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US-12617857-B2 - Chemokine receptor 4 (CXCR4) antagonist antibodies

US12617857B2US 12617857 B2US12617857 B2US 12617857B2US-12617857-B2

Abstract

This application provides isolated antibodies, and antigen-binding fragments thereof that specifically bind chemokine receptor 4 (CXCR4). These CXCR4 antibodies, or antigen-binding fragments thereof, have a high affinity for CXCR4, function to effectively block SDF-1 binding to CXCR4, thereby inhibiting forskolin stimulated cAMP with low nM IC50, are less immunogenic compared to their unmodified parent antibodies in a given species (e.g., in a human), and can be used to treat CXCR4-associated disorders while avoiding the adverse side effects associated with the current CXCR4 antagonist therapies.

Inventors

  • Hai Yan
  • John Zhang

Assignees

  • REMD BIOTHERAPEUTICS, INC.

Dates

Publication Date
20260505
Application Date
20210422

Claims (14)

  1. 1 . An isolated antibody or antigen-binding fragment thereof which specifically binds human Chemokine receptor 4 (CXCR4) and comprises: the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13 and the heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7.
  2. 2 . An isolated antibody or antigen-binding fragment thereof according to claim 1 , further comprising a set of four variable region framework regions from a human immunoglobulin (IgG).
  3. 3 . An isolated antibody or antigen-binding fragment thereof according to claim 1 that binds to CXCR4 protein with a dissociation constant (K D ) of at least about 1×10 −6 M, at least about 1×10 −7 M, at least about 1×10 −8 M, at least about 1×10 −9 M, at least about 1×10 −10 M, at least about 1×10 −11 M, or at least about 1×10 −12 M.
  4. 4 . An isolated antibody or antigen-binding fragment thereof according to claim 1 wherein the antibody or antigen-binding fragment is selected from a human antibody, a humanized antibody, chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a single chain antibody, a diabody, a triabody, a tetrabody, a Fab fragment, a Fab′ fragment, a Fab 2 fragment, a F (ab)′ 2 fragment, a domain antibody, an IgD antibody, an IgE antibody, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, or an IgG4 antibody having at least one mutation in the hinge region that alleviates a tendency to form intra H-chain disulfide bonds.
  5. 5 . An isolated humanized antibody or antigen-binding fragment thereof that specifically binds human CXCR4 and comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 24-29, and a light chain variable region sequence selected from the group consisting of SEQ ID NOs: 30-32.
  6. 6 . An isolated humanized antibody or antigen-binding fragment thereof according to claim 5 which comprises the heavy chain sequence set forth in SEQ ID NO: 33, and the light chain sequence set forth in SEQ ID NO: 35.
  7. 7 . An isolated humanized antibody or antigen-binding fragment thereof according to claim 5 which comprises the heavy chain sequence set forth in SEQ ID NO: 33, and the light chain sequence set forth in SEQ ID NO: 37.
  8. 8 . An isolated humanized antibody or antigen-binding fragment thereof according to claim 5 which comprises the heavy chain sequence set forth in SEQ ID NO: 33, and the light chain sequence set forth in SEQ ID NO: 39.
  9. 9 . A pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof according to claim 1 in admixture with a pharmaceutically acceptable carrier.
  10. 10 . A method of treating a subject suffering from a CXCR4-associated disorder, comprising administering to said subject a therapeutically effective amount of the pharmaceutical composition of claim 9 .
  11. 11 . An isolated immunoconjugate or fusion protein comprising an antibody or antigen-binding fragment thereof according to claim 1 coupled to an effector molecule.
  12. 12 . An isolated nucleic acid comprising a polynucleotide sequence encoding an antibody or antigen-binding fragment thereof according to claim 1 .
  13. 13 . A recombinant expression vector comprising the isolated nucleic acid of claim 12 .
  14. 14 . A host cell comprising the vector of claim 13 .

Description

RELATED PATENT APPLICATIONS This application is a U.S. National Stage Application pursuant to 35 U.S.C. § 371 of PCT/US2021/028583, filed Apr. 22, 2021, which claims benefit of U.S. Provisional Application No. 63/014,602, filed on Apr. 23, 2020, incorporated in its entirety by reference herein. TECHNICAL FIELD Chemokines (chemoattractant cytokines) are comprised of a family of structurally and functionally related polypeptides with 8-10 kilodaltons. Chemokines are involved in diverse biological functions including regulation of immune cell proliferation, migration, activation, differentiation and homing. The biological activities of chemokines are mediated by a family of 7-transmembrane G-protein coupled receptors (GPCRs). One member of the chemokine receptor family is CXCR4 which is specific for chemokine CXCL12 (also known as stromal-derived-factor-1 (SDF-1)). SDF-1 binding to CXCR4 activates a cascade of signaling molecules including suppression of cAMP, activation of phospholipase C and intracellular calcium flux (Mukaida et al., Exp Cell Res., 318(2):95-102, 2012). SDF-1 is expressed on a wide variety of human tissues, including liver, lungs, bone marrow, lymph nodes, stromal and endothelial cells. Similarly, CXCR4 is found in various tissues with predominant expression on lymphocytes (Zhou et al., Curr Med Chem., 26 (17): 3026-3041, 2019). Human genetic studies support the concept that increased CXCR4 activity is associated with compromised immune system and increased risks of cancer and other immunodeficiency diseases. Overexpression of CXCR4 has been found in 75% of solid and hematopoietic cancers, including pancreatic, lung, breast, ovarian, prostate and colorectal tumors, leukemias and lymphomas (Zlotnik et al., Homeostatic chemokine receptors and organ-specific metastasis. Nat Rev Immunol., 11(9):597-606, 2011). Activation of CXCR4 by CXCL12 stimulates various signaling pathways that promote chemotaxis, adhesion, migration, cell proliferation and survival (Balkwill et al., J Pathol., 226(2):148-157, 2012). The downstream effectors of CXCR4/SDF-1, including PI3 kinase, Ras, stress-activated protein kinase (SAPK), c-Jun N-terminal kinase (JNK), phospholipase C (PLC), mitogen-activated protein kinase (MAPK), p38 MAPK and AKT are involved in tumor cell proliferation, migration and metastasis. Both SDF-1 and CXCR4 were identified in cancer stem cells (CSC), a population of tumor cells that drive carcinogenesis and resistant to conventional cancer therapies (Larzabal et al., Plos one, 8(11) e79798, 2013). In clinical studies, CXCR4 has been associated with increased propensity for metastasis and decreased survival and greater expression of CXCR4 correlates with disease severity (Spano et al., Ann Oncol, 15:613-7, 2004). Immunotherapy using agonistic, antagonistic, or blocking antibodies to co-stimulatory or co-inhibitory molecules (immune checkpoints) has been an area of extensive research and clinical evaluation. Immune checkpoint proteins include CTLA-4, PD-1, PD-L1, LAG-3, and TIM-3 as well as several others (Sharpe et al., Nat Immunol, 8:239-45, 2007). Under normal physiological conditions, immune checkpoints are crucial for the maintenance of self-tolerance (that is, the prevention of autoimmunity) and protect tissues from damage when the immune system is responding to pathogenic infection. It is now also clear that tumors co-opt certain immune-checkpoint pathways as a major mechanism of immune resistance, particularly against T cells that are specific for tumor antigens (Pardoll D M., Nat Rev Cancer, 12:252-64, 2012). Accordingly, treatment utilizing antibodies to immune checkpoint molecules including, e.g., CTLA-4 (ipilimumab), PD-1 (nivolumab; pembrolizumab; pidilizumab) and PD-L1 (BMS-936559; MPLD3280A; MED14736; MSB0010718C)(see, e.g, Philips and Atkins, International Immunology, 27(1); 39-46, October 2014), and OX-40, CD137, GITR, LAGS, TIM-3, and VISTA (see, e.g., Sharon et al., Chin J Cancer., 33(9): 434-444, September 2014; Hodi et al., N Engl J Med, 2010; Topalian et al., N Engl J Med, 366:2443-54) are being evaluated as new, alternative immunotherapies to treat patients with proliferative diseases such as cancer, and in particular, patients with refractory and/or recurrent cancers. Currently, checkpoint inhibitor therapy has become the preferred second- or third-line therapy for many cancers, and PD1/PDL1 antibodies have changed the treatment paradigm for several cancers. Despite these significant advancements, there remains a major need to improve on the current state-of-the-art. For example, checkpoint inhibitor therapy remains limited by concerns over potential severe side effects and the fact that many tumors lack the targeted antigen and will therefore evade treatment. In general, about 20% of patients with various cancers respond to PD-1/PD-L1 antibodies or CTLA-4 antibodies and overall survival remains less than a year in most instances and objective response rates are modest, un