Vis and are summarized in Table 6. When the binder payload was used at ~ 11 eq, DARs approached 4.0 after 4 hours of incubation. Table 6 Characterization of ADC-B, ADC-E and ADC-F
[00164] [00164] ADC-Q and ADC-R were synthesized using site-specific conjugation technology (Dennler et al., Bioconjugate Chemistry,
[00165] [00165] The binding to tumor cells was evaluated by antibodies Ab1 and 4A5, as well as ADC constructs derived from these antibodies (ADC-A and ADC-T). First, Ab1 binding to two ROR1 positive human tumor cell lines, representing different types of cancer, was tested. The Jeko-1 cell line, of the lining cell lymphoma, is a cell line in suspension, while the cell line MDA-MB-231, of triple breast cancer
[00166] [00166] Jeko-1 cells (FIG.2A) or MDA-MB-231 cells (FIG.2B) were incubated with 0, 1, 10, 100 or 1000 ng / mL of Ab1 for 20 minutes on ice. After washing the unbound antibody, the cells were incubated with secondary anti-human IgG antibody labeled with phycerythrin (PE) for an additional 20 minutes. The amount of PE fluorescence was measured using the BDFACS Verse analyzer and FlowJo V10 software. The PE signal for the highest concentration was used as the maximum binding signal to calculate the maximum binding%. EC50 was calculated using GraphPad Prism 7. The receptor binding assay resulted in EC50 values of 13.6 ng / ml in Jeko-1 cells and 32.8 ng / ml in MDA-MB-231 cells.
[00167] [00167] Next, Ab1 binding was compared to ADC-A (FIGS. 2A and 2B). The binding was compared in the cell lines Jeko-1 and MDA-MB-231. The binding of ADC-A to Jeko-1 cells (FIG. 2A, squares) and MDA-MB-231 cells (FIG. 2B, squares) was very similar to the binding of the unconjugated parental antibody Ab1 (FIG. 2A, 17 ng / mL vs. 13.6 ng / mL and FIG. 2B, 48.5 ng / mL vs. 32.8 ng / mL). The similarity between the EC50 values of unconjugated Ab1 and ADC-A demonstrates that the conjugation of the drug had minimal impact on Ab1 binding to target cells.
[00168] [00168] Ab1 binding was compared to 4A5, an antibody that binds an epitope on ROR1 distinct from the Ab1 epitope (FIG. 3A). Ab1 (EC50 = 16.39 ng / mL; FIG. 3A, open circles) bound Jeko-1 cells with a higher affinity / avidity than 4A5 (EC50 = 120.6 ng / mL; FIG. 3A, open squares ). The binding of the ADC constructs was compared with the corresponding unconjugated parental antibodies (Ab1 versus ADC-A and 4A5 against ADC-T). ADC-A bound with an EC50 of 21.57 ng / mL, while ADC-T bound with an EC50 of 192.7 ng / mL. The similarity between the EC50 values of unconjugated antibodies and the corresponding ADC constructs again demonstrates that the conjugation of the drug had minimal impact on the binding of antibodies to the target cells.
[00169] [00169] In a different experiment, the binding of Ab1 to Jeko-1 cells was also compared to the binding of D10, a less affinity antibody that binds to the same epitope as Ab1 (FIG. 3B). Ab1 (EC50 = 16.5 ng / mL; FIG. 3B, open circles) linked Jeko-1 cells with a higher affinity / avidity than D10 (EC50 = 250 ng / mL; FIG. 3B, open triangles). In addition, the binding of the ADC constructs was compared to that of the corresponding unconjugated parental antibodies (Ab1 versus ADC-A and D10 versus ADC-S). ADC-A bound with an EC50 of 22.1 ng / mL (FIG. 3B, closed circles) while ADC-S bound with an EC50 of 150 ng / mL (FIG. 3B, closed triangles). The similarity between the EC50 values of unconjugated antibodies and the corresponding constructs of ADC demonstrates again that the conjugation of the drug had a minimal impact on the binding of antibodies to the target cells.
[00170] [00170] Similarly, the binding of all other ADC constructs to ROR1 positive cells was evaluated. All of the constructs described herein have been found to bind to ROR1 positive cells. Example 3: Internalization of antibodies and ROR1 immunoconjugates
[00171] [00171] Several methods were used to determine the internality
[00172] [00172] The cells and the antibody were incubated on ice for 20 minutes, spun at 300 x g for 4 minutes, washed twice with 200 µL of FACS buffer and resuspended in 100 µL of FACS buffer. The samples were incubated at 37 ° C for 15, 30, 60, 120 and 240 minutes, as indicated above. Internalization was ended by transferring the samples to ice. A period of time for the internalization of the antibody was generated for each test article as described above, after which the remaining antibody on the cell surface was detected using a secondary PE-labeled antibody. The cells were centrifuged at 250 x g, washed twice with FACS buffer and resuspended in 100 µL of FACS buffer. The secondary antibody (anti-goat human IgG-PE, specific for Fc-gamma - eBiosciences, Cat. No. 12-4998) was diluted 1: 2000 (strain 10X) in FACS buffer and 10 µL / tube was added to the appropriate tubes . The cells were incubated on ice for 20 minutes, washed twice with FACS buffer and resuspended in 100 µL of correction buffer (4% paraformaldehyde in PBS). Subsequently, the FACS analysis was performed. The median fluorescence intensity (MFI) was quantified and the degree of internalization of the receptor was determined using the amount of antibody or ADC present in each moment, when compared with the amount present in time = 0. The dashed line re - shows background staining (secondary antibody only).
[00173] [00173] Ab1 bound and was rapidly internalized by Jeko-1 (FIG. 4A) and MDA-MB-231 (FIG. 4B) cells. The internalization of 80-90% of the bound antibody was observed in less than 60 minutes for MDA-MB-231 or 120 minutes for Jeko-1. Notably, the addition of ligand and payload to Ab1 did not negatively affect binding or internalization, as demonstrated with ADC-A and ADC-B (FIG. 4A, compare ADC-A and ADC-B with Ab1, and FIG. 4B , compare ADC-A with Ab1).
[00174] [00174] Similarly, the internalization characteristics of other ADC constructs with different ligands and payloads were evaluated using Jeko-1 cells. All constructs exhibited similar internalization kinetics compared to Ab1, consistent with the observation above that the ligands and payloads used did not affect the characteristics of the antibody (data not shown).
[00175] [00175] The internalization properties were also evaluated
[00176] [00176] Ab1 internalization has also been characterized using conventional immunofluorescence staining methods and MDA-MB-231 cells. In this approach, Ab1 was loaded onto the cell surface and the cells were subsequently fixed and the Ab1 surface was quantified. In addition, a second sample of cells was allowed and Ab1 (surface and intracellular) quantified. In this protocol, the primary antibody was allowed to incubate without a secondary antibody, eliminating the possibility that the secondary antibody could influence internalization. Staining was observed on the surface. After permeabilization, a clear and distinctive intracellular staining of Ab1 was observed. In addition, using a specific lysosome tracker, it was demonstrated that the internalized Ab1 was co-located with the lysosomal pathway. This finding is consistent with a method of internalizing antibodies that occurs mainly via the lysosome / endosome pathway. Finally, the hospitalization rate was quantified. When cells were subjected to continuous exposure to Ab1, the initial rate was approximately twice the average rate and the terminal rate was approximately half the average rate (FIG. 5). This indicates that there is an initial rapid phase of cleaning the cell surface receptor, followed by a slower process of binding and internalizing the receptor that is recently expressed on the cell surface. The newly expressed cell surface receptor can be recycled, expressed from intracellular stocks.
[00177] [00177] Antibody internalization was measured using pulse search approaches that detected non-internalized antibody on the cell surface at various times (2, 5, 10, 15, 20, 30, 60, 120 and 240 minutes). In this study, in addition to measuring the antibody not internalized from the cell surface, the expression of ROR1 on the cell surface was quantified by (1) new staining with Ab1 and a secondary antibody or (2) using a second anti-ROR1 antibody marked that recognizes a distinct epitope of Ab1.
[00178] [00178] For the first approach, the internalization of Ab1 in Jeko-1 cells was performed as described in Example 3 (a sample was processed at each time to quantify the remaining antibody on the cell surface). In addition, a second sample at each time was quickly stained again using saturation levels of Ab1 (30 µg / mL), keeping the cells on ice. Subsequently, the cells were washed and the surface antibody was quantified using the same secondary antibody used for hospitalization measurements. As shown earlier, Ab1 was quickly internalized (FIG. 6, squares). On the other hand, while ROR1 quantification of the cell surface initially showed a small decrease in the first 10 minutes, subsequent measurements indicated that the expression of the ROR1 surface was restored to initial or slightly higher levels (FIG. 6 , circles). These data are consistent with the recycling of ROR1 to the cell surface dissociated from the antibody, or with the rapid positive regulation of ROR1 through de novo synthesis or the traffic of intracellular reserves. This experiment was repeated using Jeko-1 cells (FIG. 7A), MDA-
[00179] [00179] An alternative approach to measure the ROR1 expression of the cell surface was tested in conjunction with Ab1 internalization studies. Specifically, the expression of ROR1 on the cell surface was quantified at 0, 0.5, 1, 2 and 4 hours by staining cells directly with a labeled anti-ROR1 antibody that binds to an epitope distinct from that of Ab1. The surface expression of ROR1 in Jeko-1 cells treated with ADC-A, M, N and P was evaluated. Consistent with the results obtained using the approach described above, the expression of ROR1 was maintained or slightly increased, while the cells were being treated with ADC constructs based on Ab1 (data not shown).
[00180] [00180] These data indicate that Ab1 and its immunoconjugates can be internalized effectively by ROR1 positive cells. In addition, the persistent expression of ROR1 on the cell surface demonstrates that ROR1 is an excellent target for the delivery of cytotoxic agents to cancer cells through an ADC of the present invention. Example 5: Potency of in vitro immunoconjugates
[00181] [00181] The ADC-A, B, C, E and F immunoconjugates, which had different cytotoxic and chemical ligand portions, were analyzed for this Example. The binding of ROR1 by these conjugates was tested in cell culture to define the potency of various ligand-cytotoxic agent combinations. Three different types of cancer cell lines were tested: TMD-8, HBL-1 and DOHH2 lymphoma cell lines; triple-negative breast cancer cell lines HCC1187, MDA-MB-468, Bt549; and ovarian cancer cell lines A2008, TOV112D, JHOM1 and SKOvr3.
[00182] [00182] The cells were grown in logarithmic growth and distributed in 96 well plates. Each cell line
[00183] [00183] This example provided additional data on the potency of the various ADCs in inducing cell death in a variety of cancer cell lines. The cells were cultured and distributed in 96-well plates, as described in Example 5. Duplicate cells were incubated with a three-fold serial dilution of a specific immunoconjugate (660, 220, 73.3, 24, 4, 8.14, 2.71, 0.91 and 0.3 nM) for 72 hours at 37 ° C and 5% CO2. In some experiments, cells were incubated with an immunoconjugate for 96 hours instead of 72 hours (values highlighted with "*"). After treatment, cell viability was determined as described in Example
[00184] [00184] The MEC1 cell line, which is derived from chronic lymphocytic leukemia B in prolinocytoid transformation, was transfected with an expression vector encoding human ROR1 or a control vector, and stable cell lines were created using selection media containing G418. MEC1 cells transfected with ROR1 showed significantly higher proportions of cells in the S / G2 / M phase than the control transfected cells 16 hours after being transferred from the serum-free medium to the complete growth medium, implying that the expression of ROR1 increased the relative proportions of cells undergoing cell division. Consistent with this, ROR1 + MEC1 cells had significantly more cells ≥ 48 hours after being transferred from the serum-free medium than comparatively seeded cultures of MEC1 cells that did not express ROR1. Elevated levels of p-AKT and p-CREB were also observed in MEC1 cells produced to express ROR1 in relation to MEC1 cells transfected with a control vector.
[00185] [00185] The data in the tables demonstrate that the tested ADCs are effective against a variety of cancer cell lines with different levels of ROR1 expression and different sensitivities to various payloads. Example 7: Antigenic Dependence on Antiproliferative Effects of Exemplary ADCs
[00186] [00186] The studies described in this example evaluated the dependence of ADCs tested on ROR1 for their antiproliferative effects. Jeko-1 cells were cultured in the growth of the log phase before the installation of the experiment. For competition experiments, 5x104 cells were incubated with 100 µg / ml of Ab1 or vehicle control for 2 hours at 37 ° C at 5% CO2. Subsequently, 3, 10 and 30 µg / ml ADC-A were added and the cells were incubated for an additional 72 hours at 37 ° C and 5% CO2. Each condition was tested in duplicate. Cell viability was determined as described in Example 5.
[00187] [00187] The data show that ADC-A inhibited cell proliferation in a dose-dependent manner (FIG. 9, black bars). Pre-incubation of cells with the unconjugated parental antibody Ab1 inhibited this antiproliferative activity in all tested ADC-A concentrations (FIG. 9, gray bars), demonstrating that cell death was mediated by ADC-A binding to your destination, ROR1. Example 8: Exemplary Immunoconjugate Antitumor Activities in vivo
[00188] [00188] ROR1 has been shown to interact with the T cell leukemia oncogene (TCL1) and improve leukemogenesis in transgenic mice Em-TCL1, and treatment with an anti-ROR1 antibody can impair the graft of cells from leukemia ROR1 x TCL1 (Widhopf et al., PNAS111: 793-798 (2014)). This study evaluated the activity of ADC-A in comparison with the vehicle and the non-conjugated Ab1 in the mouse model with TCL1 x ROR1 CLL. In the study, the ROR1 x TCL1 leukemia cells were grafted into mice by injection into the tail vein, and the injected mice were randomized into five groups, 8 mice / group. The groups were vehicle control, 10 mg / kg of Ab1 and 1, 2 and 5 mg / kg of ADC-A. The mice received IV dosing weekly for a total of four doses. The results of this study are shown in Table 10 below.
[00189] [00189] Jeko-1 cells (MCL) were grafted subcutaneously into mice, and the grafted mice were randomized into five groups, 9 mice / group. The groups were vehicle control, 10 mg / kg of Ab1, 20 mg / kg of ibrutinib, 5 mg / kg of ADC-A and 5 mg / kg of ADC-Q. The rats were dosed IV q4d. The results of this study are shown in Table 10 below.
[00190] [00190] The cells of the diffuse large B-cell lymphoma of the germ cell type B cell (DLBCL-GCB) were subcutaneously grafted into mice, and the grafted mice were randomized into five groups, 6 mice / group. The groups were vehicle control, 10 mg / kg Ab1, 50 mg / kg venetoclax, 10 mg / kg Ab1 plus 50 mg / kg venetoclax and 5 mg / kg ADC-A. Ab1 and ADC-A were administered IV, qw and venetoclax was administered PO qd. The results of this study are shown in Table 10 below.
[00191] [00191] NSG mice were injected subcutaneously in both flank areas with RS9373 cells (patient-derived xenograft or PDX) in a matrix suspension of cells. When palpable tumors were observed (at approximately 50 mm3), the animals were randomized into three groups, 4 mice / group. The treatment groups were vehicle control, 2.5 mg / kg ADC-A IV q4d and 5 mg / kg ADC-A IV q4d. The mice received 3 total treatments and the tumors were harvested 24 hours after the last treatment. Mean tumor growth inhibition (TGI) was calculated using the following formula: TGI  [1   Treated (Final)  Treated (Day1) ]  100% ( Control (Final)  Control ( Day1) Figure Caption: - Treaty (Final) - Treaty (1st Day) - Control (Final) - Control (1st Day)
[00192] [00192] In most types of tumors, the heterogeneous expression of ROR1 is observed at the intratumor level. However, tumor regression was observed in groups treated with ADC-A. In the RS101 PDX model of Richter lymphoma, 20 to 30% of the cells were positive for ROR1, but complete and prolonged regressions were observed (FIG. 13). The results of this study are shown in Table 10 below.
[00193] [00193] NCR mice were injected subcutaneously into the breast fat pad with MDA-MB-231 cells (ROR1 positive triple negative human breast cancer (TNBC) cells) in a matrix suspension of cells. When palpable tumors were observed (approximately 250 mm3), the animals were randomized into three different groups, 9 mice / group: vehicle control, 1 mg / kg ADC-A IV qw and 5 mg / kg ADC- The IV qw. The mice received 5 total treatments and the tumors were harvested 24 h after the last treatment. The mean inhibition of tumor growth (TGI) was calculated using the formula above. The results of this study are shown in Table 10 below.
[00194] [00194] Two PDX models showing different levels of ROR1 expression were selected for this study. The expression of ROR1 was based on the level of ROR1 staining via IHC of a tissue microarray representing triplicate nuclei of TNBC PDX models. The average level of ROR1 expression, assessed by% of ROR1 positive cells, was 58% and 38% for human TNBC cells BR5011 and BR5015, respectively.
[00195] [00195] NOD / SCID nude mice were injected subcutaneously with BR5011 or BR5015 cells in the breast fat pad. When palpable tumors were observed (mean tumor volume of approximately 150 mm3 for BR5011 and approximately 250 mm3 for BR5015), the animals were randomized into three groups. The groups for the BR5011 PDX model were vehicle control, 1 mg / kg ADC-A IV q4d and 5 mg / kg ADC-A IV q4d. The groups for the BR5015 PDX model were vehicle control, 1 mg / kg ADC-A IV qw and 5 mg / kg ADC-A IV qw. The average TGI was calculated as described above.
[00196] [00196] Tumor inhibition and / or regression was observed in the groups treated with ADC-A. For example, in the BR5011 TNBC PDX model, which showed ROR1 expression in 58% of cancer cells, complete and prolonged regressions were observed (FIG. 15). The results of this study are shown in Table 10 below.
[00197] [00197] Jeko-1 cells (MCL) were grafted subcutaneously into mice. When the tumor size reached 100 mm3, the mice were randomized into nine groups, 9 mice / group. The groups were vehicle control; 1 mg / kg of ADC-N, ADC-P or ADC-R; or 5 mg / kg of ADC-A, ADC-L, ADC-M, ADC-S or ADC-T. The mice were dosed IV q4d. The intermediate results of this study are shown in Table 10 below. Table 10 Results of In vivo Studies with Exemplary Immunoconjugates ADC Study Animal Model Duration Results FIG. 1 ADC-A leukemia cells 1 month ADC-A inhibited the tumor load of 10 TCL1 x ROR1 leukemic cells in a dose-dependent manner (1, 2 and 5 mg / kg). 2 ADC-A 3-week xenograft ADC-A caused tumor regression, while ADC-Q 11 ADC-Q Jeko-1 MCL caused delayed tumor growth. 3 ADC-A DLBCL-GCB PDX 24 days ADC-A caused tumor regression (104%) with 12 complete regressions in all animals.
[00198] [00198] In vivo studies 1-7 here show that, despite the heterogeneity of ROR1 expression in a tumor and varying levels of ROR1 expression between types of cancer, treatment with ADC-
[00199] [00199] Current in vivo studies also show that ADC-A was effective in the treatment of drug-resistant cancers. Regressions are observed in xenografts of human tumors that were resistant to ibrutinib (model Jeko-1 MCL) or immunochemotherapy with rituximab-CHOP (Richter transformation model). Since ROR1 is a marker for advanced cancers and previous chemotherapy appears to increase ROR1 expression, our results demonstrate the potential of the immunoconjugates of the present invention in the treatment of advanced or aggressive cancers.
[00200] [00200] The in vivo efficacy of ADCs, for example, ADC-A, E, F, L, M, N, P, Q and R, can be further evaluated in a human, subcutaneous and MCL xenograft model positive for ROR1, using cells
[00201] [00201] The effects of combining ADC-A with other anti-proliferative agents were examined. Initial studies focused on the effects of ADC-A combined with a BTK inhibitor (ibrutinib, ACP-196 / acalabrutinib), Bcl-2 (ABT-199 / venetoclax), mTOR (INK128) or PI3K (idelalisib) . Additional studies have examined the effects of combining ADC-A with two additional Bcl-2 inhibitors, called Bcl-2i-1 and Bcl-2i-2 strains. The cells were tested for both distinct subtypes of DLBCL - germinal center B cell (GCB) and activated B cell (ABC). The analysis of the median effect was used to determine synergism, antagonism or additivity of inhibition of the proliferation of various cell lines treated with ADC-A combined with antiproliferative agents. The Combination Index (CI) was determined by the Chou / Talalay equation. The IC50 for each compound was determined in a 72-hour CellTiter-Glo® assay. For combination assays, drugs were used in equimolar proportions (that is, in proportion to their IC50 values). The CalcuSyn software (from Biosoft) was used to analyze the dose effect. A combination index less than 1.1 indicates that treatments are synergistic; 0.9 - 1.1 indicates that the treatments are additive; and greater than 1.1 indicates that the treatments are antagonistic.
[00202] [00202] ADC-A exhibited a synergistic effect with two different BTK inhibitors, ibrutinib (FIG. 18A) and ACP-196 / acalabrutinib (FIG. 18B). The synergistic effect was more pronounced with acalabrutinib and the MCL cell lines. Representative data for ADC-A combined with ibrutinib and acalabrutinib in the MCL Jeko-1 cell line are shown in FIGS.19A and 19B, respectively.
[00203] [00203] ADC-A also exhibited a synergistic effect with the Bcl-2 inhibitor ABT-199 / venetoclax (FIG. 20A). The synergistic effect was pronounced in the MCL and DLBCL cell lines. ADC-A activity combined with additional Bcl-2 inhibitors, Bcl2i-1 and Bcl2i-2, was examined in the Jeko-1 (FIG. 20B) and Mino (FIG. 20C) cells (both are of the MCL type). ADC-A exhibited a synergistic effect with both inhibitors on Jeko-1 cells and an additive effect with both inhibitors on Mino cells. Representative data for ADC-A combined with Jeko-1 venetoclax are shown in FIG. 21.
[00204] [00204] ADC-A exhibited a synergistic effect with the inhibitor of mTOR1 / 2 INK128 / sapanisertib (FIG. 22). The synergistic effect was observed in the MCL and DLBCL cell lines. Representative data for ADC-A combined with sapanisertib in Jeko-1 is shown in FIG. 23.
[00205] [00205] ADC-A exhibited a synergistic effect with the PI3K inhibitor CAL-101 / idelalisib (FIG. 24). The synergistic effect was observed in the MCL and DLBCL cell lines. Representative data for ADC-A combined with idelalisib demonstrating synergistic effects in both DLBCL subtypes are shown in FIG. 25A (TMD, DLBCL-ABC cells) and FIG. 25B (DOHH2, DLBCL-GCB cells). Example 10: Additional Assessment of Combination Therapy
[00206] [00206] The in vivo efficacy of ADCs in combination with a Bcl-2 inhibitor can be evaluated in a ROR1 positive human MCL xenograft model, using Jeko-1 cells or in a PDX model. Both ADC and Bcl-2 inhibitor are dosed with maximum tolerated and subideal doses, alone and in combination, as described below, in a repeated dose study. Tumor growth and body weight are measured every 2-3 days.
[00207] [00207] Pharmacokinetic analysis is performed to determine standard pharmacokinetic parameters, such as Cmax and antibody half-life. Example11: Phase 2 Clinical Study of Anti-ROR1-MMAE Immunoconjugates
[00208] [00208] The following is a protocol for a prospective open label Phase 1b / 2 clinical trial for therapy with anti-ROR1-MMAE immunoconjugate. Inclusion criteria: • Eastern Cooperative Oncology Group (ECOG) performance status of 0, 1 or 2 • Histological diagnosis of LLC / SLL or MCL as documented in medical records • CLL / SLL or MCL was treated previously and relapsed after or progressed during previous treatment • Presence of radiographically measurable lymphadenopathy or extranodal lymphoid malignancy (defined as the presence of ≥1 non-irradiated and non-biopsied lesion that measures ≥2.0 cm in the longest dimension [LD] and ≥1.0 cm in the largest perpendicular dimension [LPD] assessed by computed tomography [CT] or magnetic resonance imaging [MRI]). • Current medical need for therapy due to symptoms related to the disease, lymphadenopathy, organomegaly, extranodal organ involvement or progressive disease. • Completion of all previous therapy (including surgery, radiotherapy, chemotherapy, immunotherapy or experimental therapy) for the treatment of cancer 1 week before the start of study therapy. • All acute toxic effects of any previous anti-tumor therapy were resolved to Grade 1 prior to commencing treatment under study (with the exception of alopecia [Grade 1 or 2 allowed], or neurotoxicity [Grade 1 or 2 allowed] or selected laboratory parameters [Grade 1 or grade 2 allowed, with exceptions, as indicated below]). • Adequate bone marrow function: ○ a) Absolute neutrophil count (ANC) ≥ 1.0 × 109 / L (Grade ≤ 2). b) Platelet count ≥ 50 × 109 / L (Grade ≤ 2). b) Hemoglobin ≥ 8.0 g / dL (Grade ≤2) maintained for ≥ 1 week after any previous transfusion. • Note: Neutropenia, thrombocytopenia or anemia of grade ≥ 3 is allowed if the abnormality is related to bone marrow involvement with hematological malignancy (as documented by the bone marrow biopsy / aspirate obtained since the last previous therapy). • Adequate liver profile: ○ Alanine serum aminotransferase (ALT) ≤ 3 × upper limit of normal (ULN) (Grade ≤ 1). ○ Aspartate serum aminotransferase (AST) ≤ 3 × ULN (Grade ≤ 1).
[00209] [00209] The dose determination phase will be carried out by comparing parallel doses at 1.0, 2.0 and 3.0 mg / kg.
[00210] [00210] Although preferred embodiments of the present invention have been shown and described here, it will be obvious to those skilled in the art that such modalities are provided by way of example only. Numerous variations, changes and substitutions will now occur for those skilled in the art without departing from the invention. It is to be understood that various alternatives to the embodiments of the invention described herein can be used in the practice of the invention.
[00211] [00211] The amino acid sequences and nucleotide sequences described here are listed in Table 12 below. Table 12 Description Sequence Listing SEQUENCE SIN 1 ROR1 human fragment VATNGKEVVS STGVLFVKFG ROR1 human PC 2 fragment heavy chain EVVSSTGVLF VKFGPC 3 Ab1 QVQLQESGPGLVKPSQTLSLTCTVSGYAFTAYNIHWVR- QAPGQGLEWMGSFDPYDGGSSYNQKFKDRLTISKDTSKN- QVVLTMTNMDPVDTATYYCARGWYYFDYWGHGTLVTVSSASTKG- PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL- TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT- KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS- RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS- TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA- KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN- GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN-
4 VFSCSVMHEALHNHYTQKSLSLSPGK light chain Ab1 DIVMTQTPLSLPVTPGEPASISCRASKSISKYLAWYQQKPGQAPR- LLIYSGSTLQSGIPPRFSGSGYGTDFTLTINNIESE- DAAYYFCQQHDESPYTFGEGTKVEIKRTVAAPSVFIFPPS- DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE- QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN-
RGEC 5 VH of Ab1 QVQLQESGPGLVKPSQTLSLTCTVSGYAFTAYNIHWVR- QAPGQGLEWMGSFDPYDGGSSYNQKFKDRLTISKDTSKN-
QVVLTMTNMDPVDTATYYCARGWYYFDYWGHGTLVTVSS 6 VL by Ab1 DIVMTQTPLSLPVTPGEPASISCRASKSISKYLAWYQQKPGQAPR- LLIYSGSTLQSGIPPRFSGSGYGTDFTLTINNIES
DAAYYFCQQHDESPYTFGEGTKVEIK 7 HCDR1 by Ab1 GYAFTAYN 8 HCDR2 by Ab1 FDPYDGGS 9 HCDR3 by Ab1 GWYYFDY 10 LCDR1 by Ab1 KSISKY 11 LCDR2 by Ab1 SGS 12 LCDR3 by Ab1 QQHDESPY 13 frag. Ab1 VH SGYAFTAYNIHWVRQ 14 frag. Ab1 VH cell GSFDPYDGGSSYNQKF 15 frag. Ab1 VH YYCARGWYYFDYWGHGTLVTVSS 16 frag. of VL by Ab1 CRASKSISKYLAWY 17 frag. of VL of Ab1 LLIYSGSTLQSG 18 frag. VL Ab1 CQQHDESPYTFGEGTKVEIK 19 AAGCTTACCGCCACCATGGGCTGGAGCTGTATCATCCTCTT- chain coding sequence heavy Ab1 CCTGGTGGCGACCGCGACGGGTGTCCACTCCCAGGTGCAG- CTCCAGGAGTCCGGCCCCGGGCTTGTGAAGCCGTCACAAAC- CCTGTCCCTGACGTGCACGGTCTCCGGCTACGCCTTCACGGCC- TACAACATACATTGGGTCCGGCAGGCGCCGGGCCAGGGGCTG- GAGTGGATGGGTTCCTTCGACCCGTACGATGGCGGGAGCTCG- TACAACCAGAAGTTCAAAGACCGCCTGACGATCTCCAAGGACAC- CTCGAAAAACCAGGTCGTCCTGACCATGACCAACATGGAC- CCGGTGGACACGGCGACCTACTATTGCGCCCGCGGCTGGTAC- TACTTCGACTACTGGGGCCACGGGACCCTGGTCACCGTGTCTT-
SIN Description SEQUENCE CCGCTTCGACCAAGGGCCCCAGCGTCTTCCCGCTCGCG- CCCTCCTCGAAGTCCACCTCGGGCGGCACTGCCGCCCTGGG- CTGCCTGGTCAAGGACTACTTCCCGGAGCCGGTGACCGTCTCG- TGGAACAGCGGGGCACTCACCTCCGGCGTGCACACCTT- CCCGGCCGTGCTGCAGTCCTCGGGGCTGTATTCACTCAG- CTCGGTCGTCACCGTCCCCTCGTCGTCCCTCGGCACGCAGACG- TACATCTGCAACGTCAACCACAAGCCCTCGAACACCAAGGTG- GACAAGAAGGTCGAGCCGAAGTCCTGCGATAAGACCCACAC- CTGCCCCCCGTGCCCGGCCCCCGAGCTCCTGGGCGGTCCG- TCCGTGTTCCTCTTCCCGCCCAAGCCCAAGGACACCCTGATGAT- CAGCCGCACGCCCGAGGTGACCTGCGTCGTCGTGGACG- TCTCCCACGAGGATCCCGAGGTGAAGTTCAACTGGTACGTGGA- CGGGGTGGAGGTCCACAACGCCAAGACAAAGCCGCGGGA- AGAGCAGTACAACTCGACCTACCGCGTCGTCAGCGTGCTGA- CGGTCCTCCACCAGGACTGGCTGAACGGCAAGGAGTACAAATG- CAAGGTGTCCAACAAGGCCCTGCCCGCGCCCATCGAGAAGAC- CATCTCCAAGGCCAAGGGACAGCCGCGCGAGCCGCAGGTCTA- CACGCTGCCTCCCTCCCGGGACGAGCTCACGAAGAACCAGGTA- TCGCTCACCTGCCTCGTGAAGGGCTTCTACCCGAGCGACATCG- CCGTCGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTA- CAAAACCACGCCGCCGGTCCTCGACTCTGACGGGTCCTTCTT- CCTGTACTCCAAGCTGACCGTGGACAAGTCGCGGTGGCAGCA- GGGGAACGTGTTCTCGTGCTCGGTC ATGCACGAGGCGTTGCA- CAACCACTACACCCAGAAGTCACTCTCCCTGAGCCCGGGCAAG-
TGATAATCTAGAGTCGGGGCGGCCGGCC 20 AAGCTTACCGCCACCATGGGCTGGTCATGCATCATCCTGTT- coding sequence light chain Ab1 CCTGGTCGCCACCGCGACGGGGGTCCACAGTGATATCGT- CATGACGCAGACGCCGCTGAGCCTCCCGGTGACGCCCGG- CGAGCCCGCCAGCATCTCCTGCCGCGCTTCCAAG- TCCATCTCGAAGTACCTGGCGTGGTATCAGCAGAAGCCCGG- CCAGGCCCCGCGCCTGCTCATCTACTCTGGTTCCACGCTCCAG- TCGGGCATCCCGCCCCGGTTCTCGGGTTCGGGATACGGCAC- CGACTTCACCCTGACCATCAACAACATCGAGAGCGAAGACG- CGGCGTACTACTTCTGCCAGCAGCACGACGAGTCCCCGTACAC- CTTCGGCGAGGGGACCAAGGTCGAGATCAAGCGTACCGTCG- CGGCACCGAGCGTCTTCATCTTCCCCCCGTCCGACGAGCAGCT- CAAGTCTGGCACCGCCTCGGTCGTCTGTCTCCTGAACAACTTC- TACCCCAGGGAAGCCAAGGTCCAGTGGAAGGTGGACAACGCG- CTGCAGTCCGGGAATAGCCAGGAGTCGGTGACGGAGCAGGAC- TCCAAGGACTCCACGTACTCGCTCTCGTCCACCCTGAC- CCTCTCCAAGGCGGACTACGAAAAGCACAAGGTCTACGCCTG- CGAGGTGACGCACCAAGGCCTGTCCTCCCCAGTGACCAAGTCG- TTCAACCGCGGCGAGTGCTGATAATCTAGAGTCGGGGCGG-
21 CCGGCC coding sequence VH 1 AAGCTTACCGCCACCATGGGCTGGAGCTGTATCATCCTCTT- Ab1 CCTGGTGGCGACCGCGACGGGTGTCCACTCCCAGGTGCAG- CTCCAGGAGTCCGGCCCCGGGCTTGTGAAGCCGTCACAAAC- CCTGTCCCTGACGTGCACGGTCTCCGGCTACGCCTTCACGGCC- TACAACATACATTGGGTCCGGCAGGCGCCGGGCCAGGGGCTG- GAGTGGATGGGTTCCTTCGACCCGTACGATGGCGGGAGCTCG- TACAACCAGAAGTTCAAAGACCGCCTGACGATCTCCAAGGACAC- CTCGAAAAACCAGGTCGTCCTGACCATGACCAACATGGAC- CCGGTGGACACGGCGACCTACTATTGCGCCCGCGGCTGGTAC- TACTTCGACTACTGGGGCCACGGGACCCTGGTCACCGTG-
22 TCTTCC coding sequence of VL 1 GATATCGTCATGACGCAGACGCCGCTGAGCCTCCCGGTGACG- Ab1 CCCGGCGAGCCCGCCAGCATCTCCTGCCGCGCTTCCAAG- TCCATCTCGAAGTACCTGGCGTGGTATCAGCAGAAGCCCGG- CCAGGCCCCGCGCCTGCTCATCTACTCTGGTTCCACGCTCCAG- TCGGGCATCCCGCCCCGGTTCTCGGGTTCGGGATACGGCAC- CGACTTCACCCTGACCATCAACAACATCGAGAGCGAAGACG- CGGCGTACTACTTCTGCCAGCAGCACGACGAGTCCCCGTACAC-
23 CTTCGGCGAGGGGACCAAGGTCGAGATCAAG sequence encoding VH 2 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAG- Ab1 CCTTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGTTATG- CATTCACTGCCTACAACATACACTGGGTGCGACAGGCCCCTG- GACAAGGGCTTGAGTGGATGGGTTCTTTTGATCCTTACGATGG- TGGTAGTAGTTACAACCAGAAGTTCAAGGACAGACTCAC-
SIN Description SEQUENCE CATCTCCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGA- CCAACATGGACCCTGTGGACACAGCCACGTATTACTGTG- CAAGAGGGTGGTACTACTTTGACTACTGGGGCCACGGAAC-
24 CCTGGTCACCGTCTCCTCA coding sequence of VL 2 GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCAC- Ab1 CCCTGGAGAGCCGGCCTCCATCTCCTGCAGGGCAAGTAAGAG- CATTAGCAAATATTTAGCCTGGTACCAGCAGAAACCTGGCCAGG- CTCCCAGGCTCCTCATCTATTCTGGATCCACTTTGCAATCTGG- GATCCCACCTCGATTCAGTGGCAGCGGGTATGGAACAGATTT- TACCCTCACAATTAATAACATAGAATCTGAGGATGCTGCATAT- TACTTCTGTCAACAGCATGATGAATCCCCGTACACGTTCGG-
CGAGGGGACCAAGGTGGAAATCAAA 25 VH of D10 QVQLKESGPGLVAPSQTLSITCTVSGFSLTSYGVHWVR- QPPGKGLEWLGVIWAGGFTNYNSALKSRLSISKDNSKSQVLLKMTS-
LQTDDTAMYYCARRGSSYSMDYWGQGTSVIVSS 26 VL of D10 EIVLSQSPAITAASLGQKVTITCSASSNVSYIHWYQQRSG- TSPRPWIYEISKLASGVPVRFSGSGSGTSYSLTISSMEAEDAAY
YCQQWNYPLITFGSGTKLEIQ 27 HCDR1 HCDR2 28 GFSLTSYG D10 D10 D10 WAGGFT HCDR3 RGSSYSMDY 29 30 31 SNVSYI LCDR1 LCDR2 D10 D10 D10 EIS 32 LCDR3 QQWNYPLI 33 CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCG- coding sequence D10 VH CCCTCACAGACTCTGTCCATCACTTGCACTGTCTCTGGGTTTT- CATTAACCAGTTATGGTGTACACTGGGTTCGCCAGCCTCCAGGA- AAGGGTCTGGAGTGGCTGGGAGTAATATGGGCTGGTGGATTCA- CAAATTATAATTCGGCTCTCAAGTCCAGACTGAGCATCAGCAAA- GACAACTCCAAGAGCCAAGTTCTCTTAAAAATGACCAGTCTG - CAAACTGATGACACAGCCATGTACTACTGTGCCAGGAGAGGTAG- TTCCTATTCTATGGACTATTGGGGTCAAGGAACCTCAGTCACCG-
TCTCCTCA 34 GAAATTGTGCTCTCTCAGTCTCCAGCCATCACAGCTG- coding sequence D10 VL CATCTCTGGGCCAAAAGGTCACCATCACCTGCAGTGCCAGTT- CAAATGTAAGTTACATCCACTGGTACCAGCAGAGGTCAGGCAC- CTCCCCCAGACCATGGATTTATGAAATATCCAAACTGGCTTCTG- GAGTCCCAGTTCGCTTCAGTGGCAGTGGGTCTGGGACCTCT- TACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTG- CCATTTATTATTGTCAGCAGTGGAATTATCCTCTTATCACGTT-
CGGCTCGGGGACAAAGTTGGAAATACAA 35 VH of 4A5 EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQIPEKR- LEWVASISRGGTTYYPDSVKGRFTISRDNVRNILYLQMSSLRSE-
DTAMYYCGRYDYDGYYAMDYWGQGTSVTVSS 36 VL of 4A5 DIKMTQSPSSMYASLGERVTITCKASPDINSYLSWFQQK- PGKSPKTLIYRANRLVDGVPSRFSGGGSGQDYSLTINSLEYEDM
GIYYCLQYDEFPYTFGGGTKLEMK 37 HCDR1 4A5 GFTFSSYA 38 HCDR2 4A5 ISRGGTT 39 HCDR3 4A5 YDYDGYYAMDY 40 LCDR1 4A5 PDINSY 41 LCDR2 4A5 RAN
SIN SEQUENCE DESCRIPTION LQYDEFPYT 42 LCDR3 of 4A5 VH 43 GAAGTGAAACTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTG- coding sequence of 4A5 GAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTT- CAGTAGCTATGCCATGTCTTGGGTTCGCCAGATTCCA- GAGAAGAGGCTGGAGTGGGTCGCATCCATTAGTCGTGGTGG- TACCACCTACTATCCAGACAGTGTGAAGGGCCGATTCAC- CATCTCCAGAGATAATGTCAGGAACATCCTGTACCTGCAAA- TGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTG- GAAGATATGATTACGACGGGTACTATGCAATGGACTACTGGGGT-
CAAGGAACCTCAGTCACCGTCTCCTCA 44 GACATCAAGATGACCCAGTCTCCATCTTCCATGTATGCATCTC- VL coding sequence of 4A5 TAGGAGAGAGAGTCACTATCACTTGCAAGGCGAGTCCGGACAT- TAATAGCTATTTAAGCTGGTTCCAGCAGAAACCAGGGAAA- TCTCCTAAGACCCTGATCTATCGTGCAAACAGATTGG- TTGATGGGGTCCCATCAAGGTTCAGTGGCGGTGGATCTGGG- CAAGATTATTCTCTCACCATCAACAGCCTGGAGTATGAAGATA- TGGGAATTTATTATTGTCTACAGTATGATGAATTTCCGTACACG-
TTCGGAGGGGGGACCAAGCTGGAAATGAAAC 45 VH of 99961 EIQLQQSGPVLVKPGASVKVSCKASGYAFTAYNIHWVRQSHGKR- LEWIGSFDPYDGGSSYNQKFKDKATLTVDKSSTTAYMHLNSLTS
DSAVYYCARGWYYFDYWGHGTTLTVSS 46 VL of 99961 DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTN- KLLIYSGSTLQSGIPSRFRGSGSGTDFTLTISS-
47 LEPEDFAMYYCQQHDESPYTFGEGTKLEIKR heavy chain Ab4 QVQLQESGPGLVKPSQTLSLTCTVSGYAFTAYNIHWIR- QPPGKGLEWIGSFDPYDGGSSYNQKFKDRLTISKDTSKNQVVLTM- TNMDPVDTATYYCARGWYYFDYWGHGTLVTVSSASTKG- PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL- TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT- KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS- RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS- TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA- KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN- GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN-
VFSCSVMHEALHNHYTQKSLSLSPGK 48 VH de Ab4 QVQLQESGPGLVKPSQTLSLTCTVSGYAFTAYNIHWIR- QPPGKGLEWIGSFDPYDGGSSYNQKFKDRLTISKDTSKNQVVLTM-
49 TNMDPVDTATYYCARGWYYFDYWGHGTLVTVSS light chain of Ab4 DVVMTQSPLSLPVTLGQPASISCRASKSISKYLAWYQQK- PGKAPKLLIYSGSTLQSGIPPRFSGSGYGTDFTLTINNIESE- DAAYYFCQQHDESPYTFGEGTKVEIKRTVAAPSVFIFPPS- DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE- QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN-
RGEC 50 VL of Ab4 DVVMTQSPLSLPVTLGQPASISCRASKSISKYLAWYQQK- PGKAPKLLIYSGSTLQSGIPPRFSGSGYGTDFTLTINNIESE-
DAAYYFCQQHDESPYTFGEGTKVEIK 51 CAGGTCCAGCTGCAGGAGTCAGGTCCCGGACTGGTCAAGCCG- chain coding sequence heavy Ab4 TCGCAGACGCTGTCCCTCACCTGCACCGTGTCGGGCTACG- CCTTCACCGCCTACAACATCCACTGGATCCGTCAGCCCCCTGG- GAAGGGCCTCGAGTGGATCGGCAGCTTCGACCCGTACGACGG- CGGGTCGTCCTACAACCAGAAGTTCAAGGACCGCCTCACCAT- CAGCAAGGACACCTCCAAGAACCAGGTCGTCCTCACCATGAC- CAACATGGACCCCGTGGACACCGCCACGTACTACTGCGCG- CGGGGCTGGTACTACTTCGACTACTGGGGGCACGGCACCCTCG- TCACGGTCTCGTCGGCGAGCACCAAGGGTCCGAGCGTCTT- CCCCCTGGCCCCCTCCAGCAAGTCCACCTCGGGGGGCACCG- CCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCG- TGACCGTGAGCTGGAACTCCGGCGCCCTCACCAGCGGGG- TCCACACCTTCCCGGCGGTCCTGCAGTCATCCGGTCTCTAC- TCCTTGAGCTCAGTCGTCACCGTCCCGAGCTCCTCCCTCGGA- ACGCAGACCTACATCTGCAACGTCAACCACAAGCCGTCCAACA- CCAAGGTCGACAAGAAGGTGGAGCCCAAATCGTGCGACAAGAC-
SIN Description SEQUENCE CCACACCTGCCCGCCGTGCCCCGCCCCGGAACTGCTCGGCGG- CCCCTCGGTGTTCCTGTTCCCCCCGAAGCCCAAGGACACCCT- CATGATCTCCCGCACCCCCGAGGTCACCTGCGTGGTGGTGGA- TGTCTCCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTG- GACGGGGTCGAGGTGCACAACGCCAAGACCAAGCCCCGAGAG- GAACAGTATAACTCGACGTACCGCGTGGTCAGCGTCCTGACCG- TGCTCCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTG- CAAGGTCAGCAACAAGGCCCTGCCCGCCCCCATCGAGAAGA- CGATCTCCAAGGCGAAGGGGCAGCCGCGCGAGCCGCAGGTC- TACACCCTGCCGCCCAGCCGGGACGAGCTCACGAAGAATCAGG- TCTCGCTCACCTGCCTCGTCAAGGGTTTCTACCCGTCGGACA- TCGCGGTCGAATGGGAGTCGAACGGTCAGCCCGAGAATAACTA- CAAGACGACCCCGCCCGTCCTGGACTCGGACGGCAGCTTCTT- CCTGTACTCGAAGCTGACGGTCGACAAGTCGCGCTGGCAGCA- GGGCAACGTCTTCTCGTGCTCGGTGATGCACGAGGCCCTCCA-
CAACCACTACACACAGAAGAGCCTCTCGCTTTCGCCGGGCAAG 52 CAGGTCCAGCTGCAGGAGTCAGGTCCCGGACTGGTCAAGCCG- VH coding sequence Ab4 TCGCAGACGCTGTCCCTCACCTGCACCGTGTCGGGCTACG- CCTTCACCGCCTACAACATCCACTGGATCCGTCAGCCCCCTGG- GAAGGGCCTCGAGTGGATCGGCAGCTTCGACCCGTACGACGG- CGGGTCGTCCTACAACCAGAAGTTCAAGGACCGCCTCACCAT- CAGCAAGGACACCTCCAAGAACCAGGTCGTCCTCACCATGAC- CAACATGGACCCCGTGGACACCGCCACGTACTACTGCGCG- CGGGGCTGGTACTACTTCGACTACTGGGGGCACGGCACCCTCG-
TCACGGTCTCGTCG 53 GACGTCGTGATGACCCAGTCGCCCCTCTCCCTGCCGGTTAC- light chain coding sequence Ab4 CCTGGGCCAGCCCGCCTCCATCAGCTGCCGTGCCTCCAAG- TCCATTTCCAAGTACCTGGCCTGGTACCAGCAGAAGCCGGGGA- AGGCCCCAAAGCTCCTCATCTACTCCGGCTCCACCCTCCAGAG- CGGCATCCCCCCCCGCTTCAGCGGCTCCGGCTACGGCACCGA- CTTCACCCTCACCATCAATAACATCGAGTCGGAGGACGCCGCG- TACTACTTCTGCCAGCAGCACGACGAATCGCCGTACACCTT- CGGGGAGGGCACCAAGGTGGAGATCAAGAGGACGGTCGCCG- CGCCCTCCGTGTTCATCTTCCCCCCCTCGGACGAACAG- CTGAAGTCCGGGACCGCCTCCGTCGTCTGCCTCCTCAACAAC- TTCTACCCGCGCGAGGCCAAGGTGCAGTGGAAGGTCGACAACG- CGCTCCAGTCCGGCAACTCCCAGGAGTCGGTGACCGAGCAG- GACTCGAAGGACAGTACCTACTCGCTGAGCTCCACACTGACG- CTCTCGAAGGCCGACTACGAGAAGCACAAGGTGTACGCATG- CGAGGTGACCCACCAGGGGCTGAGCTCGCCGGTGACTAAG-
TCGTTCAACAGGGGCGAATGC 54 GACGTCGTGATGACCCAGTCGCCCCTCTCCCTGCCGGTTAC- VL coding sequence Ab4 CCTGGGCCAGCCCGCCTCCATCAGCTGCCGTGCCTCCAAG- TCCATTTCCAAGTACCTGGCCTGGTACCAGCAGAAGCCGGGGA- AGGCCCCAAAGCTCCTCATCTACTCCGGCTCCACCCTCCAGAG- CGGCATCCCCCCCCGCTTCAGCGGCTCCGGCTACGGCACCGA- CTTCACCCTCACCATCAATAACATCGAGTCGGAGGACGCCGCG- TACTACTTCTGCCAGCAGCACGACGAATCGCCGTACACCTT-
CGGGGAGGGCACCAAGGTGGAGATCAAG 55 GGFG Binding Peptide Sequence 56 ALAL Binding Peptide Sequence 57 GFLG Binding Peptide Sequence 58 LLQGA Glutamine Marker 59 LPxTG Sortase Reason 60 NPQTN Sorting Reason 61 GDDYL
SIN Description SEQUENCE 62 Human CH1 domain plus ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS- upper articulation region GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK-
PSNTKVDKKVEPKSC 63 cover constant domain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD- human NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC
64 EVTHQGLSSPVTKSFNRGEC humanized 4A5 scFv DIQMTQSPSSLSASVGDRVTITCKASPDINSYLSWFQQRPGQSPRR- LIYRANRLVDGVPDRFSGSGSGTDFTLKISRVEAE- DVGVYYCLQYDEFPYTFGQGTKVEIKGGGGSGSTSGSGK- PGSGEGSTKGGGGGSEVQLVQSGAEVKKPGESLRISCKGSGFT- FSSYAMSWIRQSPSRGLEWLGSISRGGTTYYPDSVKGRFTISRD- NAKNSLYLQMNSLRAEDTAVYYCGRYDYDGYYAMDYWGQG-
TLVTVSS 65 scFv Ab1 DIVMTQTPLSLPVTPGEPASISCRASKSISKYLAWYQQKPGQAPR- LLIYSGSTLQSGIPPRFSGSGYGTDFTLTINNIESE- DAAYYFCQQHDESPYTFGEGTKVEIKGGGGSGSTSGSGK- PGSGEGSTKGGGGGSQVQLQESGPGLVKPSQTLSLTCTVSGYAF- TAYNIHWVRQAPGQGLEWMGSFDPYDGGSSYNQKFKDRLTISK-
DTSKNQVVLTMTNMDPVDTATYYCARGWYYFDYWGHGTLVTVSS 66 scFv Ab2 DVVMTQSPLSLPVTLGQPASISCRASKSISKYLAWYQQK- PGKAPKLLIYSGSTLQSGIPPRFSGSGYGTDFTLTINNIESE- DAAYYFCQQHDESPYTFGEGTKVEIKGGGGSGSTSGSGK- PGSGEGSTKGGGGGSQVQLQESGPGLVKPSQTLSLTCTVSGYAF- TAYNIHWVRQAPGQGLEWMGSFDPYDGGSSYNQKFKDRLTISK-
DTSKNQVVLTMTNMDPVDTATYYCARGWYYFDYWGHGTLVTVSS 67 scFv of Ab3 DIVMTQTPLSLPVTPGEPASISCRASKSISKYLAWYQQKPGQAPR- LLIYSGSTLQSGIPPRFSGSGYGTDFTLTINNIESE- DAAYYFCQQHDESPYTFGEGTKVEIKGGGGSGSTSGSGK- PGSGEGSTKGGGGGSQVQLQESGPGLVKPSQTLSLTCTVSGYAF- TAYNIHWIRQPPGKGLEWIGSFDPYDGGSSYNQKFKDRLTISK-
DTSKNQVVLTMTNMDPVDTATYYCARGWYYFDYWGHGTLVTVSS 68 scFv Ab4 DVVMTQSPLSLPVTLGQPASISCRASKSISKYLAWYQQK- PGKAPKLLIYSGSTLQSGIPPRFSGSGYGTDFTLTINNIESE- DAAYYFCQQHDESPYTFGEGTKVEIKGGGGSGSTSGSGK- PGSGEGSTKGGGGGSQVQLQESGPGLVKPSQTLSLTCTVSGYAF- TAYNIHWIRQPPGKGLEWIGSFDPYDGGSSYNQKFKDRLTISK- DTSKNQVVLTMTNMDPVDTATYYCARGWYYFDYWGHGTLVTVSS * SIN: SEQ ID NO.

权利要求:
Claims (41)
[1]
1. Immunoconjugate having the formula of Ab - ((L) m– (D)) n, characterized by the fact that: Ab is an antibody or antigen-binding fragment of the same that specifically binds to the orphan receptor type human receptor tyrosine kinase 1 (ROR1); L is a linker and m is 0 or 1; D is a portion of cytotoxic drug; and n is an integer from 1 to 10.
[2]
2. Immunoconjugate according to claim 1, characterized by the fact that the cytotoxic portion of the drug is selected from the group consisting of an antitubulin agent, a DNA-binding agent, a DNA cross-linking agent , a DNA interleaving agent and an RNA polymerase II inhibitor.
[3]
3. Immunoconjugate, according to claim 2, characterized by the fact that the cytotoxic drug portion is selected from the group consisting of monomethyl auristatin E (MMAE), azo-nafide, α-amanitin, duocarmycin TM, pyrrolobenzodiazepine (PBD), PNU-159682 and pharmaceutically acceptable salts, esters and their analogs.
[4]
4. Immunoconjugate, according to any of the preceding claims, characterized by the fact that the ligand comprises a cleavable portion.
[5]
5. Immunoconjugate, according to any of the preceding claims, characterized by the fact that the ligand is branched.
[6]
6. Immunoconjugate, according to any of the preceding claims, characterized by the fact that the binder comprises one or more valine-citrulline (VC), valine-alanine (VA), para-aminobenzyloxycarbonyl (PAB), polyethylene glycol ( PEG), di-
aminopropionic (DPR), Phe-C4, C2-Gly3, C6 alkyl, dimethylethylamine (DMEA) and ethylene diamine (EDA).
[7]
7. Immunoconjugate, according to any of the preceding claims, characterized by the fact that the ligand is covalently linked to the antibody or antigen-binding fragment in a succinimide, carbonyl, cyclooctene or triazole group of the ligand.
[8]
8. Immunoconjugate, according to any of the preceding claims, characterized by the fact that the antibody or fragment is covalently linked to the ligand by reaction with a portion selected from the group consisting of: 6-maleimidocaproyl (MC) - VC-PAB; 6-MC-C6; 6-MC-PEG4-VC-PAB-DMEA; 6-MC-PEG4-VA; 6-MC-DPR-VC-PAB; 6-MC-Phe-C4-VC-PAB; 6-MC-Phe-C4-VC-PAB-DMEA; 6-MC-C2-Gly3-EDA; dibenzylcyclooctin (DBCO) - (PEG2-VC-PAB) 2; DBCO-PEG4-VC-PAB-DMEA; and N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate-VC-PAB.
[9]
9. Immunoconjugate having the formula of Ab - ((L) m– (D)) n, characterized by the fact that: Ab is an antibody or antigen-binding fragment of the same that specifically binds to the orphan receptor type human receptor tyrosine kinase 1 (ROR1); L is a cleavable linker and m is 0 or 1; D is an auristatin; and n is an integer from 1 to 10.
[10]
10. Immunoconjugate, according to claim 9, characterized by the fact that auristatin is MMAE.
[11]
11. Immunoconjugate, according to any of the preceding claims, characterized by the fact that the linker comprises: a heterocycle or carbonyl covalently linked to the antibody or antigen-binding fragment, a spacer group covalently linked to the heterocycle or carbonyl , and an ester, thioester, amide, carbonate, thiocarbonate or carbamate covalently linked to the cytotoxic drug moiety.
[12]
12. Immunonconjugate according to claim 11, characterized in that the spacer group comprises an amino acid, a polyamino acid or a benzyl amino group or a combination thereof.
[13]
13. Immunoconjugate, according to any of the preceding claims, characterized by the fact that the ligand forms a covalent bond with a cysteine or lysine residue in the antibody or fragment.
[14]
14. Immunoconjugate according to any one of claims 1 to 13, characterized by the fact that the antibody or fragment binds to the same ROR1 epitope as an antibody that comprises the amino acid sequences of the heavy chain and the light chain of SEQ ID NOs: 3 and 4, respectively.
[15]
15. Immunoconjugate, according to any of claims 1 to 13, characterized by the fact that the antibody comprises the region that determines the weight chain complementarity (CDR) 1-3 (HCDR1-3) in SEQ ID NO: 3 and the light chain CDR1-3 (LCDR1-3) in SEQ ID NO: 4.
[16]
16. Immunoconjugate according to any one of claims 1 to 13, characterized by the fact that the antibody heavy chain comprises the amino acid sequences of SEQ ID NOs: 7-9, and the antibody light chain comprises the sequences amine acid of SEQ ID NOs: 10-12.
[17]
17. Immunoconjugate, according to any of the preceding claims, characterized by the fact that the antibody or antigen-binding fragment is humanized.
[18]
18. Immunoconjugate, according to claim 17, characterized by the fact that the antibody or antigen-binding fragment has one or more of the following properties: a) it facilitates the internalization of ROR1 in a human cell; b) binds to human ROR1 with a KD of less than 100 nM; and c) inhibits the growth of human ROR1 + cancer cells in vitro with an EC50 of 300 nM or less.
[19]
19. Immunoconjugate according to claim 16, characterized by the fact that the heavy chain variable domain (VH) and the light chain variable domain (VL) of the antibody comprise the amino acid sequences of: a) SEQ ID NOs: 5 and 6, respectively; b) SEQ ID NOs: 5 and 50, respectively; c) SEQ ID NOs: 48 and 6, respectively; or d) SEQ ID NOs: 48 and 50, respectively.
[20]
20. Immunoconjugate, according to any of the preceding claims, characterized by the fact that the antibody comprises a human IgG1 constant region.
[21]
21. Immunoconjugate according to claim 16, characterized by the fact that the heavy chain and the light chain of the anti-
body comprise the amino acid sequences of: a) SEQ ID NOs: 3 and 4, respectively; b) SEQ ID NOs: 3 and 49, respectively; c) SEQ ID NOs: 47 and 4, respectively; or d) SEQ ID NOs: 47 and 49, respectively.
[22]
22. Immunoconjugate, according to any of the preceding claims, characterized by the fact that Ab is a Fab, F (ab) 2 or scFv.
[23]
23. Immunoconjugate, according to any of the preceding claims, characterized by the fact that the number of the drug portion per antibody or fragment (DAR) varies from 1 to 7.
[24]
24. Immunoconjugate, characterized by the fact that it comprises an antibody conjugated to a portion of cytotoxic drug, in which the VH and VL of the antibody comprise the amino acid sequences of SEQ ID NOs: 5 and 6, respectively, and the immunoconjugate has a structure shown in Table 3 as ADC-A, E, H, I, J, K, L, M, N, O, P, Q or R.
[25]
25. Immunoconjugate according to claim 24, characterized by the fact that the heavy chain and the light chain of the antibody comprise the amino acid sequences of SEQ ID NOs: 3 and 4, respectively.
[26]
26. Immunoconjugate, according to claim 25, characterized by the fact that the ratio of the portion of cytotoxic drug to the antibody is from 1 to 7.
[27]
27. Pharmaceutical composition, characterized in that it comprises the immunoconjugate as defined in any one of claims 1 to 26 and a pharmaceutically acceptable excipient.
[28]
28. Pharmaceutical composition according to claim 27, characterized by the fact that it further comprises an additional therapeutic agent selected from the group consisting of an inhibitor
Bruton tyrosine kinase pain (BTK), a B cell lymphoma inhibitor 2 (Bcl-2), a mammalian rapamycin target (mTOR), and an inhibitor of phosphoinositide 3 kinase (PI3K).
[29]
29. Pharmaceutical composition according to claim 28, characterized by the fact that the additional therapeutic agent is selected from the group consisting of ibrutinib, acalabrutine, venetoclax, everolimus, sapanisertib and idelalisib.
[30]
30. Method of treating cancer in a patient in need of it, characterized by the fact that it comprises administering to the patient a therapeutically effective amount of the immunoconjugate as defined in any of claims 1 to 26.
[31]
31. Method, according to claim 30, characterized by the fact that cancer is heterogeneous for the expression of ROR1.
[32]
32. Method, according to claim 30 or 31, characterized by the fact that the cancer is a leukemia, lymphoma or solid tumor.
[33]
33. Method according to claim 32, characterized by the fact that the cancer is chronic lymphocytic leukemia, T cell leukemia, lining cell lymphoma, diffuse large B cell lymphoma, Burkitt's lymphoma, multiple myeloma, marginal zone lymphoma, small lymphocytic lymphoma or non-Hodgkin's lymphoma that underwent Richter transformation.
[34]
34. Method, according to claim 32, characterized by the fact that cancer is non-small cell lung cancer, hepatocellular carcinoma, pancreatic cancer, osteosarcoma, head and neck cancer, cancer of the ovary, breast cancer or triple negative breast cancer.
[35]
35. Method according to any one of the claims
30 to 34, characterized by the fact that it also includes the administration of an additional anti-cancer therapeutic agent to the patient.
[36]
36. Method according to claim 35, characterized by the fact that the additional anti-cancer therapeutic agent is selected from the group consisting of a Brute tyrosine kinase (BTK) inhibitor, a lymphoma inhibitor 2 B cells (Bcl-2), a mammalian rapamycin target (mTOR) and a phosphoinositide 3 kinase inhibitor (PI3K).
[37]
37. Method, according to claim 36, characterized by the fact that the additional therapeutic agent is selected from the group consisting of ibrutinib, acalabrutinib, venetoclax, eveolimo, sapanisertib and idelalisib.
[38]
38. Method according to any one of claims 35 to 37, characterized by the fact that the cancer is chronic lymphocytic leukemia, lining cell lymphoma or a non-Hodgkin lymphoma that has undergone Richter's transformation.
[39]
39. Immunoconjugate as defined in any of claims 1 to 26, or a pharmaceutical composition as defined in any of claims 27 to 29, characterized in that they are for use in the treatment of cancer in a method of any of the claims 30 to 38.
[40]
40. Use of an immunoconjugate, as defined in any one of claims 1 to 26, characterized in that it is for the manufacture of a medicament for the treatment of cancer in a method as defined in any of claims 30 to
38.
[41]
41. Method for making an immunoconjugate, characterized by the fact that it comprises: providing an antibody or an antigen-binding fragment that specifically binds to the tyrosine receptor
human kinase as the orphan receptor 1 (ROR1); conjugating to the antibody a portion of cytotoxic drug selected from the group consisting of an anti-tubulin agent, a DNA alkylating agent, a DNA cross-linking agent, a DNA intercalating agent and an RNA polymerase II inhibitor; wherein the heavy chain of the antibody comprises the amino acid sequences of SEQ ID NOs: 7-9 and the light chain of the antibody comprises the amino acid sequences of SEQ ID NOs: 10-
12.
% of Maximum Connection
Antibody% of Maximum Binding
Antibody
% of Maximum Connection
Antibody% of Maximum Binding
Antibody
Connection% at t = 0
Call Time (minutes)% at t = 0
Time (minutes)
Time (minutes)
% Connection at t = 0 6/29
Cell Surface
Time (minutes)
Connection% at t = 0
Cell Surface
Call Time (hours)% at t = 0
Cell Surface
Control time (hours)%
Cell Surface
Time (hours)
% Inhibition% Inhibition%
Compound
% Inhibition
Compound% Inhibition
Compound
% Inhibition
Compound% Inhibition
Compound
% Inhibition
Compound% Inhibition
Compound
% Inhibition
Compound
% of Cell Proliferation Inhibition 13/29
Leukemic Cells in the Middle Spleen + SD le tro on
C Treatment
Vehicle
Ibrutinib Tumor Volume
Treatment Days
Tumor Size
Petition 870190137190, of 12/20/2019, p. 177/235 ROR1 expression
Control 16/29
PE-A subset
Tumor volume, mean + SEM
Isotype Control Counts Therapy Start Time, days
ROR1 expression
Petition 870190137190, of 12/20/2019, p. 178/235 Vehicle isotype control
Events 17/29
Tumor Volume, mean + SEM Implant Start of Tumor Treatment Interruption Treatment Euthanasia of Vehicle / Mice Ab1 Treatment Observation of ADC-Mice
Vehicle Tumor Volume
Dosing start
Treatment Days
Vehicle Tumor Volume
Dosing start
End of dosage
Treatment Days
Vehicle Tumor Volume
Treatment Days
Vehicle Tumor Volume
Treatment Days
ADC A + Ibrutinib Combination Index Combination Index
% Inhibition
ADC A + ibrutinib
Compound% Inhibition
Compound
Petition 870190137190, of 12/20/2019, p. 185/235 Combination Index Combination Index Combination Index 24/29
% Inhibition
Compound
Combination Index 26/29
% Inhibition
Compound
Combination Index 28/29
% Inhibition
Compound% Inhibition
Compound
Petition 870190137190, of 12/20/2019, p. 162/235 Pharmaceutical Binding Antibody
Cleavable linker group Cytotoxic drug Antibody ROR1 MMAE protease binding 1/29
Methyl Valine Dolaiso- Dolaproína Norephedrine Maleimida Valine leucine caproic acid
Maleimidocaproíla Valina Citrulline

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同族专利:

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公开号 | 公开日

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AU2018289581A1|2020-01-16|

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TW201919709A|2019-06-01|

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CA3067829A1|2018-12-27|

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KR20200062161A|2020-06-03|

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WO2018237335A1|2018-12-27|

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US20180369406A1|2018-12-27|

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EP3641830A1|2020-04-29|

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IL271575D0|2020-02-27|

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JP2020525542A|2020-08-27|

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US10335496B2|2019-07-02|

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CN111587124A|2020-08-25|

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US20200030454A1|2020-01-30|

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KR20210079427A|2021-06-29|

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法律状态:
2021-11-03| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201762524388P| true| 2017-06-23|2017-06-23|

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US201762524382P| true| 2017-06-23|2017-06-23|

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US201762524386P| true| 2017-06-23|2017-06-23|

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US62/524,382|2017-06-23|

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---