anti-sirp-alpha antibodies and related methods

专利摘要:
Anti-SIRPa antibodies, including multi-specific anti-SIRPa antibodies, are provided, as are related compositions and methods. The antibodies of the disclosure bind to SIRPa and can block the interaction of CD47 in a cell with SIRPa in a phagocytic cell.
公开号:BR112020001653A2
申请号:R112020001653-5
申请日:2018-07-25
公开日:2020-07-21
发明作者:Jie Liu；Jens-Peter Volkmer
申请人:Forty Seven, Inc.；
IPC主号:

专利说明:

[0001] [0001] This application claims the benefit of US Provisional Application No. 62 / 537,207, filed on July 26, 2017, which is incorporated into this document in its entirety by reference for all purposes. SEQUENCE LISTING
[0002] [0002] The current order contains a Sequence Listing that was submitted via EFS-Web and is incorporated in its entirety by this means for reference. Said ASCII copy, created in the month XX, 20XX, is called XXXXXUS_sequencelisting.txt and is Χ, ΧΧΧ, ΧΧΧ bytes in size. BACKGROUND OF THE INVENTION
[0003] [0003] Cell rotation starts with the induction of an apoptotic program or other cellular changes that mark them for removal and the subsequent recognition of markers by phagocytes, including macrophages, dendritic cells and the like. This process requires specific and selective removal of unwanted cells. Unlike healthy cells, unwanted / aged / dying cells exhibit markers or ligands called "eat me" signals, that is, "altered self", which in turn can be recognized by receptors in phagocytes. Healthy cells can exhibit "don’t eat me" signals that actively inhibit phagocytosis; these signals are deregulated in the dying cells, are present in an altered conformation or are replaced by the positive regulation of "eat me" or pro-phagocytic signals. The cell surface protein CD47 in healthy cells and their involvement with a phagocyte receptor, SIRP, constitute a key "eat me" signal that can disable involvement mediated by several modalities, including apoptotic cell clearance and FcR-mediated phagocytosis. Blocking CDIR-mediated involvement of SIRP in a phagocyte can cause the removal of living cells that contain "eat me" signals.
[0004] [0004] CD47 is a transmembrane glycoprotein widely expressed with a single Ig-like domain and five regions spanning the membrane, which functions as a cell linker for SIRP with mediated binding through the V-like domain of the NH2 terminal of SIRP. SIRP is expressed mainly in myeloid cells, including macrophages, granulocytes, myeloid dendritic cells (DCs), mast cells and their precursors, including hematopoietic stem cells. Structural determinants of SIRP that mediate binding to CD47 are discussed by Lee et al. (2007) J. Immunol. 179: 7741-7750; Hatherley et al. (2007) JBC 282: 14567-75; and the role of dimerization of SIRPização cis in binding to CD47 is discussed by Lee et al. (2010) J.B.C. 285: 37953-63. According to the role of CD47 in inhibiting phagocytosis in normal cells, there is evidence that it is transiently regulated in hematopoietic stem cells (HSCs) and parents immediately before and during the migratory phase, and that the level of CD47 in these cells determines the likelihood of being swallowed in vivo. SUMMARY
[0005] [0005] An isolated humanized, human or chimeric antibody is disclosed in this document which: specifically binds human SIRP; does not specifically bind human SIRP; and optionally comprises a human Fc region comprising at least one modification that reduces binding to a human Fc receptor.
[0006] [0006] In some respects, the antibody comprises: a CDR-H1 comprising the sequence set out in SEQ ID NO: 1; a CDR-H2 comprising the sequence set forth in SEQ ID NO: 2; a CDR-H3 comprising the sequence shown in SEQ ID NO: 3; a CDR-L1 comprising the sequence set forth in SEQ ID NO: 4; a CDR-L2 comprising the sequence set out in SEQ ID NO: 5; and a CDR-L3 comprising the sequence shown in SEQ ID NO: 6; or a CDR-H1 comprising the sequence set forth in SEQ ID NO: 9; a CDR-H2 comprising the sequence set forth in SEQ ID NO: 10; a CDR-H3 comprising the sequence set out in SEQ ID NO: 11; a CDR-L1 comprising the sequence set out in SEQ ID NO: 12; a CDR-L2 comprising the sequence set out in SEQ ID NO: 13; and a CDR-L3 comprising the sequence set out in SEQ ID NO: 14.
[0007] [0007] In some respects, the antibody comprises: a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 8; or a VH sequence of SEQ ID NO: 15 and a VL sequence of SEQ ID NO: 16.
[0008] [0008] In some respects, the antibody comprises: a heavy chain of SEQ ID NO: 17 and a light chain of SEQ ID NO: 18; or a SEQ ID NO: 19 heavy chain and a SEQ ID NO: 20 light chain.
[0009] [0009] Also in this disclosed document is an isolated humanized, human or chimeric antibody, comprising: a CDR-H1 comprising the sequence set out in SEQ ID NO: 1; a CDR-H2 comprising the sequence set forth in SEQ ID NO: 2; a CDR-H3 comprising the sequence shown in SEQ ID NO: 3; a CDR-L1 comprising the sequence set forth in SEQ ID NO: 4; a CDR-L2 comprising the sequence set out in SEQ ID NO: 5; and a CDR-L3 comprising the sequence set out in SEQ ID NO: 6.
[0010] [0010] An isolated humanized, human or chimeric antibody is also disclosed in this document, comprising: a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 8.
[0011] [0011] An isolated humanized, human or chimeric antibody is also disclosed in this document, comprising: a heavy chain of SEQ ID NO: 17 and a light chain of SEQ ID NO: 18.
[0012] [0012] An isolated humanized, human or chimeric antibody is also disclosed in this document, comprising: a CDR-H1 comprising the sequence set forth in SEQ ID NO: 9; a CDR-H2 comprising the sequence set forth in SEQ ID NO: 10; a CDR-H3 comprising the sequence set out in SEQ ID NO: 11; a CDR-L1 comprising the sequence set out in SEQ ID NO: 12; a CDR-L2 comprising the sequence set out in SEQ ID NO: 13; and a CDR-L3 comprising the sequence set out in SEQ ID NO: 14.
[0013] [0013] An isolated humanized, human or chimeric antibody is also disclosed in this document, comprising: a VH sequence of SEQ ID NO: 15 and a VL sequence of SEQ ID NO: 16.
[0014] [0014] An isolated humanized, human or chimeric antibody is also disclosed in this document, comprising: a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 20.
[0015] [0015] In some respects, an antibody disclosed in this document comprises a human Fc region comprising at least one modification that reduces binding to a human Fc receptor.
[0016] [0016] In some respects, an antibody disclosed in this document: (a) competes for binding to human SIRP with an antibody selected from 1H9 and 3C2; (b) does not compete for binding to human SIRP with the KWar antibody; (c) partially competes for binding to human SIRP with the KWar antibody; (d) inhibits the binding of human CD47 to human SIRP; (e) inhibits the binding of human SP-A to human SIRP; (f) inhibits the binding of human SP-D to human SIRP; (g) binds to the Rhesus monkey SIRP; (h) binds to cinomolgo SIRP (i) increases phagocytosis compared to control; or (j) is capable of any combination of (a) - (i).
[0017] [0017] In some respects, an antibody disclosed in this document is pan-specific for human SIRP isotypes. An antibody disclosed in this document, such as 1H9, can bind to several human SIRP isotypes, including one or more of V1, V2 and V1 / V5. An antibody disclosed in this document can bind to each of the human isotypes of SIRP V1 and V2. An antibody disclosed in this document can bind to the human SIRP V1 isotype, including homozygous. An antibody disclosed in this document can bind to the human SIRP isotype V2, including homozygous. An antibody disclosed in this document can bind to human V1 / V5 (heterozygous) SIRP isotypes. An antibody disclosed in this document, such as 1H9, can bind to several human SIRP isotypes, including each of V1, V2 and V1 / V5. Such antibodies can include 1H9 and 3C2. Binding to human SIRP variants can be measured using assays known in the art, including PCR and / or flow cytometry. For example, a given sample can be genotyped to determine SIRP status and binding to SIRP can be determined using flow cytometry.
[0018] [0018] In some respects, an antibody disclosed in this document is specific for a human SIRP isotype.
[0019] [0019] In some ways, human SIRP is expressed in a cell presenting a professional antigen. In some ways, human SIRP is expressed in a macrophage.
[0020] [0020] An antibody disclosed in this document, such as 1H9, can bind to human SIRP on the cell surface. The binding of an antibody disclosed in this document to the SIRP can be stable, for example, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more than 24 hours. An antibody disclosed in this document can prevent substantial internalization after binding to SIRP. Such antibodies can include 1H9 and 3C2, including humanized and / or Fc-modified versions of such antibodies. Binding to human SIRP can be measured using assays known in the art, including flow cytometry and / or IHC.
[0021] [0021] In some ways, the antibody is 1H9 or 3C2.
[0022] [0022] In some respects, the human Fc region is IgG1 or IgG4, optionally modified with a modification.
[0023] [0023] In some respects, the glycosylation of the antibody is reduced by enzymatic deglycosylation, expression in a bacterial host or modification of an amino acid residue used for glycosylation. In some respects, a modification disclosed in this document reduces the glycosylation of the human Fc region. In some respects, the modification of the human Fc region comprises a modification in the asparagine in the index position of the EU 297. In some respects, the modification of the human Fc region comprises a substitution of amino acids in the asparagine in the index position of the EU 297. In some aspects, the modification of the human Fc region comprises a substitution of amino acids N297A, numbered according to the EU index. In some respects, the modification comprises one or more amino acid substitutions at: N297A; L234A / L235A; C220S / C226S / C229S / P238S; C226S / C229S / E3233P / L234V / L235A; or L234F / L235E / P331S, numbering according to the EU index. In some respects, the modification comprises one or more amino acid substitutions at: N297; L234 / L235; C220 / C226 / C229 / P238; C226 / C229 / E3233 / L234 / L235; or L234 / L235 / P331, numbering according to the EU index. In some respects, the modification comprises one or more amino acid substitutions in the CH2 region at EU index positions 234, 235 and / or 237. In some respects, the modification comprises one or both amino acid substitutions L234A and L235A and, optionally , P331S and / or K322A and / or G237A, numbered according to the EU index. In some respects, the modification comprises the substitution of amino acids K322A, numbered according to the EU index. In some respects, the modification comprises E233P / L234V / L235A / G236 + A327G / A330S / P331S, numbered according to the EU index.
[0024] [0024] In some ways, the antibody is a monoclonal antibody.
[0025] [0025] In some ways, the antibody is multispecific. In some ways, the antibody binds to more than one antigen or to more than one epitope on a single antigen.
[0026] [0026] In some respects, the antibody comprises the heavy chain constant region of a class selected from IgG, IgA, IgD, IgE and IgM. In some respects, the antibody comprises an IgG class heavy chain constant region and a selected subclass of IgG1, IgG4, IgG2 and IgG3.
[0027] [0027] In some respects, the antibody binds human SIRP with a KD less than or equal to about 1, 1-6, 1-5, 1-4, 1-3, 2, 3, 4, 5, 6, 7 , 8, 9 or 10 x10-9 M as measured by the Biacore assay.
[0028] [0028] In some respects, an antibody disclosed in this document is for use as a medicine. In some respects, an antibody disclosed in this document is for use in the treatment of cancer or infection. In some respects, an antibody disclosed in this document is for use in the treatment of cancer, in which the cancer is selected from a solid tumor and a hematological tumor. In some respects, an antibody disclosed in this document is for use in increasing phagocytosis.
[0029] [0029] An isolated humanized, human or chimeric antibody that competes for binding to human SIRP with an antibody disclosed in this document is also disclosed in this document.
[0030] [0030] Also disclosed in this document is an isolated humanized, human or chimeric antibody that binds to the human SIRP epitope linked by an antibody disclosed in this document.
[0031] [0031] An isolated polynucleotide or set of polynucleotides encoding an isolated antibody disclosed in this document, a VH of this, a VL of this, a light chain of this, a light chain of this, a heavy chain of this or a portion of it, is also disclosed in this document binding to its antigen.
[0032] [0032] A vector or set of vectors comprising a polynucleotide or set of polynucleotides disclosed in this document is also disclosed in this document.
[0033] [0033] Also disclosed in this document is a host cell comprising a polynucleotide or set of polynucleotides disclosed in this document or a vector or set of vectors disclosed in this document.
[0034] [0034] Also disclosed in this document is a method of producing an antibody that comprises expressing the antibody with a host cell disclosed in this document and isolating the expressed antibody.
[0035] [0035] Also disclosed in this document is a pharmaceutical composition comprising an antibody disclosed in this document and a pharmaceutically acceptable excipient.
[0036] [0036] Also disclosed in this document is a method of treating or preventing a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of an antibody disclosed in this document or a pharmaceutical composition disclosed in this document.
[0037] [0037] In some aspects, the disease or condition is: cancer; infection; a viral infection; a bacterial infection; a fungal infection; fibrosis; arteriosclerosis; a parasitic infection, optionally malaria; and depletion or reduction of endogenous blood-forming stem cells from the bone marrow to allow radiation and / or chemotherapy - free or reduced conditioning for transplantation of blood-forming stem cells, optionally in combination with the anti-CKIT antibody (CD117).
[0038] [0038] In some ways, the disease or condition is a cancer, and the cancer is selected from a solid tumor and a hematological tumor.
[0039] [0039] Also disclosed in this document is a method for increasing phagocytosis in a subject in need thereof, comprising administering to the subject an effective amount of an antibody disclosed in this document or a pharmaceutical composition disclosed in this document.
[0040] [0040] Also disclosed in this document is a method of modulating an immune response in a subject in need thereof, comprising administering to the subject an effective amount of an antibody disclosed in this document or a pharmaceutical composition disclosed in this document.
[0041] [0041] In some respects, a method disclosed in this document further comprises the administration of one or more additional therapeutic agents to the subject.
[0042] [0042] In some ways, the additional therapeutic agent is an antibody. In some ways, the additional therapeutic agent is an antibody that binds a protein or proteins to the cell surface of a tumor. In some respects, the additional therapeutic agent is an antibody that binds: HER2 (ERBB2 / neu), CD52, PD-L1, VEGF, CD30, EGFR, CD38, RANKL (CD254), GD2 (ganglioside), SLAMF7 (CD319), CD20, EGFR, PDGFRa, VEGFR2, CD33, CD44, CD99, CD96, CD90, CD133, CKIT (CD117 for CKIT positive tumors); CTLA-4, PD-1, PD-L1, CD40 (agonistic), LAG3 (CD223), 41BB (agonistic CD137), OX40 (agonistic CD134); and / or CKIT (CD117) to deplete blood-forming stem cells for transplant therapy. In some respects, the additional therapeutic agent is at least one of: Rituximab, Cetuximab, Alemtuzumab (CD52), Atezolizumab (PD-L1), Avelumabe (PD-L1), Bevacizumab (VEGF), Brentuximabe (CD30), Daratumumabe (CD38 ), Denosumab (RANKL), Dinutuximab (GD2), Elotuzumab (SLAMF7), Ibritumomab (CD20), Ipilimumab (CTLA-4), Necitumumab (EGFR), Nivolumab (PD-1), Obinutuzumab (CD20), Ofatumuma (CD20), Ofatumuma , Olaratumab (PDGFRa), Panitumumab (EGFR), Pembrolizumab (PD-1), Pertuzumab (HER2), Ramucirumab (VEGFR2), Tositumomab (CD20), and Gemtuzumabe (CD33).
[0043] [0043] In some respects, the additional therapeutic agent is formulated in the same pharmaceutical composition as the antibody. In some respects, the additional therapeutic agent is formulated in a pharmaceutical composition other than the antibody.
[0044] [0044] In some respects, the additional therapeutic agent is administered prior to administration of the antibody. In some aspects, the additional therapeutic agent is administered after administration of the antibody. In some respects, the additional therapeutic agent is administered simultaneously with the antibody.
[0045] [0045] Also disclosed in this document is a kit comprising an antibody disclosed in this document or a pharmaceutical composition disclosed in this document; and instructions for use. BRIEF DESCRIPTION OF THE VARIOUS VIEWS OF THE FIGURES
[0046] [0046] These and other aspects, characteristics and advantages of the present invention will become more understandable with regard to the description that follows, and the attached figures, where:
[0047] [0047] Figure 1. Sequences of variable regions of heavy chain (A) and light (B) of 1H9. CDRs are underlined.
[0048] [0048] Figure 2. 3C2 heavy (A) and light (B) variable region sequences. CDRs are underlined.
[0049] [0049] Figure 3. 1H9 and 3C2 recognize distinct epitopes. (A) The SIRPa-Fc fusion protein was coated on a 96-well plate and incubated with 1H9 or 3C2 in the absence or presence of 50 or 100 times excessive amounts of mouse Kwar. (B) The SIRPa-Fc fusion protein was coated on a 96-well plate and incubated with mouse 1H9 in the absence or presence of excessive amounts of 5, 10, 50 and 100 times of 1H9 or 3C2. (C) The SIRPa-Fc fusion protein was coated in a 96-well plate and incubated with 3C2 mouse in the absence or presence of excessive amounts of 5, 10, 50 and 100 times of 3C2 or 1H9.
[0050] [0050] Figure 4. 1H9 and 3C2 synergize with rituximab to promote macrophage-mediated phagocytosis of Raji cells. Macrophages were differentiated from monocytes from donor A (A) and donor B (B) in the presence of human serum for 7 days. Raji cells were labeled with CFSE and incubated with macrophages in the presence of 10 µg / ml of rituximab alone or in combination with 10 µg / ml of 1H9-G4, 1H9-G1, 3C2-G4 or 3C2-G1. Two hours later, the percentage of phagocytosis was calculated by flow cytometric analysis, looking for GFP + macrophages.
[0051] [0051] Figure 5. Humanized 1H9 heavy (A) and light (B) variable region sequences. CDRs are underlined.
[0052] [0052] Figure 6. Humanized 3C2 heavy (A) and light (B) variable region sequences. CDRs are underlined.
[0053] [0053] Figure 7. Humanized 1H9 and 3C2 have the same antigen-binding specificity as their parental antibodies. (A) The fusion protein
[0054] [0054] Figure 8. Biacore affinity measurement of humanized 1H9 and 3C2.
[0055] [0055] Figure 9. Humanized 1H9 and 3C2 synergize with therapeutic antibodies to promote phagocytosis. (A) Raji cells were labeled with CFSE and incubated with macrophages derived from human monocytes in the presence of 10 µg / ml of rituximab3 alone or in combination with 10 µg / ml of Hu1H9-G1 or Hu3C2-G1. (B) HT29 cells were labeled with CFSE and incubated with macrophages derived from human monocytes in the presence of 0.1 µg / ml of cetuximab alone or in combination with 0.5 µg / ml, 5 µg / ml and 10 µg / ml Hu1H9-G1 or Hu3C2-G1. Two hours later, the percentage of phagocytosis was calculated by flow cytometric analysis, looking for GFP + macrophages.
[0056] [0056] Figure 10. Cross reactivity for SIRPB and SIRPG. (A) The binding of Kwar, 1H9 and 3C2 to the human SIRPB-His fusion protein was determined by ELISA. (B) The binding of Kwar, 1H9 and 3C2 to the human SIRPG-His fusion protein was determined by ELISA.
[0057] [0057] Figure 11. 9B11 and 7E11 synergize with rituximab to promote macrophage-mediated phagocytosis of Raji cells.
[0058] [0058] Figure 12. Link to epitope 7E11 and 9B11. 7E11 recognizes an overlapping epitope compared to Kwar (similar to 3C2) and 9B11 recognizes a very similar or identical epitope compared to Kwar.
[0059] [0059] Figure 13. Hu1H9-G1 binds to V1 and V2 variants of SIRPa in cells.
[0060] [0060] Figure 14. Hu1H9-G1 blocks the binding of CD47 to monocytes from different donors.
[0061] [0061] Figure 15. Hu1H9-G1 synchronizes with Cetuximab to promote phagocytosis in different donors.
[0062] [0062] Unless otherwise indicated, all terms of the technique, notations and other scientific or terminology used in this document are intended to have the meanings commonly understood by those versed in the technique. In some cases, terms with commonly understood meanings are defined in this document for the purpose of clarification and / or for quick reference, and the inclusion of such definitions in this document should not necessarily be construed to represent a difference over what is generally understood in the art. The techniques and procedures described or referenced in this document are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely used molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. As appropriate, procedures involving the use of commercially available kits and reagents are generally performed in accordance with the protocols and conditions defined by the manufacturer, unless otherwise indicated.
[0063] [0063] As used in this document, the singular forms "one", "one and" o (a) "include the referring plural forms, unless the context clearly indicates otherwise. The terms" include "," as "and the like are intended to convey inclusion without limitation, unless otherwise indicated.
[0064] [0064] As used herein, the term "comprising" also specifically includes modalities "consisting of" and "consisting essentially of" the recited elements, unless specifically indicated otherwise. For example, a multispecific antibody "comprising a diabody" includes a multispecific antibody "consisting of a diabody" and a multispecific antibody "consisting essentially of a diabody."
[0065] [0065] The term "about" indicates and covers an indicated value and an interval above and below that value. In certain embodiments, the term "about" indicates the designated value ± 10%, ± 5% or ± 1%. In certain embodiments, when applicable, the term "about" indicates the designated value (s) ± a standard deviation of that (s)
[0066] [0066] SIRPα1 (PTPNS1, SHPS1), is a transmembrane glycoprotein, expressed mainly in myeloid and neuronal cells. SIRPα interacts with the widely distributed membrane protein CD47. In addition to SIRP, there are two closely related proteins in the SIRP family: SIRP and SIRP. All three have three immunoglobulin superfamily (IgSF) domains in their extracellular region. In humans, the SIRPα protein is found in two main forms. One form, variant 1 or V1, has the amino acid sequence established as NCBI RefSeq NP_542970.1 (residues 27-504 constitute the mature form). Another form, variant 2 or V2, differs in 13 amino acids and has the amino acid sequence established in GenBank as CAA71403.1 (residues 30-504 constitute the mature form). These two forms of SIRPα constitute about 80% of the forms of SIRPα present in humans, and both are adopted in this document by the term "human SIRPα". Also adopted by the term "human SIRPα" are the minor secondary forms that are endogenous to humans and have the same property of triggering signal transduction through CD47 after binding to them. Sequences of human SIRPa variants can be accessed through public databases, including Genbank access numbers: ref | NP_542970.1; gb | EAX10606.1; ref | XP_005260726.1; gb | EAX10606.1; XP_005260726.1; gb | EAX10611.1; gb | EAX10609.1; dbj | BAA12974.1; gb | AAH26692.1; ref | XP_011527475.1. See, for example, Lee et al. (2007) J. Immunol. 179 (11): 7741-7750; specifically in this document incorporated by reference.
[0067] [0067] Antibodies that specifically bind to human SIRP are known and used in the art and can be adapted by using a manipulated Fc region as disclosed in this document. Exemplary antibodies include those described in international patent application WO 2015/138600; in published US application 2014/0242095 (University Health Networks); published application CN103665165 (JIANGSU KUANGYA BIOLOGICAL MEDICAL SCIENCE &TECHNOLOGY); Zhao XW et al. Proc Natl Acad Sci USA 108: 18342-7 (2011), each in this document specifically incorporated by reference. An anti-SIRP antibody can be pan specific, that is, binding to two or more different human SIRP isoforms; or it can be specific to an isoform. For example, the 1.23A antibody described by Zhang et al., Supra, is reported to be specific for the SIRP1 variant, while 12C4 antibody is pan-specific. Anti-SIRP antibodies can also be specific for SIRP and have no binding to SIRP and / or SIRP. Anti-SIRPa antibodies can be pan specific to SIRP and / or SIRP
[0068] [0068] The term "immunoglobulin" refers to a class of structurally related proteins, generally comprising two pairs of polypeptide chains: a pair of light chains (L) and a pair of heavy chains (H). In an "intact immunoglobulin", all four of these chains are interconnected by disulfide bonds. The immunoglobulin structure has been well characterized. See, for example, Paul, Fundamental Immunology, 7th ed., Chap. 5 (2013), Lippincott Williams; Wilkins, Philadelphia, PA. Briefly, each heavy chain typically comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region typically comprises three abbreviated CH1, CH2, and CH3 domains. Each light chain typically comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region typically comprises an abbreviated CL domain.
[0069] [0069] The term "antibody" is used in this document in its broadest sense and includes certain types of immunoglobulin molecules comprising one or more antigen-binding domains that specifically bind to an antigen or epitope. An antibody specifically includes intact antibodies (for example, intact immunoglobulins), antibody fragments and multispecific antibodies. In some embodiments, the antibody comprises an alternative scaffold structure. In some embodiments, the antibody consists of an alternative scaffold structure. In some embodiments, the antibody essentially consists of an alternative scaffold structure. In some embodiments, the antibody comprises an antibody fragment. In some embodiments, the antibody consists of an antibody fragment. In some embodiments, the antibody essentially consists of an antibody fragment. An "SIRP-ALPHA antibody", "anti-SIRP-ALPHA antibody" or "SIRP-ALPHA specific antibody" is an antibody, as provided in this document, which specifically binds to the SIRP-ALPHA antigen. In some embodiments, the antibody binds to the extracellular domain of SIRP-ALPHA. In certain embodiments, a SIRP-ALPHA antibody provided in this document binds to an SIRP-ALPHA epitope that is conserved between or between SIRP-ALPHA proteins of different species.
[0070] [0070] The term "scaffold structure" refers to a molecule in which one or more regions can be diversified to produce one or more antigen-binding domains that specifically bind to an antigen or epitope. In some embodiments, the antigen-binding domain binds the antigen or epitope with specificity and affinity similar to that of an antibody. Examples of alternative structures include those derived from fibronectin (eg, AdnectinsTM), β-sandwich (eg, iMab), lipocalin (eg, Anticalins®), EETI-II / AGRP, BPTI / LACI-D1 / ITI-D2 (for example, Kunitz domains), thioredoxin peptide aptamers, protein A (for example, Affibody®), ankyrin repeats (for example, DARPins), gamma-B-crystalline / ubiquitin (for example, Affilins), CTLD3 (for example, tetranectins), Finomers and (LDLR-A module) (for example, Avimers). Additional information on alternative scaffolding structure is provided in Binz et al., Nat. Biotechnol., 2005 23: 1257-1268; Skerra, Current Opin. in Biotech., 2007 18: 295-304; and Silacci et al., J. Biol. Chem., 2014, 289: 14392-14398; each of which is incorporated by reference in its entirety. An alternative scaffold structure is a type of antibody.
[0071] [0071] The term "antigen binding domain" means the portion of an antibody that is capable of specifically binding to an antigen or epitope. An example of an antigen-binding domain is an antigen-binding domain formed by a VH -VL dimer of an antibody. Another example of an antigen-binding domain is an antigen-binding domain formed by the diversification of certain loops from the tenth domain of fibronectin type III of an Adnectin. An antigen-binding domain can include CDRs 1, 2 and 3 of a heavy chain in that order; and CDRs 1, 2 and 3 of a light chain in that order.
[0072] [0072] The terms "full length antibody", "intact antibody" and "whole antibody" are used in this document interchangeably to refer to an antibody with a structure substantially similar to a naturally occurring antibody structure and with heavy chains that comprise an Fc region. For example, when used to refer to an IgG molecule, a "full length antibody" is an antibody that comprises two heavy chains and two light chains.
[0073] [0073] The term "Fc region" or "Fc" means the Terminal C region of an immunoglobulin heavy chain that, in naturally occurring antibodies, interacts with Fc receptors and certain proteins of the complement system. The structures of the Fc regions of various immunoglobulins and the glycosylation sites contained therein are known in the art. See Schroeder and Cavacini, J. Allergy Clin. Immunol., 2010, 125: S41-52, incorporated by reference in its entirety. The Fc region can be a naturally occurring Fc region or a modified Fc region as described in the art or elsewhere in this disclosure.
[0074] [0074] The VH and VL regions can be subdivided into regions of hypervariability ("hypervariable regions (HVRs)"; also called "complementarity determining regions" (CDRs)) interspersed with more conserved regions. The most conserved regions are called framework regions (FRs). Each VH and VL generally comprises three CDRs and four FRs, organized in the following order (from terminal N to terminal C): FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. CDRs are involved in binding to the antigen and influence the specificity of the antigen and the binding affinity of the antibody. See, Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. (1991), Public Health Service, National Institutes of Health, Bethesda, MD, incorporated by reference in its entirety.
[0075] [0075] The light chain of any vertebrate species can be attributed to one of two types, called kappa (κ) and lambda (λ), based on the sequence of its constant domain.
[0076] [0076] The heavy chain of any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG and IgM. These classes are also designated α, δ, ε, γ and µ, respectively. The IgG and IgA classes are divided into subclasses based on differences in sequence and function. Human beings express the following subclasses: IgG1, IgG2, IgG3,
[0077] [0077] The limits of the amino acid sequence of a CDR can be determined by one skilled in the art using any of several known numbering schemes, including those described by Kabat et al., Supra ("Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273: 927-948 ("Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262: 732-745 ("Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27: 55-77 ("IMGT" numbering scheme); and Honegge and Plückthun, J. Mol. Biol., 2001, 309: 657-70 ("AHo" numbering scheme); each of which is incorporated by reference in its entirety.
[0078] [0078] Table 1 provides the positions of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3, as identified by the schemes of Kabat and Chothia. For CDR-H1, the numbering of residues is provided using the Kabat and Chothia Numbering schemes.
[0079] [0079] CDRs can be assigned, for example, using antibody numbering software, such as Abnum, available at www.bioinf.org.uk/abs/abnum/, and described in Abhinandan and Martin, Immunology, 2008, 45 : 3832-3839, incorporated by reference in its entirety. Table 1. Residues in CDRs according to the numbering schemes of Kabat and Chothia. CDR Kabat Chothia L1 L24-L34 L24-L34 L2 L50-L56 L50-L56 L3 L89-L97 L89-L97 H1 (H31-H35B numbering H26-H32 or H34 * Kabat) H1 (H31-H35 numbering H26-H32 Chothia ) H2 H50-H65 H52-H56 H3 H95-H102 H95-H102 * The C-terminal of CDR-H1, when numbered using the Kabat Numbering convention, varies between H32 and H34, depending on the length of the CDR.
[0080] [0080] The "EU numbering scheme" is generally used when referring to a residue in an antibody heavy chain constant region (for example, as reported in Kabat et al., Supra). Unless otherwise indicated, the EU numbering scheme is used to refer to residues in the heavy antibody constant chain regions described in this document.
[0081] An "antibody fragment" comprises a portion of an intact antibody, such as antigen binding or variable region of an intact antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F (ab ') 2 fragments, Fab' fragments, scFv (sFv) fragments and scFv-Fc fragments.
[0082] [0082] "Fv" fragments comprise a non-covalently linked dimer of a heavy chain variable domain and a light chain variable domain.
[0083] [0083] The "Fab" fragments comprise, in addition to the heavy and light chain variable domains, the light chain constant domain and the first heavy chain constant domain (CH1). Fab fragments can be generated, for example, by recombinant methods or by papain digestion of a complete antibody.
[0084] [0084] The "F (ab ') 2" fragments contain two Fab' fragments joined, close to the hinge region, by disulfide bonds. F (ab ') 2 fragments can be generated, for example, by recombinant methods or by pepsin digestion of an intact antibody. F (ab ') fragments can be dissociated, for example, by treatment with ß-mercaptoethanol.
[0085] [0085] Antibody fragments "single chain Fv" or "sFv" or "scFv" comprise a VH domain and a VL domain in a single polypeptide chain. VH and VL are generally linked by a peptide linker. See Plückthun A. (1994). Any suitable peptide linker can be used. In some embodiments, the peptide linker is a (GGGGS) n (SEQ ID NO: 127). In some embodiments, n = 1, 2, 3, 4, 5, or 6. See, Escherichia coli antibodies. In Rosenberg M. & Moore GP (Eds.), The Pharmacology of Monoclonal Antibodies Vol. 113 (pp. 269-315). Springer-Verlag, New York, incorporated by reference in its entirety.
[0086] [0086] The "scFv-Fc" fragments comprise a scFv attached to an Fc domain. For example, an Fc domain can be attached to scFv's Terminal C. The Fc domain can follow either VH or VL, depending on the orientation of the variable domains in scFv (ie, VH-VL or VL -VH). Any suitable Fc domain known in the art or described in this document can be used. In some cases, the Fc domain comprises an IgG4 Fc domain.
[0087] [0087] The term "single domain antibody" refers to a molecule in which a variable domain of an antibody specifically binds to an antigen without the presence of the other variable domain. Single domain antibodies and their fragments are described in Arabi Ghahroudi et al., FEBS Letters, 1998, 414: 521-526 and Muyldermans et al., Trends in Biochem. Sci., 2001, 26: 230-245, each of which is incorporated by reference in its entirety. Single domain antibodies are also known as sdAbs or nanobodies.
[0088] [0088] A "multispecific antibody" is an antibody comprising two or more different antigen-binding domains that collectively specifically bind to two or more different epitopes. The two or more different epitopes can be epitopes on the same antigen (for example, a single SIRP-ALPHA molecule expressed by a cell) or on different antigens (for example, different SIRP-ALPHA molecules expressed by the same cell or a SIRP- ALPHA molecule and a non-SIRP-ALPHA molecule). In some ways, a multispecific antibody binds two different epitopes (that is, a "bispecific antibody"). In some respects, a multispecific antibody binds three different epitopes (that is, a "triespecific antibody").
[0089] [0089] A "monospecific antibody" is an antibody comprising one or more binding sites that specifically bind to a single epitope. An example of a monospecific antibody is a naturally occurring IgG molecule that, although divalent (i.e., having two antigen-binding domains), recognizes the same epitope in each of the two antigen-binding domains. The binding specificity can be present in any suitable valence.
[0090] [0090] The term "monoclonal antibody" refers to an antibody from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies comprises antibodies that are substantially similar and that bind to the same epitope (s), except for variants that may normally arise during the production of the monoclonal antibody. Such variants are generally present only in small quantities. A monoclonal antibody is typically obtained by a process that includes selecting a single antibody from a plurality of antibodies. For example, the selection process may be the selection of a single clone from a plurality of clones, such as a cluster of hybridoma clones, phage clones, yeast clones, bacterial clones or other recombinant DNA clones. The selected antibody can be further altered, for example, to improve affinity with the target ("affinity maturation"), humanize the antibody, improve its production in cell culture and / or reduce its immunogenicity in a subject.
[0091] [0091] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a specific source or species, while the remainder of the heavy and / or light chain is derived from a source or different species.
[0092] [0092] "Humanized" forms of non-human antibodies are chimeric antibodies that contain a minimal sequence derived from non-human antibody. A humanized antibody is generally a human antibody (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs from a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken or non-human primate antibody with a desired specificity, affinity or biological effect. In some cases, the selected residues from the recipient antibody framework region are replaced by the corresponding residues from the donor antibody framework region. Humanized antibodies can also comprise residues that are not found in the recipient antibody or the donor antibody. Such modifications can be made to further refine the function of the antibody. For more details, see Jones et al., Nature, 1986, 321: 522-525; Riechmann et al., Nature, 1988, 332: 323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2: 593-596, each of which is incorporated by reference in its entirety.
[0093] [0093] A "human antibody" is one that has an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source that uses a repertoire of human antibodies or sequences that encode antibodies (for example, example, obtained from human sources or redesigned). Human antibodies specifically exclude humanized antibodies.
[0094] [0094] "Affinity" refers to the strength of the total sum of non-covalent interactions between a single molecule binding site (eg, an antibody) and its binding partner (eg, an antigen or epitope). Unless otherwise indicated, as used herein, "affinity" refers to the intrinsic binding affinity that reflects a 1: 1 interaction between members of a binding pair (for example, antibody and antigen or epitope). The affinity of a molecule X for its partner Y can be represented by the dissociation equilibrium constant (KD). The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described in this document, such as surface plasmon resonance (SPR) technology (for example, BIACORE®) or biolayer interferometry (for example, FORTEBIO®).
[0095] [0095] Regarding the binding of an antibody to a target molecule, the terms "bind", "specific binding", "specifically binds to", "specific to", "selectively binds" and "selective "to a specific antigen (for example, a polypeptide target) or an epitope in a medium binding to the particular antigen that is measurably different from a non-specific or non-selective interaction (for example, with a non-target molecule). Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. In that case, specific binding is indicated if the binding of the antibody to the target molecule is competitively inhibited by the control molecule. In some respects, the affinity of a SIRP-ALPHA antibody for a non-target molecule is less than about 50% of the affinity for SIRP-ALPHA. In some respects, the affinity of a SIRP-ALPHA antibody for a non-target molecule is less than about 40% of the affinity for SIRP-ALPHA. In some respects, the affinity of a SIRP-ALPHA antibody for a non-target molecule is less than about 30% of the affinity for SIRP-ALPHA. In some respects, the affinity of a SIRP-ALPHA antibody for a non-target molecule is less than about 20% of the affinity for SIRP-ALPHA. In some respects, the affinity of a SIRP-ALPHA antibody for a non-target molecule is less than about 10% of the affinity for SIRP-ALPHA. In some respects, the affinity of a SIRP-ALPHA antibody for a non-target molecule is less than about 1% of the affinity for SIRP-ALPHA. In some respects, the affinity of a SIRP-ALPHA antibody for a non-target molecule is less than about 0.1% of the affinity for SIRP-ALPHA.
[0096] [0096] The term "kd" (sec-1) as used in this document, refers to the dissociation rate constant for a specific antibody-antigen interaction. This value is also referred to as the koff value.
[0097] [0097] The term "ka" (M-1 × sec-1), as used in this document, refers to the constant rate of association for a specific antibody-antigen interaction. This value is also referred to as the kon value.
[0098] [0098] The term "KD" (M), as used in this document, refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. KD = kd / ka. In some embodiments, the affinity of an antibody is described in terms of KD for an interaction between that antibody and its antigen. For clarity, as known in the art, a lower KD value indicates a higher affinity interaction, while a higher KD value indicates a lower affinity interaction.
[0099] [0099] The term "KA" (M-1), as used in this document, refers to the association equilibrium constant of a specific antibody-antigen interaction. KA = ka / kd.
[00100] [00100] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecule (s), as a therapeutic (cytokine, for example) or diagnostic agent.
[00101] [00101] "Effector functions" refer to the biological activities mediated by the Fc region of an antibody, whose activities may vary depending on the isotype of the antibody. Examples of antibody effector functions include binding to C1q to activate complement-dependent cytotoxicity (CDC), binding to the Fc receptor to activate antibody-dependent cell cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP).
[00102] [00102] When used in this document in the context of two or more antibodies, the term "competes with" or "competes with" indicates that the two or more antibodies compete for binding to an antigen (for example, SIRP-ALPHA). In an exemplary assay, SIRP-ALPHA is coated on a surface and contacted with a first SIRP-ALPHA antibody, after which a second SIRP-ALPHA antibody is added. In another exemplary assay, a first SIRP-ALPHA antibody is coated on a surface and contacted with SIRP-ALPHA and then a second SIRP-ALPHA antibody is added. If the presence of the first SIRP-ALPHA antibody reduces the binding of the second SIRP-ALPHA antibody in any of the assays, then the antibodies compete with each other. The term "competes with" also includes combinations of antibodies in which one antibody reduces the binding of another antibody, but where no competition is observed when antibodies are added in the reverse order. However, in some embodiments, the first and second antibodies inhibit each other's binding, regardless of the order in which they are added. In some embodiments, an antibody reduces the binding of another antibody to its antigen by at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% , or at least 95%. One skilled in the art can select the concentrations of antibodies used in competition assays based on the affinities of the antibodies to SIRP-ALPHA and the valence of the antibodies. The assays described in this definition are illustrative and one skilled in the art can use any suitable assay to determine whether antibodies compete with each other. Suitable assays are described, for example, in Cox et al., "Immunoassay Methods," in the Assay Guidance Manual [Internet], updated on December 24, 2014 (www.ncbi.nlm.nih.gov/books/NBK92434/ ; accessed September 29, 2015); Silman et al., Cytometry, 2001, 44: 30-37; and Finco et al., J. Pharm. Biomed. Anal., 2011, 54: 351-358; each of which is incorporated by reference in its entirety.
[00103] [00103] The term "epitope" means a portion of an antigen that specifically binds to an antibody. Epitopes often consist of surface-accessible amino acid residues and / or side chains of sugar and may have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the bond to the former, but not the latter, can be lost in the presence of denaturing solvents. An epitope may comprise amino acid residues that are directly involved in binding and other amino acid residues, which are not directly involved in binding. The epitope to which an antibody binds can be determined using known techniques for determining epitopes, such as, for example, testing for antibody binding to SIRP-ALPHA variants with different point mutations or to chimeric SIRP-ALPHA variants.
[00104] [00104] The percent "identity" between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence identical to the amino acid residues in the reference sequence, after aligning the sequences and inserting gaps, if necessary, to achieve maximum percent sequence identity. Alignment for purposes of determining the percentage of amino acid sequence identity can be achieved in many ways in the prior art, for example, using free computer software, such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW , CLUSTAL OMEGA, or MUSCLE software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the total length of the sequences being compared.
[00105] [00105] A "conservative substitution" or a "conservative substitution of amino acids" refers to the replacement of an amino acid by a chemically or functionally similar amino acid. Conservative substitution tables that provide similar amino acids are known in the art. For example, the groups of amino acids provided in Tables 2-4 are, in some embodiments, considered conservative substitutions for each other.
[00106] [00106] Table 2. Selected groups of amino acids that are considered conservative substitutions for each other, in certain modalities. DeE Acid Waste Basic Waste K, R and H Unloaded hydrophilic waste S, T, N and Q Unloaded aliphatic waste G, A, V, L and I Unloaded non-polar waste C, M and P Aromatic waste F, Y, and W
[00107] [00107] Table 3. Additional selected groups of amino acids that are considered conservative substitutions for each other, in certain modalities. Group 1 A, S and T Group 2 DeE Group 3 NeQ Group 4 ReK Group 5 I, L and M Group 6 F, Y, and W
[00108] [00108] Table 4. Additional selected groups of amino acids that are considered conservative substitutions for each other, in certain modalities. Group A AeG Group B DeE Group C NeQ Group D R, K and H Group E I, L, M, V Group F F, Y, and W Group G SeT Group H CeM
[00109] [00109] Additional conservative substitutions can be found, for example, in Creighton, Proteins: Structures and Molecular Properties, 2nd ed. (1993), W. H. Freeman & Co., New York, NY. An antibody generated by performing one or more conservative substitutions of amino acid residues in a parent antibody is referred to as a "conservatively modified variant."
[00110] [00110] The term "amino acid" refers to the twenty common naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), Glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tir; Y) and valine (Val; V).
[00111] [00111] The term "vector", as used in this document, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is attached. The term includes the vector as a self-replicating nucleic acid structure, as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to in this document as "expression vectors."
[00112] [00112] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to the cells into which exogenous nucleic acid was introduced, and the progeny of these cells. Host cells include "transformants" (or "transformed cells") and "transfectants" (or "transfected cells"), each including the transformed or transfected primary cell and the progeny derived therefrom. Such a progeny may not be completely identical in nucleic acid content to a precursor cell, and may contain mutations.
[00113] [00113] The term "treatment" (and its variations, such as "treating" or "treatment") refers to clinical intervention in an attempt to alter the natural course of a disease or condition in a subject who needs it. Treatment can be performed for prophylaxis and during the course of clinical pathology. Desirable effects of treatment include preventing the occurrence or recurrence of the disease, relieving symptoms, decreasing any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, improving or palliating the disease state, and remission or improved prognosis.
[00114] [00114] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of an antibody or pharmaceutical composition provided in this document which, when administered to a subject, is effective in treating a disease or disturb.
[00115] [00115] As used herein, the term "subject" means a mammalian subject. Examples of subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits and sheep. In specific modalities, the subject is a human being. In some embodiments, the subject has a disease or condition that can be treated with an antibody provided in this document. In some embodiments, the disease or condition is cancer. In some ways, the disease or condition is a viral infection.
[00116] [00116] The term “package insert” is used to refer to instructions normally included in commercial packaging of therapeutic or diagnostic products (for example, kits) that contain information about the indications, use, dosage, administration, combination therapy, contraindications and / or notices regarding the use of such therapeutic or diagnostic products.
[00117] [00117] The term "cytotoxic agent", as used in this document, refers to a substance that inhibits or prevents cellular function and / or causes cell death or destruction.
[00118] [00118] A "chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer. Chemotherapeutic agents include "anti-hormonal agents" or "endocrine therapies" that act to regulate, reduce, block or inhibit the effects of hormones that can promote cancer growth.
[00119] [00119] The term "cytostatic agent" refers to a compound or composition that stops the growth of a cell in vitro or in vivo. In some embodiments, a cytostatic agent is an agent that reduces the percentage of cells in the S phase. In some embodiments, a cytostatic agent reduces the percentage of cells in the S phase by at least about 20%, at least about 40%, at least about 60% or at least about 80%.
[00120] [00120] The term "tumor" refers to all growth and proliferation of neoplastic cells, malignant or benign, and to all precancerous and cancerous cells and tissues. The terms "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder" and "tumor" are not mutually exclusive, as referred to in this document. The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders that are associated with a certain degree of abnormal cell proliferation. In some embodiments, the cell proliferative disorder is a cancer. In some ways, the tumor is a solid tumor. In some respects, the tumor is a hematological neoplasm.
[00121] [00121] The term "pharmaceutical composition" refers to a preparation that is such as to allow the biological activity of an active ingredient contained therein to be effective in treating a subject and that it does not contain additional components that are unacceptably toxic to the subject in the quantities provided in the pharmaceutical composition.
[00122] [00122] The terms "co-administration", "co-administration" and "in combination with" include the administration of two or more therapeutic agents simultaneously, simultaneously or sequentially, without specific time limits. In one embodiment, the agents are present in the subject's cell or body at the same time or exert their biological or therapeutic effect at the same time. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. In certain embodiments, a first agent can be administered before (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours , 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks before), concomitantly or after (for example, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks after ) administration of a second therapeutic agent.
[00123] [00123] The terms "modular" and "modulation" refer to reducing or inhibiting or, alternatively, activating or increasing a recited variable.
[00124] [00124] The terms "increase" and "activate" refer to an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% , 95%, 100%, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 50 times, 100 times or more in a recited variable.
[00125] [00125] The terms "reduce" and "inhibit" refer to a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90 %, 95%, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 50 times, 100 times or more in a recited variable.
[00126] [00126] The term "agonize" refers to the activation of receptor signaling to induce a biological response associated with receptor activation. An "agonist" is an entity that binds and agonizes a receptor.
[00127] [00127] The term "antagonize" refers to the inhibition of receptor signaling to inhibit a biological response associated with receptor activation. An "antagonist" is an entity that binds and antagonizes a receptor. SIRP-ALPHA Antibodies
[00128] [00128] Antibodies that specifically bind to SIRP-ALPHA are provided in this document. In some ways, SIRP-ALPHA is human SIRP-ALPHA. In some embodiments, the antibodies provided in this document specifically bind to the extracellular domain of SIRP-ALPHA. SIRP-ALPHA can be expressed on the surface of any suitable target cell. In some embodiments, the target cell is a professional antigen presenting cell. In some embodiments, the target cell is a macrophage. An antibody can be pan-specific for human SIRP isotypes. An antibody can be specific for a human SIRP isotype.
[00129] [00129] In certain embodiments, an antibody is 1H9. In certain embodiments, an antibody is 3C2.
[00130] [00130] In some embodiments, the antibodies provided in this document comprise a light chain. In some ways, the light chain is a kappa light chain. In some ways, the light chain is a lambda light chain.
[00131] [00131] In some embodiments, the antibodies provided in this document comprise a heavy chain. In some ways, the heavy chain is an IgA. In some ways, the heavy chain is an IgD. In some ways, the heavy chain is an IgE. In some ways, the heavy chain is an IgG. In some ways, the heavy chain is an IgM. In some ways, the heavy chain is an IgG1. In some ways, the heavy chain is an IgG2. In some ways, the heavy chain is an IgG3. In some ways, the heavy chain is an IgG4. In some ways, the heavy chain is an IgA1. In some ways, the heavy chain is an IgA2.
[00132] [00132] In some embodiments, an antibody binds human SIRP with a KD less than or equal to about 1, 1-6, 1-5, 1-4, 1-3, 2, 3, 4, 5, 6, 7 , 8, 9 or 10 x10-9M as measured by the Biacore assay.
[00133] [00133] In some embodiments, the antibodies provided in this document comprise an antibody fragment. In some embodiments, the antibodies provided in this document consist of an antibody fragment. In some embodiments, the antibodies provided in this document consist essentially of an antibody fragment. In some respects, the antibody fragment is an Fv fragment. In some respects, the antibody fragment is a Fab fragment. In some respects, the antibody fragment is an F (ab ') 2 fragment. In some respects, the antibody fragment is a Fab 'fragment. In some ways, the antibody fragment is a scFv (sFv) fragment. In some respects, the antibody fragment is a scFv-Fc fragment. In some respects, the antibody fragment is a fragment of a single domain antibody.
[00134] [00134] In some embodiments, an antibody fragment provided in this document is derived from an illustrative antibody provided in this document. In some embodiments, an antibody fragment provided in this document is not derived from an illustrative antibody provided in this document and can, for example, be isolated again according to the methods provided in this document to obtain antibody fragments.
[00135] [00135] In some embodiments, an antibody fragment provided in this document retains the ability to antagonize SIRP-ALPHA, as measured by one or more assays or biological effects described in this document. In some embodiments, an antibody fragment provided in this document retains the ability to prevent SIRP-ALPHA from interacting with one or more of its ligands, as described in this document.
[00136] [00136] In some embodiments, an antibody fragment provided in this document competes for binding to SIRP-ALPHA with 1H9 and / or 3C2. In some embodiments, an antibody fragment provided herein binds to the same SIRP-ALPHA epitope as that antibody.
[00137] [00137] As an alternative to using an antibody comprising a human Fc region with reduced affinity for an Fcγ receptor, an antibody can be modified to have no Fc sequences, for example, producing an antibody fragment as an F (ab 'fragment) )2. To generate an F (ab) 2 fragment, the purified antibody is suspended with Pepsin of preparation Pierce F (ab ') 2 immobilized on seated resin, according to the manufacturer's instructions. Digestion with pepsin normally produces an F (ab ') 2 fragment (~ 110kDa by SDS-PAGE under non-reducing conditions) and numerous small peptides from the Fc portion. The resulting F (ab ') 2 fragment is composed of a pair of Fab' units connected by two disulfide bonds. The Fc fragment is extensively degraded and separated from F (ab ') 2 by dialysis, gel filtration or ion exchange chromatography.
[00138] [00138] In some embodiments, the antibodies provided in this document are monoclonal antibodies. In some embodiments, the antibodies provided in this document are polyclonal antibodies.
[00139] [00139] In some embodiments, the antibodies provided in this document comprise a chimeric antibody. In some embodiments, the antibodies provided in this document consist of a chimeric antibody. In some embodiments, the antibodies provided in this document consist essentially of a chimeric antibody. In some embodiments, the antibodies provided in this document comprise a humanized antibody. In some embodiments, the antibodies provided in this document consist of a humanized antibody. In some embodiments, the antibodies provided in this document consist essentially of a humanized antibody. In some embodiments, the antibodies provided in this document comprise a human antibody. In some embodiments, the antibodies provided in this document consist of a human antibody. In some embodiments, the antibodies provided in this document consist essentially of a human antibody.
[00140] [00140] In some embodiments, the antibodies provided in this document comprise an alternative scaffold structure. In some embodiments, the antibodies provided in this document consist of an alternative scaffold structure. In some embodiments, the antibodies provided in this document essentially consist of an alternative scaffold structure. Any suitable alternative scaffolding structure can be used. In some ways, the alternative scaffold structure is selected from an AdnectinTM, an iMab, anticalin®, an EETI-II / AGRP, a Kunitz domain, a thioredoxin peptide aptamer, an Affibody®, a DARPin, an Affilin, a Tetranectin, a Fynomer and an Avimer.
[00141] [00141] In some embodiments, an antibody provided in this document inhibits the binding of SIRP-ALPHA to one or more SIRP-ALPHA ligands.
[00142] [00142] In some respects, an antibody does not bind to SIRP-Gamma. In some respects, an antibody does not substantially bind to SIRP-Gamma.
[00143] [00143] In some respects, an antibody disclosed in this document is pan-specific for human SIRP isotypes. An antibody disclosed in this document, such as 1H9, can bind to several human SIRP isotypes, including one or more of V1, V2 and V1 / V5. Exemplary V1 sequence shown in SEQ ID NO:
[00144] [00144] In some respects, an antibody competes for binding to human SIRP with an antibody selected from 1H9 and 3C2. In some respects, an antibody binds to the same human SIRP epitope bound by 1H9 or 3C2. In some respects, an antibody binds to a human SIRP epitope overlaid as bound by 1H9 or 3C2. In some respects, an antibody binds to a distinct human SIRP epitope as bound by 1H9 or 3C2.
[00145] [00145] In some respects, an antibody does not compete for binding to human SIRP with the KWar antibody.
[00146] [00146] In some respects, an antibody partially competes for binding to human SIRP with the KWar antibody.
[00147] [00147] In certain respects, an antibody inhibits the binding of human CD47 to human SIRP.
[00148] [00148] In certain respects, an antibody inhibits the binding of human SP-A to human SIRP.
[00149] [00149] In certain respects, an antibody inhibits the binding of human SP-D to human SIRP.
[00150] [00150] In certain respects, an antibody binds to the Rhesus monkey SIRP.
[00151] [00151] In certain respects, an antibody binds to the cinomolgo SIRP
[00152] [00152] In certain respects, an antibody increases phagocytosis compared to control.
[00153] [00153] Also disclosed in this document is an isolated humanized, human or chimeric antibody that competes for binding to human SIRP with an antibody disclosed in this document.
[00154] [00154] Also disclosed in this document is an isolated humanized, human or chimeric antibody that binds to the human SIRP epitope linked by an antibody disclosed in this document.
[00155] [00155] In certain respects, an antibody comprises a human Fc region comprising at least one modification that reduces binding to a human Fc receptor.
[00156] [00156] In some embodiments, an antibody is an antibody that competes with an illustrative antibody provided in this document, for example, 1H9 and / or 3C2. In some respects, the antibody that competes with the illustrative antibody provided in this document binds to the same epitope as an illustrative antibody provided in this document.
[00157] [00157] It is known that when an antibody is expressed in cells, the antibody is modified after translation. Examples of post-translational modification include cleavage of lysine at the heavy chain Terminal C by a carboxypeptidase; modification of glutamine or glutamic acid at the heavy and light chain N-Terminal in pyroglutamic acid by pyroglutamylation; glycosylation; oxidation; deamidation; and glycation, and these post-translational modifications are known to occur in various antibodies (See Journal of Pharmaceutical Sciences, 2008, Vol. 97, p. 2426-2447, incorporated by reference in their entirety). In some embodiments, an antibody is an antibody or fragment that binds it to the antigen that has undergone post-translation modification. Examples of an antibody or antigen-binding fragment that has undergone post-translational modification include an antibody or its antigen-binding fragments that have undergone pyroglutamylation at the N-terminus of the heavy chain variable region and / or lysine deletion in the Heavy chain C terminal. It is known in the art that such post-translation modification due to pyroglutamylation at Terminal N and deletion of lysine at Terminal C has no influence on the activity of the antibody or its fragment (Analytical Biochemistry, 2006, Vol. 348, p 24- 39, incorporated by reference in its entirety). SIRP-ALPHA antibody sequences
[00158] [00158] An antibody can comprise: a CDR-H1 comprising the sequence set out in SEQ ID NO: 1; a CDR-H2 comprising the sequence set forth in SEQ ID NO: 2; a CDR-H3 comprising the sequence shown in SEQ ID NO: 3; a CDR-L1 comprising the sequence shown in SEQ ID NO: 4; a CDR-L2 comprising the sequence set out in SEQ ID NO: 5; and a CDR-L3 comprising the sequence set out in SEQ ID NO: 6.
[00159] [00159] An antibody can comprise: a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 8.
[00160] [00160] An antibody can comprise: a heavy chain of SEQ ID NO: 17 and a light chain of SEQ ID NO: 18.
[00161] An antibody can comprise: a CDR-H1 comprising the sequence set out in SEQ ID NO: 9; a CDR-H2 comprising the sequence set forth in SEQ ID NO: 10; a CDR-H3 comprising the sequence set out in SEQ ID NO: 11; a CDR-L1 comprising the sequence set out in SEQ ID NO: 12; a CDR-L2 comprising the sequence shown in SEQ ID NO: 13; and a CDR-L3 comprising the sequence set out in SEQ ID NO: 14.
[00162] An antibody can comprise: a VH sequence of SEQ ID NO: 15 and a VL sequence of SEQ ID NO: 16.
[00163] An antibody can comprise: a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 20.
[00164] [00164] An antibody can comprise: a CDR-H1 comprising the sequence set out in SEQ ID NO: 21; a CDR-H2 comprising the sequence set forth in SEQ ID NO: 22; a CDR-H3 comprising the sequence set out in SEQ ID NO: 23; a CDR-L1 comprising the sequence set out in SEQ ID NO: 24; a CDR-L2 comprising the sequence set out in SEQ ID NO: 25; and a CDR-L3 comprising the sequence set out in SEQ ID NO: 26.
[00165] [00165] An antibody can comprise: a VH sequence of SEQ ID NO: 27 and a VL sequence of SEQ ID NO: 28.
[00166] [00166] An antibody can comprise: a CDR-H1 comprising the sequence set forth in SEQ ID NO: 29; a CDR-H2 comprising the sequence set out in SEQ ID NO: 30; a CDR-H3 comprising the sequence set out in SEQ ID NO: 31; a CDR-L1 comprising the sequence set out in SEQ ID NO: 32; a CDR-L2 comprising the sequence set out in SEQ ID NO: 33; and a CDR-L3 comprising the sequence set out in SEQ ID NO: 34.
[00167] [00167] An antibody can comprise: a VH sequence of SEQ ID NO: 35 and a VL sequence of SEQ ID NO: 36.
[00168] [00168] In certain respects, an antibody may comprise one or more 1H9 CDRs. In some respects, an antibody can comprise all 1H9 CDRs. In certain respects, an antibody can comprise one or more variable sequences of 1H9. In certain respects, an antibody can comprise each variable sequence of 1H9. In certain respects, an antibody can comprise the 1H9 heavy chain. In some respects, an antibody can comprise the 1H9 light chain. In certain aspects, an antibody can comprise the 1H9 heavy chain and the light chain. In some ways, an antibody is 1H9.
[00169] [00169] In certain respects, an antibody can comprise one or more 3C2 CDRs. In some ways, an antibody can comprise all 3C2 CDRs. In certain respects, an antibody can comprise one or more variable 3C2 sequences. In certain respects, an antibody can comprise each variable 3C2 sequence. In certain respects, an antibody can comprise the 3C2 heavy chain. In some respects, an antibody can comprise the 3C2 light chain. In certain aspects, an antibody can comprise the 3C2 heavy chain and the light chain. In some ways, an antibody is 3C2.
[00170] [00170] In certain respects, an antibody can comprise one or more 9B11 CDRs. In some respects, an antibody can comprise all 9B11 CDRs. In certain respects, an antibody can comprise one or more variable sequences of 9B11. In certain respects, an antibody can comprise each variable sequence of 9B11. In certain aspects, an antibody can comprise the 9B11 heavy chain. In some respects, an antibody can comprise the 9B11 light chain. In certain aspects, an antibody can comprise the 9B11 heavy chain and the light chain. In some ways, an antibody is 9B11.
[00171] [00171] In certain respects, an antibody may comprise one or more
[00172] [00172] In some embodiments, an antibody provided in this document comprises a sequence with at least about 50%, 60%, 70%, 80%, 90%, 95% or 99% identity to an illustrative sequence provided in SEQ ID NOs: 1-36. In some embodiments, an antibody provided in this document comprises a sequence provided in SEQ ID NOs: 1-36, with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acid substitutions. In some ways, amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to in this document as "variants." In some embodiments, these variants are derived from a sequence provided in this document, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis or any other method known in the art or described in this document. In some embodiments, these variants are not derived from a sequence provided in this document and can, for example, be isolated again according to the methods provided in this document for obtaining antibodies. Monospecific and multispecific SIRP-ALPHA antibodies
[00173] [00173] In some embodiments, the antibodies provided in this document are monospecific antibodies.
[00174] [00174] In some embodiments, the antibodies provided in this document are multispecific antibodies.
[00175] [00175] In some embodiments, a multispecific antibody provided in this document binds to more than one antigen. In some embodiments, a multispecific antibody binds 2 antigens. In some embodiments, a multispecific antibody binds 3 antigens. In some embodiments, a multispecific antibody binds 4 antigens. In some embodiments, a multispecific antibody binds 5 antigens.
[00176] [00176] In some embodiments, a multispecific antibody provided in this document binds more than one epitope to a SIRP-ALPHA antigen. In some embodiments, a multispecific antibody binds 2 epitopes to a SIRP-ALPHA antigen. In some embodiments, a multispecific antibody binds to 3 epitopes on a SIRP-ALPHA antigen.
[00177] [00177] Many multispecific antibody constructs are known in the art, and the antibodies provided in this document can be provided in the form of any suitable multispecific construct.
[00178] [00178] In some embodiments, the multispecific antibody comprises an immunoglobulin comprising at least two different heavy chain variable regions, each paired with a common light chain variable region (i.e., a "common light chain antibody"). The common light chain variable region forms a distinct antigen-binding domain with each of the two different heavy chain variable regions. See Merchant et al., Nature Biotechnol., 1998, 16: 677-681, incorporated by reference in its entirety.
[00179] [00179] In some embodiments, the multispecific antibody comprises an immunoglobulin comprising an antibody or a fragment thereof attached to one or more of the N or C terminals of the heavy or light chains of that immunoglobulin. See Coloma and Morrison, Nature Biotechnol., 1997, 15: 159-163, incorporated by reference in its entirety. In some respects, that antibody comprises a bispecific tetravalent antibody.
[00180] [00180] In some embodiments, the multispecific antibody comprises a hybrid immunoglobulin comprising at least two different heavy chain variable regions and at least two different light chain variable regions. See Milstein and Cuello, Nature, 1983, 305: 537-540; and Staerz and Bevan, Proc. Natl. Acad. Sci. USA, 1986, 83: 1453-1457; each of which is incorporated by reference in its entirety.
[00181] [00181] In some embodiments, the multispecific antibody comprises immunoglobulin chains with changes to reduce the formation of side products that do not have multispecificity. In some respects, antibodies comprise one or more "knobs-into-holes" modifications, as described in US Patent No. 5,731,168, incorporated by reference in their entirety.
[00182] [00182] In some embodiments, the multispecific antibody comprises immunoglobulin chains with one or more electrostatic modifications to promote the assembly of Fc hetero-multimers. See WO 2009/089004, incorporated by reference in its entirety.
[00183] [00183] In some embodiments, the multispecific antibody comprises a bispecific single chain molecule. See Traunecker et al., EMBO J., 1991, 10: 3655-3659; and Gruber et al., J. Immunol., 1994, 152: 5368-5374; each of which is incorporated by reference in its entirety.
[00184] [00184] In some embodiments, the multispecific antibody comprises a heavy chain variable domain and a light chain variable domain connected by a polypeptide linker, where the length of the linker is selected to promote the assembly of the multispecific antibody with the desired multispecificity. For example, monospecific scFvs generally form when a heavy chain variable domain and a light chain variable domain are connected by a polypeptide linker with more than 12 amino acid residues. See Pat. US Nos. 4,946,778 and 5,132,405, each of which is incorporated by reference in its entirety. In some embodiments, reducing the length of the polypeptide linker to less than 12 amino acid residues prevents the pairing of variable domains of the heavy and light chains on the same polypeptide chain, thus allowing the pairing of the variable domains of the heavy and light chains of a chain with complementary domains in another chain. The resulting antibody is therefore multispecific, with the specificity of each binding site contributed by more than one polypeptide chain. Polypeptide chains comprising variable domains of the heavy and light chains that are joined by linkers between 3 and 12 amino acid residues form predominantly dimers (called diabetes). With linkers between 0 and 2 amino acid residues, trimers (called triabodies) and tetramers (called tetrabodies) are preferred. However, the exact type of oligomerization appears to depend on the composition of the amino acid residue and the order of the variable domain in the polypeptide chain (for example, VH-ligand-VL vs. VL-ligand-VH), in addition to the length of the ligand. One skilled in the art can select the appropriate binder length based on the desired multispecificity. Glycosylation and related variants
[00185] [00185] An antibody provided in this document can be changed to increase, decrease or eliminate the extent to which it is glycosylated. Polypeptide glycosylation is typically "N-linked" or "O-linked." In some respects, antibody glycosylation is reduced by enzymatic deglycosylation, expression in a bacterial host, or modification of an amino acid residue used for glycosylation. Modifications such as mutations can be used to alter glycosylation.
[00186] [00186] "N-linked" glycosylation refers to the binding of the carbohydrate fraction of a side chain to an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for the enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site.
[00187] [00187] "O-linked" glycosylation refers to the attachment of one of the N-acetylgalactosamine, galactose or xylose sugars to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.
[00188] [00188] The addition or deletion of N-linked glycosylation sites to (or from) an antibody provided in this document can be accomplished by altering the amino acid sequence so that one or more of the tripeptide sequences described above are created or removed. The addition or deletion of O-linked glycosylation sites can be performed by adding, deleting or replacing one or more serine or threonine residues in or for (as the case may be) the sequence of an antibody.
[00189] [00189] In some embodiments, an antibody provided in this document comprises a glycosylation motif that is different from a naturally occurring antibody. Any suitable naturally occurring glycosylation motif can be modified in the antibody provided in this document. The structural and glycosylation properties of immunoglobulins, for example, are known in the art and summarized, for example, in Schroeder and Cavacini, J. Allergy Clin. Immunol., 2010, 125: S41-52, incorporated by reference in its entirety.
[00190] [00190] In some embodiments, an antibody provided in this document comprises an IgG1 Fc region with modification in the oligosaccharide attached to asparagine 297 (Asn 297). Naturally occurring IgG1 antibodies produced by mammalian cells typically comprise a branched biantenary oligosaccharide that is generally linked by an N bond to Asn 297 of the CH2 domain of the Fc region. See Wright et al., TIBTECH, 1997, 15: 26-32, incorporated by reference in its entirety. The oligosaccharide attached to Asn 297 can include various carbohydrates, such as mannose, N-acetyl glucosamine (GlcNAc), galactose and sialic acid, as well as a GlcNAc-linked fucose on the "stem" of the biantenaria oligosaccharide structure.
[00191] [00191] In some embodiments, the oligosaccharide attached to Asn 297 is modified to create antibodies with altered ADCC. In some embodiments, the oligosaccharide is altered to improve ADCC. In some embodiments, the oligosaccharide is altered to reduce ADCC.
[00192] [00192] In some respects, an antibody provided in this document comprises an IgG1 domain with reduced fucose content at the Asn 297 position compared to a naturally occurring IgG1 domain. These Fc domains are known to have improved ADCC. See Shields et al., J. Biol. Chem., 2002, 277: 26733-26740, incorporated by reference in its entirety. In some respects, these antibodies do not comprise fucose at the Asn 297 position. The amount of fucose can be determined using any suitable method, for example, as described in WO 2008/077546, incorporated by reference in its entirety.
[00193] [00193] In some embodiments, an antibody provided in this document comprises a bisected oligosaccharide, such as a biantenary oligosaccharide linked to the Fc region of the antibody that is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878; Pat. US No. 6,602,684; and Pat. US Pub. 2005/0123546; each of which is incorporated by reference in its entirety.
[00194] [00194] Other illustrative glycosylation variants that can be incorporated into an antibody provided herein are described, for example, in US Patent No. Nos. 2003/0157108, 2004/0093621, 2003/0157108, 2003/0115614, 2002 / 0164328, 2004/0093621, 2004/0132140, 2004/0110704, 2004/0110282, 2004/0109865; Pat. International Pub. No. 2000/61739, 2001/29246, 2003/085119, 2003/084570, 2005/035586, 2005/035778; 2005/053742, 2002/031140; Okazaki et al., J. Mol. Biol., 2004, 336: 1239-1249; and Yamane-Ohnuki et al., Biotech. Bioeng., 2004, 87: 614-622; each of which is incorporated by reference in its entirety.
[00195] [00195] In some embodiments, an antibody provided in this document comprises an Fc region with at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants can have improved CDC function. Examples of such antibody variants are described, for example, in WO 1997/30087; WO 1998/58964; and WO 1999/22764; each of which is incorporated by reference in its entirety.
[00196] [00196] Examples of cell lines capable of producing defucosylated antibody include Lec13 CHO cells, which are deficient in protein fucosylation (see Ripka et al., Arch. Biochem. Biophys., 1986, 249: 533-545; Pat. US Pub 2003/0157108; WO 2004/056312; each of which is incorporated by reference in its entirety) and knockout cell lines, such as the alpha-1,6-fucosyltransferase gene or FUT8 CHO knockout cells (see Yamane- Ohnuki et al., Biotech. Bioeng., 2004, 87: 614-622; Kanda et al., Biotechnol. Bioeng., 2006, 94: 680-688; and WO 2003/085107; each of which is incorporated by reference in your totality).
[00197] [00197] In some embodiments, an antibody provided in this document is an aglycosylated antibody. An aglycosylated antibody can be produced using any method known in the art or described in this document. In some respects, an aglycosylated antibody is produced by modifying the antibody to remove all glycosylation sites. In some respects, the glycosylation sites are removed only from the Fc region of the antibody. In some respects, an aglycosylated antibody is produced by expressing the antibody in an organism that is not capable of glycosylation, such as E. coli, or by expressing the antibody in a cell-free reaction mixture.
[00198] [00198] In some embodiments, an antibody provided in this document has a constant region with reduced effector function compared to a native IgG1 antibody. In some embodiments, the affinity of an Fc region constant of an antibody provided herein for the Fc receptor is less than the affinity of a native IgG1 constant region for that Fc receptor. Variants and Region Fc
[00199] [00199] In certain embodiments, an antibody provided in this document comprises an Fc region. In certain embodiments, an antibody provided in this document comprises an Fc region with one or more amino acid substitutions, insertions or deletions compared to a naturally occurring Fc region. In some respects, these substitutions, insertions or deletions produce antibodies with altered stability, glycosylation or other characteristics. In some ways, these substitutions, insertions or deletions produce aglycosylated antibody.
[00200] [00200] A "variant Fc region" or "engineered Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by virtue of at least one amino acid modification, preferably one or more substitution (s) of amino acids. Preferably, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or an origin polypeptide Fc region, for example, from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region in this document preferably will have at least about 80% homology with a native Fc sequence region and / or with a Fc region of a polypeptide of origin and, more preferably, at least about 90% homology to it, more preferably at least about 95% homology to it.
[00201] [00201] The variant Fc sequences for "dead Fc" can include three amino acid substitutions in the CH2 region to reduce the binding of FcRI to EU index positions 234, 235 and 237 (see Duncan et al., (1988 ) Nature 332: 563). Two amino acid substitutions at the complement C1q binding site at index positions EU 330 and 331 reduce complement fixation (see Tao et al., J. Exp. Med. 178: 661 (1993) and Canfield and Morrison, J Exp. Med. 173: 1483 (1991)). Substitution in human IgG1 of residues of IgG2 at positions 233-236 and residues of IgG4 at positions 327, 330 and 331 greatly reduces ADCC and CDC (see, for example, Armor KL. Et al., 1999 Eur J Immunol. 29 ( 8): 2613-24; and Shields RL. Et al., 2001. J Biol Chem. 276 (9): 6591-604).
[00202] [00202] IgG binding to the FcRs or C1q depends on residues located in the hinge region and the CH2 domain. Two regions of the CH2 domain are critical for binding Fc Rs and C1q, and have unique sequences in IgG2 and IgG4. Substitutions in human IgG1 or IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330 and 331 have been shown to significantly reduce ADCC and CDC. Numerous mutations have been made in the CH2 domain of human IgG1.
[00203] [00203] The triple substitution of amino acids L234A, L235A and G237A largely eliminates FcR and complements effector functions (see, for example, US20100266505).
[00204] [00204] In some embodiments, the Fc region was modified by choosing the expression host, enzymatic treatment of amino acid substitutions to have reduced glycosylation and binding to FcR, in relation to the native protein. Mutations that reduce binding to FcR include, without limitation, modification of glycosylation in asparagine 297 of the Fc domain, which is known to be necessary for the ideal interaction of FcR. For example, known amino acid substitutions include N297A or N297G, which results in the loss of a glycosylation site in the protein. Enzymatically deglycosylated Fc domains, recombinantly expressed antibodies in the presence of a glycosylation inhibitor and the expression of Fc domains in bacteria have a similar loss of glycosylation and consequent binding to FcRs.
[00205] [00205] The LALA variant, L234A / L235A, also significantly reduced the binding to FcR; as well as E233P / L234V / L235A / G236 + A327G / A330S / P331S. See, for example, Armor et al. (1999) Eur J Immunol. 29 (8): 2613-24. The set of mutations: K322A, L234A and L235A are sufficient to almost completely abolish the binding of FcR and C1q. A set of three mutations, L234F / L235E / P331S (called TM), has a very similar effect.
[00206] [00206] Other Fc variants are possible, including, without limitation, one in which a region capable of forming a disulfide bond is deleted, or in which certain amino acid residues are eliminated at the N-terminus in a native Fc form or is a methionine residue is added.
[00207] [00207] Fc can be in the form of native sugar chains, increased sugar chains compared to a native form or decreased sugar chains compared to the native form, or it can be in an aglycosylated or deglycosylated form. The increase, decrease, removal or other modification of the sugar chains can be achieved by methods common in the art, such as a chemical method, an enzymatic method or by expressing them in a genetically modified cell line. Such cell lines include microorganisms, for example, Pichia Pastoris, and mammalian cell lines, for example, CHO cells, which naturally express glycosylating enzymes. In addition, microorganisms or cells can be designed to express glycosylation enzymes, or they can be rendered unable to express glycosylation enzymes (see for example, Hamilton, et al, Science, 313: 1441 (2006); Kanda, et al, J Biotechnology, 130: 300 (2007); Kitagawa, et al, J. Biol Chem., 269 (27): 17872 (1994); Ujita-Lee et al., J. Biol. Chem., 264 (23): 13848 (1989); Imai-Nishiya, et al, BMC Biotechnology 7:84 (2007); and WO 07/055916). As an example of a cell engineered to have altered sialylation activity, the alpha-2,6-sialyltransferase 1 gene has been modified in Chinese hamster ovary cells and sf9 cells. The antibodies expressed by these engineered cells are thus sialylated by the exogenous gene product. An additional method for obtaining Fc molecules, having a modified amount of sugar residues, as compared to a plurality of native molecules includes separating said plurality of molecules into glycosylated and non-glycosylated fractions, for example, using affinity chromatography of lectin (see for example, WO 07/117505). The presence of particular glycosylation fractions has been shown to alter the function of immunoglobulins. For example, removal of sugar chains from an Fc molecule results in a marked decrease in binding affinity to the C1q part of the first C1 complement component and a decrease or loss in antibody-dependent cell-mediated cytotoxicity (ADCC) or dependent cytotoxicity complement (CDC), thereby not inducing unnecessary immune responses in vivo. Additional important modifications include sialylation and fucosylation: the presence of sialic acid in IgG has been correlated with anti-inflammatory activity (see for example, Kaneko, et al, Science 313: 760 (2006)), while the removal of fucose from IgG leads to improved ADCC activity (see, for example, Shoj-Hosaka, et al, J. Biochem., 140: 777 (2006)).
[00208] [00208] The term "antibody comprising the Fc region" refers to an antibody comprising an Fc region. Terminal C lysine (residue 447 according to the EU numbering system) from the Fc region can be removed, for example, during antibody purification or by recombinant engineering of the nucleic acid encoding the antibody. Consequently, an antibody having an Fc region can comprise an antibody with or without K447.
[00209] [00209] In some respects, the Fc region of an antibody provided in this document is modified to produce an antibody with altered affinity for an Fc receptor or an antibody that is more immunologically inert. In some embodiments, the antibody variants provided in this document have some, but not all, effector functions. Such antibodies can be useful, for example, when the half-life of the antibody is important in vivo, but when certain effector functions (for example, complement activation and ADCC) are unnecessary or deleterious.
[00210] [00210] In some embodiments, the Fc region of an antibody provided in this document is a human IgG4 Fc region comprising one or more of the S228P and L235E hinge stabilizing mutations. See Aalberse et al., Immunology, 2002, 105: 9-19, incorporated by reference in its entirety. In some embodiments, the IgG4 Fc region comprises one or more of the following mutations: E233P, F234V and L235A. See Armor et al., Mol. Immunol., 2003, 40: 585-593, incorporated by reference in its entirety. In some embodiments, the Fc IgG4 region comprises a deletion at the G236 position.
[00211] [00211] In some embodiments, the Fc region of an antibody provided herein is a human IgG1 Fc region comprising one or more mutations to reduce binding to the Fc receptor. In some respects, one or more mutations are in the selected residues of S228 (for example, S228A), L234 (for example, L234A), L235 (for example, L235A), D265 (for example, D265A) and N297 (for example , N297A). In some ways, the antibody comprises a PVA236 mutation. PVA236 means that the ELLG amino acid sequence, from amino acid position 233 to 236 of IgG1 or EFLG of IgG4, is replaced by PVA. See Pat. US No. 9,150,641, incorporated by reference in its entirety.
[00212] [00212] In some embodiments, the Fc region of an antibody provided in this document is modified as described in Armor et al., Eur. J. Immunol., 1999, 29: 2613-2624; WO 1999/058572; and / or Pat. U.K. App. No. 98099518; each of which is incorporated by reference in its entirety.
[00213] [00213] In some embodiments, the Fc region of an antibody provided herein is a human IgG2 Fc region comprising one or more of the A330S and P331S mutations.
[00214] [00214] In some embodiments, the Fc region of an antibody provided in this document has an amino acid substitution at one or more selected positions of 238, 265, 269, 270, 297, 327 and 329. See Pat. US No. 6,737,056, incorporated by reference in its entirety. Such Fc mutants include Fc mutants with substitutions at two or more of the amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 with alanine. See Pat. US No. 7,332,581, incorporated by reference in its entirety. In some embodiments, the antibody comprises an alanine at the amino acid position 265. In some embodiments, the antibody comprises an alanine at the amino acid position 297.
[00215] [00215] In certain embodiments, an antibody provided in this document comprises an Fc region with one or more amino acid substitutions that improve ADCC, such as a substitution at one or more of positions 298, 333 and 334 of the Fc region. In some embodiments, an antibody provided in this document comprises an Fc region with one or more amino acid substitutions at positions 239, 332 and 330, as described in Lazar et al., Proc. Natl. Acad. Sci. USA, 2006, 103: 4005-4010, incorporated by reference in its entirety.
[00216] [00216] In some embodiments, an antibody provided in this document comprises one or more changes that improve or decrease C1q and / or CDC binding. See Pat. US No. 6,194,551; WO 99/51642; and Idusogie et al., J. Immunol., 2000, 164: 4178-4184; each of which is incorporated by reference in its entirety.
[00217] [00217] In some embodiments, an antibody provided in this document comprises one or more changes to increase the half-life. Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn) are described, for example, in Hinton et al., J. Immunol., 2006, 176: 346-356; and Pat. US Pub. 2005/0014934; each of which is incorporated by reference in its entirety. These Fc variants include those with substitutions in one or more residues in the Fc region: 238, 250, 256, 265, 272, 286, 303, 305, 307, 311, 312, 314, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, 428 and 434 of an IgG.
[00218] [00218] In some embodiments, an antibody provided in this document comprises one or more variants of the Fc region, as described in Pat. US No.
[00219] [00219] Isolated nucleic acids encoding SIRP-ALPHA antibodies, vectors comprising nucleic acids and host cells comprising vectors and nucleic acids, as well as recombinant techniques for the production of antibodies are also provided.
[00220] [00220] In some embodiments, a nucleic acid sequence that encodes a sequence with at least about 50%, 60%, 70%, 80%, 90%, 95% or 99% identity is provided for an illustrative sequence provided in SEQ ID NOs: 1-36. In some embodiments, a nucleic acid sequence is provided that encodes a sequence provided in SEQ ID NOs: 1-36, with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acid substitutions. In some embodiments, an antibody provided in this document comprises a sequence with at least about 50%, 60%, 70%, 80%, 90%, 95% or 99% identity to an illustrative sequence provided in SEQ ID NOs: 37 -44. In some embodiments, an antibody provided in this document comprises a sequence provided in SEQ ID NOs: 37-44, with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 mutations. In some ways, amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to in this document as "variants." In some embodiments, these variants are derived from a sequence provided in this document, for example, by affinity maturation, site-directed mutagenesis, random mutagenesis or any other method known in the art or described in this document. In some embodiments, these variants are not derived from a sequence provided in this document and can, for example, be isolated again according to the methods provided in this document for obtaining antibodies.
[00221] [00221] For recombinant production of an antibody, the nucleic acids that encode it can be isolated and inserted into a replicable vector for later cloning (that is, amplification of DNA) or expression. In some aspects, the nucleic acid can be produced by homologous recombination, for example, as described in US Patent No. 5,204,244, incorporated by reference in its entirety.
[00222] [00222] Many of the different vectors are known in the art. The vector components generally include one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter and a transcription termination sequence, for example, as described in the US Patent No. 5,534,615, incorporated by reference in its entirety.
[00223] [00223] Illustrative examples of suitable host cells are provided below. These host cells are not intended to limit, and any suitable host cell can be used to produce the antibodies provided in this document.
[00224] Suitable host cells include any prokaryotic (for example, bacterial), lower eukaryotic (for example, yeast) or higher (for example, mammals) cells. Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia (E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (S. typhimurium), Serratia (S. marcescans), Shigella, Bacilli (B. subtilis and B. licheniformis), Pseudomonas (P. aeruginosa), and Streptomyces. A useful E. coli cloning host is E. coli 294, although other strains such as E. coli B, E. coli X1776, and E. coli W3110 are also suitable.
[00225] [00225] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeasts, are also suitable cloning or expression hosts for SIRP-ALPHA antibody coding vectors. Saccharomyces cerevisiae, or common baker's yeast, is a commonly used lower eukaryotic host microorganism. However, several other genera, species and lineages are available and useful, such as Schizosaccharomyces pombe, Kluyveromyces (K. lactis, K. fragilis, K. bulgaricus K. wickerhamii, K. waltii, K. drosophilarum, K. thermotolerans, and K. marxianus), Yarrowia, Pichia pastoris, Candida (C. albicans), Trichoderma reesia, Neurospora crassa, Schwanniomyces (S. occidentalis), and filamentous fungi such as, for example Penicillium, Tolypocladium, and Aspergillus (A. nidulans and A . niger).
[00226] [00226] Useful mammalian host cells include COS-7 cells, HEK293 cells; baby hamster kidney cells (BHK); Chinese hamster ovary (CHO); mouse Sertoli cells; African green monkey kidney cells (VERO-76) and the like.
[00227] [00227] Host cells used to produce a SIRP-ALPHA antibody can be grown in a variety of media. Commercially available media, such as, for example, Ham's F10, Minimum Essential Medium (MEM), RPMI-1640 and Dulbecco's Modified Eagle Medium (DMEM) are suitable for culturing host cells. In addition, any of the means described in
[00228] [00228] Any of these media can be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics, trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range) and glucose or an equivalent energy source. All other necessary supplements can also be included in appropriate concentrations that will be known to those skilled in the art.
[00229] [00229] Culture conditions, such as temperature, pH and the like, are those used previously with the host cell selected for expression, and will be evident to the person ordinarily skilled in the art.
[00230] [00230] When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space or directly secreted in the medium. If the antibody is produced intracellularly, as a first step, particulate debris, host cells or lysed fragments are removed, for example, by centrifugation or ultrafiltration. For example, Carter et al. (Bio / Technology, 1992, 10: 163-167; incorporated by reference in its entirety) describes a procedure for isolating antibodies that are secreted into the E. coli periplasmic space. Briefly, the cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 min. Cell debris can be removed by centrifugation.
[00231] [00231] In some embodiments, the antibody is produced in a cell-free system. In some aspects, the cellless system is an in vitro transcription and translation system, as described in Yin et al., MAbs, 2012, 4: 217- 225, incorporated by reference in its entirety. In some ways, the cell-free system uses a cell-free extract from a eukaryotic cell or a prokaryotic cell. In some ways, the prokaryotic cell is E. coli. The cell-free expression of the antibody can be useful, for example, where the antibody accumulates in a cell as an insoluble aggregate, or where the yields of periplasmic expression are low.
[00232] [00232] When the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon® or Millipore® Pellcon® ultrafiltration unit. A protease inhibitor, such as PMSF, can be included in any of the previous steps to inhibit proteolysis and antibiotics can be included to prevent the growth of adventitious contaminants.
[00233] [00233] The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, with affinity chromatography being a particularly useful purification technique. The suitability of protein A as an affinity linker depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies comprising human humanas1, 2 or 4 heavy chains (Lindmark et al., J. Immunol. Meth., 1983, 62: 1-13, incorporated by reference in its entirety ). G protein is useful for all mouse isotypes and for human γ3 (Guss et al., EMBO J., 1986, 5: 1567-1575, incorporated by reference in its entirety).
[00234] [00234] The matrix to which the affinity binder is attached is very often agarose, but other matrices are available. Mechanically stable matrices, such as controlled pore glass or poly (styrenodivinyl) benzene, allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, BakerBond ABX® resin is useful for purification.
[00235] [00235] Other techniques for protein purification are also available, such as fractionation on an ion exchange column, ethanol precipitation, reverse phase HPLC, silica chromatography, chromatography on Sepharose® heparin precipitation, chromatography, SDS-PAGE and precipitation with ammonium sulfate, and can be applied by one skilled in the art.
[00236] [00236] After any preliminary purification step (s), the mixture comprising the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2, 5 to about 4.5, generally carried out at low salt concentrations (for example, from about 0 to about 0.25 M of salt). SIRP-ALPHA Antibody Manufacturing Methods Preparation of the SIRP-ALPHA Antigen
[00237] [00237] The SIRP-ALPHA antigen used for isolation or creation of the antibodies provided in this document can be intact SIRP-ALPHA or a fragment of SIRP-ALPHA. The SIRP-ALPHA antigen can be, for example, in the form of an isolated protein or a protein expressed on the surface of a cell.
[00238] [00238] In some embodiments, the SIRP-ALPHA antigen is a non-naturally occurring variant of SIRP-ALPHA, such as a SIRP-ALPHA protein with an amino acid sequence or post-translational modification that does not occur in nature.
[00239] [00239] In some embodiments, the SIRP-ALPHA antigen is truncated by removing, for example, intracellular or membrane extension sequences or signal sequences. In some embodiments, the SIRP-ALPHA antigen is fused at Terminal C therefrom to a human IgG1 Fc domain or to a polyhistidine tag. Monoclonal Antibody Manufacturing Methods
[00240] [00240] Monoclonal antibodies can be obtained, for example, using the hybridoma method first described by Kohler et al., Nature, 1975, 256: 495-497 (incorporated by reference in its entirety) and / or by methods recombinant DNA (see, for example, US Patent No. 4,816,567, incorporated by reference in its entirety). Monoclonal antibodies can also be obtained, for example, using phage or yeast based libraries. See, for example, US Patent Nos. 8,258,082 and 8,691,730, each of which is incorporated by reference in its entirety.
[00241] [00241] In the hybridoma method, a mouse or other appropriate host animal is immunized to cause lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent, such as polyethylene glycol, to form a hybridoma cell. See Goding J.W., Monoclonal Antibodies: Principles and Practice, 3rd ed. (1986), Academic Press, San Diego, CA, incorporated by reference in its entirety.
[00242] [00242] Hybridoma cells are thus seeded and grown in a suitable culture medium, a medium that contains one or more substances that inhibit the growth or survival of the unfused precursor myeloma cells. For example, if precursor myeloma cells lack the hypoxanthine guanine phosphoribosyl transferase enzyme (HGPRT or HPRT), the culture medium for hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), substances that prevent the growth of HGPRT-deficient cells.
[00243] [00243] Useful myeloma cells are those that fuse efficiently, support stable high-level antibody production by selected antibody-producing cells and are sensitive media conditions, such as the presence or absence of HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOP-21 and MC-11 mouse tumors (available from the Salk Institute Cell Distribution Center, San Diego, CA) and SP cells -2 or X63-Ag8-653 (available from the American Type Culture Collection, Rockville, MD). Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies. See, for example, Kozbor, J. Immunol., 1984, 133: 3001, incorporated by reference in its entirety.
[00244] [00244] After identifying hybridoma cells that produce antibodies of the desired specificity, affinity and / or biological activity, the selected clones can be subcloned by limiting dilution procedures and cultured by standard methods. See Goding, supra. Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be cultured in vivo as ascites tumors in an animal.
[00245] [00245] DNA encoding the monoclonal antibody can be easily isolated and sequenced using conventional procedures (for example, using oligonucleotide probes that are able to specifically bind to genes encoding the heavy and light chains of the monoclonal antibodies). Thus, hybridoma cells can serve as a useful source of antibodies that encode DNA with the desired properties. Once isolated, DNA can be placed in expression vectors, which are then transfected into host cells such as bacteria (eg, E. coli), yeast (eg, Saccharomyces or Pichia Sp., COS cells, cells from Chinese hamster ovary (CHO) or myeloma cells that do not produce antibodies, to produce monoclonal antibodies.
[00246] [00246] Illustrative methods for producing chimeric antibodies are described, for example, in US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 1984, 81: 6851-6855; each of which is incorporated by reference in its entirety. In some embodiments, a chimeric antibody is produced using recombinant techniques to combine a non-human variable region (for example, a variable region derived from a mouse, rat, hamster, rabbit or non-human primate, such as a monkey) with a human constant region . Humanized Antibody Manufacturing Methods
[00247] [00247] Humanized antibodies can be generated by replacing most or all of the structural fractions of a non-human monoclonal antibody with the corresponding human antibody sequences. Consequently, a hybrid molecule is generated in which only the antigen-specific variable, or CDR, is composed of a non-human sequence. Methods for obtaining humanized antibodies include those described in, for example, Winter and Milstein, Nature, 1991, 349: 293-299; Rader et al., Proc. Nat. Acad. Sci. U.S.A., 1998, 95: 8910-8915; Steinberger et al., J. Biol. Chem., 2000, 275: 36073- 36078; Queen et al., Proc. Natl. Acad. Sci. U.S.A., 1989, 86: 10029-10033; US Patent No. 5,585,089; 5,693,761; 5,693,762; and 6,180,370; each of which is incorporated by reference in its entirety. Methods of making human antibodies
[00248] [00248] Human antibodies can be generated by a variety of techniques known in the art, for example using transgenic animals (for example, humanized mice). See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 1993, 90: 2551; Jakobovits et al., Nature, 1993, 362: 255-258; Bruggermann et al., Year in Immuno., 1993, 7:33; and US Patent Nos. 5,591,669,
[00249] [00249] The antibody fragments provided in this document can be produced by any suitable method, including the illustrative methods described in this document or those known in the art. Suitable methods include recombinant techniques and proteolytic digestion of complete antibodies. Illustrative methods for producing antibody fragments are described, for example, in Hudson et al., Nat. Med., 2003, 9: 129-134, incorporated by reference in their entirety. Methods for producing scFv antibodies are described, for example, in Plückthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994); WO 93/16185; and Pat. US Nos. 5,571,894 and 5,587,458; each of which is incorporated by reference in its entirety. Alternative scaffolding manufacturing methods
[00250] [00250] The alternative scaffolding structures provided in this document can be made by any suitable method, including the illustrative methods described in this document or those known in the art. For example, methods of preparing AdnectinsTM are described in Emanuel et al., MAbs, 2011, 3: 38-48, incorporated by reference in their entirety. Methods of preparing iMabs are described in U.S. Pat. US Pub. No. 2003/0215914, incorporated by reference in its entirety. Methods of preparing Anticalins® are described in Vogt and Skerra, Chem. Biochem., 2004, 5: 191-199, incorporated by reference in its entirety. Methods for preparing Kunitz domains are described in Wagner et al., Biochem. & Biophys. Comm., 1992, 186: 118-1145, incorporated by reference in its entirety. Methods of preparing thiorretoxin peptide aptamers are provided in Geyer and Brent, Meth. Enzymol., 2000, 328: 171-208, incorporated by reference in its entirety. Methods of preparing Affibodies are provided in Fernandez, Curr. Opinion in Biotech., 2004, 15: 364-373, incorporated by reference in its entirety. Methods of preparing DARPins are provided in Zahnd et al., J. Mol. Biol., 2007, 369: 1015-1028, incorporated by reference in their entirety. The methods of preparing Affilins are provided in Ebersbach et al., J. Mol. Biol., 2007, 372: 172-185, incorporated by reference in their entirety. Methods of preparing Tetranectins are provided in Graversen et al., J. Biol. Chem., 2000, 275: 37390-37396, incorporated by reference in its entirety. The methods of preparing Avimers are provided in Silverman et al., Nature Biotech., 2005, 23: 1556-1561, incorporated by reference in their entirety. Methods for preparing Fynomers are provided in Silacci et al., J. Biol. Chem., 2014, 289: 14392-14398, incorporated by reference in its entirety. Additional information on alternative scaffolding structures is provided in Binz et al., Nat. Biotechnol., 2005 23: 1257-1268; and Skerra, Current Opin. in Biotech., 2007 18: 295-304, each of which is incorporated by reference in its entirety. Methods of making multispecific antibodies
[00251] [00251] The alternative multispecific antibodies provided in this document can be made by any suitable method, including the illustrative methods described in this document or those known in the art. Methods for producing common light chain antibodies are described in Merchant et al., Nature Biotechnol., 1998, 16: 677-681, incorporated by reference in their entirety. Methods for producing bispecific tetravalent antibodies are described in Coloma and Morrison, Nature Biotechnol., 1997, 15: 159-163, incorporated by reference in their entirety. Methods for producing hybrid immunoglobulins are described in Milstein and Cuello, Nature, 1983, 305: 537-540; and Staerz and Bevan, Proc. Natl. Acad. Sci. USA, 1986, 83: 1453-1457; each of which is incorporated by reference in its entirety. Methods of producing immunoglobulins with modification of knobs-into-holes are described in US Patent No. 5,731,168, incorporated by reference in their entirety. Methods of producing immunoglobulins with electrostatic modifications are provided in WO 2009/089004, incorporated by reference in their entirety. Methods for producing bispecific single chain antibodies are described in Traunecker et al., EMBO J., 1991, 10: 3655-3659; and Gruber et al., J. Immunol., 1994, 152: 5368-5374; each of which is incorporated by reference in its entirety. Methods of producing single chain antibodies, the length of which may vary, are described in US Patent No. 4,946,778 and
[00252] [00252] Any suitable method can be used to introduce variability into a polynucleotide sequence encoding an antibody, including error-prone PCR, chain shuffling and oligonucleotide-directed mutagenesis, such as trinucleotide-directed mutagenesis (TRIM). In some respects, several CDR residues (for example, 4-6 residues at a time) are randomized. The CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted by mutation.
[00253] [00253] The introduction of diversity in variable regions and / or CDRs can be used to produce a secondary library. The secondary library is then searched to identify antibody variants with enhanced affinity. Affinity maturation through the construction and reselection of secondary libraries has been described, for example, in Hoogenboom et al., Methods in Molecular Biology, 2001, 178: 1-37, incorporated by reference in its entirety. Essay
[00254] [00254] A variety of assays known in the art can be used to identify and characterize a SIRP-ALPHA antibody provided in this document. Binding, competition and epitope mapping assays
[00255] [00255] The specific antigen-binding activity of an antibody provided in this document can be evaluated by any suitable method, including the use of SPR, BLI, RIA and MSD-SET, as described elsewhere in this disclosure. In addition, antigen-binding activity can be assessed by ELISA assays and Western blot assays.
[00256] [00256] Assays to measure competition between two antibodies, or an antibody and another molecule (for example, one or more SIRP-ALPHA ligands) are described elsewhere in this disclosure and, for example, in Harlow and Lane, Antibodies : A Laboratory Manual, chapter 14, 1988, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, incorporated by reference in its entirety.
[00257] [00257] The assays for mapping the epitopes to which an antibody provided in this document bind are described, for example, in Morris "Epitope Mapping Protocols", in Methods in Molecular Biology vol. 66, 1996, Humana Press, Totowa, NJ, incorporated by reference in its entirety. In some embodiments, the epitope is determined by peptide competition. In some embodiments, the epitope is determined by mass spectrometry. In some embodiments, the epitope is determined by crystallography. Tests for Effector Functions
[00258] [00258] The effector function after treatment with an antibody provided in this document can be evaluated using a variety of in vitro and in vivo assays known in the art, including those described in Ravetch and Kinet, Annu. Rev. Immunol., 1991, 9: 457-492; U.S. Pat. Nos. 5,500,362 and 5,821,337; Hellstrom et al., Proc. Nat’l Acad. Sci. USA, 1986, 83: 7059-7063; Hellstrom et al., Proc. Nat’l Acad. Sci. USA, 1985, 82: 1499-1502; Bruggemann et al., J. Exp. Med., 1987, 166: 1351-1361; Clynes et al., Proc. Nat’l Acad. Sci. USA, 1998, 95: 652-656; WO 2006/029879; WO 2005/100402; Gazzano-Santoro et al., J. Immunol. Methods, 1996, 202: 163-171; Cragg et al., Blood, 2003, 101: 1045-1052; Cragg et al. Blood, 2004, 103: 2738-2743; and Petkova et al., Int'l. Immunol., 2006, 18: 1759-1769; each of which is incorporated by reference in its entirety. Pharmaceutical Compositions
[00259] [00259] An antibody provided in this document can be formulated in any appropriate pharmaceutical composition and administered by any suitable route of administration. Suitable routes of administration include, but are not limited to, intra-arterial, intradermal, intramuscular, intraperitoneal, intravenous, nasal, parenteral, pulmonary and subcutaneous routes.
[00260] [00260] The pharmaceutical composition can comprise one or more pharmaceutical excipients. Any suitable pharmaceutical excipient can be used, and one skilled in the art is able to select suitable pharmaceutical excipients. Consequently, the pharmaceutical excipients provided below are intended to be illustrative and not limiting. Additional pharmaceutical excipients include, for example, those described in the Handbook of Pharmaceutical Excipients, Rowe et al. (Eds.), 6th Ed. (2009), incorporated by reference in its entirety.
[00261] [00261] In some embodiments, the pharmaceutical composition comprises an anti-foam agent. Any suitable antifoaming agent can be used. In some respects, the antifoam agent is selected from an alcohol, an ether, an oil, a wax, a silicone, a surfactant and combinations of these. In some respects, the antifoaming agent is selected from a mineral oil, a vegetable oil, ethylene bis-stearamide, a paraffin wax, an ester wax, a fatty alcohol wax, a long-chain fatty alcohol, a soap fatty acid, a fatty acid ester, a silicon glycol, a fluorosilicone, a polyethylene glycol-polypropylene glycol copolymer, polydimethylsiloxane-silicon dioxide, ether, octy alcohol, caprylic alcohol, sorbitan trioleate, ethyl alcohol, 2-ethylhexanol , dimethicone, oleyl alcohol, simethicone, and combinations thereof.
[00262] [00262] In some embodiments, the pharmaceutical composition comprises a cosolvent. Illustrative examples of cosolvents include ethanol, poly (ethylene) glycol, butylene glycol, dimethylacetamide, glycerin, propylene glycol and combinations thereof.
[00263] [00263] In some embodiments, the pharmaceutical composition comprises a buffer. Illustrative examples of buffers include acetate, borate, carbonate, lactate, malate, phosphate, citrate, hydroxide, diethanolamine, monoethanolamine, glycine, methionine, guar gum, monosodium glutamate and combinations thereof.
[00264] [00264] In some embodiments, the pharmaceutical composition comprises a carrier or filler. Illustrative examples of carriers or fillers include lactose, maltodextrin, mannitol, sorbitol, chitosan, stearic acid, xanthan gum and guar gum.
[00265] [00265] In some embodiments, the pharmaceutical composition comprises a surfactant. Illustrative examples of surfactants include d-alpha tocopherol, benzalkonium chloride, benzethonium chloride, cetrimide, cetylpyridinium chloride, sodium docusate, glyceryl behenate, glyceryl monooleate, lauric acid, macrogol hydroxystearate 15, myristyl alcohol, phospholipids, ether of polyoxyethylene alkyl, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, polyoxylglycerides, sodium lauryl sulfate, sorbitan esters and vitamin E polyethylene (glycol) succinate
[00266] [00266] In some embodiments, the pharmaceutical composition comprises an anti-caking agent. Illustrative examples of anti-caking agents include calcium phosphate (tribasic), hydroxymethylcellulose, hydroxypropylcellulose, magnesium oxide and combinations thereof.
[00267] [00267] Other excipients that can be used with the pharmaceutical compositions include, for example, albumin, antioxidants, antibacterial agents, antifungal agents, bioabsorbable polymers, chelating agents, controlled release agents, diluents, dispersing agents, dissolution enhancers, emulsifying agents , gelling agents, ointment bases, penetration enhancers, preservatives, solubilizing agents, solvents, stabilizing agents, sugars and combinations thereof. Specific examples of each of these agents are described, for example, in the Handbook of Pharmaceutical Excipients, Rowe et al. (Eds.), 6th Ed. (2009), The Pharmaceutical Press, incorporated by reference in its entirety.
[00268] [00268] In some embodiments, the pharmaceutical composition comprises a solvent. In some respects, the solvent is saline, such as sterile isotonic saline or dextrose solution. In some ways, the solvent is water for injection.
[00269] [00269] In some embodiments, the pharmaceutical compositions are in a particulate form, such as a microparticle or a nanoparticle. Microparticles and nanoparticles can be formed from any suitable material, such as a polymer or a lipid. In some respects, microparticles or nanoparticles are micelles, liposomes or polymerases.
[00270] [00270] Anhydrous pharmaceutical compositions and dosage forms comprising an antibody are also provided in this document, since water can facilitate the degradation of some antibodies.
[00271] [00271] The anhydrous pharmaceutical compositions and dosage forms provided in this document can be prepared using anhydrous or low humidity containing ingredients and low humidity or low humidity conditions. Pharmaceutical compositions and dosage forms that comprise lactose and at least one active ingredient that comprises a primary or secondary amine can be anhydrous if substantial contact with hydration and / or moisture during manufacture, packaging and / or storage is expected.
[00272] [00272] An anhydrous pharmaceutical composition must be prepared and stored in such a way that its anhydrous nature is maintained. Thus, anhydrous compositions can be packaged using known materials to prevent exposure to water, such that they can be included in kits of appropriate formulas. Examples of suitable packaging include, but are not limited to, hermetically sealed sheets, plastics, unit dose containers (eg vials), blister packs and strip packs.
[00273] [00273] In certain embodiments, an antibody provided in this document is formulated as parenteral dosage forms. Parenteral dosage forms can be administered to subjects in a variety of ways, including, but not limited to, subcutaneous, intravenous (including infusions and bolus injections), intramuscular and intraarterial. Because their administration typically exceeds the subjects' natural defenses against contaminants, parenteral dosage forms are typically sterile or capable of being sterilized prior to administration to a subject. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products (i.e., lyophilized) ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection and emulsions.
[00274] [00274] Suitable vehicles that can be used to provide parenteral dosage forms are well known to those skilled in the art. Examples include, but are not limited to: Water for Injection USP; aqueous vehicles such as, but not limited to Sodium Chloride Injection, Ringer Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer Injection; water miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
[00275] [00275] Excipients that increase the solubility of one or more of the antibodies disclosed in this document can also be incorporated into parenteral dosage forms.
[00276] [00276] In some embodiments, the parenteral dosage form is lyophilized. Exemplary lyophilized formulations are described, for example, in US Patent Nos. 6,267,958 and 6,171,586; and WO 2006/044908; each of which is incorporated by reference in its entirety.
[00277] [00277] In human therapy, the doctor will determine the dosage that he considers most appropriate according to a preventive or curative treatment and according to the age, weight, condition and other factors specific to the subject to be treated.
[00278] [00278] In certain embodiments, a composition provided in this document is a pharmaceutical composition or a single unit dosage form. The pharmaceutical compositions and single dosage forms provided herein comprise a prophylactic or therapeutically effective amount of one or more prophylactic or therapeutic antibodies.
[00279] [00279] The amount of antibody or composition that will be effective in preventing or treating a disorder or one or more symptoms thereof will vary with the nature and severity of the disease or condition and the route through which the antibody is administered. The frequency and dosage also vary according to the specific factors for each subject, depending on the specific therapy (for example, therapeutic or prophylactic agents) administered, the severity of the disorder, disease or condition, the route of administration, as well as the age , body weight, response and the subject's past medical history. Effective doses can be extrapolated from dose-response curves derived from in vitro test systems or animal models.
[00280] [00280] Different therapeutically effective amounts may be applicable for different diseases and conditions, as will be easily known to those skilled in the art. Likewise, amounts sufficient for the prevention, administration, treatment or amelioration of such disorders, but insufficient to cause or sufficient to reduce the adverse effects associated with the antibodies provided in this document are also covered by the dosage amounts and dose frequency schedules provided in this document. In addition, when a subject is administered multiple dosages of a composition provided in this document, not all dosages need to be the same. For example, the dosage administered to the subject can be increased to improve the prophylactic or therapeutic effect of the composition, or it can be decreased to reduce one or more side effects that a particular subject is experiencing.
[00281] [00281] In certain modalities, treatment or prevention can be started with one or more loading doses of an antibody or composition provided in this document, followed by one or more maintenance doses.
[00282] [00282] In certain embodiments, a dose of an antibody or composition provided in this document can be administered to achieve a steady-state concentration of the antibody in the subject's blood or serum. The steady-state concentration can be determined by measurement according to the techniques available to those skilled in the art, or it can be based on the subject's physical characteristics, such as height, weight and age.
[00283] [00283] As discussed in more detail elsewhere in this disclosure, an antibody provided in this document can optionally be administered with one or more additional agents useful to prevent or treat a disease or disorder. The effective amount of these additional agents may depend on the amount of antibody present in the formulation, the type of disorder or treatment and the other factors known in the art or described in this document. Therapeutic Applications
[00284] [00284] For therapeutic applications, the antibodies of the invention are administered to a mammal, generally a human being, in a pharmaceutically acceptable dosage form such as those known in the art and those discussed above. For example, the antibodies of the invention can be administered to a human intravenously as a bolus or by continuous infusion over a period of time, intramuscularly, intraperitoneally, intra-cerebrospinally, subcutaneously, intra-articularly, intrasynovially, intrathecally or intratumoral. Antibodies are also adequately administered via peritumoral, intralesional or perilesional routes, to exert local and systemic therapeutic effects. The intraperitoneal route can be particularly useful, for example, in the treatment of ovarian tumors.
[00285] [00285] The antibodies provided in this document may be useful for the treatment of any disease or condition involving SIRP-ALPHA. In some embodiments, the disease or condition is a disease or condition that can benefit from treatment with an anti-SIRP-ALPHA antibody. In some embodiments, the disease or condition is a tumor. In some embodiments, the disease or condition is a cell proliferative disorder. In some embodiments, the disease or condition is cancer. In some embodiments, the disease or condition is an infection.
[00286] [00286] Examples of symptoms, illnesses and / or diseases that can be treated with an anti-SIRPα antibody in question include, but are not limited to, cancer (any form of cancer, including but not limited to: carcinomas, tissue tumors moles, sarcomas, teratomas, melanomas, leukemias, lymphomas, brain cancers, solid tumors, mesothelioma (MSTO), etc.); infection (for example, chronic infection); and an immune disease or disorder (e.g., an inflammatory disease) (e.g., multiple sclerosis, arthritis and the like, e.g., for immunosuppressive therapy). An anti-SIRPα antibody in question can also be used for transplant conditioning (for example, stem cell transplantation, bone marrow transplantation, etc.) (for example, to destroy malignant cells, to provide immunosuppression to prevent the body from rejects stem / donor cells, etc.). For example, in some cases, a combination of the antibodies in question or a bispecific antibody (for example, anti-SIRPα in combination with anti-CD117) finds use in transplant conditioning. For example, a combination of the antibodies in question or bispecific antibody (for example, anti-SIRPα in combination with anti-CD117) can be used for conditioning bone marrow transplantation. In some cases, an anti-SIRPα antibody in question (for example, a combination of antibodies) can be used for immunosuppressive therapy.
[00287] [00287] In some embodiments, the antibodies provided in this document are provided for use as a medicine. In some embodiments, the antibodies provided in this document are provided for use in the manufacture or preparation of a drug. In some modalities, the drug is for the treatment of a disease or condition that may benefit from an anti-SIRP-ALPHA antibody. In some embodiments, the disease or condition is a tumor. In some embodiments, the disease or condition is a cell proliferative disorder. In some embodiments, the disease or condition is cancer. In some embodiments, the disease or condition is an infection. A disease or condition can be: cancer; infection; a viral infection; a bacterial infection; a fungal infection; fibrosis; arteriosclerosis; a parasitic infection, optionally malaria; and / or depletion or reduction of endogenous bone marrow-forming stem cells to allow radiation and / or chemotherapy - free or reduced conditioning for transplantation of blood-forming stem cells, optionally in combination with the anti-CKIT antibody (CD117 ).
[00288] [00288] In some embodiments, a method of treating a disease or condition in a subject in need thereof is provided in this document by administering an effective amount of an antibody provided in this document to the subject. In some embodiments, the disease or condition is cancer. In some ways, the disease or condition is an infection.
[00289] [00289] In some embodiments, a method of treating a disease or condition in a subject in need of this subject is provided in this document by administering an effective amount of an antibody provided in this document to the subject, in which the disease or condition is a cancer and the cancer is selected from a solid tumor and a hematological tumor.
[00290] [00290] In some embodiments, a method of increasing phagocytosis in a subject in need thereof is provided in this document, comprising administering to the subject an effective amount of an antibody disclosed in this document or a pharmaceutical composition disclosed in this document.
[00291] [00291] In some embodiments, a method of modulating the immune response in a subject in need thereof is provided in this document, comprising administering to the subject an effective amount of an antibody disclosed in this document or a pharmaceutical composition disclosed in this document.
[00292] [00292] Any suitable cancer can be treated with the antibodies provided in this document.
[00293] [00293] For example, any cancer, in which cancer cells exhibit increased expression of CD47 compared to non-cancer cells, is a cancer suitable to be treated by the methods and compositions in question. As used herein, "cancer" includes any form of cancer, including, but not limited to, solid cancers of tumors (e.g., lung, prostate, breast, bladder, colon, ovary, pancreas,
[00294] [00294] Carcinomas are malignant diseases that originate in the epithelial tissues. Epithelial cells cover the outer surface of the body, line the internal cavities and form the lining of glandular tissues. Examples of carcinomas include, but are not limited to: adenocarcinoma (cancer that begins in glandular (secretory) cells), for example, breast, pancreas, lung, prostate and colon cancer can be adenocarcinomas; adrenocortical carcinoma; hepatocellular carcinoma; renal cell carcinoma; ovarian carcinoma; carcinoma in situ; ductal carcinoma; breast carcinoma; Basal cell carcinoma; squamous cell carcinoma; transition cell carcinoma; colon carcinoma; nasopharyngeal carcinoma; multilocular cystic renal cell carcinoma; oat grain cell carcinoma; large cell lung carcinoma; small cell lung carcinoma; non-small cell lung carcinoma; and the like. Carcinomas can be found in the prostate, pancreas, colon, brain (usually as secondary metastases), lung, breast, skin, etc.
[00295] [00295] Soft tissue tumors are a highly diverse group of rare tumors derived from connective tissue. Examples of soft tissue tumors include, but are not limited to: soft tissue alveolar sarcoma; fibrous angiomatoid histiocytoma; chondromyxoid fibroma; skeletal chondrosarcoma; extra-skeletal myxoid chondrosarcoma; clear cell sarcoma; desmoplastic small round cell tumor; protruding dermatofibrosarcoma; endometrial stromal tumor; Ewing's sarcoma; fibromatosis
[00296] [00296] A sarcoma is a rare type of cancer that arises in cells of mesenchymal origin, for example, in the bones or soft tissues of the body, including cartilage, fat, muscle, blood vessels, fibrous tissue or other connective or supporting tissue . Different types of sarcoma are based on where the cancer forms. For example, osteosarcoma forms in bone, liposarcoma forms in fat and rhabdomyosarcoma forms in muscle. Examples of sarcomas include, but are not limited to: Askin's tumor; botryoid sarcoma; chondrosarcoma; Ewing's sarcoma; malignant hemangioendothelioma; malignant schwannoma; osteosarcoma; soft tissue sarcomas (for example, alveolar soft tissue sarcoma; angiosarcoma; phylloid cystosarcoma; protuberant dermatofibrosarcoma (DFSP); desmoid tumor; small round cell desmoplastic tumor; epithelioid sarcoma; extraskeletal chondrosomal tumor; GIST); hemangiopericytoma; hemangiosarcoma (more commonly referred to as "angiosarcoma"); Kaposi's sarcoma; leiomyosarcoma; liposarcoma; lymphangiosarcoma; malignant tumor of the peripheral nerve sheath (MPNST); neurofibrosarcoma; similar synovial sarcoma;
[00297] [00297] A teratoma is a type of germ cell tumor that may contain several different types of tissue (for example, it may include tissues derived from any and / or all three germ layers: endoderm, mesoderm and ectoderm), including, for example, hair, muscle and bone. Teratomas occur most often in women's ovaries, testicles in men and coccyx in children.
[00298] [00298] Melanoma is a form of cancer that begins in melanocytes (cells that produce the pigment melanin). It can start on a wart (skin melanoma), but also on other pigmented tissues, such as the eye or the intestine.
[00299] [00299] Leukemias are cancers that start in blood-forming tissue, such as bone marrow, and cause a large number of abnormal blood cells to be produced and enter the bloodstream. For example, leukemias can originate in cells derived from bone marrow that normally mature in the bloodstream. Leukemias are named for how quickly the disease develops and progresses (for example, acute versus chronic) and the type of white blood cell that is made (for example, myeloid versus lymphoid). Myeloid leukemias are also called myelogenous or myeloblastic leukemias. Lymphoid leukemias are also called lymphoblastic or lymphocytic leukemia. Lymphoid leukemia cells can accumulate in the lymph nodes, which can become swollen. Examples of leukemias include, but are not limited to: acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL).
[00300] [00300] Lymphomas are cancers that start in the cells of the immune system. For example, lymphomas can originate in cells derived from bone marrow that normally mature in the lymphatic system. There are two basic categories of lymphoma. One type is Hodgkin's lymphoma (HL), marked by the presence of a type of cell called the Reed-Sternberg cell. Currently, there are 6 recognized types of HL. Examples of Hodgkin's lymphomas include: classic nodular sclerosis (CHL) Hodgkin's lymphoma, mixed cellularity CHL, lymphocyte depletion CHL, lymphocyte-rich CHL and predominant nodular lymphocyte HL.
[00301] [00301] The other category of lymphoma is non-Hodgkin's lymphoma (NHL), which includes a large and diverse group of immune cell cancers. Non-Hodgkin's lymphomas can be divided into cancers with an indolent (slow-growing) course and those with an aggressive (fast-growing) course. Currently, there are 61 recognized types of NHL. Examples of non-Hodgkin's lymphomas include, but are not limited to: AIDS-related lymphomas, anaplastic large cell lymphoma, angioimmunoblastic lymphoma, blast NK cell lymphoma, Burkitt's lymphoma, Burkitt-type lymphoma (small non-cleaved lymphoma), chronic lymphocytic leukemia / small lymphocytic lymphoma, cutaneous T cell lymphoma, diffuse large B cell lymphoma, enteropathy T cell lymphoma, follicular lymphoma, hepatosplenic gamma-delta T cell lymphoma, T cell leukemia, lymphoblastic lymphoma, lymphoma mantle cell, marginal zone lymphoma, nasal T cell lymphoma, pediatric lymphoma, peripheral T cell lymphoma, primary central nervous system lymphoma, transformed lymphoma, treatment-related T cell lymphoma and Waldenstrom macroglobulinemia.
[00302] [00302] Brain cancers include any cancer of brain tissues. Examples of brain cancer include, but are not limited to: gliomas (e.g., glioblastomas, astrocytomas, oligodendrogliomas, ependymomas and the like), meningiomas, pituitary adenomas, vestibular schwannomas, primitive neuroectodermal tumors (medulloblastomas), etc.
[00303] [00303] As used in this document, the term "infection" refers to any condition in at least one cell of an organism (i.e., a subject) that is infected by an infectious agent (for example, a subject has an infection by intracellular pathogen, for example, a chronic intracellular pathogen infection). As used herein, the term "infectious agent" refers to a foreign biological entity (i.e., a pathogen) that induces the expression of CD47 (e.g., increased expression of CD47) in at least one cell in the infected organism. For example, infectious agents include, but are not limited to, bacteria, viruses, protozoa and fungi. Intracellular pathogens are of particular interest. Infectious diseases are disorders caused by infectious agents. Some infectious agents do not cause recognizable symptoms or illness under certain conditions, but have the potential to cause symptoms or illness under altered conditions. The methods in question can be used in the treatment of chronic pathogenic infections, for example, including but not limited to viral infections, for example, retrovirus, lentivirus, hepadna virus, herpes virus, pox virus, human papilloma virus, etc .; intracellular bacterial infections, for example Mycobacterium, Chlamydophila, Ehrlichia, Rickettsia, Brucella, Legionella, Francisella, Listeria, Coxiella, Neisseria, Salmonella, Yersinia sp, etc .; and intracellular protozoan pathogens, for example, Plasmodium sp, Trypanosoma sp., Giardia sp., Toxoplasma sp., Leishmania sp., etc.
[00304] [00304] In some embodiments, a method of antagonizing SIRP-ALPHA in a target cell of a subject in need thereof is provided in this document by administering an effective amount of an antibody provided in this document to the subject.
[00305] [00305] In some embodiments, a method of enhancing the immune response in a subject in need of this subject is provided in this document by administering an effective amount of an antibody provided in this document to the subject.
[00306] [00306] In some embodiments, this method provides a method of delaying the onset of a tumor in a subject in need of it, by administering an effective amount of an antibody provided in this document to the subject.
[00307] [00307] In some embodiments, a method of preventing the recurrence or beginning of a tumor in a subject in need of this document is provided in this document, by administering an effective amount of an antibody provided in this document to the subject.
[00308] [00308] In some embodiments, this method provides a method of delaying the onset of cancer in a subject in need of it, by administering an effective amount of an antibody provided in this document to the subject.
[00309] [00309] In some embodiments, a method of preventing the recurrence or onset of cancer in a subject in need is provided in this document by administering an effective amount of an antibody provided in this document to the subject.
[00310] [00310] In some embodiments, this method provides a method of reducing the size of a tumor in a subject in need of it, by administering an effective amount of an antibody provided in this document to the subject.
[00311] [00311] In some embodiments, a method of reducing the number of metastases in a subject in need of this subject is provided in this document by administering an effective amount of an antibody provided in this document to the subject.
[00312] [00312] In some embodiments, a method of delaying the onset of infection in a subject in need of this is provided in this document by administering an effective amount of an antibody provided in this document to the subject.
[00313] [00313] In some embodiments, a method of preventing the recurrence or onset of infection in a subject in need of this subject is provided in this document by administering an effective amount of an antibody provided in this document to the subject.
[00314] [00314] In some embodiments, this method provides a method of reducing the viral titer in a subject in need of it, by administering an effective amount of an antibody provided in this document to the subject.
[00315] [00315] In some embodiments, a method of eliminating a virus in a subject in need of this one is provided in this document by administering an effective amount of an antibody provided in this document to the subject.
[00316] [00316] In some embodiments, a method of extending the overall survival period, mean survival time or progression-free survival in a subject in need of this subject is provided in this document by administering an effective amount of an antibody provided in this document to the subject. Combination Therapies
[00317] [00317] In some embodiments, an antibody provided in this document is administered with at least one additional therapeutic agent. Any suitable additional therapeutic agent can be administered with an antibody provided in this document. In some respects, the additional therapeutic agent is selected from radiation, a cytotoxic agent, a chemotherapeutic agent, a cytostatic agent, an antihormonal agent, an EGFR inhibitor, an immunostimulating agent, an anti-angiogenic agent and combinations of these.
[00318] [00318] In some embodiments, the additional therapeutic agent comprises an immunostimulating agent.
[00319] [00319] In some embodiments, the additional therapeutic agent is an antibody.
[00320] [00320] Anti-SIRPα antibodies can be used therapeutically in combination with a second antibody or agent that selectively binds to a target cell. The term "target cell" can be used in different ways, depending on the context. Typically, a "target cell" is a cell that will be phagocyted by a phagocytic cell (for example, a phagocyte), where phagocytosis is potentiated as a result of the administration of an anti-SIRPα antibody in question. Thus, the term "target cell" can refer to a cell that expresses CD47, because an anti-SIRPα antibody in question, by inhibiting the interaction between the cell that expresses CD47 and the phagocytic cell that expresses SIRPα, facilitates cell phagocytosis that expresses CD47.
[00321] [00321] However, in some cases, the target cell does not need to express high levels of CD47 (and, in some cases, does not need to express CD47) for a multispecific antibody in question to induce phagocytosis of the target cell. For example, in the context of a multispecific antibody (for example, bispecific), the SIRPα binding region (the first binding region) of a multispecific antibody in question (for example, bispecific) binds SIRPα in a phagocytic cell ( for example, a macrophage), which allows the multispecific antibody to act as a cord to bring the phagocytic cell into the vicinity of a cell that expresses an antigen (for example, a cancer cell marker) that is recognized by (specifically bound por) a second multispecific antibody binding region (e.g., the second bispecific antibody binding region). Therefore, in the context of a multispecific antibody, a target cell can be a cell that does not express high levels of CD47 (and it can also be a cell that does not express CD47). In some embodiments, a target cell is a mammalian cell, for example a human cell. A target cell can be any individual (e.g., patient, subject and the like), as described below.
[00322] [00322] In some cases, a target cell is an "inflicted" cell (for example, a cell of an "inflicted" individual), where the term "inflicted" is used in this document to refer to a subject with symptoms, a disease or a disease that can be treated with an anti-SIRPα antibody in question. An "inflicted" subject may have cancer, may harbor an infection (for example, a chronic infection) and / or may have other hyperproliferative conditions, for example, sclerosis, fibrosis, the like, etc. "Inflicted cells" can be those that cause symptoms, illness or disease. As non-limiting examples, the cells inflicted on an inflicted patient can be cancer cells, infected cells, inflammatory cells, immune cells and the like that express CD47. An indication that a disease or illness can be treated with an anti-SIRPα antibody in question is that the cells involved (ie, the inflicted cells, for example, cancer cells, infected cells, inflammatory cells, immune cells , etc.) express CD47 (for example, in some cases, an increased level of CD47 compared to normal cells of the same cell type).
[00323] [00323] In some embodiments, the additional therapeutic agent is an antibody that binds a protein or proteins to the cell surface of a tumor.
[00324] [00324] For the treatment of cancer, the anti-SIRPα antibody can be combined with one or more antibodies specific to a tumor antigen. Of these, tumor-associated antigens (TAAs) are relatively restricted to tumor cells, while specific tumor antigens (TSAs) are exclusive to tumor cells. TSAs and TAAs are typically portions of intracellular molecules expressed on the cell surface as part of the major histocompatibility complex.
[00325] [00325] Tissue specific differentiation antigens are molecules present in tumor cells and their normal cellular counterparts. The tumor-associated antigens that are known to be recognized by therapeutic mAbs are in several different categories. Hematopoietic differentiation antigens are glycoproteins that are normally associated with differentiation groups (CD) and include CD20, CD30, CD33 and CD52. Cell surface differentiation antigens are a diverse group of glycoproteins and carbohydrates that are found on the surface of normal and tumor cells. The antigens that are involved in growth and differentiation signaling are often growth factors and growth factor receptors. Growth factors that are targets for antibodies in cancer patients include CEA, epidermal growth factor receptor (EGFR; also known as ERBB1), ERBB2 (also known as HER2), ERBB3, MET (also known as HGFR), insulin-like growth receptor 1 (IGF1R), ephrin receptor A3 (EPHA3), tumor necrosis factor (TNF) -related apoptosis-inducing receptor 1 (TRAILR1; also known as TNFRSF10A), TRAILR2 (also known as TNFRSF10B) and nuclear factor-κB ligand receptor activator (RANKL; also known as TNFSF11). The antigens involved in angiogenesis are usually proteins or growth factors that support the formation of new microvasculature, including the vascular endothelial growth factor (VEGF), the VEGF receptor (VEGFR), the αVβ3 integrin and the α5β1 integrin. The tumor stroma and the extracellular matrix are indispensable support structures for a tumor. The stromal and extracellular matrix antigens that are therapeutic targets include fibroblast activating protein (FAP) and tenascin.
[00326] [00326] Examples of therapeutic antibodies useful in bispecific configurations or as combination therapy include, without limitation, rituximab; Ibritumomab; thiuxethane; tositumomab; Brentuximab; vedotine; Gemtuzumab; ozogamycin; Alemtuzumab; IGN101; adecatumumab; Labetuzumab; huA33; Pemtumomab; oregovomab; CC49 (minetumomab); cG250; J591; MOv18;
[00327] [00327] For the treatment of cancer, the anti-SIRP antibody can be combined with one or more antibodies that inhibit the proteins of the immunological checkpoint. Of particular interest are the immunological checkpoint proteins displayed on the surface of a tumor cell. The immune checkpoint receptors that have been most actively studied in the context of clinical cancer immunotherapy, antigen 4 associated with cytotoxic T lymphocytes (CTLA4; also known as CD152) and programmed cell death protein 1 (PD1; also known as CD279 ) - are both inhibitory receptors. The clinical activity of antibodies that block any of these receptors implies that antitumor immunity can be increased at multiple levels and that combinatorial strategies can be intelligently designed, guided by mechanistic considerations and preclinical models.
[00328] [00328] The two ligands for PD1 are PD1 ligand 1 (PDL1; also known as B7-H1 and CD274) and PDL2 (also known as B7-DC and CD273). PDL1 is expressed in cancer cells and by binding to its PD1 receptor in T cells it inhibits T cell activation / function. See, for example, Avelumab as a therapeutic antibody.
[00329] [00329] Agents that agonize an immune co-stimulatory molecule are also useful in the methods disclosed in this document. These agents include agonists or CD40 and OX40. CD40 is a co-stimulatory protein found in antigen presenting cells (APCs) and is necessary for its activation. These APCs include phagocytes (macrophages and dendritic cells) and B cells. CD40 is part of the TNF receptor family. The main signaling molecules that activate CD40 are the ligand IFNγ and CD40 (CD40L). Stimulation through CD40 activates macrophages.
[00330] [00330] The anti-CCR4 (CD194) antibodies of interest include humanized monoclonal antibodies directed against the C-C 4 chemokine receptor (CCR4) with potential anti-inflammatory and antineoplastic activities.
[00331] [00331] In some respects, the additional therapeutic agent is an antibody that binds: HER2 (ERBB2 / neu), CD52, PD-L1, VEGF, CD30, EGFR, CD38, RANKL (CD254), GD2 (ganglioside), SLAMF7 (CD319), CD20, EGFR, PDGFRa, VEGFR2, CD33, CD44, CD99, CD96, CD90, CD133, CKIT (CD117 for CKIT positive tumors); CTLA-4, PD-1, PD-L1, CD40 (agonistic), LAG3 (CD223), 41BB (agonistic CD137), OX40 (agonistic CD134); and / or CKIT (CD117) to deplete blood-forming stem cells for transplant therapy.
[00332] [00332] In some embodiments, the additional therapeutic agent is at least one of: Rituximab, Cetuximab, Alemtuzumab (CD52), Atezolizumab (PD-L1), Avelumabe (PD-L1), Bevacizumabe (VEGF), Brentuximabe (CD30), Daratumumab (CD38), Denosumab (RANKL), Dinutuximab (GD2), Elotuzumab (SLAMF7), Ibritumomabe (CD20), Ipilimumab (CTLA-4), Necitumumabe (EGFR), Nivolumabe (PD-1), Obinutuzumabe (20) (CD20), Olaratumab (PDGFRa), Panitumumab (EGFR), Pembrolizumab (PD-1), Pertuzumab (HER2), Ramucirumab (VEGFR2), Tositumomab (CD20), and Gemtuzumab (CD33).
[00333] [00333] The additional therapeutic agent can be administered by any suitable means. In some embodiments, an antibody provided herein and the additional therapeutic agent are included in the same pharmaceutical composition. In some embodiments, an antibody provided herein and the additional therapeutic agent are included in different pharmaceutical compositions.
[00334] [00334] In the embodiments in which an antibody provided herein and the additional therapeutic agent are included in different pharmaceutical compositions, administration of the antibody can occur before, simultaneously and / or after the administration of the additional therapeutic agent. In some respects, administration of an antibody provided herein and the additional therapeutic agent occur within about a month of each other. In some aspects, administration of an antibody provided herein and the additional therapeutic agent occur within about a week of each other. In some respects, administration of an antibody provided herein and the additional therapeutic agent occur within about one day of each other. In some respects, administration of an antibody provided herein and the additional therapeutic agent occur within twelve hours of each other. In some respects, administration of an antibody provided herein and the additional therapeutic agent occur within about an hour of each other. Diagnostic Methods
[00335] [00335] Methods are also provided to detect the presence of SIRP-ALPHA in a subject's cells. Such methods can be used, for example, to predict and evaluate the responsiveness to treatment with an antibody provided in this document.
[00336] [00336] In some embodiments, a blood sample is obtained from a subject and the fraction of cells that express SIRP-ALPHA is determined. In some ways, the relative amount of SIRP-ALPHA expressed by these cells is determined. The fraction of cells that express SIRP-ALPHA and the relative amount of SIRP-ALPHA expressed by these cells can be determined by any suitable method. In some embodiments, flow cytometry is used to make these measurements. In some embodiments, fluorescence-assisted cell classification (FACS) is used to make this measurement. See Li et al., J. Autoimmunity, 2003, 21: 83-92 for methods of assessing the expression of SIRP-ALPHA in peripheral blood. Kits
[00337] [00337] Kits are also provided comprising an antibody provided in this document. The kits can be used for the treatment, prevention and / or diagnosis of a disease or disorder, as described in this document.
[00338] [00338] In some embodiments, the kit comprises a container and a label or package insert in or associated with the container. Suitable containers include, for example, bottles, vials, syringes and bags of IV solution. The containers can be formed from a variety of materials, such as glass or plastic. The container contains a composition that is by itself, or when combined with another composition, effective in the treatment, prevention and / or diagnosis of a disease or disorder. The container can have a sterile access door. For example, if the container is a pouch or vial of intravenous solution, it may have a port that can be pierced by a needle. At least one active agent in the composition is an antibody provided in this document. The label or package insert indicates that the composition is used to treat the selected clinical condition.
[00339] [00339] In some embodiments, the kit comprises (a) a first container with a first composition contained therein, wherein the first composition comprises an antibody provided herein; and (b) a second container with a second composition contained therein, wherein the second composition comprises another therapeutic agent. The kit in this embodiment can also comprise a package insert that indicates that the compositions can be used to treat a specific condition.
[00340] [00340] Alternatively, or in addition, the kit may further comprise a second (or third) container comprising a pharmaceutically acceptable excipient. In some respects, the excipient is a buffer. The kit can also include other materials desirable from a commercial and user point of view, including filters, needles and syringes. EXAMPLES
[00341] [00341] Examples of specific modalities for carrying out the present invention are described below. The examples are presented for illustrative purposes only and are not intended to impose limitations on the scope of the present invention, under any circumstances. Efforts have been made to ensure accuracy with respect to the numbers used (for example, quantities, temperature, etc.), but some experimental errors and deviations must, of course, be considered.
[00342] [00342] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemical protein, biochemistry, DNA recombination and pharmacological techniques, within the scope of the technique. These techniques are fully explained in the literature. See for example, T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook,
[00343] [00343] Generation of antibodies. A human SIRPa cDNA fragment encoding the extracellular domain was synthesized and fused to the mouse Fc to generate a fusion SIRPa-Fc protein, which was used to immunize mice to produce anti-human monoclonal anti-human CD47 antibodies. Hybridomas were generated using standard protocols. In summary, Balb / c mice 4-6 weeks of age were immunized with a purified recombinant fusion SIRPa-Fc protein twice a week for a total of 4 weeks. Titers were subsequently evaluated and the spleen cells were fused with SP2 / 0 cells. Hybridomas were selected and the supernatants of the resulting clones were screened by enzyme-linked immunosorbent assay (ELISA).
[00344] [00344] Cloning and sequencing of antibody V. The cloning strategy used in this document involved an initial isolation of RNA from hybridoma cells (Qiagen). The cDNA sequences encoding the variable regions of the heavy and light chains of monoclonal antibodies 1H9 and 3C2 were obtained using RACE-PCR 5 'techniques (Clontech) and were sequenced using standard DNA sequencing techniques.
[00345] [00345] Molecular modeling and humanization of antibodies. The humanization of 1H9 and 3C2 was carried out by installing CDR residues of mouse antibodies in human germline frameworks (FRs). Briefly, the 1H9 and 3C2 mice were humanized by careful recruitment of the corresponding CDR residues. The differences between the 1H9 and 3C2 mice and human RF residues were modeled individually to investigate their possible influence on CDR conformation.
[00346] [00346] Cell transfection. 293F cells were cultured in FreeStyle ™ 293 expression medium (Invitrogen). Transient transfection was performed by cotransfecting expression vectors encoding antibody heavy chain and light chain using 293fectin transfection reagent (Invitrogen), according to the manufacturer's instructions. Four to five days later, the supernatants from the transfected cells were collected and tested for antibody secretion by ELISA. Briefly, 96-well plates (Nunc, Roskilde, Denmark) were coated with 1 µg / ml of goat anti-human Fc gamma antibody in phosphate buffered saline (PBS) for 16 hours at 4 ° C. After blocking for 1 hour with 0.4% BSA in PBS at room temperature, the isolated supernatants were added in 1/3 sequential dilutions and incubated for 1 hour at room temperature. The plates were subsequently washed three times and incubated with HRP-conjugated goat-specific kappa anti-human antibody for 1 hour at room temperature. After washing, the plates were developed with TMB. The reaction was stopped with 2M H2SO4 and OD was measured at 450 nM.
[00347] [00347] Purification and characterization of antibodies. The culture supernatant was applied to protein A Sepharose columns (GE Healthcare). The column was washed with PBS and the protein was then eluted with elution buffer (0.1 M sodium citrate buffer, pH 3.0). The collected fractions were neutralized with 1 M Tris at pH 9.0. Finally, purified samples were dialyzed against PBS. The purity of the eluted antibody fraction was analyzed by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE) in 10% gels under reducing or non-reducing conditions. The bands were visualized by bright blue Coomassie staining.
[00348] [00348] Measurement of antibody affinity. The human SIRPa-His fusion protein was produced by fusing the extracellular domain of human SIRPa to His-tag and used to measure the affinity of monomeric binding to 1H9 and 3C2. Binding experiments were carried out in Biacore 3000 at 25 ° C. The goat anti-human capture antibody was immobilized (as indicated in the table) on the chip surface by direct immobilization using EDC / NHS coupling chemistry in flow cell 2, 3 and 4 of the CM5 chip. The vacant sites were blocked with 1M ethanolamine. Flow cell 1 was not treated and used as a reference for subtracting any non-specific binding of Ag to the chip surface. Test abs were captured in flow cell 2, 3 and 4 in an RU, as indicated. The antigen was drained over the chip at the concentration of single analyte. The binding of the antigen to the ligand was monitored in real time to obtain association (ka) and dissociation (kd) rates. The equilibrium constant (KD) was calculated from the ka and kd observed. For the interactions of the rapid dissociation rate, KD was determined by steady-state kinetic analysis.
[00349] [00349] Phagocytosis assay in vitro. The cancer cells Raji and HT29 were washed and counted, and then 25 µL containing 1 x 105 cells in IMDM without serum were added to each well. Antibody treatment (in 25 µL) with a final concentration of 10 µg / mL of 1H9, 3C2 (otherwise indicated in the figures), rituximab or 0.1 µg / mL of cetuximab was added to the wells and incubated at 37 ° C for 30 minutes. At 30 minutes, macrophages that were previously collected with TrypLE were counted and plated with 5 x 104 cells in 50 µL of IMDM without serum. The plates were incubated at 37 ° C for 2 hours (Effector: Target = 1: 2). The percentage of phagocytosis was calculated by flow cytometric analysis, looking for GFP + macrophages.
[00350] [00350] Genotyping of SIRPa variants. Genomic DNA was isolated from blood samples from human donors using the QIAamp DNA isolation kit (Qiagen). PCR was performed using isolated genomic DNA and TAG AAT ACA GGC TCA TGT TGC AGG T and GCC TTC AGC AAA TAG CAT GAC GT primers. The PCR fragments were purified and sequenced. Different variants of SIRPa were analyzed and identified according to the reference sequences of SIRPa (Polymorphism in Sirpa modulates engraftment of human hematopoietic stem cells. Nature Immunology, 8; 1313, 2007).
[00351] [00351] Blocking the binding of human CD47 to monocytes isolated from human donors. Human peripheral blood mononuclear cells (PBMCs) were isolated from human blood using Ficoll. 5x105 cells were incubated with 1 µg / ml of human fusion CD47-Fc protein conjugated to AF488 in the absence or presence of increasing concentrations of hHu1H9-G1. CD47 binding in cells was measured and analyzed by flow cytometry.
[00352] [00352] Internalization of Hu1H9-G1. The internalization of humanized 1H9 was tested by incubating 10 µg / ml of the antibody with macrophage cells differentiated from normal human blood at 37 ° C. The cells were fixed and permeabilized at each moment (0, 20 min, 1h, 2h, 4h, 6h and 24h). PE-labeled anti-human IgG1 antibody was used to detect 1H9. DAPI was used to color cores. The 4C incubation was used as a control for the 1H9 surface staining. Example 1: Generation of anti-SIRPa monoclonal antibodies and epitope mapping
[00353] [00353] A human SIRPa cDNA fragment encoding the extracellular domain was synthesized and fused to the mouse Fc to generate a SIRPa-Fc fusion protein (SEQ ID NO: 45), which was used to immunize mice to produce SIRPa antibodies anti-human monoclonal mice. The specificity of selected hybridoma clones was examined by ELISA binding to human SIRPa. Two of the positive clones were obtained and designated as 1H9 and 3C2. The variable regions of the heavy and light chains were cloned and sequenced, and the VH and VL sequences of 1H9 (Figure 1) and 3C2 (Figure 2) were determined.
[00354] [00354] To determine the epitopes recognized by 1H9 and 3C2, the human fusion SIRPa-Fc protein was coated on a 96-well plate. The binding of SIRPa to 1H9 and 3C2 was measured in the absence or presence of increasing concentrations of an anti-SIRPa antibody, KWar (disclosed in International Application WO 2015/138600, incorporated herein specifically by reference; Vh and Vl sequences shown in SEQ ID NOs 46-47). As shown in Figure 3A, Kwar did not compete with 1H9 for binding to SIRPa, indicating that 1H9 recognizes a distinct epitope from KWar. In contrast, Kwar competed in 3C2 for the SIRPa link; however, 3C2 binding to SIRPa was partially blocked even when excessive amounts of 100 times KWar were used. This indicates that 3C2 probably recognizes an overlapping, but not identical, epitope compared to KWar (Figure 3A). Competitive binding was also performed between 1H9 and 3C2, and it was shown that the binding of 1H9 to SIRPa was competed by 1H9 itself in a dose-dependent manner, but not by 3C2 (Figure 3B). Likewise, 3C2 competed in a dose-dependent manner, but not for 1H9 (Figure 3C). As such, 1H9 and 3C2 recognize distinct epitopes on SIRP-alpha. Example 2: Selection of antibody isotype for 1H9 and 3C2
[00355] [00355] Chimeric 1H9 and 3C2 were constructed by fusing their light and heavy chain variable domains to the constant regions of human kappa, human IgG4 or human IgG1 that has an N297A mutation to cancel the interaction with FcgR. The resulting antibodies were then tested in an in vitro phagocytosis assay in combination with rituximab (Rx). The effects of donor variation were observed. 1H9 synergized with rituximab to promote phagocytosis equally well in the formats of human IgG4 (1H9-G4) and IgG1 N297A (1H9-G1) using differentiated monocyte macrophages from some donors (Figure 4A). While using monocytes differentiated from monocytes from different donors, 1H9-G1 triggered a better synergy with rituximab than 1H9-G4 (Figure 4B). Similar results were also seen with 3C2 (Figure 4A-B). It is possible that different allelic variations in FcgRs expressed in macrophages may cause the variations observed in the in vitro phagocytosis assay. These results demonstrate the general benefit of the killed Fc construct for anti-SIRPα antibodies, in reducing the variability of responsiveness, that is, in reducing the number of individuals who do not respond to the improvement of phagocytosis when combined with an antibody directed to cells. Example 3: Humanization 1H9 and 3C2
[00356] [00356] The humanization of 1H9 and 3C2 was performed by CDR grafting, and the humanized VH and VL sequences of 1H9 and 3C2 are shown in Figures 5 and 6, respectively. Complete strings are shown in SEQ ID NOs 37-
[00357] [00357] To assess the specificity of binding to the humanized 1H9 and 3C2 antigen, the competitive binding between humanized and parental mouse 1H9 or 3C2 was performed by ELISA. It demonstrated that humanized 1H9 and 3C2 competed with mouse 1H9 and 3C2 for binding to SIRPa in a dose-dependent manner, respectively (Figure 7). Thus, humanized 1H9 and 3C2 have the same antigen-binding specificity as their parental antibodies. The binding affinities to the humanized 1H9 and 3C2 antigen were then measured using surface plasmon resonance. The humanized 1H9 bound to the monomeric human SIRPa antigen with a KD of 1.15x10-9 M and the humanized 3C2 bound to the monomeric human SIRPa with a KD of 5.53x10-9 M (Figure 8). Example 4: 1H9 and humanized 3C3 synergize with therapeutic antibodies to promote macrophage-mediated phagocytosis
[00358] [00358] Next, we investigated the ability of humanized 1H9 and 3C2 to allow phagocytosis of human cancer cells by peripheral macrophages derived from human blood in combination with therapeutic antibodies. Humanized 1H9 or 3C2 alone did not substantially induce phagocytosis; however, when combined with rituximab (Rx), both antibodies induced greater phagocytic activity in Raji cells than that of rituximab alone (Figure 9A). In addition, humanized 1H9 and 3C2 synergized with cetuximab (Cx) to induce phagocytosis of a human colorectal adenocarcinoma cell line HT-29 and synergistic activity was observed in a range of humanized 1H9 and 3C2 concentrations that were tested (Figure 9B) . Example 5: Cross reactivity of 1H9 and 3C2 for family members SIRP
[00359] [00359] In addition to SIRP alpha, there are two closely related proteins in the SIRP family, namely (SIRPB, accession number NM_001083910.3) and SIRP gamma (SIRPG, accession number NM_001039508.1). SIRPB, although closely related to SIRPa, does not appear to bind to CD47 and does not have cytoplasmic ITIMs or any other recognizable cytosolic motifs for signaling. Instead, SIRPB contains a transmembrane region with a positively charged lysine residue that mediates the association with DAP12, an adapter protein that carries an ITAM. The phosphorylation of DAP12 ITAM mediates the recruitment of the protein tyrosine kinase Syk and the consequent activation of the MAPK pathway that regulates various functions. Firing the murine SIRPB receptor, for example, which also complexes with DAP12, promotes phagocytosis in macrophages. SIRPG, the third member of the human SIRP family, is expressed in T cells and activated NK cells. It can connect to CD47,
[00360] [00360] The His-SIRPB and SIRPG fusion protein was generated and the binding of 1H9 and 3C2 to SIRPB and SIRPG was tested. As shown in Figure 10, 1H9 and 3C2 bound to SIRPB compared to Kwar (Figure 10A). Unlike Kwar, no 1H9 or 3C2 binding to SIRPG was detected (Figure 10B). The lack of binding to SIRPG by 1H9 and 3C2 confers the following potential advantages to these antibodies over the anti-SIRPA antibodies of the prior art: (1) SIRPA is more restricted in relation to SIRPG, which reduces the risk of outside the target (2) there is a decreased risk of toxicity, for example, given the increased specificity for SIRPA, (3) there is a decreased risk of developing an "antigen sinking" phenomenon when the antibody is assayed in a subject and ( 4) there is a decreased risk of interference with T cells and / or B cell function by the antibody. Example 6: Generation and testing of additional anti-SIRP-alpha antibodies
[00361] [00361] Additional antibodies were created for human SIRPa by immunizing mice as described above. Two clones of monoclonal antibodies were designated - 9B11 and 7E11, respectively. See SEQ ID NOs 21-36 and 41-44. The variable regions of the mouse were joined as a chimera to the human IgG4 Fc region (designated as 7E11-G4 or 9B11-G4), or to a human IgG1 Fc region comprising the N297A mutation to cancel the interaction with human FcRs (designated as 7E11 -G1 or 9B11-G1).
[00362] [00362] As was discovered with 1H9 and 3C2, antibodies 9B11 and 7E11 showed a synergistic response in the potentiation of phagocytosis of cancer cells when combined with Rituximab. Shown in Figure 11, macrophages were differentiated from monocytes from donor A (A) and donor B (B) in the presence of human serum for 7 days. Raji cells were labeled with CFSE and incubated with macrophages in the presence of 10 µg / ml of rituximab (Rx)
[00363] [00363] The data show that while antibodies in the IgG4 format and antibodies in the mutated IgG1 format can provide a synergistic response, the mutated IgG1 format provided a more consistent response among donors.
[00364] [00364] To determine the epitopes recognized by 9B11 and 7E11, the human fusion SIRPa-Fc protein was coated on a 96-well plate. The binding of SIRPa to 9B11 and 7E11 was measured in the absence or presence of increasing concentrations of an anti-SIRPa antibody, KWar (disclosed in International Application WO 2015/138600, incorporated in this document specifically by reference). As shown in Figure 12, 7E11 recognizes an overlapping epitope compared to Kwar (similar to 3C2) and 9B11 recognizes a very similar or identical epitope compared to Kwar. Example 7: Binding of Hu1H9-G1 to different variants of SIRP-alpha in primary human cells
[00365] [00365] Human SIRP-α is highly polymorphic in the IgV domain, however, most variants are variant 1 (V1) and variant 2 (V2).
[00366] [00366] 22 normal human donors were screened and genotyped and donors were identified with homozygous V1, homozygous V2 and heterozygous V1 / V5 status for SIPR-alpha. (Polymorphism in Sirpa modulates engraftment of human hematopoietic stem cells. Nature Immunology, 8; 1313, 2007; For reference: V1 sequence shown in SEQ ID NO: 48; V2 sequence shown in SEQ ID NO: 49) The humanized 1H9 has been tested and ligated to each of the V1, V2 and V1 / V5 alleles using donor monocytes and macrophages (Figure 13). These data indicate that humanized 1H9 can be used in a wide range of humans, given its ability to bind to multiple distinct variants of SIRP-alpha in primary human donor cells. Example 8: Hu1H9-G1 blocks the binding of CD47 to monocytes from different donors
[00367] [00367] The humanized 1H9-G1 was then tested to determine whether it can block the interaction of CD47 and SIRP-alpha which is expressed as different variants. Monocytes were isolated from donors expressing V1, V2 and V1 / V5 and incubated with the fusion CD47-Fc protein in the absence or in the presence of increasing concentrations of humanized 1H9 (Figure 14). The data show that humanized 1H9 blocked the interaction of CD47 and SIRP-alpha in a dose-dependent manner, and the blocking activities were comparable between the different variants of SIRP-alpha tested. Example 9: Hu1H9-G1 synchronizes with Cetuximab to promote phagocytosis in different donors
[00368] [00368] Phagocytoses were performed in vitro using differentiated macrophages from different donors. Humanized 1H9-G1 synergized with cetuximab to promote phagocytosis in donors with V1, V2 and V1 / V5 variants (Figure 15). Example 10: Internalization of Hu1H9-G1
[00369] [00369] The internalization of the humanized 1H9 was tested by incubating 10 µg / ml of the antibody with macrophage cells differentiated from normal human blood at 37 ° C. The cells were fixed and permeabilized at each moment (0, 20 min, 1h, 2h, 4h, 6h and 24h). PE-labeled anti-human IgG1 antibody was used to detect 1H9. DAPI was used to color cores. The 4C incubation was used as a control for the 1H9 surface staining.
[00370] [00370] The data show that humanized 1H9 does not internalize in the cells and the staining of the surface of 1H9 was detectable at all times, including 24 hours (data not shown). These data indicate that humanized 1H9 is stable on the cell surface, which may be indicative of greater therapeutic efficacy in vivo.
[00371] [00371] Although this invention has been particularly shown and described with reference to a preferred modality and several alternative modalities, it will be understood by those skilled in the art that various changes in form and details can be made without departing from the spirit and scope of the invention .
[00372] [00372] All references, issued patents and patent applications cited in the body of this specification are incorporated in this document by reference in their entirety, for all purposes. TABLE A - SEQUENCES SEQ ID String
ID NO 1 SYWIT 1H9 CDR-H1 2 1H9 CDR-H2 DIYPGSGSTNHIEKFKS 3 1H9 CDR-H3 GYGSSYGYFDY 4 5 1H9 CDR-L1 RASENIYSYLA 1H9 CDR-L2 TAKTLAE 6 7 1H9 CDR-L3 QHQYGPPFT 1H9 VH QVQLVQSGAE VKKPGASVKV SCKASGYTFT Humanizad SYWITWVKQA PGQGLEWIGD IYPGSGSTNH the IEKFKSKATL TVDTSISTAY MELSRLRSDD
TAVYYCATGY GSSYGYFDYW GQGTLVTVSS 8 1H9 VL DIQMTQSPSS LSASVGDRVT ITCRASENIY Humanizad SYLAWYQQKP GKAPKLLIYT AKTLAEGVPS o RFSGSGSGTD FTLTISSYQC EDFAT
QYGPPFTFGQ GTKLEIK 9 3C2 CDR- SYWMH H1 10 3C2 CDR- NIDPSDSDTHYNQKFKD H2 11 3C2 CDR- GYSKYYAMDY H3 12 3C2 CDR- RSSQSIVHSYGNTYLE L1
13 3C2 CDR- KVSNRFS L2 14 3C2 CDR- FQGSHVPYT L3 15 3C2 VH QVQLVQSGAE VKKPGASVKV SCKASGYTFT Humanizad SYWMHWVRQA PGQGLEWMGN IDPSDSDTHY the NQKFTDRVTTM
TAVYYCARGY SKYYAMDYWG QGTLVTVSS 16 3C2 VL DIVMTQTPLS LSVTPGQPAS ISCRSSQSIV Humanizad HSYGNTYLEW YLQKPGQSPQ LLIYKVSNRF o SGVPDRFSGS GSGTDFTLKI SRVEA
YYCFQGSHVP YTFGQGTKLE 1H9 HC IK 17 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWITW Humanizad VKQAPGQGLEWIGDIYPGSGSTNHIEKFKSKATLTVD the TSISTAYMELSRLRSDDTAVYYCATGYGSSYGYFDY (full GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG nt WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL compresses) LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH
18 NHYTQKSLSLSPG 1H9 LC DIQMTQSPSSLSASVGDRVTITCRASENIYSYL Humanizad AWYQQKPGKAPKLLIYTAKTLAEGVPSRFSGSGSGT the DFTLTISSLQPEDFATYYCQHQYGPPFTFGQGTKLEIK (full KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT nt RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA compresses) LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
19 3C2 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMH Humanizad WVRQAPGQGLEWMGNIDPSDSDTHYNQKFKDRVTM the TRDTSTSTVYMELSSLRSEDTAVYYCARGYSKYYAM (full ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS nt DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA compresses) SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
LHNHYTQKSLSLSPG 20 3C2 LC DIVMTQTPLSLSVTPGQPASISCRSSQSIVHSYGNTYL Humanizad EWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGT the DFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEI (compresses KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE nto complete AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL) TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 21 9B11 DYYIH CDR-H1 22 9B11 RIDPEDGETKYAPKFQG CDR-H2 23 9B11 GGFAY CDR-H3 24 9B11 ASSSVSSSYLY CDR-L1 25 9B11 STSNLAS CDR-L2 26 9B11 HQWSSHPYT CDR-L3
27 9B11 VH EVQLQQSGAELVKPGASVKLSCTASGFNIKDYYIHWV KQRTEQGLEWIGRIDPEDGETKYAPKFQGKATITADT SSNTAYLQLNSLTSEDTAVYSCAKGGFAYWGQGTLV
TVSA 28 9B11 VL QIVLTQSPAIMSASPGEKVTLTCSASSSVSSSYLYWY QQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYS
LTISSMEAEDAASYFCHQWSSHPYTFGGGTKLEIK 29 CDR-H1 SYWMH 7E11 30 7E11 31 NIDPSDSDTHYNQKFKD CDR-H2 CDR-H3 SYGNYGENAMDY 7E11 32 7E11 33 RSSQSIVHSYGNTYLE CDR-L1, CDR L2 KVSNRFS 7E11 34 7E11 35 FQGSHVPFT CDR-L3 VH 7E11 QVKLQESGAELVRPGSSVKLSCKASGYTFTSYWMH WVKQRPIQGLEWIGNIDPSDSDTHYNQKFKDKATLTV DNSSSTAYMQLSSLTSEDSAVYYCASYGNYGENAMD
YWGQGTSVTVSS 36 7E11 VL DILMTQTPLSLPVSLGDQASISCRSSQSIVHSYGNTYL EWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGT DFTLKISRVEAEDLGVYYCFQGSHVPFTFGSGTKLEIK
37 CAGGTTCAGTTGGTTCAGTCTGGCGCCGAAGTGAA nucleic acid GAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAAGG 1H9 CTTCCGGCTACACCTTTACCAGCTACTGGATCACCT humanizad GGGTCAAGCAGGCTCCTGGACAGGGACTCGAGTG the heavy chain GATCGGCGATATCTATCCTGGCTCCGGCTCCACCA ACCACATCGAGAAGTTCAAGTCCAAGGCTACCCTG ACCGTGGACACCTCCATCTCCACCGCCTACATGGA ACTGTCCCGGCTGAGATCTGACGACACCGCCGTGT ACTATTGCGCTACCGGCTACGGCTCCTCCTACGGC TACTTTGATTATTGGGGCCAGGGCACCCTGGTCAC CGTGTCCTCTGCTTCTACCAAGGGACCCAGCGTGT TCCCTCTGGCTCCTTCCAGCAAGTCTACCTCTGGC GGAACAGCTGCTCTGGGCTGCCTGGTCAAGGACTA CTTTCCTGAGCCTGTGACCGTGTCTTGGAACTCTG GCGCTCTGACATCTGGCGTGCACACATTCCCTGCT GTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTC TGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACCC AGACCTACATCTGCAATGTGAACCACAAGCCTTCC AACACCAAGGTGGACAAGAAGGTGGAACCCAAGTC CTGCGACAAGACCCACACCTGTCCTCCATGTCCTG CTCCAGAACTGCTCGGCGGACCTTCCGTGTTTCTG TTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCT CGGACCCCTGAAGTGACCTGCGTGGTGGTGGATG TGTCTCACGAGGACCCAGAAGTGAAGTTCAATTGG TACGTGGACGGCGTGGAAGTGCACAACGCCAAGA CCAAGCCTAGAGAGGAACAGTACGCCTCCACCTAC AGAGTGGTGTCCGTGCTGACAGTGCTGCACCAGG ATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTG TCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGAC CATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCC CAGGTTTACACCCTGCCACCTAGCCGGGAAGAGAT GACCAAGAACCAGGTGTCCCTGACCTGCCTCGTGA AGGGCTTCTACCCTTCCGATATCGCTGTGGAATGG GAGAGCAACGGCCAGCCTGAGAACAACTACAAGAC AACCCCTCCTGTGCTGGACTCCGACGGCTCATTCT TTCTGTACTCCAAGCTGACTGTGGACAAGTCCAGA TGGCAGCAGGGCAACGTGTTCTCCTGCAGCGTGAT GCACGAGGCCCTGCACAATCACTACACACAGAAGT
CTCTGTCTCTGAGCCCCGGC 38 GACATCCAGATGACCCAGTCTCCATCCTCTCTGTC nucleic acid CGCCTCTGTGGGCGACAGAGTGACCATCACCTGTC 1H9 GGGCCTCCGAGAACATCTACTCCTACCTGGCCTGG humanizad TATCAGCAGAAGCCTGGCAAGGCTCCCAAGCTGCT the light chain GATCTACACCGCTAAGACACTGGCCGAGGGCGTG CCCTCTAGATTTTCTGGCTCTGGAAGCGGCACCGA CTTTACCCTGACAATCTCCAGCCTGCAGCCTGAGG ACTTCGCCACCTACTACTGCCAGCACCAGTACGGC CCTCCATTCACCTTTGGCCAGGGCACCAAGCTGGA AATCAAGCGGACAGTGGCCGCTCCTTCCGTGTTCA TCTTCCCACCTTCCGACGAGCAGCTGAAGTCTGGC ACAGCCTCTGTCGTGTGCCTGCTGAACAACTTCTA CCCTCGGGAAGCCAAGGTGCAGTGGAAGGTGGAC AATGCCCTGCAGTCCGGCAACTCCCAAGAGTCTGT GACCGAGCAGGACTCCAAGGACAGCACCTACAGC CTGTCCTCCACACTGACCCTGTCCAAGGCCGACTA CGAGAAGCACAAGGTGTACGCCTGCGAAGTGACC CATCAGGGCCTGTCTAGCCCTGTGACCAAGTCTTT CAACCGGGGCGAGTGC
39 CAGGTTCAGTTGGTTCAGTCTGGCGCCGAAGTGAA nucleic acid GAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAAGG 3C2 CTTCCGGCTACACCTTTACCAGCTACTGGATGCAC humanizad TGGGTCCGACAGGCTCCAGGACAAGGCTTGGAGT GGATGGGCAACATCGACCCCTCTGACAGCGACAC the heavy chain CCACTACAACCAGAAATTCAAGGACCGCGTGACCA TGACCAGAGACACCTCCACCAGCACCGTGTACATG GAACTGTCCAGCCTGAGATCCGAGGACACCGCCG TGTACTACTGTGCCAGAGGCTACTCCAAGTACTAC GCCATGGACTACTGGGGCCAGGGCACACTGGTTA CCGTGTCCTCTGCTTCCACCAAGGGACCCTCTGTG TTCCCTCTGGCTCCTTCCAGCAAGTCTACCTCTGG CGGAACAGCTGCTCTGGGCTGCCTGGTCAAGGAC TACTTTCCTGAGCCTGTGACCGTGTCTTGGAACTCT GGCGCTCTGACATCTGGCGTGCACACATTCCCTGC TGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCT CTGTCGTGACCGTGCCTTCCAGCTCTCTGGGAACC CAGACCTACATCTGCAATGTGAACCACAAGCCTTC CAACACCAAGGTGGACAAGAAGGTGGAACCCAAGT CCTGCGACAAGACCCACACCTGTCCTCCATGTCCT GCTCCAGAACTGCTCGGCGGACCTTCCGTGTTTCT GTTCCCTCCAAAGCCTAAGGACACCCTGATGATCT CTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGA TGTGTCCCACGAAGATCCAGAAGTGAAGTTCAATT GGTACGTGGACGGCGTGGAAGTGCACAACGCCAA GACCAAGCCTAGAGAGGAACAGTACGCCTCCACCT ACAGAGTGGTGTCCGTGCTGACAGTGCTGCACCAG GATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGT GTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGA CCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACC CCAGGTTTACACCCTGCCTCCAAGCCGGGAAGAGA TGACCAAGAACCAGGTGTCCCTGACCTGCCTCGTG AAGGGCTTCTACCCTTCCGATATCGCCGTGGAATG GGAGAGCAATGGCCAGCCAGAGAACAACTACAAGA CAACCCCTCCTGTGCTGGACTCCGACGGCTCATTC TTTCTGTACTCCAAGCTGACCGTGGACAAGTCCAG ATGGCAGCAGGGCAACGTGTTCTCCTGCAGCGTGA TGCACGAGGCCCTGCACAATCACTATACCCAGAAG
TCCCTGTCTCTGTCCCCTGGC 40 GACATCGTGATGACCCAGACACCTCTGAGCCTGAG nucleic acid CGTGACACCTGGACAGCCTGCCTCCATCTCCTGCA 3C2 GATCCTCTCAGTCCATCGTGCACTCCTACGGCAAC humanizad ACCTACCTGGAATGGTATCTGCAGAAGCCCGGCCA the light chain GTCTCCTCAGCTGCTGATCTACAAGGTGTCCAACC GGTTCTCTGGCGTGCCCGACAGATTTTCCGGCTCT GGCTCTGGCACCGACTTCACCCTGAAGATCTCCAG AGTGGAAGCCGAGGACGTGGGCGTGTACTACTGC TTCCAAGGCTCTCACGTGCCCTACACCTTTGGCCA GGGCACCAAGCTGGAAATCAAGCGGACAGTGGCC GCTCCTTCCGTGTTCATCTTCCCACCTTCCGACGA GCAGCTGAAGTCCGGCACAGCTTCTGTCGTGTGCC TGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTG CAGTGGAAGGTGGACAATGCCCTGCAGTCCGGCA ACTCCCAAGAGTCTGTGACCGAGCAGGACTCCAAG GACAGCACCTACAGCCTGTCCAGCACACTGACCCT GTCCAAGGCCGACTACGAGAAGCACAAGGTGTAC GCCTGCGAAGTGACCCATCAGGGCCTGTCTAGCC CTGTGACCAAGTCTTTCAACCGGGGCGAGTGC
41 GAGGTTCAGCTGCAGCAGTCTGGGGCAGAGCTTG nucleic acid TGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACA 9B11 VH GCTTCTGGCTTCAACATTAAAGACTACTATATACAC TGGGTGAAGCAGAGGACTGAACAGGGCCTGGAGT GGATTGGAAGGATTGATCCTGAGGATGGTGAAACT AAATATGCCCCGAAATTCCAGGGCAAGGCCACTAT AACAGCAGACACATCCTCCAACACAGCCTACCTGC AGCTCAACAGCCTGACATCTGAGGACACTGCCGTC TATTCCTGTGCTAAGGGGGGGTTTGCTTACTGGGG
CCAAGGGACTCTGGTCACTGTCTCTGCA 42 CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCT nucleic acid GCATCTCCTGGGGAGAAGGTCACCTTGACCTGCAG 9B11 VL TGCCAGTTCAAGTGTAAGTTCCAGCTACTTGTACTG GTACCAGCAGAAGCCAGGATCCTCCCCCAAACTCT GGATTTATAGCACATCCAACCTGGCTTCTGGAGTC CCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTC TTACTCTCTCACAATCAGCAGCATGGAGGCTGAAG ATGCTGCCTCTTATTTCTGCCATCAGTGGAGTAGTC ACCCGTACACGTTCGGAGGGGGGACCAAGCTGGA
AATAAAA 43 CAGGTCAAGCTGCAGGAGTCTGGGGCTGAGCTGG nucleic acid TGAGGCCTGGGTCTTCAGTGAAGCTGTCCTGCAAG 7E11 VH GCTTCTGGCTACACCTTCACCAGCTACTGGATGCA TTGGGTGAAGCAGAGGCCTATACAAGGCCTTGAAT GGATTGGTAACATTGACCCTTCTGATAGTGATACTC ACTACAATCAAAAGTTCAAGGACAAGGCCACATTGA CTGTGGACAACTCCTCCAGCACAGCCTACATGCAG CTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTA TTACTGTGCAAGCTATGGTAACTACGGGGAGAATG CTATGGACTACTGGGGTCAAGGAACCTCAGTCACC GTCTCCTCA
44 GATATTTTGATGACCCAAACTCCACTCTCCCTGCCT nucleic acid GTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAG 7E11 VL ATCTAGTCAGAGCATTGTACATAGTTATGGAAACAC CTATTTAGAATGGTACCTGCAGAAACCAGGCCAGT CTCCAAAACTCCTGATCTACAAAGTTTCCAACCGAT TTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGA TCAGGTACAGATTTCACACTCAAGATCAGCAGAGT GGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTC AAGGTTCACATGTTCCATTCACGTTCGGCTCGGGG
ACAAAGTTGGAAATAAAA 45 SIRPa EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQW FRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDF SIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTEL
SVRA 46 KWar VH EVQLVQSGAEVKKPGATVKISCKVSGFNIKDYYIHWV QQAPGKGLEWIGRIDPEDGETKYAPKFQDRATITADT STDTAYMELSSLRSEDTAVYYCARWGAYWGQGTLV
TVSS 47 KWar VL QIVLTQSPPTLSLSPGERVTLTCSASSSVSSSYLYWY QQKPGQAPKLWIYSTSNLASGVPARFSGSGSGTSYT
LTISSLQPEDFAVYFCHQWSSYPRTFGAGTKLEIK 48 SIRPa V1 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQW FRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDF SIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTEL
SVRA 49 SIRPa V2 EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQW FRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDF SISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELS VRA

权利要求:
Claims (63)
[1]
1. Humanized, human or isolated chimeric antibody characterized by the fact that: (a) it specifically binds to human SIRPα; (b) links each of the human SIRPα alleles V1 and V2; (c) does not specifically bind human SIRPγ; and (d) optionally comprises a human Fc region comprising at least one modification that reduces binding to a human Fc receptor.
[2]
2. Isolated antibody according to claim 1, characterized by the fact that it comprises: (a) a CDR-H1 comprising the sequence set out in SEQ ID NO: 1; a CDR-H2 comprising the sequence set forth in SEQ ID NO: 2; a CDR-H3 comprising the sequence set out in SEQ ID NO: 3; a CDR-L1 comprising the sequence set forth in SEQ ID NO: 4; a CDR-L2 comprising the sequence set out in SEQ ID NO: 5; and a CDR-L3 comprising the sequence set out in SEQ ID NO: 6; or (b) a CDR-H1 comprising the sequence set forth in SEQ ID NO: 9; a CDR-H2 comprising the sequence shown in SEQ ID NO: 10; a CDR-H3 comprising the sequence set out in SEQ ID NO: 11; a CDR-L1 comprising the sequence set out in SEQ ID NO: 12; a CDR-L2 comprising the sequence set out in SEQ ID NO: 13; and a CDR-L3 comprising the sequence set out in SEQ ID NO: 14.
[3]
3. Isolated antibody according to any one of the preceding claims, characterized in that: (a) the antibody of claim 2 (a) comprises a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 8; or (b) the antibody of claim 2 (b) comprises a VH sequence of SEQ ID NO: 15 and a VL sequence of SEQ ID NO: 16.
[4]
4. Isolated antibody according to any one of the preceding claims, characterized in that: (a) the antibody of claim 3 (a) comprises a heavy chain of SEQ ID NO: 17 and a light chain of SEQ ID NO: 18; or
(b) the antibody of claim 3 (b) comprises a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 20.
[5]
5. Isolated humanized, human or chimeric antibody characterized by the fact that it comprises: a CDR-H1 comprising the sequence set out in SEQ ID NO: 1; a CDR-H2 comprising the sequence set forth in SEQ ID NO: 2; a CDR-H3 comprising the sequence set out in SEQ ID NO: 3; a CDR-L1 comprising the sequence set forth in SEQ ID NO: 4; a CDR-L2 comprising the sequence set out in SEQ ID NO: 5; and a CDR-L3 comprising the sequence set out in SEQ ID NO: 6.
[6]
6. Isolated humanized, human or chimeric antibody, characterized by the fact that it comprises: a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 8.
[7]
7. Humanized, human or chimeric antibody isolated, characterized by the fact that it comprises: a heavy chain of SEQ ID NO: 17 and a light chain of SEQ ID NO: 18.
[8]
8. Isolated humanized, human or chimeric antibody characterized by the fact that it comprises: a CDR-H1 comprising the sequence set out in SEQ ID NO: 9; a CDR-H2 comprising the sequence set forth in SEQ ID NO: 10; a CDR-H3 comprising the sequence set out in SEQ ID NO: 11; a CDR-L1 comprising the sequence set out in SEQ ID NO: 12; a CDR-L2 comprising the sequence set out in SEQ ID NO: 13; and a CDR-L3 comprising the sequence set out in SEQ ID NO: 14.
[9]
9. Humanized, human or chimeric antibody isolated, characterized by the fact that it comprises: a VH sequence of SEQ ID NO: 15 and a VL sequence of SEQ ID NO: 16.
[10]
10. Humanized, human or chimeric antibody isolated, characterized by the fact that it comprises: a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 20.
[11]
11. Isolated antibody according to any one of the preceding claims, characterized in that the antibody comprises a human Fc region with reduced Fc dependent function (s), optionally comprising at least one modification that reduces binding to a human Fc receptor.
[12]
12. Isolated antibody according to any of the above claims, characterized by the fact that: (a) it competes for binding to human SIRPα with an antibody selected from 1H9 and 3C2; (b) does not compete for binding to human SIRPα with the KWar antibody; (c) partially competes for binding to human SIRPα with the KWar antibody; (d) inhibits the binding of human CD47 to human SIRPα; (e) inhibits the binding of human SP-A to human SIRPα; (f) inhibits the binding of human SP-D to human SIRPα; (g) binds to the rhesus monkey SIRPα; (h) binds to cynomolgus SIRPα (i) increases phagocytosis compared to control; (j) links each of the human SIRPα alleles V1 and V2; (k) links each of the human SIRPα alleles V1, V2 and V1 / V5; (l) links the human SIRPα allele V1; (m) binds the human SIRPα allele V2; or (n) is capable of any combination of (a) - (m).
[13]
13. Isolated antibody according to any of the preceding claims, characterized by the fact that the antibody is pan-specific for isotypes of human SIRPα.
[14]
14. Isolated antibody according to any one of the preceding claims, characterized by the fact that the antibody is specific for a human SIRPα isotype.
[15]
15. Isolated antibody according to any one of the preceding claims, characterized by the fact that human SIRPα is expressed in a professional antigen presenting cell.
[16]
16. Isolated antibody according to any one of the preceding claims, characterized by the fact that human SIRPα is expressed in a macrophage.
[17]
17. Isolated antibody according to any of the preceding claims, characterized by the fact that the antibody is 1H9 or 3C2.
[18]
18. Isolated antibody according to any of the preceding claims, characterized by the fact that the antibody does not have an Fc region.
[19]
19. Isolated antibody according to any of the preceding claims, characterized by the fact that the human Fc region is IgG1 or IgG4.
[20]
20. Isolated antibody according to any one of the preceding claims, characterized in that the glycosylation of the antibody is reduced by enzymatic deglycosylation, expression in a bacterial host or modification of an amino acid residue used for glycosylation.
[21]
21. Isolated antibody according to any of the preceding claims, characterized by the fact that the modification reduces the glycosylation of the human Fc region.
[22]
22. Isolated antibody according to any of the preceding claims, characterized by the fact that the modification of the human Fc region comprises a modification in the EU index position of asparagine 297.
[23]
23. Isolated antibody according to any one of the preceding claims, characterized by the fact that the modification of the human Fc region comprises an amino acid substitution at the position of the EU index of asparagine 297.
[24]
24. Isolated antibody according to any one of the preceding claims, characterized by the fact that the modification of the human Fc region comprises a substitution of amino acids N297A, numbered according to the EU index.
[25]
25. Isolated antibody according to any of the preceding claims, characterized by the fact that the modification comprises one or more amino acid substitutions at: N297A; L234A / L235A; C220S / C226S / C229S / P238S; C226S / C229S / E3233P / L234V / L235A; or L234F / L235E / P331S, numbering according to the EU index.
[26]
26. Isolated antibody according to any one of the above claims, characterized by the fact that the modification comprises one or more amino acid substitutions at: N297; L234 / L235; C220 / C226 / C229 / P238; C226 / C229 / E3233 / L234 / L235; or L234 / L235 / P331, numbering according to the EU index.
[27]
27. Isolated antibody according to any of the above claims, characterized by the fact that the modification comprises one or more amino acid substitutions in the CH2 region at EU index positions 234, 235 and / or 237.
[28]
28. Isolated antibody according to any one of the preceding claims, characterized in that the modification comprises one or both amino acid substitutions L234A and L235A and, optionally, P331S and / or K322A and / or G237A, numbered according to the EU index.
[29]
29. Isolated antibody according to any one of the preceding claims, characterized by the fact that the modification of the human Fc region comprises a substitution of amino acids K322A, numbered according to the EU index.
[30]
30. Isolated antibody according to any one of the preceding claims, characterized by the fact that the modification comprises E233P / L234V / L235A / G236 + A327G / A330S / P331S, numbered according to the EU index.
[31]
31. Isolated antibody according to any one of the preceding claims, characterized by the fact that the antibody is a monoclonal antibody.
[32]
32. Isolated antibody according to any of the preceding claims, characterized by the fact that the antibody is multispecific.
[33]
33. Isolated antibody according to any one of the preceding claims, characterized by the fact that the antibody binds to more than one antigen or to more than one epitope on a single antigen.
[34]
34. Isolated antibody according to any one of the preceding claims, characterized by the fact that the antibody comprises the heavy chain constant region of a class selected from IgG, IgA, IgD, IgE and IgM.
[35]
35. Isolated antibody according to any one of the preceding claims, characterized by the fact that the antibody comprises an IgG class heavy chain constant region and a selected subclass of IgG1, IgG4, IgG2 and IgG3.
[36]
36. Isolated antibody according to any one of the preceding claims, characterized by the fact that the antibody binds to human SIRPα with a KD less than or equal to about 1, 1-6, 1-5, 1-4, 1- 3, 2, 3, 4, 5, 6, 7, 8, 9 or 10 x 10-9 M, as measured by the Biacore assay.
[37]
37. Antibody isolated from any of the claims above characterized by the fact that it is for use as a medicine.
[38]
38. Antibody isolated from any of the above claims for use in the treatment of cancer or infection.
[39]
39. Isolated antibody according to any one of the above claims, characterized by the fact that it is for use in the treatment of cancer, in which the cancer is selected from a solid tumor and a hematological tumor.
[40]
40. Antibody isolated from any of the claims above characterized by the fact that it is for use in increasing phagocytosis.
[41]
41. Isolated humanized, human or chimeric antibody characterized by the fact that it competes for binding to human SIRPα with the antibody isolated from any of the above claims.
[42]
42. Isolated humanized, human or chimeric antibody characterized by the fact that it binds the epitope of human SIRPα bound to the antibody isolated from any of the above claims.
[43]
43. Isolated polynucleotide or set of polynucleotides encoding the isolated antibody according to any one of the preceding claims, a VH of this, a VL of this, a light chain of this, a light chain of this, a heavy chain of it or a binding portion to its antigen.
[44]
44. Vector or set of vectors characterized by the fact that it comprises the polynucleotide or set of polynucleotides, according to claim 43.
[45]
45. Host cell characterized by the fact that it comprises the polynucleotide or set of polynucleotides of claim 43 or the vector or set of vectors of claim 44.
[46]
46. Method for producing an antibody characterized in that it expresses the antibody with the host cell of claim 45 and isolates the expressed antibody.
[47]
47. Pharmaceutical composition characterized by the fact that it comprises the antibody of any one of claims 1-42 and a pharmaceutically acceptable excipient.
[48]
48. Method for treating or preventing a disease or condition in a subject in need, characterized in that it comprises administering to the subject an effective amount of the antibody of any one of claims 1-42 or a pharmaceutical composition of claim 47.
[49]
49. Method, according to claim 48, characterized by the fact that the disease or condition is selected from: (a) cancer; (b) infection; (c) a viral infection; (d) a bacterial infection; (e) a fungal infection; (f) fibrosis; (g) arteriosclerosis; (h) a parasitic infection, optionally malaria; and (i) depletion or reduction of bone marrow-forming endogenous stem cells to allow radiation and / or chemotherapy - free of, or - reduced for transplantation of blood-forming stem cells, optionally in combination with anti-CKIT antibody (CD117).
[50]
50. Method according to claim 49, characterized by the fact that the disease or condition is a cancer, and the cancer is selected from a solid tumor and a hematological tumor.
[51]
51. Method for treating or preventing a disease or condition in a subject in need, characterized in that it comprises administering to the subject an effective amount of the antibody of any one of claims 1-42 or a pharmaceutical composition of claim 47.
[52]
52. Method for modulating an immune response in a subject in need thereof, characterized in that it comprises administering to the subject an effective amount of the antibody of any one of claims 1-42 or a pharmaceutical composition of claim 47.
[53]
53. Method according to any one of claims 48 to 52, characterized in that it further comprises the administration of one or more additional therapeutic agents to the subject.
[54]
54. Method according to claim 53, characterized in that the additional therapeutic agent is an antibody that binds,
[55]
55. Method according to claim 54, characterized in that the additional therapeutic agent is an antibody that binds a protein or proteins to the cell surface of a tumor.
[56]
56. Method according to claim 54, characterized in that the additional therapeutic agent is an antibody that binds: (a) HER2 (ERBB2 / neu), CD52, PD-L1, VEGF, CD30, EGFR, CD38 , RANKL (CD254), GD2 (ganglioside), SLAMF7 (CD319), CD20, EGFR, PDGFRa, VEGFR2, CD33, CD44, CD99, CD96, CD90, CD133, CKIT (CD117 for CKIT positive tumors); (b) CTLA-4, PD-1, PD-L1, CD40 (agonistic), LAG3 (CD223), 41BB (agonistic CD137), OX40 (CD134, agonistic); and / or (c) CKIT (CD117) to deplete blood-forming stem cells for transplant therapy.
[57]
57. Method according to claim 54, characterized by the fact that the antibody is at least one of: Rituximab, Cetuximab, Alemtuzumab (CD52), Atezolizumab (PD-L1), Avelumab (PD-L1), Bevacizumab (VEGF ), Brentuximab (CD30), Daratumumab (CD38), Denosumab (RANKL), Dinutuximab (GD2), Elotuzumab (SLAMF7), Ibritumomab (CD20), Ipilimumab (CTLA-4),
Necitumumab (EGFR), Nivolumab (PD-1), Obinutuzumab (CD20), Ofatumumab (CD20), Olaratumab (PDGFRa), Panitumumab (EGFR), Pembrolizumab (PD-1), Pertuzumab (HER2), Ramuciruma (V2), Ramuciruma (CD20), and Gemtuzumab (CD33).
[58]
58. Method according to any one of claims 53 to 57, characterized in that the additional therapeutic agent is formulated in the same pharmaceutical composition as the antibody.
[59]
59. Method according to any one of claims 53 to 57, characterized in that the additional therapeutic agent is formulated in a pharmaceutical composition other than the antibody.
[60]
60. Method according to any one of claims 53 to 57, characterized in that the additional therapeutic agent is administered prior to administration of the antibody.
[61]
61. Method according to any one of claims 53 to 57, characterized in that the additional therapeutic agent is administered after administration of the antibody.
[62]
62. Method according to any one of claims 53 to 61, characterized in that the additional therapeutic agent is administered contemporaneously with the antibody.
[63]
63. Kit characterized by the fact that it comprises the antibody of any of claims 1-42 or a pharmaceutical composition of claim 47 and instructions for use.

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法律状态:
2020-12-15| B25G| Requested change of headquarter approved|Owner name: FORTY SEVEN, INC. (US) |

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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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US201762537207P| true| 2017-07-26|2017-07-26|

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US62/537,207|2017-07-26|

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PCT/US2018/043699|WO2019023347A1|2017-07-26|2018-07-25|Anti-sirp-alpha antibodies and related methods|

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