Antibodies

Abstract

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

Description

Field of the invention

[0001] Described herein are antibodies or antigen-binding fragments that specifically bind CXCR4 (C-X-C motif chemokine receptor 4), and their use in treating various diseases or conditions. BACKGROUND

[0003] CXCR4 (also known as C-X-C motif chemokine receptor 4, 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 is part of the chemokine receptor family. CXCR4 shares the common barrel structure with GPCRs and contains 7 transmembrane a-helices, 3 extracellular loops, 3 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).

[0004] GPCRs are the largest family of membrane receptors in humans and many other species. Furthermore, GPCRs are considered the largest family of approved drugs 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 have contributed to the widespread use of GPCR-targeted drugs, including their druggability, interaction with multiple types of chemokines and other ligands, and expression in the plasma membrane, allowing extracellular targeting with molecules such as antibodies. This has led to more than 35% of approved drugs targeting GPCRs and related 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 challenging.The crystal structure of CXCR4 was solved in 2010 and is the first crystal structure of a peptide GPCR (Wu et al., (2010), Zhu et al., (2013), both supra).

[0005] The endogenous ligand of CXCR4 is CXCL12, which is also known as SDF-1 (Uniprot ID: P48061). CXCL12 is a highly conserved chemokine with 99% homology between human CXCL12 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 isoforms of human CXCL12, such as alpha, beta, gamma, delta, epsilon, theta, and isoform 7. Additional ligands for CXCR4 have also been found, including macrophage migration inhibitory factor (IMF) (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).

[0006] CXCL12 binding promotes a three-dimensional CXCR4 conformation that favors G αi Proteolysis into a and βγ subunits. CXCL12 binding confers different cellular activities, such as inhibition of cAMP formation via inhibition of adenylyl cyclase activity, activation of phospholipase C (PLC)-β, production of diacylglycerol and inositol 1,4,5 triphosphate (IP3), which control intracellular Ca 2+release. While inhibiting adenylate cyclase, the receptor activates the NF-kβ, JAK-STAT and PI3K-AKT pathways and mTOR and JNK / p38MAPK, which modulate 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). Recruitment of β-arrestin desensitizes the receptor by internalization upon 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 Signalling and Receptor Internalization, and This Implicates Multiple Interaction Sites between β-Arrestin and CXCR4. J Biol Chem. 275(4):2479-2485).

[0007] CXCR4 is expressed in many organs of the body and is expressed highest on cells of the immune system, thus also found to be highly expressed in the bone marrow niche from which these mature immune cells originate (e.g. myeloid hematopoietic cells, erythroblasts, lymphocytes, monocytes and blast cells) (data from The Human Protein Atlas version 18.1, updated 15 / 11 / 2018 https: / / www.proteinatlas.org / ). CXCR4 is also found to be increased in endocrine-derived tissues such as adrenal and thyroid tissue. CXCR4 expression has been found on various cancer cells, such as breast, kidney, pancreatic, ovarian, endometrial, head and neck, colorectal, gastric and lung cancer cells (data from The Human Protein Atlas version 18.1, updated 15 / 11 / 2018 https: / / www.proteinatlas.org / ). CXCR4 is also involved in late-stage 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 CXC motif chemokine receptor 4 inhibits the proliferation, migration and angiogenesis of lung cancer cells. Oncol Lett. 16(3):3976-3982).

[0008] In addition to the role of CXCR4 in cancer, mutations in CXCR4 (primarily found in the C-terminus) are associated with WHIM syndrome (warts, hypogammaglobulinemia, infections, and myelokathexis) ((McDermott & Murphy (2019). WHIM syndrome: Immunopathogenesis, treatment and cure strategies. Immunol Rev. 287(1):91-102). Moreover, CXCR4 is a coreceptor for HIV-1 and has been studied 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

[0009] Antibodies or antigen binding fragments

[0010] Disclosed herein are antibodies and antigen-binding fragments thereof that specifically bind to CXCR4 (e.g., human CXCR4). In one embodiment, the antibody or its antigen-binding fragment specifically binds to surface-expressed CXCR4. In another embodiment, the antibody or its antigen-binding fragment inhibits the binding of CXCL12 to CXCR4. In one embodiment, the antibody or its antigen-binding fragment inhibits the binding of CXCL12 to human CXCR4.

[0011] In one aspect, the application 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 of each of which comprises 3, 2 or 1 amino acid substitution(s) of SEQ ID NO: 10 or 13; (ii) the CDRH2 amino acid sequence of SEQ ID NO: 11 or 14, or of each of which comprises 3, 2 or 1 amino acid substitution(s) of SEQ ID NO: 11 or 14; (iii) the CDRH3 amino acid sequence of SEQ ID NO: 12 or 15, or of each of which comprises 3, 2 or 1 amino acid substitution(s) of SEQ ID NO: 12 or 15; and wherein the antibody or antigen-binding fragment thereof comprises a VL domain, wherein the VL domain comprises: (i) the CDRL1 amino acid sequence of SEQ ID NO: 20 or 23, or of each of which comprises 3, 2 or 1 amino acid substitution(s) of SEQ ID NO: 20 or 23; (ii) the CDRL2 amino acid sequence of SEQ ID NO: 21 or 24, or of each of which comprises 3, 2 or 1 amino acid substitution(s) of SEQ ID NO: 21 or 24; and (iii) the CDRL3 amino acid sequence of SEQ ID NO: 22 or 25, or of each of which comprises 3, 2 or 1 amino acid substitution(s) of SEQ ID NO: 22 or 25.

[0012] In one embodiment, the VH domain comprises: (i) the CDRH1 amino acid sequence of SEQ ID NO: 10 or 13; (ii) the 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 VL domain comprises: (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, wherein each of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 comprises at most one amino acid substitution, optionally a conservative amino acid substitution.

[0013] In one embodiment, the VH domain comprises: (i) the CDRH1 amino acid sequence of SEQ ID NO: 10 or 13; (ii) the 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 VL domain comprises: (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, wherein at most one of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprises at most one amino acid substitution, optionally a conservative amino acid substitution.

[0014] In one embodiment, the VH domain comprises: (i) the CDRH1 amino acid sequence of SEQ ID NO: 10 or 13; (ii) the 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 VL domain comprises: (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.

[0015] In one embodiment, the VH domain comprises: (i) the CDRH1 amino acid sequence of SEQ ID NO: 10 or 13; (ii) the 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 VL domain comprises: (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, wherein at most one of 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; (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 at most one amino acid substitution, optionally a conservative amino acid substitution.

[0017] 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; (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 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.

[0018] 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; (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.

[0019] In one embodiment, the VH domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 16; and / or the VL domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 26.

[0020] In one embodiment, the VH domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 16; and / or the VL domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 26.

[0021] In one embodiment, the antibody or fragment is human, humanized, or chimeric.

[0022] 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).

[0023] In one embodiment, the antibody or fragment specifically binds to human, rhesus, cynomolgus, and / or rodent CXCR4, optionally human and cynomolgus CXCR4, optionally wherein the binding is determined by surface plasmon resonance (SPR), flow cytometry, live cell imaging, ELISA, or radioligand binding.

[0024] 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 IgGl constant region, such as an IgGl constant region comprising a mutation that reduces binding to Fc-gamma receptor and / or Clq 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.

[0025] In one embodiment, the antibody or fragment comprises a heavy chain and a light chain, and the heavy chain amino acid sequence comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 18, and / or the light chain amino acid sequence comprises an amino acid sequence that is 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.

[0026] In one embodiment, the antibody or fragment inhibits binding of CXCL12 to CXCR4.

[0027] In one embodiment, the antibody or fragment inhibits CXCL12-mediated inhibition of cAMP stimulated by forskolin with an IC50 of 1 to 100 nM (e.g., 1 to 30 nM), optionally wherein the CXCR4-dependent CXCL12-mediated inhibition of cAMP is determined using a forskolin-stimulated cAMP signaling assay.

[0028] In an embodiment, the antibody or fragment inhibits recruitment of β-arrestin to CXCR4 with an IC50 of 0.01 to 15 nM (e.g., 0.01 to 0.2 nM), optionally wherein β-arrestin recruitment is determined using a functional cell-based reporter assay.

[0029] In an embodiment, the antibody or fragment inhibits binding of CXCL12 to CXCR4 with an IC50 of 0.2 to 4 nM (e.g., 0.2 to 1 nM), optionally wherein CXCL12 inhibition is determined using an HTRF assay.

[0030] In an embodiment, the antibody or fragment does not induce apoptosis in T cells (optionally CD8+ T cells), optionally wherein apoptosis is determined using flow cytometry.

[0031] In an embodiment, the antibody or fragment binds to cynomolgus CXCR4 with an EC50 of 0.5 to 10 nM (e.g., 0.5 to 5 nM), optionally wherein cynomolgus CXCR4 binding is determined using flow cytometry.

[0032] In an 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), optionally wherein binding affinity is determined using surface plasmon resonance (SPR).

[0033] In an embodiment, the antibody or fragment does not bind to CXCR7 (optionally wherein 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.

[0034] In an embodiment, the antibody or fragment inhibits CXCL12-mediated T cell responses with an IC50 of 0.5 to 20 nM (e.g., 0.5 to 5 nM), wherein inhibition is determined in vitro using a label-free dynamic mass redistribution assay.

[0035] In an 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), wherein inhibition is determined using live cell imaging or flow cytometry.

[0036] In an embodiment, the antibody or fragment increases average CD45+ cell mobilization compared to PBS, optionally wherein mobilization is determined using flow cytometry.

[0037] In one embodiment, the antibody or fragment binds to a CXCR4 homodimer.

[0038] In one embodiment, the antibody or fragment enables CD8+ T cells to infiltrate a tumor.

[0039] One aspect of the application provides an antibody or antigen binding fragment as described herein for use in therapy.

[0040] Yet another aspect of the application 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.

[0041] Yet another aspect of the application provides an antibody or fragment as described herein for use in the treatment of a solid tumor.

[0042] In one embodiment, the treatment further comprises administration of a further therapy, optionally wherein the further therapy comprises one or more further 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 further therapy is selected from chemotherapy, radiotherapy and / or surgical resection of a tumor.

[0043] In one embodiment, the treatment further comprises administration of a further 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).

[0044] Yet another aspect of the application 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 further therapeutic agents.

[0045] Yet another aspect of the application provides a nucleic acid encoding (a) a VH domain and / or a VL domain of an antibody or fragment as described herein, or (b) a heavy chain and / or a light chain of an antibody or fragment as described herein.

[0046] Yet another aspect of the application provides a vector comprising a nucleic acid as described herein, optionally wherein the vector is a CHO or HEK293 vector.

[0047] Yet another aspect of the application provides a host cell comprising a nucleic acid as described herein or a vector as described herein. BRIEF DESCRIPTION OF DRAWINGS

[0049] 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. Related positive (human anti-CD20 antibody) controls. The graph shows the internalization rates at increasing concentrations of antibodies. mAb conc; antibody concentration used in the assay, nanomolar.

[0050] Figure 2 Figure 2. Binding of anti-CXCR4 antibodies (in IgG4-PE format), benchmark antibodies (in IgG4-PE format) and IgG4-PE isotype control antibodies to cynomolgus CXCR4. Representative data of anti-CXCR4 antibody binding to cynomolgus CXCR4 expressed on CHO cells as assessed by flow cytometry. Data are representative of four independent experiments. Binding is expressed as geometric (Geo) mean.

[0051] Figure 3 Figure 3. Anti-CXCR4 (in IgG4-PE format), benchmark (in IgG4-PE format), IgG4-PE isotype control, anti-CXCR7 mAb #11G8 and mouse IgGl isotype control antibodies do not bind to CXCR7. Representative data of anti-CXCR4 antibody binding to CXCR7 expressed on CHO cells as assessed by flow cytometry. Data are representative of two independent experiments. Binding is expressed as geometric (Geo) mean.

[0052] Figure 4 Figure 4. Mean percentage inhibition (± SD) obtained with anti-CXCR4 antibodies (in IgG4-PE format), benchmark antibodies (in IgG4-PE format) and IgG4-PE isotype control antibodies when incubated with Jurkat T cells stimulated to migrate towards 30 nM CXCL12. Data are representative of three independent experiments.

[0053] Figure 5 Figure 5. Summary of anti-CXCR4-mediated inhibition of primary naive T cell migration. This graph represents the mean (± S.D.) of the maximum migration percentage of primary naive T cells towards 6.25 nM CXCL12 when pre-treated with anti-CXCR4 antibodies (in IgG4-PE format) and IgG4-PE isotype control at different concentrations in 3 to 5 independent donors.

[0054] Figure 6Inhibition of primary naive T cells by benchmark anti-CXCR4 antibody. This graph represents the mean (± S.D.) of the percent of maximum migration of naive T cells towards 6.25 nM CXCL12 pre-treated with benchmark anti-CXCR4 antibody (in IgG4-PE format) and IgG4-PE isotype control at different concentrations for three independent donors.

[0055] Figure 7 Percentage of CCRF-HSB-2 infiltrating into IFNy-treated HT-29 tumor spheroids after treatment with anti-CXCR4 antibody (in IgG4-PE format) or IgG4-PE isotype control. Data represent five independent experiments (mean ± SD).

[0056] Figure 8 Mobilization of human CD45+ cells in NSG mice when treated with anti-CXCR4 antibody (in IgG4-PE format). Total number of human CD45+ cells detected in each blood sample well as recorded by flow cytometer. + +

[0057] Figure 9 Schematic representation of the proposed mechanism of action of T cell exclusion from tumors. Cancer-associated fibroblasts (CAFs) located in the stroma respond to local pro-tumoral conditions by secreting elevated levels of CXCL12. CXCR4 + T cells react to the elevated CXCL12 gradient and are prevented from following other less defined gradients (e.g. CXCL9, CXCL10 and / or CXCL11), which results in T cells remaining in the stroma. CXCL9, CXCL10 and / or CXCL11 gradients are believed to encourage T cells to enter the tumor and kill malignant tumor cells.

[0058] Figure 10 Inhibition of CXCR4 on T cells by anti-CXCR4 antibody prevents the cells from reacting to CXCL12 within the stroma. T cells are able to follow other T cell-related chemokine gradients (e.g. CXCL9, CXCL10 and / or CXCL11), which can allow T cells to successfully infiltrate into the tumor and subsequently kill tumor cells.

[0059] Figure 11 Combination treatment with anti-CXCR4 antibody and PD-1 inhibitor delays tumor growth compared to vehicle control. DETAILED DESCRIPTION

[0060] 1. DEFINITIONS

[0061] ​​Unless otherwise defined, scientific and technical terms used in this document shall have the meanings as commonly understood by one of ordinary skill in the art. Also, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0062] The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to mean "and" unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin phrase "exempli gratia," and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[0063] In the description and claims, the term "about" is used to indicate that exact values as used in, for example, amounts of ingredients in a composition, concentrations, volumes, treatment temperatures, treatment times, yields, flow rates, pressures, and like values, and ranges thereof, are not necessarily used in the practice of the application. The term "about" refers to, for example, a numerical value which can vary from the nominal by a margin that takes into account experimental error, or that the process, such as for manufacturing a compound, composition, concentrate, or use formulation, is variable over time. The term "about" also encompasses variations that occur due to aging of a formulation having a particular initial concentration or mixture, as well as variations that occur due to mixing or handling of a formulation having a particular initial concentration or mixture. When the term "about" is used, equivalents of such amounts are included in the claims.

[0064] As used herein, "administer" 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) that is present outside of the body into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. When a disease (or condition) or symptom thereof is to be treated, the substance is typically administered after the onset of the disease (or condition) or symptom thereof. When a disease (or condition) or symptom thereof is to be prevented, the substance is typically administered before the onset of the disease (or condition) or symptom thereof.

[0065] As used herein, the term "affinity," "binding affinity," or the like refers to the degree or tendency of two molecules to bind to each other. In one embodiment, affinity or binding affinity is designated as K D or EC 50The skilled person will be able to calculate these values from data generated in appropriate assays, for example surface plasmon resonance (SPR) (using a Biacore TM or using ProteOn XPR36 TM (Bio-Rad TM )), KinExA TM (Sapidyne Instruments, Inc), ForteBio Octet (Pall ForteBio Corp.), flow cytometry (e.g. Mirrorball TM fluorocytometer), live cell imaging, enzyme-linked immunosorbent assay (ELISA) or radioligand binding (preferably SPR, flow cytometry, live cell imaging, ELISA or radioligand binding); optionally 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 of binding of an individual paratope (antigen-binding site) on an antibody or antigen-binding fragment to an epitope (of an antigen, e.g. a receptor) or the strength of interaction of a receptor (e.g. CXCR4) and a ligand (e.g. CXCL12). In a preferred embodiment, 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, the term “K D ” means the equilibrium dissociation constant for a particular antibody-antigen interaction. “K a ” is the association rate constant (also known as K on ), and “K d ” is the dissociation rate constant (also known as K off ).

[0066] As used herein, the terms "antagonist," "antagonism," or 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 one or more binding partners with the target antigen. In the case of CXCR4, an antagonist (e.g., an antibody or its antigen-binding fragment) is able to antagonize the binding of a ligand (e.g., CXCL12) and prevent the biological effects of the ligand on the receptor (e.g., signal transduction of the ligand, conformational changes of the receptor, activation of the receptor, or interaction of the receptor with other cellular molecules). Known ligands for CXCR4 include CXCL12 (also known as SDF-1), ubiquitin, and MIF. Other CXCR4 ligands may not yet be determined. The functional activity of CXCR4 can be measured in cAMP assays, β-arrestin assays, CXCL12 inhibition assays, internalization assays, apoptosis assays, DMR assays, chemotaxis assays, T cell infiltration assays, migration assays, or disease-related models of cancer, 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 its antigen-binding fragment is an antagonist, such as a complete antagonist. In another embodiment, the antibody or antigen-binding fragment thereof is a partial antagonist.A partial inhibitor partially inhibits (ie, does not completely inhibit) the interaction of one or more binding partners with the target antigen.

[0067] The terms "antibody," "immunoglobulin," or "Ig" are 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 of the foregoing, through at least one antigen recognition site in 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, which is composed of four polypeptide chains: two light (L) chains and two heavy (H) chains. The five types of mammalian Ig heavy chain constant regions are further described below. There are two types of immunoglobulin light chains in mammals: λ and κ. 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 or antigen-binding fragment. The variable domains of the heavy and light chains may be referred to as "V H ” and “V L These domains are usually the most variable parts of an antibody (relative to other antibodies of the same class) and contain the antigen-binding site. H and V L Attached to the heavy chain (C H ) constant region and light chain (C L ) constant region.

[0068] The antibodies or antigen-binding fragments described herein can be oligoclonal, polyclonal, monoclonal (including full-length monoclonal antibodies), camelized, chimeric (e.g., mouse variable and human constant regions, or human variable and mouse constant regions), CDR-grafted, multispecific, bispecific, catalytic, humanized, human, fully human, anti-idiotypic, including antibodies and fragments, variants, fusions or derivatives thereof that can be labeled in soluble or bound form, as well as any other modified immunoglobulin comprising an antigen-binding site, as 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 can be 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 bound to other molecules, such as toxins, radioisotopes, etc.

[0069] The terms "antigen-binding domain," "antigen-binding region," "antigen-binding fragment," and like terms refer to a portion of an antibody that comprises amino acid residues (e.g., complementarity determining regions (CDRs)) that interact with an antigen and confer specificity and affinity for the antigen to the binding agent. The antigen-binding region can be derived from any animal species, such as rodents (e.g., mouse, rabbit, rat, or hamster), chicken, and human. Preferably, the antigen-binding region is of human origin. Throughout this specification, the term "fragment" in the context of an antibody is taken to mean an antigen-binding fragment of such an antibody.

[0070] 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')2 fragments, antibody fragments that exhibit the desired biological activity, disulfide-linked variable region (dsFv), dimeric variable region (diabody), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to 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, the antibodies and antibody fragments described herein can include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Papain digestion of antibodies produces two identical antigen binding fragments (also referred to as "Fab" fragments) and one "Fc" fragment, which has no antigen binding activity but does have the ability to crystallize. As used herein, "Fab" refers to a fragment of an antibody that includes one constant and one variable domain of each of the heavy and light chains. The term "Fc region" is used herein to define a 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 disulfides. The effector functions of antibodies are determined by sequences in the Fc region that are also recognized by the Fc receptors (FcR) found on certain types of cells. Digestion of antibodies with the enzyme, pepsin, results in F(ab')2 fragments in which the two arms of the antibody remain linked and contain both antigen binding sites. F(ab')2 fragments have the ability to cross-link antigen.

[0071] As used herein, "authorization number" or "marketing authorization number" refers to a number issued by a regulatory agency in deciding that a particular medical product and / or composition can be marketed and / or offered for sale within the jurisdiction of that agency. As used herein, "regulatory agency" refers to one of the agencies responsible for assessing the safety and efficacy of, for example, medical products and / or compositions, and controlling the sale / marketing of such products and / or compositions in a given area. The U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) are just two examples of such regulatory agencies. Other non-limiting examples can include SDA, MPA, MHPRA, IMA, ANMAT, Hong Kong Department of Health Drug Office, CDSCO, Medsafe, and KFDA.

[0072] The term "bispecific antibody" denotes an antibody comprising specificities for two target molecules. The term "bispecific antibody" includes formats such as bis-cognate antibody, DVD-Ig (see DiGiammarino et al., "Design and generation of DVD-Ig TMmolecules for dual-specific targeting”, Meth. Mo. Biol., 2012, 889, 145-156), mAb 2 (see WO2008 / 003103), FIT-Ig (see WO2015 / 103072), mAb-dAb, dock and 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, TandAb, scDiabody, scDiabody-CH3, diabody-CH3, triple body), Miniantibody, minibody, TriBi minibody, scFv-CH3KIH, scFv-CH-CL-scFv, F(ab')2-scFv, scFv-KIH, Fab-scFv-Fc, Tetravalent HCab, ImmTAC, in-hole knob, in-hole knob with common light chain, in-hole knob with common light chain and charge pair, charge pair, charge pair with 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 zybody. For a review of bispecific formats, see Spiess, C., et al., Mol. Immunol. (2015).

[0073] As used herein, the term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant), excipient, or vehicle administered with a therapeutic agent. Such pharmaceutical carriers can be sterile liquids such as water and oils, including oils of petroleum, animal, plant, 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. Saline solutions, as well as 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 adjuvants.

[0074] The term "CDR region" or "CDR" refers to the regions of an antibody or antigen binding fragment variable domain that are hypervariable in sequence and / or form structurally defined loops. Generally, the antigen binding site of an antibody includes six CDR regions: three in the V H (CDRH1, CDRH2, CDRH3) and three in the V L (CDRL1, CDRL2, CDRL3). These regions of the heavy and light chains of an antibody 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, which are the system designed by Chothia et al. (see Chothia, C. & Lesk, A. M., 1987, "Canonical structures for the hypervariable regions of immunoglobulins", J. Mol. Biol., 196, 901-917). Other systems for determining CDRs include 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 Pluckthun (2001). Yet another numbering scheme for immunoglobulin variable domains: an automatic modelling 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. Nucelic Acids Res. 40 (web server issue): W521-W524) numbering schemes. The term CDR is used herein to indicate one or more of these regions. A person skilled in the art is able to easily compare different naming systems and determine whether a particular sequence can be defined as a CDR.

[0075] The term "chemotherapeutic agent" or "chemotherapy" refers to a therapeutic agent whose primary purpose is to destroy cancer cells, usually by interfering with the ability of tumor cells to grow or multiply. There are multiple types of chemotherapeutic agents, with over 50 approved chemotherapy drugs. 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 and prevent cells from growing and multiplying. An example of an antimetabolite drug is 5-fluorouracil (5-FU). Antitumor antibiotics are made from natural substances in the soil, such as fungi. They interfere with important cellular functions, including the production of DNA and cellular proteins. Doxorubicin and bleomycin are in this class 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 cancers that rely on hormones. For example, tamoxifen is used to treat breast cancer that relies 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, for example, platinum therapy such as oxaliplatin. In one embodiment, the chemotherapy is a cytotoxic chemotherapy that is standard of care for the cancer being treated.

[0076] Examples of chemotherapeutic agents include Adriamycin, doxorubicin, 5-fluorouracil, Ara-C, cyclophosphamide, Thiotepa, Taxotere (docetaxel), Busulfan, a cytotoxin, Gemcitabine, Paclitaxel, doxorubicin, Taxol, methotrexate, cisplatin, Melphalan, vinblastine, bleomycin, Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincristine, Vinorelbine, Carboplatin, Teniposide, Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycin, Esperamicins (see U.S. Patent No. 4,675,187), Melphalan, and other related nitrogen mustards. Suitable toxins and chemotherapeutic agents are described in Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co., 1995), and Goodman and Gilman's The Pharmacological Basis of Therapeutics, 7th Ed. (MacMillan Publishing Co., 1985). Other examples of chemotherapeutic agents include Irinotecan (liposomal), which is a topoisomerase I inhibitor. Other suitable toxins and / or chemotherapeutic agents will be apparent to those skilled in the art. Leucovorin (folic acid) is used in combination with chemotherapeutic drugs to enhance the effectiveness of the drug or to act as a chemoprotective agent, e.g., Leucovorin in combination with 5-fluorouracil or methotrexate.

[0077] As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients (e.g., the antibodies or antigen-binding fragments of the application) in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts.

[0078] As used herein, the term "comprising" or "comprises" is used in reference to a composition, method, use, or process, that comprises an identified element or step. Use of "comprising" means "including, but not limited to," and is not intended to exclude, for example, additional conjugate species, conjugate species that are not specifically listed or processes that consist of steps in addition to those recited.

[0079] The term "consisting of' refers to an antibody, antigen binding fragment, use, composition, method, and respective components thereof as described herein, exclusive of any element not specified in the description of the embodiment.

[0080] The term "cross-react," "cross-reactivity," or like terms refers 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 targets and / or ligands described elsewhere herein) 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 to the desired target antigen (e.g., hCXCR4), as measured, e.g., by SPR. In another embodiment, an antibody or antigen binding fragment cross-reacts with a similar target if binding is greater than about 80% (e.g., 85%, 90%, 95%) of the binding of the antibody or antigen binding to the desired target antigen (e.g., hCXCR4) or binds the same.

[0081] In the context of polypeptides, the term "derivative" as used herein 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, which has been altered by the introduction of amino acid residue substitutions, deletions or additions. The term "derivative" as used herein 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 covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, 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 by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. The derivative is modified in a manner different from the naturally occurring or starting peptide or polypeptide, whether 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 a CXCR4 polypeptide or a CXCR4 antibody or antigen-binding fragment can be chemically modified by chemical modification using techniques known to those of skill in the art, including but not limited to specific chemical cleavage, acetylation, formulation, tunicamycin metabolic synthesis, etc. Also, derivatives of a CXCR4 polypeptide or an anti-CXCR4 antibody or antigen-binding fragment can contain one or more non-canonical amino acids. Polypeptide derivatives have similar or identical function as the CXCR4 polypeptide or anti-CXCR4 antibody or antigen-binding fragment described herein.

[0082] An "effective amount" refers to an amount effective to achieve a desired effect, including therapeutic or prophylactic results. A "therapeutically effective amount" refers to the minimum concentration needed to affect a measurable improvement or prophylactic effect on a particular disorder. A therapeutically effective amount herein can vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody or antigen-binding fragment to elicit a desired response in the individual. A therapeutically effective amount is also one in which toxic or detrimental effects of the antibody or antigen-binding fragment are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective to achieve a desired prophylactic result. In some embodiments, an effective amount of an antibody or antigen-binding fragment of the application 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, 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 a range therein). In some embodiments, as used herein, an "effective amount" also refers to the amount of an antibody or antigen-binding fragment of the application to achieve a particular result, e.g., inhibition of CXCR4 biological activity.

[0083] As used herein, the term "epitope" refers to a localized region on the surface of an antigen, such as a CXCR4 polypeptide, that is capable of binding to one or more antigen binding regions of an antibody or antigen binding fragment, and which has antigenic or immunogenic activity in an animal, preferably a mammal, most preferably in a human, capable of eliciting an immune response. An epitope that has immunogenic activity is a portion of a polypeptide that elicits an antibody response in an animal. An epitope that has antigenic activity is a portion of a polypeptide to which an antibody or antigen binding fragment specifically binds, as determined by any method well known in the art (e.g., by immunoassay), some of which are described herein. An antigenic epitope is not necessarily immunogenic. Epitopes are generally composed of chemical groups on the surface of a molecule, 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 a polypeptide, or the epitope can be from two or more non-contiguous regions of a polypeptide. An epitope can or can 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 can be a monomer, a dimer (e.g., a homo- or hetero-dimer), a trimer, a tetramer, or a multimer. In a preferred embodiment, a CXCR4 epitope is a three-dimensional surface feature of a monomeric or homo-dimeric CXCR4 polypeptide. In another embodiment, a CXCR4 epitope is a three-dimensional surface feature of a hetero-dimeric CXCR4 polypeptide, as further described herein. In other embodiments, a CXCR4 epitope is a linear feature of a monomeric or homo-dimeric CXCR4 polypeptide.

[0084] As used herein, the term "excipient" refers to an inert substance generally used as a diluent, vehicle, preservative, binder or stabilizer in a pharmaceutical, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (such as aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (such as sulfonic acid alkyl ester, octanoate, etc.), surfactants (e.g., SDS, polysorbate, non-ionic 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., the entire contents of which are incorporated herein by reference.

[0085] As used herein, the term "fusion protein" refers to a polypeptide comprising an amino acid sequence of an antibody or antigen binding fragment and an 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 a CXCR4 or anti-CXCR4 antibody, the term "fusion" refers to the linkage of a peptide or polypeptide, or fragment, variant and / or derivative thereof, to a heterologous peptide or polypeptide. Preferably, the fusion protein retains the biological activity of the CXCR4 or anti-CXCR4 antibody. In certain embodiments, the fusion protein comprises a CXCR4 antibody V H domain, V L domain, V H CDR (one, two or three V H CDRs) and / or V L CDR (one, two or three V L CDRs), wherein the fusion protein specifically binds to a CXCR4 epitope.

[0086] When used in reference to an antibody, the term "heavy chain" refers to the five different types, designated alpha, delta, epsilon, gamma, and mu, based on amino acid sequence of the constant domains of the heavy chains. These different types of heavy chains are well known and give rise to the five classes of antibodies, IgA, IgD, IgE, IgG, and IgM, including the 4 subclasses of IgG (i.e., IgGl, IgG2, IgG3, and IgG4), and the two subclasses of IgA (i.e., IgAl and IgA2), respectively. One 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. The heavy chain comprises a variable region (V H) and a heavy chain constant region. The numbering of amino acid positions in the heavy chain constant region in this specification is in accordance with the EU index (Kabat, E. A. (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).

[0087] As used herein, the term "host cell" or like term refers to a particular cell of interest that has been transfected with a nucleic acid molecule, as well as the progeny and potential progeny of such a cell. Progeny of such a cell can not be identical to the parent cell transfected with the nucleic acid molecule due to mutations that occur sometime during replication or due to the potential insertion 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.

[0088] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or has been made using any of the techniques for making human antibodies.

[0089] The term "humanized antibody" refers to a subset of chimeric antibodies in which "CDR regions" of a non-human immunoglobulin (donor antibody) replace residues in CDR regions of a human immunoglobulin (recipient antibody). Generally, a humanized antibody will include substantially all of one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the framework regions are those of a human immunoglobulin sequence, although the framework regions can include one or more substitutions to improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc.

[0090] The terms "IMGT numbering" and like terms are art-recognized and refer to a system of numbering amino acid residues for the purpose of comparing variable region sequences of immunoglobulins and T cell receptors (Lefranc, M.P., 1997, "Unique database numbering system for immunogenetic analysis", Immunol. Today, 18, 50). The IMGT numbering system relies on the high degree of conservation of variable region structure. The IMGT numbering system takes into account and incorporates the definition of framework regions (FRs) and complementarity determining regions (CDRs).

[0091] As used herein, the terms "inhibits", "inhibition", "inhibiting" and like terms refer to the ability of an antibody or antigen binding fragment to bind to an epitope that partially, substantially, or completely prevents a ligand (e.g., CXCL12) from binding to a receptor (e.g., CXCR4). If the epitope bound by the antibody or antigen binding fragment completely blocks the binding site for the ligand, then ligand binding is completely prevented (either by physical blocking (in the case of identical or overlapping epitopes) or steric blocking (where the antibody or antigen binding fragment is so large that it prevents the ligand from binding to its unique epitope)), and the ligand is not removed from circulation. Thus, the concentration of circulating ligand can appear to increase. If the epitope bound by the antibody or antigen binding fragment partially blocks the binding site for the ligand, then the ligand can be able to bind, but only weakly (in the case of partial inhibition), or in a different orientation than the native interaction. In this case, some ligand can be removed from circulation, but not as much as if the binding site for the ligand were completely free and available for binding. Thus, inhibition refers to the physical interaction of the ligand and the receptor. IC 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 in Mathis (1995) Clinical Chemistry 41(9), 1391-1397. Inhibition can also be measured by a time-resolved fluorescence energy transfer (TR-FRET) based cell-based ligand inhibition assay (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 “inhibition” is interchangeable with the term “neutralization.” In any embodiment herein, “inhibition” means that the % specific binding at a given concentration or the maximum % specific binding achieved is not substantially different from (or the same [within standard error] of) that achieved when no ligand (e.g., CXCL12) is provided in the assay. Thus, in one embodiment, inhibition provides a reduction in the % specific binding of a ligand (e.g., CXCL12) (at 50 nM, 30 nM, 10 nM, 1 nM, or 0.1 nM [antibody or antigen binding fragment]) in the range of 50-100%, 60-100%, 70-100%, 80-100%, e.g., 85-100%, or 90-100% compared to an isotype control. In another embodiment, inhibition provides a reduction in the % specific binding of a ligand (e.g., CXCL12) (at 50 nM, 30 nM, 10 nM, 1 nM, or 0.1 nM [antibody or antigen binding fragment]) in the range of 95-100% compared to an isotype control. In another embodiment, inhibition is a 100% reduction in the % specific binding of a ligand (e.g., CXCL12) compared to an isotype control. In another embodiment, inhibition provides a reduction in the maximum % specific binding of a ligand (e.g., CXCL12) in the range of 80-100%, e.g., 85-100%, or 90-100% compared to an isotype control. In another embodiment, inhibition provides a reduction in the maximum % specific binding of a ligand (e.g., CXCL12) in the range of 95-100% compared to an isotype control. In another embodiment, complete neutralization is a 100% reduction in the maximum % 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 a partial antagonist, etc.) or an inverse agonist.A reverse 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 that induced by the agonist (e.g., if the agonist decreases cAMP levels, the reverse agonist can increase cAMP levels). Reverse agonism can be exhibited when the receptor has constitutive activity, producing a pharmacological response in the absence of an agonist. A reverse agonist can decrease the pharmacological response of the receptor below the level of basal activity in the absence of an agonist. In one embodiment, an anti-CXCR4 antibody (e.g., a CXCR4 reverse agonist) can decrease the pharmacological response of the receptor via one pathway and can not affect other pathways by which the receptor signals. In another embodiment, an anti-CXCR4 antibody (e.g., a CXCR4 reverse agonist) can increase cAMP levels and also affect other CXCR4 signaling pathways compared to basal signaling of the receptor.

[0092] As used herein, an "injection device" refers to a device designed to perform an injection, which includes the step of temporarily fluidly connecting the injection device to a tissue of a human, typically subcutaneous tissue. An injection also includes the administration of an amount of a liquid medicament into the tissue and the separation or removal of the injection device from the tissue. In some embodiments, the injection device can be an intravenous device or IV device, which is a type of injection device used when the target tissue is blood within the circulatory system, e.g., blood intravenously. A common, but non-limiting example of an injection device is a needle and syringe.

[0093] As used herein, "instructions" refers to written, printed, or graphic content displayed on the immediate container of an article, such as written material displayed on a vial containing a pharmaceutically active agent, or detailed information about the composition and use of a product of interest housed in a kit containing a composition of interest. The instructions set forth the method of treatment intended to be administered or performed.

[0094] An "isolated" or "purified" antibody, antigen-binding fragment, protein, or nucleic acid is one which has been identified, separated and / or recovered from a component of its production environment (e.g., natural or recombinant). For example, an antibody, antigen-binding fragment, protein, or nucleic acid is substantially free of cell 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 chemical precursors or other chemicals when chemically synthesized. The phrase "substantially free of cellular material" includes preparations of an antibody, antigen-binding fragment, protein, or nucleic acid in which the desired protein is separated from the cellular components of the cells from which the protein is derived 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 protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as "contaminating protein"). When the antibody, antigen-binding fragment, protein, or nucleic acid is recombinantly produced, it is preferably also substantially free of culture medium, i.e., culture medium comprises less than about 20%, 10%, or 5% of the volume of the protein preparation. When the antibody, antigen-binding fragment, protein, or nucleic acid is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals involved in the synthesis of the protein. Thus, such preparations have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the desired material. In a preferred embodiment, the antibody or antigen-binding fragment of the application is isolated and / or purified.

[0095] As used herein, the term "isotype control" or similar terms is an antibody or antigen-binding fragment that is typically of the same species (or mixture of species in the case of a chimera), and preferably has the same constant region as the test antibody or antigen-binding fragment, but in which the V H domains and V L domains are not specific for the target antigen of interest (i.e., CXCR4), or are specific for an antigen other than the target antigen of interest (i.e., CXCR4), or are V H and V L non-binding combination for any other human target. When testing human variable-mouse constant chimeras, the isotype control can be a commercially available mouse IgGl isotype control (e.g., available from Sigma Aldrich, catalog number M9269). When testing fully human IgG4-PE format antibodies, the isotope control is preferably an antibody having human variable regions and human IgG4-PE constant regions. Those of skill in the art will be able to identify a suitable antibody as an isotype control.

[0096] The terms "Kabat numbering" and like terms are art-recognized and refer to a system of numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the heavy chain variable region of a comparison antibody or antigen-binding fragment 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, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable regions generally range from amino acid positions 31 to 35 of CDR1, amino acid positions 50 to 65 of CDR2, and amino acid positions 95 to 102 of CDR3.

[0097] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possibly naturally occurring mutations and / or post-translation modifications (e.g., isomerization, amidation) that can be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic determinant or epitope. In contrast, polyclonal antibody preparations typically include different antibodies directed against different antigenic determinants (or epitopes). The term "monoclonal antibody" as used herein encompasses both intact and full-length monoclonal antibodies (e.g., full-length, four-chain monoclonal antibodies), as well as antigen-binding fragments thereof, as described elsewhere herein. Moreover, "monoclonal antibody" refers to such antibodies made in a variety of ways including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals. Monoclonal antibodies of the application can include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical with a corresponding sequence in a antibody derived from a different species or belonging to a different antibody class or subclass, while the remainder of the chain(s) is identical to that of a antibody derived from a different species or belonging to a different antibody class or subclass, as well as fragments of such antibodies, that exhibit the desired biological activity.

[0098] The term "naturally occurring" or "native" when used in connection with biological material such as nucleic acid molecules, polypeptides, host cells, and the like, refers to those that are found in nature and have not been manipulated by the hand of man (also referred to as wild type). Naturally occurring CXCR4 ligands include CXCL12 (SDF-1), MIF, and ubiquitin.

[0099] As used herein, "packaging" refers to how components are organized and / or confined within a unit suitable for distribution and / or use. Packaging can include, for example, boxes, pouches, syringes, ampules, vials, tubes, clamshell packages, barriers and / or containers for maintaining sterility, labels, and the like.

[0100] The terms "percent identity," "percent amino acid sequence identity" (or "percent nucleotide sequence identity"), "percent identical to," "identity," and the like, in reference to a peptide, polypeptide, antibody or antigen binding fragment (or nucleotide) sequence, are defined as the percentage of amino acid residues (or nucleotide bases) in a candidate sequence that are identical with the amino acid residues (or nucleotide bases) in a specified peptide or polypeptide sequence, when the sequences are aligned for maximum comparison, and gaps introduced in the alignment of a sequence for optimal comparison are not considered as part of either sequence. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as Needleman-Wunsch (the NEEDLE program of Emboss), Basic Local Alignment Search Tool (BLAST™), BLAST-2, ALIGN or MEGALIGN TM (DNASTAR) software. In one embodiment, % 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, alignment can be achieved using Needleman-Wunsch (the NEEDLE program of Emboss), for example, using a BLOSUM60 matrix, with gap open of 10, and gap extend of 0.5.

[0101] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0102] As used herein, the terms "polynucleotide," "nucleotide," "nucleic acid," "nucleic acid molecule," and other like terms are used interchangeably and include DNA, RNA, mRNA, and the like.

[0103] As used herein, the terms "prevent," "preventing," and "prevention" refer to the complete or partial inhibition of the development, recurrence, onset, or spread of a CXCR4-mediated disease (or condition) and / or symptoms associated therewith due to the administration of a therapy or combination of therapies provided herein (e.g., a combination of prophylactic agents or therapeutic agents, such as an antibody or antigen-binding fragment of the application).

[0104] The term "radiotherapy" refers to a treatment that uses radiation (e.g., ionizing radiation) to kill cancer cells and / or reduce tumor mass. Radiotherapy can be used in combination with other therapeutic agents. Radiotherapy can be used in combination with chemotherapy. Radiotherapy can be used as a treatment after surgical removal of a malignant tumor. Radiotherapy can be applied externally to an organism (e.g., a human, dog, cat, mouse, etc.) by an implant (also known as brachytherapy) or by a radioisotope administered by injection, capsule, drink, or implant. Radiotherapy is a term known in the art and includes three-dimensional conformal radiotherapy (3D-CRT), image-guided radiotherapy (IGRT), intensity-modulated radiotherapy (IMRT), helical tomotherapy, photon beam radiotherapy, proton beam radiotherapy, intraoperative radiotherapy (IORT), stereotactic radiotherapy, and stereotactic body radiotherapy (SBRT). Radiotherapy can be administered externally by a linear accelerator (LINAC) (also known as a linear particle accelerator).

[0105] The term "rodent" refers to a mammal of the order Rodentia. In one embodiment, a rodent is a mammal having a pair of continuously growing incisors in each of the upper and lower jaws. In one embodiment, the term "rodent" refers to one or more rodent selected from the group consisting of a mouse, a rat, a squirrel, a chipmunk, a chinchilla, a paca, a porcupine, a beaver, a caiman, a gerbil, a hamster, and a guinea pig. In one embodiment, the rodent is a murine. In one embodiment, the rodent is a murine. In a preferred embodiment, the rodent is a mouse or a rat.

[0106] The terms "specifically binds" and like are used herein to refer to an antibody or antigen binding fragment that binds to a protein of interest (e.g., CXCR4) in a specific manner, as opposed to sticking through polar interactions or other non-specific (but observable) binding. For example, an anti-CXCR4 antibody or antigen binding fragment specifically binds to CXCR4 by virtue of its CDR regions interacting with its antigenic determinants (epitopes) on CXCR4. Antibodies or antigen binding fragments that specifically bind to a protein of interest (e.g., a CXCR4 antigen) can be identified, for example, by immunoassays (e.g., enzyme-linked immunosorbent assay (ELISA)), plate-based arrays (e.g., Meso Scale Discovery™ platform), SPR (e.g., Biacore™), flow cytometry (e.g., Mirrorball™ fluorescence cytometer), live cell imaging, radioimmunoassay (RIA), radioligand binding, or other techniques known to one of skill in the art. In a particular embodiment, binding is determined using flow cytometry, radioligand binding, SPR, live cell imaging, or ELISA. Typically, a specific reaction will be at least twice the background signal or noise, more typically more than 10 times background (e.g., more than 15 times, more than 20 times, more than 50 times, or more than 100 times). See, e.g., Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York at pages 332-336 for a discussion of antibody specificity.

[0107] The term "subject" or "patient" refers to any animal, including but not limited to mammals, such as non-human mammals. As used herein, the term "mammal" refers to any vertebrate that 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 and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals such as mice, rats (including cotton rats), hamsters, and guinea pigs; birds, including domestic birds, wild birds, and racing birds, such as chickens, turkeys and other galliform birds, ducks, geese and the like. In a particular embodiment, the subject is a human patient.

[0108] As used herein, "substantially," "substantially all," or "substantially the" means 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%.

[0109] As used herein, the term "therapeutic agent" refers to any agent that can be used to prevent, control, treat, and / or ameliorate a CXCR4-mediated disease (or disorder) and / or a symptom associated therewith, which is described elsewhere herein. In certain embodiments, the term "therapeutic agent" refers to an antibody or antigen-binding fragment of the application. In certain other embodiments, the term "therapeutic agent" refers to an agent other than an antibody or antigen-binding fragment of the application. Preferably, the therapeutic agent is an agent known to be useful, or has been or is currently used, to treat, control, or ameliorate a CXCR4-mediated disease (or disorder) or one or more symptoms associated therewith, e.g., a therapeutic agent that a health care professional considers to be standard of care.

[0110] As used herein, the term "therapy" refers to any regimen, method, and / or agent that can be used to prevent, control, treat, and / or ameliorate a CXCR4-mediated disease or disorder, e.g., a cancer or any other disease or disorder described herein. In certain embodiments, the terms "therapies" and "therapy" refer to biological therapies, supportive therapies, and / or other therapies known to those of skill in the art, such as medical professionals, to prevent, control, treat, and / or ameliorate a CXCR4-mediated disease or disorder.

[0111] 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 disorder, which is described elsewhere herein, as a result of 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 antigen-binding fragment of the application or antigen-binding fragment thereof. In particular 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 disorder, as described elsewhere herein, e.g., a cancer.

[0112] The term "variable region" or "variable domain" refers to the portion of the light and heavy chains that is generally the amino-terminal portion of about 120 to 130 amino acids in the heavy chain and of about 100 to 110 amino acids in the light chain that varies greatly in sequence among antibodies, and that is involved in binding and specificity of each particular antibody to its particular antigen, e.g., CXCR4. The variable sequences are concentrated in the CDRs, while the more highly conserved regions in the variable domains are called the framework regions (FRs). In preferred embodiments, the variable region is a human variable region.

[0113] Definitions for common terms in the art of 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.

[0114] The present application uses, unless otherwise stated, as for example Sambrook et al., Molecular Cloning: A Laboratory Manual (4ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012); Molecular Biology 3rd Edition, David Clark et al., Elsevier Science Publishing, Inc., New York, USA (2018); Molecular Biology of the Cell, 6th Edition, 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 8th 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.Standard procedures described in 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) are followed, which are incorporated herein by reference in their entirety.

[0115] Other terms are defined in the specification of various embodiments of the invention.

[0116] 2. CXCR4 antibodies

[0117] 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 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 4-chain antibody comprising 2 heavy chains and 2 light chains.

[0118] The inventors have identified antibodies specific for CXCR4 that have many potential uses and benefits over existing anti-CXCR4 antibodies. For example, the anti-CXCR4 antibodies or antigen-binding fragments described herein can have one or more of the following improved or beneficial properties:

[0119] a. selectivity for inhibiting only one of the ligands of CXCR4 (e.g., inhibiting CXCL12 / CXCR4 interaction, but not CXCR4 interaction with other ligands (e.g., MIF or ubiquitin))

[0120] b. specificity for CXCR4, inhibiting binding of multiple ligands (e.g., CXCL12, MIF, and ubiquitin; CXCL12 and ubiquitin; or CXCL12 and MIF)

[0121] c. inducing increased levels of T cell infiltration into tumors (e.g., solid tumors), thereby shrinking tumor volume

[0122] d. inducing low levels of T cell apoptosis

[0123] e. improved or reduced immunogenicity / side effects

[0124] f. improved solubility

[0125] g. improved stability

[0126] h. ease of formulation

[0127] i. frequency of dosing and / or route of administration

[0128] j. manufacturability (e.g., expression, ease of purification, isoform, affinity of column during purification (e.g., protein A or nickel), heavy and light chain non-dissociation, heavy chain dissociation, increased yield, reduced protein cleavage / truncation)

[0129] k. improved efficacy and / or as part of a combination therapy

[0130] The anti-CXCR4 antibodies or antigen-binding fragments of the present application, 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 will be described in detail below, are described in terms of embodiments and configurations described herein. Unless otherwise indicated, all embodiments and configurations should be interpreted as being combinable with any other embodiment or configuration, unless such a combination is not technically meaningful or otherwise explicitly stated. In particular, reference to a particular embodiment includes reference to any sub-embodiment (e.g., reference to embodiment 3 in a subsequent embodiment specifically includes reference to embodiment 3a, embodiment 3b, etc.), unless otherwise apparent from the context.

[0131] Throughout this specification, reference to CXCR4 in one embodiment refers to a mammalian CXCR4. Examples of mammalian species include, but are not limited to, humans and other primates, including non-human primates such as chimpanzees and other apes, and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; experimental mammals including rodents such as mice, rats (including cotton rats), hamsters, gerbils, and guinea pigs. In one embodiment, the CXCR4 is a rodent CXCR4 (e.g. mouse or rat CXCR4). In another embodiment, the CXCR4 is a 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 a human CXCR4 (optionally wherein the human CXCR4 is selected from SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3). In another embodiment, the CXCR4 is a cell surface expressed CXCR4.

[0132] CXCR4 is a GPCR and like most GPCRs exists in multiple isoforms. In one embodiment, the antibody or antigen-binding fragment thereof is capable of interacting 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 isoforms of hCXCR4 have been identified in the Uniprot database: isoform 1 is 352 amino acids in length, while isoform 2 is 356 amino acids in length (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 (at the N-terminus of the receptor) in isoform 1 are replaced by MSIPLPLLQ in isoform 2. In a preferred embodiment, the antibody or antigen-binding fragment thereof specifically binds to isoform 1 (SEQ ID NO: 2) or isoform 2. In a preferred embodiment, the antibody or antigen-binding fragment thereof specifically binds to both isoforms 1 and 2. It will be appreciated that not all isoforms have been identified.

[0133] In another embodiment, the CXCR4 protein has undergone a post-translational modification (e.g., glycosylation, phosphorylation, ubiquitination, or sulfation). In one embodiment, the CXCR4 has undergone glycosylation (N-linked and / or O-linked) at amino acids N11, S18, and / or N176 (Reference Sequence: Isoform 1).

[0134] Throughout the embodiments herein, the CXCR4 can be wild-type human CXCR4. Additionally or alternatively, the human CXCR4 can be a variant CXCR4, such as a SNP, a polymorphic variant, a truncated variant, a mutated variant, or a variant with a frameshift mutation. In one embodiment, the variant CXCR4 is encoded by an amino acid sequence comprising one or more single nucleotide polymorphisms (SNPs) (e.g., selected from 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 variants that are commonly found in warts, hypogonatoidism, infection, and myelokathexis (WHIM) or related diseases (e.g., Waldenstrom's macroglobulinemia, WHIM syndrome, and related diseases, such as leukocyte adhesion deficiency (LAD) type 2, and leukocyte adhesion deficiency (LAD) type 1, respectively). macroglobulinaemia). Additionally or alternatively, the variant CXCR4 can be encoded by an amino acid sequence comprising one or more mutations, such as a nonsense mutation (resulting in truncation of the amino acid sequence, such as E343K, E343X, S338X, G336X and R334X (where X is a stop 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). A frameshift mutation is due to the insertion or deletion of one or more nucleotides, resulting 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 to a stopper nucleic acid sequence). In another embodiment, the variant CXCR4 is encoded by an amino acid sequence comprising a frameshift mutation (e.g. S341 frameshift, S339 frameshift, H315 frameshift, T318 frameshift, R322 frameshift, L326 frameshift, K327 frameshift and I328 frameshift) ((Liu et al., (2012) supra, and Poulain et al., (2016). Genomic Landscape of CXCR4 Mutations in WHIM Syndrome. Human Mutation 37(10): 1010-1019). Macroglobulinemia. Clin. Cancer Res. 22(6): 1480-1488)). In another embodiment, the variant CXCR4 is encoded by an amino acid sequence comprising 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 can express any of these variant CXCR4 proteins, and thus antibodies or antigen-binding fragments that inhibit these variant CXCR4 can be useful in 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, certain CXCR4-mediated diseases or disorders can be treated or prevented when the anti-CXCR4 antibody or antigen-binding fragment binds to a variant CXCR4 having a mutation independently selected from a deletion of amino acids 334-352, 338-352, or 343-352 (Reference Sequence: Isoform 1); or a variant CXCR4 encoded by an amino acid sequence comprising one or more SNPs selected from CXCR4-I261I, CXCR4-K68K, and CXCR4-F93S; or a variant CXCR4 encoded by an amino acid sequence comprising one or more nonsense mutations selected from E343K, E343X, S338X, G336X, or R334X; or a variant CXCR4 encoded by an amino acid sequence comprising one or more frame-shift mutations selected from S341 frame-shift, S339 frame-shift, H315 frame-shift, T318 frame-shift, R322 frame-shift, L326 frame-shift, K327 frame-shift, and I328 frame-shift.

[0135] In one example, the binding site of the 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 4-chain antibodies or fragments thereof (e.g., Fabs or scFvs). Suitable methods for generating a plurality of binding sites for screening include phage display (generating a phage display library of antibody binding sites), ribosome display (generating a ribosome display library of antibody binding sites), yeast display (generating a yeast display library of antibody binding sites), mammalian display, covalent display (e.g., cis display), or non-human vertebrate (e.g., a rodent, such as a mouse or rat, e.g., a Velocimouse™, Intelliselect™ transgenic mouse, Xenomouse™, Aliva Mouse™, HuMab Mouse™, Omnmouse™, Omnirat™ MeMoMouse™), OmniFlic™, OmniChicken™ and OmniClic TM ) immunization with hCXCR4 protein or hCXCR4 epitope, followed by isolation of a repertoire of antibody-producing cells (e.g., B cells, plasma cells, or plasmablasts) and / or a library of isolated antibodies or antigen binding fragments.

[0136] The inventors provide the following anti-CXCR4 antibodies, each of which specifically binds to CXCR4:

[0137] CL-82574 has a heavy chain variable region (V H ) amino acid sequence of SEQ ID NO: 16, comprising a CDRH1 amino acid sequence of SEQ ID NO: 10 (IMGT) or SEQ ID NO: 13 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 11 (IMGT) or SEQ ID NO: 14 (Kabat), and a CDRH3 amino acid sequence of SEQ ID NO: 12 (IMGT) or SEQ ID NO: 15 (Kabat).V H The heavy chain nucleic acid sequence of the variable domain is SEQ ID NO: 17. CL-82574 has a light chain variable region (V L ) amino acid sequence of SEQ ID NO: 26, comprising a CDRL1 amino acid sequence of SEQ 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).VL The light chain nucleic acid sequence of the V domain is SEQ ID NO: 27. H The V domain can be combined with any of the heavy chain constant region sequences described herein, e.g., 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, e.g., 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 is SEQ ID NO: 19). The full-length light chain amino acid sequence is SEQ ID NO: 28 (light chain nucleic acid sequence is SEQ ID NO: 29). The heavy chain variable region was 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 was generated by recombination of human IGKV1-17*01 (SEQ ID NO: 320) and IGKJ3*01 (SEQ ID NO: 323) gene segments.

[0138] CL-82458 has a heavy chain variable region (V H) an amino acid sequence comprising a CDRH1 amino acid sequence of SEQ ID NO: 30 (IMGT) or SEQ ID NO: 33 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 31 (IMGT) or SEQ ID NO: 34 (Kabat), and a CDRH3 amino acid sequence of SEQ ID NO: 32 (IMGT) or SEQ ID NO: 35 (Kabat).V H The heavy chain nucleic acid sequence of the VL-82458 domain is SEQ ID NO: 37. CL-82458 has a light chain variable region (VL) amino acid sequence of SEQ ID NO: 46.V L ) an amino acid sequence comprising 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).V L The light chain nucleic acid sequence of the VL-82458 domain is SEQ ID NO: 47.V H The VL-82458 domain can be combined with any of the heavy chain constant region sequences described herein, e.g., 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.V 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 was made 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 was made by recombination of human IGKV1-17*01 (SEQ ID NO:320) and IGKJ4*01 (SEQ ID NO:326) gene segments.

[0139] CL-82558 has a heavy chain variable region (V H ) amino acid sequence of SEQ ID NO:50 (IMGT) or SEQ ID NO:53 (Kabat) CDRH1 amino acid sequence, SEQ ID NO:51 (IMGT) or SEQ ID NO:54 (Kabat) CDRH2 amino acid sequence, and SEQ ID NO:52 (IMGT) or SEQ ID NO:55 (Kabat) CDRH3 amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO:57. CL-82558 has a light chain variable region (V L ) amino acid sequence of SEQ ID NO:60 (IMGT) or SEQ ID NO:63 (Kabat) CDRL1 amino acid sequence, SEQ ID NO:61 (IMGT) or SEQ ID NO:64 (Kabat) CDRL2 amino acid sequence, and SEQ ID NO:62 (IMGT) or SEQ ID NO:65 (Kabat) CDRL3 amino acid sequence. LThe light chain nucleic acid sequence of the domain is SEQ ID NO: 67.V 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.V 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 was made 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 was made by recombination of human IGKV2-28*01 (SEQ ID NO: 322) and IGKJ1*01 (SEQ ID NO: 325) gene segments.

[0140] CL-82658 has a heavy chain variable region (V H) amino acid sequence comprising 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). V H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 77. CL-82658 has a light chain variable region (V L ) amino acid sequence comprising the CDRL1 amino acid sequence of SEQ ID NO: 80 (IMGT) or SEQ ID NO: 83 (Kabat), the CDRL2 amino acid sequence of SEQ ID NO: 81 (IMGT) or SEQ ID NO: 84 (Kabat), and the CDRL3 amino acid sequence of SEQ ID NO: 82 (IMGT) or SEQ ID NO: 85 (Kabat). V L The light chain nucleic acid sequence of the V domain is SEQ ID NO: 87. H The V domain can be combined with any of the heavy chain constant region sequences described herein, e.g., 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: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 was made 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 was made by recombination of human IGKV1-17*01 (SEQ ID NO:320) and IGKJ3*01 (SEQ ID NO:323) gene segments.

[0141] CL-83083 has a heavy chain variable region (V H ) amino acid sequence of SEQ ID NO:96, including a CDRH1 amino acid sequence of SEQ ID NO:90 (IMGT) or SEQ ID NO:93 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO:91 (IMGT) or SEQ ID NO:94 (Kabat), and a CDRH3 amino acid sequence of SEQ ID NO:92 (IMGT) or SEQ ID NO:95 (Kabat). H The heavy chain nucleic acid sequence of the domain is SEQ ID NO:97. CL-83083 has a light chain variable region (V L ) amino acid sequence of SEQ ID NO:106, including a CDRL1 amino acid sequence of SEQ ID NO:100 (IMGT) or SEQ ID NO:103 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO:101 (IMGT) or SEQ ID NO:104 (Kabat), and a CDRL3 amino acid sequence of SEQ ID NO:102 (IMGT) or SEQ ID NO:105 (Kabat). LThe light chain nucleic acid sequence of the domain is SEQ ID NO: 107.V 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.V 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 was made 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 was made by recombination of human IGLV10-54*02 (SEQ ID NO: 321) and IGLJ3*02 (SEQ ID NO: 324) gene segments.

[0142] CL-83083-2 has a heavy chain variable region (V H) an amino acid sequence comprising a CDRH1 amino acid sequence of SEQ ID NO: 90 (IMGT) or SEQ ID NO: 93 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 91 (IMGT) or SEQ ID NO: 94 (Kabat), and a CDRH3 amino acid sequence of SEQ ID NO: 92 (IMGT) or SEQ ID NO: 95 (Kabat).V H The heavy chain nucleic acid sequence of the VL domain is SEQ ID NO: 97. CL-83083-2 has a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 106. L ) an amino acid sequence comprising a CDRL1 amino acid sequence of SEQ ID NO: 100 (IMGT) or SEQ ID NO: 103 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 101 (IMGT) or SEQ ID NO: 104 (Kabat), and a CDRL3 amino acid sequence of SEQ ID NO: 102 (IMGT) or SEQ ID NO: 105 (Kabat).V L The light chain nucleic acid sequence of the VL domain is SEQ ID NO: 107. The VH domain can be combined with any of the heavy chain constant region sequences described herein, e.g., 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.V LThe domain can be combined with any of the light chain constant region sequences described herein, e.g., 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 is SEQ ID NO: 99). The full-length light chain amino acid sequence is SEQ ID NO: 110 (light chain nucleic acid sequence is SEQ ID NO: 111). The heavy chain variable region was 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 was generated by recombination of human IGLV10-54*02 (SEQ ID NO: 321) and IGLJ3*02 (SEQ ID NO: 324) gene segments.

[0143] CL-82583 has a heavy chain variable region (V H ) amino acid sequence, comprising from V H The amino acid sequence of SEQ ID NO: 112 is the amino acid sequence of CDRH1, CDRH2 and CDRH3 as defined by IMGT or Kabat. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 113. CL-82583 has a light chain variable region (V L ) amino acid sequence comprising the CDRL1, CDRL2 and CDRL3 amino acid sequences as defined by IMGT or Kabat from the VL amino acid sequence SEQ ID NO: 116. L The light chain nucleic acid sequence of the V domain is SEQ ID NO: 117. 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. The VL 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 was made 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 was made by recombination of human IGKV1-17*01 (SEQ ID NO: 320) and IGKJ3*01 (SEQ ID NO: 323) gene segments.

[0144] CL-82580 has a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 120, comprising CDRH1, CDRH2, and CDRH3 amino acid sequences as defined by IMGT or Kabat from the VH amino acid sequence of SEQ ID NO: 120. H ) amino acid sequence of SEQ ID NO: 124. CL-82580 has a light chain variable region (VL) amino acid sequence of SEQ ID NO: 122, comprising CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat from the VL amino acid sequence of SEQ ID NO: 122. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 121. CL-82580 has a light chain variable region (VL) amino acid sequence of SEQ ID NO: 122, comprising CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat from the VL amino acid sequence of SEQ ID NO: 122. L) amino acid sequence comprising the CDRL1, CDRL2 and CDRL3 amino acid sequences as defined by IMGT or Kabat from the VL amino acid sequence SEQ ID NO: 124. L The light chain nucleic acid sequence of the V domain is SEQ ID NO: 125. H The V domain can be combined with any of the heavy chain constant region sequences described herein, e.g., 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, e.g., 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 is SEQ ID NO: 123). The full-length light chain amino acid sequence is SEQ ID NO: 126 (light chain nucleic acid sequence is SEQ ID NO: 127). The heavy chain variable region was generated 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 was generated by recombination of human IGKV1-5*03 and IGKJ1*01 (SEQ ID NO: 325) gene segments.

[0145] CL-82577 has a heavy chain variable region (V H) amino acid sequence comprising the CDRH1, CDRH2 and CDRH3 amino acid sequences as defined by IMGT or Kabat from the VH amino acid sequence SEQ ID NO: 128. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 129. CL-82577 has a light chain variable region (V L ) amino acid sequence comprising the CDRL1, CDRL2 and CDRL3 amino acid sequences as defined by IMGT or Kabat from the VL amino acid sequence SEQ ID NO: 132. L The light chain nucleic acid sequence of the V domain is SEQ ID NO: 133. H The V domain can be combined with any of the heavy chain constant region sequences described herein, e.g., 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 was made 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 was made by recombination of human IGKV2-28*01 (SEQ ID NO:322) and IGKJ1*01 (SEQ ID NO:325) gene segments.

[0146] CL-82571 has a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 136, which includes CDRH1, CDRH2, and CDRH3 amino acid sequences as defined by IMGT or Kabat, from VH amino acid sequence SEQ ID NO: 136. H ) amino acid sequence of SEQ ID NO: 140, which includes CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat, from VL amino acid sequence SEQ ID NO: 140. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 137. CL-82571 has a light chain variable region (VL) amino acid sequence of SEQ ID NO: 140, which includes CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat, from VL amino acid sequence SEQ ID NO: 140. L The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 137. CL-82571 has a light chain variable region (VL) amino acid sequence of SEQ ID NO: 140, which includes CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat, from VL amino acid sequence SEQ ID NO: 140. L The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 137. CL-82571 has a light chain variable region (VL) amino acid sequence of SEQ ID NO: 140, which includes CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat, from VL amino acid sequence SEQ ID NO: 140. 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.V 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: 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 was made by recombination of human IGHV3-21*03, IGHD5-18*01, and IGHJ6*02 (SEQ ID NO: 318) gene segments. The light chain variable region was made by recombination of human IGKV1D-13*d01 and IGKJ4*01 (SEQ ID NO: 326) gene segments.

[0147] CL-82562 has a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 144, which includes CDRH1, CDRH2, and CDRH3 amino acid sequences as defined by IMGT or Kabat from the VH amino acid sequence of SEQ ID NO: 144. H ) amino acid sequence of SEQ ID NO: 148, which includes CDRH1, CDRH2, and CDRH3 amino acid sequences as defined by IMGT or Kabat from the VH amino acid sequence of SEQ ID NO: 144. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 145. CL-82562 has a light chain variable region (VL) amino acid sequence of SEQ ID NO: 148, which includes CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat from the VL amino acid sequence of SEQ ID NO: 148. L ) amino acid sequence of SEQ ID NO: 148, which includes CDRH1, CDRH2, and CDRH3 amino acid sequences as defined by IMGT or Kabat from the VH amino acid sequence of SEQ ID NO: 144. LCDRL1, CDRL2, and CDRL3 amino acid sequences of amino acid sequence SEQ ID NO: 148 as defined by IMGT or Kabat.V L The light chain nucleic acid sequence of the domain is SEQ ID NO: 149.V 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.V 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: 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 was made 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 was made by recombination of human IGKV1-17*01 (SEQ ID NO: 320) and IGKJ1*01 (SEQ ID NO: 325) gene segments.

[0148] CL-82556 has a heavy chain variable region (V H) the amino acid sequence of CDRH1, CDRH2, and CDRH3 as defined by IMGT or Kabat from the VH amino acid sequence of SEQ ID NO: 152. The heavy chain nucleic acid sequence of the VL domain is SEQ ID NO: 153. CL-82556 has a light chain variable region (VL) with the amino acid sequence of SEQ ID NO: 156.V H ) the amino acid sequence of CDRH1, CDRH2, and CDRH3 as defined by IMGT or Kabat from the VH amino acid sequence of SEQ ID NO: 152. The heavy chain nucleic acid sequence of the VL domain is SEQ ID NO: 153. CL-82556 has a light chain variable region (VL) with the amino acid sequence of SEQ ID NO: 156.V L ) the amino acid sequence of CDRH1, CDRH2, and CDRH3 as defined by IMGT or Kabat from the VH amino acid sequence of SEQ ID NO: 152. The heavy chain nucleic acid sequence of the VL domain is SEQ ID NO: 153. CL-82556 has a light chain variable region (VL) with the amino acid sequence of SEQ ID NO: 156.V LThe domain can be combined with any of the light chain constant region sequences described herein, e.g., 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 is SEQ ID NO: 155). The full-length light chain amino acid sequence is SEQ ID NO: 158 (light chain nucleic acid sequence is SEQ ID NO: 159). The heavy chain variable region was 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 was generated by recombination of human IGKV1-17*01 (SEQ ID NO: 320) and IGKJ3*01 (SEQ ID NO: 323) gene segments.

[0149] CL-82551 has a heavy chain variable region (V H ) amino acid sequence comprising the CDRH1, CDRH2 and CDRH3 amino acid sequences as defined by IMGT or Kabat from the VH amino acid sequence SEQ ID NO: 160. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 161. CL-82551 has a light chain variable region (V L ) amino acid sequence, comprising from V L The amino acid sequence of CDRL1, CDRL2 and CDRL3 as defined by IMGT or Kabat of amino acid sequence SEQ ID NO: 164. L The light chain nucleic acid sequence of the V domain is SEQ ID NO: 165. 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.V 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: 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 was made 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 was made by recombination of human IGKV1-5*03 and IGKJ1*01 (SEQ ID NO: 325) gene segments.

[0150] CL-82541 has a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 168, which includes CDRH1, CDRH2, and CDRH3 amino acid sequences as defined by IMGT or Kabat from the VH amino acid sequence of SEQ ID NO: 168.V H ) The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 169. CL-82541 has a light chain variable region (VL) amino acid sequence of SEQ ID NO: 172, which includes CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat from the VL amino acid sequence of SEQ ID NO: 172. H ) The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 169. CL-82541 has a light chain variable region (VL) amino acid sequence of SEQ ID NO: 172, which includes CDRL1, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat from the VL amino acid sequence of SEQ ID NO: 172. L) the amino acid sequence of a CDRL1, CDRL2, and CDRL3 as defined by IMGT or Kabat from the VL amino acid sequence of SEQ ID NO: 172.V L The light chain nucleic acid sequence of the domain is SEQ ID NO: 173.V 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.V 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: 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 was made 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 was made by recombination of human IGKV2-28*01 (SEQ ID NO: 322) and IGKJ1*01 (SEQ ID NO: 325) gene segments.

[0151] CL-82523 has a heavy chain variable region (V H ) the amino acid sequence of a CDRL1, CDRL2, and CDRL3 as defined by IMGT or Kabat from the VL amino acid sequence of SEQ ID NO: 172.V HCDRH1, CDRH2, and CDRH3 amino acid sequences of the amino acid sequence of SEQ ID NO: 176 as defined by IMGT or Kabat.V H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 177. CL-82523 has a light chain variable region (V L ) amino acid sequence of SEQ ID NO: 180, comprising CDRLL, CDRL2, and CDRL3 amino acid sequences from the V L CDRH1, CDRH2, and CDRH3 amino acid sequences of the amino acid sequence of SEQ ID NO: 176 as defined by IMGT or Kabat.V L The light chain nucleic acid sequence of the domain is SEQ ID NO: 181.V H The domain can be combined with any of the heavy chain constant region sequences described herein, e.g., 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.V LThe domain can be combined with any of the light chain constant region sequences described herein, e.g., 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 is SEQ ID NO: 179). The full-length light chain amino acid sequence is SEQ ID NO: 182 (light chain nucleic acid sequence is SEQ ID NO: 183). The heavy chain variable region was 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 was generated by recombination of human IGKV1-17*01 (SEQ ID NO: 320) and IGKJ4*01 (SEQ ID NO: 326) gene segments.

[0152] CL-82517 has a heavy chain variable region (V H ) amino acid sequence comprising V from SEQ ID NO: 184 H The amino acid sequences of CDRH1, CDRH2 and CDRH3 as defined by IMGT or Kabat. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 185. CL-82517 has a light chain variable region (V L ) amino acid sequence comprising V from SEQ ID NO: 188 L The amino acid sequences of CDRL1, CDRL2 and CDRL3 as defined by IMGT or Kabat. L The light chain nucleic acid sequence of the V domain is SEQ ID NO: 189. 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.V 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: 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 was made 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 was made by recombination of human IGKV1-5*03 and IGKJ1*01 (SEQ ID NO: 325) gene segments.

[0153] CL-82495 has a heavy chain variable region (V H ) amino acid sequence of SEQ ID NO: 192, including CDRH1, CDRH2, and CDRH3 amino acid sequences as defined by IMGT or Kabat from the V H H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 193. CL-82495 has a light chain variable region (V L ​) amino acid sequence comprising V L CDRL1, CDRL2, and CDRL3 amino acid sequences of the amino acid sequence as defined by IMGT or Kabat. L The light chain nucleic acid sequence of the domain 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. 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: 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 was made 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 was made by recombination of human IGKV1-17*01 (SEQ ID NO: 320) and IGKJ1*01 (SEQ ID NO: 325) gene segments.

[0154] CL-82473 has a heavy chain variable region (V H ) amino acid sequence comprising V HCDRH1, CDRH2, and CDRH3 amino acid sequences of the amino acid sequence of SEQ ID NO: 200 as defined by IMGT or Kabat.V H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 201. CL-82473 has a light chain variable region (VL) amino acid sequence of SEQ ID NO: 204, which includes CDRLL, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat from the V L ) amino acid sequence of SEQ ID NO: 204.V L The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 201. CL-82473 has a light chain variable region (VL) amino acid sequence of SEQ ID NO: 204, which includes CDRLL, CDRL2, and CDRL3 amino acid sequences as defined by IMGT or Kabat from the V L The light chain nucleic acid sequence of the domain is SEQ ID NO: 205.V H The domain can be combined with any of the heavy chain constant region sequences described herein, e.g., 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.V 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 was made 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 was made by recombination of human IGKV2-30*01 and IGKJ3*01 (SEQ ID NO: 323) gene segments.

[0155] CL-82455 has a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 208, including CDRH1, CDRH2, and CDRH3 amino acid sequences from the VH amino acid sequence of SEQ ID NO: 208, as defined by IMGT or Kabat. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 209. CL-82455 has a light chain variable region (VL) amino acid sequence of SEQ ID NO: 212, including CDRL1, CDRL2, and CDRL3 amino acid sequences from the VL amino acid sequence of SEQ ID NO: 212, as defined by IMGT or Kabat. L The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 209. CL-82455 has a light chain variable region (VL) amino acid sequence of SEQ ID NO: 212, including CDRL1, CDRL2, and CDRL3 amino acid sequences from the VL amino acid sequence of SEQ ID NO: 212, as defined by IMGT or Kabat. L The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 209. CL-82455 has a light chain variable region (VL) amino acid sequence of SEQ ID NO: 212, including CDRL1, CDRL2, and CDRL3 amino acid sequences from the VL amino acid sequence of SEQ ID NO: 212, as defined by IMGT or Kabat. L The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 209. CL-82455 has a light chain variable region (VL) amino acid sequence of SEQ ID NO: 212, including CDRL1, CDRL2, and CDRL3 amino acid sequences from the VL amino acid sequence of SEQ ID NO: 212, as defined by IMGT or Kabat. 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.V 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: 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 was made 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 was made by recombination of human IGKV3-15*01 and IGKJ1*01 (SEQ ID NO: 325) gene segments.

[0156] CL-83158 has a heavy chain variable region (V H ) amino acid sequence of SEQ ID NO: 216, including CDRH1, CDRH2, and CDRH3 amino acid sequences as defined by IMGT or Kabat from the V H H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 217. CL-83158 has a light chain variable region (V L ​) amino acid sequence comprising V L CDRL1, CDRL2, and CDRL3 amino acid sequences of the amino acid sequence as defined by IMGT or Kabat. L The light chain nucleic acid sequence of the domain 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. 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: 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 was made 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 was made by recombination of human IGLV3-19*01 and IGLJ3*02 (SEQ ID NO: 324) gene segments.

[0157] CL-148712 has a heavy chain variable region (V H ) amino acid sequence comprising V HCDRH1, CDRH2, and CDRH3 amino acid sequences of the amino acid sequence of SEQ ID NO: 224 as defined by IMGT or Kabat.V H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 225. CL-148712 has a light chain variable region (VL) amino acid sequence of SEQ ID NO: 228, which includes CDRLL, CDRLL, and CDRL3 amino acid sequences from the VL amino acid sequence of SEQ ID NO: 228 as defined by IMGT or Kabat.V L ) amino acid sequence of SEQ ID NO: 228. The VL amino acid sequence of SEQ ID NO: 228 includes CDRLL, CDRLL, and CDRL3 amino acid sequences as defined by IMGT or Kabat.V L The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 225. CL-148712 has a light chain variable region (VL) amino acid sequence of SEQ ID NO: 228, which includes CDRLL, CDRLL, and CDRL3 amino acid sequences from the VL amino acid sequence of SEQ ID NO: 228 as defined by IMGT or Kabat.V L The light chain nucleic acid sequence of the domain is SEQ ID NO: 229.V H The domain can be combined with any of the heavy chain constant region sequences described herein, e.g., 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.V LThe domain can be combined with any of the light chain constant region sequences described herein, e.g., 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 is SEQ ID NO: 227). The full-length light chain amino acid sequence is SEQ ID NO: 230 (light chain nucleic acid sequence is SEQ ID NO: 231). The heavy chain variable region was 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 was generated by recombination of human IGKV1-17*01 (SEQ ID NO: 320) and IGKJ4*01 (SEQ ID NO: 326) gene segments.

[0158] CL-148729 has a heavy chain variable region (V H ) amino acid sequence, comprising from V H The amino acid sequence of SEQ ID NO: 232 is the amino acid sequence of CDRH1, CDRH2 and CDRH3 as defined by IMGT or Kabat. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 233. CL-148729 has a light chain variable region (V L ) amino acid sequence comprising V from SEQ ID NO: 236 L The amino acid sequences of CDRL1, CDRL2 and CDRL3 as defined by IMGT or Kabat. L The light chain nucleic acid sequence of the V domain is SEQ ID NO: 237. 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.V 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: 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 was made by recombination of human IGHV3-73*02 (SEQ ID NO: 309), IGHD6-25*01, and IGHJ4*02 gene segments. The light chain variable region was made by recombination of human IGLV1-51*01 and IGLJ3*02 (SEQ ID NO: 324) gene segments.

[0159] The antibody or antigen binding fragment of the application (as described herein) specifically binds to CXCR4; the specific binding to CXCR4 and / or the binding affinity for 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 human CXCR4 and cynomolgus monkey CXCR4.

[0160] In one embodiment, SPR is performed using nanodiscs, cells or virus-like particles (VLPs) to present all or part of a receptor (e.g., CXCR4). In another embodiment, nanodiscs, cells or VLPs are used to present a mutant (e.g., thermostabilized) or variant receptor (e.g., CXCR4). In one embodiment, SPR is performed using a configuration-thermostabilized receptor, e.g., CXCR4 which is a mutant (thermostabilized) CXCR4 receptor, has been generated and selected according to the methods described in WO 2009 / 081136 (see also: Magnani et al (2008). Co-evolving stability and conformational homogeneity of the human adenosine A2a 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), which is optionally presented using nanodiscs, cells or VLPs.

[0161] 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 at pH 7.6 (e.g., using Hepes Buffered Saline at pH 7.6 (also known as 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 a detergent content of no more than 0.05% by volume, e.g., in the presence of 0.05% P20 (polysorbate 20; e.g., Tween-20™) and 3 mM EDTA.

[0162] In one example, SPR is performed at 25 °C and using 10 nM Hepes, 150 mM NaCl, 0.1% w / v BSA as running buffer at pH 7.3. In one example, SPR is performed at 25 °C or 37 °C in a buffer of pH 7.6, 150 mM NaCl, 0.05% detergent (e.g. P20) and 3 mM EDTA. The buffer can contain 10 mM Hepes. In one example, SPR is performed at 25 °C or 37 °C in HBS-EP. In one example, the affinity of an antibody or antigen binding fragment is determined using SPR by:

[0163] 1. Coupling of anti-human (or other relevant human, rat or non-human vertebrate antibody constant region matched species) IgG (e.g. Biacore™ BR-1008-38) to a biosensor chip (e.g. a GLM chip), such as by primary amine coupling;

[0164] 2. Exposing the anti-human IgG (or other antibody matched species) to the test IgG antibody (e.g. anti-CXCR4 antibody or antigen binding fragment) to capture the test antibody to the chip;

[0165] 3. Passing the test antigen over the capture surface of the chip at 1024 nM, 256 nM, 64 nM, 16 nM, 4 nM and 0 nM (i.e. buffer alone); and

[0166] 4. And determining the affinity of the test antibody binding to the test antigen using surface plasmon resonance, e.g. under the SPR conditions discussed above (e.g. at 25 °C in a physiological buffer). SPR can be performed using any standard SPR device, such as by Biacore TM or using ProteOn XPR36 TM (Bio-Rad TM ).

[0167] In another embodiment, the antigen can be presented on a biosensor chip (rather than steps 1 and 2), and then the antigen is exposed to the antibody or antigen binding fragment (rather than chip 3). In another embodiment, biotinylated CXCR4 virus-like particles are captured on the active flow channel of a SA chip, and then purified anti-CXCR4 antibodies or antigen binding fragments (e.g. Fab fragments) are used as analytes and flowed over the captured CXCR4 VLPs at 1.23 nM, 3.7 nM, 11.11 nM, 33.33 nM and 100 nM.

[0168] In another embodiment, SPR performed using cells can be analyzed using a technique that measures repetitive differential measurements of surface-associated proteins (e.g. Ligand Tracer™ technology (Ridgeview)).

[0169] Regeneration of the capture surface can be performed using 10 mM glycine at pH 1.7. This removes the captured antibody or antigen binding fragment and allows the surface to be used for other interactions. Binding data can be fitted to the intrinsic 1 : 1 model using standard techniques, e.g. using ProteOn XPR36 TM the model inherent to the analysis software.

[0170] Antibodies or antigen-binding fragments thereof that specifically bind to a CXCR4 antigen can cross-react with related antigens, particularly CXCR4 of different species (e.g. rhesus, cynomolgus, rodent and / or human). Thus, in one embodiment, the antibody or antigen-binding fragment specifically binds to human, rhesus, cynomolgus and / or rodent (e.g. mouse or rat) CXCR4. In one embodiment, the antibody or antigen-binding fragment specifically binds to human and / or cynomolgus CXCR4.

[0171] In a preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to human CXCR4 antigen does not cross-react with other antigens, e.g., an antigen selected from the group consisting of 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 to, or does not cross-react with, CXCR7, e.g., does not detectably bind to CXCR7 (optionally wherein CXCR7 is human and further optionally selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6). In a preferred embodiment, the antibody or antigen-binding fragment does not bind to CXCR7 (optionally wherein CXCR7 is human and further optionally selected from the group consisting of 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 to, or does not cross-react with, CCR5, e.g., does not detectably bind to CCR5. In another embodiment, the antibody or antigen-binding fragment does not bind to, or does not cross-react with, CXCR3, e.g., does not detectably bind to CXCR3. In one embodiment, cross-reactivity is determined in an assay substantially as described herein in Example 12.

[0172] In one embodiment, the antibody or antigen-binding fragment specifically binds to CXCR4 (e.g., human CXCR4). In one embodiment, the specific binding can be measured by binding affinity, which can be expressed as an EC 50 or K D In one embodiment, the antibody or antigen-binding fragment specifically binds to human CXCR4 with a K D In one embodiment, the binding affinity is determined by SPR, e.g., performed as described elsewhere herein or substantially as described herein in Example 11.

[0173] In another embodiment, the antibody or antigen-binding fragment specifically binds to cynomolgus monkey CXCR4 with an EC 50 In one embodiment, the binding affinity is determined by flow cytometry, e.g., performed as described elsewhere herein or substantially as described herein in Example 10.

[0174] In one embodiment, the antibody or antigen binding fragment (substantially) inhibits the binding of a ligand (such as a natural ligand as described above) to CXCR4. In a preferred embodiment, the ligand is CXCL12 (such as human CXCL12, in particular human aCXCL12 (SEQ ID NO: 327)). In one embodiment, the CXCL12 has a signal peptide (optionally 21 amino acids in length) cleaved. In a preferred embodiment, the antibody or antigen binding fragment (substantially) inhibits the binding of CXCL12 to CXCR4. In one embodiment, the antibody or antigen binding fragment completely inhibits CXCL12 binding to CXCR4 (such as human CXCL12 binding to human CXCR4 and / or cynomolgus CXCL12 binding to cynomolgus CXCR4). In one embodiment, the antibody or antigen binding fragment partially inhibits CXCL12 binding to CXCR4. In one embodiment, the antibody or antigen binding fragment partially or completely inhibits CXCL12 binding to CXCR4 but does not show any detectable inhibition of CXCR4 ligand (such as MIF or ubiquitin) binding to CXCR4.

[0175] In a preferred embodiment, the antibody or antigen binding fragment inhibits CXCL12 (such as human or cynomolgus CXCL12) binding to CXCR4 (such as human or cynomolgus CXCR4) with an IC50of 0.2 to 4 nM, 0.2 to 3 nM, 0.2 nM to 2 nM, or preferably 0.2 to 1 nM. 50 In a preferred embodiment, the antibody or antigen binding fragment inhibits CXCL12 (such as human or cynomolgus CXCL12) binding to CXCR4 (such as human or cynomolgus CXCR4) with an IC50of 0.2 to 4 nM, 0.2 to 3 nM, 0.2 nM to 2 nM, or preferably 0.2 to 1 nM. 50 In a preferred embodiment, the antibody or antigen binding fragment inhibits human CXCL12 binding to human CXCR4 (optionally selected from SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3). In one embodiment, the CXCL12 inhibition is determined using a HTRF assay as described elsewhere herein or a HTRF assay performed essentially as described herein in Example 7.

[0176] 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 binds to the receptor of interest (i.e. CXCR4) or the ligand of interest (e.g. CXCL12). The acceptor molecule binds to the binding partner of the receptor / ligand to which the donor molecule binds. The donor molecule or the acceptor molecule can be linked to, for example, an anti-mouse Fc antibody, an anti-human Fc antibody, an anti-Flag antibody, an anti-His antibody, directly to streptavidin [each of which binds to 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 (665 nm emission). In one embodiment, the concentration of the antibody or antigen-binding fragment thereof is determined in a titrated manner to provide a % specific binding curve (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, a plurality of data points are taken, 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)).

[0177] In one example, the % specific binding of a ligand (e.g. CXCL12) is determined using HTRF by:

[0178] 1. Recombinant ligand (e.g. CXCL12 (labelled with AlexaFlour 647)) is combined with the antibody or antigen-binding fragment in a plate;

[0179] 2. Cells expressing the receptor (e.g. CXCR4 (SNAP labelled with europium cryptate)) are added to the plate;

[0180] 3. The test antibody or antigen-binding fragment is added and left for a sufficient time to reach equilibrium (e.g. about 1 or 2 hours at room temperature);

[0181] 4. reading the plate using a plate reader capable of exciting the donor molecule (e.g. at 335 nm) and capable of measuring the emission wavelengths of the donor and acceptor fluorescent molecules (e.g. at 620 nm and 665 nm) (examples of plate readers include EnVision™ (PerkinElmer), PHERAstar™ FS, PHERAstar™ FSX, CLARIOstar™ and FLUOstar™, and POLARstar™ Omega); and

[0182] 5. determining the % specific binding of the test molecule (e.g. CXCL12), for example using Equation 6; and / or

[0183] 6. determining the IC50 of the ligand (e.g. CXCL12), for example using Equation 5 50 In one embodiment, the (e.g. inhibition) data (e.g. % specific binding) can be fitted using standard analysis techniques (e.g. using GraphPad / PRISM analysis software).

[0184] In one embodiment, an anti-CXCR4 antibody or antigen-binding fragment described herein does not induce apoptosis (e.g., CXCR4-mediated apoptosis) in T cells (optionally CD8+ T cells), optionally wherein apoptosis is determined using flow cytometry. In one embodiment, an anti-CXCR4 antibody or antigen-binding fragment described herein does not induce CXCR4-mediated apoptosis (whether by acting directly on CXCR4 or by inhibiting one or more of its natural ligands), e.g., in vitro in CXCR4-expressing cells, optionally 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 can be CD8+ T cells, CD4+ T cells, or regulatory T cells (Tregs), preferably CD8+ T cells. In one embodiment, a suitable 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, an antibody or antigen-binding fragment does not induce apoptosis in T cells if it has a percent apoptosis induction that is less than 50%, less than 40%, less than 30%, or less than 20% of the maximum specific induction (optionally expressed as a percent of the anti-Fas response or staurosporine-induced apoptosis). In another embodiment, the percent apoptosis induction that is less than 10%, less than 8%, less than 5%, or less than 4%, particularly less than 5% of the maximum specific induction (optionally expressed as a percent of the anti-Fas response or staurosporine-induced apoptosis). In another embodiment, the percent apoptosis induction that is less than 3%, less than 2%, less than 1%, or less than 0.8% of the maximum specific induction (optionally expressed as a percent of the anti-Fas response or staurosporine-induced apoptosis). In another embodiment, the percent apoptosis induction that is less than 0.5%, less than 0.3%, less than 0.2%, or less than 0.1% of the maximum specific induction (optionally expressed as a percent of the anti-Fas response or staurosporine-induced apoptosis). In a preferred embodiment, the percent apoptosis induction that is the maximum specific induction is determined using flow cytometry, e.g., as described herein or substantially as described in Example 9A or 9B herein. In another embodiment, the antibody (e.g., an antibody with reduced or no effector function) or antigen-binding fragment does not induce CXCR4-mediated apoptosis of B cells.

[0185] Apoptosis is a programmed form of cell death that involves the degradation of cellular components by a group of cysteine ​​proteases called apoptotic proteases. Apoptotic proteases can be activated through 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 perforins by 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.

[0186] In one embodiment, the anti-CXCR4 antibodies or antigen-binding fragments described herein (substantially) inhibit CXCL12 (e.g., human or cynomolgus macaque CXCL12) signaling via CXCR4 (e.g., human or cynomolgus macaque CXCR4), e.g., inhibit CXCL12-mediated CXCR4 signaling via cAMP. In one embodiment, the antibodies or antigen-binding fragments (substantially) inhibit CXCL12-mediated inhibition of forskolin-stimulated cAMP, e.g., using a forskolin-stimulated cAMP signaling assay (e.g., AlphaScreen TM 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 IC of 1 to 100 nM, 1 to 90 nM, 1 to 80 nM, 1 to 70 nM, 1 to 60 nM, 1 to 50 nM, 1 to 40 nM, or preferably 1 to 30 nM. 50 Inhibition of CXCL12-Mediated Inhibition of Forskolin-Stimulated cAMP In one embodiment, cAMP is determined using a forskolin-stimulated cAMP signaling assay, performed essentially as described herein in Example 6 (human CXCR4) and Example 10B (cynomolgus monkey CXCR4).

[0187] In one embodiment, the anti-CXCR4 antibodies or antigen-binding fragments described herein (substantially) inhibit β-arrestin recruitment to CXCR4 (e.g., hCXCR4 or cynomolgus monkey CXCR4), optionally wherein the β-arrestin recruitment is induced by CXCL12 (e.g., hCXCL12 or cynomolgus monkey CXCL12). In one embodiment, the antibody or antigen-binding fragment has an IC of 0.01 to 15 nM, 0.01 to 10 nM, 0.01 to 5 nM, 0.01 to 4 nM, 0.01 to 3 nM, 0.01 to 2 nM, 0.01 to 1.5 nM, 0.01 to 1 nM, 0.01 to 0.5 nM, or preferably 0.01 to 0.2 nM.50 Inhibition of recruitment of beta-arrestin to CXCR4. In one embodiment, inhibition of recruitment of beta-arrestin to CXCR4 is performed in a functional cell-based reporter assay (e.g., using Tango CXCR4-bla U2OS cells). In one embodiment, the inhibition of beta-arrestin recruitment is measured using 30 nM of CXCL12 to induce beta-arrestin. In one embodiment, the concentration of the antibody or antigen-binding fragment is 100 nM to 0.1 pM (e.g., 50 nM to 0.5 pM or 40 nM to 0.5 pM). In one embodiment, the functional cell-based reporter assay is performed essentially as described in Example 6C herein.

[0188] In one embodiment, an anti-CXCR4 antibody or antigen-binding fragment described herein, upon binding to the cell surface (e.g., upon binding to CXCR4 on the cell surface), is (significantly) internalized into the cell, and optionally wherein the 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 to total overlaid target area that is 1.2-fold to 10-fold, 1.2-fold to 5-fold, 1.2-fold to 4-fold, 1.2-fold to 3-fold, or 1.2-fold to 2-fold greater than an isotype control. In one embodiment, the maximum signal to total overlaid target area is 2-fold to 10-fold, 2-fold to 5-fold, 2-fold to 4-fold, 2-fold to 3-fold, 3-fold to 4-fold, or 3-fold to 5-fold, particularly 2-fold to 4-fold, greater than an isotype control. In one embodiment, the isotype control is IgG4-PE. In one embodiment, the internalization is determined using fluorescence imaging technology (e.g., IncuCyte™ S3 Live Cell Analysis System (Essen BioScience)) or ELISA, particularly fluorescence imaging technology. In one embodiment, the internalization of an anti-CXCR4 antibody or antigen-binding fragment is determined essentially as described in Example 8 herein.

[0189] In one embodiment, the fluorescence imaging technology comprises live cell analysis, which measures real-time quantification of live cell imaging and analysis. This technology enables visualization and quantification of cellular behavior over time, and optionally automatically collects and analyzes images in a lab 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 infiltration into 3D cell environments (e.g., tumor spheroids). Internalization results in a positive readout of secondary antibody (red fluorescence). TM S3Live Cell AnalysisSystem(Essen BioScience), it is used to measure receptor or antibody (or antigen-binding fragment) internalization, chemotaxis, apoptosis, or cell infiltration into 3D cell environments (e.g., tumor spheroids). Internalization results in a positive readout of secondary antibody (red fluorescence).

[0190] Without being bound by theory, the antibodies or antigen-binding fragments of the application can be internalized and recycled back to the cell surface with the receptor (e.g., CXCR4). Alternatively, they can remain bound to the receptor (e.g., CXCR4) within the cell, which prevents their degradation by the usual cellular machinery. Alternatively, the antibody or antigen-binding fragment bound to the receptor is internalized, and the receptor is recycled back to the cell surface without the antibody or antigen-binding fragment. Because the antibodies or antigen-binding fragments of the application are not internalized as quickly as the benchmark antibody, the inventors speculate that they are bound to the receptor on the cell surface for a longer period of time, inhibiting the ligand (e.g., CXCL12) for a longer period of time, and thus the antibodies or antigen-binding fragments of the application can have a higher off-rate constant (Kd) value, even though the overall affinity appears similar. Another theory is that the antibodies or antigen-binding fragments of the application bind to monomeric CXCR4, and due to steric hindrance, a single antibody or antigen-binding fragment does not bind to more than one CXCR4 protein (monomer). Thus, the antibodies or antigen-binding fragments of the application can not cross-link two receptors, and thus the internalization of the antibody or antigen-binding fragment:receptor complex is slower.

[0191] In one embodiment, the anti-CXCR4 antibodies or antigen-binding fragments described herein (substantially) inhibit ligand-mediated (e.g., CXCL12, MIF, and / or ubiquitin, particularly CXCL12) T cell responses, e.g., as measured by a change in cell shape. In such an assay, the observed change in cell shape is caused by a redistribution of cellular material or a change in the cell membrane, or a change in the location of intracellular components. In one embodiment, ligand-mediated T cell responses are determined using electrical conductance or using a label-free dynamic mass redistribution (DMR) assay. In one embodiment, the antibody or antigen-binding fragment inhibits ligand-mediated T cell responses with an IC50 of 0.5 to 20 nM, 0.5 to 15 nM, 0.5 to 13 nM, 0.15 to 10 nM, 0.5 to 8 nM, or preferably 0.5 to 5 nM. 50 CXCL12-mediated T cell responses. In one embodiment, the DMR assay is performed substantially as described herein in Example 13.

[0192] In one embodiment, the anti-CXCR4 antibodies or antigen-binding fragments described herein (substantially) inhibit the chemotaxis of CXCR4+ T cells (e.g., human T cells, such as primary human T cells extracted from human blood) to a CXCR4 ligand (such as CXCL12). In one embodiment, live cell imaging (e.g., IncuCyte®) is used to determine the chemotaxis of CXCR4+ T cells to a CXCR4 ligand (such as CXCL12). TMS3 Chemotaxis Assay System) or flow cytometry (e.g., a transwell-based flow cytometry method), optionally using Jurkat T cells, to determine chemotaxis. In one embodiment, 30 nM or 6.25 nM of CXCL12 is used to induce chemotaxis in the assay (as a chemoattractant). In one embodiment, the antibody or antigen-binding fragment is at an IC 50 CXCR4+T cells to CXCL12. In one embodiment, chemotaxis assays are performed essentially as described herein in Example 14.

[0193] In one embodiment, the anti-CXCR4 antibodies or antigen-binding fragments described herein (essentially or substantially) enable T cells (e.g., CD8+T cells) to infiltrate (migrate into) tumors (e.g., solid tumors), optionally as measured in tumor spheroid assays. T cell infiltration into mouse tumors can be measured in vivo by using FACS or immunohistochemistry (IHC) (taking sections of treated and untreated tumors, applying reagents to identify T cells, and comparing the two) by comparing samples from untreated mice and mice treated with the anti-CXCR4 antibodies or antigen-binding fragments described herein. Tumors (e.g., central tumor mass) are surrounded by tumor stroma (see, e.g., Figure 1). Figure 9 and 10 In one preferred embodiment, the anti-CXCR4 antibodies or antigen-binding fragments described herein (essentially or substantially) enable T cells (e.g., CD8+T cells) to infiltrate tumors from the tumor stroma. Once the T cells enter the tumor (e.g., central tumor mass), they are able to effect cell killing and are able to destroy malignant cells. Figure 10 The underlying mechanisms are shown in Figure 2. Without being limited by theory, in the absence of the anti-CXCR4 antibodies described herein, cancer-associated fibroblasts (CAFs) that appear in the stroma secrete CXCL12 and establish a stronger gradient away from the tumor cells that T cells prefer to migrate towards. Figure 9 This is schematically illustrated in Figure 1.

[0194] Tumor infiltration can be measured in vitro using spheroid assays. Spheroids are 3D cell culture models that can be used to mimic some of the characteristics of solid tumors. Compared to 2D cell culture, 3D cell culture has many advantages, such as improved cell-cell contact, better representation of the tumor, and creation of tumor environmental conditions (such as 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). Spheroids contain tumor cells (such as 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 exceed the CXCL9, CXCL10 and CXCL11 pro-infiltration chemokine gradient secreted by the tumor, thus more accurately reflecting the in vivo conditions. This assay uses CCRF-HSB-2 cells (T cell lymphoblasts) expressing CXCR4 and simulates T cells in the tumor environment. It is believed that the inhibition of CXCL12 signaling by anti-CXCR4 antibodies causes the CXCR3 chemotaxis of CXCL9, CXCL10 and / or CXCL11 released by T cell lymphoblasts to the spheroid to recover, and causes an increase in the degree of T cell infiltration, so that the tumor volume is reduced. Therefore, in one embodiment, the anti-CXCR4 antibodies or antigen-binding fragments described herein (significantly) induce an increase in the infiltration of T cell lymphoblasts into tumor spheroids. In one embodiment, the tumor spheroid comprises cells selected from one or more of HT-29 cells, HS-5 cells, MS-5 cells and MEF cells, such as HT-29 cells. In one embodiment, the antibody or antigen-binding fragment induces the infiltration of T cell lymphoblasts (e.g., CCRF-HSB-2 cells) into tumor spheroids to a maximum infiltration percentage of 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, or 97% to 100%. In one embodiment, the maximum infiltration percentage is 10 to 100%, 20 to 90%, 30 to 90%, 40 to 90%, 50 to 90%, 60 to 90%, 70 to 90%, 80% to 90%, 90 to 90%, 95 to 90%, or 97% to 90%, particularly 50% to 100%.In an embodiment, T cell lymphocytosis into spheroids is measured using live cell analysis (e.g., IncuCyte™ S3 Live Cell Analysis System, as described elsewhere herein). In an embodiment, modulation of T cell infiltration into cancer spheroids is performed substantially as described in Example 15 herein.

[0195] In an embodiment, an anti-CXCR4 antibody or antigen-binding fragment described herein (significantly) increases cell mobilization, such as CD45+ cell (leukocyte) mobilization, compared to a buffer (e.g., phosphate buffered saline (PBS)) control. In an embodiment, cell mobilization is measured by a cell counter or flow cytometry, particularly flow cytometry. In an embodiment, increased cell mobilization is measured as an average increase in CD45+ cells compared to a control (e.g., a vehicle control (such as PBS), or an isotype control), optionally wherein the antibody or antigen-binding fragment significantly increases the average increase in CD45+ cell mobilization. In an embodiment, CD45+ cell mobilization is performed substantially as described in Examples 16-18 herein.

[0196] In an embodiment, the antibody or antigen-binding fragment comprises a constant region (e.g., C H and / or C L ), e.g., as described elsewhere herein. In an embodiment, the constant region is a mammalian (e.g., human, cynomolgus monkey, rhesus monkey, horse, dog, cat, mouse, rat, etc.) constant region (e.g., CH and / or CL), particularly a rodent constant region, e.g., a murine constant region, such as a mouse or rat constant region. In a preferred embodiment, the antibody or antigen-binding fragment comprises a human constant region (e.g., C H and / or C L ).

[0197] In an embodiment, the heavy chain constant region can be altered (e.g., mutated) to modify the properties of the antibody, e.g., 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. Pub. No. 2012 / 0251531; U.S. Pat. Nos. 9,133,274 and 10,239,944; and PCT Pub. Nos. WO 2014 / 065945, WO 2015 / 150447, and WO 2021 / 016571, each of which is incorporated herein by reference in its entirety.

[0198] In some embodiments, the heavy chain constant region lacks Fc effector function, i.e., an effectorless constant region. In one embodiment, the heavy chain constant region comprises mutations that reduce binding to Fc-gamma receptors and / or Clq compared to wild type. In one embodiment, the heavy chain constant region can lack measurable binding to human FcyRI, FcyRIIa, FcyRIIIa, and FcyRIIIb receptors, but maintains binding to human FcyRIIb receptor and optionally maintains binding to human FcRn receptor. FcyRI, FcyRIla, FcyRIIIa, and FcyRIIIb are examples of activating receptors. FcyRIIb is an example of an inhibitory receptor. FcRn is an example of a recycling receptor.

[0199] In certain embodiments, the antibody or antigen-binding fragment comprises a human IgG4 constant region, e.g., comprising the amino acid sequence of SEQ ID NO: 258 to 263. In one embodiment, the IgG4 heavy chain constant region comprises mutations that reduce binding to Fc-gamma receptors and / or Clq compared to wild type. For example, the constant region can comprise a Leu235Glu (using EU index) mutation. Another optional mutation for the IgG4 heavy chain constant region is a Ser228Pro (using EU index) mutation, which can improve stability. In one embodiment, the human IgG4 constant region can comprise both a Leu235Glu and a Ser228Pro (using EU index) mutation. This "IgG4-PE" heavy chain constant region is effectorless. 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 in SEQ ID NO: 264 to 266 or 304.

[0200] In certain embodiments, the antibody or antigen-binding fragment comprises a human IgGl constant region comprising mutations that reduce effector function (i.e., an inactivated IgGl constant region). In one embodiment, the IgGl constant region comprises mutations that reduce binding to Fc-gamma receptor and / or Clq compared to wild-type, such as substitution of one or more of the amino acids selected from Leu234, Leu235, Gly237, Asp265, Asp270, Asn297, Ala327, Pro329, and Pro331 (using EU index). In one embodiment, the IgGl constant region comprises Leu235Ala and / or Gly237Ala (using EU index) mutations (e.g., amino acid sequence SEQ ID NO: 249, optionally encoded by nucleotide sequence SEQ ID NO: 248). In another embodiment, the IgGl constant region comprises Leu234Phe, Leu235Glu, and Pro331Ser (using EU index) mutations (e.g., amino acid sequence SEQ ID NO: 307, optionally encoded by nucleotide sequence SEQ ID NO: 306).

[0201] Any of the antibodies or antigen-binding fragments of the application can comprise a constant region, such as a rodent constant region or a human constant region (e.g., an IgG4 constant region or an IgGl constant region), e.g., an effectorless human constant region (e.g., IgG4), optionally wherein the constant region is IgG4-PE (SEQ ID NO: 267 or SEQ ID NO: 305) or disabled IgGl (e.g., SEQ ID NO: 249 or SEQ ID NO: 307).

[0202] Constant regions, such as IgG4 or IgGl constant regions as described elsewhere herein, typically end in a C-terminal lysine, which is sometimes cleaved, e.g., during protein production or downstream processing. Any antibody sequence described herein comprising a C-terminal lysine is also intended to encompass embodiments in which such lysine is post-translationally deleted (i.e., cleaved). Thus, in one embodiment, the constant region is IgG4 (e.g., fully human IgG4 or a mutant IgG4), and the IgG4 constant region comprises a C-terminal lysine or does not comprise a C-terminal lysine (lysine-cleaved). In a preferred embodiment, the constant region is IgG4-PE and comprises a C-terminal lysine (e.g., SEQ ID NO: 267) or does not contain a C-terminal lysine (lysine-cleaved) (e.g., SEQ ID NO: 305).

[0203] In another embodiment, the constant region is a wild-type human IgGl, optionally selected from SEQ ID NOs: 241, 243, 245, and 247. For example, the constant region is an effector- enabled IgGl constant region, optionally with ADCC and / or CDC activity. In one embodiment, the constant region is engineered for enhanced ADCC and / or CDC and / or ADCP. The mechanism of antibody-dependent cellular phagocytosis (ADCP) is discussed in Gül et al.,“Antibody-Dependent Phagocytosis of Tumour Cells by Macrophages: A Potent Effector Mechanism of Monoclonal Antibody Therapy of Cancer”, Cancer Res., 75(23), December 1, 2015.

[0204] Without being limited by theory, the antibodies or antigen-binding fragments of the application, which comprise a heavy chain constant region comprising Leu235Glu and / or Ser228Pro mutations (using the EU index) (e.g., an IgG4-PE constant region, optionally SEQ ID NO: 267 or 305) can provide for improved yield of the antibody or antigen-binding fragment during production, or can enhance the biological properties of the antibody or antigen-binding fragment, e.g., in any of the assays described herein. For example, these mutations can reduce the level of apoptosis induced by the antibody or antigen-binding fragment (e.g., by acting directly on CXCR4 or by inhibiting the action of 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., IgGl, disabled IgGl, IgGl mutant, or IgG4). The constant region sequence can contribute to the internalization profile of the antibody or antigen-binding fragment, or can result in the 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., IgGl, IgG4, disabled IgGl, or IgGl mutant).

[0205] 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. Kappa light chain constant region amino acid sequences 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. Lambda light chain constant region amino acid sequences and nucleotide sequences are set forth in SEQ ID NOs: 280-303.

[0206] In one embodiment, the antibody comprises two full-length heavy chains and two full-length light chains (each comprising V H and C H and V L and C L ).

[0207] In one embodiment, the antibody or antigen-binding fragment is a multispecific (e.g., bispecific) antibody or fusion protein, e.g., a bispecific antibody as described above.

[0208] In one embodiment, the antibody or antigen-binding fragment thereof comprises a V H domain comprising a CDRH3, wherein

[0209] a) the CDRH3 amino acid sequence is selected from the group consisting of SEQ ID NOs: 12, 15, 32, 35, 52, 55, 72, 75, 92, and 95, each of which comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s);

[0210] b) the CDRH3 amino acid sequence is identical to a sequence selected from the group consisting of SEQ ID NOs: 12, 15, 32, 35, 52, 55, 72, 75, 92, and 95;

[0211] c) the CDRH3 amino acid sequence is as defined by IMGT or Kabat and from a V H domain selected from the group consisting of SEQ ID NOs: 112, 120, 128, 136, 144, 152, 160, 168, 176, 184, 192, 200, 208, 216, 224, and 232, wherein the CDRH3 sequences each comprise 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s); or

[0212] d) the CDRH3 amino acid sequence is identical to a CDRH3 amino acid sequence as defined by IMGT or Kabat and from a V H domain selected from the group consisting of SEQ ID NOs: 112, 120, 128, 136, 144, 152, 160, 168, 176, 184, 192, 200, 208, 216, 224, and 232.

[0213] In one embodiment, the antibody or antigen-binding fragment comprises a V H domain, wherein

[0214] a. the V H domain comprises:

[0215] i. a CDRH3 amino acid sequence of SEQ ID NO: 12 or 15, or of each thereof comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) of SEQ ID NO: 12 or 15; and / or

[0216] ii. a CDRH1 amino acid sequence of SEQ ID NO: 10 or 13, or of each thereof comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) of SEQ ID NO: 10 or 13; and / or

[0217] iii. a CDRH2 amino acid sequence of SEQ ID NO: 11 or 14, or of each thereof comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) of SEQ ID NO: 11 or 14,

[0218] b. V H the domain comprises:

[0219] i. a CDRH3 amino acid sequence of SEQ ID NO: 32 or 35, or of each thereof comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) of SEQ ID NO: 32 or 35; and / or

[0220] ii. a CDRH1 amino acid sequence of SEQ ID NO: 30 or 33, or of each thereof comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) of SEQ ID NO: 30 or 33; and / or

[0221] iii. a CDRH2 amino acid sequence of SEQ ID NO: 31 or 34, or of each thereof comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) of SEQ ID NO: 31 or 34,

[0222] c. V H the domain comprises:

[0223] i. a CDRH3 amino acid sequence of SEQ ID NO: 52 or 55, or of each thereof comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) of SEQ ID NO: 52 or 55; and / or

[0224] ii. a CDRH1 amino acid sequence of SEQ ID NO: 50 or 53, or of each thereof comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) of SEQ ID NO: 50 or 53; and / or

[0225] iii. a CDRH2 amino acid sequence of SEQ ID NO: 51 or 54, or of each that comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) of SEQ ID NO: 51 or 54,

[0226] d. the VH domain comprises:

[0227] i. a CDRH3 amino acid sequence of SEQ ID NO: 72 or 75, or of each that comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) of SEQ ID NO: 72 or 75; and / or

[0228] ii. a CDRH1 amino acid sequence of SEQ ID NO: 70 or 73, or of each that comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) of SEQ ID NO: 70 or 73; and / or

[0229] iii. a CDRH2 amino acid sequence of SEQ ID NO: 71 or 74, or of each that comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) of SEQ ID NO: 71 or 74,e.V H the VH domain comprises:

[0230] i. a CDRH3 amino acid sequence of SEQ ID NO: 92 or 95, or of each that comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) of SEQ ID NO: 92 or 95; and / or

[0231] ii. a CDRH1 amino acid sequence of SEQ ID NO: 90 or 93, or of each that comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) of SEQ ID NO: 90 or 93; and / or

[0232] iii. a CDRH2 amino acid sequence of SEQ ID NO: 91 or 94, or of each that comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) of SEQ ID NO: 91 or 94,f.V H the VH domain comprises:

[0233] i. a CDRH3 amino acid sequence as defined by IMGT or Kabat and from a VH domain selected from the group consisting of SEQ ID NO: 112 H a VH domain, or of each that comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) of SEQ ID NO: 112 Hdomain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s); and / or

[0234] ii. the CDRH1 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain, or wherein the CDRH1 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s); and / or

[0235] iii. the CDRH2 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain, or wherein the CDRH2 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s),g.V H domain comprises:

[0236] i. the CDRH3 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain, or wherein the CDRH3 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s); and / or

[0237] ii. the CDRH1 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain, or wherein the CDRH1 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s); and / or

[0238] iii. the CDRH2 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain, or wherein the CDRH2 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s),h.VH the domain comprises:

[0239] i. a CDRH3 amino acid sequence as defined by IMGT or Kabat and from a V H the domain, or wherein the CDRH3 amino acid sequence as defined by IMGT or Kabat and from a V H the domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0240] ii. a CDRH1 amino acid sequence as defined by IMGT or Kabat and from a V H the domain, or wherein the CDRH1 amino acid sequence as defined by IMGT or Kabat and from a V H the domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0241] iii. a CDRH2 amino acid sequence as defined by IMGT or Kabat and from a V H the domain, or wherein the CDRH2 amino acid sequence as defined by IMGT or Kabat and from a V H the domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution, i. a V H the domain comprises:

[0242] i. a CDRH3 amino acid sequence as defined by IMGT or Kabat and from a V H the domain, or wherein the CDRH3 amino acid sequence as defined by IMGT or Kabat and from a VH domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0243] ii. a CDRH1 amino acid sequence as defined by IMGT or Kabat and from a V H the domain, or wherein the CDRH1 amino acid sequence as defined by IMGT or Kabat and from a V H the domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0244] iii. the CDRH2 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain, or wherein the CDRH2 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution, j.V H domain comprises:

[0245] i. the CDRH3 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain, or wherein the CDRH3 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0246] ii. the CDRH1 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain, or wherein the CDRH1 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0247] iii. the CDRH2 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain, or wherein the CDRH2 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution, k.V H domain comprises:

[0248] i. the CDRH3 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain, or wherein the CDRH3 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0249] ii. a CDRH1 amino acid sequence as defined by IMGT or Kabat and from a V H domain, or wherein the CDRH1 amino acid sequence as defined by IMGT or Kabat and from a V H domain and comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0250] iii. a CDRH2 amino acid sequence as defined by IMGT or Kabat and from a V H domain, or wherein the CDRH2 amino acid sequence as defined by IMGT or Kabat and from a V H domain and comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution, l.V H domain comprises:

[0251] i. a CDRH3 amino acid sequence as defined by IMGT or Kabat and from a V H domain, or wherein the CDRH3 amino acid sequence as defined by IMGT or Kabat and from a V H domain and comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0252] ii. a CDRH1 amino acid sequence as defined by IMGT or Kabat and from a V H domain, or wherein the CDRH1 amino acid sequence as defined by IMGT or Kabat and from a V H domain and comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0253] iii. a CDRH2 amino acid sequence as defined by IMGT or Kabat and from a V H domain, or wherein the CDRH2 amino acid sequence as defined by IMGT or Kabat and from a V H domain and comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution, m.V H domain comprises:

[0254] i. the CDRH3 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain, or wherein the CDRH3 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain selected from SEQ ID NO: 168 and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0255] ii. the CDRH1 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain, or wherein the CDRH1 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain selected from SEQ ID NO: 168 and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0256] iii. the CDRH2 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain, or wherein the CDRH2 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain selected from SEQ ID NO: 168 and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution, n. V H domain comprises:

[0257] i. the CDRH3 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain, or wherein the CDRH3 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain selected from SEQ ID NO: 176 and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0258] ii. the CDRH1 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain, or wherein the CDRH1 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain selected from SEQ ID NO: 176 and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0259] iii. the CDRH2 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain, or wherein the CDRH2 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution, o.V H domain comprises:

[0260] i. the CDRH3 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain, or wherein the CDRH3 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0261] ii. the CDRH1 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain, or wherein the CDRH1 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0262] iii. the CDRH2 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain, or wherein the CDRH2 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution, p.V H domain comprises:

[0263] i. the CDRH3 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain, or wherein the CDRH3 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0264] ii. a CDRH1 amino acid sequence as defined by IMGT or Kabat and from a V H domain, or wherein the CDRH1 amino acid sequence as defined by IMGT or Kabat and from a V H domain and comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0265] iii. a CDRH2 amino acid sequence as defined by IMGT or Kabat and from a V H domain, or wherein the CDRH2 amino acid sequence as defined by IMGT or Kabat and from a V H domain and comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution, q. H domain comprises:

[0266] i. a CDRH3 amino acid sequence as defined by IMGT or Kabat and from a V H domain, or wherein the CDRH3 amino acid sequence as defined by IMGT or Kabat and from a V H domain and comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0267] ii. a CDRH1 amino acid sequence as defined by IMGT or Kabat and from a V H domain, or wherein the CDRH1 amino acid sequence as defined by IMGT or Kabat and from a V H domain and comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0268] iii. a CDRH2 amino acid sequence as defined by IMGT or Kabat and from a V H domain, or wherein the CDRH2 amino acid sequence as defined by IMGT or Kabat and from a V H domain and comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution, r. H domain comprises:

[0269] i. the CDRH3 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain as defined by IMGT or Kabat and is from a V H domain selected from SEQ ID NO: 208 and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0270] ii. the CDRH1 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain as defined by IMGT or Kabat and is from a V H domain selected from SEQ ID NO: 208 and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0271] iii. the CDRH2 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain as defined by IMGT or Kabat and is from a V H domain selected from SEQ ID NO: 208 and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution, s.V H domain comprises:

[0272] i. the CDRH3 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain as defined by IMGT or Kabat and is from a V H domain selected from SEQ ID NO: 216 and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0273] ii. the CDRH1 amino acid sequence is as defined by IMGT or Kabat and is from a V H domain as defined by IMGT or Kabat and is from a V H domain selected from SEQ ID NO: 216 and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0274] iii. CDRH2 amino acid sequence as defined by IMGT or Kabat and from a V selected from SEQ ID NO: 216 H domain, or wherein the CDRH2 amino acid sequence is as defined by IMGT or Kabat and is selected from the group consisting of V domains of SEQ ID NO: 216 H domain and contains 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions, tV H The domains include:

[0275] i. CDRH3 amino acid sequence as defined by IMGT or Kabat and selected from V of SEQ ID NO: 224 H domain, or wherein the CDRH3 amino acid sequence is as defined by IMGT or Kabat and is selected from the group consisting of V domains of SEQ ID NO: 224 H domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions; and / or

[0276] ii CDRH1 amino acid sequence as defined by IMGT or Kabat and from a V selected from SEQ ID NO: 224 H domain, or wherein the CDRH1 amino acid sequence is as defined by IMGT or Kabat and is selected from the group consisting of SEQ

[0277] ID NO:224's V H domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions; and /

[0278] or

[0279] iii. CDRH2 amino acid sequence as defined by IMGT or Kabat and from a V selected from SEQ ID NO: 224 H domain, wherein the CDRH2 amino acid sequence is as defined by IMGT or Kabat and is selected from the group consisting of SEQ ID

[0280] NO:224's V H domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions, or

[0281] uV H The domains include:

[0282] i. CDRH3 amino acid sequence as defined by IMGT or Kabat and selected from V of SEQ ID NO: 232 H domain, or wherein the CDRH3 amino acid sequence is as defined by IMGT or Kabat and is selected from the group consisting of SEQ

[0283] ID NO:232 V H domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions; and /

[0284] or

[0285] ii. CDRH1 amino acid sequence as defined by IMGT or Kabat and from a V selected from SEQ ID NO: 232 H domain, or wherein the CDRH1 amino acid sequence is as defined by IMGT or Kabat and is selected from the group consisting of SEQ

[0286] ID NO:232 V H domain and comprises 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions; and /

[0287] or

[0288] iii. CDRH2 amino acid sequence as defined by IMGT or Kabat and from a V selected from SEQ ID NO: 232 H domain, or wherein the CDRH2 amino acid sequence is as defined by IMGT or Kabat and is selected from the group consisting of SEQ

[0289] ID NO:232 V H domain and contain 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.

[0290] In one embodiment, the antibody or antigen-binding fragment comprises or further comprises a V comprising CDRL3. L domain, in which

[0291] a. a CDRL3 amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 25, 42, 45, 62, 65, 82, 85, 102, and 105, each of which comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions;

[0292] b. the CDRL3 amino acid sequence is identical to a sequence selected from SEQ ID NO: 22, 25, 42, 45, 62, 65, 82, 85, 102, and 105;

[0293] c. CDRL3 amino acid sequence as defined by IMGT or Kabat and from a V sequence selected from SEQ ID 116, 124, 132, 140, 148, 156, 164, 172, 180, 188, 196, 204, 212, 220, 228 and 236 Ldomains, wherein each of the CDRH3 sequences comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; or

[0294] d. the CDRL3 amino acid sequence is identical to a CDRL3 amino acid sequence as defined by IMGT or Kabat and is from a V L domain.

[0295] In one embodiment, the antibody or antigen binding fragment comprises or further comprises a V L domain, wherein

[0296] a. the V L domain comprises:

[0297] i. a CDRL3 amino acid sequence of SEQ ID NO: 22 or 25, or of each of which comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution of the CDRL3 amino acid sequence of SEQ ID NO: 22 or 25; and / or

[0298] ii. a CDRL1 amino acid sequence of SEQ ID NO: 20 or 23, or of each of which comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution of the CDRL1 amino acid sequence of SEQ ID NO: 20 or 23; and / or

[0299] iii. a CDRL2 amino acid sequence of SEQ ID NO: 21 or 24, or of each of which comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution of the CDRL2 amino acid sequence of SEQ ID NO: 21 or 24,

[0300] b. the V L domain comprises:

[0301] i. a CDRL3 amino acid sequence of SEQ ID NO: 42 or 45, or of each of which comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution of the CDRL3 amino acid sequence of SEQ ID NO: 42 or 45; and / or

[0302] ii. a CDRL1 amino acid sequence of SEQ ID NO: 40 or 43, or of each of which comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution of the CDRL1 amino acid sequence of SEQ ID NO: 40 or 43; and / or

[0303] iii. a CDRL2 amino acid sequence of SEQ ID NO: 41 or 44, or of each of which comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) of SEQ ID NO: 41 or 44,

[0304] c. V L The domain comprises:

[0305] i. a CDRL3 amino acid sequence of SEQ ID NO: 62 or 65, or of each of which comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) of SEQ ID NO: 62 or 65; and / or

[0306] ii. a CDRL1 amino acid sequence of SEQ ID NO: 60 or 63, or of each of which comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) of SEQ ID NO: 60 or 63; and / or

[0307] iii. a CDRL2 amino acid sequence of SEQ ID NO: 61 or 64, or of each of which comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) of SEQ ID NO: 61 or 64,d. V L The domain comprises:

[0308] i. a CDRL3 amino acid sequence of SEQ ID NO: 82 or 85, or of each of which comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) of SEQ ID NO: 82 or 85; and / or

[0309] ii. a CDRL1 amino acid sequence of SEQ ID NO: 80 or 83, or of each of which comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) of SEQ ID NO: 80 or 83; and / or

[0310] iii. a CDRL2 amino acid sequence of SEQ ID NO: 81 or 84, or of each of which comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) of SEQ ID NO: 81 or 84,e. V L The domain comprises:

[0311] i. a CDRL3 amino acid sequence of SEQ ID NO: 102 or 105, or of each of which comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) of SEQ ID NO: 102 or 105; and / or

[0312] ii. a CDRL1 amino acid sequence as defined by IMGT or Kabat and from a V

[0313] iii. a CDRL2 amino acid sequence as defined by IMGT or Kabat and from a V L domain comprises:

[0314] i. a CDRL3 amino acid sequence as defined by IMGT or Kabat and from a V L domain, or wherein the CDRL3 amino acid sequence is as defined by IMGT or Kabat and from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0315] ii. a CDRL1 amino acid sequence as defined by IMGT or Kabat and from a V L domain, or wherein the CDRL1 amino acid sequence is as defined by IMGT or Kabat and from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0316] iii. a CDRL2 amino acid sequence as defined by IMGT or Kabat and from a V L domain, or wherein the CDRL2 amino acid sequence is as defined by IMGT or Kabat and from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or L domain comprises:

[0317] i. a CDRL3 amino acid sequence as defined by IMGT or Kabat and from a V L domain, or wherein the CDRL3 amino acid sequence is as defined by IMGT or Kabat and from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution; and / or

[0318] ii. the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 124; and / or

[0319] iii. the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 124; and / or L domain comprises:

[0320] i. the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 132; and / or

[0321] ii. the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 132; and / or

[0322] iii. the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 132; and / or L domain comprises:

[0323] i. the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 140; and / or

[0324] ii. the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 140; and / or

[0325] iii. the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 140; and / or L domain comprises:

[0326] i. the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 148; and / or

[0327] ii. the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 148; and / or

[0328] iii. the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 148, k. V L domain comprises:

[0329] i. the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 156; and / or

[0330] ii. the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 156; and / or

[0331] iii. the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 156, l. V L domain comprises:

[0332] i. the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 164; and / or

[0333] ii. the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 164; and / or

[0334] iii. the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 164; and / or L domain comprises:

[0335] i. the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 172; and / or

[0336] ii. the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 172; and / or

[0337] iii. the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 172; and / or L domain comprises:

[0338] i. the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 180; and / or

[0339] ii. the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 180; and / or

[0340] iii. the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 180, o. the V L domain comprises:

[0341] i. the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 188; and / or

[0342] ii. the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 188; and / or

[0343] iii. the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 188 L domain comprises:

[0344] i. the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 196; and / or

[0345] ii. the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 196; and / or

[0346] iii. the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 196 L domain comprises:

[0347] i. the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 204; and / or

[0348] ii. CDRL1 amino acid sequence as defined by IMGT or Kabat and from a V selected from SEQ ID NO: 204 L domain, or wherein the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is derived from a V domain selected from SEQ ID NO: 204 comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L domain; and / or

[0349] iii. CDRL2 amino acid sequence as defined by IMGT or Kabat and from a V selected from SEQ ID NO: 204 L domain, or wherein the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is derived from a V domain selected from SEQ ID NO: 204 comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L domain,

[0350] rV L The domains include:

[0351] i. CDRL3 amino acid sequence as defined by IMGT or Kabat and from a V selected from SEQ ID NO: 212 L domain, or wherein the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is derived from a V domain selected from SEQ ID NO: 212 comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L domain; and / or

[0352] ii. CDRL1 amino acid sequence as defined by IMGT or Kabat and from a V selected from SEQ ID NO: 212 L domain, or wherein the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is derived from a V domain selected from SEQ ID NO: 212 comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L domain; and / or

[0353] iii. CDRL2 amino acid sequence as defined by IMGT or Kabat and from a V selected from SEQ ID NO: 212 L domain, or wherein the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is derived from a V domain selected from SEQ ID NO: 212 comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. L domain,

[0354] sV LThe domain comprises:

[0355] i. a CDRL3 amino acid sequence as defined by IMGT or Kabat and from a V L domain, or wherein the CDRL3 amino acid sequence is as defined by IMGT or Kabat and from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 220; and / or

[0356] ii. a CDRL1 amino acid sequence as defined by IMGT or Kabat and from a V L domain, or wherein the CDRL1 amino acid sequence is as defined by IMGT or Kabat and from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 220; and / or

[0357] iii. a CDRL2 amino acid sequence as defined by IMGT or Kabat and from a V L domain, or wherein the CDRL2 amino acid sequence is as defined by IMGT or Kabat and from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 220,

[0358] t. a V L domain comprises:

[0359] i. a CDRL3 amino acid sequence as defined by IMGT or Kabat and from a V L domain, or wherein the CDRL3 amino acid sequence is as defined by IMGT or Kabat and from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 228; and / or

[0360] ii. a CDRL1 amino acid sequence as defined by IMGT or Kabat and from a V L domain, or wherein the CDRL1 amino acid sequence is as defined by IMGT or Kabat and from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 228; and / or

[0361] iii. the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 228,

[0362] u. the V L domain comprises:

[0363] i. the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL3 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 236; and / or

[0364] ii. the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL1 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 236; and / or

[0365] iii. the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain, or wherein the CDRL2 amino acid sequence is as defined by IMGT or Kabat and is from a V L domain comprising 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s) selected from SEQ ID NO: 236.

[0366] According to embodiments herein, a specified CDR (e.g., CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3) is as defined by Kabat or IMGT. In one embodiment, a specified CDR comprises one amino acid substitution, optionally a conservative amino acid substitution. In one embodiment, a specified CDR comprises one or two (e.g., two) amino acid substitutions, optionally conservative amino acid substitutions. In one embodiment, a specified CDR comprises one, two, or three (e.g., three, or two or three) amino acid substitutions, optionally conservative amino acid substitutions. In one embodiment, a specified CDR comprises 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 specified CDR comprises one, two, three, four, or five (e.g., five, four or five, three or five, two or five, or one or five) amino acid substitutions, optionally conservative amino acid substitutions. In one embodiment, a specified CDR comprises one, two, three, four, five, or six (e.g., six, five or six, four or six, three or six, two or six, or one or six) amino acid substitutions, optionally conservative amino acid substitutions. An amino acid substitution includes a change in which an amino acid is replaced by a different naturally occurring amino acid residue. Such substitutions can be classified as "conservative" in which case an amino acid residue contained in the polypeptide is replaced with another naturally occurring amino acid that has similar characteristics in polarity, side chain functionality or size. Such conservative substitutions are well known in the art. In one embodiment, an amino acid substitution is a conservative amino acid substitution, optionally wherein the conservative substitution is a replacement of an amino acid from group (1) to (8) with an amino acid from the same group:

[0367] 1) glycine (G), alanine (A);

[0368] 2) serine (S), threonine (T), cysteine (C), methionine (M);

[0369] 3) aspartic acid (D), glutamic acid (E);

[0370] 4) asparagine (N), glutamine (Q);

[0371] 5) aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q);

[0372] 6) arginine (R), lysine (K), histidine (H);

[0373] 7) glycine (G), alanine (A), isoleucine (I), leucine (L), valine (V);

[0374] 8) Isoleucine (I), Leucine (L), Methionine (M), Valine (V), Proline (P); and

[0375] 9) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0376] 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; and wherein 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. H 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; and wherein 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. L 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; and wherein 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.

[0377] 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; and wherein 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.

[0378] 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; and wherein 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, wherein one of the CDRs (e.g., CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and / or CDRL3) comprises at most one amino acid substitution, which is optionally a conservative amino acid substitution.

[0379] In one embodiment, the antibody or antigen-binding fragment comprises V H domain, in which

[0380] aV H The domain comprises an amino acid sequence that is identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 16;

[0381] bV H a domain comprising an amino acid sequence that is identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 36;

[0382] cV H a domain comprising an amino acid sequence that is identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 56;

[0383] dV H a domain comprising an amino acid sequence that is identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 76;

[0384] eV H a domain comprising an amino acid sequence that is identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 96;

[0385] fV H The domain comprises an amino acid sequence that is identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 112;

[0386] gV H a domain comprising an amino acid sequence that is identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 120;

[0387] hV Hthe domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 128;

[0388] i. V H the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 136;

[0389] j. V H the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 144;

[0390] k. V H the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 152;

[0391] l. V H the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 160;

[0392] m. V H the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 168;

[0393] n. V H the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 176;

[0394] o. V H the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 184;

[0395] p. V H the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 192;

[0396] q. V H the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 200;

[0397] r. V H the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 208;

[0398] s. V H the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 216;

[0399] t.V H the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 224;

[0400] or

[0401] u.V H the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 232.

[0402] In one embodiment, the antibody or antigen binding fragment comprises or further comprises a V L domain, wherein

[0403] a.V L the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 26;

[0404] b.V L the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 46;

[0405] c.V L the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 66;

[0406] d.V L the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 86;

[0407] e.V L the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 106;

[0408] f.V L the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 116;

[0409] g.V L the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 124;

[0410] h.V L the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 132;

[0411] i.V Lthe domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 148;

[0412] j. V L the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 148;

[0413] k. V L the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 156;

[0414] l. V L the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 164;

[0415] m. V L the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 172;

[0416] n. V L the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 180;

[0417] o. V L the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 188;

[0418] p. V L the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 196;

[0419] q. V L the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 204;

[0420] r. V L the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 212;

[0421] s. V L the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 220;

[0422] t. V L the domain comprises an amino acid sequence that is the same as or at least 90%, 95%, or 98% identical to SEQ ID NO: 228;

[0423] or

[0424] u. The V L domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 236.

[0425] For the avoidance of doubt, as used throughout these embodiments, the term "at least X% identical" is intended to refer to "% identity" as defined herein.

[0426] In one embodiment, the antibody or antigen binding fragment comprises a V H domain and a V L domain, wherein

[0427] a. The V H domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 16; and the V L domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 26;

[0428] b. The V H domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 36; and the V L domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 46;

[0429] c. The V H domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 56; and the V L domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 66;

[0430] d. The V H domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 76; and the V L domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 86;

[0431] e. The V H domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 96; and the V L domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 106;

[0432] f. The VH domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 112; and the VL domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 116. H f. The VH domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 112; and the VL domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 116. L f. The VH domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 112; and the VL domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 116.

[0433] f. The VH domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 112; and the VL domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 116. H f. The VH domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 112; and the VL domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 116. L f. The VH domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 112; and the VL domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 116.

[0434] f. The VH domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 112; and the VL domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 116. H f. The VH domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 112; and the VL domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 116. L f. The VH domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 112; and the VL domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 116.

[0435] f. The VH domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 112; and the VL domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 116. H f. The VH domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 112; and the VL domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 116. L f. The VH domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 112; and the VL domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 116.

[0436] f. The VH domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 112; and the VL domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 116.

[0437] f. The VH domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 112; and the VL domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 116. H f. The VH domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 112; and the VL domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 116. L f. The VH domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 112; and the VL domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 116.

[0438] f. The VH domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 112; and the VL domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 116. H f. The VH domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 112; and the VL domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 116. L f. The VH domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 112; and the VL domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to SEQ ID NO: 116.

[0439] mV H The domain comprises an amino acid sequence that is identical or at least 90%, 95% or 98% identical to SEQ ID NO: 168; and V L a domain comprising an amino acid sequence that is identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 172;

[0440] nV H The domain comprises an amino acid sequence that is identical or at least 90%, 95% or 98% identical to SEQ ID NO: 176; and V L a domain comprising an amino acid sequence that is identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 180;

[0441] oV H The domain comprises an amino acid sequence that is identical or at least 90%, 95% or 98% identical to SEQ ID NO: 184; and V L a domain comprising an amino acid sequence that is identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 188;

[0442] pV H The domain comprises an amino acid sequence that is identical or at least 90%, 95% or 98% identical to SEQ ID NO: 192; and V L a domain comprising an amino acid sequence that is identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 196;

[0443] qV H The domain comprises an amino acid sequence that is identical or at least 90%, 95% or 98% identical to SEQ ID NO: 200; and V L a domain comprising an amino acid sequence that is identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 204;

[0444] rV H The domain comprises an amino acid sequence that is identical or at least 90%, 95% or 98% identical to SEQ ID NO: 208; and V L a domain comprising an amino acid sequence that is identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 212;

[0445] sV H The domain comprises an amino acid sequence that is identical or at least 90%, 95% or 98% identical to SEQ ID NO: 216; and V L a domain comprising an amino acid sequence that is identical or at least 90%, 95%, or 98% identical to SEQ ID NO: 220;

[0446] t. V H the domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to, SEQ ID NO: 224; and L the domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to, SEQ ID NO: 228; or

[0447] u. V H the domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to, SEQ ID NO: 232; and L the domain comprises an amino acid sequence that is identical to, or at least 90%, 95%, or 98% identical to, SEQ ID NO: 236.

[0448] In one embodiment, the antibody or antigen binding fragment comprises a heavy chain and a light chain, wherein

[0449] 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;

[0450] b. the heavy chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 18, and the light chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 28;

[0451] 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;

[0452] d. the heavy chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 38, and the light chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 48;

[0453] 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;

[0454] f. the heavy chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 58, and the light chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 68;

[0455] 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;

[0456] h. the heavy chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 78, and the light chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 88;

[0457] 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;

[0458] j. the heavy chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 98, and the light chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 108;

[0459] 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;

[0460] l. the heavy chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 98, and the light chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 110;

[0461] 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;

[0462] n. the heavy chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 114, and the light chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 118;

[0463] 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;

[0464] p. the heavy chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 122, and the light chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 126;

[0465] 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;

[0466] r. the heavy chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 130, and the light chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 134;

[0467] 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;

[0468] t. the heavy chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 138, and the light chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 142;

[0469] 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;

[0470] v. the heavy chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 146, and the light chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 150;

[0471] 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;

[0472] x. the heavy chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 154, and the light chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 158;

[0473] 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;

[0474] z. the heavy chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 162, and the light chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 166;

[0475] 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;

[0476] bb. the heavy chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 170, and the light chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 174;

[0477] 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;

[0478] dd. the heavy chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 178, and the light chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 182;

[0479] 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;

[0480] ff. the heavy chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 186, and the light chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 190;

[0481] 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;

[0482] hh. the heavy chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 194, and the light chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 198;

[0483] 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;

[0484] jj. the heavy chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 202, and the light chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 206;

[0485] 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;

[0486] ll. the heavy chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 210, and the light chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 214;

[0487] 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;

[0488]

[0489] nn. the heavy chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 218, and the light chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 222;

[0490] 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;

[0491] pp. the heavy chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 226, and the light chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 230;

[0492] 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

[0493] ​rr. the heavy chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 234, and the light chain amino acid sequence comprises an amino acid sequence that is at least 90%, 95%, or 98% identical to SEQ ID NO: 238.

[0494] In any of the embodiments described herein, the % identity can be replaced by any of the % identities selected from the group consisting of 70% identical, 75% identical, 80% identical, 85% identical, or 90% identical. In any of the embodiments herein, the % identity can be replaced by any of the % identities selected from the group consisting of 91% identical, 92% identical, 93% identical, 94% identical, or 95% identical, in particular 95% identical. In any of the embodiments herein, the % identity is replaced by any of the % identities selected from the group consisting of 96% identical, 97% identical, 98% identical, 99% identical, or 100% identical, in particular 98% identical.

[0495] In one embodiment, the anti-CXCR4 antibody or antigen binding fragment described herein comprises a V H domain, the V H domain comprises a CDRH3 of 5 to 27 amino acids in length, 5 to 25 amino acids in length, 5 to 24 amino acids in length, 5 to 23 amino acids in length, 5 to 21 amino acids in length, 10 to 25 amino acids in length, 10 to 24 amino acids in length, 10 to 23 amino acids in length, 10 to 21 amino acids in length, 12 to 25 amino acids in length, 12 to 23 amino acids in length, 12 to 21 amino acids in length, 14 to 25 amino acids in length, 14 to 23 amino acids in length, or 14 to 21 amino acids in length. In another embodiment, the CDRH3 is 6, 14, 15, 17, 19, 21, or 23 amino acids in length.

[0496] In one embodiment, the anti-CXCR4 antibody or antigen binding fragment described herein comprises a V H gene segment, a human D gene segment, and a human J H gene segment, wherein the human V H 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 H gene segment is IGHJ3 (e.g., IGHJ3*02), optionally wherein the antibody or antigen binding fragment comprises a V H domain comprising a CDRH3 of 12 to 23 amino acids in length.

[0497] In one embodiment, the anti-CXCR4 antibody or antigen binding fragment described herein comprises a V Hgene segments, human D gene segments and human J H A VH domain recombined with gene segments, wherein the human VH gene segment is IGHV3-30 (eg, IGHV3-30*18), and / or the human D gene segment is IGHD3-16 (eg, IGHD3-16*02), and / or the human J H The gene segment is IGHJ6 (eg, IGHJ6*02), optionally wherein the antibody or antigen-binding fragment comprises a V sequence comprising a CDRH3 of 12 to 23 amino acids in length. H domain.

[0498] In one embodiment, the anti-CXCR4 antibodies or antigen-binding fragments described herein comprise a polypeptide derived from human V H gene segments, human D gene segments and human J H V gene segment recombination H domain, in which human V H The gene segment is IGHV3-73 (eg, IGHV3-73*02), and / or the human D gene segment is IGHD3-9 (eg, IGHD3-9*01), and / or the human J H The gene segment is IGHJ6 (eg, IGHJ6*02), optionally wherein the antibody or antigen-binding fragment comprises a V sequence comprising a CDRH3 of 12 to 23 amino acids in length. H domain.

[0499] In one embodiment, the anti-CXCR4 antibodies or antigen-binding fragments described herein comprise a polypeptide derived from human V H gene segments, human D gene segments and human J H The VH domain of gene segment recombination, in which human V H The gene segment is IGHV4-31 (eg, IGHV4-31*03), and / or the human D gene segment is IGHD3-10 (eg, IGHD3-10*01), and / or the human J H The gene segment is IGHJ6 (eg, IGHJ6*02), optionally wherein the antibody or antigen-binding fragment comprises a V sequence comprising a CDRH3 of 12 to 23 amino acids in length. H domain.

[0500] In one embodiment, the anti-CXCR4 antibodies or antigen-binding fragments described herein comprise a polypeptide derived from human V H gene segments, human D gene segments and human J H V gene segment recombination H domain, in which human V HThe 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 H The gene segment is IGHJ6 (e.g., IGHJ6*02), optionally wherein the antibody or antigen binding fragment comprises a V H domain comprising a CDRH3 of 12 to 23 amino acids in length.

[0501] In one embodiment, an anti-CXCR4 antibody or antigen binding fragment described herein comprises a V H domain comprising a CDRH3, wherein the CDRH3 comprises the amino acid motif AX1GQLERR or X2YGX3DV, wherein X1, X2, and X3 are independently any amino acid and optionally wherein the CDRH3 is 12 to 23 amino acids in length. 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).

[0502] In one embodiment, an anti-CXCR4 antibody or antigen binding fragment described herein comprises a V L domain comprising a CDRL3, the CDRL3 comprising the motif LQX1NX2YPX3T, 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).

[0503] In one embodiment, an 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, especially 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 have any of the sequences of the CDRs, variable regions, and / or full length heavy and light chains as described above. In one embodiment, competition is by the SPR assay as described elsewhere herein. Such competition can be due to, for example, antibodies or antigen binding fragments that bind to the same or overlapping epitopes of CXCR4. In another embodiment, it is determined by ELISA, HTRF, fluorescence activated cell sorting (FACS), or techniques apparent to one of skill in the art and described elsewhere herein. In one embodiment, competition is determined by biolayer interferometry (BLI), which is apparent to one of skill in the art. In one embodiment, the antibody or antigen binding fragment competes (e.g., in a dose dependent manner) with CXCL12 (or a fusion protein thereof) for binding to cell surface expressed CXCR4.

[0504] In one embodiment, an anti-CXCR4 antibody or antigen binding fragment described herein binds to an epitope specifically bound by an antibody or antigen binding fragment selected from the group consisting of 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 (especially CL-82574 or CL-83083-2). In one embodiment, the epitope is identified by site-directed mutagenesis (e.g., alanine scanning), overlapping peptide scanning (e.g., peptidescan), or by X-ray crystallography. In one embodiment, there is provided an antibody or antigen binding fragment that specifically binds to an epitope that is (substantially) similar (or identical) to an epitope specifically bound by any one of 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 of an antibody or antigen binding fragment with an antigen can be determined by various methods known to those of skill in the art. In one embodiment, successive replacement of amino acids of the antigen sequence (mutating the DNA of the antigen-encoding sequence using standard molecular biology techniques), in this case CXCR4, with alanine (also known as alanine scanning) or another inconsequential amino acid, can provide residues that when mutated will decrease or eliminate 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 (which are described elsewhere herein). Other substitutions can be made to improve the disruption of binding, such as changing the charge on the side chain of an amino acid of the antigen sequence (e.g., lysine to glutamic acid), switching a polar and nonpolar residue (e.g., serine to leucine). The alanine scanning or other amino acid substitution methods can be performed directly on cells using transient or stable expression of the mutated forms.When alanine scanning or other amino acid substitution methods are performed using ELISA or HTRF, an amino acid residue is identified as contributing to the epitope if there is at least a 25% or 30% reduction in signal. In one embodiment, there is at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 55%, or at least a 60% reduction in signal. In one embodiment, there is at least a 70%, at least a 75%, at least a 80%, at least a 85%, or at least a 90% reduction in signal. When alanine scanning or other amino acid substitution methods are performed at SPR, an amino acid residue is identified as contributing to the epitope if there is at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 30-fold, at least a 40-fold, at least a 50-fold, or at least a 100-fold. In one embodiment, X-ray crystallography is used to determine the contact residues between the antibody or antigen binding fragment and the antigen (i.e., to determine the epitope bound by the antibody or antigen binding fragment), crystallography allows direct visualization of the contact residues involved in the antibody-antigen interaction; optionally, wherein the 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 the binding of the antibody or antigen binding fragment to these peptides can be assessed using standard techniques (e.g., SPR, HTRF, or ELISA). The epitope can be further mapped by performing alanine scanning on any of the peptides that show binding. As an alternative to linear peptides, Pepscan technology (http: / / www.pepscan.com / ) can be used, which utilizes peptide chemistry to link peptides to a scaffold for conformational scanning (including overlapping peptide scanning), which has been used to determine discontinuous epitopes for 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.).

[0505] In one embodiment, the polypeptide (e.g., CXCR4) is a monomer. In another embodiment, the polypeptide (e.g., CXCR4) forms a homodimer with another identical polypeptide. In another embodiment, the 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, the 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 more than two polypeptides. The multimer consists of multiple identical polypeptides or a combination of different polypeptides or 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., signaling, signaling profile). In one embodiment, dimerization can be determined by proximity-based assays 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 Cross-Linking, and Co-Immunoprecipitation.

[0506] In one embodiment, the anti-CXCR4 antibody or antigen-binding fragment described herein (specifically or additionally) binds to a CXCR4 homodimer. In one embodiment, the antibody or antigen-binding fragment (specifically or additionally) binds to a CXCR4 monomer. In one embodiment, the antibody or antigen-binding fragment (specifically or additionally) binds to a CXCR4 multimer. In one embodiment, the antibody or antigen-binding fragment (specifically or additionally) binds 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 the group consisting of CXCR1, CXCR2, CXCR5, or CXCR6. In another embodiment, the heterodimer comprises CXCR4 and CCR5. In another embodiment, the antibody or antigen-binding fragment (specifically or additionally) binds to CXCR4 complexed with beta-arrestin.

[0507] In one embodiment, a nucleic acid encoding a CDRH3 of an anti-CXCR4 antibody or antigen-binding fragment thereof as described herein is provided.

[0508] In one embodiment, a nucleic acid encoding a V H domain and / or V L domain of an anti-CXCR4 antibody or antigen-binding fragment thereof as described herein is provided. The V H and V L domain nucleic acid sequences of the application are provided in the Sequence Listing.

[0509] In one embodiment, a nucleic acid is provided comprising a nucleotide sequence that is at least 80%, 85%, or 90% identical to:

[0510] a. SEQ ID NO: 19 and / or SEQ ID NO: 29;

[0511] b. SEQ ID NO: 39 and / or SEQ ID NO: 49;

[0512] c. SEQ ID NO: 59 and / or SEQ ID NO: 69;

[0513] d. SEQ ID NO: 79 and / or SEQ ID NO: 89;

[0514] e. SEQ ID NO: 99 and / or SEQ ID NO: 109;

[0515] f. SEQ ID NO: 99 and / or SEQ ID NO: 111 ;

[0516] g. SEQ ID NO: 115 and / or SEQ ID NO: 119;

[0517] h. SEQ ID NO: 123 and / or SEQ ID NO: 127;

[0518] i. SEQ ID NO: 131 and / or SEQ ID NO: 135;

[0519] j. SEQ ID NO: 139 and / or SEQ ID NO: 143;

[0520] k. SEQ ID NO: 147 and / or SEQ ID NO: 151 ;

[0521] 1. SEQ ID NO: 155 and / or SEQ ID NO: 159;

[0522] m. SEQ ID NO: 163 and / or SEQ ID NO: 167;

[0523] n. SEQ ID NO: 171 and / or SEQ ID NO: 175;

[0524] o. SEQ ID NO: 179 and / or SEQ ID NO: 183;

[0525] p. SEQ ID NO: 187 and / or SEQ ID NO: 191 ;

[0526] q. SEQ ID NO: 195 and / or SEQ ID NO: 199;

[0527] r. SEQ ID NO: 203 and / or SEQ ID NO: 207;

[0528] s. SEQ ID NO: 211 and / or SEQ ID NO: 215;

[0529] t. SEQ ID NO: 219 and / or SEQ ID NO: 223;

[0530] u. SEQ ID NO: 227 and / or SEQ ID NO: 231 ; or

[0531] v. SEQ ID NO: 235 and / or SEQ ID NO: 239.

[0532] In one embodiment, there is provided a nucleic acid encoding a heavy chain and / or a light chain of an anti-CXCR4 antibody or antigen-binding fragment thereof as described herein.

[0533] 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 the specified sequence (e.g. defined by a SEQ ID NO). In any of the embodiments herein, the nucleic acid is an isolated and purified nucleic acid.

[0534] In one embodiment, there is provided a vector comprising a nucleic acid as described herein; optionally wherein the vector is a CHO vector or a HEK293 vector.

[0535] In one embodiment, there is provided a host cell comprising a nucleic acid as described herein or a vector as described herein. In one embodiment, the host cell is a mammalian. In one embodiment, the host cell is a CHO (Chinese hamster ovary) cell (e.g. CHO-S cell) or a HEK (human embryonic kidney) cell (e.g. HEK293 or HEK293T cell).

[0536] 3. Uses of the antibodies

[0537] Therapeutic agents

[0538] Any of the anti-CXCR4 antibodies or antigen-binding fragments as described herein can be used for the therapeutic modulation of the CXCL12 / CXCR4 pathway, and thus can be used for the treatment or prevention of a CXCR4-mediated disease or disorder.

[0539] Thus, in one embodiment, there is provided an anti-CXCR4 antibody or antigen-binding fragment as described herein for use in therapy. In one embodiment, there is provided the use of an anti-CXCR4 antibody or antigen-binding fragment as described herein in the manufacture of a medicament for therapy. In another embodiment, there is provided an anti-CXCR4 antibody or antigen-binding fragment as described herein for use in the treatment or prevention of a CXCR4-mediated disease or disorder as described below.

[0540] In another embodiment, there is provided the use of an anti-CXCR4 antibody or antigen-binding fragment as described herein in the manufacture of a medicament for the treatment or prevention of a CXCR4-mediated disease or disorder as described below.

[0541] In another embodiment, a method of treating or preventing a CXCR4-mediated disease or disorder as described below in a patient is provided, comprising administering to the patient a therapeutically or prophylactically effective amount of an anti-CXCR4 antibody or antigen-binding fragment as described herein, wherein the CXCR4-mediated disease or disorder is thereby treated or prevented.

[0542] In another embodiment, a pharmaceutical composition comprising an anti-CXCR4 antibody or antigen-binding fragment as described herein for use in therapy, or for use in the treatment or prevention of a CXCR4-mediated disease or disorder as described below is provided.

[0543] Throughout this specification, a CXCR4-mediated disease or disorder is any disease or disorder that involves abnormal regulation of the CXCR4 pathway, e.g., CXCR4 signaling, downstream effects of CXCR4, and / or overexpression of CXCR4.

[0544] In a preferred embodiment, the CXCR4-mediated disease or disorder is a cancer, in particular a solid tumor. In yet another embodiment, the cancer is characterized by the presence of CD8+ T cells, e.g., T cells that are unable to infiltrate the tumor, e.g., the central tumor mass.

[0545] In one embodiment, the CXCR4-mediated disease or disorder is one or more diseases or disorders independently selected from the group consisting of small cell lung cancer, non-small cell lung cancer (e.g., adenocarcinoma, squamous cell carcinoma, and large cell lung cancer), breast cancer, ovarian cancer (e.g., metastatic ovarian cancer), kidney cancer, stomach cancer, pancreatic cancer, pancreatic ductal carcinoma, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, brain cancer, esophageal cancer, liver cancer, lung cancer, prostate cancer, uterine cancer, brain 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 multiforme (GBM)), medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, neurofibrosarcoma, osteosarcoma, meningioma, neuroblastoma, retinoblastoma, chordoma, CNS lymphoma, brain stem glioma, mixed glioma, optic nerve glioma, subependymal glioma, meningioma, metastatic brain tumor, oligodendroglioma, pituitary tumor, primitive neuroectodermal (PNET) tumor, juvenile pilocytic astrocytoma (JPA), pineal tumor, rhabdomyoma, and human papillomavirus (HPV)-associated cancer (wherein the HPV-positive tumor and / or lesion is cervical intraepithelial neoplasm 1, cervical intraepithelial neoplasm 2, cervical intraepithelial neoplasm 3, cervical carcinoma (in situ and / or invasive), head and neck cancer, HPV papillomatosis, anal papillomatosis, anal carcinoma, vaginal carcinoma, vulvar carcinoma, and / or penile carcinoma).

[0546] In another embodiment, the CXCR4-mediated disease or disorder is a cancer selected from the group consisting of a carcinoma, a sarcoma, a myeloma, a leukemia, and / or a lymphoma. In one embodiment, the CXCR4-mediated disease or disorder is a carcinoma, e.g., an adenocarcinoma and / or a squamous cell carcinoma. In one embodiment, the CXCR4-mediated disease or disorder is an adenocarcinoma (which develops in a gland or an organ), e.g., one or more adenocarcinomas selected from the group consisting of a colon cancer, a breast cancer, a lung cancer, a prostate cancer, an esophageal cancer, a stomach cancer, and a pancreatic cancer. In one embodiment, the CXCR4-mediated disease or disorder is a squamous cell carcinoma, e.g., one or more squamous cell carcinomas selected from the group consisting of a melanoma, a cervical cancer, a head and neck cancer, a vaginal cancer, a thyroid cancer, an esophageal cancer, a lung cancer, a penile cancer, a bladder cancer, a liver cancer, and an anal cancer. In one embodiment, the CXCR4-mediated disease or disorder is a sarcoma, e.g., one or more sarcomas selected from the group consisting of a bladder cancer, a rhabdomyosarcoma, a glioma, and a astrocytoma (Grade I - pilocytic astrocytoma, Grade II - low-grade astrocytoma, Grade III - anaplastic astrocytoma, or Grade IV - glioblastoma multiforme (GBM)). In another embodiment, the CXCR4-mediated disease or disorder is a myeloma, e.g., a multiple myeloma. In another embodiment, the CXCR4-mediated disease or disorder is a lymphoma, e.g., a Hodgkin's lymphoma and / or a non-Hodgkin's lymphoma. In another embodiment, the CXCR4-mediated disease or disorder is a leukemia, e.g., one or more lymphomas selected from the group consisting of a myeloid leukemia, a granulocytic leukemia, a lymphatic leukemia, a lymphocytic leukemia, a lymphoblastic leukemia, a polycythemia vera, and a red cell anemia. In another embodiment, the CXCR4-mediated disease or disorder is a mixed cancer (e.g., wherein the cancer comprises two or more different cancers), e.g., one or more mixed cancers selected from the group consisting of an adenosquamous carcinoma, a carcinoid tumor, a carcinosarcoma, and a teratocarcinoma.

[0547] In one embodiment, the CXCR4-mediated disease or disorder is one or more diseases or disorders independently selected from the group consisting of ovarian cancer (e.g., metastatic ovarian cancer), pancreatic cancer, glioblastoma (GBM), and primitive neuroectodermal (PNET) tumors. In one embodiment, the CXCR4-mediated disease or disorder is one or more diseases or disorders independently selected from the group consisting of ovarian cancer (e.g., metastatic ovarian cancer), pancreatic cancer, glioblastoma (GBM), primitive neuroectodermal (PNET) tumors, small cell lung cancer, non-small cell lung cancer, breast cancer, kidney cancer, pancreatic ductal carcinoma, and colorectal cancer. In one embodiment, the CXCR4-mediated disease or disorder is one or more diseases or disorders independently selected from the group consisting of pancreatic cancer, pancreatic ductal carcinoma, prostate cancer, and small cell lung cancer. In one embodiment, the CXCR4-mediated disease or disorder is one or more diseases or disorders independently selected from the group consisting of 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.

[0548] In one embodiment, the CXCR4-mediated disease or disorder is one or more diseases or disorders independently selected from the group consisting of warts, hypogammaglobulinemia, infection, and myelokathexis (WHIM) (also known as WHIM syndrome), Waldenstrom's macroglobulinemia, human immunodeficiency virus (HIV), thrombocytopenia, ischemic heart failure, post-myocardial infarction recovery, post-myeloablative allogeneic transplantation, bronchiolitis obliterans, neutropenia, malaria, opioid-induced hyperalgesia, rheumatoid arthritis, any of the blood genetic diseases such as sickle cell anemia, hemophilia A, hemophilia B, alpha-thalassemia, beta-thalassemia, delta-thalassemia, von Willebrand Disease, pernicious anemia, Fanconi anemia, thrombocytopenic purpura, thrombophilia, and any of the primary immunodeficiency diseases. In one embodiment, the CXCR4-mediated disease or disorder comprises a blood genetic disease such as any of sickle cell anemia, hemophilia A, hemophilia B, alpha-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 disorder is WHIM or HIV.

[0549] In another embodiment, an anti-CXCR4 antibody or antigen binding fragment described herein is used in a method of 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 disorder is post-myeloablative allogeneic transplantation.

[0550] In another embodiment, an anti-CXCR4 antibody or antigen binding fragment disclosed herein is used in a method of internal radiotherapy. In another embodiment, an anti-CXCR4 antibody or antigen binding fragment disclosed herein is used in a method of wound healing.

[0551] In another embodiment, the CXCR4-mediated disease or disorder comprises a CXCL12-mediated disease or disorder. A CXCL12-mediated disease or disorder comprises any disease or disorder involving abnormal regulation of the CXCL12 pathway (e.g., CXCL12 signaling, downstream effects of CXCL12, and / or overexpression of CXCL12). A CXCL12-mediated disease or disorder includes, but is not limited to, one or more selected from the group consisting of: germ cell tumors and metastasis (e.g., testicular germ cell tumors), pregnancy-related diseases (e.g., pre-eclampsia), and adult-onset Sturge-Weber disease.

[0552] In another embodiment, an anti-CXCR4 antibody or antigen binding fragment disclosed herein is used in combination with radiotherapy to reduce or treat residual tumors, e.g., the antibody or antigen binding fragment is administered after radiotherapy (e.g., 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, or 2 weeks after radiotherapy). In one embodiment, an anti-CXCR4 antibody or antigen binding fragment disclosed herein is used in combination with radiotherapy to reduce or prevent angiogenesis and / or tumor recurrence. This can be by reducing or preventing CXCR4 + CXCR4 +immune cells; e.g., macrophages or T cells) infiltrate into the irradiated (via radiotherapy) tumor to reach. In one embodiment, the residual tumor is a remaining tumor mass. In one embodiment, the tumor is any tumor listed in the “3. Uses of the antibodies” section. In a preferred embodiment, the tumor is a glioblastoma and / or a cervical cancer. Macrophage Exclusion after Radiation Therapy (MERT): A First in Human Phase I / II Trial using a CXCR4 Inhibitor in Glioblastoma. Thomas et al (2019). Clin Cancer Res. 25(23):6948-6957 further describes Macrophage Exclusion after Radiation Therapy in Glioblastoma, MERT.

[0553] In one embodiment, there is provided a method of inhibiting CXCR4 activity in a patient or in vitro, comprising administering an effective amount of an anti-CXCR4 antibody or antigen binding fragment as described herein.

[0554] In one embodiment, there is provided a method of reducing the size or volume of a (malignant) tumor in a patient, comprising administering to the patient an effective amount of an anti-CXCR4 antibody or antigen binding fragment as described herein.

[0555] In one embodiment, there is provided a method or use as described above, further comprising administering (to the patient) a further therapy, optionally wherein the further therapy comprises one or more (e.g., more than one, such as two or three) further therapeutic agents independently selected from:

[0556] a) an immune checkpoint inhibitor (such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIM-3 antibody, an anti-CTLA-4 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-VISTA antibody, and an anti-LAG-3 antibody);

[0557] b) an immune stimulator (such as an anti-OX40 antibody, an anti-HVEM antibody, an anti-CD27 antibody, an anti-CD28 antibody, an anti-CD137L antibody, an anti-OX40L antibody, an anti-GITRL antibody, an anti-ICOSL antibody, an anti-PD-L2 antibody, an anti-GITR antibody, an anti-CD137 antibody, an anti-ICOS antibody, and an anti-CD40 antibody);

[0558] c) Anti-CSF1R antibodies, anti-CXCR2 antibodies, anti-CD200 antibodies, anti- GARP antibodies, anti-SIRPa antibodies, anti-CXCL9 antibodies, anti-CXCL10 antibodies, anti-CXCL11 antibodies, and anti-CD155 antibodies;

[0559] d) Chemokine receptor antagonists (such as CCR4 and CXCR2 chemokine receptors);

[0560] e) Somatosin receptor, EP2 / EP4, A2AR, CD39, CD73 receptor antagonists

[0561] f) Targeted kinase inhibitors (such as CSF-1R, EGFR, or VEGFR inhibitors);

[0562] g) Angiogenesis inhibitors (such as anti-VEGF-A or Delta-like ligand-4);

[0563] h) Immunostimulatory peptides or chemokines (such as CXCL9 or CXCL10);

[0564] i) Cytokines (such as 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, particularly IL-2, IL-12, IL-15, IL-21) and interferons (such as IFN-a, IFN-b, and IFN-g, particularly IFN-g);

[0565] j) Bi-specific T cell engagers (BiTEs) with at least one specificity for CD3 (e.g., CD3 / CD19

[0566] BiTE) and NK cell engagers (e.g., CD16);

[0567] k) Other bi-specific molecules (e.g., IL-2 or IL-15 containing molecules targeting tumor associated antigens such as EGFR, Her-2, New York esophageal cancer-1

[0568] (NY-ESO-1), GD2, EpCAM, or melanoma-associated antigen 3 (MAGE-A3));

[0569] l) oncolytic viruses (such as HSV viruses (optionally secreting GMCSF), Newcastle disease virus, vaccinia virus, adenovirus, NCD virus (paramyxovirus), Sindbis virus, respiratory syncytial virus, measles virus, coxsackie virus, vesicular stomatitis virus, Mengo virus, reovirus, parvovirus, Maraba virus (rhabdovirus), and Echovirus);

[0570] m) vaccination against tumor-associated antigens (such as New York esophageal cancer-1 [NY-ESO-1], melanoma-associated antigen-3

[0571] [MAGE-3], BCMA);

[0572] n) cell-based therapies (such as, for example, 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);

[0573] o) adoptive transfer of tumor-specific T cells (e.g., autologous tumor infiltrating lymphocytes (TILs)) or LAK cells; and

[0574] p) tumor-associated antigen antibodies (such as anti-CEA, anti-GD2, anti- glypican-3, anti-glypican-2, anti-junctional adhesion molecule-4, and anti-mesothelin);

[0575] and / or optionally, wherein yet another therapy is selected from chemotherapy, radiation therapy, and / or surgical resection of the tumor.

[0576] In one embodiment, the chemokine receptor antagonist is an antagonist of the adenosine pathway (e.g., an antibody targeting the adenosine pathway). In another embodiment, the further therapeutic agent is selected from the group consisting of an anti-CD47, anti-SIRPa, anti-KIR, anti-CD70, and anti-GARP antibody. In another embodiment, the further therapeutic agent is selected from the group consisting of an anti-HER2, anti-CD20, anti-NKG2A, anti-TRAIL, and anti-Notch antibody. In another embodiment, the further therapeutic agent is selected from the group consisting of an anti-PVRIG, anti-ILDR2, and anti-CD55 antibody. In one embodiment, the further therapeutic agent is in combination with a further therapy selected from the group consisting of radiation therapy, chemotherapy, and / or an antibody drug conjugate (ADC). In another embodiment, the further therapeutic agent is a small molecule selected from the group consisting of 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, a FGFR-1 / 3, and / or a PDGFR inhibitor. In one embodiment, the further therapeutic agent is selected from the group consisting of ImmTac, an antisense / siRNA / shRNA, a ProTAC, and a prodrug conjugate.

[0577] In one embodiment, there is provided a method or use as described herein, comprising administering to a patient (e.g., a human) an anti-CXCR4 antibody or antigen binding fragment as described, in addition to chemotherapy or radiation therapy. In one embodiment, there is provided a method or use as described herein, comprising administering to a patient an anti-CXCR4 antibody or antigen binding fragment as described herein prior to or after administration of chemotherapy and / or radiation therapy, wherein the antibody or antigen binding fragment increases the efficacy of the chemotherapy and / or radiation therapy (e.g., as compared to when the antibody or antigen binding fragment is not administered). It can be beneficial to administer an anti-CXCR4 antibody or antigen binding fragment as described herein after administration of chemotherapy and / or radiation therapy, as the cancer cells can be more susceptible to the treatment.

[0578] In a preferred embodiment, the further 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 further therapy is a combination of a checkpoint inhibitor (as described above) and chemotherapy. In one embodiment, the further therapeutic agent is a combination of two checkpoint inhibitors (as described above).

[0579] In one embodiment, there is provided a further therapeutic agent comprising an antibody or antigen binding fragment comprising an antigen binding site that specifically binds to an antigen as described in detail below.

[0580] In one embodiment, the additional therapeutic agent is an anti-PD-L1 antibody or antigen binding fragment thereof that specifically binds PD-L1 (e.g., hPD-L1). In one embodiment, the anti-PD-L1 antibody or antigen binding fragment thereof comprises any one of the anti-PD-L1 antibodies selected from atezolizumab (MPDL3280A) (Roche), avelumab (MSB0010718C) (Merck), BMS-936559 / MDX-1105 (BMS), durvalumab (Medi4736) (Medimmune), KN-035, CA-170, FAZ-053 M7824, ABBV-368, LY-3300054, GNS-1480, YW243.55.S70, REGN3504, and WO2019 / 129136, WO2019 / 129211, WO2019 / 132533, WO2019 / 072869, WO2019 / 075097, WO2019 / 085238, WO2019 / 040780, WO2019 / 005634, WO2019 / 005635, WO2019 / 005636, WO2019 / 005637, WO2019 / 005638, WO2019 / 005639, WO2019 / 005642, WO2019 / 005641, WO2019 / 005640, WO2018 / 222949, WO2018 / 194496, WO2018 / 195226, WO2018 / 162430, WO2018 / 162446, WO2018 / 162749, WO2018 / 181064, WO2018 / 153320, WO2018 / 133873, WO2018 / 136553, WO2018 / 119475, WO2018 / 080812, WO2018 / 054940, WO2018 / 034225, WO2018 / 009894, WO2018 / 024237, WO2018 / 026249, WO2018 / 005682, WO2017 / 072280, WO2017 / 215590, WO2017 / 072273, WO2017 / 218435, WO2017 / 196867, WO2017 / 197667, WO2017 / 148424, WO2017 / 118321, WO2017 / 132562, WO2017 / 097407, WO2017 / 084495, WO2017 / 087547, WO2017 / 091429, WO2017 / 034916,WO2017 / 020291, WO2017 / 020858, WO2017 / 020802, WO2017 / 020801, WO2016 / 111645, WO2016 / 050721, WO2016 / 197367, WO2016 / 061142, WO2016 / 149201, WO2016 / 000619, WO2016 / 160792, WO2016 / 022630, WO2016 / 007235, WO2015 / 036499, WO2015 / 179654, WO2015 / 173267, WO2015 / 181342, WO2015 / 109124, WO2015 / 195163, WO2015 / 112805, WO2015 / 061668, WO2014 / 159562, 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 are incorporated herein by reference.

[0581] In one embodiment, the additional therapeutic agent is an anti-PD-1 antibody or antigen binding fragment thereof that specifically binds PD-1 (e.g., hPD-1). In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof comprises any one of the anti-PD-1 antibodies selected from the group consisting of pembrolilzumab (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, TSR-042 ANA011, AUNP-12, KD033, MCLA-134, mDX400, muDX400, STI-A1110, AB011, 244C8, 388D4, XCE853, or pidilizumab (CT-011), or any one of the anti-PD-1 antibodies described in WO2015 / 112800 & US2015 / 0203579 (including antibodies in Tables 1-3), US9,394,365, US5,897,862, and US7,488,802, WO2017 / 087599 (including antibodies SSI-361 and SHB-617), WO2017 / 079112, WO2017 / 071625 (including deposit C2015132, hybridoma LT004, and antibodies 6F5 / 6F5(Re), 6F5H1L1, and 6F5H2L2), WO2017 / 058859 (including PD1AB-1 to PD1AB-6), WO2017 / 058115 (including 67D9, c67D9, and hu67D9), WO2017 / 055547 (including 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375, and 13112.15380), WO2017 / 040790 (including AGEN2033w, AGEN2034w, AGEN2046w, AGEN2047w, AGEN2001w, and AGEN2002w), WO2017 / 025051, and WO2017 / 024515 (including 1.7.3 hAb, 1.49.WO2017 / 025016 & WO2017 / 024465 (including Antibody A to Antibody I), WO2017 / 020858 & WO2017 / 020291 (including 1.4.1, 1.14.4, 1.20.15 and 1.46.11), WO2017 / 019896 and WO2015 / 112900 and US2015 / 0210769 (including BAP049-hum01 to BAP049-hum16 and BAP049-Clone-A to BAP049-Clone-E), WO2017 / 019846 (including PD-1 mAb 1 to PD-1 mAb 15), WO2017 / 016497 (including MHC723, MHC724, MHC725, MHC728, MHC729, m136-M13, m136-M19, m245-M3, m245-M5 and m136-M14), WO2016 / 201051 (including 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), WO2016 / 197497 (including DFPD1-1 to DFPD1-13), WO2016 / 197367 (including 2.74.15 and 2.74.15.hAb4 to 2.74.15.hAb8), WO2016 / 196173 (including Table 5, and antibodies in Figures 1 to 5), WO2016 / 127179 (including R3A1, R3A2, R4B3 and R3D6), WO2016 / 077397 (including antibodies described in Example 9 Table 1), WO2016 / 106159 (including murine antibodies of Example 2 Table 3 and humanized antibodies of Example 3 Tables 7, 8 and 9), WO2016 / 092419 (including C1, C2, C3, EH12.1, mAb7-G4, mAb15-G4, mAb-AAA, mAb15-AAA), WO2016 / 068801 (including clone A3 and variants thereof, and other antibodies described in Figures 1 to 4), WO2016 / 014688 (including 10D1, 4C10, 7D3, 13F1, 15H5, 14A6, 22A5, 6E1, 5A8, 7A4 and 7A4D, and humanized antibodies of Examples 9-10), WO2016 / 015685 (including 10F8, BA08-1, BA-08-2 and 15H6), WO2015 / 091911 & WO2015 / 091910 (including anti-canine PD-1 antibodies of Examples 2, 3 and 4), WO2015 / 091914 (including anti-canine PD-1 antibodies of Table 3), WO2015 / 085847 (including mAb005, H005-1 to H005-4), WO2015 / 058573 (including cAB7), WO2015 / 036394 (including LOPD180), WO2015 / 035606 (including antibodies of Example 2 Table 1, Example 7 Tables 14, 15 and 16, and Example 1 1 Tables 20, 21 and 22), WO2014 / 194302 (including GA2, RG1 B3, RG1 H10, RG2A7, RG2H10, SH-A4, RG4A6, GA1, GB1, GB6, GH1, A2, C7, H7, SH-A4, SH-A9, RG1 H11, and RG6B), WO2014 / 179664 (including 9A2, 10B11, 6E9, APE1922, APE1923, APE1924, APE1950, APE1963 and APE2058), WO2014 / 206107 (including clones 1, 10, 1 1, 55, 64, 38, 39, 41 and 48), WO2012 / 135408 (including h409A11, h409A16, and h409A17), WO2012 / 145493 (including antibodies 1 E3, 1 E8, 1 H3 and h1 H3Var 1 to h1 H3Var 14), WO201 1 / 1 10621 (including antibodies 949 and modified forms disclosed in Figures 1 to 1 1 ), WO201 1 / 1 10604 (including. Figures 3 to 11The antibody 948 disclosed in WO2010 / 089411 (including CNCM deposit no. 1-4122, 1-4080 or 1-4081), WO2010 / 036959 (including the antibody in Example 1, Table 1), WO2010 / 029435 & WO2010 / 029434 (including clones 2, 10 and 19), WO2008 / 156712 (including hPD-1.08A, hPD-1.09A, h409A11, h409A16 and h409A17 and the antibodies described in Example 2, Table H, Example 4 and Table IV), WO2006 / 121168 (including clones 17D8, 4H1, 5C4, 4A11, 7D3, 5F4 and 2D3), WO2004 / 004771 or WO2004 / 056875 (including PD1-17, PD1-28, PD1-33, PD1-35, PD1-F2 and the Abs described in Table 1); the sequences and characteristics of anti-PD-1 antibodies are incorporated herein by reference.

[0582] In one embodiment, the anti-CXCR4 antibody or antigen binding fragment described herein is 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 disorder (as described herein), wherein one, more or all of a-i apply:

[0583] a. increased sensitivity of cancer cells to host immune responses;

[0584] b. decreased immunosuppression in the tumor;

[0585] c. T cells cause apoptosis of tumor cells

[0586] d. enhanced recognition of cancer cells within the tumor;

[0587] e. inhibition of cancer cell growth;

[0588] f. elimination of cancer cells;

[0589] g. reduction in tumor mass;

[0590] h. the tumor comprises FAP+ stromal cells; or

[0591] i. the tumor is resistant to immunotherapy.

[0592] Yet another embodiment includes a method of treating a proliferation- 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 as described herein. Without being limited by theory, this can be achieved by inhibiting the exclusion of T cells from tumors and / or stroma driven by CXCL12 or by inhibiting tumor suppressor cells (e.g., myelin-derived suppressor cells or T-regs) from tumors 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 chemoresistance.

[0593] detecting

[0594] In another embodiment, the antibodies or antigen-binding fragments can be used to detect the presence, absence, and / or amount of CXCR4 expression in a sample. CXCR4 expression can be detected in vivo and / or in vitro and can be used to aid in the diagnosis of a disease or condition involving CXCR4 expression and / or overexpression. In another embodiment, the anti-CXCR4 antibodies or antigen-binding fragments described herein are used to detect a disease or condition, for example, imaging (i.e., PET).

[0595] Guiding patient selection

[0596] In one embodiment, the 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 aid in the selection of patients for therapeutic antibody treatment with an anti-CXCR4 antibody or antigen-binding fragment. In some cases, higher levels of CXCR4 can indicate a successful treatment, while lower levels can indicate a reduced likelihood of success. Preferential expression of splice variants and / or protein processing can result in unique protein mixture profiles that can affect a patient’s response to treatment or can change after treatment. These profiles can aid in identifying patients and defining subsets of patients who should receive treatment, continue treatment, or should receive alternative treatment. In another embodiment, the antibodies or antigen-binding fragments thereof can be used to detect CXCR4 isoforms. Patient samples can include, for example, blood, plasma, serum, sputum, saliva, urine, CSF, tears, exhaled extracellular particles samples, cell supernatants, cells or tissue lysates, or tissue samples.

[0597] 4. Pharmaceutical compositions

[0598] In one embodiment, a pharmaceutical composition is provided comprising an effective amount of an anti-CXCR4 antibody or antigen-binding fragment as described herein and a pharmaceutically acceptable excipient, diluent, or carrier. The therapeutically effective amount of the anti-CXCR4 antibody or antigen-binding fragment employed will depend, for example, on the subject, the route of administration, and the condition of the patient. In one embodiment, the composition includes other excipients or stabilizers.

[0599] Pharmaceutically acceptable carriers are known and include carriers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often a physiologically acceptable carrier is an aqueous pH buffered solution. 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 nonionic surfactants such as TWEEN TM , polyethylene glycol (PEG), and PLURONICS TM .

[0600] An anti-CXCR4 antibody or antigen-binding fragment as described herein or a pharmaceutical composition as described herein can be administered intravenously, for example, as a liquid or a powder aerosol (lyophilized) or by nasal, pulmonary administration. The antibody, antigen-binding fragment, or composition can also be administered parenterally or subcutaneously. When administered systemically, the composition should be sterile, pyrogen-free, and in a physiologically acceptable solution with due consideration of pH, isotonicity, and stability. These conditions are well known to those skilled in the art.

[0601] Methods of administering prophylactic or therapeutic agents, e.g., anti-CXCR4 antibodies or antigen-binding fragments as described herein, or pharmaceutical compositions 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 a particular embodiment, prophylactic or therapeutic agents, e.g., anti-CXCR4 antibodies or antigen-binding fragments as described herein, or pharmaceutical compositions are administered intranasally, intramuscularly, intravenously, or subcutaneously, particularly intravenously. The prophylactic or therapeutic agents or compositions can be administered by any convenient route, e.g., by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, nasal mucosa, rectal mucosa, and intestinal mucosa, etc.), and can be administered, for example, with other therapeutic or biologically active agents. Administration can be systemic or local. Each dose can or can not be administered by the same route of administration. In one embodiment, an anti-CXCR4 antibody or antigen-binding fragment thereof as described herein can be administered via a variety of routes of administration concurrently or subsequently with other doses of the same or different anti-CXCR4 antibody or antigen-binding fragment thereof as described.

[0602] A variety of delivery systems are known and can be used to administer prophylactic or therapeutic agents, e.g., anti-CXCR4 antibodies or antigen-binding fragments thereof as 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 as a part of a retroviral or other vector. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosol. 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.

[0603] In a particular embodiment, it can be desirable to administer a prophylactic or therapeutic agent or pharmaceutical composition as described herein locally to an area in need of treatment. This can be achieved by, for example, local infusion during surgery, by injection, by means of a topical application such as a cream, by means of a gel, or by means of an implant, the implant being 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., anti-CXCR4 antibodies or antigen-binding fragments as described herein, care must be taken to employ a material that does not absorb the antibody or antigen-binding fragment.

[0604] In one embodiment, a pharmaceutical composition is provided comprising an anti-CXCR4 antibody or antigen binding fragment as described herein and a pharmaceutically acceptable excipient, diluent, or carrier, and further comprising one or more (e.g., more than one, or e.g., two) therapeutic agents independently selected from:

[0605] a) an immune checkpoint inhibitor (such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIM-3 antibody, an anti-CTLA-4 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-VISTA antibody, and an anti-LAG-3 antibody);

[0606] b) an immune stimulator (such as an anti-OX40 antibody, an anti-HVEM antibody, an anti-CD27 antibody, an anti-CD28 antibody, an anti-CD137L antibody, an anti-OX40L antibody, an anti-GITRL antibody, an anti-ICOSL antibody, an anti-PD-L2 antibody, an anti-GITR antibody, an anti-CD137 antibody, an anti-ICOS antibody, and an anti-CD40 antibody);

[0607] c) an anti-CSF1R antibody, an anti-CCR4 antibody, an anti-CD39 antibody, an anti-CD73 antibody, an anti-CD96 antibody, an anti-CXCR2 antibody, an anti-CD200 antibody, an anti-GARP antibody, an anti-SIRPa antibody, an anti-CXCL9 antibody, an anti-CXCL10 antibody, an anti-CXCL11 antibody, and an anti-CD155 antibody;

[0608] d) a chemokine receptor antagonist (such as a CCR4 and CXCR2 chemokine receptor);

[0609] e) a somatostatin receptor, EP2 / EP4, A2AR, CD39, CD73 antagonist;

[0610] f) a targeted kinase inhibitor (such as a CSF-1R, EGFR, or VEGFR inhibitor);

[0611] g) an angiogenesis inhibitor (such as an anti-VEGF-A or Delta-like ligand-4);

[0612] h) an immune-stimulating peptide or chemokine (such as CXCL9 or CXCL10);

[0613] i) cytokines (such as 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, in particular IL-2, IL-12, IL-15, IL-21) and interferons (such as IFN-a, IFN-b and IFN-g, in particular IFN-g);

[0614] j) bispecific T cell engagers (BiTEs) with at least one specificity for CD3 (e.g. CD3 / CD19

[0615] BiTE) and NK cell engagers (e.g. CD16);

[0616] k) other bispecific molecules (e.g. IL-2 or IL-15 containing molecules targeting tumor associated antigens such as epidermal growth factor receptor, such as EGFR, Her-2, New York esophageal-1

[0617] (NY-ESO-1), GD2, EpCAM or melanoma-associated antigen 3 (MAGE-A3));

[0618] l) oncolytic viruses (such as HSV viruses (optionally secreting GMCSF), Newcastle disease virus, vaccinia virus, adenovirus, NCD virus (paramyxovirus), Sindbis virus, respiratory syncytial virus, measles virus, coxsackie virus, vesicular stomatitis virus, Mengo virus, Rio virus, parvovirus, Maraba virus (rhabdovirus) and Echn virus);

[0619] m) vaccination against tumor associated antigens (such as New York esophageal-1 [NY-ESO-1], melanoma-associated antigen-3

[0620] [MAGE-3], BCMA);

[0621] n) cell-based therapies (such as 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);

[0622] o) adoptive transfer of tumor specific T cells or LAK cells;

[0623] p) tumor associated antigen antibodies (such as anti-CEA, anti-GD2, anti- glypican-3, anti-glypican-2, anti-junctional adhesion molecule-4 and anti-mesothelin); and

[0624] q) chemotherapy.

[0625] In one embodiment, there is provided a pharmaceutical composition as described herein, or a kit comprising a pharmaceutical composition as described herein, wherein the composition is for use in therapy; or for use in the treatment and / or prevention of a CXCR4-mediated disease or condition as described above.

[0626] In one embodiment, there is provided a pharmaceutical composition as described herein (in combination with a label or instructions) or a kit comprising a label or instructions for use in treating and / or preventing a CXCR4-mediated disease or condition in a patient as described herein; optionally, wherein the label or instructions include a marketing authorization number (e.g. FDA or EMA authorization number). Optionally, the label or instructions can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of drugs or biological products, which reflects approval by the agency of manufacture, use or sale of the drug or biological product 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 the anti-CXCR4 antibody or antigen binding fragment as 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 as described herein is contained in a medical container, e.g. 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 in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, compositions can also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. However, such compositions can be administered by other routes. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed by a drip infusion using a sterile medical grade water or saline solution. Where the composition is to be administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration. Yet another therapeutic agent of the present application can be delivered by any method well known to those of ordinary skill in the art. For example, yet another therapeutic agent can be delivered orally, systemically or locally (to the tumor environment). In one embodiment, yet another therapeutic agent is delivered orally. In one embodiment, yet another therapeutic agent is delivered systemically (e.g. intravenously). In one embodiment, yet another therapeutic agent is delivered locally to the tumor environment. In another embodiment, the anti-CXCR4 antibody and / or other therapeutic agent is delivered locally to the site after surgical resection of all or (substantially) a portion of the tumor.

[0627] 5. Kits and articles of manufacture

[0628] In another embodiment, an article of manufacture is provided comprising a container containing a composition described herein comprising an anti-CXCR4 antibody or antigen binding fragment and a pharmaceutical package insert or label indicating that the composition is useful for treating a disease or disorder characterized by CXCR4 expression or overexpression. In one embodiment, a kit for treating and / or preventing a CXCR4-mediated disease or disorder is provided, the kit comprising an anti-CXCR4 antibody or antigen binding fragment as described in any embodiment or combination of embodiments herein, optionally in combination with a label or instructions for use in treating and / or preventing the disease or disorder in a human; optionally wherein the label or instructions comprise a marketing authorization number (e.g. FDA or EMA authorization number); optionally wherein the kit comprises 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 within a container or IV bag. In another embodiment, the container or IV bag is a sterile container or sterile IV bag. In another embodiment, the antibody or antigen binding fragment is formulated as a pharmaceutical composition contained within a (sterile) container or contained within a (sterile) IV bag. In yet another embodiment, the kit further comprises instructions for use.

[0629] 6. Additional embodiments

[0630] In one embodiment, an antibody or antigen binding fragment thereof is provided that specifically binds to CXCR4, wherein the antibody or antigen binding fragment enables CD8+ T cells to infiltrate a tumor.

[0631] In one embodiment, an antibody or antigen binding fragment thereof is provided that specifically binds to CXCR4 and comprises a V H domain comprising CDRH3 sequences identical to the CDRH3 sequences of an antibody or antigen binding fragment as described herein, or comprising 3, 2, or 1 amino acid substitution(s).

[0632] In one embodiment, an antibody or antigen binding fragment thereof is provided that specifically binds to CXCR4 and comprises a V H domain comprising CDRH1-3 sequences identical to the CDRH1-3 sequences of an antibody or antigen binding fragment as described herein, or comprising 3, 2, or 1 amino acid substitution(s).

[0633] In one embodiment, an antibody or antigen binding fragment thereof is provided that specifically binds to CXCR4 and comprises a V H domain comprising a V H domain comprising a VH The amino acid sequences are identical or at least 90% identical to each other.

[0634] In one embodiment, an antibody or antigen-binding fragment thereof is provided that specifically binds to CXCR4 and comprises a V region containing CDRL3. L domain, wherein the CDRL3 sequence is identical to the CDRL3 sequence of an antibody or antigen-binding fragment as described herein, or comprises 3, 2, or 1 amino acid substitutions.

[0635] In one embodiment, an antibody or antigen-binding fragment thereof is provided that specifically binds to CXCR4 and comprises a V region comprising CDRL1, CDRL2, and CDRL3. L domain, wherein the CDRL1-3 sequence is identical to the CDRL1-3 sequence of an antibody or antigen-binding fragment as described herein, or comprises 3, 2, or 1 amino acid substitutions.

[0636] In one embodiment, an antibody or antigen-binding fragment thereof is provided that specifically binds to CXCR4 and comprises V L domain, in which V L The domain comprises a V domain that is identical to an antibody or antigen-binding fragment as described herein. L The amino acid sequences are identical or at least 90% identical to each other.

[0637] In one embodiment, an antibody or antigen-binding fragment thereof is provided that specifically binds to CXCR4 and comprises V H domain and V L domain, in which V H The domain comprises a V domain that is identical to an antibody or an...

Claims

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

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

26.

3. The antibody or fragment according to any one of the preceding claims, wherein: a) CXCR4 is human (optionally selected from SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3); b) CXCR4 is rhesus and / or cynomolgus macaque (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 monkey, cynomolgus monkey and / or rodent CXCR4, optionally human and cynomolgus monkey CXCR4; Optionally wherein the binding is determined by surface plasmon resonance (SPR), flow cytometry, live cell imaging, ELISA or radioligand binding.

4. The antibody or fragment according to any one of the preceding claims, wherein the antibody or fragment comprises a constant region (C H and / or C L ), optionally wherein: a)C H 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 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)C L It is the kappa light chain constant region.

5. The antibody or fragment according to any one of the preceding claims, 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 according to any one of the preceding claims, wherein the antibody or fragment inhibits the binding of CXCL12 to CXCR4.

7. The antibody or fragment according to any one of the preceding claims, wherein the antibody or fragment does not induce apoptosis in T cells (optionally CD8+ T cells), optionally wherein apoptosis is determined using flow cytometry.

8. The antibody or fragment according to any one of the preceding claims, wherein the antibody or fragment has an EC of 0.5 to 10 nM (e.g. 0.5 to 5 nM). 50 Binding to cynomolgus monkey CXCR4, optionally wherein cynomolgus monkey CXCR4 binding is determined using flow cytometry.

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

10. The antibody or fragment of any preceding claim, wherein the antibody or fragment does not bind to CXCR7 (optionally, wherein 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. The antibody or fragment according to any one of the preceding claims, wherein the antibody or fragment has an IC of 0.5 to 20 nM (e.g. 0.5 to 5 nM). 50 Inhibits CXCL12-mediated T cell responses, measured in vitro using a label-free dynamic mass redistribution assay.

12. The antibody or fragment according to any one of the preceding claims, wherein the antibody or fragment has an IC of 0.01 to 5 nM (eg 0.01 to 1 nM). 50 Inhibits chemotaxis of CXCR4+ T cells toward CXCL12, where the inhibition is measured using live cell imaging or flow cytometry.

13. The antibody or fragment according to any one of the preceding claims, wherein the antibody or fragment increases mean CD45+ cell mobilization compared to PBS, optionally wherein the mobilization is determined using flow cytometry.

14. The antibody or fragment of any preceding claim, wherein the antibody or fragment binds to a CXCR4 homodimer.

15. The antibody or fragment of any preceding claim, wherein the antibody or fragment enables CD8+ T cells to infiltrate a tumor.

16. An antibody or fragment according to any preceding claim for use in therapy.

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

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

19. The antibody or fragment for use according to any one of claims 16 to 18, wherein the treatment further comprises administering a further therapy, optionally wherein the further therapy comprises one or more further 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 further therapy is selected from chemotherapy, radiotherapy and / or surgical resection of the tumor.

20. The antibody or fragment for use according to any one of claims 16 to 19, further comprising administering a further therapeutic agent which 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. A pharmaceutical composition comprising the antibody or fragment according to any one of claims 1 to 15 and a pharmaceutically acceptable excipient, diluent or carrier, and optionally further comprising one or more further therapeutic agents.

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

23. A vector comprising the nucleic acid according to claim 22; optionally wherein the vector is a CHO or HEK293 vector.

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

Images