Antibody

Abstract

The present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to CXCR4. The present invention also relates to said antibody for use in therapy, for use in the treatment of cancer, and for use in the treatment of solid tumors. The present invention also relates to pharmaceutical compositions comprising said antibody, nucleic acids encoding said antibody, vectors comprising said nucleic acids, and host cells comprising said nucleic acids or vectors.

Description

[Technical Field]

[0001] Antibodies or antigen-binding fragments that specifically bind to CXCR4 (CXC motif chemokine receptor 4) and their uses in treating various diseases or conditions are described. [Background technology]

[0002] CXCR4 (CXC motif chemokine receptor 4, also known as CD184, FB22, fusin, HM89, LCR1, leukocyte-derived seven transmembrane domain receptor (LESTR), lipopolysaccharide-associated protein 3 (LAP-3) and stromal cell-derived factor 1 (SDF-1) receptor) (Uniprot ID: P61073) is a class A, G-protein coupled receptor (GPCR) and part of the chemokine receptor family. CXCR4 shares a barrel-like structure common to GPCRs, containing seven transmembrane alpha helices, three extracellular loops, three intracellular loops, an extracellular N-terminus, and an intracellular C-terminus (Wu et al., (2010). Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists. Science. 330(6007):1066-1071; Zhu et al., (2013). Structure-based studies of chemokine receptors. Curr Opin Struct Biol. 23(4):539-546).

[0003] GPCRs are the largest family of membrane receptors in humans and many other species. In addition, GPCRs are considered the largest family of approved drug targets (Allen and Roth, (2011). Strategies to discover unexpected targets for drugs active at G protein-coupled receptors. Annu Rev Pharmacol Toxicol. 51:117-144; Rask-Andersen et al., (2014). The druggable genome: Evaluation of drug targets in clinical trials suggests major shifts in molecular class and indication. Annu Rev Pharmacol Toxicol. 54:9-26; Santos et al., (2017) and Oprea et al., (2018). Unexplored therapeutic opportunities in the human genome. Nat Rev Drug Discov. 17:317-332. A comprehensive map of molecular drug targets. Nat Rev Drug Discov. 16(1):19-34). Many factors contribute to the widespread utility of GPCR-targeted drugs, including their druggability, interactions with numerous types of chemokines and other ligands, and expression at the plasma membrane, which allows for extracellular targeting by molecules such as antibodies. This has led to over 35% of approved drugs targeting GPCRs and associated upstream or downstream ligands or signaling pathways.Approximately 12% directly target GPCRs (Hopkins and Groom, (2002). The druggable genome. Nat Rev Drug Discov. 1(9):727-730; Sriram and Insel, (2018). GPCRs as targets for approved drugs: How many targets and how many drugs?. Mol Pharmacol. 93(4):251-258). However, GPCRs are dynamic transmembrane proteins, making structural studies difficult. The CXCR4 crystal structure, solved in 2010, was the first crystal structure of a peptide GPCR (Wu et al., (2010), Zhu et al., (2013), both cited above).

[0004] The endogenous ligand for CXCR4 is CXCL12, also known as SDF-1 (Uniprot ID: P48061). CXCL12 is a highly conserved chemokine with 99% homology between human and mouse CXCL12 (Schabath et al., (1999). The murine chemokine receptor CXCR4 is tightly regulated during T cell development and activation. Journal of Leukocyte Biology. 66(6):996-1004). There are many human CXCL12 isoforms, including alpha, beta, gamma, delta, epsilon, theta, and isoform 7. Additional ligands for CXCR4 have also been identified, including macrophage migration inhibitory factor (MIF) (Bernhagen et al., (2007). MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment. Nat Med. 13(5):587-596) and ubiquitin (Saini et al., (2010). CXC chemokine receptor 4 is a cell surface receptor for extracellular ubiquitin. J Biol Chem. 14;285(20):15566-15576).

[0005] CXCL12 binding is mediated by G αi CXCL12 binding promotes a three-dimensional CXCR4 conformation that facilitates protein dissociation. CXCL12 binding inhibits cAMP formation via inhibition of adenylyl cyclase activity, activation of phospholipase C (PLC)-β, and the release of diacylglycerol and intracellular Ca. 2+CXCR4 mediates various cellular activities, including the generation of inositol 1,4,5 trisphosphate (IP3), which regulates the release of CXCR4. By inhibiting adenylyl cyclase, the receptor activates the NF-kB, JAK-STAT, and PI3K-AKT pathways, as well as mTOR and JNK / p38 MAPK, regulating cell survival, proliferation, and chemotaxis (Scala (2015). Molecular Pathways: Targeting the CXCR4-CXCL12 Axis—Untapped Potential in the Tumor Microenvironment. Clin Cancer Res. 21(19):4278-4285). Beta-arrestin recruitment allows desensitization of the receptor by internalization following CXCL12 binding (Teicher & Fricker (2010). CXCL12 (SDF-1) / CXCR4 pathway in cancer. Clin Cancer Res. 16(11):2927-2931; Cheng et al. (2000). β-Arrestin Differentially Regulates the Chemokine Receptor CXCR4-Mediated Signaling and Receptor Internalization, and This Implicates Multiple Interaction Sites between β-Arrestin and CXCR4. J Biol Chem. 275(4):2479-2485).

[0006] CXCR4 is expressed in many organs of the body, most highly in cells of the immune system, and consequently, is also highly expressed in the bone marrow niche from which these mature immune cells originate (e.g., myeloid cells, erythropoietic cells, lymphocytes, monocytes, and blast cells) (Data from The Human Protein Atlas, Version 18.1, Updated 15 / 11 / 2018, https: / / www.proteinatlas.org / ). CXCR4 has also been found to be elevated in tissues of endocrine origin (e.g., adrenal and thyroid tissues). CXCR4 expression has been identified in multiple cancer cells, including breast, renal, pancreatic, ovarian, endometrial, head and neck, colorectal, gastric, and lung cancers (Data from The Human Protein Atlas, Version 18.1, Updated 15 / 11 / 2018, https: / / www.proteinatlas.org / ). CXCR4 is also involved in the subsequent proliferation and metastasis of cancer cells (Guo et al., (2014). CXCL12-CXCR4 Axis Promotes Proliferation, Migration, Invasion, and Metastasis of Ovarian Cancer. Oncol Res. 22(5-6):247-258; Wei et al., (2018). Targeting the C-X-C motif chemokine receptor 4 inhibits lung cancer cell proliferation, migration, and angiogenesis. Oncol Lett. 16(3):3976-3982).

[0007] In addition to the role of CXCR4 in cancer, mutations in CXCR4 (mainly found in the C-terminus) have been associated with WHIM syndrome (warts, hypogammaglobulinemia, infections, and myeloid cell pooling) (McDermott & Murphy (2019). WHIM syndrome: Immunopathogenesis, treatment, and cure strategies. Immunol Rev. 287(1):91-102). CXCR4 is also a coreceptor for HIV-1 and is being investigated as a therapeutic target for HIV treatment (Chen (2019). Molecular Mechanism of HIV-1 Entry. Trends Microbiol. doi:10.1016 / j.tim.2019.06.002. [Epub ahead of print]). Summary of the Invention [Means for solving the problem]

[0008] Antibody or antigen-binding fragment Disclosed herein are antibodies and antigen-binding fragments thereof that specifically bind to CXCR4, e.g., human CXCR4. In one embodiment, the antibody or antigen-binding fragment thereof specifically binds to surface-expressed CXCR4. In another embodiment, the antibody or antigen-binding fragment thereof inhibits binding of CXCL12 to CXCR4. In one embodiment, the antibody or antigen-binding fragment thereof inhibits binding of CXCL12 to human CXCR4.

[0009] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CXCR4, wherein the antibody or antigen-binding fragment thereof comprises a VH domain, wherein the VH domain comprises: (i) the CDRH1 amino acid sequence of SEQ ID NO: 10 or 13, or SEQ ID NO: 10 or 13, each comprising three, two, or one amino acid substitutions; (ii) the CDRH2 amino acid sequence of SEQ ID NO: 11 or 14, or SEQ ID NO: 11 or 14, each comprising three, two, or one amino acid substitutions; (iii) SEQ ID NO: 12 or 15, or SEQ ID NO: 12 or 15, each comprising three, two, or one amino acid substitutions. and (iii) a CDRL3 amino acid sequence of SEQ ID NO: 22 or 25 or a CDRL4 amino acid sequence of SEQ ID NO: 22 or 25, each of which contains three, two, or one amino acid substitutions.

[0010] In one embodiment, the VH domain comprises (i) a CDRH1 amino acid sequence of SEQ ID NO: 10 or 13, (ii) a CDRH2 amino acid sequence of SEQ ID NO: 11 or 14, and (iii) a CDRH3 amino acid sequence of SEQ ID NO: 12 or 15, and the VL domain comprises (i) a CDRL1 amino acid sequence of SEQ ID NO: 20 or 23, (ii) a CDRL2 amino acid sequence of SEQ ID NO: 21 or 24, and (iii) a CDRL3 amino acid sequence of SEQ ID NO: 22 or 25, and each of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 contains up to one amino acid substitution, optionally a conservative amino acid substitution.

[0011] In one embodiment, the VH domain comprises (i) a CDRH1 amino acid sequence of SEQ ID NO: 10 or 13, (ii) a CDRH2 amino acid sequence of SEQ ID NO: 11 or 14, and (iii) a CDRH3 amino acid sequence of SEQ ID NO: 12 or 15; the VL domain comprises (i) a CDRL1 amino acid sequence of SEQ ID NO: 20 or 23, (ii) a CDRL2 amino acid sequence of SEQ ID NO: 21 or 24, and (iii) a CDRL3 amino acid sequence of SEQ ID NO: 22 or 25; and up to one CDR selected from CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprises up to one amino acid substitution, optionally a conservative amino acid substitution.

[0012] In one embodiment, the VH domain comprises (i) a CDRH1 amino acid sequence of SEQ ID NO: 10 or 13, (ii) a CDRH2 amino acid sequence of SEQ ID NO: 11 or 14, and (iii) a CDRH3 amino acid sequence of SEQ ID NO: 12 or 15, and the VL domain comprises (i) a CDRL1 amino acid sequence of SEQ ID NO: 20 or 23, (ii) a CDRL2 amino acid sequence of SEQ ID NO: 21 or 24, and (iii) a CDRL3 amino acid sequence of SEQ ID NO: 22 or 25.

[0013] In one embodiment, the VH domain comprises (i) a CDRH1 amino acid sequence of SEQ ID NO: 13, (ii) a CDRH2 amino acid sequence of SEQ ID NO: 14, and (iii) a CDRH3 amino acid sequence of SEQ ID NO: 15, and the VL domain comprises (i) a CDRL1 amino acid sequence of SEQ ID NO: 23, (ii) a CDRL2 amino acid sequence of SEQ ID NO: 24, and (iii) a CDRL3 amino acid sequence of SEQ ID NO: 25, wherein each of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprises 0 to 3 amino acid substitutions, optionally conservative amino acid substitutions.

[0014] In one embodiment, the VH domain comprises (i) a CDRH1 amino acid sequence of SEQ ID NO: 13, (ii) a CDRH2 amino acid sequence of SEQ ID NO: 14, and (iii) a CDRH3 amino acid sequence of SEQ ID NO: 15, and the VL domain comprises (i) a CDRL1 amino acid sequence of SEQ ID NO: 23, (ii) a CDRL2 amino acid sequence of SEQ ID NO: 24, and (iii) a CDRL3 amino acid sequence of SEQ ID NO: 25, wherein each of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 contains up to one amino acid substitution, optionally a conservative amino acid substitution.

[0015] In one embodiment, the VH domain comprises (i) a CDRH1 amino acid sequence of SEQ ID NO: 13, (ii) a CDRH2 amino acid sequence of SEQ ID NO: 14, and (iii) a CDRH3 amino acid sequence of SEQ ID NO: 15; the VL domain comprises (i) a CDRL1 amino acid sequence of SEQ ID NO: 23, (ii) a CDRL2 amino acid sequence of SEQ ID NO: 24, and (iii) a CDRL3 amino acid sequence of SEQ ID NO: 25; and at most one CDR selected from CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprises at most one amino acid substitution, optionally a conservative amino acid substitution.

[0016] In one embodiment, the VH domain comprises (i) a CDRH1 amino acid sequence of SEQ ID NO: 13, (ii) a CDRH2 amino acid sequence of SEQ ID NO: 14, and (iii) a CDRH3 amino acid sequence of SEQ ID NO: 15, and the VL domain comprises (i) a CDRL1 amino acid sequence of SEQ ID NO: 23, (ii) a CDRL2 amino acid sequence of SEQ ID NO: 24, and (iii) a CDRL3 amino acid sequence of SEQ ID NO: 25.

[0017] In one embodiment, the VH domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 16, and / or the VL domain comprises an amino acid sequence at least 90% identical to SEQ ID NO:26.

[0018] In a further embodiment, the VH domain comprises the amino acid sequence of SEQ ID NO:16 and the VL domain comprises the amino acid sequence of SEQ ID NO:26.

[0019] In one embodiment, the antibody or fragment is human, humanized, or chimeric.

[0020] In one embodiment, the CXCR4 is human (optionally selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3). In one embodiment, the CXCR4 is rhesus and / or cynomolgus (optionally selected from SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9).

[0021] In one embodiment, the antibody or fragment specifically binds to human, rhesus, cynomolgus and / or rodent CXCR4, optionally human and cynomolgus CXCR4, optionally wherein binding is determined by surface plasmon resonance (SPR), flow cytometry, live cell imaging, ELISA or radioligand binding.

[0022] In one embodiment, the antibody or fragment comprises a constant region (CH and / or CL). In a further embodiment, the CH is (i) an IgG4 constant region, such as an IgG4-PE constant region (e.g., SEQ ID NO: 267 or SEQ ID NO: 305), or (ii) an IgG1 constant region, such as an IgG1 constant region comprising mutations that reduce binding to Fc-γ receptors and / or C1q compared to wild-type (e.g., SEQ ID NO: 249 or 307). In a further embodiment, the CL is a kappa light chain constant region.

[0023] In one embodiment, the antibody or fragment comprises a heavy chain and a light chain, wherein the heavy chain amino acid sequence comprises an amino acid sequence at least 90% identical to SEQ ID NO: 18 and / or the light chain amino acid sequence comprises an amino acid sequence at least 90% identical to SEQ ID NO: 28. In a further embodiment, the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 18 and the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 28.

[0024] In one embodiment, the antibody or fragment inhibits binding of CXCL12 to CXCR4.

[0025] In one embodiment, the antibody or fragment inhibits CXCL12-mediated inhibition of forskolin-stimulated cAMP with an IC50 of 1 to 100 nM (e.g., 1 to 30 nM), and optionally, CXCR4-dependent CXCL12-mediated inhibition of cAMP is determined using a forskolin-stimulated cAMP signaling assay.

[0026] In one embodiment, the antibody or fragment inhibits beta-arrestin recruitment to CXCR4 with an IC50 of 0.01 to 15 nM (e.g., 0.01 to 0.2 nM), and optionally, beta-arrestin recruitment is determined using a functional cell-based reporter gene assay.

[0027] In one embodiment, the antibody or fragment inhibits binding of CXCL12 to CXCR4 with an IC50 of 0.2 to 4 nM (eg, 0.2 to 1 nM), optionally CXCL12 inhibition is determined using an HTRF assay.

[0028] In one embodiment, the antibody or fragment does not induce apoptosis in T cells (optionally CD8+ T cells), and optionally, apoptosis is determined using flow cytometry.

[0029] In one embodiment, the antibody or fragment binds to cynomolgus CXCR4 with an EC50 of 0.5-10 nM (eg, 0.5-5 nM), and optionally, cynomolgus CXCR4 binding is determined using flow cytometry.

[0030] In one embodiment, the antibody or fragment binds to human CXCR4 with a KD of 0.2 to 2 nM, (e.g., 0.4 to 0.8 nM), and optionally, the binding affinity is determined using surface plasmon resonance (SPR).

[0031] In one embodiment, the antibody or fragment does not bind to CXCR7 (optionally, CXCR7 is human and further optionally selected from SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6), and optionally CXCR7 binding is determined using flow cytometry, radioligand binding, surface plasmon resonance (SPR), live cell imaging, or ELISA.

[0032] In one embodiment, the antibody or fragment inhibits a CXCL12-mediated response of T cells with an IC50 of 0.5 to 20 nM (e.g., 0.5 to 5 nM), where inhibition is determined using an in vitro label-free dynamic mass redistribution assay.

[0033] In one embodiment, the antibody or fragment inhibits CXCR4+ T cell chemotaxis to CXCL12 with an IC50 of 0.01 to 5 nM (e.g., 0.01 to 1 nM), where inhibition is determined using live cell imaging or flow cytometry.

[0034] In one embodiment, the antibody or fragment increases mean CD45+ cell recruitment compared to PBS, optionally where recruitment is determined using flow cytometry.

[0035] In one embodiment, the antibody or fragment binds to a CXCR4 homodimer.

[0036] In one embodiment, the antibody or fragment enables CD8+ T cells to infiltrate the tumor.

[0037] One aspect of the invention provides an antibody or antigen-binding fragment described herein for use in therapy.

[0038] A further aspect of the invention provides an antibody or fragment as described herein for use in the treatment of cancer, optionally wherein the cancer is pancreatic cancer or pancreatic ductal carcinoma.

[0039] A further aspect of the invention provides an antibody or fragment as described herein for use in the treatment of a solid tumor.

[0040] In one embodiment, the treatment further comprises administering an additional therapy, optionally wherein the additional therapy comprises one or more additional therapeutic agents independently selected from the group consisting of an anti-PD-1 antibody or antigen-binding fragment thereof and an anti-PD-L1 antibody or antigen-binding fragment thereof, and / or optionally wherein the additional therapy is selected from chemotherapy, radiation therapy, and / or surgical removal of the tumor.

[0041] In one embodiment, the treatment further comprises administering an additional therapeutic agent that is a PD-1 / PD-L1 signaling inhibitor (e.g., a PD-1 antibody or antigen-binding fragment thereof, or a PD-L1 antibody or antigen-binding fragment thereof).

[0042] A further aspect of the invention provides a pharmaceutical composition comprising an antibody or fragment as described herein and a pharmaceutically acceptable excipient, diluent or carrier, and optionally further comprising one or more additional therapeutic agents.

[0043] A further aspect of the invention provides nucleic acids encoding (a) the VH domain and / or the VL domain of an antibody or fragment described herein, or (b) the heavy and / or light chain of an antibody or fragment described herein.

[0044] A further aspect of the invention provides a vector comprising a nucleic acid as described herein, which is optionally a CHO or HEK293 vector.

[0045] A further aspect of the invention provides a host cell comprising a nucleic acid described herein or a vector described herein. [Brief explanation of the drawings]

[0046] [Figure 1-1]Figure 1. Time-dependent internalization rates of anti-CXCR4 antibodies (A. CL-82458, B. CL-82558, C. CL-82574, D. CL-82658, E. CL-83083, F. Benchmark 1c, G. negative (IgG4-PE isotype) and H. relevant positive (human anti-CD20 antibody) controls. The figure shows the internalization rate with increasing concentrations of antibody. mAb conc; concentration of antibody used in the assay, nanomolar. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 2] Binding of anti-CXCR4 antibodies (IgG4-PE format), benchmark antibodies (IgG4-PE format), and IgG4-PE isotype control antibodies to cynomolgus monkey CXCR4. Representative data of anti-CXCR4 antibodies binding to cynomolgus monkey CXCR4 expressed on CHO cells as assessed by flow cytometry. Data are representative of four independent experiments. Binding expressed as geometric (Geo) mean. [Figure 3] Non-binding of anti-CXCR4 (IgG4-PE format), benchmark (IgG4-PE format), IgG4-PE isotype control, anti-CXCR7 mAb #11G8, and mouse IgG1 isotype control antibody to CXCR7. Representative data of anti-CXCR4 antibodies that do not bind to CXCR7 expressed on CHO cells, as assessed by flow cytometry. Data are representative of two independent experiments. Binding expressed as geometric (Geo) mean. [Figure 4] Mean percent inhibition (±SD) obtained for anti-CXCR4 antibodies (IgG4-PE format), benchmark antibodies (IgG4-PE format), and IgG4-PE isotype control antibodies incubated with Jurkat T cells stimulated to migrate toward 30 nM CXCL12. Data are representative of three independent experiments. [Figure 5]Summary of anti-CXCR4-mediated inhibition of primary naive T cell migration. Plots represent the mean (±SD) of the maximum percentage of naive primary T cells' migration toward 6.25 nM CXCL12 when pretreated with different concentrations of anti-CXCR4 antibodies (IgG4-PE format) and IgG4-PE isotype control in 3-5 independent donors. [Figure 6] Inhibition of primary naive T cells by benchmark anti-CXCR4 antibodies. The plot represents the mean (±SD) of the maximum percentage migration towards 6.25 nM CXCL12 achieved for naive T cells pretreated with different concentrations of benchmark anti-CXCR4 antibodies (IgG4-PE format) and IgG4-PE isotype control for three independent donors. [Figure 7] Percentage of CCRF-HSB-2 invasion into IFNγ-treated HT-29 tumor spheroids after treatment with anti-CXCR4 antibody (IgG4-PE format) or IgG4-PE isotype control. Data represent five independent experiments (mean ± SD). [Figure 8] Mobilization of human CD45+ cells from NSG mice upon treatment with anti-CXCR4 antibody (IgG4-PE format). The total number of human CD45+ cells detected in each blood sample well was recorded by flow cytometry. [Figure 9] Schematic of the proposed mechanism of action for T cell exclusion from tumors. In response to local tumor-promoting conditions, cancer-associated fibroblasts (CAFs) localized in the stroma secrete high levels of CXCL12. CXCR4+ T cells respond to the rising CXCL12 gradient and are prevented from following other, less established gradients (e.g., CXCL9, CXCL10, and / or CXCL11), resulting in the T cells remaining in the stroma. The CXCL9, CXCL10, and / or CXCL11 gradients are thought to promote T cell entry into the tumor, leading to the killing of malignant tumor cells. [Figure 10]Blockade of CXCR4 on T cells with anti-CXCR4 antibodies prevents the cells from responding to CXCL12 in the stroma. T cells can follow other T cell-associated chemokine gradients (e.g., CXCL9, CXCL10, and / or CXCL11), which may allow successful T cell infiltration into tumors and subsequent tumor cell killing. [Figure 11] Combination treatment using an anti-CXCR4 antibody and a PD-1 inhibitor slows tumor growth compared to vehicle controls. DETAILED DESCRIPTION OF THE INVENTION

[0047] 1.Definition Unless otherwise defined herein, scientific and technical terms shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular.

[0048] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the term "or" is intended to include "and" unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used to practice or test the present disclosure, suitable methods and compositions are described below. The abbreviation "eg" is derived from the Latin "exempli gratia" and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example."

[0049] In the present specification and claims, the term "about" is used to modify values ​​and ranges thereof, such as the amount, concentration, volume, process temperature, process time, yield, flow rate, and pressure of a component in a composition, used in describing an embodiment of the present disclosure. The term "about" refers to variations in numerical quantities that may occur due to approximation considerations, such as typical measuring and handling procedures used to prepare a compound, composition, concentrate, or use formulation, inadvertent errors in these procedures, differences in the manufacture, source, or purity of starting materials or components used to carry out the method, etc. The term "about" also encompasses amounts that vary with aging of a formulation having a particular initial concentration or mixture, and amounts that vary with mixing or processing of a formulation having a particular initial concentration or mixture. When modified by the term "about," the claims appended hereto include amounts equivalent to these amounts.

[0050] As used herein, "administering" or "administration" refers to the act of injecting or otherwise physically delivering a substance (e.g., an anti-CXCR4 antibody or antigen-binding fragment provided herein) to a patient if the substance is present outside the body, such as by mucosal, intradermal, intravenous, intramuscular delivery and / or any other method of physical delivery described herein or known in the art. When a disease (or condition) or a symptom thereof is being treated, administration of the substance typically occurs after the onset of the disease (or condition) or a symptom thereof. When a disease (or condition) or a symptom thereof is being prevented, administration of the substance usually occurs before the onset of the disease (or condition) or a symptom thereof.

[0051] As used herein, the terms "affinity," "binding affinity," and the like refer to the degree or tendency of two molecules to bind to one another. In one embodiment, affinity or binding affinity is measured by the K D or EC 50and one of skill in the art would be able to calculate these values ​​from data generated in a suitable assay, such as surface plasmon resonance (SPR) (using Biacore™ or ProteOn XPR36™ (Bio-Rad™)), KinExA™ (Sapidyne Instruments, Inc.), ForteBio Octet (Pall ForteBio Corp.), flow cytometry (e.g., a Mirrorball™ fluorocytometer), live-cell imaging, enzyme-linked immunosorbent assay (ELISA), or radioligand binding (preferably SPR, flow cytometry, live-cell imaging, ELISA, or radioligand binding) (optionally using in combination with a program such as GraphPad Prism). In one embodiment, "affinity" or "binding affinity" is a measure of how strongly an antibody or antigen-binding fragment binds to an antigen, e.g., a target receptor (e.g., CXCR4). In one embodiment, "affinity" or "binding affinity" is the strength with which an individual paratope (antigen binding site) on an antibody or antigen-binding fragment binds to an epitope (of an antigen, e.g., a receptor), or the strength with which a receptor (e.g., CXCR4) interacts with a ligand (e.g., CXCL12). In preferred embodiments, the affinity or binding affinity of an antibody or antigen-binding fragment for a receptor is in the nanomolar (nM) range or in the picomolar (pM) range. As used herein, "K" refers to the affinity of an antibody or antigen-binding fragment to an epitope (e.g., an antigen, e.g., a receptor). D The term "K" is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction. a ” is the association rate constant (K オン Also known as "K d ” is the dissociation rate constant (K オフ (also known as

[0052] As used herein, the terms "antagonist," "antagonize," and the like refer to the ability of an antibody or its antigen-binding fragment to bind to a target antigen and inhibit the interaction of the target antigen with one or more binding partners. In the case of CXCR4, an antagonist (e.g., an antibody or its antigen-binding fragment) can antagonize the binding of a ligand (e.g., CXCL12) and prevent the biological effects of the ligand on the receptor (e.g., ligand signaling, receptor conformational changes, receptor activation, or receptor interaction with other cellular molecules). Known ligands for CXCR4 include CXCL12 (also known as SDF-1), ubiquitin, and MIF. Other CXCR4 ligands may yet be identified. CXCR4 functional activity can be measured using cAMP assays, beta-arrestin assays, CXCL12 inhibition assays, internalization assays, apoptosis assays, DMR assays, chemotaxis assays, T cell infiltration assays, migration assays, or disease-relevant models of cancer, all of which are well known to those skilled in the art. Other assays are known to those skilled in the art. In a preferred embodiment, the antibody or antigen-binding fragment thereof is an antagonist, e.g., a full antagonist. In another embodiment, the antibody or antigen-binding fragment thereof is a partial antagonist. A partial antagonist partially inhibits (i.e., does not completely inhibit) the interaction of one or more binding partners with a target antigen.

[0053] The terms "antibody," "immunoglobulin," or "Ig" may be used interchangeably herein and refer to an immunoglobulin molecule that recognizes and specifically binds to a target antigen, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof, via at least one antigen recognition site within the variable region of the immunoglobulin molecule. The term "antibody" also refers to a Y-shaped glycoprotein with a molecular weight of approximately 150 kDa, composed of four polypeptide chains (two light (L) chains and two heavy (H) chains). There are five types of mammalian Ig heavy chain constant regions, further described below. In mammals, there are two types of immunoglobulin light chains, lambda and kappa. The "variable region" or "variable domain" of an antibody or antigen-binding fragment refers to the amino-terminal domain of the heavy or light chain of the antibody. The heavy and light chain variable domains are referred to as "V" and "V" respectively. H " and "V L These domains are generally the most variable parts of an antibody (compared to other antibodies of the same class) and contain the antigen-binding sites. H and V L are the heavy chains (C H ) and light chain (C L ) bound to the constant region.

[0054] The antibody or antigen-binding fragment may be oligoclonal, polyclonal, monoclonal (including full-length monoclonal antibodies), camelized, chimeric (e.g., mouse variable regions and human constant regions or human variable regions and mouse constant regions), CDR-grafted, multispecific, bispecific, catalytic, humanized, human, fully human, anti-idiotype, and fragments, variants, fusions, or derivatives thereof, including antibodies that may be labeled in soluble or bound form, as well as any other modified immunoglobulin molecule containing an antigen-binding site, so long as the antibody or antigen-binding fragment, alone or in combination with other amino acid sequences provided by known techniques, exhibits the desired biological activity. In one embodiment, the antibody or antigen-binding fragment is human, fully human, humanized, or chimeric. In a preferred embodiment, the antibody or antigen-binding fragment is a human antibody or antigen-binding fragment, optionally with non-human post-translational modifications (e.g., glycosylation). The antibody or antigen-binding fragment may be derived from any species. In one embodiment, the antibody or antigen-binding fragment is a mammalian or rodent antibody or antigen-binding fragment. The antibodies or antigen-binding fragments described herein can be naked or conjugated to other molecules, such as toxins, radioisotopes, etc.

[0055] The terms "antigen-binding domain," "antigen-binding region," "antigen-binding fragment," and the like refer to the portion of an antibody (e.g., the complementarity-determining region (CDR)) that comprises the amino acid residues that interact with an antigen and confer on the binder its specificity and affinity for the antigen. The antigen-binding region may be derived from any animal species, including rodent (e.g., mouse, rabbit, rat, or hamster), chicken, and human. Preferably, the antigen-binding region will be of human origin. Throughout this specification, the term "fragment," in reference to an antibody, should be considered to be an antigen-binding fragment of such an antibody.

[0056] The term "antigen-binding fragment" can include single-chain Fv (scFv), single-chain antibodies, single-domain antibodies, domain antibodies, Fv fragments, Fab fragments, F(ab') fragments, F(ab') fragments, antibody fragments exhibiting the desired biological activity, disulfide-stabilized variable regions (dsFv), dimeric variable regions (diabodies), anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against antibodies), intrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments and epitope-binding fragments of any of the above. In particular, antibodies and antibody fragments described herein can include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing an antigen-binding site. Digestion of antibodies with the enzyme papain produces two identical antigen-binding fragments, also known as "Fab" fragments, and an "Fc" fragment, which lacks antigen-binding activity but is capable of crystallization. As used herein, "Fab" refers to an antibody fragment containing one constant domain and one variable domain of each of the heavy and light chains. The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. An "Fc fragment" refers to the carboxy-terminal portions of both H chains held together by disulfide bonds. The effector functions of an antibody are determined by the sequence of the Fc region, which is also recognized by Fc receptors (FcRs) found on certain types of cells. Digestion of an antibody with the enzyme pepsin produces an F(ab')2 fragment, in which the two arms of the antibody molecule remain linked and contain two antigen-binding sites. The F(ab')2 fragment retains the ability to cross-link antigens.

[0057] As used herein, "approval number" or "marketing approval number" refers to a number issued by a regulatory agency when the regulatory agency determines that a particular medical product and / or composition may be sold and / or offered for sale within that regulatory agency's jurisdiction. As used herein, "regulatory agency" refers to, for example, one of the agencies responsible for evaluating the safety and effectiveness of medical products and / or compositions and controlling the sale / marketing of such products and / or compositions in a given territory. The Food and Drug Administration (FDA) in the United States and the European Medicines Agency (EPA) in Europe are just two examples of such regulatory agencies. Other non-limiting examples include the SDA, MPA, MHPRA, IMA, ANMAT, Hong Kong Department of Health-Drug Office, CDSCO, Medsafe, and KFDA.

[0058] The term "bispecific antibody" refers to an antibody that contains specificity for two target molecules. The term "bispecific antibody" refers to a biparatopic antibody, DVD-Ig (DiGiammarino et al., "Design and generation of DVD-Ig"). TM molecules for dual-specific targeting”, Meth. Molecules. Biol., 2012, 889, 145-156), mAb 2(see WO 2008 / 003103), FIT-Ig (see WO 2015 / 103072), mAb-dAb, Dock & Lock, Fab arm exchange, SEEDbody, Triomab, Luz-Y, Fcab, κλ-body, orthogonal Fab, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandemAb, scDiabody, scDiabody-CH3, Diabody-CH3, triplebody, miniantibody, minibody, TriBi minibody, scFv-CH3 These formats include KIH, scFv-CH-CL-scFv, F(ab')2-scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCab, ImmTAC, knobs-in-hole, knobs-in-hole with a common light chain, knobs-in-hole with a common light chain and charge pair, charge pair, charge pair with a common light chain, DT-IgG, DutaMab, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, and zybodies. For a review of bispecific formats, see Spiess, C., et al., Mol. Immunol. (2015).

[0059] As used herein, the term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant), excipient, or vehicle used in administering a therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as water and oils, such as those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Physiological saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Preferred carriers are lipid A-based carriers and Gerbu's adjuvant.

[0060] The term "CDR region" or "CDR" refers to the region of an antibody or antigen-binding fragment variable domain that is hypervariable in sequence and / or forms structurally defined loops. Generally, the antigen-binding site of an antibody comprises the following six CDR regions: H Three of these (CDRH1, CDRH2, CDRH3) and V LThese regions of the antibody heavy and light chains confer antigen-binding specificity to the antibody. CDRs can be defined according to the Kabat system or the IMGT system. Other systems can be used to define CDRs, such as the system devised by Chothia et al. (see Chothia, C. & Lesk, AM, 1987, "Canonical structures for the hypervariable regions of immunoglobulins", J. Mol. Biol., 196, 901-917). Other systems for determining CDRs include the Martin (Enhanced Chothia) (Abhinandan and Martin (2008). Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains. Mol Immunol. 45(14):3832-3839), AHo (Honegger and Plueckthun (2001). Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool. J Mol Biol. 309(3):657-670), and Paratome (Kunik et al., (2012). Paratome: an online tool for systematic identification of antigen-binding regions in antibodies based on sequence or structure. Nucleic Acids Res. 40 (Web Server Issue):W521-W524) numbering systems. The term CDR is used herein to refer to one or several of these regions. Those skilled in the art can easily compare different nomenclature systems and determine whether a particular sequence can be defined as a CDR.

[0061] The terms "chemotherapeutic agent" or "chemotherapy" refer to therapeutic agents whose primary purpose is to destroy cancer cells, typically by interfering with their ability to grow or proliferate. There are many different types of chemotherapy drugs, with over 50 approved chemotherapy drugs available. Chemotherapy drugs can be classified based on how they work. Alkylating drugs kill cancer cells by directly attacking DNA, the genetic material of genes. Cyclophosphamide is an alkylating drug. Antimetabolites interfere with the production of DNA, preventing cell growth and proliferation. An example of an antimetabolite is 5-fluorouracil (5-FU). Antitumor antibiotics are produced from natural substances, such as fungi in the soil. They interfere with important cellular functions, including the production of DNA and cellular proteins. Doxorubicin and bleomycin belong to this group of chemotherapy drugs. Plant alkaloids prevent cells from dividing normally. Vinblastine and vincristine are plant alkaloids obtained from the periwinkle plant. Steroid hormones slow the growth of some hormone-dependent cancers. For example, tamoxifen is used to treat breast cancer, which depends on the hormone estrogen for growth. DNA damage response (DDR) inhibitors, such as PARP inhibitors, block DNA repair mechanisms after single- or double-strand breaks. In another embodiment, the chemotherapeutic agent is an agent that induces immunogenic cell death, e.g., platinum therapy, such as oxaliplatin. In one embodiment, the chemotherapy is a standard-of-care cytotoxic chemotherapy for the cancer being treated.

[0062] Examples of chemotherapeutic agents include adriamycin, doxorubicin, 5-fluorouracil, cytosine arabinoside (Ara-C), cyclophosphamide, thiotepa, taxotere (docetaxel), busulfan, cytoxin, gemcitabine, paclitaxel, doxorubicin, taxol, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycin, esperamicin (see U.S. Pat. No. 4,675,187), melphalan, and other related nitrogen mustards. Suitable toxins and chemotherapeutic agents are described in Remington's Pharmaceutical Sciences, 1999. th Ed. (Mack Publishing Co., 1995) and Goodman and Gilman's The Pharmacological Basis of Therapeutics, 7 th Ed. (MacMillan Publishing Co., 1985). Other examples of chemotherapeutic agents include irinotecan (liposomal), a topoisomerase I inhibitor. Other suitable toxins and / or chemotherapeutic agents are known to those skilled in the art. Leucovorin (folic acid) is used in combination with chemotherapy drugs to either enhance the effectiveness of the drug or to function as a chemoprotectant; for example, leucovorin is used in combination with 5-fluorouracil or methotrixate.

[0063] As used herein, the term "composition" is intended to encompass a product containing specified components (e.g., an antibody or antigen-binding fragment of the invention), optionally in specified amounts, as well as any product resulting directly or indirectly from the combination of specified components, optionally in specified amounts.

[0064] As used herein, the terms "comprising" or "comprises" are used in reference to antibodies, antigen-binding fragments, uses, compositions, methods and their respective components that are essential to the methods or compositions of the invention, but are open to including non-specified elements (whether essential or not).

[0065] The term "consisting of" refers to the antibodies, antigen-binding fragments, uses, compositions, methods and each component thereof described herein, and excludes any element not recited in the description of that embodiment.

[0066] "Cross-react," "cross-reactive," or similar terms refer to specific binding between an antibody or antigen-binding fragment (e.g., an anti-hCXCR4 antibody) and a similar target (e.g., CXCR4 of another species) or an unrelated target (e.g., CXCR7, CXCR3, CCR5, or other target and / or ligand) that is not the target antigen (e.g., human CXCR4). In one embodiment, an antibody or antigen-binding fragment does not cross-react with an unrelated target if binding is less than about 10% (e.g., 7%, 5%, 3%, or 1%) of the binding of the antibody or antigen-binding fragment to the desired target antigen (e.g., hCXCR4), as measured, for example, by SPR. In another embodiment, an antibody or antigen-binding fragment cross-reacts with a similar target if binding is greater than or equal to about 80% (e.g., 85%, 90%, 95%) of the binding of the antibody or antigen-binding fragment to the desired target antigen (e.g., hCXCR4), as measured, for example, by SPR.

[0067] The term "derivative" as used herein in reference to a polypeptide refers to a polypeptide comprising the amino acid sequence of a CXCR4 polypeptide or an antibody or antigen-binding fragment that specifically binds to a CXCR4 polypeptide, altered by the introduction of amino acid residue substitutions, deletions, or additions. As used herein, the term "derivative" also refers to a CXCR4 polypeptide or an antibody or antigen-binding fragment that specifically binds to a CXCR4 polypeptide, which has been chemically modified, for example, by the covalent attachment of any type of molecule to the polypeptide. For example, but not limited to, a CXCR4 polypeptide, a fragment of a CXCR4 polypeptide, or a CXCR4 antibody can be chemically modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, attachment to a cellular ligand or other protein, etc. Derivatives are naturally occurring or modified in a manner different from the starting peptide or polypeptide, either in the type or location of the attached molecule. Derivatives further include deletion of one or more chemical groups naturally occurring on the peptide or polypeptide. Derivatives of CXCR4 polypeptides or CXCR4 antibodies or antigen-binding fragments can be chemically modified using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Additionally, derivatives of CXCR4 polypeptides or anti-CXCR4 antibodies or antigen-binding fragments can contain one or more non-classical amino acids. Polypeptide derivatives have similar or identical functions as the CXCR4 polypeptides or anti-CXCR4 antibodies or antigen-binding fragments described herein.

[0068] An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired effect (including therapeutic or prophylactic results). A "therapeutically effective amount" refers to the minimum concentration necessary to affect a measurable improvement or prevention of a particular disorder. A therapeutically effective amount may vary depending on factors such as the patient's condition, age, sex, and weight, and the ability of the antibody or antigen-binding fragment to elicit a desired response in an individual. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or detrimental effects of the antibody or antigen-binding fragment. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. In some embodiments, an effective amount of an antibody or antigen-binding fragment of the invention is from about 0.1 mg / kg (mg of antibody or antigen-binding fragment per kg of subject body weight) to about 100 mg / kg. In certain embodiments, an effective amount of an antibody or antigen-binding fragment is about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 3 mg / kg, 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg (or within these ranges). In some embodiments, "effective amount," as used herein, also refers to the amount of an antibody or antigen-binding fragment of the invention to achieve a particular result (e.g., inhibition of CXCR4 biological activity).

[0069] As used herein, the term "epitope" refers to a localized region on the surface of an antigen, e.g., a CXCR4 polypeptide, that can bind to one or more antigen-binding regions of an antibody or antigen-binding fragment, has antigenic or immunogenic activity, and can elicit an immune response in an animal, preferably a mammal, most preferably a human. An epitope with immunogenic activity is a portion of a polypeptide that elicits an antibody response in an animal. An epitope with antigenic activity is a portion of a polypeptide to which an antibody or antigen-binding fragment specifically binds, as determined by any method known in the art, such as immunoassays, some of which are described herein. An antigenic epitope is not necessarily immunogenic. Epitopes usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. The region of a polypeptide that contributes to an epitope can be contiguous amino acids of the polypeptide, or an epitope can be formed by the assembly of two or more noncontiguous regions of the polypeptide. An epitope may or may not be a three-dimensional surface feature of an antigen. In certain embodiments, a CXCR4 epitope is a three-dimensional surface feature of a CXCR4 polypeptide, which may be a monomer, dimer (e.g., homodimer or heterodimer), trimer, tetramer, or multimer. In preferred embodiments, a CXCR4 epitope is a three-dimensional surface feature of a monomeric or homodimeric CXCR4 polypeptide. In another embodiment, a CXCR4 epitope is a three-dimensional surface feature of a heterodimeric CXCR4 polypeptide, as further described herein. In other embodiments, a CXCR4 epitope is a linear feature of a monomeric or homodimeric CXCR4 polypeptide.

[0070] The term "excipient" as used herein refers to an inert substance commonly used as a diluent, vehicle, preservative, binder, or stabilizer for a drug, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkylsulfonates, caprylates, etc.), surfactants (e.g., SDS, polysorbates, nonionic surfactants, etc.), sugars (e.g., sucrose, maltose, trehalose, etc.), and polyols (e.g., mannitol, sorbitol, etc.). See also Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa., which is incorporated herein by reference in its entirety.

[0071] As used herein, the term "fusion protein" refers to a polypeptide comprising the amino acid sequence of an antibody or antigen-binding fragment and the amino acid sequence of a heterologous polypeptide or protein (i.e., a polypeptide or protein that is not normally part of an antibody or antigen-binding fragment (e.g., a non-anti-CXCR4 antibody or antigen-binding fragment)). When used in reference to CXCR4 or an anti-CXCR4 antibody, the term "fusion" refers to the association of a peptide or polypeptide, or a fragment, variant, and / or derivative thereof, with a heterologous peptide or polypeptide. Preferably, the fusion protein retains the biological activity of CXCR4 or an anti-CXCR4 antibody. In certain embodiments, the fusion protein comprises a CXCR4 antibody V H Main, V L Domain, V H CDR (1, 2 or 3 V H CDR) and / or V L CDR (1, 2 or 3 V L CDRs), and the fusion protein specifically binds to a CXCR4 epitope.

[0072] The term "heavy chain," when used in reference to antibodies, refers to five different types, called alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the heavy chain constant domain. These different types of heavy chains are well known and give rise to five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, respectively, which include four subclasses of IgG, namely, IgG1, IgG2, IgG3, and IgG4, and two subclasses of IgA, namely, IgA1 and IgA2. A preferred embodiment is the IgG isotype. In one embodiment, the heavy chain is a rodent heavy chain. In a preferred embodiment, the heavy chain is a human heavy chain. A heavy chain comprises a variable region (V H ) and the heavy chain constant region. The numbering of amino acid positions used in the heavy chain constant region herein is according to the EU index (Kabat, EA (1991) Sequences of Proteins of Immunological Interest: Tabulation and Analysis of Amino Acid and Nucleic Acid Sequences of Precursors, V-Regions, C-Regions, J-Chain, T-Cell Receptors for Antigen, T-Cell Surface Antigens, [Beta]2-Microglobulins, Major Histocompatibility Antigens, Thy-1, Complement, C-Reactive Protein, Thymopoietin, Integrins, Post-Gamma Globulin, [Alpha]2-Macroglobulins, and Other Related Proteins, 5th ed., National Institutes of Health).

[0073] As used herein, the term "host cell" or like terms refers to a particular subject cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in subsequent generations or upon integration of the nucleic acid molecule into the host cell genome. In one embodiment, the "host cell" is non-human. In one embodiment, the "host cell" is not a human totipotent cell.

[0074] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human and / or made using any of the techniques for making human antibodies; specifically excluding humanized antibodies that comprise non-human antigen-binding residues.

[0075] The term "humanized antibody" refers to a subset of chimeric antibodies in which "CDR regions" from a non-human immunoglobulin (donor antibody) substitute residues from the CDR regions of a human immunoglobulin (recipient antibody). Generally, a humanized antibody will comprise substantially all of at least one, and usually two, variable domains, in which all or substantially all of the CDR regions correspond to those of non-human immunoglobulin sequences and all or substantially all of the framework regions are those of human immunoglobulin sequences, although the framework regions may contain one or more substitutions which improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc.

[0076] The term "IMGT numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues for comparing immunoglobulin and T-cell receptor variable region sequences (Lefranc, MP, 1997, "Unique database numbering system for immunogenetic analysis", Immunol. Today, 18, 50). The IMGT numbering system relies on the high conservation of variable region structure. The IMGT numbering system takes into account and combines the definitions of framework (FR) and complementarity-determining regions (CDR).

[0077] As used herein, the terms "inhibit," "inhibition," "inhibiting," and the like refer to the ability of an antibody or antigen-binding fragment that binds to an epitope that partially, substantially, or completely prevents the binding of a ligand (e.g., CXCL12) to a receptor (e.g., CXCR4). If the epitope bound by the antibody or antigen-binding fragment completely blocks the binding site of the ligand, ligand binding is completely prevented (this may be physical blocking, in the case of identical or overlapping epitopes, or steric blocking, if the antibody or antigen-binding fragment is so large as to prevent ligand binding to its separate epitope), and the ligand is not removed from circulation. Thus, the concentration of circulating ligand may appear to increase. If the epitope bound by the antibody or antigen-binding fragment partially blocks the binding site of the ligand, the ligand may still bind, but weakly (in the case of partial inhibition) or in an orientation different from the natural binding interaction. In this case, some of the ligand may be removed from circulation, but not as strongly as if the ligand-binding site were completely free and available for binding. Thus, inhibition refers to the physical interaction between the ligand and the receptor. I C 50is the half-maximal inhibitory concentration of an inhibitor (e.g., an antibody or antigen-binding fragment disclosed herein). Inhibition can be measured by HTRF, which is described in detail elsewhere herein and by Mathis (1995) Clinical Chemistry 41(9), 1391-1397. Inhibition can also be measured by time-resolved fluorescence energy transfer (TR-FRET) cell-based ligand inhibition assays (Zwier et al., (2010). A fluorescent ligand-binding alternative using Tag-lite Technology. J Biomol Screen. 15(10):1248-1259). Throughout this specification, the term "inhibit" is interchangeable with the term "neutralize." In any of the embodiments herein, "inhibited" means that the % specific binding at a given concentration or the % maximum specific binding reached is equivalent (or the same [within standard error]) to that achieved in the absence of the ligand (e.g., CXCL12) provided in the assay. Thus, in one embodiment, inhibition provides a reduction in % specific binding of a ligand (e.g., CXCL12) (at either 50 nM, 30 nM, 10 nM, 1 nM, or 0.1 nM [antibody or antigen-binding fragment]) compared to an isotype control by 50-100%, 60-100%, 70-100%, 80%-100%, e.g., 85%-100%, or 90%-100%. In another embodiment, inhibition provides a reduction in % specific binding of a ligand (e.g., CXCL12) (at either 50 nM, 30 nM, 10 nM, 1 nM, or 0.1 nM [antibody or antigen-binding fragment]) compared to an isotype control by 95%-100%. In another embodiment, inhibition is a 100% reduction in % specific binding of a ligand (e.g., CXCL12) compared to an isotype control. In another embodiment, inhibition provides a reduction in % maximal specific binding of a ligand (e.g., CXCL12) in the range of 80% to 100%, for example 85% to 100% or 90% to 100% compared to an isotype control, hi another embodiment, inhibition provides a reduction in % maximal specific binding of a ligand (e.g., CXCL12) in the range of 95% to 100% compared to an isotype control.In another embodiment, full neutralization is a 100% reduction in the % maximal specific binding of a ligand (e.g., CXCL12) compared to an isotype control. In one embodiment, an antibody or antigen-binding fragment that inhibits CXCL12 binding to CXCR4 is an antagonist (e.g., a full antagonist or partial antagonist) or an inverse agonist. An inverse agonist is an antibody or antigen-binding fragment that binds to the same target receptor as an agonist ligand (e.g., CXCR4) but induces a pharmacological response opposite to the agonist-induced response (e.g., if an agonist reduces cAMP levels, an inverse agonist can increase cAMP levels). An inverse agonist can be indicated when the receptor is constitutively active (produces a pharmacological response in the absence of an agonist). An inverse agonist can reduce the pharmacological response of a receptor in the absence of an agonist to below the basal activity level. In one embodiment, an anti-CXCR4 antibody (e.g., a CXCR4 inverse agonist) may reduce the pharmacological response of the receptor through one pathway without affecting other pathways through which the receptor signals. In another embodiment, an anti-CXCR4 antibody (e.g., a CXCR4 inverse agonist) may increase cAMP levels relative to basal receptor signaling and may also affect other CXCR4 signaling pathways.

[0078] As used herein, "injection device" refers to a device designed to perform injections, which includes temporarily fluidly connecting the injection device to human tissue, usually subcutaneous tissue. The injection further includes administering a quantity of liquid medication into the tissue and disconnecting or removing the injection device from the tissue. In some embodiments, the injection device may be an intravenous or IV device, which is a type of injection device used when the target tissue is blood in the circulatory system, e.g., blood in a vein. A common, but non-limiting, example of an injection device is a needle and a syringe.

[0079] As used herein, "instructions" refers to written, printed, or graphical indication of contents on an article's immediate container, e.g., written indication on a vial containing a pharmaceutically active agent, or details of the composition and use of a product of interest included in a kit containing the composition of interest. The instructions describe a method of treatment that is intended to be performed or carried out.

[0080] An "isolated" or "purified" antibody, antigen-binding fragment, protein, or nucleic acid is one that has been identified, separated, and / or recovered from components of its production environment (e.g., natural or recombinant). For example, an antibody, antigen-binding fragment, protein, or nucleic acid is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody, antigen-binding fragment, protein, or nucleic acid is derived, or, if chemically synthesized, substantially free of chemical precursors or other chemicals. The term "substantially free of cellular material" includes preparations of antibodies, antigen-binding fragments, proteins, or nucleic acids in which the desired product is separated from cellular components of the cells from which the antibody, antigen-binding fragment, protein, or nucleic acid is isolated or recombinantly produced. Thus, an antibody, antigen-binding fragment, protein, or nucleic acid that is substantially free of cellular material includes preparations of the desired material that contain less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as "contaminating protein"). If the antibody is recombinantly produced, it is preferably also substantially free of culture medium. That is, the culture medium represents less than about 20%, less than about 10%, or less than about 5% of the volume of the protein preparation. When antibodies are produced by chemical synthesis, they are preferably substantially free of chemical precursors or other chemicals, i.e., separated from chemical precursors or other chemicals involved in the synthesis of the protein. Thus, such preparations contain less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the desired substance. In preferred embodiments, the antibodies or antigen-binding fragments of the invention are isolated and / or purified.

[0081] As used herein, the term "isotype control" or similar terms refers to an antibody or antigen-binding fragment, generally of the same species (or mixture of species in the case of chimeras), preferably having the same constant region as the test antibody or antigen-binding fragment, but with a V H Domain and V L The domain may have no specificity for the target antigen of interest (i.e., CXCR4), or may have specificity for an antigen unrelated to the target antigen of interest (i.e., CXCR4), or may have specificity for any other human target. H and V L When testing a human variable / mouse constant chimera, the isotype control can be a commercially available mouse IgG1 isotype control (e.g., available from Sigma Aldrich, catalog number M9269). When testing a full IgG4-PE format antibody, the isotype control is preferably an antibody with human variable regions and a human IgG4-PE constant region. One skilled in the art would be able to identify an antibody suitable as an isotype control.

[0082] The term "Kabat numbering" and similar terms are art-recognized and refer to a numbering system for amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in an antibody heavy chain variable region or antigen-binding portion thereof (Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). In the heavy chain variable region, the hypervariable regions typically range from amino acid positions 31 to 35 in CDR1, amino acid positions 50 to 65 in CDR2, and amino acid positions 95 to 102 in CDR3.

[0083] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., an individual antibody comprising the population that is identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic determinant or epitope. In contrast, polyclonal antibody preparations typically contain different antibodies directed against different antigenic determinants (or epitopes). As used herein, the term "monoclonal antibody" encompasses both intact and full-length monoclonal antibodies (e.g., full-length, four-chain monoclonal antibodies), as well as antigen-binding fragments as defined elsewhere herein. Furthermore, "monoclonal antibody" refers to such antibodies produced by a variety of methods, including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals. Monoclonal antibodies as used herein include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, which exhibit the desired biological activity.

[0084] The terms "naturally occurring" or "native," when used in reference to biological material such as a nucleic acid molecule, polypeptide, host cell, etc., refer to one found in nature and not manipulated by man (also known as wild-type). Natural CXCR4 ligands include CXCL12 (SDF-1), MIF, and ubiquitin.

[0085] As used herein, "packaging" refers to the manner in which components are grouped and / or secured together into a unit suitable for distribution and / or use. Packaging can include, for example, boxes, bags, syringes, ampoules, vials, tubes, clamshell packages, barriers and / or containers to maintain sterility, labeling, etc.

[0086] The terms "percent identity," "percent amino acid sequence identity" (or "percent nucleotide sequence identity"), "percent identical to," and the like, with respect to peptide, polypeptide, antibody, or antigen-binding fragment (or nucleotide) sequences, are defined as the percentage of amino acid residues (or nucleotide bases) in a candidate sequence that are identical to those in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve best alignment, and without considering conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways that are within the purview of those skilled in the art, for example, using publicly available computer software such as Needleman-Wunsch (the NEEDLE program from Emboss), Basic Local Alignment Search Tool (BLAST™), BLAST-2, ALIGN, or MEG ALIGN™ (DNASTAR) software. In one embodiment, the percent identity is the number of identical amino acid residues divided by the length of the alignment (X+Y), where the length of the alignment is the length of the longest sequence (e.g., X) plus the number of gap residues in that sequence (Y). In a preferred embodiment, the alignment can be achieved using Needleman-Wunsch (NEEDLE program from Emboss), e.g., using the BLOSUM60 matrix with a gap opening of 10 and a gap extension of 0.5.

[0087] As used herein, the term "pharmaceutically acceptable," as used herein in reference to a pharmaceutical composition, means approved by a state or federal regulatory agency or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias for use in animals, particularly humans.

[0088] As used herein, the terms "polynucleotide," "nucleotide," "nucleic acid," "nucleic acid molecule," and other similar terms are used interchangeably and include DNA, RNA, mRNA, and the like.

[0089] As used herein, "prevent," "preventing," "prevention," and "prophylaxis" refer to the total or partial inhibition of the onset, recurrence, development, or spread of a CXCR4-mediated disease (or condition) and / or symptoms associated therewith resulting from the administration of a therapy or combination of therapies provided herein (e.g., a combination of prophylactic or therapeutic agents, such as an antibody or antigen-binding fragment of the invention).

[0090] The term "radiotherapy" refers to treatment that uses radiation (e.g., ionizing radiation) to kill cancer cells and / or reduce tumor mass. Radiation therapy can be used in combination with other therapeutic agents. Radiation therapy can be used in combination with chemotherapy. Radiation therapy can be used as a post-surgical treatment to remove malignant tumors. Radiation therapy can be applied to an organism (e.g., a human, dog, cat, mouse, etc.) externally, via an implant (also known as brachytherapy), or by radioisotopes that can be administered by injection, capsule, drink, or implant. Radiation therapy is a term known in the art and includes three-dimensional conformal radiation therapy (3D-CRT), image-guided radiation therapy (IGRT), intensity-modulated radiation therapy (IMRT), helical tomotherapy, photon beam radiation therapy, proton beam radiation therapy, intraoperative radiation therapy (IORT), stereotactic radiosurgery, and stereotactic body radiation therapy (SBRT). Radiation therapy can be applied externally by a linear accelerator (LINAC) (also known as a linear fractional accelerator).

[0091] The term "rodent" refers to a mammal of the order Rodentia. In one embodiment, a rodent is a mammal with a single pair of continuously growing incisors on each of the upper and lower jaws. In one embodiment, the term "rodent" refers to one or more rodents selected from mouse, rat, squirrel, prairie dog, chipmunk, chinchilla, porcupine, beaver, capybara, gerbil, hamster, and guinea pig. In one embodiment, the rodent is of the family Muridae. In one embodiment, the rodent is a murine. In a preferred embodiment, the rodent is a mouse or a rat.

[0092] The term "specifically binds" and the like, as used herein, refers to an antibody or antigen-binding fragment that binds to a protein of interest (e.g., CXCR4) in a specific manner, rather than adhering through polar interactions or other non-specific (but observable) binding. For example, an anti-CXCR4 antibody or antigen-binding fragment specifically binds to CXCR4 through its CDR regions that interact with their antigenic determinants (epitopes) on CXCR4. Antibodies or antigen-binding fragments thereof that specifically bind to a protein of interest (e.g., a CXCR4 antigen) can be identified by, for example, immunoassays (e.g., enzyme-linked immunosorbent assays (ELISAs)), plate-based multiarrays (e.g., the Meso Scale Discovery™ platform), SPR (e.g., Biacore™), flow cytometry (e.g., the Mirrorball™ fluorocytometer), live-cell imaging, radioimmunoassays (RIAs), radioligand binding, or other techniques known to those of skill in the art. In certain embodiments, binding is determined using flow cytometry, radioligand binding, SPR, live-cell imaging, or ELISA. Typically, a specific response will be at least twice the background signal or noise, and more typically greater than 10 times background (e.g., greater than 15 times, greater than 20 times, greater than 50 times, or greater than 100 times). For a discussion of antibody specificity, see, e.g., Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York at pages 332-336.

[0093] The term "subject" or "patient" refers to any animal, including, but not limited to, mammals, e.g., non-human mammals. As used herein, the term "mammal" refers to a vertebrate that either suckles its young and gives birth to live young (eutherian or placental mammals) or lays eggs (metatherian or non-placental mammals). Examples of mammalian species include, but are not limited to, humans and other primates, including non-human primates such as chimpanzees, other ape and monkey species, farm animals such as cattle, sheep, pigs, goats, and horses, domestic mammals such as dogs and cats, laboratory animals such as rodents, e.g., mice, rats (including cotton rats), hamsters, and guinea pigs, birds, e.g., domestic birds, wild birds, and game birds, e.g., chickens, turkeys, and other poultry, ducks, geese, etc. In certain embodiments, the subject is a human patient.

[0094] As used herein, the terms "substantially," "substantially all," or "substantially" refer to at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.

[0095] As used herein, the term "therapeutic agent" refers to any agent that can be used in the treatment, management, or amelioration of a CXCR4-mediated disease (or condition) and / or symptoms associated therewith, where the disease (or condition) is described elsewhere herein. In certain embodiments, the term "therapeutic agent" refers to an antibody or antigen-binding fragment of the invention. In certain other embodiments, the term "therapeutic agent" refers to an agent other than an antibody or antigen-binding fragment of the invention. Preferably, the therapeutic agent is an agent that is known to be useful in, or that has been or is currently being used to, treat, manage, or ameliorate a CXCR4-mediated disease (or condition) or one or more symptoms associated therewith, e.g., a therapeutic agent that is considered by medical professionals to be the standard of care.

[0096] As used herein, the term "therapy" refers to any protocol, method, and / or agent that can be used in the prevention, management, treatment, and / or amelioration of a CXCR4-mediated disease or condition (e.g., cancer or any of the other diseases or conditions described herein). In certain embodiments, the term "therapy" refers to biological therapy, supportive therapy, and / or other therapies useful in the prevention, management, treatment, and / or amelioration of a CXCR4-mediated disease or condition known to those of skill in the art, such as medical professionals.

[0097] The terms "treat," "treatment," and "treating" refer to the reduction or amelioration of the progression, severity, and / or duration of a CXCR4-mediated disease or condition (as described elsewhere herein) brought about by the administration of one or more therapies (including, but not limited to, the administration of one or more therapeutic agents, such as an antibody or antibody-binding fragment thereof of the invention). In certain embodiments, such terms refer to the reduction or inhibition of binding of a CXCR4 ligand (e.g., CXCL12) to CXCR4 and / or the amelioration, reduction, or inhibition of one or more symptoms associated with a CXCR4-mediated disease or condition (e.g., cancer, as described elsewhere herein).

[0098] The term "variable region" or "variable domain" refers to portions of the light and heavy chains, typically the amino-terminal 120-130 amino acids of the heavy chain and approximately 100-110 amino acids of the light chain, which vary significantly in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen (e.g., CXCR4). Sequence variability is concentrated in the CDR regions, while the more highly conserved regions in variable domains are called framework regions (FRs). In a preferred embodiment, the variable regions are human variable regions.

[0099] Definitions of common terms in cell biology and molecular biology can be found in "The Merck Manual of Diagnosis and Therapy", 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0); Robert S. Porter et al., (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10:0763766321); Kendrew et al., (Eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds.

[0100] Unless otherwise indicated, the present disclosure is based on and incorporates the principles of the present invention, as defined in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (4 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012); Molecular Biology 3 rd Edition,David Clark et al.,Elsevier Science Publishing,Inc.,New York,USA(2018);Molecular Biology of the Cell,6 thEdition,Alberts et al.,Garland Science,New York,USA(2014);Culture of Animal Cells:A Manual of Basic Technique and Specialized Applications,R.Ian Freshney,John Wiley&Sons,New Jesrsey USA(2011);Rang&Dale's Pharmacology 8 th Edition,Rang et al.,Elsevier Churchill Livingstone.,England(2016);Davis et al.,Basic Methods in Molecular Biology,Elsevier Science Publishing,Inc.,New York,USA(1995);Current Protocols in Protein Science(CPPS)(John E.Coligan,et al.,ed.,John Wiley and Sons,Inc.);Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et al., ed., John Wiley and Sons, Inc.); Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005) and Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998), all of which are incorporated herein by reference in their entireties.

[0101] Other terms are defined herein within the description of various embodiments of the present disclosure.

[0102] 2.CXCR4 antibody The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CXCR4 (e.g., human CXCR4 [hCXCR4]). In a preferred embodiment, the antibody or antigen-binding fragment is a monoclonal antibody or antigen-binding fragment thereof (such as a fully human monoclonal antibody) that specifically binds to CXCR4 (e.g., hCXCR4). In one embodiment, the antibody or antigen-binding fragment thereof is a full-length antibody. In a preferred embodiment, the antibody or antigen-binding fragment is a four-chain antibody comprising two heavy chains and two light chains.

[0103] The present inventors have identified antibodies with specificity for CXCR4 that have many potential utilities and advantages over existing anti-CXCR4 antibodies. For example, the anti-CXCR4 antibodies or antigen-binding fragments described herein may have the following improved or beneficial properties: a. Selectivity for inhibiting only one of the ligands of CXCR4 (e.g., inhibiting the CXCL12 / CXCR4 interaction but not the interaction of CXCR4 with other ligands (e.g., MIF or ubiquitin)) b. Specificity for CXCR4 that inhibits binding of multiple ligands (e.g., CXCL12, MIF and ubiquitin, CXCL12 and ubiquitin, or CXCL12 and MIF). c. Inducing increased levels of T cell infiltration into tumors (e.g., solid tumors) that allow for a reduction in tumor volume Induction of low-level apoptosis in dT cells e. Improved or reduced immunogenicity / absence of side effects f.Improved solubility g. Stability improvements h. Ease of formulation i. Frequency of administration and / or route of administration j. Manufacturability (e.g., expression, ease of purification, isoforms, affinity for column during purification (e.g., Protein A or Nickel), lack of dissociation of heavy and light chains, dissociation of heavy chains, improved yield, reduced protein cleavage / clipping) k. Improved efficacy and / or improved efficacy as part of a combination therapy

[0104] The anti-CXCR4 antibodies or antigen-binding fragments of the invention (including CL-82574, CL-82458, CL-82558, CL-82658, CL-83083, CL-83083-2, CL-82583, CL-82580, CL-82577, CL-82571, CL-82562, CL-82556, CL-82551, CL-82541, CL-82523, CL-82517, CL-82495, CL-82473, CL-82455, CL-83158, CL-148712, and CL-148729, which are described in detail below) are described with respect to the embodiments and configurations described herein. Unless otherwise stated, all embodiments and configurations should be read as being combinable with any other embodiment or configuration (unless such a combination makes no technical sense or is explicitly stated otherwise). In particular, reference to a particular embodiment includes reference to any sub-embodiments unless otherwise clear from the context (e.g., reference to Embodiment 3 in subsequent embodiments specifically includes reference to Embodiment 3a, Embodiment 3b, etc.).

[0105] Throughout this specification, in one embodiment, reference to CXCR4 refers to mammalian CXCR4. Examples of mammalian species include, but are not limited to, humans and other primates, including non-human primates such as chimpanzees, other apes, and monkey species; livestock animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; and laboratory animals such as rodents, including mice, rats (including cotton rats), hamsters, gerbils, and guinea pigs. In one embodiment, the CXCR4 is rodent CXCR4 (e.g., mouse or rat CXCR4). In another embodiment, the CXCR4 is rhesus and / or cynomolgus CXCR4 (optionally selected from SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9). In a preferred embodiment, the CXCR4 is human CXCR4 (optionally selected from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3). In another embodiment, the CXCR4 is cell-surface-expressed CXCR4.

[0106] CXCR4 is a GPCR, and like most GPCRs, multiple isoforms exist. In one embodiment, the antibody or antigen-binding fragment thereof can interact with all isoforms of hCXCR4. The amino acid sequence of human CXCR4 (hCXCR4) can be found in Uniprot ID: P61073, SEQ ID NO: 2. To date, two hCXCR4 isoforms have been identified in the Uniprot database. Isoform 1 is 352 amino acids long, while isoform 2 is 356 amino acids long (Duquenne et al., (2014). The Two Human CXCR4 Isoforms Display Different HIV Receptor Activities: Consequences for the Emergence of X4 Strains. J Immunol. 193(8):4188-4194). Amino acids 1-5 MEGIS (N-terminus of the receptor) of isoform 1 are replaced by MSIPLPLLQ in isoform 2. In preferred embodiments, the antibody or antigen-binding fragment thereof specifically binds to either isoform 1 (SEQ ID NO: 2) or isoform 2. In preferred embodiments, the antibody or antigen-binding fragment thereof specifically binds to both isoforms 1 and 2. It will be understood that all isoforms may not yet be identified.

[0107] In another embodiment, the CXCR4 protein is post-translationally modified (e.g., glycosylated, phosphorylated, ubiquitinated, or sulfation). In one embodiment, CXCR4 is glycosylated (N-linked and / or O-linked) at amino acids N11, S18, and / or N176 (Reference Sequence: Isoform 1).

[0108] Throughout the embodiments herein, CXCR4 may be wild-type human CXCR4. Additionally or alternatively, human CXCR4 may be a variant CXCR4, such as a variant having a SNP, a polymorphic variant, a truncated variant, a mutation variant, or a frameshift mutation. In one embodiment, the variant CXCR4 is encoded by an amino acid sequence containing one or more single nucleotide polymorphisms (SNPs) (e.g., selected from the group consisting of CXCR4-I261I, CXCR4-K68K, and CXCR4-F93S) (Petersen et al., (2005). Risk for HIV-1 Infection Associated with a Common CXCL12 (SDF1) Polymorphism and CXCR4 Variation in an African Population, J. Acquir. Immune Defic. Syndr. 40(5):521-526). Single-point mutations in the intracellular C-terminal region of CXCR4 can result in the production of variants common in warts, hypogammaglobulinemia, infections, and myeloid cellular imbalance (WHIM) or related disorders (e.g., Waldenström's macroglobulinemia). Additionally or alternatively, variant CXCR4 can be encoded by an amino acid sequence containing one or more mutations, such as nonsense mutations (leading to truncation of the amino acid sequence, e.g., E343K, E343X, S338X, G336X, and R334X (where X terminates the sequence)) (Liu et al., (2012) WHIM syndrome caused by a single amino acid substitution in the carboxy-tail of chemokine receptor CXCR4. Blood. 120(1):181-189). Frameshift mutations are caused by the insertion or deletion of one or more nucleotides, which results in translation of the genetic code in a non-natural reading frame from the position of the mutation (e.g., to the end of the reading frame or a terminator nucleic acid sequence).In another embodiment, the variant CXCR4 is encoded by an amino acid sequence that includes a frameshift mutation (e.g., an S341 frameshift, an S339 frameshift, an H315 frameshift, a T318 frameshift, an R322 frameshift, an L326 frameshift, a K327 frameshift, and an I328 frameshift (Liu et al., (2012) supra and Poulain et al., (2016). Genomic Landscape of CXCR4 Mutations in Waldenstroem Macroglobulinemia. Clin. Cancer Res. 22(6):1480-1488)). In another embodiment, the variant CXCR4 is encoded by an amino acid sequence containing a deletion between amino acids 334-352, 338-352, or 343-352 (reference sequence: isoform 1) (Hernandez et al., (2003). Mutations in the chemokine receptor gene CXCR4 are associated with WHIM syndrome, a combined immunodeficiency disease. Nat Genet. 34:70-74; Treon et al., (2014). Somatic mutations in MYD88 and CXCR4 are determinants of clinical presentation and overall survival in Waldenstrom macroglobulinemia. Blood. 123:2791-2796.). Some patients may express any of these variant CXCR4 proteins, and therefore antibodies or antigen-binding fragments capable of inhibiting these variant CXCR4s may be useful for treating or preventing a different or broader range of patients (e.g., a broader range of patients if the antibody or antigen-binding fragment is cross-reactive between wild-type CXCR4 and variant CXCR4).Thus, in one embodiment, the particular CXCR4-mediated disease or condition is treated with a CXCR4 antibody or antigen-binding fragment containing a variant CXCR4 having a mutation independently selected from a deletion between amino acids 334-352, 338-352, or 343-352 (reference sequence: isoform 1), or a variant CXCR4 encoded by an amino acid sequence containing one or more SNPs selected from the group consisting of CXCR4-I261I, CXCR4-K68K, and CXCR4-F93S, or E343K, E343X, S338X, G3 When the antibody binds to a variant CXCR4 encoded by an amino acid sequence containing one or more nonsense mutations selected from the group consisting of S36X or R334X, or a variant CXCR4 encoded by an amino acid sequence containing one or more frameshift mutations selected from the group consisting of S341 frameshift, S339 frameshift, H315 frameshift, T318 frameshift, R322 frameshift, L326 frameshift, K327 frameshift and I328 frameshift, the antibody can be treated or prevented.

[0109] In one example, the binding site of an antibody or antigen-binding fragment is selected from a plurality (e.g., a library) of binding sites. For example, the plurality of binding sites comprises or consists of a plurality of four-chain antibodies or fragments thereof, such as Fabs or scFvs. Suitable methods for generating a plurality of binding sites for screening include phage display (generation of a phage-displayed library of antibody binding sites), ribosome display (generation of a ribosome-displayed library of antibody binding sites), yeast display (generation of a yeast-displayed library of antibody binding sites), mammalian display, covalent display (e.g., cis-display) or non-human vertebrate (e.g., rodent, e.g., mouse or rat, e.g., Velocimouse™, Intelliselect™ transgenic mouse, Xenomouse™, Aliva Mouse™, HuMab Mouse™, Omnimouse™, Omnirat™, MeMouse™, etc.). Mouse™), OmniFlic™, OmniChicken™, and OmniClic™) with hCXCR4 protein or hCXCR4 epitopes, followed by isolation of a repertoire of antibody-producing cells (e.g., a B cell, plasma cell, or plasmablast cell repertoire) and / or a repertoire of isolated antibodies or antigen-binding fragments.

[0110] The present inventors provide the following anti-CXCR4 antibodies, each of which specifically binds to CXCR4.

[0111] CL-82574 contains a heavy chain variable region (V) of SEQ ID NO: 16, which contains the CDRH1 amino acid sequence of SEQ ID NO: 10 (IMGT) or SEQ ID NO: 13 (Kabat), the CDRH2 amino acid sequence of SEQ ID NO: 11 (IMGT) or SEQ ID NO: 14 (Kabat), and the CDRH3 amino acid sequence of SEQ ID NO: 12 (IMGT) or SEQ ID NO: 15 (Kabat). H ) amino acid sequence. HThe heavy chain nucleic acid sequence of the domain is SEQ ID NO: 17. CL-82574 contains a light chain variable region (V) of SEQ ID NO: 26, which contains a CDRL1 amino acid sequence of SEQ ID NO: 20 (IMGT) or SEQ ID NO: 23 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 21 (IMGT) or SEQ ID NO: 24 (Kabat), and a CDRL3 amino acid sequence of SEQ ID NO: 22 (IMGT) or SEQ ID NO: 25 (Kabat). L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 27. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:305, or SEQ ID NO:307. L The domain can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:284, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301 or SEQ ID NO:303. The full-length heavy chain amino acid sequence is SEQ ID NO:18 (heavy chain nucleic acid sequence SEQ ID NO:19). The full-length light chain amino acid sequence is SEQ ID NO:28 (light chain nucleic acid sequence SEQ ID NO:29). The heavy chain variable region is generated by recombination of human IGHV3-33*01 (SEQ ID NO:311), IGHD1-20*01 (SEQ ID NO:316), and IGHJ3*02 (SEQ ID NO:319) gene segments. The light chain variable region is generated by recombination of human IGKV1-17*01 (SEQ ID NO: 320) and IGKJ3*01 (SEQ ID NO: 323) gene segments.

[0112] CL-82458 contains a heavy chain variable region (V) of SEQ ID NO: 36, which contains the CDRH1 amino acid sequence of SEQ ID NO: 30 (IMGT) or SEQ ID NO: 33 (Kabat), the CDRH2 amino acid sequence of SEQ ID NO: 31 (IMGT) or SEQ ID NO: 34 (Kabat), and the CDRH3 amino acid sequence of SEQ ID NO: 32 (IMGT) or SEQ ID NO: 35 (Kabat). H ) amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 37. CL-82458 contains a light chain variable region (V) of SEQ ID NO: 46, which contains a CDRL1 amino acid sequence of SEQ ID NO: 40 (IMGT) or SEQ ID NO: 43 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 41 (IMGT) or SEQ ID NO: 44 (Kabat), and a CDRL3 amino acid sequence of SEQ ID NO: 42 (IMGT) or SEQ ID NO: 45 (Kabat). L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 47. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:305, or SEQ ID NO:307. LThe domain can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:284, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301 or SEQ ID NO:303. The full-length heavy chain amino acid sequence is SEQ ID NO:38 (heavy chain nucleic acid sequence SEQ ID NO:39). The full-length light chain amino acid sequence is SEQ ID NO:48 (light chain nucleic acid sequence SEQ ID NO:49). The heavy chain variable region is generated by recombination of human IGHV3-23*04 (SEQ ID NO:312), IGHD1-1*01 (SEQ ID NO:317), and IGHJ6*02 (SEQ ID NO:318) gene segments. The light chain variable region is generated by recombination of human IGKV1-17*01 (SEQ ID NO: 320) and IGKJ4*01 (SEQ ID NO: 326) gene segments.

[0113] CL-82558 contains a heavy chain variable region (V) of SEQ ID NO: 56, which contains the CDRH1 amino acid sequence of SEQ ID NO: 50 (IMGT) or SEQ ID NO: 53 (Kabat), the CDRH2 amino acid sequence of SEQ ID NO: 51 (IMGT) or SEQ ID NO: 54 (Kabat), and the CDRH3 amino acid sequence of SEQ ID NO: 52 (IMGT) or SEQ ID NO: 55 (Kabat). H ) amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 57. CL-82558 contains a light chain variable region (V) of SEQ ID NO: 66, which contains a CDRL1 amino acid sequence of SEQ ID NO: 60 (IMGT) or SEQ ID NO: 63 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 61 (IMGT) or SEQ ID NO: 64 (Kabat), and a CDRL3 amino acid sequence of SEQ ID NO: 62 (IMGT) or SEQ ID NO: 65 (Kabat). L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 67. HThe domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:305, or SEQ ID NO:307. L The domain can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:284, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301 or SEQ ID NO:303. The full-length heavy chain amino acid sequence is SEQ ID NO:58 (heavy chain nucleic acid sequence SEQ ID NO:59). The full-length light chain amino acid sequence is SEQ ID NO:68 (light chain nucleic acid sequence SEQ ID NO:69). The heavy chain variable region is generated by recombination of human IGHV4-31*03 (SEQ ID NO:310), IGHD3-10*01 (SEQ ID NO:315), and IGHJ6*02 (SEQ ID NO:318) gene segments. The light chain variable region is generated by recombination of human IGKV2-28*01 (SEQ ID NO: 322) and IGKJ1*01 (SEQ ID NO: 325) gene segments.

[0114] CL-82658 contains a heavy chain variable region (V) of SEQ ID NO: 76, which contains the CDRH1 amino acid sequence of SEQ ID NO: 70 (IMGT) or SEQ ID NO: 73 (Kabat), the CDRH2 amino acid sequence of SEQ ID NO: 71 (IMGT) or SEQ ID NO: 74 (Kabat), and the CDRH3 amino acid sequence of SEQ ID NO: 72 (IMGT) or SEQ ID NO: 75 (Kabat). H ) amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 77. CL-82658 contains a light chain variable region (V) of SEQ ID NO: 86, which contains a CDRL1 amino acid sequence of SEQ ID NO: 80 (IMGT) or SEQ ID NO: 83 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 81 (IMGT) or SEQ ID NO: 84 (Kabat), and a CDRL3 amino acid sequence of SEQ ID NO: 82 (IMGT) or SEQ ID NO: 85 (Kabat).L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 87. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:305, or SEQ ID NO:307. L The domain can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:284, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301 or SEQ ID NO:303. The full-length heavy chain amino acid sequence is SEQ ID NO:78 (heavy chain nucleic acid sequence SEQ ID NO:79). The full-length light chain amino acid sequence is SEQ ID NO:88 (light chain nucleic acid sequence SEQ ID NO:89). The heavy chain variable region is generated by recombination of human IGHV3-30*18 (SEQ ID NO:308), IGHD3-16*02 (SEQ ID NO:313), and IGHJ6*02 (SEQ ID NO:318) gene segments. The light chain variable region is generated by recombination of human IGKV1-17*01 (SEQ ID NO: 320) and IGKJ3*01 (SEQ ID NO: 323) gene segments.

[0115] CL-83083 contains a heavy chain variable region (V) of SEQ ID NO: 96, which contains the CDRH1 amino acid sequence of SEQ ID NO: 90 (IMGT) or SEQ ID NO: 93 (Kabat), the CDRH2 amino acid sequence of SEQ ID NO: 91 (IMGT) or SEQ ID NO: 94 (Kabat), and the CDRH3 amino acid sequence of SEQ ID NO: 92 (IMGT) or SEQ ID NO: 95 (Kabat). H ) amino acid sequence. HThe heavy chain nucleic acid sequence of the domain is SEQ ID NO: 97. CL-83083 contains a light chain variable region (VL) of SEQ ID NO: 106, which contains the CDRL1 amino acid sequence of SEQ ID NO: 100 (IMGT) or SEQ ID NO: 103 (Kabat), the CDRL2 amino acid sequence of SEQ ID NO: 101 (IMGT) or SEQ ID NO: 104 (Kabat), and the CDRL3 amino acid sequence of SEQ ID NO: 102 (IMGT) or SEQ ID NO: 105 (Kabat). L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 107. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:305, or SEQ ID NO:307. L The domain can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:284, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301 or SEQ ID NO:303. The full-length heavy chain amino acid sequence is SEQ ID NO:98 (heavy chain nucleic acid sequence SEQ ID NO:99). The full-length light chain amino acid sequence is SEQ ID NO:108 (light chain nucleic acid sequence SEQ ID NO:109). The heavy chain variable region is generated by recombination of human IGHV3-73*02 (SEQ ID NO:309), IGHD3-9*01 (SEQ ID NO:314), and IGHJ6*02 (SEQ ID NO:318) gene segments. The light chain variable region is generated by recombination of human IGLV10-54*02 (SEQ ID NO: 321) and IGLJ3*02 (SEQ ID NO: 324) gene segments.

[0116] CL-83083-2 contains a heavy chain variable region (V) of SEQ ID NO: 96, which contains the CDRH1 amino acid sequence of SEQ ID NO: 90 (IMGT) or SEQ ID NO: 93 (Kabat), the CDRH2 amino acid sequence of SEQ ID NO: 91 (IMGT) or SEQ ID NO: 94 (Kabat), and the CDRH3 amino acid sequence of SEQ ID NO: 92 (IMGT) or SEQ ID NO: 95 (Kabat). H ) amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 97. CL-83083-2 contains a light chain variable region (VL) of SEQ ID NO: 106, which contains the CDRL1 amino acid sequence of SEQ ID NO: 100 (IMGT) or SEQ ID NO: 103 (Kabat), the CDRL2 amino acid sequence of SEQ ID NO: 101 (IMGT) or SEQ ID NO: 104 (Kabat), and the CDRL3 amino acid sequence of SEQ ID NO: 102 (IMGT) or SEQ ID NO: 105 (Kabat). L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 107. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:305, or SEQ ID NO:307. LThe domain can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:284, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, or SEQ ID NO:303. The full-length heavy chain amino acid sequence is SEQ ID NO:98 (heavy chain nucleic acid sequence SEQ ID NO:99). The full-length light chain amino acid sequence is SEQ ID NO:110 (light chain nucleic acid sequence SEQ ID NO:111). The heavy chain variable region is generated by recombination of human IGHV3-73*02 (SEQ ID NO:309), IGHD3-9*01 (SEQ ID NO:314), and IGHJ6*02 (SEQ ID NO:318) gene segments. The light chain variable region is generated by recombination of human IGLV10-54*02 (SEQ ID NO: 321) and IGLJ3*02 (SEQ ID NO: 324) gene segments.

[0117] CL-82583 is V H The heavy chain variable region (V) of SEQ ID NO: 112, including the CDRH1, CDRH2, and CDRH3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 112. H ) amino acid sequence. H The heavy chain nucleic acid sequence of domain V is SEQ ID NO: 113. L The light chain variable region (V) of SEQ ID NO: 116, comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 116. L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 117. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:305, or SEQ ID NO:307. LThe domain can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:284, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301 or SEQ ID NO:303. The full-length heavy chain amino acid sequence is SEQ ID NO:114 (heavy chain nucleic acid sequence SEQ ID NO:115). The full-length light chain amino acid sequence is SEQ ID NO:118 (light chain nucleic acid sequence SEQ ID NO:119). The heavy chain variable region is generated by recombination of human IGHV3-33*01 (SEQ ID NO:311), IGHD1-1*01 (SEQ ID NO:317), and IGHJ3*02 (SEQ ID NO:319) gene segments. The light chain variable region is generated by recombination of human IGKV1-17*01 (SEQ ID NO: 320) and IGKJ3*01 (SEQ ID NO: 323) gene segments.

[0118] CL-82580 is V H The heavy chain variable region (V) of SEQ ID NO: 120, including the CDRH1, CDRH2, and CDRH3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 120. H ) amino acid sequence. H The heavy chain nucleic acid sequence of domain V is SEQ ID NO: 121. L The light chain variable region (V) of SEQ ID NO: 124, comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 124. L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 125. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:305, or SEQ ID NO:307. LThe domain can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:284, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, or SEQ ID NO:303. The full-length heavy chain amino acid sequence is SEQ ID NO:122 (heavy chain nucleic acid sequence SEQ ID NO:123). The full-length light chain amino acid sequence is SEQ ID NO:126 (light chain nucleic acid sequence SEQ ID NO:127). The heavy chain variable region is produced by recombination of human IGHV1-8*01, IGHD3-10*01 (SEQ ID NO:315), and IGHJ6*02 (SEQ ID NO:318) gene segments. The light chain variable region is produced by recombination of human IGKV1-5*03 and IGKJ1*01 (SEQ ID NO:325) gene segments.

[0119] CL-82577 is V H The heavy chain variable region (V) of SEQ ID NO: 128, comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 128. H ) amino acid sequence. H The heavy chain nucleic acid sequence of domain V is SEQ ID NO: 129. L The light chain variable region (V) of SEQ ID NO: 132, comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 132. L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 133. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:305, or SEQ ID NO:307. LThe domain can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:284, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, or SEQ ID NO:303. The full-length heavy chain amino acid sequence is SEQ ID NO:130 (heavy chain nucleic acid sequence SEQ ID NO:131). The full-length light chain amino acid sequence is SEQ ID NO:134 (light chain nucleic acid sequence SEQ ID NO:135). The heavy chain variable region is generated by recombination of human IGHV4-31*03 (SEQ ID NO:310), IGHD3-10*01 (SEQ ID NO:315), and IGHJ6*02 (SEQ ID NO:318) gene segments. The light chain variable region is generated by recombination of human IGKV2-28*01 (SEQ ID NO: 322) and IGKJ1*01 (SEQ ID NO: 325) gene segments.

[0120] CL-82571 is V H The heavy chain variable region (V) of SEQ ID NO: 136, comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 136. H ) amino acid sequence. H The heavy chain nucleic acid sequence of domain V is SEQ ID NO: 137. L The light chain variable region (V) of SEQ ID NO: 140, comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 140. L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 141. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:305, or SEQ ID NO:307. LThe domain can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:284, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, or SEQ ID NO:303. The full-length heavy chain amino acid sequence is SEQ ID NO:138 (heavy chain nucleic acid sequence SEQ ID NO:139). The full-length light chain amino acid sequence is SEQ ID NO:142 (light chain nucleic acid sequence SEQ ID NO:143). The heavy chain variable region is produced by recombination of human IGHV3-21*03, IGHD5-18*01, and IGHJ6*02 (SEQ ID NO:318) gene segments. The light chain variable region is produced by recombination of human IGKV1D-13*d01 and IGKJ4*01 (SEQ ID NO:326) gene segments.

[0121] CL-82562 is V H The heavy chain variable region (V) of SEQ ID NO: 144, including the CDRH1, CDRH2, and CDRH3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 144. H ) amino acid sequence. H The heavy chain nucleic acid sequence of domain V is SEQ ID NO: 145. L The light chain variable region (V) of SEQ ID NO: 148, comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 148. L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 149. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:305, or SEQ ID NO:307. LThe domain can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:284, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, or SEQ ID NO:303. The full-length heavy chain amino acid sequence is SEQ ID NO:146 (heavy chain nucleic acid sequence SEQ ID NO:147). The full-length light chain amino acid sequence is SEQ ID NO:150 (light chain nucleic acid sequence SEQ ID NO:151). The heavy chain variable region is generated by recombination of human IGHV3-30*18 (SEQ ID NO:308), IGHD1-20*01 (SEQ ID NO:316), and IGHJ6*02 (SEQ ID NO:318) gene segments. The light chain variable region is generated by recombination of human IGKV1-17*01 (SEQ ID NO: 320) and IGKJ1*01 (SEQ ID NO: 325) gene segments.

[0122] CL-82556 is V H The heavy chain variable region (V) of SEQ ID NO: 152, comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 152. H ) amino acid sequence. H The heavy chain nucleic acid sequence of domain CL-82556 is SEQ ID NO: 153. L The light chain variable region (V) of SEQ ID NO: 156, comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 156. L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 157. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:305, or SEQ ID NO:307. LThe domain can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:284, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301 or SEQ ID NO:303. The full-length heavy chain amino acid sequence is SEQ ID NO:154 (heavy chain nucleic acid sequence SEQ ID NO:155). The full-length light chain amino acid sequence is SEQ ID NO:158 (light chain nucleic acid sequence SEQ ID NO:159). The heavy chain variable region is generated by recombination of human IGHV3-33*01 (SEQ ID NO:311), IGHD1-20*01 (SEQ ID NO:316), and IGHJ3*02 (SEQ ID NO:319) gene segments. The light chain variable region is generated by recombination of human IGKV1-17*01 (SEQ ID NO: 320) and IGKJ3*01 (SEQ ID NO: 323) gene segments.

[0123] CL-82551 is V H The heavy chain variable region (V) of SEQ ID NO: 160, comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 160. H ) amino acid sequence. H The heavy chain nucleic acid sequence of domain V is SEQ ID NO: 161. L The light chain variable region (V) of SEQ ID NO: 164, comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 164. L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 165. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:305, or SEQ ID NO:307. LThe domain can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:284, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, or SEQ ID NO:303. The full-length heavy chain amino acid sequence is SEQ ID NO:162 (heavy chain nucleic acid sequence SEQ ID NO:163). The full-length light chain amino acid sequence is SEQ ID NO:166 (light chain nucleic acid sequence SEQ ID NO:167). The heavy chain variable region is produced by recombination of human IGHV1-8*01, IGHD3-10*01 (SEQ ID NO:315), and IGHJ6*02 (SEQ ID NO:318) gene segments. The light chain variable region is produced by recombination of human IGKV1-5*03 and IGKJ1*01 (SEQ ID NO:325) gene segments.

[0124] CL-82541 is V H The heavy chain variable region (V) of SEQ ID NO: 168, comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 168. H ) amino acid sequence. H The heavy chain nucleic acid sequence of domain V is SEQ ID NO: 169. L The light chain variable region (V) of SEQ ID NO: 172, comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 172. L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 173. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:305, or SEQ ID NO:307. LThe domain can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:284, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, or SEQ ID NO:303. The full-length heavy chain amino acid sequence is SEQ ID NO:170 (heavy chain nucleic acid sequence SEQ ID NO:171). The full-length light chain amino acid sequence is SEQ ID NO:174 (light chain nucleic acid sequence SEQ ID NO:175). The heavy chain variable region is generated by recombination of human IGHV4-31*03 (SEQ ID NO:310), IGHD3-10*01 (SEQ ID NO:315), and IGHJ6*02 (SEQ ID NO:318) gene segments. The light chain variable region is generated by recombination of human IGKV2-28*01 (SEQ ID NO: 322) and IGKJ1*01 (SEQ ID NO: 325) gene segments.

[0125] CL-82523 is V H The heavy chain variable region (V) of SEQ ID NO: 176, comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 176. H ) amino acid sequence. H The heavy chain nucleic acid sequence of domain V is SEQ ID NO: 177. L The light chain variable region (V) of SEQ ID NO: 180, comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 180. L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 181. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:305, or SEQ ID NO:307. LThe domain can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:284, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301 or SEQ ID NO:303. The full-length heavy chain amino acid sequence is SEQ ID NO:178 (heavy chain nucleic acid sequence SEQ ID NO:179). The full-length light chain amino acid sequence is SEQ ID NO:182 (light chain nucleic acid sequence SEQ ID NO:183). The heavy chain variable region is generated by recombination of human IGHV3-23*04 (SEQ ID NO:312), IGHD1-20*01 (SEQ ID NO:316), and IGHJ6*02 (SEQ ID NO:318) gene segments. The light chain variable region is generated by recombination of human IGKV1-17*01 (SEQ ID NO: 320) and IGKJ4*01 (SEQ ID NO: 326) gene segments.

[0126] CL-82517 is V H The heavy chain variable region (V) of SEQ ID NO: 184, including the CDRH1, CDRH2, and CDRH3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 184. H ) amino acid sequence. H The heavy chain nucleic acid sequence of domain V is SEQ ID NO: 185. L The light chain variable region (V) of SEQ ID NO: 188, comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 188. L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 189. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:305, or SEQ ID NO:307. LThe domain can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:284, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, or SEQ ID NO:303. The full-length heavy chain amino acid sequence is SEQ ID NO:186 (heavy chain nucleic acid sequence SEQ ID NO:187). The full-length light chain amino acid sequence is SEQ ID NO:190 (light chain nucleic acid sequence SEQ ID NO:191). The heavy chain variable region is produced by recombination of human IGHV3-73*02 (SEQ ID NO:309), IGHD3-9*01 (SEQ ID NO:314), and IGHJ6*02 (SEQ ID NO:318) gene segments. The light chain variable region is produced by recombination of human IGKV1-5*03 and IGKJ1*01 (SEQ ID NO:325) gene segments.

[0127] CL-82495 is V H The heavy chain variable region (V) of SEQ ID NO: 192, comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 192. H ) amino acid sequence. H The heavy chain nucleic acid sequence of domain V is SEQ ID NO: 193. L The light chain variable region (V) of SEQ ID NO: 196, comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 196. L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 197. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:305, or SEQ ID NO:307. LThe domain can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:284, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301 or SEQ ID NO:303. The full-length heavy chain amino acid sequence is SEQ ID NO:194 (heavy chain nucleic acid sequence SEQ ID NO:195). The full-length light chain amino acid sequence is SEQ ID NO:198 (light chain nucleic acid sequence SEQ ID NO:199). The heavy chain variable region is generated by recombination of human IGHV3-33*01 (SEQ ID NO:311), IGHD3-10*01 (SEQ ID NO:315), and IGHJ6*02 (SEQ ID NO:318) gene segments. The light chain variable region is generated by recombination of human IGKV1-17*01 (SEQ ID NO: 320) and IGKJ1*01 (SEQ ID NO: 325) gene segments.

[0128] CL-82473 is V H The heavy chain variable region (V) of SEQ ID NO: 200, including the CDRH1, CDRH2, and CDRH3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 200. H ) amino acid sequence. H The heavy chain nucleic acid sequence of domain CL-82473 is SEQ ID NO: 201. L The light chain variable region (V) of SEQ ID NO: 204, comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 204. L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 205. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:305, or SEQ ID NO:307. LThe domain can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:284, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, or SEQ ID NO:303. The full-length heavy chain amino acid sequence is SEQ ID NO:202 (heavy chain nucleic acid sequence SEQ ID NO:203). The full-length light chain amino acid sequence is SEQ ID NO:206 (light chain nucleic acid sequence SEQ ID NO:207). The heavy chain variable region is produced by recombination of human IGHV3-33*01 (SEQ ID NO:311), IGHD3-10*01 (SEQ ID NO:315), and IGHJ4*02 gene segments. The light chain variable region is produced by recombination of human IGKV2-30*01 and IGKJ3*01 (SEQ ID NO:323) gene segments.

[0129] CL-82455 is V H The heavy chain variable region (V) of SEQ ID NO: 208, comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 208. H ) amino acid sequence. H The heavy chain nucleic acid sequence of domain V is SEQ ID NO: 209. L The light chain variable region (V) of SEQ ID NO: 212, comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 212. L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 213. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:305, or SEQ ID NO:307. LThe domain can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:284, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301 or SEQ ID NO:303. The full-length heavy chain amino acid sequence is SEQ ID NO:210 (heavy chain nucleic acid sequence SEQ ID NO:211). The full-length light chain amino acid sequence is SEQ ID NO:214 (light chain nucleic acid sequence SEQ ID NO:215). The heavy chain variable region is generated by recombination of human IGHV3-33*01 (SEQ ID NO:311), IGHD3-10*01 (SEQ ID NO:315), and IGHJ6*02 (SEQ ID NO:318) gene segments. The light chain variable region is generated by recombination of human IGKV3-15*01 and IGKJ1*01 (SEQ ID NO: 325) gene segments.

[0130] CL-83158 is V H The heavy chain variable region (V) of SEQ ID NO: 216, comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 216. H ) amino acid sequence. H The heavy chain nucleic acid sequence of domain CL-83158 is SEQ ID NO: 217. L The light chain variable region (V) of SEQ ID NO: 220, comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 220. L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 221. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:305, or SEQ ID NO:307. LThe domain can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:284, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, or SEQ ID NO:303. The full-length heavy chain amino acid sequence is SEQ ID NO:218 (heavy chain nucleic acid sequence SEQ ID NO:219). The full-length light chain amino acid sequence is SEQ ID NO:222 (light chain nucleic acid sequence SEQ ID NO:223). The heavy chain variable region is produced by recombination of human IGHV4-34*01, IGHD3-10*01 (SEQ ID NO:315), and IGHJ6*02 (SEQ ID NO:318) gene segments. The light chain variable region is produced by recombination of human IGLV3-19*01 and IGLJ3*02 (SEQ ID NO:324) gene segments.

[0131] CL-148712 is V H The heavy chain variable region (V) of SEQ ID NO: 224, including the CDRH1, CDRH2, and CDRH3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 224. H ) amino acid sequence. H The heavy chain nucleic acid sequence of domain V is SEQ ID NO: 225. L The light chain variable region (V) of SEQ ID NO: 228, comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 228. L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 229. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:305, or SEQ ID NO:307. LThe domain can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:284, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, or SEQ ID NO:303. The full-length heavy chain amino acid sequence is SEQ ID NO:226 (heavy chain nucleic acid sequence SEQ ID NO:227). The full-length light chain amino acid sequence is SEQ ID NO:230 (light chain nucleic acid sequence SEQ ID NO:231). The heavy chain variable region is generated by recombination of human IGHV3-23*04 (SEQ ID NO:312), IGHD1-1*01 (SEQ ID NO:317), and IGHJ6*02 (SEQ ID NO:318) gene segments. The light chain variable region is generated by recombination of human IGKV1-17*01 (SEQ ID NO: 320) and IGKJ4*01 (SEQ ID NO: 326) gene segments.

[0132] CL-148729 is V H The heavy chain variable region (V) of SEQ ID NO: 232, comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 232. H ) amino acid sequence. H The heavy chain nucleic acid sequence of domain CL-148729 is SEQ ID NO: 233. L The light chain variable region (V) of SEQ ID NO: 236, comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat from amino acid sequence SEQ ID NO: 236. L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 237. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:305, or SEQ ID NO:307. LThe domain can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:284, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, or SEQ ID NO:303. The full-length heavy chain amino acid sequence is SEQ ID NO:234 ​​(heavy chain nucleic acid sequence SEQ ID NO:235). The full-length light chain amino acid sequence is SEQ ID NO:238 (light chain nucleic acid sequence SEQ ID NO:239). The heavy chain variable region is produced by recombination of human IGHV3-73*02 (SEQ ID NO:309), IGHD6-25*01, and IGHJ4*02 gene segments. The light chain variable region is produced by recombination of human IGLV1-51*01 and IGLJ3*02 (SEQ ID NO:324) gene segments.

[0133] The antibodies or antigen-binding fragments of the invention (described herein) specifically bind to CXCR4, and specific binding and / or binding affinity to CXCR4 can be determined by SPR or other techniques known in the art. In one embodiment, the antibody or antigen-binding fragment specifically binds to human CXCR4. In a preferred embodiment, the antibody or antigen-binding fragment specifically binds to both human CXCR4 and cynomolgus monkey CXCR4.

[0134] In one embodiment, SPR is performed using Nanodiscs, cells, or virus-like particles (VLPs) to display all or part of a receptor (e.g., CXCR4). In another embodiment, Nanodiscs, cells, or VLPs are used to display a mutant (e.g., thermostabilized) or variant receptor (e.g., CXCR4). In one embodiment, SPR is performed using a conformational thermostabilized receptor, e.g., CXCR4 StaR®, which is a mutant (thermostabilized) CXCR4 receptor generated and selected according to the methods described in WO 2009 / 081136 (Magnani et al. (2008). Co-evolving stability and conformational homogeneity of the human adenosine triphosphate (ADP) receptor. 2a receptor. Proc Natl Acad Sci USA. 105(31):10744-10749; Serrano-Vega et al (2008). Conformational thermostabilization of the beta1-adrenergic receptor in a detergent-resistant form. Proc Natl Acad Sci USA. 105(3):877-882 and Shibata et al (2009). Thermostabilization of the neurotensin receptor NTS1. J Mol Biol. 390(2):262-277), optionally displayed using nanodiscs, cells or VLPs.

[0135] In one embodiment, SPR is performed at 25°C or 37°C. In one embodiment, SPR is performed at physiological pH, such as about pH 7 or pH 7.6 (e.g., using HEPES-buffered saline at pH 7.6 (also called HBS-EP, available from Teknova Inc, California, catalog number H8022)). In one embodiment, SPR is performed at physiological salt levels, e.g., 150 mM NaCl. In one embodiment, SPR is performed at detergent levels of 0.05% by volume or less, e.g., in the presence of 0.05% P20 (polysorbate 20, e.g., Tween-20™) and 3 mM EDTA.

[0136] In one example, SPR is performed at 25° C. using 10 nM Hepes, 150 mM NaCl, 0.1% w / v BSA as a running buffer at pH 7.3. In one example, SPR is performed at 25° C. or 37° C. in a pH 7.6 buffer, 150 mM NaCl, 0.05% detergent (e.g., P20), and 3 mM EDTA. The buffer may contain 10 mM HEPE. In one example, SPR is performed in HBS-EP at 25° C. or 37° C. In one example, the affinity of an antibody or antigen-binding fragment is determined using SPR by: 1. Anti-human (or species-matched other relevant human, rat, or non-human vertebrate antibody constant region) IgG (e.g., Biacore™ BR-1008-38) is coupled to a biosensor chip (e.g., a GLM chip) such as by primary amine coupling; 2. Exposing human IgG (or other compatible species antibody) to a test IgG antibody (e.g., an anti-CXCR4 antibody or antigen-binding fragment) to capture the test antibody on the chip; 3. Passing test antigen over the capture surface of the chip at 1024 nM, 256 nM, 64 nM, 16 nM, 4 nM, 0 nM (i.e., buffer only); and 4. Determine the binding affinity of the test antibody to the test antigen using, for example, surface plasmon resonance under the SPR conditions discussed above (e.g., in physiological buffer at 25°C). SPR can be performed using any standard SPR instrument, such as with a Biacore™ or a ProteOn XPR36™ (Bio-Rad™).

[0137] In another embodiment, antigen can be presented on a biosensor chip (instead of steps 1 and 2), and then the antigen is exposed to an antibody or antigen-binding fragment (instead of chip 3). In another embodiment, biotinylated CXCR4 virus-like particles are captured on the active flow channel of an SA chip, and then purified anti-CXCR4 antibodies or antigen-binding fragments (e.g., Fab fragments) are used as analytes and splashed over the captured CXCR4 VLPs at 1.23 nM, 3.7 nM, 11.11 nM, 33.33 nM, and 100 nM.

[0138] In another embodiment, SPR performed using cells can be analyzed using a technology that measures repetitive differential measurements of surface-bound proteins (e.g., LigandTracer™ technology (Ridgeview)).

[0139] Regeneration of the capture surface can be performed with 10 mM glycine at pH 1.7. This removes the captured antibody or antigen-binding fragment, making the surface available for another interaction. Binding data can be fitted to a specific 1:1 model using standard techniques, for example, using the model intrinsic to ProteOn XPR36™ analysis software.

[0140] An antibody or antigen-binding fragment thereof that specifically binds to the CXCR4 antigen may be cross-reactive with related antigens, particularly CXCR4 from different species, such as rhesus monkeys, cynomolgus monkeys, rodents, and / or humans. Thus, in one embodiment, the antibody or antigen-binding fragment specifically binds to human, rhesus monkeys, cynomolgus monkeys, and / or rodents (e.g., mice or rats) CXCR4. In one embodiment, the antibody or antigen-binding fragment specifically binds to human and / or cynomolgus monkey CXCR4.

[0141] In a preferred embodiment, an antibody or antigen-binding fragment thereof that specifically binds to the human CXCR4 antigen does not cross-react with other antigens, such as antigens selected from CXCR1, CXCR2, CXCR3, CXCR5, CXCR6, CXCR7, CXCR8, CXCR9, CXCR10, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, and CB2, or any combination thereof. In one embodiment, the antibody or antigen-binding fragment does not bind or cross-react with CXCR7, e.g., does not detectably bind to CXCR7 (optionally, CXCR7 is human and further optionally selected from SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6), when compared to a positive control (e.g., an anti-CXCR7 antibody). In another embodiment, the antibody or antigen-binding fragment does not bind or cross-react with CCR5, e.g., does not detectably bind to CCR5. In another embodiment, the antibody or antigen-binding fragment does not bind or cross-react with CXCR3, e.g., does not detectably bind to CXCR3. In one embodiment, cross-reactivity is determined essentially as described in Example 12 herein.

[0142] In one embodiment, the antibody or antigen-binding fragment specifically binds to CXCR4 (e.g., human CXCR4). 50 or K DIn one embodiment, the antibody or antigen-binding fragment has a K of 0.2-2 nM, 0.2-1 nM, 0.2-0.8 nM, or preferably 0.4-0.8 nM. D In one embodiment, the binding affinity is determined by SPR, e.g., as described elsewhere herein, or essentially as described in Example 11 herein.

[0143] In another embodiment, the antibody or antigen-binding fragment has an EC 50 In one embodiment, binding affinity is determined by flow cytometry, e.g., as described elsewhere herein, or essentially as described in Example 10 herein.

[0144] In one embodiment, the antibody or antigen-binding fragment (substantially) inhibits binding of a ligand (e.g., a natural ligand as described above) to CXCR4. In a preferred embodiment, the ligand is CXCL12 (e.g., human CXCL12, particularly human alpha-CXCL12 (SEQ ID NO: 327)). In one embodiment, CXCL12 has a truncated (optionally 21 amino acid long) signal peptide. In a preferred embodiment, the antibody or antigen-binding fragment (substantially) inhibits binding of CXCL12 to CXCR4. In one embodiment, the antibody or antigen-binding fragment completely inhibits binding of CXCL12 to CXCR4 (e.g., binding of human CXCL12 to human CXCR4 and / or binding of cynomolgus monkey CXCL12 to cynomolgus monkey CXCR4). In one embodiment, the antibody or antigen-binding fragment partially inhibits binding of CXCL12 to CXCR4. In one embodiment, the antibody or antigen-binding fragment partially or completely inhibits the binding of CXCL12 to CXCR4, but does not exhibit detectable inhibition of the binding of other CXCR4 ligands (e.g., MIF or ubiquitin) to CXCR4.

[0145] In preferred embodiments, the antibody or antigen-binding fragment has an IC of 0.2-4 nM, 0.2-3 nM, 0.2-2 nM, or preferably 0.2-1 nM. 50 In a preferred embodiment, the antibody or antigen-binding fragment inhibits binding of human CXCL12 (e.g., human or cynomolgus CXCL12) to CXCR4 (e.g., human or cynomolgus CXCR4) with an IC of 0.4 to 4 nM. 50 In a preferred embodiment, CXCL12 inhibition is determined using an HTRF assay, for example as described elsewhere herein, or essentially as described in Example 7 herein.

[0146] In one embodiment, the HTRF assay is performed at room temperature (e.g., 22 or 25°C). In another embodiment, the assay is performed in the dark, particularly during the incubation period. In any HTRF assay, an appropriate donor-acceptor fluorescent pair is used. The donor molecule is conjugated to either the receptor of interest (i.e., CXCR4) or the ligand of interest (e.g., CXCL12). The acceptor molecule is conjugated to a binding partner of the receptor / ligand to which the donor molecule is conjugated. The donor or acceptor molecule can be bound, for example, to an anti-mouse Fc antibody, an anti-human Fc antibody, an anti-Flag antibody, an anti-His antibody, streptavidin (each conjugated to either the ligand or receptor of interest (e.g., CXCL12 or CXCR4)), or directly to the ligand or receptor of interest (e.g., CXCL12 or CXCR4). In one example, the donor molecule is a cryptate (620 nM emission) and the acceptor molecule is selected from D2 (647 nM emission) and Alexaflour 647 (emission 665 nM). In one embodiment, the concentration of the antibody or antigen-binding fragment thereof is titrated to provide a curve of percent specific binding (see Example 7, Equation 6). In one embodiment, the concentration of the antigen or antigen-binding fragment ranges from about 1 mM to 1 pM (e.g., 500 nM to 15 pM or 50 nM to 5 pM). In another embodiment, multiple data points are obtained, for example, 6, 7, 8, 9, 10, 11, 12, or 13 data points, particularly 10 or 11 data points. In one embodiment, the HTRF assay is performed in HTRF buffer (e.g., PBS (Sigma) + 0.53 M KF (Sigma) + 0.1% w / v BSA (Sigma)).

[0147] In one example, % specific binding of a ligand (eg, CXCL12) is determined using HTRF by the following: 1. Combining recombinant ligand (e.g., CXCL12 (tagged with AlexaFlour647)) with an antibody or antigen-binding fragment in a plate; 2. Add receptor-expressing cells (e.g., CXCR4 (SNAP-tagged with europium cryptate)) to the plate; 3. Add the test antibody or antigen-binding fragment and allow a sufficient period of time to equilibrate (e.g., about 1 or 2 hours at room temperature); 4. Read the plate using a reader that can excite the donor molecule (e.g., at 335 nM) and measure the emission wavelengths of both the donor and acceptor fluorescent molecules (e.g., at 620 nM and 665 nM) (examples of plate readers include EnVision™ (Perkin Elmer), PHERAstar™ FS, PHERAstar™ FSX, CLARIOstar™, and FLUOstar™, and POLARstar™ Omega), and 5. Determine the % specific binding of the test molecule (e.g., CXCL12), e.g., using Equation 6, and / or 6. Calculate the IC of a ligand (e.g., CXCL12) using, for example, Equation 5 50 In one embodiment, (e.g., inhibition) data (e.g., % specific binding) can be fitted using standard analytical techniques, for example, using GraphPad / Prism analysis software.

[0148] In one embodiment, the anti-CXCR4 antibodies or antigen-binding fragments described herein target T cells (optionally CD8 +In one embodiment, the anti-CXCR4 antibody or antigen-binding fragment described herein does not induce CXCR4-mediated apoptosis (e.g., CXCR4-mediated apoptosis) in cells expressing CXCR4 (e.g., T cells), optionally as determined using flow cytometry. In one embodiment, the anti-CXCR4 antibody or antigen-binding fragment described herein does not induce CXCR4-mediated apoptosis (e.g., by a direct effect on CXCR4 or the effect of inhibiting one or more of its natural ligands) in cells expressing CXCR4 in vitro, optionally as determined using flow cytometry. In one embodiment, apoptosis is determined using Jurkat T cells or primary naive T cells (e.g., mammalian or human). In one embodiment, the T cells are CXCR4-expressing T cells. The T cells are CD8 + T cells, CD4 + T cells or regulatory T cells (Treg), preferably CD8 +The apoptosis may be in T cells. In one embodiment, an appropriate isotype control (e.g., IgG4-PE) and a positive control (e.g., an anti-Fas antibody or staurosporine) are used in the assay to determine apoptosis. In one embodiment, the antibody or antigen-binding fragment does not induce apoptosis in T cells if the maximum percentage specifically induced apoptosis (optionally expressed as a percentage of the anti-Fas response or staurosporine-induced apoptosis) is less than 50%, less than 40%, less than 30%, or less than 20%. In another embodiment, the maximum percentage specifically induced apoptosis (optionally expressed as a percentage of the anti-Fas response or staurosporine-induced apoptosis) is less than 10%, less than 8%, less than 5%, or less than 4%, particularly less than 5%. In another embodiment, the maximum percentage specifically induced apoptosis (optionally expressed as a percentage of the anti-Fas response or staurosporine-induced apoptosis) is less than 3%, less than 2%, less than 1%, or less than 0.8%. In another embodiment, the maximum percentage specifically induced apoptosis (optionally expressed as a percentage of the anti-Fas response or staurosporine-induced apoptosis) is less than 0.5%, less than 0.3%, less than 0.2%, or less than 0.1%. In a preferred embodiment, the maximum percentage specifically induced apoptosis is determined using, e.g., flow cytometry as described herein, or performed essentially as described in Examples 9A or 9B herein. In another embodiment, the antibody (e.g., the antibody has reduced or no effector function) or antigen-binding fragment does not induce CXCR4-mediated apoptosis of B cells.

[0149] Apoptosis is a form of programmed cell death that involves the degradation of cellular components by a group of cysteine ​​proteases called caspases. Caspases can be activated through either the intrinsic (mitochondrial-mediated) or extrinsic (e.g., death receptor and effector cell-mediated) apoptotic pathways. Examples of extrinsic activation can be through Fas activation or the release of granzymes and perforin from effector cells. In one embodiment, the anti-CXCR4 antibodies or antigen-binding fragments described herein do not induce extrinsic (e.g., death receptor and effector cell-mediated) and / or intrinsic (mitochondrial-mediated) apoptosis.

[0150] In one embodiment, the anti-CXCR4 antibody or antigen-binding fragment described herein (substantially) inhibits CXCL12 (e.g., human or cynomolgus CXCL4) signaling via CXCR4, e.g., human or cynomolgus CXCL4, e.g., inhibits CXCL12-mediated CXCR4 signaling via cAMP. In one embodiment, the antibody or antigen-binding fragment (substantially) inhibits CXCL12-mediated inhibition of forskolin-stimulated cAMP, e.g., using a forskolin-stimulated cAMP signaling assay (e.g., AlphaScreen™ cAMP assay). In one embodiment, the antibody or antigen-binding fragment completely or substantially inhibits CXCL12-mediated inhibition of forskolin-stimulated cAMP. In another embodiment, the antibody or antigen-binding fragment partially inhibits CXCL12-mediated inhibition of forskolin-stimulated cAMP. In one embodiment, the antibody or antigen-binding fragment has an IC50 of 1-100 nM, 1-90 nM, 1-80 nM, 1-70 nM, 1-60 nM, 1-50 nM, 1-40 nM, or preferably 1-30 nM. 50 In one embodiment, cAMP is determined using a forskolin-stimulated cAMP signaling assay, which is performed essentially as described in Example 6 (human CXCR4) and Example 10B (cynomolgus monkey CXCR4) herein.

[0151] In one embodiment, an anti-CXCR4 antibody or antigen-binding fragment described herein (substantially) inhibits beta-arrestin recruitment to CXCR4 (e.g., hCXCR4 or cynomolgus CXCR4), and optionally, said beta-arrestin recruitment is induced by CXCL12 (e.g., hCXCL12 or cynomolgus CXCL12). In one embodiment, the antibody or antigen-binding fragment has an IC of 0.01-15 nM, 0.01-10 nM, 0.01-5 nM, 0.01-4 nM, 0.01-3 nM, 0.01-2 nM, 0.01-1.5 nM, 0.01-1 nM, 0.01-0.5 nM, or preferably 0.01-0.2 nM. 50 In one embodiment, the inhibition of beta-arrestin recruitment to ICXCR4 is measured using a functional cell-based reporter gene assay (e.g., using Tango CXCR4-bla U2OS cells). In one embodiment, the inhibition of beta-arrestin recruitment is measured using 30 nM CXCL12 to induce beta-arrestin. In one embodiment, the concentration of the antibody or antigen-binding fragment is between 100 nM and 0.1 pM (e.g., between 50 nM and 0.5 pM or between 40 nM and 0.5 pM). In one embodiment, the functional cell-based reporter gene assay is performed essentially as described in Example 6C herein.

[0152] In one embodiment, the anti-CXCR4 antibody or antigen-binding fragment described herein is (significantly) internalized into the cell upon binding to the cell surface, e.g., upon binding to CXCR4 on the cell surface; optionally, internalization is determined using fluorescence microscopy. In one embodiment, the antibody or antigen-binding fragment is internalized into the cell upon binding to CXCR4 on the cell surface with a maximum signal over a total overlap range of 1.2-10x, 1.2-5x, 1.2-4x, 1.2-3x, or 1.2-2x that of the isotype control. In one embodiment, the maximum signal over a total overlap range is 2-10x, 2-5x, 2-4x, 2-3x, 3-4x, or 3-5x, particularly 2-4x, that of the isotype control. In one embodiment, the isotype control is IgG4-PE. In one embodiment, internalization is determined using a fluorescent imaging technique (e.g., IncuCyte™ S3 Live Cell Analysis System (Essen BioScience) or ELISA, particularly a fluorescent imaging technique. In one embodiment, internalization of the anti-CXCR4 antibody or antigen-binding fragment is determined essentially as described in Example 8 herein.

[0153] In one embodiment, the fluorescence imaging technology includes live cell analysis, which measures real-time quantification of live cell imaging and analysis. This technology allows visualization and quantification of cell behavior over time, optionally automatically collecting and analyzing images in a laboratory incubator. One example of a live cell analysis system is the IncuCyte™ S3 Live Cell Analysis System (Essen BioScience), which is used to measure receptor or antibody (or antigen-binding fragment) internalization, chemotaxis, apoptosis, or cell invasion into 3D cellular environments (e.g., tumor spheroids). Internalization results in a positive readout of the secondary antibody (red fluorescence).

[0154] Without being bound by theory, antibodies or antigen-binding fragments of the present invention may be internalized and recycled to the cell surface together with their receptors (e.g., CXCR4). Alternatively, they may remain bound to their receptors (e.g., CXCR4) within the cell, thereby preventing their degradation by normal cellular mechanisms. Instead, the receptor-bound antibodies or antigen-binding fragments are internalized, and the receptors are recycled to the cell surface without the antibodies or antigen-binding fragments. Because the antibodies or antigen-binding fragments of the present invention are not internalized as rapidly as the benchmark antibodies, the inventors hypothesize that they remain bound to their receptors on the cell surface longer and inhibit their ligands (e.g., CXCL12) longer; therefore, despite appearing to have similar overall affinities, the antibodies or antigen-binding fragments of the present invention may have higher dissociation rate constant (Kd) values. An alternative theory is that the antibodies or antigen-binding fragments of the present invention bind to monomeric CXCR4, and a single antibody or antigen-binding fragment does not bind to more than one CXCR4 protein (monomer) due to steric hindrance. Thus, the antibodies or antigen-binding fragments of the invention do not cross-link the two receptors, resulting in slower internalization of the antibody or antigen-binding fragment:receptor complex.

[0155] In one embodiment, the anti-CXCR4 antibody or antigen-binding fragment described herein (substantially) inhibits a ligand-mediated (e.g., CXCL12, MIF, and / or ubiquitin, particularly CXCL12) response of T cells, as measured, for example, by changes in cell shape. In such assays, the observed changes in cell shape are caused by either a redistribution of cellular material, or a change in the cell membrane, or a change in the location of intracellular components. In one embodiment, the ligand-mediated response of T cells is determined using conductivity or a label-free dynamic mass redistribution (DMR) assay. In one embodiment, the antibody or antigen-binding fragment has an IC of 0.5-20 nM, 0.5-15 nM, 0.5-13 nM, 0.15-10 nM, 0.5-8 nM, or preferably 0.5-5 nM. 50 In one embodiment, the DMR assay is performed essentially as described in Example 13 herein.

[0156] In one embodiment, the anti-CXCR4 antibody or antigen-binding fragment described herein is + The antibody or antigen-binding fragment (e.g., antibody or antigen-binding fragment) inhibits (substantially) the chemotaxis of T cells (e.g., human T cells, such as primary human T cells extracted from human blood) toward a CXCL4 ligand, e.g., CXCL12. In one embodiment, chemotaxis is determined using live cell imaging (e.g., the IncuCyte™ S3 Chemotaxis Assay System) or flow cytometry (e.g., a transwell-based flow cytometry method), optionally using Jurkat T cells. In one embodiment, 30 nM or 6.25 nM CXCL12 is used to induce chemotaxis (as a chemoattractant) in the assay. In one embodiment, the antibody or antigen-binding fragment has an IC of 0.01-5 nM, 0.01-3 nM, 0.01-2 nM, or preferably 0.01-1 nM. 50 CXCR4 + Inhibiting T cell chemotaxis to CXCL12 In one embodiment, a chemotaxis assay is performed essentially as described in Example 14 herein.

[0157] In one embodiment, the anti-CXCR4 antibodies or antigen-binding fragments described herein inhibit the proliferation of T cells (e.g., CD8 + The anti-CXCR4 antibodies or antigen-binding fragments described herein are capable of (substantially or significantly) infiltrating (migrating) T cells (e.g., CD8 T cells) into tumors (e.g., solid tumors). Tumor infiltration can be measured in vivo by assessing T cell infiltration into tumors in mice (by comparing samples from untreated mice with mice treated with an anti-CXCR4 antibody or antigen-binding fragment described herein) using FACS or immunohistochemistry (IHC) (taking multiple cross-sections of treated and untreated tumors, applying a reagent that identifies T cells, and comparing the two). The tumor (e.g., central tumor mass) is surrounded by tumor stroma (see Figures 9 and 10). In preferred embodiments, the anti-CXCR4 antibodies or antigen-binding fragments described herein are capable of infiltrating T cells (e.g., CD8 T cells) into tumors (e.g., solid tumors). +This allows (substantially or significantly) T cells (e.g., T cells) to infiltrate the tumor from the tumor stroma. Once T cells are within the tumor (e.g., central tumor mass), they can effect cell killing and destroy malignant cells. A potential mechanism for this is shown in Figure 10. Without being bound by theory, in the absence of the anti-CXCR4 antibodies described herein, cancer-associated fibroblasts (CAFs) present in the stroma secrete CXCL12, creating a stronger gradient away from tumor cells toward which T cells preferentially migrate. This is shown schematically in Figure 9.

[0158] Tumor invasion can be measured in vitro using the spheroid assay. Spheroids are 3D cell culture models that can be used to mimic some characteristics of solid tumors. 3D cell culture offers many advantages over 2D cell culture, such as improved cell-cell contact that is more representative of tumors and creates tumor environmental conditions (e.g., hypoxia and necrosis). Tumor spheroids can be used as an in vitro model for screening therapeutic agents (Costa et al., (2016). 3D tumor spheroids: an overview on the tools and techniques used for their analysis. Biotechnol Adv. 34(8):1427-1441). These spheroids contain tumor cells (e.g., HT-29 cells) that secrete CXCR3 ligands (CXCL9, CXCL10, and CXCL11) when stimulated with interferon gamma (IFNγ). Recombinant CXCL12 is added to the culture medium to represent CXCL12 found within the tumor stroma. The added concentration is large enough to overcome the tumor-secreted CXCL9, CXCL10, and CXCL11 invasion-promoting chemokine gradient and thus more accurately reflect in vivo conditions. This assay uses CXCR4-expressing CCRF-HSB-2 cells, which are T cell lymphoblasts and mimic T cells in the tumor environment. Inhibition of CXCL12 signaling with an anti-CXCR4 antibody is believed to restore CXCR3 chemotaxis of T cell lymphoblasts to CXCL9, CXCL10, and / or CXCL11 released by the spheroid, resulting in increased T cell infiltration levels and allowing for a reduction in tumor volume. Thus, in one embodiment, the anti-CXCR4 antibody or antigen-binding fragment described herein induces (significantly) increased T cell lymphoblast infiltration into tumor spheroids. In one embodiment, the tumor spheroids comprise cells selected from one or more of HT-29 cells, HS-5 cells, MS-5 cells, and MEF cells, e.g., HT-29 cells.In one embodiment, the antibody or antigen-binding fragment induces a percentage of maximal infiltration of T-cell lymphoblasts (e.g., CCRF-HSB-2 cells) into tumor spheroids of 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 95-100%, or 97-100%, in one embodiment, a percentage of maximal infiltration of 10-100%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, 80-90%, 90-90%, 95-90%, or 97-90%, particularly 50-100%. In one embodiment, T cell lymphoblast infiltration into the spheroids is measured using live cell analysis (e.g., the IncuCyte™ S3 live cell analysis system described elsewhere herein). In one embodiment, modulation of T cell infiltration into cancer spheroids is performed essentially as described in Example 15 herein.

[0159] In one embodiment, the anti-CXCR4 antibodies or antigen-binding fragments described herein bind to CD45 + In one embodiment, the increase in cell recruitment is measured by a cell counter or flow cytometry, particularly flow cytometry. In one embodiment, the increase in cell recruitment is measured by a decrease in CD45 expression compared to a control (e.g., a vehicle control such as PBS or an isotype control). + and optionally, the antibody or antigen-binding fragment is a CD45 + In one embodiment, CD45 + Cell recruitment is performed essentially as described in Examples 16-18 herein.

[0160] In one embodiment, the antibody or antigen-binding fragment comprises a constant region (e.g., C), e.g., as described elsewhere herein. H and / or C. L In one embodiment, the constant region comprises a mammalian (e.g., human, cynomolgus monkey, rhesus monkey, horse, dog, cat, mouse, rat, etc.) constant region (e.g., CH and / or C. L ), particularly a rodent constant region, e.g., a mouse constant region such as a mouse or rat constant region. In a preferred embodiment, the antibody or antigen-binding fragment contains a human constant region (e.g., C H and / or C. L ) is included.

[0161] In one embodiment, the heavy chain constant region can be altered to modify the properties of the antibody, for example to increase or decrease one or more of the following: Fc receptor binding, antibody glycosylation, effector cell function, and / or complement function. See, e.g., EP 2654790, U.S. Patent Application Publication Nos. 2012 / 0251531, U.S. Patent Nos. 9,133,274 and 10,239,944, and PCT Publication Nos. WO 2014 / 065945, WO 2015 / 150447, and WO 2021 / 016571, each of which is incorporated by reference in its entirety.

[0162] In some embodiments, the heavy chain constant region lacks Fc effector function, i.e., is an effector-null constant region. In one embodiment, the heavy chain constant region comprises mutations that reduce binding to Fc-γ receptors and / or C1q compared to wild-type. In one embodiment, the heavy chain constant region may lack measurable binding to human FcyRI, FcyRI1a, FcyRIIIa, and FcyRIIIb receptors, but maintain binding to human FcyRIIb receptors, and optionally maintain binding to human FcRn receptors. FcyRI, FcyRI1a, FcyRIIIa, and FcyRIIIb are examples of activating receptors. FcyRIIb is an example of an inhibitory receptor. FcRn is an example of a recycling receptor.

[0163] In certain embodiments, the antibody or antigen-binding fragment comprises a human IgG4 constant region comprising, for example, the amino acid sequence of SEQ ID NO: 258-263. In one embodiment, the IgG4 heavy chain constant region comprises a mutation that reduces binding to Fc-γ receptors and / or C1q compared to the wild type. For example, the constant region may comprise a Leu235Glu (using the EU index) mutation. Another optional mutation in the IgG4 heavy chain constant region is a Ser228Pro (using the EU index) mutation, which increases stability. In one embodiment, the human IgG4 constant region comprises both the Leu235Glu and Ser228Pro (using the EU index) mutations. This "IgG4-PE" heavy chain constant region is effector-null. In a preferred embodiment, the constant region is an IgG4-PE constant region (optionally selected from SEQ ID NO: 267 or 305). In a preferred embodiment, the IgG4-PE constant region is encoded by any of the nucleic acid sequences of SEQ ID NO: 264-266 or 304.

[0164] In certain embodiments, the antibody or antigen-binding fragment comprises a human IgG1 constant region comprising a mutation that reduces effector function (i.e., a disabled IgG1 constant region). In one embodiment, the IgG1 constant region comprises a mutation that reduces binding to an Fc-γ receptor and / or C1q compared to wild-type, e.g., a substitution at one or more amino acids selected from the group consisting of Leu234, Leu235, Gly237, Asp265, Asp270, Asn297, Ala327, Pro329, and Pro331 (using the EU index). In one embodiment, the IgG1 constant region comprises a Leu235Ala and / or Gly237Ala (using the EU index) mutation (e.g., amino acid SEQ ID NO: 249, optionally encoded by nucleotide SEQ ID NO: 248). In another embodiment, the IgG1 constant region comprises a Leu234Phe, Leu235Glu, and Pro331Ser (using the EU index) mutation (e.g., amino acid SEQ ID NO: 307, optionally encoded by nucleotide SEQ ID NO: 306).

[0165] Any of the antibodies or antigen-binding fragments of the invention can comprise a constant region such as a rodent constant region or a human constant region (e.g., an IgG4 constant region or an IgG1 constant region), e.g., an effector-null human constant region (e.g., IgG4), and optionally the constant region is IgG4-PE (SEQ ID NO: 267 or SEQ ID NO: 305) or a disabled IgG1 (e.g., SEQ ID NO: 249 or SEQ ID NO: 307).

[0166] Constant regions, such as the IgG4 or IgG1 constant regions described elsewhere herein, often end with a C-terminal lysine, which may be clipped, for example, during protein production or downstream processing. Any antibody sequence described herein that includes a C-terminal lysine is intended to encompass embodiments in which such lysine is deleted (i.e., clipped) after translation. Thus, in one embodiment, the constant region is an IgG4 (e.g., fully human IgG4 or a mutated IgG4), and the IgG4 constant region includes or does not include the C-terminal lysine (lysine-clipped). In a preferred embodiment, the constant region is an IgG4-PE, and includes or does not include the C-terminal lysine (e.g., SEQ ID NO: 267) (e.g., SEQ ID NO: 305) (lysine-clipped).

[0167] In another embodiment, the constant region is wild-type human IgG1 (optionally selected from SEQ ID NOs: 241, 243, 245, and 247). For example, the constant region is an effector-capable IgG1 constant region, optionally with ADCC and / or CDC activity. In one embodiment, the constant region is engineered to enhance ADCC and / or CDC and / or ADCP. The antibody-dependent cellular phagocytosis (ADCP) mechanism is discussed in Guel et al., "Antibody-Dependent Phagocytosis of Tumor Cells by Macrophages: A Potent Effector Mechanism of Monoclonal Antibody Therapy of Cancer", Cancer Res., 75(23), December 1, 2015.

[0168] Without being bound by theory, antibodies or antigen-binding fragments of the invention comprising a heavy chain constant region comprising a Leu235Glu and / or Ser228Pro (using the EU index) mutation (e.g., an IgG4-PE constant region, optionally SEQ ID NO: 267 or 305) may provide improved yields of the antibody or antigen-binding fragment during production or may enhance the biological properties of, for example, any of the antibodies or antigen-binding fragments described herein. For example, these mutations may reduce the level of apoptosis induced by the antibody or antigen-binding fragment (e.g., by a direct effect on CXCR4 or the effect of inhibiting one or more of its natural ligands) compared to the same antibody or antigen-binding fragment variable region comprising a different heavy chain constant region (e.g., an IgG1, a disabled IgG1, an IgG1 mutant, or an IgG4). The constant region sequence may contribute to the internalization profile of the antibody or antigen-binding fragment, or may result in an antibody or antigen-binding fragment having a shorter half-life than the same antibody or antigen-binding fragment variable region comprising a different constant region (e.g., an IgG1, an IgG4, a disabled IgG1, or an IgG1 mutant).

[0169] In one embodiment, the antibody or antigen-binding fragment comprises a light chain, such as a kappa light chain. In one embodiment, the antibody or antigen-binding fragment comprises a light chain constant region, such as a kappa light chain constant region. The kappa light chain constant region amino acid and nucleotide sequences are set forth in SEQ ID NOs: 271-279. In another embodiment, the antibody or antigen-binding fragment comprises a lambda light chain and / or a lambda light chain constant region. The lambda light chain constant region amino acid and nucleotide sequences are set forth in SEQ ID NOs: 280-303.

[0170] In one embodiment, the antibody comprises two full-length heavy chains and two full-length light chains (V H and C H and V L and C L (including

[0171] In one embodiment, the antibody or antigen-binding fragment is a multispecific (e.g., bispecific) antibody or fusion protein, such as the bispecific antibodies described above.

[0172] In one embodiment, the antibody or antigen-binding fragment thereof comprises a V H Includes the domain, a) the CDRH3 amino acid sequence is selected from SEQ ID NOs: 12, 15, 32, 35, 52, 55, 72, 75, 92, and 95, each of which contains 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; b) the CDRH3 amino acid sequence is identical to a sequence selected from SEQ ID NOs: 12, 15, 32, 35, 52, 55, 72, 75, 92, and 95; c) the CDRH3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NOs: 112, 120, 128, 136, 144, 152, 160, 168, 176, 184, 192, 200, 208, 216, 224, and 232 H domain, and the CDRH3 sequence contains 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, respectively; or d) the CDRH3 amino acid sequence is identical to the CDRH3 amino acid sequence defined by IMGT or Kabat and is selected from SEQ ID NOs: 112, 120, 128, 136, 144, 152, 160, 168, 176, 184, 192, 200, 208, 216, 224, and 232; H It comes from the domain.

[0173] In one embodiment, the antibody or antigen-binding fragment has H Includes the domain, aV H The domain is i. SEQ ID NO: 12 or 15 or the CDRH3 amino acid sequence of SEQ ID NO: 12 or 15, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, and / or ii. SEQ ID NO: 10 or 13 or the CDRH1 amino acid sequence of SEQ ID NO: 10 or 13, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions; and / or iii. SEQ ID NO: 11 or 14 or the CDRH2 amino acid sequence of SEQ ID NO: 11 or 14, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions Including, bV H The domain is i. SEQ ID NO: 32 or 35 or the CDRH3 amino acid sequence of SEQ ID NO: 32 or 35, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions; and / or ii. SEQ ID NO: 30 or 33 or the CDRH1 amino acid sequence of SEQ ID NO: 30 or 33, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions; and / or iii. SEQ ID NO: 31 or 34 or the CDRH2 amino acid sequence of SEQ ID NO: 31 or 34, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions Including, cV H The domain is i. SEQ ID NO: 52 or 55 or the CDRH3 amino acid sequence of SEQ ID NO: 52 or 55, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions; and / or ii. SEQ ID NO: 50 or 53 or the CDRH1 amino acid sequence of SEQ ID NO: 50 or 53, respectively, comprising 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions; and / or iii. SEQ ID NO: 51 or 54 or the CDRH2 amino acid sequence of SEQ ID NO: 51 or 54, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions Including, dV H The domain is i. SEQ ID NO: 72 or 75 or the CDRH3 amino acid sequence of SEQ ID NO: 72 or 75, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions; and / or ii. SEQ ID NO: 70 or 73 or the CDRH1 amino acid sequence of SEQ ID NO: 70 or 73, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions; and / or iii. SEQ ID NO: 71 or 74 or the CDRH2 amino acid sequence of SEQ ID NO: 71 or 74, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions Including, eV H The domain is i. SEQ ID NO: 92 or 95 or the CDRH3 amino acid sequence of SEQ ID NO: 92 or 95, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, and / or ii. SEQ ID NO: 90 or 93 or the CDRH1 amino acid sequence of SEQ ID NO: 90 or 93, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions; and / or iii. SEQ ID NO: 91 or 94 or the CDRH2 amino acid sequence of SEQ ID NO: 91 or 94, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions Including, fV H The domain includes: i. The CDRH3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 112 H The CDRH3 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 112. H and / or ii. The CDRH1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 112. H The CDRH1 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 112. H and / or iii. The CDRH2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 112. HThe CDRH2 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 112. H and / or gV H The domain includes: i. The CDRH3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 120 H The CDRH3 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 120. H and / or ii. The CDRH1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 120 H The CDRH1 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 120. H and / or iii. The CDRH2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 120. H The CDRH2 amino acid sequence is derived from a V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 120. H and / or hV H The domain includes: i. The CDRH3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 128 HThe CDRH3 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 128. H and / or ii. The CDRH1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 128 H The CDRH1 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 128. H and / or iii. The CDRH2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 128 H The CDRH2 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 128. H and / or iV H The domain includes: i. The CDRH3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 136 H The CDRH3 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 136. H and / or ii. The CDRH1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 136 H The CDRH1 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 136. Hand / or iii. The CDRH2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 136 H The CDRH2 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 136. H and / or jV H The domain includes: i. The CDRH3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 144 H The CDRH3 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 144. H and / or ii. The CDRH1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 144 H The CDRH1 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 144. H and / or iii. The CDRH2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 144 H The CDRH2 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 144. H and / or kV H The domain includes: i. The CDRH3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 152 H The CDRH3 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 152. H and / or ii. The CDRH1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 152 H The CDRH1 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 152. H and / or iii. The CDRH2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 152 H The CDRH2 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 152. H and / or lV H The domain includes: i. The CDRH3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 160 H The CDRH3 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 160. H and / or ii. The CDRH1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 160 HThe CDRH1 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 160. H and / or iii. The CDRH2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 160. H The CDRH2 amino acid sequence is derived from a V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 160. H and / or mV H The domain includes: i. The CDRH3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 168 H The CDRH3 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 168. H and / or ii. The CDRH1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 168 H The CDRH1 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 168. H and / or iii. The CDRH2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 168 H The CDRH2 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 168. Hand / or nV H The domain includes: i. The CDRH3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 176 H The CDRH3 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 176. H and / or ii. The CDRH1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 176 H The CDRH1 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 176. H and / or iii. The CDRH2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 176 H The CDRH2 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 176. H and / or oV H The domain includes: i. The CDRH3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 184 H The CDRH3 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 184. H and / or ii. The CDRH1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 184 H The CDRH1 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 184. H and / or iii. The CDRH2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 184. H The CDRH2 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 184. H and / or pV H The domain includes: i. The CDRH3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 192 H The CDRH3 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 192. H and / or ii. The CDRH1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 192 H The CDRH1 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 192. H and / or iii. The CDRH2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 192 HThe CDRH2 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 192. H and / or qV H The domain includes: i. The CDRH3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 200 H The CDRH3 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 200. H and / or ii. The CDRH1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 200 H The CDRH1 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 200. H and / or iii. The CDRH2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 200 H The CDRH2 amino acid sequence is derived from a V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 200. H and / or rV H The domain includes: i. The CDRH3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 208 HThe CDRH3 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 208. H and / or ii. The CDRH1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 208 H The CDRH1 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 208. H and / or iii. The CDRH2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 208 H The CDRH2 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 208. H and / or sV H The domain includes: i. The CDRH3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 216 H The CDRH3 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 216. H and / or ii. The CDRH1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 216 H The CDRH1 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 216. Hand / or iii. The CDRH2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 216 H The CDRH2 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 216. H and / or tV H The domain includes: i. The CDRH3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 224 H The CDRH3 amino acid sequence is derived from the V domain or as defined by IMGT or Kabat and is selected from SEQ ID NO: 224. H and / or ii. The CDRH1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 224 H or the CDRH1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 224 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. H Originates from the domain, and / or iii. The CDRH2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 224 H or the CDRH2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 224 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. H Originates from the domain, and / or uV H The domain includes: i. The CDRH3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 232 H or the CDRH3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 232 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. H Originates from the domain, and / or ii. The CDRH1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 232 H or the CDRH1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 232 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. H Originates from the domain, and / or iii. The CDRH2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 232 H or the CDRH2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 232 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. H It comes from the domain.

[0174] In one embodiment, the antibody or antigen-binding fragment comprises a V L including or further including a domain, a. the CDRL3 amino acid sequence is selected from SEQ ID NOs: 22, 25, 42, 45, 62, 65, 82, 85, 102, and 105, each of which contains 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; b. the CDRL3 amino acid sequence is identical to a sequence selected from SEQ ID NOs: 22, 25, 42, 45, 62, 65, 82, 85, 102, and 105; c. The CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NOs: 116, 124, 132, 140, 148, 156, 164, 172, 180, 188, 196, 204, 212, 220, 228, and 236. L domain, and the CDRL3 sequence contains 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, respectively; or d. The CDRL3 amino acid sequence is identical to the CDRL3 amino acid sequence defined by IMGT or Kabat and is selected from SEQ ID NOs: 116, 124, 132, 140, 148, 156, 164, 172, 180, 188, 196, 204, 212, 220, 228, and 236. L It comes from the domain.

[0175] In one embodiment, the antibody or antigen-binding fragment has L including or further including a domain, aV L The domain is i. SEQ ID NO: 22 or 25 or the CDRL3 amino acid sequence of SEQ ID NO: 22 or 25, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, and / or ii. SEQ ID NO: 20 or 23 or the CDRL1 amino acid sequence of SEQ ID NO: 20 or 23, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions, and / or iii. SEQ ID NO: 21 or 24 or the CDRL2 amino acid sequence of SEQ ID NO: 21 or 24, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions Including, bV L The domain is i. SEQ ID NO: 42 or 45 or the CDRL3 amino acid sequence of SEQ ID NO: 42 or 45, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions, and / or ii. SEQ ID NO: 40 or 43 or the CDRL1 amino acid sequence of SEQ ID NO: 40 or 43, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions, and / or iii. SEQ ID NO: 41 or 44 or the CDRL2 amino acid sequence of SEQ ID NO: 41 or 44, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions Including, cV L The domain is i. SEQ ID NO: 62 or 65 or the CDRL3 amino acid sequence of SEQ ID NO: 62 or 65, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, and / or ii. SEQ ID NO: 60 or 63 or the CDRL1 amino acid sequence of SEQ ID NO: 60 or 63, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions; and / or iii. SEQ ID NO: 61 or 64 or the CDRL2 amino acid sequence of SEQ ID NO: 61 or 64, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions Including, dV L The domain is i. SEQ ID NO: 82 or 85 or the CDRL3 amino acid sequence of SEQ ID NO: 82 or 85, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, and / or ii. SEQ ID NO: 80 or 83 or the CDRL1 amino acid sequence of SEQ ID NO: 80 or 83, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions, and / or iii. SEQ ID NO: 81 or 84 or the CDRL2 amino acid sequence of SEQ ID NO: 81 or 84, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions Including, eV L The domain is i. SEQ ID NO: 102 or 105 or the CDRL3 amino acid sequence of SEQ ID NO: 102 or 105, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions, and / or ii. SEQ ID NO: 100 or 103 or the CDRL1 amino acid sequence of SEQ ID NO: 100 or 103, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions; and / or iii. SEQ ID NO: 101 or 104 or the CDRL2 amino acid sequence of SEQ ID NO: 101 or 104, respectively, containing 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions Including, fV L The domain includes: i. the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 116 L or the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 116 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or ii. The CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 116 L or the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 116 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or iii. The CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 116. L or the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 116 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L It comes from the domain gVL The domain includes: i. The CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 124 L or the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 1294 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or ii. The CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 124 L or the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 124 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or iii. The CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 124 L or the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 124 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L It comes from the domain hV L The domain includes: i. the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 132 L or the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 132 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or ii. The CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 132 Lor the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 132 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or iii. The CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 132 L or the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 132 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L It comes from the domain iV L The domain includes: i. the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 140 L or the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 140 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or ii. the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 140 L or the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 140 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or iii. The CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 140 L or the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 140 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. LIt comes from the domain jV L The domain includes: i. the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 148 L or the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 148 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or ii. the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 148 L or the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 148 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or iii. The CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 148 L or the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 148 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L It comes from the domain kV L The domain includes: i. the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 156 L or the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 156 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or ii. The CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 156 L or the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 156 containing 3, 2 or 1 amino acid substitutions. L Originates from the domain, and / or iii. The CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 156 L or the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 156 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L It comes from the domain lV L The domain includes: i. the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 164 L or the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 164 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or ii. The CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 164 L or the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 164 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or iii. The CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 164. Lor the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 164 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L It comes from the domain mV L The domain includes: i. the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 172 L or the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 172 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or ii. The CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 172 L or the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 172 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or iii. The CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 172 L or the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 172 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L It comes from the domain nV L The domain includes: i. the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 180 Lor the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 180 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or ii. The CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 180 L or the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 180 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or iii. The CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 180. L or the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 180 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L It comes from the domain oV L The domain includes: i. the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 188 L or the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 188 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or ii. The CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 188 L or the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 188 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. LOriginates from the domain, and / or iii. The CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 188 L or the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 188 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L It comes from the domain pV L The domain includes: i. the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 196 L or the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 196 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or ii. The CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 196 L or the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 196 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or iii. The CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 196 L or the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 196 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L It comes from the domain qV L The domain includes: i. the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 204 L or the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 204 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or ii. The CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 204 L or the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 204 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or iii. The CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 204 L or the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 204 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L It comes from the domain rV L The domain includes: i. The CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 212 L or the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 212 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or ii. The CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 212 Lor the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 212 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or iii. The CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 212 L or the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 212 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L It comes from the domain sV L The domain includes: i. the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 220 L or the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 220 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or ii. The CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 220 L or the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 220 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or iii. The CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 220 L or the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 220 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. LIt comes from the domain tV L The domain includes: i. the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 228 L or the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 228 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or ii. The CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 228 L or the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 228 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or iii. The CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 228 L or the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 228 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L It comes from the domain, or uV L The domain includes: i. The CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 236 L or the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 236 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or ii. The CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 236 L or the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 236 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L Originates from the domain, and / or iii. The CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 236 L or the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is selected from SEQ ID NO: 236 containing 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L It comes from the domain.

[0176] According to embodiments herein, the designated CDRs (e.g., CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and / or CDRL3) are as defined by Kabat or IMGT. In one embodiment, the designated CDRs comprise one amino acid substitution, optionally a conservative amino acid substitution. In one embodiment, the designated CDRs comprise one or two (e.g., two) amino acid substitutions, optionally conservative amino acid substitutions. In one embodiment, the designated CDRs comprise one, two or three (e.g., three or two or three) amino acid substitutions, optionally conservative amino acid substitutions. In one embodiment, the designated CDRs comprise one, two, three or four (e.g., four, three or four, one or four or two or four) amino acid substitutions, optionally conservative amino acid substitutions. In one embodiment, a designated CDR comprises 1, 2, 3, 4, or 5 (e.g., 5, 4 or 5, 3 or 5, 2 or 5, or 1 or 5) amino acid substitutions, optionally conservative amino acid substitutions. In one embodiment, a designated CDR comprises 1, 2, 3, 4, 5, or 6 (e.g., 6, 5 or 6, 4 or 6, 3 or 6, 2 or 6, or 1 or 6) amino acid substitutions, optionally conservative amino acid substitutions. Amino acid substitutions include modifications in which an amino acid is replaced with a different naturally occurring amino acid residue. Such substitutions can be classified as "conservative," in which an amino acid residue in a polypeptide is substituted with another naturally occurring amino acid having similar properties in terms of polarity, side chain functionality, or size. Such conservative substitutions are well known in the art. A compound selected from: In one embodiment, the amino acid substitution is a conservative amino acid substitution, optionally a conservative substitution in which an amino acid of group (1) to (8) is replaced with an amino acid of the same group: 1) Glycine (G), Alanine (A), 2) serine (S), threonine (T), cysteine ​​(C), methionine (M), 3) Aspartic acid (D), glutamic acid (E), 4) Asparagine (N), Glutamine (Q), 5) Aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q), 6) Arginine (R), Lysine (K), Histidine (H), 7) Glycine (G), Alanine (A), Isoleucine (I), Leucine (L), Valine (V), 8) Isoleucine (I), Leucine (L), Methionine (M), Valine (V), Proline (P), and 9) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0177] In one embodiment, the antibody or antigen-binding fragment comprises a VH domain, wherein the VH domain comprises a CDRH1 amino acid sequence of SEQ ID NO: 10 or 13, a CDRH2 amino acid sequence of SEQ ID NO: 11 or 14, and a CDRH3 amino acid sequence of SEQ ID NO: 12 or 15; the antibody or antigen-binding fragment comprises a VL domain, wherein the VL domain comprises a CDRL1 amino acid sequence of SEQ ID NO: 20 or 23, a CDRL2 amino acid sequence of SEQ ID NO: 21 or 24, and a CDRL3 amino acid sequence of SEQ ID NO: 22 or 25; and one or more CDRs (e.g., CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and / or CDRL3) comprise at most one amino acid substitution, optionally a conservative amino acid substitution.

[0178] In one embodiment, the antibody or antigen-binding fragment comprises a VH domain, wherein the VH domain comprises the CDRH1 amino acid sequence of SEQ ID NO: 13, the CDRH2 amino acid sequence of SEQ ID NO: 14, and the CDRH3 amino acid sequence of SEQ ID NO: 15; the antibody or antigen-binding fragment comprises a VL domain, wherein the VL domain comprises the CDRL1 amino acid sequence of SEQ ID NO: 23, the CDRL2 amino acid sequence of SEQ ID NO: 24, and the CDRL3 amino acid sequence of SEQ ID NO: 25; and one or more CDRs (i.e., CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3) comprise at most one amino acid substitution, optionally a conservative amino acid substitution.

[0179] In one embodiment, the antibody or antigen-binding fragment comprises a VH domain, wherein the VH domain comprises the CDRH1 amino acid sequence of SEQ ID NO: 13, the CDRH2 amino acid sequence of SEQ ID NO: 14, and the CDRH3 amino acid sequence of SEQ ID NO: 15; the antibody or antigen-binding fragment comprises a VL domain, wherein the VL domain comprises the CDRL1 amino acid sequence of SEQ ID NO: 23, the CDRL2 amino acid sequence of SEQ ID NO: 24, and the CDRL3 amino acid sequence of SEQ ID NO: 25; and one CDR (i.e., CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3) comprises at most one amino acid substitution, optionally a conservative amino acid substitution.

[0180] In one embodiment, the antibody or antigen-binding fragment has H Includes the domain, aV H the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 16; bV H the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 36; cV H the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 56; dV H the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 76; eV H the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 96; fV H the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 112; gV H the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 120; hV H the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 128; iV H the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 136; jV H the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 144; kV H the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 152; lV H the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 160; mV H the domain comprises an amino acid sequence identical or at least 90%, 95% or 98% identical to SEQ ID NO: 168; nV H the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 176; oV H the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 184; pV H the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 192; qV H the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 200; rV H the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 208; sV H the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 216; tV H the domain comprises an amino acid sequence identical or at least 90%, 95% or 98% identical to SEQ ID NO: 224; or uV HA domain comprises an amino acid sequence that is identical or at least 90%, 95%, or 98% identical to SEQ ID NO:232.

[0181] In one embodiment, the antibody or antigen-binding fragment thereof has a V L including or further including a domain, aV L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 26; bV L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 46; cV L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 66; dV L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 86; eV L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 106; fV L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 116; gV L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 124; hV L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 132; iV L the domain comprises an amino acid sequence identical or at least 90-80%, 95% or 98% identical to SEQ ID NO: 140; jV L the domain comprises an amino acid sequence identical or at least 90%, 95% or 98% identical to SEQ ID NO: 148; kV Lthe domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 156; lV L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 164; mV L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 172; nV L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 180; oV L the domain comprises an amino acid sequence identical or at least 90%, 95% or 98% identical to SEQ ID NO: 188; pV L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 196; qV L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 204; rV L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 212; sV L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 220; tV L the domain comprises an amino acid sequence identical or at least 90%, 95% or 98% identical to SEQ ID NO: 228; or uV L A domain comprises an amino acid sequence that is identical or at least 90%, 95%, or 98% identical to SEQ ID NO:236.

[0182] For the avoidance of doubt, the term "at least X% identical" as used throughout these embodiments is intended to refer to "% identity" as defined herein.

[0183] In one embodiment, the antibody or antigen-binding fragment has H Domain and V L Includes the domain, aV H The domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 16, and L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 26; bV H The domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 36, and L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 46; cV H The domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 56, and L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 66; dV H The domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 76, and L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 86; eV H The domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 96, and L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 106; fV H The domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 112, and L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 116; gV HThe domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 120, and L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 124; hV H The domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 128, and L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 132; iV H The domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 136, and L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 140; jV H The domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 144, and L the domain comprises an amino acid sequence identical or at least 90%, 95% or 98% identical to SEQ ID NO: 148; kV H The domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 152, and L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 156; lV H The domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 160, and L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 164; mV H The domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 168, and L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 172; nV H The domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 176, and L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 180; oV H The domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 184, and L the domain comprises an amino acid sequence identical or at least 90%, 95% or 98% identical to SEQ ID NO: 188; pV H The domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 192, L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 196; qV H The domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 200, and L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 204; rV H The domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 208, and L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 212; sV H The domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 216, L the domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 220; tV H The domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 224, Lthe domain comprises an amino acid sequence identical or at least 90%, 95% or 98% identical to SEQ ID NO: 228; or uV H The domain comprises an amino acid sequence identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 232, L A domain comprises an amino acid sequence that is identical or at least 90%, 95%, or 98% identical to SEQ ID NO:236.

[0184] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain and a light chain, a. the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 18 and the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 28; b. the heavy chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 18, and the light chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 28; c. the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 38 and the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 48; d. the heavy chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 38, and the light chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 48; e. the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO:58 and the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO:68; f. the heavy chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO:58, and the light chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO:68; g. the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 78 and the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 88; h. the heavy chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO:78, and the light chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO:88; i. the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 98 and the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 108; j. the heavy chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 98, and the light chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 108; k. the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 98 and the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 110; l. the heavy chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 98, and the light chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 110; m. the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 114 and the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 118; n. the heavy chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 114, and the light chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 118; o. the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 122 and the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 126; p. the heavy chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 122, and the light chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 126; q. the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 130 and the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 134; r. the heavy chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 130, and the light chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 134; s. the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 138 and the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 142; t. the heavy chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 138, and the light chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 142; u. the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 146 and the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 150; v. the heavy chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 146, and the light chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 150; w. the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 154 and the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 158; x. the heavy chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 154, and the light chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 158; y. the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 162 and the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 166; z. the heavy chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 162, and the light chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 166; aa. the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 170 and the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 174; bb. the heavy chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 170, and the light chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 174; cc. the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 178, and the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 182; dd. the heavy chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 178, and the light chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 182; ee. the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 186 and the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 190; ff. the heavy chain amino acid sequence comprises an amino acid sequence at least 90%, 95% or 98% identical to SEQ ID NO: 186, and the light chain amino acid sequence comprises an amino acid sequence at least 90%, 95% or 98% identical to SEQ ID NO: 190; gg. the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 194 and the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 198; hh. the heavy chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 194, and the light chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 198; ii. the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 202 and the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 206; jj. the heavy chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 202, and the light chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 206; kk. the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 210, and the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 214; ll. the heavy chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 210, and the light chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 214; mm. the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 218, and the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 222; nn. the heavy chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 218, and the light chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 222; oo. the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 226 and the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 230; pp. the heavy chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 226, and the light chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO: 230; qq. the heavy chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 234 and the light chain amino acid sequence comprises the amino acid sequence of SEQ ID NO: 238; or rr. The heavy chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO:234, and the light chain amino acid sequence comprises an amino acid sequence at least 90%, 95%, or 98% identical to SEQ ID NO:238.

[0185] In any of the embodiments described herein, % identity may be replaced with any % identity selected from the following: 70% identity, 75% identity, 80% identity, 85% identity, or 90% identity. In any of the embodiments herein, % identity may be replaced with any % identity selected from the following: 91% identity, 92% identity, 93% identity, 94% identity, or 95% identity, particularly 95% identity. In any of the embodiments herein, % identity may be replaced with any % identity selected from the following: 96% identity, 97% identity, 98% identity, 99% identity, or 100% identity, particularly 98% identity.

[0186] In one embodiment, the anti-CXCR4 antibody or antigen-binding fragment described herein comprises a V domain comprising a CDRH3 having a length of 5 to 27 amino acids, 5 to 25 amino acids, 5 to 24 amino acids, 5 to 23 amino acids, 5 to 21 amino acids, 10 to 25 amino acids, 10 to 24 amino acids, 10 to 23 amino acids, 10 to 21 amino acids, 12 to 25 amino acids, 12 to 23 amino acids, 12 to 21 amino acids, 14 to 25 amino acids, 14 to 23 amino acids, or 14 to 21 amino acids. H In another embodiment, CDRH3 is 6, 14, 15, 17, 19, 21, or 23 amino acids in length.

[0187] In one embodiment, the anti-CXCR4 antibody or antigen-binding fragment described herein is human V H gene segments, human D gene segments and human J H V derived from recombination of gene segments H domain, human V H The gene segment is IGHV3-33 (e.g., IGHV3-33*01), and / or the human D gene segment is IGHD1-20 (e.g., IGHD1-20*01), and / or the human J gene segment is H The gene segment is IGHJ3 (e.g., IGHJ3*02), and optionally, the antibody or antigen-binding fragment comprises a V comprising a CDRH3 having a length of 12 to 23 amino acids. H Includes the domain.

[0188] In one embodiment, the anti-CXCR4 antibody or antigen-binding fragment described herein is human V H gene segments, human D gene segments and human J H V derived from recombination of gene segments H domain, human V H The gene segment is IGHV3-30 (e.g., IGHV3-30*18), and / or the human D gene segment is IGHD3-16 (e.g., IGHD3-16*02), and / or the human J gene segment is HThe gene segment is IGHJ6 (e.g., IGHJ6*02), and optionally, the antibody or antigen-binding fragment comprises a V that includes a CDRH3 having a length of 12 to 23 amino acids. H Includes the domain.

[0189] In one embodiment, the anti-CXCR4 antibody or antigen-binding fragment described herein is human V H gene segments, human D gene segments and human J H V derived from recombination of gene segments H domain, human V H The gene segment is IGHV3-73 (e.g., IGHV3-73*02), and / or the human D gene segment is IGHD3-9 (e.g., IGHD3-9*01), and / or the human J gene segment is IGHD3-9 (e.g., IGHD3-9*01). H The gene segment is IGHJ6 (e.g., IGHJ6*02), and optionally, the antibody or antigen-binding fragment comprises a V that includes a CDRH3 having a length of 12 to 23 amino acids. H Includes the domain.

[0190] In one embodiment, the anti-CXCR4 antibody or antigen-binding fragment described herein is human V H gene segments, human D gene segments and human J H V derived from recombination of gene segments H domain, human V H The gene segment is IGHV4-31 (e.g., IGHV4-31*03), and / or the human D gene segment is IGHD3-10 (e.g., IGHD3-10*01), and / or the human J gene segment is IGHD3-10 (e.g., IGHD3-10*01). H The gene segment is IGHJ6 (e.g., IGHJ6*02), and optionally, the antibody or antigen-binding fragment comprises a V that includes a CDRH3 having a length of 12 to 23 amino acids. H Includes the domain.

[0191] In one embodiment, the anti-CXCR4 antibody or antigen-binding fragment described herein is human V H gene segments, human D gene segments and human J HV derived from recombination of gene segments H domain, human V H The gene segment is IGHV3-23 (e.g., IGHV3-23*04), and / or the human D gene segment is IGHD1-1 (e.g., IGHD1-1*01), and / or the human J gene segment is H The gene segment is IGHJ6 (e.g., IGHJ6*02), and optionally, the antibody or antigen-binding fragment comprises a V that includes a CDRH3 having a length of 12 to 23 amino acids. H Includes the domain.

[0192] In one embodiment, the anti-CXCR4 antibody or antigen-binding fragment described herein comprises a V H domain, wherein CDRH3 comprises the amino acid motif AX1GQLERR or X2YGX3DV, wherein X1, X2, and X3 are independently any amino acid; optionally, CDRH3 has a length of 12-23 amino acids. In one embodiment, X1 is an amino acid selected from histidine, lysine, and arginine (preferably selected from lysine and arginine). In one embodiment, X2 is an amino acid selected from serine, threonine, and cysteine, or an amino acid selected from phenylalanine, tyrosine, and tryptophan (preferably selected from serine and tyrosine). In one embodiment, X3 is an amino acid selected from valine, leucine, isoleucine, methionine, or proline (preferably selected from methionine or leucine). In one embodiment, the amino acid substitution is a conservative substitution (as described elsewhere herein).

[0193] In one embodiment, the anti-CXCR4 antibody or antigen-binding fragment described herein comprises a V CXCR4 antibody or V CXCR4 antibody comprising a CDRL3 containing the motif LQX1NX2YPX3T. Ldomain, wherein X1, X2, and X3 are independently any amino acid. In one embodiment, X1 is an amino acid selected from alanine or glycine, or an amino acid selected from serine, threonine, or cysteine. In one embodiment, X2 is an amino acid selected from asparagine or glutamine, or an amino acid selected from histidine, lysine, or arginine. In one embodiment, X3 is an amino acid selected from tyrosine, tryptophan, or phenylalanine, or an amino acid selected from valine, leucine, isoleucine, methionine, or proline. In one embodiment, the amino acid substitution is a conservative substitution (as described elsewhere herein).

[0194] In one embodiment, the anti-CXCR4 antibody or antigen-binding fragment described herein competes for binding to CXCR4 with an antibody or antigen-binding fragment selected from CL-82574, CL-82458, CL-82558, CL-82658, CL-83083, CL-83083-2, CL-82583, CL-82580, CL-82577, CL-82571, CL-82562, CL-82556, CL-82551, CL-82541, CL-82523, CL-82517, CL-82495, CL-82473, CL-82455, CL-83158, CL-148712, and CL-148729 (particularly CL-82574 or CL-83083-2). Antibodies CL-82574, CL-82458, CL-82558, CL-82658, CL-83083, CL-83083-2, CL-82583, CL-82580, CL-82577, CL-82571, CL-82562, CL-82556, CL-82551, CL-82541, CL-82523, CL-82517, CL-82495, CL-82473, CL-82455, CL-83158, CL-148712, and CL-148729 are as described herein above and have any of the CDRs, variable regions, and / or full-length heavy and light chain sequences as described. In one embodiment, competition is determined by SPR as described elsewhere herein. Such competition can be due, for example, to antibodies or antigen-binding fragments that bind to the same or overlapping epitopes of CXCR4. In another embodiment, competition is determined by ELISA, HTRF, fluorescence-activated cell sorting (FACS), or such techniques readily apparent to one of skill in the art, and are described elsewhere herein. In one embodiment, competition is determined by biolayer interferometry (BLI), such techniques readily apparent to one of skill in the art. In one embodiment, the antibody or antigen-binding fragment competes (e.g., dose-dependently) with CXCL12 (or a fusion protein thereof) for binding to cell surface-expressed CXCR4.

[0195] In one embodiment, the anti-CXCR4 antibody or antigen-binding fragment described herein binds to an epitope (the same as) that specifically binds an antibody or antigen-binding fragment selected from CL-82574, CL-82458, CL-82558, CL-82658, CL-83083, CL-83083-2, CL-82583, CL-82580, CL-82577, CL-82571, CL-82562, CL-82556, CL-82551, CL-82541, CL-82523, CL-82517, CL-82495, CL-82473, CL-82455, CL-83158, CL-148712, and CL-148729 (particularly CL-82574 or CL-83083-2). In one embodiment, epitopes are identified by site-directed mutagenesis (eg, alanine scanning), overlapping peptide scanning (eg, pepscan), or X-ray crystallography. In one embodiment, an antibody or antigen-binding fragment is provided that specifically binds to an epitope that is (substantially) similar (or identical) to the epitope specifically bound by any of the following antibodies: CL-82574, CL-82458, CL-82558, CL-82658, CL-83083, CL-83083-2, CL-82583, CL-82580, CL-82577, CL-82571, CL-82562, CL-82556, CL-82551, CL-82541, CL-82523, CL-82517, CL-82495, CL-82473, CL-82455, CL-83158, CL-148712, and CL-148729. Contact amino acid residues involved in the interaction between an antibody or antigen-binding fragment and an antigen can be determined by various methods known to those skilled in the art. In one embodiment, amino acids of the antigen sequence (using standard molecular biology techniques to mutate the DNA of the coding sequence of the antigen), in this case CXCR4, can be sequentially substituted with alanine (also known as alanine scanning) or another unrelated amino acid to provide residues whose mutations reduce the ability of the antibody or antigen-binding fragment to recognize the antigen in question. In one embodiment, binding is assessed using standard techniques such as, but not limited to, SPR, HTRF, ELISA (described elsewhere herein).Other substitutions can be made to enhance disruption of binding, such as altering the charge on the side chain of an amino acid in the antigen sequence (e.g., changing lysine to glutamic acid) or switching polar and nonpolar residues (e.g., changing serine to leucine). Alanine scanning or other amino acid substitution methods can be performed directly on cells using transient or stable expression of the mutant versions. When alanine scanning or other amino acid substitution methods are performed in either ELISA or HTRF, an amino acid residue is identified as contributing to the epitope if the signal is reduced by at least 25% or 30%. In one embodiment, the signal reduction is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60%. In one embodiment, the signal reduction is at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%. When alanine scanning or other amino acid substitution methods are performed with SPR, amino acid residues are identified as contributing to the epitope if they are at least 10-fold, at least 15-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or at least 100-fold more likely to contribute to the epitope. In one embodiment, X-ray crystallography is used to determine contact residues between an antibody or antigen-binding fragment and the antigen (i.e., to determine the epitope to which the antibody or antigen-binding fragment binds), where crystallography allows direct visualization of contact residues involved in the antibody-antigen interaction, and optionally, X-ray crystallography uses a thermostabilized receptor. In one embodiment, if the antibody or antigen-binding fragment recognizes a linear epitope, short peptides based on the antigen sequence can be generated, and binding of the antibody or antigen-binding fragment to these peptides can be assessed using standard techniques (e.g., SPR, HTRF, or ELISA). Further investigation of the epitope can be provided by performing an alanine scan on any peptides that show binding.Instead of linear peptides, conformational scans (including overlapping peptide scans) could be performed using Pepscan technology (http: / / www.pepscan.com / ), using chemical conjugation of peptides on scaffolds that have been used to determine discontinuous epitopes on CD20-targeting antibodies (Niederfellner, Gerhard, et al., "Epitope characterization and crystal structure of GA101 provide insights into the molecular basis for type I / II distinction of CD20 antibodies.", Blood, 118.2, (2011), 358-367.).

[0196] In one embodiment, a polypeptide (e.g., CXCR4) is a monomer. In another embodiment, a polypeptide (e.g., CXCR4) forms a homodimer with another identical polypeptide. In another embodiment, a polypeptide (e.g., CXCR4) forms a heterodimer with a different polypeptide (e.g., CXCR1, CXCR2, CXCR3, CXCR5, CXCR6, CXCR7, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CB2, ADCYAP1R1, ADORA2B, ADORA3, ADRB2, APLNR, C5AR1, CALCR, CHRM1, GALR1, EDNRB, HRH1, MLNR, NTSR1, PTGER2, PTGER3, SSTR2, or TACR3). In one embodiment, a polypeptide (e.g., CXCR4) forms a heterodimer with CXCR7 or CXCR3. In another embodiment, the polypeptide (e.g., CXCR4) forms a heterodimer with CCR5 or CB2. In another embodiment, the polypeptide (e.g., CXCR4) forms a trimer or tetramer. In another embodiment, the polypeptide (e.g., CXCR4) forms a multimer, which is an association of three or more polypeptides. A multimer can consist of multiple identical or different polypeptides, or a combination of both (e.g., multiple CXCR4 proteins, or two or more CXCR4 proteins and one or more CXCR7 proteins). Dimerization or oligomerization can affect receptor structure or function (e.g., signal transduction, signaling profiles). In one embodiment, dimerization can be determined by a proximity-based assay selected from the group consisting of bimolecular fluorescence complementation (BiFC), proximity ligation assay (PLA), fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), cysteine ​​bridging, and co-immunoprecipitation.

[0197] In one embodiment, the anti-CXCR4 antibody or antigen-binding fragment described herein binds (specifically or additionally) to a CXCR4 homodimer. In one embodiment, the antibody or antigen-binding fragment binds (specifically or additionally) to a CXCR4 monomer. In one embodiment, the antibody or antigen-binding fragment binds (specifically or additionally) to a CXCR4 multimer. In one embodiment, the antibody or antigen-binding fragment binds (specifically or additionally) to a CXCR4 heterodimer. In one embodiment, the heterodimer is a CXCR4 and CXCR7 heterodimer. In another embodiment, the heterodimer comprises CXCR4 and CXCR3 or CXCR4 and CXCR2. In another embodiment, the heterodimer comprises CXCR4 and another GPCR selected from CXCR1, CXCR2, CXCR5, or CXCR6. In another embodiment, the heterodimer comprises CXCR4 and CCR5. In another embodiment, the antibody or antigen-binding fragment binds (specifically or additionally) to CXCR4 in a complex with a β-arrestin.

[0198] In one embodiment, a nucleic acid encoding the CDRH3 of an anti-CXCR4 antibody or antigen-binding fragment thereof described herein is provided.

[0199] In one embodiment, the V of an anti-CXCR4 antibody or antigen-binding fragment thereof described herein H Domain and / or V L Nucleic acids encoding the V domains of the present invention are provided. H and V L The domain nucleic acid sequences are provided in the sequence listing.

[0200] In one embodiment, a nucleic acid is provided that comprises a nucleotide sequence that is at least 80%, 85% or 90% identical to the following sequence: a. SEQ ID NO: 19 and / or SEQ ID NO: 29; b. SEQ ID NO: 39 and / or SEQ ID NO: 49; c. SEQ ID NO: 59 and / or SEQ ID NO: 69; d. SEQ ID NO: 79 and / or SEQ ID NO: 89; e. SEQ ID NO: 99 and / or SEQ ID NO: 109; f. SEQ ID NO: 99 and / or SEQ ID NO: 111; g. SEQ ID NO: 115 and / or SEQ ID NO: 119, h. SEQ ID NO: 123 and / or SEQ ID NO: 127; i. SEQ ID NO: 131 and / or SEQ ID NO: 135, j. SEQ ID NO: 139 and / or SEQ ID NO: 143, k. SEQ ID NO: 147 and / or SEQ ID NO: 151, l. SEQ ID NO: 155 and / or SEQ ID NO: 159, m. SEQ ID NO: 163 and / or SEQ ID NO: 167, n. SEQ ID NO: 171 and / or SEQ ID NO: 175, o. SEQ ID NO: 179 and / or SEQ ID NO: 183; p. SEQ ID NO: 187 and / or SEQ ID NO: 191, q. SEQ ID NO: 195 and / or SEQ ID NO: 199, r. SEQ ID NO: 203 and / or SEQ ID NO: 207; s. SEQ ID NO: 211 and / or SEQ ID NO: 215; t. SEQ ID NO: 219 and / or SEQ ID NO: 223; u. SEQ ID NO: 227 and / or SEQ ID NO: 231, or v. SEQ ID NO: 235 and / or SEQ ID NO: 239.

[0201] In one embodiment, nucleic acids encoding the heavy and / or light chains of an anti-CXCR4 antibody or antigen-binding fragment thereof described herein are provided.

[0202] In any of the embodiments herein, the nucleic acid sequence is at least 70%, 75%, 80%, 85%, 90%, 91%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to a designated sequence (e.g., as defined by a SEQ ID NO:). In any of the embodiments herein, the nucleic acid is an isolated and purified nucleic acid.

[0203] In one embodiment, a vector is provided comprising a nucleic acid described herein, optionally wherein the vector is a CHO or HEK293 vector.

[0204] In one embodiment, a host cell is provided comprising a nucleic acid described herein or a vector described herein. In one embodiment, the host cell is a mammalian cell. In one embodiment, the host cell is a CHO (Chinese Hamster Ovary) cell (e.g., a CHO-S cell) or a HEK (Human Embryonic Kidney) cell (e.g., a HEK293 or HEK293T cell).

[0205] 3. Use of Antibodies therapeutic Any of the anti-CXCR4 antibodies or antigen-binding fragments described herein are useful for therapeutic modulation of the CXCL12 / CXCR4 pathway and therefore for the treatment or prevention of a CXCR4-mediated disease or condition.

[0206] Thus, in one embodiment, there is provided an anti-CXCR4 antibody or antigen-binding fragment described herein for use in therapy. In one embodiment, there is provided use of an anti-CXCR4 antibody or antigen-binding fragment described herein in the manufacture of a medicament for therapy. In another embodiment, there is provided an anti-CXCR4 antibody or antigen-binding fragment described herein for use in treating or preventing a CXCR4-mediated disease or condition as described herein below.

[0207] In another embodiment, there is provided the use of an anti-CXCR4 antibody or antigen-binding fragment described herein in the manufacture of a medicament for treating or preventing a CXCR4-mediated disease or condition described herein below.

[0208] In another embodiment, there is provided a method for treating or preventing a CXCR4-mediated disease or condition described herein below in a patient, comprising administering to said patient a therapeutically effective amount of an anti-CXCR4 antibody or antigen-binding fragment described herein, whereby the CXCR4-mediated disease or condition is treated or prevented.

[0209] In another embodiment, there is provided a pharmaceutical composition comprising an anti-CXCR4 antibody or antigen-binding fragment described herein for use in therapy or in the treatment or prevention of a CXCR4-mediated disease or condition described herein below.

[0210] Throughout this specification, a CXCR4-mediated disease or condition is any disease or condition involving aberrant regulation of the CXCR4 pathway (eg, CXCR4 signaling, downstream effects of CXCR4 and / or overexpression of CXCR4).

[0211] In a preferred embodiment, the CXCR4-mediated disease or condition is cancer, particularly a solid tumor. + It is characterized by the presence of T cells, eg, T cells that are unable to infiltrate the tumor (eg, the central tumor mass).

[0212] In one embodiment, the CXCR4-mediated disease or condition is one or more diseases or conditions independently selected from the following: small cell lung cancer, non-small cell lung cancer (e.g., adenocarcinoma, squamous cell carcinoma, and large cell carcinoma), breast cancer, ovarian cancer (e.g., metastatic ovarian cancer), renal cancer, gastric cancer, pancreatic cancer, pancreatic ductal carcinoma, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, cerebral cancer, esophageal cancer, liver cancer, lung cancer, prostate cancer, uterine cancer, brain cancer. cancer), bladder cancer, sarcoma, adenocarcinoma, multiple myeloma, melanoma, glioma, astrocytoma (Grade I - pilocytic astrocytoma, Grade II - low-grade astrocytoma, Grade III - anaplastic astrocytoma or Grade IV - glioblastoma (GBM)), medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, neurofibrosarcoma, osteosarcoma, meningioma, neuroblastoma, retinoblastoma, chordoma, CNS lymphoma, brainstem glioma, mixed glioma, optic nerve Transependymal glioma, subependymoma, meningioma, metastatic brain tumor, oligodendroglioma, pituitary tumor, primitive neuroectodermal (PNET) tumor, juvenile pilocytic astrocytoma (JPA), pineal tumor, rhabdoid tumor, and human papillomavirus (HPV)-associated cancer (HPV-positive tumors and / or lesions include cervical intraepithelial neoplasia 1, cervical intraepithelial neoplasia 2, cervical intraepithelial neoplasia 3, cervical carcinoma (intraepithelial and / or invasive), head and neck cancer, HPV papilloma, anal papillomatosis, anal cancer, vaginal cancer, vulvar cancer, and / or penile cancer).

[0213] In another embodiment, the CXCR4-mediated disease or condition is a cancer selected from carcinoma, sarcoma, myeloma, leukemia, and / or lymphoma. In one embodiment, the CXCR4-mediated disease or condition is a carcinoma, e.g., adenocarcinoma and / or squamous cell carcinoma. In one embodiment, the CXCR4-mediated disease or condition is an adenocarcinoma (which develops in an organ or gland), e.g., one or more adenocarcinomas selected from colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, and pancreatic cancer. In one embodiment, the CXCR4-mediated disease or condition is a squamous cell carcinoma, e.g., one or more cancers selected from melanoma, cervical cancer, head and neck cancer, vaginal cancer, thyroid cancer, esophageal cancer, lung cancer, penile cancer, bladder cancer, liver cancer, and anal cancer. In one embodiment, the CXCR4-mediated disease or condition is a sarcoma, e.g., one or more sarcomas selected from bladder cancer, rhabdomyosarcoma, glioma, and astrocytoma (e.g., Grade I-pilocytic astrocytoma, Grade I-low-grade astrocytoma, Grade II-low-grade astrocytoma, or Grade IV-glioblastoma (GBM)). In another embodiment, the CXCR4-mediated disease or condition is a myeloma, e.g., multiple myeloma. In another embodiment, the CXCR4-mediated disease or condition is a lymphoma, e.g., Hodgkin's lymphoma and / or non-Hodgkin's lymphoma. In another embodiment, the CXCR4-mediated disease or condition is a leukemia, e.g., one or more lymphomas selected from myeloid leukemia, granulocytic leukemia, lymphocytic leukemia, lymphocytic leukemia, lymphoblastic leukemia, polycythemia vera, and erythrocyte malignancies. In another embodiment, the CXCR4-mediated disease or condition is a mixed cancer (e.g., the cancer comprises two or more different cancers), such as one or more mixed cancers selected from adenosquamous carcinoma, decidual mesothelial tumor, carcinosarcoma, and teratocarcinoma.

[0214] In one embodiment, the CXCR4-mediated disease or condition is one or more diseases or conditions independently selected from the following: ovarian cancer (e.g., metastatic ovarian cancer), pancreatic cancer, glioblastoma (GBM), and primitive neuroectodermal (PNET) tumor. In one embodiment, the CXCR4-mediated disease or condition is one or more diseases or conditions independently selected from the following: ovarian cancer (e.g., metastatic ovarian cancer), pancreatic cancer, glioblastoma (GBM), primitive neuroectodermal (PNET) tumor, small cell lung cancer, non-small cell lung cancer, breast cancer, renal cancer, pancreatic ductal carcinoma, and colorectal cancer. In one embodiment, the CXCR4-mediated disease or condition is one or more diseases or conditions independently selected from the following: pancreatic cancer, pancreatic ductal carcinoma, prostate cancer, and small cell lung cancer. In one embodiment, the CXCR4-mediated disease or condition is one or more diseases or conditions independently selected from the following: pancreatic cancer, pancreatic ductal carcinoma, prostate cancer, small cell lung cancer, glioma, melanoma, head and neck cancer, non-small cell lung cancer, colorectal cancer, and esophageal cancer.

[0215] In one embodiment, the CXCR4-mediated disease or condition is one or more diseases or conditions independently selected from the following: warts, hypogammaglobulinemia, infections and myeloid cellular pooling (also known as WHIM), Waldenstrom's macroglobulinemia, human immunodeficiency virus (HIV), thrombocytopenia, ischemic heart failure, recovery after myocardial infarction, allogeneic transplantation after bone marrow rupture, bronchiolitis obliterans, neutropenia, malaria, opioid-induced hyperalgesia, rheumatoid arthritis, genetic disorders of the blood, such as sickle cell anemia, hemophilia A, hemophilia B, alpha-thalassemia, beta-thalassemia, delta-thalassemia, von Willebrand's disease, pernicious anemia, Fanconi anemia, thrombocytopenic purpura, thrombophilia, any and all primary immunodeficiency diseases. In one embodiment, the CXCR4-mediated disease or condition includes any of the genetic disorders of the blood, such as sickle cell anemia, hemophilia A, hemophilia B, α-thalassemia, beta-thalassemia, delta-thalassemia, von Willebrand disease, pernicious anemia, Fanconi anemia, thrombocytopenic purpura, idiopathic pulmonary fibrosis, and thrombophilia. In a preferred embodiment, the CXCR4-mediated disease or condition is WHIM or HIV.

[0216] In another embodiment, the anti-CXCR4 antibodies or antigen-binding fragments described herein are used for stem cell mobilization (with or without granulocyte colony-stimulating factor) for the treatment of severe ischemic diseases, including myocardial infarction, limb ischemia, ischemic stroke, and acute kidney injury. In one embodiment, the CXCR4-mediated disease or condition is allogeneic transplantation after myeloablative transplantation.

[0217] In another embodiment, the anti-CXCR4 antibodies or antigen-binding fragments disclosed herein are used in methods of internal radiation therapy. In another embodiment, the anti-CXCR4 antibodies or antigen-binding fragments disclosed herein are used in methods of wound healing.

[0218] In another embodiment, the CXCR4-mediated disease or condition includes a CXCL12-mediated disease or condition. CXCL12-mediated diseases or conditions include any disease or condition involving aberrant regulation of the CXCL12 pathway (e.g., CXCL12 signaling, downstream effects of CXCL12, and / or overexpression of CXCR12). CXCL12-mediated diseases or conditions include, but are not limited to, one or more selected from the following: germ cell tumors and metastases (e.g., testicular germ cell tumors), pregnancy-related disorders (e.g., pre-eclampsia), and adult-onset Still's disease.

[0219] In another embodiment, the anti-CXCR4 antibodies or antigen-binding fragments disclosed herein are used in combination with radiation therapy to reduce or treat residual tumors, e.g., the antibodies or antigen-binding fragments are administered after radiation therapy (e.g., 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, or 2 weeks). In one embodiment, the anti-CXCR4 antibodies or antigen-binding fragments disclosed herein are used in combination with radiation therapy to reduce or prevent angiogenesis and / or tumor recurrence. This is achieved by targeting CXCR4 to irradiated tumors (by radiation therapy). + Cells (e.g., CXCR4 +This can be achieved by reducing or preventing the infiltration of immune cells, such as macrophages or T cells. In one embodiment, the residual tumor is a residual tumor mass. In one embodiment, the tumor is any tumor listed in Section 3. Use of antibodies. In a preferred embodiment, the tumor is glioblastoma and / or cervical cancer. Macrophage exclusion after radiation therapy (MERT) in glioblastoma is further described in Thomas et al. (2019). Macrophage Exclusion after Radiation Therapy (MERT): A First in Human Phase I / II Trial using a CXCR4 Inhibitor in Glioblastoma. Clin Cancer Res. 25(23):6948-6957.

[0220] In one embodiment, there is provided a method for inhibiting CXCR4 activity in a patient or in vitro, comprising administering an effective amount of an anti-CXCR4 antibody or antigen-binding fragment described herein.

[0221] In one embodiment, there is provided a method of reducing the size or volume of a (malignant) tumor in a patient, comprising administering to said patient an effective amount of an anti-CXCR4 antibody or antigen-binding fragment described herein.

[0222] In one embodiment there is provided a method or use as described above, further comprising administering (to a patient) a further therapy, optionally wherein the further therapy comprises: a) immune checkpoint inhibitors (anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-TIM-3 antibodies, anti-CTLA-4 antibodies, anti-TIGIT antibodies, anti-BTLA antibodies, anti-VISTA antibodies, and anti-LAG-3 antibodies, etc.); b) immunostimulants (anti-OX40 antibody, anti-HVEM antibody, anti-CD27 antibody, anti-CD28 antibody, anti-CD137L antibody, anti-OX40L antibody, anti-GITRL antibody, anti-ICOSL antibody, anti-PD-L2 antibody, anti-GITR antibody, anti-CD137 antibody, anti-ICOS antibody and anti-CD40 antibody, etc.), c) anti-CSF1R antibody, anti-CCR4 antibody, anti-CD39 antibody, anti-CD73 antibody, anti-CD96 antibody, anti-CXCR2 antibody, anti-CD200 antibody, anti-GARP antibody, anti-SIRPα antibody, anti-CXCL9 antibody, anti-CXCL10 antibody, anti-CXCL11 antibody and anti-CD155 antibody, d) chemokine receptor antagonists (such as CCR4 and CXCR2 chemokine receptors); e) somatocin receptor, EP2 / EP4, A2AR, CD39, CD73 receptor antagonists; f) targeted kinase inhibitors (such as CSF-1R, EGFR or VEGFR inhibitors); g) angiogenesis inhibitors (such as anti-VEGF-A or delta-like ligand-4); h) immunostimulatory peptides or chemokines (such as CXCL9 or CXCL10); i) Cytokines (e.g. IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL_12, IL-13, IL_14, IL-15, IL-16, IL -17, IL-18, IL-19, IL-20 and IL-21, especially IL-2, IL-12, IL-15, IL-21) and interferons (such as IFN-α, IFN-β and IFN-γ, especially IFN-γγ), j) bispecific T cell engagers with at least one specificity for CD3 (e.g., CD3 / CD19 BiTE) and NK cell engagers (e.g., CD16); k) other bispecific molecules (e.g., IL-2- or IL-15-containing molecules targeting tumor-associated antigens such as epidermal growth factor receptor, e.g., EGFR, Her-2, New York esophageal carcinoma-1 (NY-ESO-1), GD2, EpCAM, or melanoma-associated antigen-3 (MAGE-A3)); l) oncolytic viruses (e.g., HSV virus (optionally secreting GMCSF), Newcastle disease virus, vaccinia virus, adenovirus, NCD virus (paramyxovirus), Sindbis virus, respiratory syncytial virus, measles virus, coxsackievirus, vesicular stomatitis virus, mengovirus, reovirus, parvovirus, maraba virus (rhabdovirus) and echovirus), m) vaccination with tumor-associated antigens (e.g., New York esophageal carcinoma-1 [NY-ESO-1], melanoma-associated antigen-3 [MAGE-3], BCMA); n) cell-based therapies (e.g., anti-CD19 expressing chimeric antigen receptor-T cells (CAR-T), chimeric antigen receptor natural killer cells (CAR-NK), anti-EpCam, anti-VEGF / VEGFR or anti-mesothelin, etc.); o) adoptive transfer of tumor-specific T cells (e.g., autologous tumor-infiltrating lymphocytes (TILs)) or LAK cells, and p) Tumor-associated antigen antibodies (anti-CEA, anti-GD2, anti-glypican-3, anti-glypican-2, anti-nectin-4, anti-mesothelin, etc.) and / or optionally, the additional treatment is selected from chemotherapy, radiation therapy, and / or surgical removal of the tumor.

[0223] In one embodiment, the chemokine receptor antagonist is an antagonist of the adenosine pathway (e.g., an antibody that targets the adenosine pathway). In another embodiment, the additional therapeutic agent is selected from anti-CD47, anti-SIRPα, anti-KIR, anti-CD70, and anti-GARP antibodies. In another embodiment, the additional therapeutic agent is selected from anti-HER2, anti-CD20, anti-NKG2A, anti-TRAIL, and anti-Notch antibodies. In another embodiment, the additional therapeutic agent is selected from anti-PVRIG, anti-ILDR2, and anti-CD55 antibodies. In one embodiment, the additional therapeutic agent is combined with an additional therapy selected from radiation therapy, chemotherapy, and / or an antibody drug conjugate (ADC). In another embodiment, the additional therapeutic agent is a small molecule selected from an IDO inhibitor, a PARP inhibitor, a BTK inhibitor, a MAPK inhibitor, a STING agonist, a TLR agonist (e.g., TLR7 / 9, TLR8), an A2aR antagonist, a CXCR2 antagonist, a STAT3 transcription factor inhibitor, an EGFR inhibitor, a BRAF inhibitor, a Wee1 tyrosine kinase inhibitor, an FGFR-1 / 3 inhibitor, and / or a PDGFR inhibitor. In one embodiment, the additional therapeutic agent is selected from ImmTac, antisense / siRNA / shRNA, ProTAC, and a prodrug conjugate.

[0224] In one embodiment, a method or use is provided herein that comprises administering to a patient (e.g., a human) an anti-CXCR4 antibody or antigen-binding fragment described herein in addition to chemotherapy or radiation therapy. In one embodiment, a method or use is provided herein that comprises administering to a patient an anti-CXCR4 antibody or antigen-binding fragment described herein before or after administration of chemotherapy and / or radiation therapy, wherein the antibody or antigen-binding fragment enhances the efficacy of chemotherapy and / or radiation therapy (compared to when the antibody or antigen-binding fragment is not administered). Administering an anti-CXCR4 antibody or antigen-binding fragment as described herein after administration of chemotherapy and / or radiation therapy can be beneficial because cancer cells may be sensitized to the treatment.

[0225] In a preferred embodiment, the additional therapeutic agent is a PD-1 / PD-L1 signaling inhibitor (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody). In one embodiment, the additional therapy is a combination of a checkpoint inhibitor (as described above) and chemotherapy. In one embodiment, the additional therapeutic agent is a combination of two checkpoint inhibitors (as described above).

[0226] In one embodiment, an additional therapeutic agent is provided that comprises an antibody or antigen-binding fragment that comprises an antigen-binding site that specifically binds to an antigen, as detailed below.

[0227] In one embodiment, the additional therapeutic agent is an anti-PD-L1 antibody or antigen-binding fragment thereof that specifically binds to PD-L1, e.g., hPD-L1. In one embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof is atezolizumab / MPDL3280A (Roche), avelumab / MSB0010718C (Merck), BMS-936559 / MDX-1105 (BMS), durvalumab / Medi4736 (Medimmune), KN-035, CA-170, FAZ-053 any one of anti-PD-L1 antibodies selected from M7824, ABBV-368, LY-3300054, GNS-1480, YW243.55.S70, and REGN3504, and the compounds disclosed in WO 2019 / 129136, WO 2019 / 129211, WO 2019 / 132533, WO 2019 / 072869, and WO 2019 / 075097 Brochure, International Publication No. 2019 / 085238, International Publication No. 2019 / 040780, International Publication No. 2019 / 005634, International Publication No. 2019 / 005635, International Publication No. 2019 / 005636, International Publication No. 2019 / 005637, International Publication No. 2019 / 005638, International Publication No. 2019 / 005639 , International Publication No. 2019 / 005642, International Publication No. 2019 / 005641, International Publication No. 2019 / 005640, International Publication No. 2018 / 222949, International Publication No. 2018 / 194496, International Publication No. 2018 / 195226, International Publication No. 2018 / 162430, International Publication No. 2018 / 162446 , International Publication No. 2018 / 162749 pamphlet, International Publication No. 2018 / 181064 pamphlet, International Publication No. 2018 / 153320 pamphlet, International Publication No. 2018 / 133873 pamphlet, International Publication No. 2018 / 136553 pamphlet, International Publication No. 2018 / 119475 pamphlet, International Publication No. 2018 / 080812 pamphlet, International Publication No. 2018 / 054940 pamphlet,International Publication No. 2018 / 034225, International Publication No. 2018 / 009894, International Publication No. 2018 / 024237, International Publication No. 2018 / 026249, International Publication No. 2018 / 005682, International Publication No. 2017 / 072280, International Publication No. 2017 / 215590, International Publication No. 2017 / 072273, International Publication No. 2017 / 218435, International Publication No. 2017 / 196867, International Publication No. 2 International Publication No. 017 / 197667, International Publication No. 2017 / 148424, International Publication No. 2017 / 118321, International Publication No. 2017 / 132562, International Publication No. 2017 / 097407, International Publication No. 2017 / 084495, International Publication No. 2017 / 087547, International Publication No. 2017 / 091429, International Publication No. 2017 / 034916, International Publication No. 2017 / 020291, International Publication No. 2017 / 02 International Publication No. 0858, International Publication No. 2017 / 020802, International Publication No. 2017 / 020801, International Publication No. 2016 / 111645, International Publication No. 2016 / 050721, International Publication No. 2016 / 197367, International Publication No. 2016 / 061142, International Publication No. 2016 / 149201, International Publication No. 2016 / 000619, International Publication No. 2016 / 160792, International Publication No. 2016 / 022630 Brochure, International Publication No. 2016 / 007235 pamphlet, International Publication No. 2015 / 036499 pamphlet, International Publication No. 2015 / 179654 pamphlet, International Publication No. 2015 / 173267 pamphlet, International Publication No. 2015 / 181342 pamphlet, International Publication No. 2015 / 109124 pamphlet, International Publication No. 2015 / 195163 pamphlet, International Publication No. 2015 / 112805 pamphlet, International Publication No. 2015 / 061668 pamphlet, International Publication No. 2014 / 159562 pamphlet,and any of the PD-L1 antibodies described in WO2014 / 165082, WO2014 / 100079, WO2014 / 055897, WO2013 / 181634, WO2013 / 173223, WO2013 / 079174, WO2012 / 145493, WO2011 / 066389, WO2010 / 077634, WO2010 / 036959, WO2010 / 089411 or WO2007 / 005874 (which antibodies are incorporated herein by reference).

[0228] In one embodiment, the additional therapeutic agent is an anti-PD-1 antibody or antigen-binding fragment thereof that specifically binds to PD-1, eg, hPD-1. In one embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof is selected from the group consisting of pembrolizumab (Keytruda™ / MK-3475), nivolumab (Opdivo™ / BMS-936558 / MDX-1106), MEDI-0680 / AMP514, PDR001, lambrolizumab, BMS-936558, REGN2810, BGB-A317, BGB-108, PDR-001, SHR-1210, JS-001, JNJ-63723283, AGEN-2034, PF-06801591, genolimzumab, MGA-012, IBI-308, BCD-100, and TSR-042. ANA011, AUNP-12, KD033, MCLA-134, mDX400, muDX400, STI-A1110, AB011, 244C8, 388D4, XCE853 or pidilizumab / CT-011, or from WO 2015 / 112800 and U.S. Patent No. 2015 / 0203579 (including antibodies in Tables 1 to 3), U.S. Patent No. 9,394,366 No. 5, U.S. Pat. Nos. 5,897,862 and 7,488,802, WO 2017 / 087599 (including antibodies SSI-361 and SHB-617), WO 2017 / 079112, WO 2017 / 071625 (deposit C2015132, hybridoma LT004 and antibodies 6F5 / 6 F5(Re), 6F5H1 L1 and 6F5 H2L2), International Publication No. 2017 / 058859 (including PD1AB-1 to PD1AB-6), International Publication No. 2017 / 058115 (including 67D9, c67D9 and hu67D9), International Publication No. 2017 / 055547 (including 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375 and 13112.15380), International Publication No. 2017 / 040790 (AGEN2033w, AGEN2034w, AGEN2046w,AGEN2047w, AGEN2001w and AGEN2002w), WO 2017 / 025051 and WO 2017 / 024515 (1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.103.11-v2 hAb, 1.139.15 hAb and 1.153.7 hAb), WO 2017 / 025016 and WO 2017 / 024465 (including Antibody A to Antibody I), WO 2017 / 020858 and WO 2017 / 020291 (including 1.4.1, 1.14.4, 1.20.15, and 1.46.11), WO 2017 / 019896 and WO 2015 / 112900, and U.S. Patent No. 2015 / 0210769 (including BAP049-hum01 to BAP049-hum16 and BAP049-Clone-A to BAP049-Clone-E), WO 2017 / 019846 (PD-1 mAb 1 to PD-1 mAb 15), WO 2017 / 016497 (including MHC723, MHC724, ​​MHC725, MHC728, MHC729, m136-M13, m136-M19, m245-M3, m245-M5 and m136-M14), WO 2016 / 201051 (antibody EH12.2H7, antibody hPD-1 mAb2, antibody hPD-1 mAb7, antibody hPD-1 mAb9, antibody hPD-1 mAb15 or an anti-PD-1 antibody selected from Table 1), WO 2016 / 197497 (including DFPD1-1 to DFPD1-13), WO 2016 / 197367 (including 2.74.15 and 2.74.15.hAb4 to 2.74.15.hAb8), WO 2016 / 196173 (including antibodies in Table 5 and Figures 1 to 5), WO 2016 / 127179 (including R3A1, R3A2, R4B3 and R3D6), WO 2016 / 077397 (including antibodies described in Table 1 of Example 9), WO 2016 / 106159 (including the mouse antibodies in Table 3 of Example 2 and Table 7 of Example 3,WO 2016 / 092419 (including humanized antibodies C1, C2, C3, EH12.1, mAb7-G4, mAb15-G4, mAb-AAA, mAb15-AAA), WO 2016 / 068801 (including clone A3 and its variants and other antibodies described in Figures 1 to 4), WO 2016 / 014688 (including 10D1, 4C10, 7D3, 13F1, 15H5, 14A6, 22A5, 6E1, 5A8, 7A4 and 7A4D and the humanized antibodies of Examples 9 / 10). antibody), WO 2016 / 015685 (including 10F8, BA08-1, BA-08-2, and 15H6), WO 2015 / 091911 and WO 2015 / 091910 (including the anti-canine PD-1 antibodies of Examples 2, 3, and 4), WO 2015 / 091914 (including the anti-canine PD-1 antibodies of Table 3), WO 2015 / 085847 (including mAb005, H005-1 to H005-4), WO 2015 / 058573 (including cAB7), WO 2015 / 036394 (including LOPD180), WO 2015 / 035606 (including the antibodies of Table 1 in Example 2, Tables 14, 15 and 16 in Example 7, and Tables 20, 21 and 22 in Example 11), WO 2014 / 194302 (including GA2, RG1B3, RG1H10, RG2A7, RG2H10, SH-A4, RG4A6, GA1, GB1, GB6, GH1, A2, C7, H7, SH-A4, SH-A9, RG1H11 and RG6B ... Publication No. 2014 / 179664 (including 9A2, 10B11, 6E9, APE1922, APE1923, APE1924, APE1950, APE1963, and APE2058), WO 2014 / 206107 (including clones 1, 10, 11, 55, 64, 38, 39, 41, and 48), WO 2012 / 135408 (including h409A11, h409A16, and h409A17), WO 2012 / 145493 (including antibodies 1E3, 1E8, 1H3, and h1H3 Var 1 to h1H3 Var 14),No. WO 2011 / 110621 (including antibody 949 and modified versions disclosed in Figures 1 to 11), WO 2011 / 110604 (including antibody 948 and modified versions disclosed in Figures 3 to 11), WO 2010 / 089411 (including CNCM accession numbers 1-4122, 1-4080 or 1-4081), WO 2010 / 036959 (including the antibodies in Table 1 of Example 1), WO 2010 / 029435 and WO 2010 / 029434 (including clones 2, 10 and 19), WO 2008 / 156712 (hPD-1.08A, hPD-1.08B, hPD-1.08C, hPD-1.08D, hPD-1.08E, hPD-1.08F, hPD-1.08G, hPD-1.08H ... and any one of the anti-PD-1 antibodies selected from any one of the anti-PD-1 antibodies described in WO 2006 / 121168 (including clones 17D8, 4H1, 5C4, 4A11, 7D3, 5F4, and 2D3), WO 2004 / 004771, or WO 2004 / 056875 (including PD1-17, PD1-28, PD1-33, PD1-35, PD1-F2, and the Abs described in Table 1) (the sequences and characteristics of the anti-PD-1 antibodies are incorporated herein by reference).

[0229] In one embodiment, the anti-CXCR4 antibodies or antigen-binding fragments described herein are used in combination with a PD-1 or PD-L1 signaling inhibitor (such as an anti-PD-1 antibody or antigen-binding fragment or an anti-PD-L1 antibody or antigen-binding fragment) for use in treating or preventing a CXCR4-mediated disease or condition (as described herein), wherein one, more or all of a-i apply: a. Increased susceptibility of cancer cells to the host immune response; b. Reduced immunosuppression in tumors; cT cells induce apoptosis in tumors; d. Increased cancer cell recognition within the tumor; e. Cancer cell growth is inhibited; f. Elimination of cancer cells, g. A reduction in tumor mass; h. the tumor contains FAP+ stromal cells, or i. The tumor is resistant to immunotherapy.

[0230] Yet another embodiment includes a method of treating a proliferative or invasion-related disease in a mammal by administering to the animal a therapeutically effective dose of an anti-CXCR4 antibody or antigen-binding fragment described herein. Without being bound by theory, this may be achieved by inhibiting CXCL12-driven T cell exclusion from the tumor and / or stroma, or by inhibiting tumor suppressor cells (e.g., myelin-derived suppressor cells or T-regs) from the tumor and / or stroma. Thus, in one embodiment, any of the anti-CXCR4 antibodies or antigen-binding fragments described herein reduce metastatic invasion and adhesion. In another embodiment, the anti-CXCR4 antibodies or antigen-binding fragments described herein reduce tumor angiogenesis. In another embodiment, the anti-CXCR4 antibodies or antigen-binding fragments described herein reduce tumor cell proliferation and chemotherapy resistance.

[0231] detection In another embodiment, the antibodies or antigen-binding fragments can be used to detect the presence, absence, and / or level of CXCR4 expression in a sample. CXCR4 expression can be detected in vivo and / or in vitro and is useful for diagnosing diseases or conditions associated with CXCR4 expression and / or overexpression. In another embodiment, the anti-CXCR4 antibodies or antigen-binding fragments described herein are used to detect diseases or conditions, for example, for imaging (i.e., PET).

[0232] Patient Selection Guide In one embodiment, detection of CXCR4 expression using any of the anti-CXCR4 antibodies or antigen-binding fragments described herein can be used to guide patient selection. In one embodiment, the anti-CXCR4 antibodies or antigen-binding fragments thereof described herein can be used to assist in patient selection for therapeutic antibody treatment with anti-CXCR4 antibodies or antigen-binding fragments. In some cases, higher levels of CXCR4 may indicate successful therapy, while lower levels may indicate a decreased likelihood of success. Preferential expression of splice variants and / or protein processing may generate unique protein mixture profiles that may affect a patient's response to therapy or change after treatment. These profiles may help identify patients and define patient subsets that should receive therapy, continue therapy, or receive alternative therapies. In another embodiment, antibodies or antigen-binding fragments thereof can be used to detect CXCR4 isoforms. Patient samples may include, for example, blood, plasma, serum, sputum, saliva, urine, CSF, tears, exhaled exogenous particle samples, cell supernatants, cell or tissue lysates, or tissue samples.

[0233] 4. Pharmaceutical Compositions In one embodiment, a pharmaceutical composition is provided comprising an effective amount of an anti-CXCR4 antibody or antigen-binding fragment described herein and a pharmaceutically acceptable excipient, diluent, or carrier. The effective amount of an antibody described herein to be used therapeutically will depend, for example, on the therapeutic objectives, route of administration, and condition of the patient. In one embodiment, the composition includes other excipients or stabilizers.

[0234] Pharmaceutically acceptable carriers are known and include carriers that are non-toxic to cells or mammals exposed to the cells or mammals at the dosages and concentrations used. Often, physiologically acceptable carriers are aqueous pH-buffered solutions. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as ethylenediaminetetraacetic acid (EDTA); sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0235] The anti-CXCR4 antibodies or antigen-binding fragments described herein or pharmaceutical compositions described herein can be administered intravenously, through the nose or lungs, for example, as a liquid or powder aerosol (lyophilized). The antibodies, antigen-binding fragments, or compositions can also be administered parenterally or subcutaneously. When administered systemically, the compositions must be sterile, pyrogen-free, and in a physiologically acceptable solution, with due consideration given to pH, isotonicity, and stability. These conditions are known to those skilled in the art.

[0236] Methods of administering a prophylactic or therapeutic agent (e.g., an anti-CXCR4 antibody or antigen-binding fragment described herein) or pharmaceutical composition include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes). In certain embodiments, a prophylactic or therapeutic agent (e.g., an anti-CXCR4 antibody or antigen-binding fragment described herein) or pharmaceutical composition is administered intranasally, intramuscularly, intravenously, or subcutaneously, particularly intravenously. The prophylactic or therapeutic agent or composition may be administered by any convenient route, such as infusion or bolus injection, absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, intranasal mucosa, rectal mucosa, intestinal mucosa, etc.), and may be administered together with other therapeutic or biologically active agents. Administration may be systemic or local. Each dose may or may not be administered by the same route of administration. In one embodiment, the anti-CXCR4 antibodies or antigen-binding fragments thereof described herein may be administered via multiple routes of administration simultaneously or subsequent to other doses of the same or different anti-CXCR4 antibodies or antigen-binding fragments thereof described herein.

[0237] A variety of delivery systems are known and can be used to administer a prophylactic or therapeutic agent (e.g., an anti-CXCR4 antibody or antigen-binding fragment thereof described herein), including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or antigen-binding fragment, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retrovirus or other vector, etc. In addition, pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer and formulation with an aerosolizing agent. See, e.g., U.S. Patent Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078, and PCT Publication Nos. WO 92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346, and WO 99 / 66903, each of which is incorporated herein by reference in its entirety.

[0238] In certain embodiments, it may be desirable to administer a prophylactic or therapeutic agent or pharmaceutical composition described herein locally to the area in need of treatment. This can be achieved, for example, by local infusion, topical administration (e.g., intranasal spray), injection, or implant, where the implant is comprised of a porous, non-porous, or gelatinous material, including membranes such as sialastic membranes or fibers. When administering a prophylactic or therapeutic agent (e.g., an anti-CXCR4 antibody or antigen-binding fragment described herein), care should be taken to use a material to which the antibody or antigen-binding fragment does not absorb.

[0239] In one embodiment, a pharmaceutical composition is provided comprising an anti-CXCR4 antibody or antigen-binding fragment described herein and a pharmaceutically acceptable excipient, diluent, or carrier, and further comprising one or more (e.g., two or more, or e.g., two) additional therapeutic agents independently selected from the group consisting of: a) immune checkpoint inhibitors (anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-TIM-3 antibodies, anti-CTLA-4 antibodies, anti-TIGIT antibodies, anti-BTLA antibodies, anti-VISTA antibodies, and anti-LAG-3 antibodies, etc.); b) immunostimulants (anti-OX40 antibody, anti-HVEM antibody, anti-CD27 antibody, anti-CD28 antibody, anti-CD137L antibody, anti-OX40L antibody, anti-GITRL antibody, anti-ICOSL antibody, anti-PD-L2 antibody, anti-GITR antibody, anti-CD137 antibody, anti-ICOS antibody and anti-CD40 antibody, etc.), c) anti-CSF1R antibody, anti-CCR4 antibody, anti-CD39 antibody, anti-CD73 antibody, anti-CD96 antibody, anti-CXCR2 antibody, anti-CD200 antibody, anti-GARP antibody, anti-SIRPα antibody, anti-CXCL9 antibody, anti-CXCL10 antibody, anti-CXCL11 antibody and anti-CD155 antibody, d) chemokine receptor antagonists (such as CCR4 and CXCR2 chemokine receptors); e) somatocin receptor, EP2 / EP4, A2AR, CD39, CD73 antagonists; f) targeted kinase inhibitors (such as CSF-1R, EGFR or VEGFR inhibitors); g) angiogenesis inhibitors (such as anti-VEGF-A or delta-like ligand-4); h) immunostimulatory peptides or chemokines (such as CXCL9 or CXCL10); i) Cytokines (e.g. IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL_12, IL-13, IL_14, IL-15, IL-16, IL -17, IL-18, IL-19, IL-20 and IL-21, especially IL-2, IL-12, IL-15, IL-21) and interferons (such as iIFN-α, IFN-β and IFN-γ, especially IFN-γγ), j) bispecific T cell engagers with at least one specificity for CD3 (e.g., CD3 / CD19 BiTE) and NK cell engagers (e.g., CD16); k) other bispecific molecules (e.g., IL-2- or IL-15-containing molecules targeting tumor-associated antigens such as epidermal growth factor receptor, e.g., EGFR, Her-2, New York esophageal carcinoma-1 (NY-ESO-1), GD2, EpCAM, or melanoma-associated antigen-3 (MAGE-A3)); l) oncolytic viruses (e.g., HSV virus (optionally secreting GMCSF), Newcastle disease virus, vaccinia virus, adenovirus, NCD virus (paramyxovirus), Sindbis virus, respiratory syncytial virus, measles virus, coxsackievirus, vesicular stomatitis virus, mengovirus, reovirus, parvovirus, maraba virus (rhabdovirus) and echovirus), m) vaccination with tumor-associated antigens (e.g., New York esophageal carcinoma-1 [NY-ESO-1], melanoma-associated antigen-3 [MAGE-3], BCMA); n) cell-based therapies (e.g., anti-CD19 expressing chimeric antigen receptor-T cells (CAR-T), chimeric antigen receptor natural killer cells (CAR-NK), anti-EpCam, anti-VEGF / VEGFR or anti-mesothelin, etc.); o) adoptive transfer of tumor-specific T cells or LAK cells; p) Tumor-associated antigen antibodies (such as anti-CEA, anti-GD2, anti-glypican-3, anti-glypican-2, anti-nectin-4, and anti-mesothelin), and q)Chemotherapy.

[0240] In one embodiment, a pharmaceutical composition as described herein or a kit comprising a pharmaceutical composition as described herein is provided, wherein the composition is for the treatment of, or for the treatment and / or prevention of, a CXCR4-mediated disease or condition as described above.

[0241] In one embodiment, a pharmaceutical composition or kit as described herein is provided in combination with a label or instructions for use in treating and / or preventing said CXCR4-mediated disease or condition in a patient as described herein, optionally wherein the label or instructions include a marketing approval number (e.g., an FDA or EMA approval number). Optionally, the label or instructions may be a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, the notice reflecting agency approval, e.g., an approval number, of the manufacture, use, or sale for human administration. In one embodiment, the kit comprises an anti-CXCR4 antibody or antigen-binding fragment described herein and instructions for administering the antibody or antigen-binding fragment to a patient in need of treatment. In one embodiment, the kit comprises an IV or injection device comprising said anti-CXCR4 antibody or antigen-binding fragment described herein. In one embodiment, the antibody or antigen-binding fragment is administered intravenously. In one embodiment, the antibody or antigen-binding fragment is administered subcutaneously. In one example, the anti-CXCR4 antibody or antigen-binding fragment described herein is contained in a medical container, such as a vial, syringe, IV container, or injection device (such as an intraocular or intravitreal injection device). In one example, the anti-CXCR4 antibody or antigen-binding fragment is in vitro, e.g., in a sterile container. In one embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, a composition for intravenous administration is a solution in sterile isotonic aqueous buffer. Optionally, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the injection site. However, such compositions may be administered by routes other than intravenous. Generally, the components of the composition are supplied separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. When the composition is administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical-grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.The additional therapeutic agent of the present disclosure may be delivered by any method, which methods are well known to those of skill in the art. For example, the additional therapeutic agent may be delivered orally, systemically, or locally (to the tumor environment). In one embodiment, the additional therapeutic agent is delivered orally. In one embodiment, the additional therapeutic agent is delivered systemically (e.g., intravenously). In one embodiment, the additional therapeutic agent is delivered locally to the tumor environment. In another embodiment, the anti-CXCR4 antibody and / or additional therapeutic agent is delivered locally to the site after surgical removal of all or a substantial portion of the tumor.

[0242] 5. Kits and Products In another embodiment, an article of manufacture is provided, comprising a container containing a composition comprising an antibody described herein and a package insert or label indicating that the composition can be used to treat a disease or condition characterized by CXCR4 expression or overexpression. In one embodiment, a kit for treating and / or preventing a CXCR4-mediated disease or condition is provided, comprising an anti-CXCR4 antibody or antigen-binding fragment disclosed herein in any embodiment or combination of embodiments herein (and optionally an additional therapeutic agent described elsewhere herein), optionally in combination with a label or instructions for treating and / or preventing the disease or condition in a human, optionally with a marketing authorization number (e.g., an FDA or EMA approval number), and optionally with an IV or injection device comprising the antibody or antigen-binding fragment. In another embodiment, the kit comprises the antibody or antigen-binding fragment contained in a container or IV bag. In another embodiment, the container or IV bag is a sterile container or IV bag. In another embodiment, the antibody or antigen-binding fragment is contained in a (sterile) container or formulated into a pharmaceutical composition contained in a (sterile) IV bag. In a further embodiment, the kit further comprises instructions for use.

[0243] 6. Additional Embodiments In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to CXCR4 is provided, wherein the antibody or antigen-binding fragment specifically binds to CD8 +It allows T cells to infiltrate tumors.

[0244] In one embodiment, a V that specifically binds CXCR4 and comprises CDRH3 H Antibodies or antigen-binding fragments thereof comprising the domain are provided, wherein the CDRH3 sequence is identical to the CDRH3 sequence of an antibody or antigen-binding fragment described herein or contains three, two, or one amino acid substitutions.

[0245] In one embodiment, a V receptor that specifically binds CXCR4 and comprises CDRH1, CDRH2, and CDRH3. H Antibodies or antigen-binding fragments thereof comprising the domain are provided, wherein the CDRH1-3 sequences are identical to or contain 3, 2 or 1 amino acid substitutions with the CDRH1-3 sequences of the antibodies or antigen-binding fragments described herein.

[0246] In one embodiment, the antibody specifically binds to CXCR4 and V H An antibody or antigen-binding fragment thereof comprising a V domain is provided. H A domain is a V domain of an antibody or antigen-binding fragment described herein. H It contains an amino acid sequence that is identical or at least 90% identical to the amino acid sequence.

[0247] In one embodiment, a V that specifically binds CXCR4 and comprises CDRL3 L Antibodies or antigen-binding fragments thereof comprising a domain are provided, wherein the CDRL3 sequence is identical to the CDRL3 sequence of an antibody or antigen-binding fragment described herein or contains three, two or one amino acid substitutions.

[0248] In one embodiment, a V receptor that specifically binds CXCR4 and comprises CDRL1, CDRL2, and CDRL3. L Antibodies or antigen-binding fragments thereof comprising a domain are provided, wherein the CDRL1-3 sequences are identical to the CDRL1-3 sequences of an antibody or antigen-binding fragment described herein or contain three, two or one amino acid substitutions.

[0249] In one embodiment, the antibody specifically binds to CXCR4 and VL An antibody or antigen-binding fragment thereof comprising a V domain is provided. L A domain is a V domain of an antibody or antigen-binding fragment described herein. L It contains an amino acid sequence that is identical or at least 90% identical to the amino acid sequence.

[0250] In one embodiment, the antibody specifically binds to CXCR4 and V H Domain and V L An antibody or antigen-binding fragment thereof comprising a V domain is provided. H A domain is a V domain of an antibody or antigen-binding fragment described herein. H an amino acid sequence identical or at least 90% identical to the amino acid sequence V L A domain is a V domain of an antibody or antigen-binding fragment described herein. L It includes amino acid sequences that are identical or at least 90% identical to the amino acid sequence.

[0251] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to CXCR4 is provided, comprising a heavy chain and a light chain, wherein the heavy chain amino acid sequence is identical or at least 90% identical to the heavy chain amino acid sequence of an antibody or antigen-binding fragment described herein, and the light chain amino acid sequence is identical or at least 90% identical to the light chain amino acid sequence of an antibody or antigen-binding fragment described herein.

[0252] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to CXCR4 and competes with an antibody or antigen-binding fragment described herein for binding to CXCR4 is provided.

[0253] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to CXCR4 is provided, wherein the antibody or antigen-binding fragment binds to the same epitope as the antibody or antigen-binding fragment specifically bound by the antibody or antigen-binding fragment described herein.

[0254] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to CXCR4 is provided, wherein the antibody or fragment binds to T cells (optionally CD8 +It does not induce apoptosis in T cells.

[0255] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to CXCR4 is provided, wherein the antibody or fragment has an EC 50 It binds to cynomolgus monkey CXCR4.

[0256] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to CXCR4 is provided, wherein the antibody or antigen-binding fragment does not bind to CXCR7.

[0257] In one embodiment, there is provided an antibody or antigen-binding fragment thereof described herein for use in treating a CXCR4-mediated disease or condition described herein.

[0258] In one embodiment, there is provided the use of an antibody or antigen-binding fragment described herein in the manufacture of a medicament for treating a CXCR4-mediated disease or condition described herein.

[0259] In one embodiment, a method of treating a CXCR4-mediated disease or condition described herein in a patient is provided, comprising administering to the patient (e.g., a human) a therapeutically effective amount of an antibody or antigen-binding fragment described herein, thereby treating the CXCR4-mediated disease or condition.

[0260] In one embodiment, a kit is provided that includes a pharmaceutical composition containing an anti-CXCR4 antibody or antigen-binding fragment described herein, wherein the composition is for treating a CXCR4-mediated disease or condition described herein.

[0261] In one embodiment, a nucleic acid sequence encoding an anti-CXCR4 antibody or antigen-binding fragment disclosed herein, or any portion thereof, is provided.

[0262] In one embodiment, a vector is provided that comprises a nucleic acid encoding an anti-CXCR4 antibody or antigen-binding fragment disclosed herein, or any portion thereof.

[0263] In one embodiment, a host cell is provided comprising a nucleic acid encoding an anti-CXCR4 antibody or antigen-binding fragment or any portion thereof disclosed herein, or a vector as described herein.

[0264] [Table 1]

[0265] [Table 2]

[0266] [Table 3]

[0267] [Table 4]

[0268] [Table 5]

[0269] [Table 6]

[0270] [Table 7]

[0271] [Table 8]

[0272] [Table 9]

[0273] Table 10

[0274] Table 11

[0275] Table 12

[0276] Table 13

[0277] Table 14

[0278] Table 15

[0279] Table 16

[0280] Table 17

[0281] Table 18

[0282] Table 19

[0283] Table 20

[0284] Table 21

[0285] Table 22

[0286] Table 23

[0287] Table 24

[0288] Table 25

[0289] Table 26

[0290] Table 27

[0291] Table 28

[0292] Table 29

[0293] Table 30

[0294] Table 31

[0295] Table 32

[0296] Table 33

[0297] Table 34

[0298] Table 35

[0299] Table 36

[0300] Table 37

[0301] Table 38

[0302] Table 39

[0303] Table 40

[0304] Table 41

[0305] Table 42

[0306] Table 43

[0307] Table 44

[0308] Table 45

[0309] Table 46

[0310] Table 47

[0311] Table 48

[0312] Table 49

[0313] Table 50

[0314] Table 51

[0315] [Table 52]

[0316] [Table 53]

[0317] [Table 54]

[0318] [Table 55]

[0319] [Table 56] [Example]

[0320] The following example describes the generation, characterization, and performance of anti-CXCR4 antibodies. The antibodies were generated using Kymouse™ (AKA IntelliSelect™ transgenic mice), a transgenic mouse platform capable of generating antibodies with human variable domains generated from human V(D) and J gene segments and mouse constant domains. The Kymouse™ (AKA IntelliSelect™ transgenic mice) system is described in Lee et al., "Complete humanization of the mouse immunoglobulin loci enables efficient therapeutic antibody discovery," Nature Biotechnology, 32(4):356-363, WO 2011 / 004192, WO 2011 / 158009, and WO 2013 / 061098. The Kymouse™ (AKA IntelliSelect™ transgenic mice) HK and HL strains were used in this project. In the HK strain, the immunoglobulin heavy chain loci and the light chain kappa loci are humanized, and in the HL strain, the immunoglobulin heavy chain loci and the light chain lambda loci are humanized.

[0321] Kymice™ (AKA IntelliSelect™ transgenic mice) were immunized with various types of CXCR4-overexpressing cell lines using a conventional 12-week cell-expressed antigen immunization schedule consisting of multiple boosts throughout each immunization (Table 1) to identify antibodies that bound to human CXCR4. Antibodies were subjected to primary and secondary screens to identify a lead panel, as further detailed below.

[0322] Unless otherwise specified, all proteins or peptides (eg, CXCR4, CXCR7 and CXCL12) are human.

[0323] Example 1 (A) Preparation of MEF, DC2.4, CHO-S, and HEK-293 cells expressing CXCR4 or StaR® CXCR4 or CXCR7 for immunization and screening Stably transfected cell lines were generated. Full-length DNA sequences encoding codon-optimized (for mammalian expression) CXCR4 (SEQ ID NO: 1) (histidine-tagged human CXCR4), cynomolgus monkey (cyno) CXCR4 (SEQ ID NO: 7), CXCR7 (SEQ ID NO: 4), and StaR® CXCR4 were ordered as synthetic DNA oligonucleotides and cloned into expression vectors under the control of a CMV or EF1 promoter flanked by 3′ and 5′ piggyBac-specific terminal repeats that promote stable integration into the cellular genome (see “A hyperactive piggyBac transposase for mammalian applications”; Yusa K., et al., Proc. Natl. Acad. Sci. USA., 108(4):1531-6, 2011 Jan 25). To generate CXCR4-expressing cell lines, human embryonic kidney-293 (HEK-293), Chinese hamster ovary-S (CHO-S) cells, mouse dendritic cells (DC2.4), and mouse embryonic fibroblast (MEF) cells were transfected using FreeStyle Max transfection reagent (Invitrogen) according to the manufacturer's instructions. To generate cynomolgus CXCR4, CXCR7, and StaR®CXCR4 cell lines, CMV promoter-driven cynomolgus CXCR4, CXCR7, and StaR®CXCR4 expression vectors were cotransfected into CHO-S cells using piggyBac transposase to generate cells that solely express the cynomolgus CXCR4, CXCR7, and StaR®CXCR4 antigens, respectively. CXCR4 or StaR® protein expression was assessed by flow cytometry using an anti-CXCR4-phycoerythrin (PE)-conjugated antibody (FAB173P, R&D Systems). Cynomolgus monkey CXCR4 expression was assessed by flow cytometry using an anti-CXCR4-PE-conjugated antibody 12G5 (R&D Systems). CXCR7 expression was assessed by flow cytometry using an anti-CXCR7-PE fluorophore-conjugated antibody 8F11-M16 (Biolegend).After evaluation of antigen expression, stable CXCR4-expressing HEK-293 and CHO-S cells and CXCR4 StaR® CHO-S cells were FACS-sorted for high CXCR4 expression. Stably expressing CXCR4 MEF cells were generated into clonal cell lines using serial dilution, and single clones were scaled up with high CXCR4 expression.

[0324] Example 2 - Immunization Kymouse™ (AKA IntelliSelect™ transgenic mice) HK and HL strains were immunized with CXCR4-expressing cells (HEK-293, DC2.4, or CHO) or StaR® DNA or StaR® protein (used alone or in combination for immunization) according to the regimens shown in Table 1. Table 1 details the Kymouse™ strains and immunogens used at each immunization step (prime and boost).

[0325] [Table 57]

[0326] Priming of KM173-B1 with CXCR4 StaR® DNA was achieved using hydrodynamic tail vein injection. The Sigma adjuvant system was used for KM157-B1 whole and KM173-B1 StaR® protein boosts. Sigma adjuvant was used only for KM161-B1 priming. The interval between immunizations and resting was usually 2-3 weeks. Serum from serial blood samples was analyzed for the presence of specific antibodies by flow cytometry, and titer data (when available) were used to select mice for B cell sorting.

[0327] Example 3 – Generation of B cell selection antigens and antibody detection and expression (A) Generation, purification, and Nanodisc integration of CXCR4 StaR® for B cell sorting. To generate a fluorescent sorting reagent for labeling and isolating B cells expressing the CXCR4-reactive B cell receptor, we generated, expressed, purified, and incorporated into Nanodiscs a CXCR4-stabilized receptor protein (StaR®) fused to green fluorescent protein (GFP). Thermostable CXCR4 variants were generated by site-directed mutagenesis (essentially as described in WO 2009 / 081136) and then cloned into an insect cell expression vector to generate recombinant proteins fused to green fluorescent protein (GFP, StaR®). The fluorescent StaR® proteins were then expressed in insect cells using a baculovirus expression system and purified in detergent using affinity and size-exclusion chromatography. The detergent-purified receptor was then incorporated into lipid-protein nanodiscs (see "Preparation Of Nanodisc Reconstitution Mixtures"; www.sigmaaldrich.com / technical-documents / articles / materialsscience / nanomaterials / nanodizc-protocols.html), and the detergent was removed to generate a receptor reagent compatible for use in live cells.

[0328] (B) Selection of B cells expressing anti-CXCR4 antibodies B cells expressing anti-CXCR4 antibodies were recovered from immunized mice using techniques substantially similar to those described in Example 1 of WO 2015 / 040401. Immunized mice were selected from each immunization campaign based on their titer response to CXCR4. Briefly, spleen and lymph node cells isolated from mice within the immunization regimen were stained with a mixture of fluorophore-conjugated antibodies and markers (CD19-PB, IgM-APC-Cy7, IgD-APC-Cy7, 7-AAD) and fluorescently tagged StaR® CXCR4 Nanodiscs-GFP to allow identification of cells of interest for fluorescence-activated cell sorting (FACS). Single cells were selected based on their forward and side scatter characteristics and analyzed by 7-AAD. +Dead cells were exc...

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to CXCR4, Ｈ domain, Ｈ The domain is i. a CDRH1 amino acid sequence of SEQ ID NO: 10 or 13; ii. a CDRH2 amino acid sequence of SEQ ID NO: 11 or 14; iii. The CDRH3 amino acid sequence of SEQ ID NO: 12 or 15 and the antibody or antigen-binding fragment thereof comprises V Ｌ domain, Ｌ The domain is i. the CDRL1 amino acid sequence of SEQ ID NO: 20 or 23; ii. the CDRL2 amino acid sequence of SEQ ID NO: 21 or 24, and iii. The CDRL3 amino acid sequence of SEQ ID NO: 22 or 25 An antibody or antigen-binding fragment thereof comprising:

2. The V Ｈ The domain comprises the amino acid sequence of SEQ ID NO: 16, and The V Ｌ The antibody or fragment of claim 1, wherein the domain comprises the amino acid sequence of SEQ ID NO:

26.

3. a) the CXCR4 is human (optionally selected from SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3); or b) said CXCR4 is rhesus monkey and / or cynomolgus monkey (optionally selected from SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9); or c) the antibody or fragment specifically binds to human, rhesus, cynomolgus and / or rodent CXCR4, optionally human and cynomolgus CXCR4; 3. The antibody or fragment of claim 1 or 2, optionally wherein the binding is determined by surface plasmon resonance (SPR), flow cytometry, live cell imaging, ELISA, or radioligand binding.

4. Constant region (C Ｈ and / or C Ｌ ), optionally including a) the above C Ｈ is (i) an IgG4 constant region, e.g., an IgG4-PE constant region (e.g., SEQ ID NO: 267 or SEQ ID NO: 305), or (ii) an IgG1 constant region, e.g., an IgG1 constant region comprising a mutation that reduces binding to an Fc-γ receptor and / or C1q compared to wild-type (e.g., SEQ ID NO: 249 or 307), and / or b) the above C Ｌ The antibody or fragment according to any one of claims 1 to 3, wherein is a kappa light chain constant region.

5. The antibody or fragment of any one of claims 1 to 4, wherein the antibody comprises a heavy chain and a light chain, and the heavy chain comprises the amino acid sequence of SEQ ID NO: 18, and the light chain comprises the amino acid sequence of SEQ ID NO:

28.

6. The antibody or fragment of any one of claims 1 to 5, which inhibits the binding of CXCL12 to CXCR4.

7. T cells (optionally CD8 ＋ 7. The antibody or fragment of claim 1, wherein the antibody or fragment does not induce apoptosis in T cells, and optionally apoptosis is determined using flow cytometry.

8. EC50 of 0.5-10 nM (e.g., 0.5-5 nM) ５０ 8. The antibody or fragment of any one of claims 1 to 7, which binds to cynomolgus CXCR4 at 200 ng / mL, and optionally cynomolgus CXCR4 binding is determined using flow cytometry.

9. K of 0.2 to 2 nM (e.g., 0.4 to 0.8 nM) Ｄ 9. The antibody or fragment of any one of claims 1 to 8, which binds to human CXCR4 at a binding affinity of 1 to 8, optionally wherein the binding affinity is determined using surface plasmon resonance (SPR).

10. 10. The antibody or fragment of any one of claims 1 to 9, which does not bind to CXCR7 (optionally, CXCR7 is human, and further optionally, selected from SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6), optionally wherein CXCR7 binding is determined using flow cytometry, radioligand binding, surface plasmon resonance (SPR), live cell imaging, or ELISA.

11. IC of 0.5-20 nM (e.g., 0.5-5 nM) ５０ 11. The antibody or fragment of any one of claims 1 to 10, wherein the antibody or fragment inhibits a CXCL12-mediated response of T cells at 200-250°C, said inhibition being determined using an in vitro label-free dynamic mass redistribution assay.

12. IC of 0.01 to 5 nM (e.g., 0.01 to 1 nM) ５０ CXCR4 against CXCL12 ＋ 12. The antibody or fragment of any one of claims 1 to 11, which inhibits T cell chemotaxis, said inhibition being determined using live cell imaging or flow cytometry.

13. Mean CD45 compared to PBS ＋ The antibody or fragment of any one of claims 1 to 12, which increases cell recruitment, optionally wherein recruitment is determined using flow cytometry.

14. The antibody or fragment of any one of claims 1 to 13, which binds to a CXCR4 homodimer.

15. CD8 ＋ The antibody or fragment of any one of claims 1 to 14, which enables T cells to infiltrate tumors.

16. An antibody or fragment according to any one of claims 1 to 15 for use in therapy.

17. 16. The antibody or fragment of any one of claims 1 to 15 for use in the treatment of cancer, optionally wherein the cancer is pancreatic cancer or pancreatic ductal carcinoma.

18. The antibody or fragment of any one of claims 1 to 15 for use in the treatment of solid tumors.

19. the treatment further comprises administering an additional therapy, optionally wherein the additional therapy comprises one or more additional therapeutic agents independently selected from the group consisting of an anti-PD-1 antibody or antigen-binding fragment thereof and an anti-PD-L1 antibody or antigen-binding fragment thereof; and / or Optionally, the further treatment is selected from chemotherapy, radiotherapy and / or surgical removal of the tumor.

20. 20. The antibody or fragment for use of any one of claims 16 to 19, further comprising administering an additional therapeutic agent that is a PD-1 / PD-L1 signaling inhibitor (e.g., a PD-1 antibody or antigen-binding fragment thereof, or a PD-L1 antibody or antigen-binding fragment thereof).

21. 16. A pharmaceutical composition comprising the antibody or fragment of any one of claims 1 to 15 and a pharmaceutically acceptable excipient, diluent or carrier, and optionally further comprising one or more additional therapeutic agents.

22. (a) V of the antibody or fragment according to any one of claims 1 to 15 Ｈ Domain and / or V Ｌ A domain, or (b) a nucleic acid encoding the heavy and / or light chain of the antibody or fragment of any one of claims 1 to 15.

23. 23. A vector comprising the nucleic acid of claim 22, optionally a CHO or HEK293 vector.

24. 24. A host cell comprising the nucleic acid of claim 22 or the vector of claim 23.

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