Anti-CXCR2 Antibodies and Uses Thereof

Abstract

Anti-CXCR2 antibodies and antigen-binding fragments thereof are provided. The antibodies or fragments thereof specifically bind to the N-terminal, extracellular domain of the CXCR2 protein. In various examples, the antibodies or fragments thereof comprise the VH CDR1 of SEQ ID NO: 1, the VH CDR2 of SEQ ID NO: 2, the VH CDR3 of SEQ ID NO: 3, or any one of SEQ ID NOs: 7-14, the VL CDR1 of SEQ ID NO: 4, the VL CDR2 of SEQ ID NO: 5, and the VL CDR3 of SEQ ID NO: 6, or variants thereof. Methods of using the antibodies or fragments thereof for the treatment and diagnosis of diseases, such as cancer and inflammatory diseases, are also provided.

Description

Priority claim

[0001] This invention claims priority to PCT / CN2019 / 104336, filed September 4, 2019, the contents of which are incorporated herein in their entirety. [Technical field]

[0002] The G protein-coupled receptor (GPCR) superfamily is one of the largest families in the human genome, consisting of more than 800 members, and is widely distributed in various organs and tissues, including the central nervous system, immune system, and cardiovascular system. GPCRs are widely involved in human physiological and pathological processes. Approximately 40% of approved drugs mediate their actions through GPCRs. [Background technology]

[0003] Most GPCRs can be classified into five major families based on their evolutionary homology and physiological ligand properties: rhodopsin, adhesion, secretin, glutamate, and Frizzled / TAS2. Rhodopsin is the largest and most heterogeneous family, which is further divided into four subfamilies: α, β, γ, and δ. These proteins have a similar structure: an extracellular N-terminus, seven transmembrane domains, and a cytoplasmic C-terminus. They sense various extracellular stimuli through interactions with various ligands, such as amino acids, nucleic acids, peptides, and proteins, and activate intracellular signaling pathways through ligand-induced conformational changes. In canonical signaling, GPCR signals are transmitted by the recruitment of intracellular GTP-dependent proteins (G proteins) to specific cytoplasmic domains at the C-terminus, the so-called G protein-dependent signaling. Then, downstream pathways, such as the cAMP pathway or the PIP2 pathway, are activated depending on the specific G protein involved.

[0004] Furthermore, recruitment of other scaffolding proteins within the cell, such as β-arrestins, has been shown to activate G protein-independent signaling. Multiple signaling allows GPCRs to have multiple functions within the cell, making it difficult to design relevant assays that link any single target receptor to specific downstream cellular activities. Developing highly effective therapeutics against these important targets remains a major challenge for the pharmaceutical industry. Also, although antibodies have emerged as a rising source of new therapeutics, only one therapeutic antibody against a GPCR has been developed yet. This is mainly due to the difficulty in generating antibodies that bind to the functional conformation of these membrane proteins.

[0005] Combinatorial antibodies have emerged as a powerful tool in drug discovery. Over 81 antibodies derived from phage panning have entered clinical trials, with over 11 of them receiving marketing approval. The combinatorial antibody library approach exploits the enormous diversity of a repertoire of up to 1014 different binding molecules. Antibodies selected from such libraries display diverse mechanisms and can be neutralizing or function as agonists, antagonists, or inverse agonists. In some cases, they have been shown to exert functions beyond the range of the native ligand.

[0006] CXCR2 (CXC motif-type chemokine receptor 2) is a member of the chemokine receptor family and is mainly expressed on neutrophils. CXCR2 has been shown to be involved in the chemotaxis of neutrophils following inflammatory stimuli. However, in various diseases involving inflammation, such as colitis, chronic obstructive pulmonary disease (COPD), asthma, and glomerulonephritis, unwanted migration of neutrophils can play a large part in the pathology. For example, previous studies have reported that depletion of CXCR2 protects the lungs from cigarette smoke-induced inflammation and injury. Furthermore, CXCR2 has been shown to be involved in the progression of various types of cancer, playing a key role in tumor cell proliferation, survival, and metastasis, and affecting the entire tumor microenvironment.

[0007] Based on these clinical findings, inhibition of CXCR2-mediated neutrophil migration has been considered to be an important, yet unrealized, strategy for treating neutrophil-associated inflammatory diseases and some cancers. Although several small molecules targeting CXCR2 have already shown significant inhibitory effects on the receptor in vitro or animal experiments, they have not shown therapeutic efficacy in clinical settings. This is believed to be due to non-functional binding to CXCR2 and off-target effects.

[0008] In contrast, antibodies could overcome these clinical challenges in that they have high specificity, high serum stability, high safety, and because of their size they can block ligand binding to receptors. However, it has been difficult to identify functional antibodies targeting GPCRs because of the difficulty in preparing sufficient amounts of stable antigen in the native conformation for selection. Summary of the Invention [Problem to be solved by the invention]

[0009] The present disclosure provides antibodies and antigen-binding fragments that can bind to CXCR2 and effectively or completely inhibit its binding by IL-8, thereby inhibiting IL-8-induced neutrophil chemotaxis. These antibodies and fragments exhibit excellent affinity (e.g., picomolar level), overcoming the problem that IL8 itself binds with high affinity (nanomolar level), resulting in complete inhibition of IL8-induced cellular functions. Furthermore, these antibodies exhibit biased agonism effects, activating CXCR2 endocytosis and more efficiently suppressing CXCR2-mediated pathological events.

[0010] Further experiments showed that the tested anti-CXCR2 antibodies interacted with CXCR2 primarily through the side chains of some of the amino acid residues at the extracellular N-terminus of the human CXCR2 protein (residues 1-48, especially residues 9-19). That such an interaction was sufficient for inhibition of IL8 binding and IL8-induced cellular functions was surprising and unexpected, at least because it had previously been suggested that CXCR2 has three other extracellular domains (loops) that are also involved in IL8 binding. [Means for solving the problem]

[0011] Thus, according to one embodiment of the present disclosure, there is provided an antibody or fragment thereof capable of specifically binding to human CXC motif-type chemokine receptor 2 (CXCR2) protein, said binding involving at least one of amino acid residues within residues 9-19 of SEQ ID NO:15. In some embodiments, the binding comprises at least one of D13, F14 and W15. In some embodiments, the binding comprises at least F14 and W15. In some embodiments, the binding comprises at least D13, F14, and W15. In some embodiments, the binding does not comprise any amino acid residues outside the 48 N-terminal residues. In some embodiments, the antibody or fragment thereof inhibits binding between CXCR2 and IL-8 protein.

[0012] Another embodiment is an antibody or fragment thereof having specificity for human C-X-C motif-type chemokine receptor 2 (CXCR2) protein, comprising a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3, and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO:1 or a variant having an amino acid substitution from SEQ ID NO:1; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:2 or a variant having an amino acid substitution from SEQ ID NO:2; the VH CDR3 comprises the amino acid sequence of SEQ ID NO:3, a variant having an amino acid substitution from SEQ ID NO:3, or a variant selected from the group consisting of SEQ ID NOs:7-14; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:4 or a variant having an amino acid substitution from SEQ ID NO:4; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:5 or a variant having an amino acid substitution from SEQ ID NO:6, the VL CDR3 comprises the amino acid sequence of SEQ ID NO:6, or a variant having an amino acid substitution from SEQ ID NO:6;

[0013] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO:1; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:2; the VH CDR3 comprises the amino acid sequence of SEQ ID NO.3, or a variant selected from the group consisting of SEQ ID NOs:7-14; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:4; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:5; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the VH CDR3 comprises the amino acid sequence of SEQ ID NO:3.

[0014] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:17 or 18, or a peptide having at least 90% sequence identity to SEQ ID NO:17 or 18. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:18, or a peptide having at least 90% sequence identity to SEQ ID NO:18. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO:19, or a peptide having at least 90% sequence identity to SEQ ID NO:19.

[0015] Therapeutic methods and uses are also provided. In one embodiment, a method for treating cancer or inflammatory disease in a patient in need of treatment is provided, comprising administering to the patient an effective amount of an antibody or fragment thereof of the present disclosure. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is selected from the group consisting of bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, esophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer, and thyroid cancer.

[0016] In some embodiments, the inflammatory disease is one or more of Parkinson's disease, arthritis, rheumatoid arthritis, multiple sclerosis, psoriasis, psoriatic arthritis, Crohn's disease, inflammatory bowel disease, ulcerative colitis, lupus, systemic lupus erythematosus, juvenile rheumatoid arthritis, juvenile idiopathic arthritis, Grave's disease, Hashimoto's thyroiditis, Addison's disease, celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis, pernicious anemia, Sjogren's syndrome, type I diabetes, vasculitis, uveitis, arteriosclerosis, and ankylosing spondylitis. [Brief description of the drawings]

[0017] [Figure 1]Figure 1 shows epitope-guided selection of antibodies with tight binding to hCXCR2. Figure 1A is a schematic diagram of antibodies selected by phage panning based on the IL-8 epitope on the extracellular N-terminus of human CXCR2; Figure 1B is a diagram showing the binding affinity measurements (fitted curves & KD values ​​of sensorgram plots) of two combinatorial antibodies abN48-IgG1 and abN48-2-IgG1 by SPR; Figure 1C is a diagram showing the results of HDX-MS showing the binding sites of selected antibodies to the N-terminus of CXCR2; Figure 1D is a resolved crystal structure diagram showing the three-dimensional structures of abN48-IgG1 and abN48-2-IgG1 (gray and purple, respectively) complexed with the epitope sequence (green and red lines) on the N-terminus of human CXCR2. [Diagram 2] Figure 2 shows the species and subtype specificity of abN48-IgG1 and ab48-2-IgG1. Figure 2A shows the colocalization of abN48-IgG1 or abN48-2-IgG1 with hCXCR2, hCXCR1, and mCXCR2 overexpressed on the membrane of U2OS cells (all tested receptor proteins were fused with mCherry fluorescent tag, and immunofluorescence images were taken by confocal microscopy); Figure 2B shows the surface interaction of abN48-IgG1 and abN48-2-IgG1 with hCXCR2, hCXCR1, mCXCR2, rCXCR2, rabbit CXCR2, and macaca CXCR2 overexpressed on U2OS cells (flow cytometry was used to measure the interaction of antibodies with CXCR2 receptor proteins). [Diagram 3]Figure 3 shows the intracellular signaling pathway mediated by CXCR2. Figure 3A shows activation of β-arrestin signaling by IL-8, GROα (CXCL1), and abN48 antibody ligand using Tango reporter gene assay; Figure 3B shows inhibition of chemokine-induced β-arrestin signaling by abN48-IgG1 and abN48-2-IgG1 (inducing concentrations of IL-8 (left) and CXCL1 (right) were corresponding EC80 values); Figure 3C shows activation of Ca2+ influx by IL-8, GROα (CXCL1), and abN48 antibody ligand using FLIPR measurement; Figure 3D shows inhibition of Ca2+ influx by abN48-IgG1 and abN48-2-IgG1 using Tango reporter gene assay; Figure 3E shows inhibition of Ca2+ influx by abN48-IgG1 and abN48-2-IgG1 using Tango reporter gene assay; Figure 3F shows inhibition of Ca2+ influx by abN48-IgG1 and abN48-2-IgG1 using Tango reporter gene assay; Figure 3G shows inhibition of Ca2+ influx by abN48-IgG1 and abN48-2-IgG1 using Tango reporter gene assay; Figure 3H ... FIG. 3B shows inhibition of chemokine-induced Ca2+ influx by abN48-IgG1 and abN48-2-IgG1 (inducing concentrations of IL-8 (left) and CXCL1 (right) were the corresponding EC90 values. Data are presented as mean ± SD. EC50 and IC50 calculations are shown next to the fitted curves); FIG. 3E shows activation of CXCR2 internalization by abN48 antibody (U2OS cells with or without stably overexpressing CXCR2 were used with abN48-IgG1 and immunotoxin conjugates of toxin, antibody, or toxin alone. Data are presented as mean ± SD). [Figure 4] Figure 4 shows that abN48-IgG1 and abN48-2-IgG1 potently inhibit IL-8-induced neutrophil chemotaxis. Figure 4A shows that abN48-IgG1 and abN48-2-IgG1 (FITC-conjugated) bind specifically to human primary neutrophils to the same extent. Figure 4B shows that the inhibitory effect of abN48-IgG1 and abN48-2-IgG1 on IL8 (10 nM)-induced neutrophil chemotaxis was measured by chemotaxis assay. Blank: medium without IL8 or inhibitor, IL8: 10 nM IL8, iso: 10 nM irrelevant human IgG1 antibody, MK: 1 μM small molecule CXCR2 inhibitor MK7123. The percentage (%) values ​​above the blue and green bars represent the percentage of inhibition of IL8-induced neutrophil chemotaxis in various concentrations of abN48-2-IgG2 and 1 μM MK7123, respectively. [Diagram 5]Selection of combinatorial antibodies targeting hCXCR2. Figure 5A shows that nine positive clones obtained from library panning were expressed as secreted combinatorial scFv antibodies, where H8 is also designated as abN48. Binding of these clones to pepN48 was confirmed by ELISA. Figure 5B shows that binding of abN48(H8) to cell surface expressed hCXCR2 was measured by FACS. Figure 5C shows that binding affinity of abN48(H8) to pepN48 was determined by SPR assay on Biacore T20. [Figure 6] Figure 6A shows the construction of full-length IgG1 format antibodies from abN48 to abN48-IgG, and SDS-PAGE of abN48 and abN48-IgG1. Figure 6B shows the Weblogo analysis of mutant CDR3 sequences in affinity maturation. [Figure 7] Figure 1 shows the homogeneity analysis of recombinant combinatorial antibodies by SEC-HPLC. The shoulder peak highlighted in red box represents an impurity arising when the antibody protein was incubated at 42°C. [Figure 8] Figure 1 shows the inverse agonist effect of combinatorial antibodies on CXCR2-mediated β-arrestin signaling. Endogenous β-arrestin recruitment (represented as red bars labeled Basal) induced by membrane expression of hCXCR2 was significantly reduced in the presence of different concentrations of abN48-IgG1 (green bars) and abN48-2-IgG1 (blue bars). [Figure 9] Figure 9 shows truncation mutagenesis of the N-terminus of hCXCR2 to map the epitope region: Figure 9A is a schematic diagram of the designed N- or C-terminal truncated synthetic peptides of the CXCR2-N-terminus; Figure 9B shows the interaction between the N-terminal peptide of hCXCR2 and abN48-IgG1 (gray) or abN48-2-IgG1 (black). [Figure 10]Overall structure of abN48 / NT9-19 protein complexes. Figure 10A shows two abN48s in one asymmetric unit as cartoon models, with the bound NT9-19 peptide depicted as yellow stick models surrounded by 2Fo-Fc electron density maps contoured at 1.1σ. The left abN48 is colored gray, the others as follows: light chain, cyan; heavy chain, light; light chain CDR, tv_green; heavy chain CDR, tv_red. Figure 10B shows that in one abN48 / NT9-19 protein complex, NT9-19 residues EDFWK12-16aa were well traced from the electron density (left), whereas in the other, only the electron density for residues FW14-15aa was visible (right). Residues traced from the electron density were depicted as yellow stick models surrounded by marine (left) or purple (right) density maps contoured at 1.1σ. [Figure 11]Residues EDFWK12-16aa of CXCR2_N interact with the CDR loops of abN48 mainly through hydrophobic interactions. Figure 11A shows the side view structure (left) and top view structure (right) of the complex of NT9-19 and abN48. The CDR loops of the heavy chain are colored in tv_red and the light chain in tv_green, the rest of the abN48 light chain in cyan, and the rest of the abN48 heavy chain in light. Residues EDFWK12-16aa of NT9-19 can be traced from the electron density and are shown as yellow stick models surrounded by a 2Fo-Fc electron density map contoured at 1.1σ. The rest of abN48 is made transparent, except for the CDR loops. Figure 11B shows that residues FW14-15aa of NT9-19 fit snugly into the large hydrophobic pocket formed by the CDR loops at the top of abN48. The electrostatic surface map of abN48 is colored depending on the local electrostatic potential, ranging from +10 V (dark blue) to -10 V (dark red). Figure 11C shows that F14 and W15 of NT9-19 insert their bulky aromatic side chains into the hydrophobic cavity formed by the CDR loops of abN48 and form strong π-π stacking interactions mainly with residues of abN48. Residues EDFWK12-16aa of NT9-19 and related residues of abN48 are all drawn as stick models and colored as in Figure 11A. [Figure 12] Figure 12 shows site-directed mutagenesis for epitope mapping. Amino acid residues (D13, F14, W15, K16) in the N-terminal IL-8 binding region of hCXCR2 were subjected to alanine-scanning site-directed mutagenesis. Point mutants of hCXCR2 were expressed as mCherry fusion proteins in U2OS cells and stained with abN48-IgG1 antibody. Figure 12A shows the ICC results indicating that D13, F14, and W15 are the three key residues for antibody binding; Figure 12B shows the FACS results indicating that D13, F14, and W15 are the three key residues for antibody binding. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] definition It should be noted that the term "a" or "an" entity refers to one or more of that entity. For example, "an antibody" is understood to represent one or more antibodies. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0019] As used herein, the term "polypeptide" is intended to encompass the singular "polypeptide" and the plural "polypeptides" and refers to a molecule consisting of monomers (amino acids) linked in a linear chain by amide bonds (also called peptide bonds). The term "polypeptide" also refers to any chain of two or more amino acids and does not refer to a specific length. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with these terms. The term "polypeptide" is also intended to refer to the product of post-expression modifications of a polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but not necessarily translated from a specified nucleic acid sequence. It may be produced by any method, including chemical synthesis.

[0020] "Homology" or "identity" or "similarity" refers to the similarity of sequences between two peptides or two nucleic acid molecules. Homology can be determined by comparing the positions of each sequence that are aligned for purposes of comparison. If a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, preferably less than 25% identity, with one of the sequences of the present disclosure.

[0021] The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence that has a degree of homology, i.e., sequence identity, with the nucleotide sequence of the nucleic acid or its complement. A homolog of a double-stranded nucleic acid is intended to include a nucleic acid having a nucleotide sequence that has a degree of homology with it or its complement. In one embodiment, a homolog of a nucleic acid is capable of hybridizing to a nucleic acid or its complement. Similarly, an "equivalent polypeptide" refers to a polypeptide that has a degree of homology, i.e., sequence identity, with the amino acid sequence of a reference polypeptide. In some embodiments, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some embodiments, an equivalent polypeptide or polynucleotide has one, two, three, four, or five additions, deletions, substitutions, and combinations thereof, compared to the reference polypeptide or polynucleotide. In some embodiments, an equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridges) of the reference sequence.

[0022] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen-binding fragment thereof or a single chain. Thus, the term "antibody" includes any protein or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Examples include, but are not limited to, a heavy or light chain complementarity determining region (CDR) or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, or at least a portion of a binding protein.

[0023] The term "antibody fragment" or "antigen-binding fragment" as used herein refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, and scFv. Regardless of structure, an antibody fragment binds with the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes synthetic or genetically engineered proteins that act like antibodies by binding to a specific antigen to form a complex.

[0024] "Single chain variable region fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin. In some embodiments, these regions are connected by a short linker peptide of 10 to about 25 amino acids. The linker is glycine-rich for flexibility and contains serine or threonine for solubility, and can connect the N-terminus of VH to the C-terminus of VL, or vice versa. The protein retains the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of the linker. ScFv molecules are known in the art, for example, U.S. Pat. No. 5,892,019.

[0025] The term antibody encompasses a wide variety of classes of biochemically distinguishable polypeptides. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (, , , ), with several subclasses (e.g., l-4) between them. The nature of the chain determines the "class" of the antibody, IgG, IgM, IgA IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and are known to result in functional differentiation. Modified versions of each of these classes and isotypes are readily discernible to one of skill in the art in view of the instant disclosure, and are therefore within the scope of the instant disclosure. All immunoglobulin classes are expressly within the scope of the instant disclosure, and the following discussion shall be generally directed to the IgG class of immunoglobulin molecules. For IgG, a standard immunoglobulin molecule contains two identical light polypeptide chains of molecular weight approximately 23,000 daltons, and two identical heavy polypeptide chains of molecular weight 53,000-70,000. The four chains are usually joined by disulfide bonds in a "Y" configuration, with the light chains joining the heavy chains from the mouth of the "Y" to the variable region.

[0026] Antibodies, antigen-binding polypeptides, variants, or derivatives thereof of the present disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments such as Fab, Fab', and F(ab')2, Fd, Fv, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (sdFv), fragments comprising either the VK or VH domains, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (such as, for example, anti-Id antibodies to the LIGHT antibodies disclosed herein). Immunoglobulin or antibody molecules of the present disclosure may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclass of immunoglobulin molecule.

[0027] Light chains are classified as either kappa or lambda (,). Each heavy chain class can associate with either kappa or lambda light chains. Generally, light and heavy chains are covalently linked to each other, and when immunoglobulins are produced by either hybridomas, B cells or genetically engineered host cells, the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chains, the amino acid sequence runs from the N-terminus at the forked end of the Y to the C-terminus at the bottom of each chain.

[0028] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be understood that the variable domains of both the light (VK) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light (CK) and heavy (CH1, CH2 or CH3) chains confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, and complement binding. The constant region domains are usually numbered increasing as they move away from the antigen-binding site or amino terminus of the antibody. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CK domains actually comprise the carboxy termini of the heavy and light chains, respectively.

[0029] As indicated above, the variable region allows the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the VK domain and the VH domain, or a subset of the complementarity determining regions (CDRs) of an antibody, combine to form the variable region that defines the three-dimensional antigen-binding site. The quaternary structure of the antibody forms the antigen-binding site present at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3) on each of the VH and VK chains. In some instances, for example, certain immunoglobulin molecules derived from camelid species or designed based on camelid immunoglobulins may have a complete immunoglobulin molecule consisting of only heavy chains and no light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).

[0030] In naturally occurring antibodies, each antigen-binding domain contains six "complementarity determining regions" or "CDRs", short non-contiguous sequences of amino acids that are specifically arranged to form the antigen-binding domain when the antibody assumes a three-dimensional structure in an aqueous environment. The remaining amino acids in the antigen-binding domain are called "framework" regions and show little variation between molecules. The framework regions are largely in a one-sheet conformation, and the CDRs form loops that connect the one-sheet structure and in some cases form part of the one-sheet structure. Thus, the framework regions act to form a scaffold for the correct orientation of the CDRs through interchain non-covalent interactions. The antigen-binding domain formed by the arranged CDRs defines a surface that is complementary to the epitope of the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids which constitute the CDRs and framework regions, respectively, have been precisely defined and can be readily identified by one of skill in the art for any given heavy or light chain variable region (see "Sequences of Proteins of Immunological Interest," Kabat, E., et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol., 196:901-917 (1987)).

[0031] In the event that there is more than one definition of a term that is used and / or accepted in the art, the definition of the term used herein is intended to include all such meanings unless expressly stated to the contrary. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-contiguous antigen binding sites found in the variable regions of both heavy and light chain polypeptides. This particular region is described in Kabat et al., US Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al., J. MoI. Biol. 196:901-917 (1987), which are incorporated herein by reference in their entirety. The definitions of CDRs by Kabat and Chothia include overlapping amino acid residues or subsets when compared to each other. Nevertheless, it is intended that the application of either definition to refer to the CDRs of an antibody or variants thereof is within the scope of the term as defined and used herein. The appropriate amino acid residues that encompass the CDRs defined by each of the above cited documents are listed in the following table for comparison. The exact residue numbers that encompass a particular CDR vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues constitute a particular CDR given the amino acid sequence of the variable region of an antibody.

[0032] [Table 1]

[0033] Kabat et al. also define a numbering system for variable domain sequences that is applicable to any antibody. One of skill in the art can unambiguously assign this system of "Kabat numbering" to any variable domain sequence without relying on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described in Kabat et al., US Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0034] In addition to the above table, according to the Kabat numbering system, the CDR regions are described as follows: CDR-H1 begins at about amino acid 31 (i.e., about 9 residues after the first cysteine ​​residue), includes about 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the 15th residue from the end of CDR-H1, includes about 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at about the 30th amino acid residue from the end of CDR-H2, includes 3-25 amino acids, and ends with the sequence WGXG (where X is any amino acid). CDR-L1 begins at about residue 24 (i.e., following the cysteine ​​residue), includes about 10-17 residues, and ends at the next tryptophan residue. CDR-L2 begins at about the 16th residue from the end of CDR-L1, and includes about 7 residues. CDR-L3 begins about the 30th residue from the end of CDR-L2 (ie, following the cysteine ​​residue), includes about 7-11 residues, and ends with the sequence F or WGXG (where X is any amino acid).

[0035] The antibodies disclosed herein may be from any animal, including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region may be of chondrichthyan origin (e.g., from sharks).

[0036] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region includes at least one of a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in the present disclosure may include a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the present disclosure includes a polypeptide chain comprising a CH3 domain. Additionally, an antibody for use in the present disclosure may lack at least a portion of a CH2 domain (e.g., all or a portion of a CH2 domain). As noted above, it will be appreciated by those skilled in the art that the heavy chain constant regions may be modified such that they differ in amino acid sequence from naturally occurring immunoglobulin molecules.

[0037] The heavy chain constant region of the antibody disclosed herein may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of the polypeptide may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain constant region may comprise a hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule. In another example, the heavy chain portion may comprise a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule.

[0038] As used herein, the term "light chain constant region" includes an amino acid sequence derived from an antibody light chain. Preferably, the light chain constant region includes at least one of the constant kappa domain or the constant lambda domain.

[0039] A "light chain-heavy chain pair" refers to an assembly of a light chain and a heavy chain that can form a dimer via disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0040] As indicated above, the subunit structures and three-dimensional configurations of the constant regions of various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term "CH1 domain" includes the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is amino-terminal to the hinge region of the immunoglobulin heavy chain molecule.

[0041] As used herein, the term "CH2 domain" includes, for example, that portion of an antibody heavy chain molecule extending from about residue 244 to residue 360, using conventional numbering schemes (residues 244-360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat et al., US Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983)). The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains in an intact native IgG molecule. Additionally, the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and is well known to include about 108 residues.

[0042] As used herein, the term "hinge region" includes the portion of a heavy chain molecule that connects the CH1 and CH2 domains. This hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upper, middle, and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).

[0043] As used herein, the term "disulfide bond" includes the covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CK regions are linked by a disulfide bond, and the two heavy chains are linked by two disulfide bonds at positions that correspond to 239 and 242 using the Kabat numbering system (positions 226 or 229, EU numbering system).

[0044] As used herein, the term "chimeric antibody" will be taken to mean any antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial or modified in accordance with the immediate disclosure) is obtained from a second species. In certain embodiments, the target binding region or site will be obtained from a non-human source (e.g., mouse or primate) and the constant region is human.

[0045] As used herein, "humanization rate" is calculated by determining the number of framework amino acid differences (i.e., non-CDR differences) between the humanized domain and the germline domain, subtracting that number from the total number of amino acids, dividing it by the total number of amino acids and multiplying by 100.

[0046] Generally, "specifically binds" or "has specificity" means that an antibody binds to an epitope through its antigen-binding domain with some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody "specifically binds" to an epitope when it binds to that epitope through its antigen-binding domain more readily than it binds to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which an antibody binds to an epitope. For example, antibody "A" may be considered to have a higher specificity for a given epitope than antibody "B," or antibody "A" may be said to bind epitope "C" with higher specificity than to related epitope "D."

[0047] As used herein, the term "treat" or "treatment" refers to both therapeutic procedures and prophylactic or preventative measures, the purpose of which is to prevent or slow down (alleviate) an undesirable physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in extent of disease, stabilized (i.e., not worsening) state of disease, delayed or slowed disease progression, improvement or mitigation of disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not treated. Those in need of treatment include those already with a condition or disorder, as well as those prone to having the condition or disorder, and those in whom the condition or disorder is to be prevented.

[0048] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject for which diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include humans, livestock, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, and cows.

[0049] As used herein, phrases such as "in a patient in need of treatment" or "subject in need of treatment" include subjects, such as mammalian subjects, who would benefit from the administration of an antibody or composition of the disclosure for use in detection, diagnostic procedures and / or treatment.

[0050] Anti-CXCR2 antibodies and fragments thereof The human CXCR2 protein is a transmembrane protein with four extracellular fragments, the N-terminus and each of the three extracellular loops of which are thought to be involved in binding to IL8. For example, Ahuja, SK et al., (1996) The Journal of biological chemistry 271, 225-232; Katancik, JA, et al., (1997) BioChemical and Biophysical Research Communications 232, 663-668; Luo, ZW, et al., (1997) Protein Engineering 10, 1039-1045; Berkamp, S., et al., (2017) Journal of Biomolecular Nmr 69, 111-121; Park, SH et al., (2017) Biophysical Journal 113, 2695-2705; Park, SH et al., (2011) J Mol Biol 414, 194-203; and Park, SH et al., (2012) Nature 491, 779-783 Please refer to.

[0051] Through sequence analysis and testing, the inventors identified the N-terminal extracellular fragment as an epitope for antibody screening. Surprisingly, no antibody was identified from a large-scale screen (10 11A group of antibodies showing selective binding to the N-terminus (with picomolar potency) obtained from a candidate library of 1000 antibodies were able to effectively block IL8 binding and strongly inhibit IL8-induced neutrophil chemotaxis. This excellent affinity overcame the problem of IL8 itself binding with high affinity (nanomolar), and IL8-induced cellular functions were completely inhibited. Furthermore, these antibodies even showed an inverse agonist effect, significantly inhibiting endogenous β-arrestin recruitment by CXCR2 expression. Furthermore, it was found that these antibodies showed a biased agonist effect, activating CXCR2 endocytosis, which may more efficiently inhibit CXCR2-mediated pathological events.

[0052] Experimental data indicates that these antibodies and antigen-binding fragments thereof, along with antibodies obtained through additional screening, may be useful in treating diseases and conditions associated with CXCR2 signaling, such as cancer, infectious diseases, inflammatory and autoimmune diseases.

[0053] According to one embodiment of the present disclosure, an antibody or fragment thereof having binding specificity for human CXC motif-type chemokine receptor 2 (CXCR2) protein is provided. In some embodiments, the binding comprises one or more amino acid residues within the extracellular N-terminus (i.e., MEDFNMESDS FEDFWKGEDL SNYSYSSTLP PFLLDAAPCE PESLEINK; SEQ ID NO:15). In some embodiments, the binding comprises at least one or more amino acid residues within residues 9-19 of SEQ ID NO:15 (i.e., DSFEDFWKGED, SEQ ID NO:16). In some embodiments, the binding does not involve, or at least does not require, any amino acid residues outside the 48 N-terminal residues, such as those within the three extracellular loops.

[0054] In some embodiments, the linkage comprises (in other words the antibody or fragment binds to) at least one of D13, F14, and W15 of SEQ ID NO:15. In some embodiments, the linkage comprises at least D13. In some embodiments, the linkage comprises at least F14. In some embodiments, the linkage comprises at least W15. In some embodiments, the linkage comprises at least D13 and F14. In some embodiments, the linkage comprises at least D13 and W15. In some embodiments, the linkage comprises at least F14 and W15. In some embodiments, the linkage comprises at least another residue in SEQ ID NO:15 in addition to D13, F14 and / or W15.

[0055] In some embodiments, the binding is effective to inhibit, compete with, or completely eliminate IL8 binding, hi some embodiments, the antibodies or fragments thereof of the present disclosure can inhibit IL8-mediated CXCR2 signaling, or even endogenous CXCR2 signaling.

[0056] Some embodiments of the present disclosure provide an antibody or fragment thereof having binding specificity for human CXC motif-type chemokine receptor 2 (CXCR2) protein. The antibody or fragment comprises a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3, and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3. In some embodiments, the CDRs comprise sequences as shown in Table A, and variants thereof. Table B also lists some variants of VH CDR3 that were tested in the experimental examples.

[0057] [Table 2]

[0058] [Table 3]

[0059] In some embodiments, as shown in Table A, VH CDR1 comprises the amino acid sequence of SEQ ID NO:1; VH CDR2 comprises the amino acid sequence of SEQ ID NO:2, or a variant with an amino acid substitution from SEQ ID NO:2; VH CDR3 comprises the amino acid sequence of SEQ ID NO.3, or a variant selected from the group consisting of SEQ ID NOs:7-14; VL CDR1 comprises the amino acid sequence of SEQ ID NO:4; VL CDR2 comprises the amino acid sequence of SEQ ID NO:5; and VL CDR3 comprises the amino acid sequence of SEQ ID NO:6.

[0060] In some embodiments, the anti-CXCR2 antibodies of the disclosure comprise the VH and VL CDRs listed in Table A with one, two or three additional modifications. Such modifications may be amino acid additions, deletions or substitutions. The substitutions, in some embodiments, are conservative amino acid substitutions.

[0061] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, non-essential amino acid residues in immunoglobulin polypeptides are preferably replaced with another amino acid residue from the same side chain family. In another embodiment, the string of amino acids can be replaced with structurally similar strings that differ in the order and / or composition of the side chain family members.

[0062] Non-limiting examples of conservative amino acid substitutions are shown in the following table, where a similarity score of 0 or greater indicates a conservative substitution between the two amino acids.

[0063] [Table 4]

[0064] [Table 5]

[0065] In some embodiments, the anti-CXCR2 antibodies of the disclosure comprise a VH of SEQ ID NO:17 or 18, a VL of SEQ ID NO:19, or a biological equivalent thereof. A biological equivalent of a VH or VL is a sequence that contains the specified amino acids while having 80%, 85%, 90%, 95%, 98% or 99% overall sequence identity. A biological equivalent of SEQ ID NO:18 can be, for example, a VH that has 80%, 85%, 90%, 95%, 98% or 99% overall sequence identity to SEQ ID NO:18 but retains the CDRs (SEQ ID NOs:1-6 or variants thereof). In one embodiment, the VH has the amino acid sequence of SEQ ID NO:18 and the VL has the amino acid sequence of SEQ ID NO:19. In one embodiment, the VH has the amino acid sequence of SEQ ID NO:17 and the VL has the amino acid sequence of SEQ ID NO:19.

[0066] It will also be understood by those of skill in the art that antibodies as disclosed herein may be modified such that they differ in amino acid sequence from the naturally occurring binding polypeptide from which they are derived. For example, a polypeptide or amino acid sequence derived from a designated protein may be similar to the starting sequence, e.g., may have a certain percentage identity, e.g., may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the starting sequence.

[0067] In certain embodiments, the antibody comprises an amino acid sequence or one or more moieties that are not normally associated with antibodies. Exemplary modifications are described in more detail below. For example, the antibody of the present disclosure may comprise a flexible linker sequence or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label).

[0068] The antibodies, variants, or derivatives thereof of the present disclosure include derivatives that have been modified, i.e., modified by covalent attachment of any type of molecule to the antibody such that the covalent attachment does not prevent the antibody from binding to the epitope. For example, but not limited to, the antibodies may be modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, protein cleavage, linkage to cellular ligands or other proteins, etc. Any of a number of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the antibodies may contain one or more non-classical amino acids.

[0069] In some embodiments, the antibody may be conjugated to a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent, or PEG.

[0070] The antibody may be conjugated or fused to a therapeutic agent including a detectable label such as a radioactive label, an immunomodulatory agent, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxin which may be a drug or a toxin, an ultrasound enhancing agent, a non-radioactive label, combinations of these and other such agents known in the art.

[0071] Polynucleotides encoding antibodies and methods for preparing said antibodies The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding the antibodies, variants or derivatives thereof of the present disclosure. The polynucleotides of the present disclosure may encode the entire heavy and light chain variable regions of the antigen-binding polypeptide, variants or derivatives thereof, on the same polynucleotide molecule or on separate polynucleotide molecules. Furthermore, the polynucleotides of the present disclosure may encode portions of the heavy and light chain variable regions of the antigen-binding polypeptide, variants or derivatives thereof, on the same polynucleotide molecule or on separate polynucleotide molecules.

[0072] Methods for producing antibodies are well known in the art and are described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptide of the present disclosure are fully human. Fully human antibodies can be produced using techniques described in the art and as described herein. For example, fully human antibodies against a particular antigen can be prepared by administering the antigen to a transgenic animal whose endogenous locus is disabled, but which has been modified to produce such antibodies in response to antigen challenge. Exemplary techniques that can be used to make such antibodies are described in U.S. Patent Nos. 6,150,584; 6,458,592; and 6,420,140, ​​which are incorporated by reference in their entirety.

[0073] In certain embodiments, the prepared antibodies do not elicit adverse immune responses in the treated animal, e.g., human. In one embodiment, the antigen-binding polypeptides, variants, or derivatives thereof of the present disclosure are modified using art-recognized techniques to reduce their immunogenicity. For example, the antibodies can be humanized, primatized, deimmunized, or chimeric antibodies can be made. These types of antibodies are derived from non-human antibodies, typically mouse or primate antibodies, and retain or substantially retain the antigen-binding properties of the parent antibody, but are less immunogenic in humans. This can be achieved by a variety of methods, such as (a) grafting the entire non-human variable domain onto a human constant region to generate a chimeric antibody, (b) grafting at least a portion of one or more of the non-human complementarity determining regions (CDRs) onto a human framework and constant region, with or without retaining key framework residues, or (c) grafting the entire non-human variable domain, but "cloaking" it with human-like sections by replacement of surface residues. Such methods are disclosed in Morrison et al., ProC. Natl. Acad. Sci. USA 57:6851-6855 (1984); Morrison et al., Adv. Immunol. 44:65-92 (1988); Verhoeyen et al., Science 239:1534-1536 (1988); Padlan, Molec. Immun. 25:489-498 (1991); Padlan, Molec. Immun. 31:169-217 (1994), as well as U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,190,370, all of which are incorporated by reference in their entireties.

[0074] Deimmunization can also be used to reduce the immunogenicity of antibodies. As used herein, the term "deimmunization" includes modification of antibodies to modify T cell epitopes (see, for example, WO 9852976 and WO 0034317). For example, the variable heavy and variable light chain sequences from the starting antibody are analyzed and a human T cell epitope "map" is generated from each V region that indicates the location of the epitope in relation to the complementarity determining regions (CDRs) and other important residues in the sequence. Individual T cell epitopes are analyzed from the T cell epitope map to identify alternative amino acid substitutions that have a low risk of altering the activity of the final antibody. A variety of alternative heavy and light chain variable sequences are designed, including combinations of amino acid substitutions, and these sequences are then incorporated into various binding polypeptides. Typically, 12 to 24 mutant antibodies are generated and tested for binding and / or function. The complete heavy and light chain genes containing the modified variable regions and human constant regions are then cloned into expression vectors and the subsequent plasmids are introduced into cell lines to produce the whole antibody. These antibodies are then compared in appropriate biochemical and biological assays to identify the optimal variants.

[0075] The binding specificity of an antigen-binding polypeptide of the disclosure can be determined by in vitro assays such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0076] Alternatively, techniques described for the production of single-chain units (U.S. Pat. No. 4,694,778; Bird, Science 242:423-442 (1988); Huston et al., ProC. Natl. Acad. Sci. USA 55:5879- 5883 (1988); and Ward et al., Nature 334:544-554 (1989)) can be adapted to produce the single-chain units of the present disclosure. The single-chain units are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single-chain fusion peptide. Techniques for the assembly of functional Fv fragments in E. coli can also be used (Skerra et al., Science 242: 1038-1041 (1988)).

[0077] Examples of techniques that can be used to produce single chain Fvs (scFvs) and antibodies are described in U.S. Patents 300,587, 4,946,778 and 5,258,498, Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., ProC. Natl. Sci. USA 90:1995-1999 (1993); and Skerra et al., Science 240:1038-1040 (1988). For some applications, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. Chimeric antibodies are molecules in which different portions of the antibody are derived from different animal species, such as antibodies with a variable region derived from a mouse monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods 125:191-202 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated by reference in their entireties.

[0078] A humanized antibody is an antibody molecule derived from a non-human species antibody that binds to a desired antigen and has one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be replaced with corresponding residues from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, such as modeling the interactions of CDR and framework residues to identify framework residues important for antigen binding, and sequence comparison to identify unusual framework residues at specific positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties). Antibodies can be modified by, for example, CDR grafting (EP 239400; WO 91 / 09967; U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (EP 592106; 519596; Padlan, Molecular Immunology 28(4 / 5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., ProC. Natl. Sci. USA 91:969-973 (1993)). (1994)), and chain shuffling (U.S. Pat. No. 5,565,332, which is incorporated by reference in its entirety).

[0079] For the treatment of human patients, fully human antibodies are particularly desirable. Human antibodies can be produced by various methods known in the art, such as phage display methods using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887 and 4,716,111; and WO 98 / 46645, 98 / 50433, 98 / 24893, 98 / 16654, 96 / 34096, 96 / 33735, and 91 / 10741, each of which is incorporated herein by reference in its entirety.

[0080] It is also possible to generate human antibodies using transgenic mice that cannot express functional endogenous immunoglobulins, but can express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable, constant, and diversity regions can be introduced into mouse embryonic stem cells in addition to human heavy and light chain genes. Mouse heavy and light chain immunoglobulin genes can be made non-functional separately or simultaneously when human immunoglobulin loci are introduced by homologous recombination. In particular, homozygous deletion of the JH region abolishes endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are bred to generate homozygous offspring that express human antibodies. The transgenic mice are immunized in the usual manner with a selected antigen, for example, all or part of a desired target polypeptide. Monoclonal antibodies directed against antigens can be obtained from immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes carried by the transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, such technology can be used to generate therapeutically useful IgG, IgA, IgM and IgE antibodies. For a review of this technology for generating human antibodies, see Lonberg and Huszar Int. Rev. Immunol. 73:65-93 (1995). For a detailed discussion of this technology for generating human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., WO 98 / 24893; WO 96 / 34096; WO 96 / 33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598, which are incorporated by reference in their entireties.In addition, companies such as Abgenix, Inc. (Fremont, Calif.) and GenPharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

[0081] Fully human antibodies that recognize a selected epitope can also be generated by a technique called "guided selection," in which a selected non-human monoclonal antibody, e.g., a murine antibody, is used to guide the selection of a fully human antibody that recognizes the same epitope. (Jespers et al., Bio / Technology 72:899-903 (1988); see also U.S. Pat. No. 5,565,332, which is incorporated by reference in its entirety.

[0082] In another embodiment, DNA encoding the desired monoclonal antibody can be readily isolated and sequenced by conventional procedures (e.g., using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies). The isolated and subcloned hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into an expression vector, which is then transfected into prokaryotic or eukaryotic host cells, such as E. coli cells, Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulins. More specifically, the isolated DNA (which may be synthesized as described herein) can be used to clone constant and variable region sequences for the manufactured antibody, as described in U.S. Patent No. 5,658,570, filed January 25, 1995, to Newman et al., which is incorporated herein by reference. Essentially, this involves extraction of RNA from the selected cells, conversion to cDNA, and amplification by PCR using Ig-specific primers. Suitable primers for this purpose are also described in U.S. Patent No. 5,658,570. As discussed in more detail below, transformed cells expressing the desired antibodies can be grown in relatively large quantities to provide clinical and commercial supplies of immunoglobulin.

[0083] Furthermore, one or more CDRs of an antigen-binding polypeptide of the present disclosure can be inserted into a framework region, e.g., into a human framework region, to humanize a non-human antibody, using routine recombinant DNA techniques. The framework region can be a naturally occurring framework region or a consensus framework region, preferably a human framework region (see, e.g., Chothia et al., J. Mol. Biol. 278:457-479 (1998) for a list of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds to at least one epitope of a desired polypeptide, e.g., LIGHT. Preferably, one or more amino acid substitutions can be made in the framework region, and preferably, the amino acid substitutions improve binding of the antibody to its antigen. Furthermore, such methods can be used to make amino acid substitutions or deletions of one or more variable region cysteine ​​residues involved in intrachain disulfide bonds to generate an antibody molecule lacking one or more intrachain disulfide bonds. Other modifications to the polynucleotide are encompassed by the present disclosure and are within the purview of one of ordinary skill in the art.

[0084] Also, techniques developed to make "chimeric antibodies" by splicing genes from a mouse antibody molecule with the appropriate antigen specificity with genes from a human antibody molecule with the appropriate biological activity (Morrison et al. Natl. Acad. Sci. USA:851-855(1984); Neuberger et al., Nature 372:604-608(1984); Takeda et al., Nature 314:452-454(1985)) can be used. As used herein, a chimeric antibody is a molecule in which different portions are derived from different animal species, e.g., having a variable region derived from a mouse monoclonal antibody and a human immunoglobulin constant region.

[0085] Yet another highly efficient means for producing recombinant antibodies has been disclosed by Newman, Biotechnology 10: 1455-1460 (1992). Specifically, this technique results in the production of primatized antibodies containing monkey variable domains and human constant sequences, which is incorporated herein by reference in its entirety. Additionally, this technique is described in commonly assigned U.S. Patent Nos. 5,658,570, 5,693,780, and 5,756,096, each of which is incorporated herein by reference.

[0086] Alternatively, antibody-producing cell lines can be selected and cultured using techniques well known to those skilled in the art. Such techniques are described in a variety of laboratory manuals and primary publications. In this regard, techniques suitable for use in the present disclosure, as described below, are described in Current Protocols in Immunology, Coligan et al., Eds., Green Publishing AssoCiates and Wiley-Interscience, John Wiley and Sons, New York (1991), which is incorporated herein by reference in its entirety, including any supplements.

[0087] Furthermore, mutations can be introduced into the nucleotide sequence encoding the antibody of the present disclosure using standard techniques known to those skilled in the art, including but not limited to site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions.Preferably, the mutant (including derivative) encodes less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions with respect to the reference variable heavy chain region, CDR-H1, CDR-H2, CDR-H3, variable light chain region, CDR-L1, CDR-L2, or CDR-L3.Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity.

[0088] Cancer Treatment As described herein, the antibodies, variants, or derivatives of the present disclosure may be used in certain therapeutic and diagnostic methods.

[0089] The present disclosure is further directed to antibody-based therapeutics comprising administering an antibody or fragment of the present disclosure to a patient, such as a human patient, to treat one or more disorders or conditions described herein. Therapeutic agents of the present disclosure include, but are not limited to, antibodies of the present disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding antibodies of the present disclosure (including variants and derivatives thereof as described herein).

[0090] The antibodies and fragments of the present disclosure can be used to treat or inhibit cancer. The chemokine receptor CXCR2 and its ligands are involved in the progression of tumors and various inflammatory diseases. Activation of the CXCL / CXCR2 axis activates multiple signaling pathways, including the PI3K, p38 / ERK, and JAK pathways, and regulates cell survival and migration. The CXCL / CXCR2 axis plays an important role in the recruitment of neutrophils to the tumor microenvironment and inflammatory sites. Extensive infiltration of neutrophils in chronic inflammation is one of the most important pathogenic factors in various inflammatory diseases.

[0091] Tumors that may be suitably treated with the antibodies or fragments of the present disclosure include bladder cancer, non-small cell lung cancer, renal cancer, breast cancer, urethral cancer, colon cancer, head and neck cancer, squamous cell carcinoma, Merkel cell carcinoma, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, and small cell lung cancer, and thus the antibodies disclosed herein can be used to treat any one or more of such cancers.

[0092] Also provided in the present disclosure is a cell therapy, such as chimeric antigen receptor (CAR) T cell therapy. Appropriate cells can be used that are contacted with the anti-CXCR2 antibody of the present disclosure (or alternatively engineered to express the anti-CXCR2 antibody of the present disclosure). After such contact or engineering, the cells can then be introduced into a cancer patient that needs treatment. The cancer patient can have any type of cancer as disclosed herein. The cells (e.g., T cells) can be, but are not limited to, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or combinations thereof.

[0093] In some embodiments, the cells are isolated from the cancer patient themselves. In some embodiments, the cells are provided from a donor or from a cell bank. If the cells are isolated from the cancer patient, unwanted immune responses can be minimized.

[0094] Additional diseases or conditions associated with increased cell viability that may be treated, prevented, diagnosed and / or prognosed using the disclosed antibodies or variants, or derivatives thereof, include leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), erythroblastoma, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors (including, but not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, and / or lymphangiosarcoma). , synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, prostate cancer, ovarian cancer, prostate cancer, squamous cell carcinoma. Progression and / or metastasis of malignant tumors and related diseases, including, but not limited to, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatic carcinoma, bile duct carcinoma, choriocarcinoma, testicular cancer, germ cell carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendritic glioma, hemangioma, melanoma, neuroblastoma, and retinoblastoma.

[0095] Treatment of inflammatory diseases As demonstrated in the experimental examples, the antibodies of the present disclosure can alter immune responses and therefore may be useful in the treatment of inflammatory diseases and conditions, autoimmune diseases, and infectious diseases.

[0096] In some embodiments, inflammatory diseases or conditions to be treated by the disclosed antibodies, fragments, and compositions include one or more of Alzheimer's disease, Addison's disease, atherosclerotic spondylitis, ankylosing spondylitis, arthritis, osteoarthritis (OA), rheumatoid arthritis (RA), psoriatic arthritis (PA), ankylosing spondylitis, asthma, arteriosclerosis, chronic obstructive pulmonary disease (COPD), Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome (IBS), systemic lupus erythematosus (SLE), nephritis, Parkinson's disease (PD), vasculitis, and ulcerative colitis.

[0097] In some embodiments, the autoimmune disease or condition to be treated by the disclosed antibodies, fragments, and compositions comprises one or more of alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes mellitus (type 1), celiac disease, autoimmune juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, autoimmune myocarditis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjogren's syndrome, systemic lupus erythematosus, autoimmune thyroiditis, Hashimoto's thyroiditis, autoimmune uveitis, vitiligo, and granulomatosis with polyangiitis (Wegener's disease).

[0098] Rheumatoid arthritis (RA) is a long-term autoimmune disease that primarily affects the joints. Generally, the joints become hot, swollen, and painful. The pain and stiffness often worsen with rest. It most commonly affects the wrists and hands, usually in the same joints on both sides of the body. The disease can also affect other parts of the body. The cause of RA is unclear, but a combination of genetic and environmental factors is thought to be involved. The underlying mechanism involves the body's immune system attacking the joints. This results in inflammation and thickening of the joint capsule. The goal of treatment is to reduce pain, suppress inflammation, and improve overall body function. Painkillers, steroids, and NSAIDs are frequently used to help with symptoms. A group of drugs called disease-modifying antirheumatic drugs (DMARDs), such as hydroxychloroquine and methotrexate, may also be used to slow the progression of the disease.

[0099] Osteoarthritis (OA) is a type of joint disease caused by the destruction of articular cartilage and the underlying bone. The most common symptoms are joint pain and stiffness. At first, symptoms may occur only after exercise, but over time, they may occur all the time. Other symptoms may include joint swelling and reduced range of motion, and weakness or numbness in the hands and feet if the hips are affected. Causes include previous joint injury, abnormal development of the joint or limb, and genetic factors. People who are overweight, have different lengths of legs, or have jobs that place high levels of stress on the joints are at higher risk. Osteoarthritis is thought to be caused by mechanical stress on the joints and a low-grade inflammatory process. Treatment includes exercise, efforts to reduce stress on the joints, support groups, and painkillers.

[0100] Multiple sclerosis (MS) is a demyelinating disease in which the insulating covering of nerve cells in the brain and spinal cord is damaged. This damage impairs the ability of parts of the nervous system to communicate with each other, resulting in a variety of signs and symptoms, including physical, mental, and sometimes psychiatric problems. Specific symptoms may include double vision, blindness in one eye, muscle weakness, sensory impairment, or impaired coordination. The cause is unclear, but destruction by the immune system or damage to myelin-producing cells is thought to be the underlying mechanism. There is no known cure for multiple sclerosis. Treatment attempts to improve function after an attack and prevent new attacks.

[0101] Asthma is a common, long-lasting inflammatory disease of the airways of the lungs. It is characterized by recurrent episodes of various symptoms, reversible airflow obstruction, and bronchospasm. Symptoms include wheezing, coughing, chest tightness, and shortness of breath. Asthma is thought to be caused by a combination of genetic and environmental factors. Environmental factors include exposure to air pollution and allergens. Asthma is classified by the frequency of symptoms, the forced expiratory volume in 1 second (FEV1), and peak expiratory flow rate. It may also be classified as atopic or nonatopic, with atopic referring to a predisposition to type 1 hypersensitivity reactions. There is no cure for asthma. Symptoms can be prevented by avoiding triggers such as allergens and irritants and by using inhaled corticosteroids. If asthma symptoms are not controlled, long-acting beta-agonists (LABAs) or anti-leukotrienes may be used in addition to inhaled corticosteroids. If symptoms rapidly worsen, treatment usually involves inhaled short-acting beta-2 agonists such as salbutamol and oral corticosteroids, but in very severe cases intravenous corticosteroids, magnesium sulfate, and hospitalization may be required.

[0102] Chronic obstructive pulmonary disease (COPD) is a type of obstructive lung disease characterized by reduced airflow over a long period of time. There are two main forms of COPD: emphysema and chronic bronchitis. In emphysema, the walls between many air sacs are damaged. As a result, the air sacs become misshapen and flat. This damage can also destroy the walls of the air sacs, resulting in fewer, larger air sacs instead of many small ones. This reduces the amount of gas exchange in the lungs. In chronic bronchitis, the lining of the airways is constantly irritated and inflamed, causing the lining to swell. A lot of thick mucus forms in the airways, making it difficult to breathe. There is no known cure for COPD, but the symptoms can be treated and its progression can be slowed.

[0103] Administration Methods of administration of antibodies, variants include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. Antigen-binding polypeptides or compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucosal linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents. Thus, pharmaceutical compositions comprising antigen-binding polypeptides of the present disclosure may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (by powder, ointment, drop or transdermal patch), intranasally, or bucally, i.e., as an oral or nasal spray.

[0104] The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrathoracic, subcutaneous and intraarticular injection and infusion.

[0105] Administration can be systemic or local.In addition, it may be desirable to introduce the antibody of the present disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by, for example, an intraventricular catheter attached to a reservoir, such as an Ommaya reservoir.Pulmonary administration can also be employed, for example, by using an inhaler or nebulizer, and by formulating with an aerosolizing agent.

[0106] It may be desirable to administer an antibody polypeptide or composition of the disclosure locally to the area in need of treatment, for example, but not limited to, by local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by catheter, by suppository, or by implant, said implant being a membrane, such as a salivary gland membrane, or a porous, non-porous, or gelatinous material, including fibers. Preferably, when administering proteins, including antibodies of the disclosure, care must be taken to use materials to which the proteins do not absorb.

[0107] In another embodiment, the antibody or composition may be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

[0108] As a general proposition, the dosage of an antigen-binding polypeptide of the present disclosure given to a patient is typically 0.1 mg / kg to 100 mg / kg of the patient's body weight, 0.1 mg / kg to 20 mg / kg of the patient's body weight, or 1 mg / kg to 10 mg / kg of the patient's body weight. In general, human antibodies have a longer half-life in the human body than antibodies from other species due to immune responses to foreign polypeptides. Thus, it is often possible to administer lower doses of human antibodies less frequently. Additionally, the dosage and frequency of administration of the antibodies of the present disclosure may be reduced by enhancing antibody uptake and tissue penetration (e.g., into the brain) by modifications such as, for example, lipidation.

[0109] Methods for treating infectious or malignant diseases, conditions, or disorders involving administration of the disclosed antibodies, variants, or derivatives thereof are typically tested in vitro and then in vivo in acceptable animal models for the desired therapeutic or prophylactic activity prior to use in humans. Suitable animal models, including transgenic animals, are well known to those of skill in the art. For example, in vitro assays for demonstrating the therapeutic utility of the antigen-binding polypeptides described herein include the effect of the antigen-binding polypeptides on cell lines or patient tissue samples. The effect of the antigen-binding polypeptides on cell lines and / or tissue samples can be determined using techniques known to those of skill in the art, such as the assays disclosed elsewhere herein. In vitro assays that can be used to determine whether administration of a particular antigen-binding polypeptide is indicated in accordance with the present disclosure include in vitro cell culture assays in which a patient tissue sample is grown in culture, exposed to or otherwise administered a compound, and the effect of such compound on the tissue sample is observed.

[0110] Various delivery systems are known and can be used to administer an antibody of the disclosure or a polynucleotide encoding an antibody of the disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a retrovirus or other vector, etc.

[0111] Diagnostic methods In some embodiments, the antibodies of the present disclosure can be used for diagnostic and prognostic purposes. In some embodiments, a method is provided for detecting expression of CXCR2 in a sample, comprising contacting the sample with the antibody or fragment thereof under conditions for the antibody or fragment thereof to bind to CXCR2, and detecting binding indicative of expression of CXCR2 in the sample.

[0112] Suitably, the sample containing cells can be taken from a patient, which may be a cancer patient or a patient who wishes to be diagnosed. The cells can be cells of tumor tissue or tumor block, blood samples, urine samples, or any sample from a patient. With optional pretreatment of the sample, the sample can be incubated with the antibody of the present disclosure under conditions that allow the antibody to interact with the CXCR2 protein potentially present in the sample. Methods such as ELISA can be used to detect the presence of CXCR2 protein in a sample, utilizing anti-CXCR2 antibodies.

[0113] The presence of CXCR2 protein in a sample (and optionally its amount or concentration) can be used to diagnose cancer, as an indication that a patient is suitable for treatment with an antibody, or as an indication that a patient has responded (or not responded) to cancer treatment. As a prognostic method, detection can be performed once, twice or more at specific stages of cancer treatment to indicate the progress of the treatment.

[0114] composition The present disclosure also provides pharmaceutical compositions. Such compositions include an effective amount of an antibody and an acceptable carrier. In some embodiments, the composition further includes a second anti-cancer agent (e.g., an immune checkpoint inhibitor).

[0115] In specific embodiments, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, and more particularly in humans. Moreover, a "pharmaceutically acceptable carrier" will generally be any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary.

[0116] The term "carrier" refers to a diluent, adjuvant, excipient, or solvent with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous dextrose and aqueous glycerol solutions can also be utilized as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, and ethanol. The composition can, if desired, also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates, or phosphates. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and osmolality adjusters such as sodium chloride or glucose are also contemplated. These compositions can take the form of solutions, suspensions, emulsions, tablets, capsules, powders, and sustained release formulations. The compositions can be formulated as suppositories, using traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with an appropriate amount of the carrier so as to provide the form for proper administration to the patient. The formulation should be appropriate for the mode of administration. The parent formulation can be enclosed in glass or plastic ampoules, disposable syringes or multiple dose vials.

[0117] In an embodiment, the composition is formulated according to a conventional procedure as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions dissolved in sterile isotonic aqueous buffer. If necessary, the composition can also include a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the injection site. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example as a dry lyophilized powder or water-free concentrate, in a sealed container, such as an ampoule or sachet indicating the amount of active agent. If the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. If the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0118] The compounds of the present disclosure can be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. EXAMPLES

[0119] Example 1: Experimental Procedure cell culture HEK293F cells (#R79007; Thermo Fisher Scientific) were cultured in FreeStyle 293 Expression Medium (#12338-026; Thermo Fisher Scientific). U2OS cell line (ATCC-HTB96; cell bank of Chinese Academy of Science, Shanghai) was maintained in McCoy's 5A medium (#16600-082, Gibco) containing 10% (vol / vol) FBS. Tango™ CXCR2-bla U2OS cell line (#K1807; Thermo Fisher Scientific) was maintained in growth medium based on McCoy's 5A medium according to the manufacturer's instructions. CHO-K1 cells were cultured in F12K medium (#21127022; Thermo Fisher Scientific) containing 10% (v / v) FBS.

[0120] Combinatorial antibody library panning A 48-amino acid peptide (pepN48) identical to the N-terminal extracellular domain of human CXCR2 (MEDFNMESDSFEDFWKEDLSNYSSTLPPFLDAAPCEPESLEINK; SEQ ID NO: 15) labeled with N-terminus biotin was synthesized as an antigen for phage panning (Chinese Peptide). The panning procedure followed a modified protocol as previously described. Briefly, ~10 11Phage particles displaying a combinatorial scFv antibody library with a diversity of 100 were incubated with the antigen pepN48. Streptavidin-coated magnetic beads (#21925; Pierce) were then added to the solution to pull down the phage-bound biotinylated antigens. Bound phages were eluted with glycine-HCl (pH 2.0) after washing away unbound phages and used to infect XL-1 blue cells (#200228; Agilent). The infected cells were used to generate phage particles for the next round of panning with the help of helper phage VCSM13 (#200251; Agilent). After three rounds of enrichment, colonies were picked and tested by phage ELISA, and then all positive clones were sequenced. The international ImMunoGeneTics information system (IMGT) was used for contig analysis of the sequences of these colonies. Nine different scFv sequences were highly enriched.

[0121] ELISA Avidin (#21121; Pierce) was diluted in Carbonate-Bicarbonate buffer (#C3041; Sigma) to a final concentration of 2ng / μl. 96-well ELISA plates (Corning Costar) were coated with avidin solution (25μl / well) overnight at 4°C. Wells were washed once with 150μl / well of PBST buffer (0.05% TWEEN20 in PBS). 50ng of antigen (2ng / μl) dissolved in PBS was added to each well and incubated at room temperature for 30 minutes. Wells were washed three times with PBST and blocked with M-PBST (3% milk in PBST, 150μl / well) for 5 minutes at 37°C. After removing the M-PBST buffer, 25μl of antibody solution (diluted in M-PBST buffer at the appropriate concentration) was added to each well and incubated at room temperature for 1 hour, followed by five repeated washes with PBST. Anti-M13 HRP-conjugated secondary antibody (1:3,000 dilution; #27-9421-01, GE) or anti-human Fc HRP-conjugated secondary antibody (1:3,000 dilution; #A0170, Sigma) was added to the wells and incubated for 1 h at room temperature. The wells were then washed 5 times with PBST and incubated with 50 μl / well ABTS solution (#11684302001; RoChe) for 20 min at room temperature. The absorbance of each well was measured at 405 nm on a plate reader (EnSpire; PerkinElmer).

[0122] Antibody expression and purification DNA sequences encoding scFv antibody candidates were cloned into the pFuse expression vector (#pfuse-hg1fc2; InvivoGen) and used to express scFv-Fc proteins carrying the entire Fc domain of human IgG1. For full-length antibodies in IgG1 format, the heavy and light chain variable regions (V H &V L ) were respectively divided into the entire heavy and light chain constant domains (C H &C L) into a plasmid carrying the scFv-Fc gene. This scFv-Fc expression plasmid was transfected into HEK293F cells, or equimolar heavy and light chain plasmids were co-transfected for full-length antibodies, and the cells were then cultured for 5 days to express the antibodies. The antibodies in the medium were purified using a HiTrap Protein A HP column (#17-0403-03; GE Healthcare) with an AKTAxpress purifier (GE Healthcare). The purified antibodies were concentrated and stored at -80°C in PBS buffer (pH 7.4).

[0123] Size Exclusion Column HPLC (SEC-HPLC) SEC-HPLC experiments were performed to examine the thermal stability and homogeneity of the purified recombinant antibodies. Briefly, the antibody solution was first concentrated to 20 mg / mL and incubated at 42°C for 5 days. The resulting antibody solution was analyzed by HPLC using a SEC column (Nanofilm SEC-250) with a running buffer of 0.05% DDM / 0.01% CHS in Tris (pH 8.1). Aggregation and degradation were evaluated in the results of protein homogeneity.

[0124] Flow cytometry Cells were transfected with an expression plasmid containing the target membrane protein. After 24 hours, cells were detached from the dish and resuspended in FACS buffer (0.5% BSA, 2 mM EDTA in PBS). 500.000 cells per tube were incubated with the corresponding antibody at 2 μg / mL in FACS buffer for 15 min at 4°C. The resulting cells were washed twice with FACS buffer and incubated with the secondary antibody, Alexa FluorTM 488 goat anti-human IgG(H+L) (#A11013; Life Technologies), at 2 μg / mL for 15 min at 4°C. After washing twice with FACS buffer, cells were resuspended in PBS and analyzed on a CytoFLEX S (Beckman Coulter).

[0125] Immunofluorescence assay Cells were plated and cultured on poly-D-lysine-coated glass-bottom 96-well microtiter plates (PerkinElmer). Plasmids expressing target membrane proteins were transfected into cells using Lipofectamine 3000. 24 hours after transfection, cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature, and then washed three times with PBS for 5 minutes each. Cells were then blocked with 1% BSA (dissolved in PBS) for 30 minutes at 37°C. After blocking, cells were incubated with the corresponding antibodies (2 μg / ml, diluted in 1% BSA / PBS) overnight at 4°C. Secondary antibodies, Alexa FluorTM 488 goat anti-human IgG(H+L) (#A11013; Life Technologies), at 2 μg / mL, were then mixed with cells in 1% BSA PBS buffer for 1 hour at room temperature. DAPI (#10236276001; RoChe) was added for nuclear staining. After washing with PBS three times, the stained cells were kept in PBS and subjected to immunofluorescence analysis under a confocal microscope (ZEISS LSM710).

[0126] Surface Plasmon Resonance (SPR) Biotinylated antigens (purified proteins or peptides) were placed on the streptavidin-coated surface of an SA biosensor chip (GE Healthcare) to form a monolayer. Antibody-antigen interactions were performed on a T200 system (GE Healthcare). Briefly, test antibodies were serially diluted to different concentrations of 0.5, 1, 2, 5, and 10 nM in the running buffer HBS-EP+ buffer (GE Healthcare) and flowed over the chip surface to detect intermolecular interactions between antigens and antibodies. Binding / dissociation kinetics were measured and fitted to an appropriate protein-protein interaction model to obtain the corresponding binding constants (K D ) was calculated.

[0127] Affinity maturation using yeast display The DNA sequences of the heavy and light chains of the selected antibody (abN48-IgG1) were cloned into the yeast surface display vector pYD-HA. Primers containing the randomly mutated H-CDR3 region were used to generate the CDR3-diversified heavy chain by overlap PCR. The CDR3-diversified heavy chain fragment and the linearized backbone plasmid were transfected into EBY100 yeast cells by electroporation, and the construction of the yeast-based Fab mutagen library was completed using the endogenous homologous recombination strategy of yeast cells.

[0128] EBY100 yeast cells transformed with the mutagen library were incubated with SD / Trp - OD 600 Cells were grown to a pH of 10.5 = 1 and induced with shaking in SG / R-CAA medium for 18-24 h at 20°C. After washing the cells with FACS buffer (0.5% BSA in PBS, containing 2 mM EDTA), pepN48 and anti-c-myc chicken IgY moiety (#A21281; Life Technologies) were added to the cell suspension (1:500 dilution) and incubated at room temperature for 30 min. Cells were washed three times with FACS buffer, after which R-phycoerythrin-conjugated streptavidin (#21627, Thermo Fisher Scientific) and FITC-conjugated goat anti-chicken secondary antibody (#PA1-28794; Invitrogen) were added to the cell suspension (1:500 dilution) and incubated at 4°C for 30 min. Finally, after three washes with FACS buffer, the cells were analyzed using a flow cytometer (CytoFLEX S; Beckman Coulter). For library screening, fluorescently labeled yeast cells from each round were sorted using a BD FACSAria III flow cytometer (FACSAria III; BD). The concentration of pepN48 was gradually decreased from 1 nM to 0.25 nM in each selection round.

[0129] Tango Assay In the Tango assay, hCXCR2 was fused to an exogenous transcription factor, which was linked to a specific cleavage sequence of a non-natural protease fused to β-arrestin. When a ligand binds to hCXCR2 and causes membrane desensitization, the intracellular arrestin-protease fusion protein is recruited to the activated receptor, where the fused transcription factor is cleaved by the protease and enters the nucleus, activating a reporter gene that emits fluorescence at 520 nm. The Tango assay was performed according to the manufacturer's instructions for the LiveBLAzer FRET-B / G Loading Kit (#K1030; Invitrogen). Briefly, TangoCXCR2-bla U2OS cells were plated at 20,000 cells / well on CellCarrier-96 (PerkinElmer) plates and cultured overnight in medium at 37°C / 5% CO2. The resulting cells were reseeded in the assay medium provided with the kit and cultured for 48 h at 37°C / 5% CO2. Different concentrations of ligands, such as IL8, Groα (CXCL1), and antibody ligands, were mixed with the above cells and cultured overnight at 37°C / 5% CO2 to measure their agonistic effects.

[0130] For inhibition studies of abN48-IgG1 and abN48-2-IgG1, cells were first treated with different concentrations of antibodies for 30 min at 37°C / 5% CO2, followed by treatment with 20 nM IL8 or Groα (CXCL1) (EC 80 ) was added and incubated overnight at 37°C / 5% CO2. Before cell lysis, detection substrate mix was added and incubated for 2 hours at room temperature. Fluorescence was recorded and quantified using a fluorescence plate reader (EnVision, PerkinElmer) with excitation at 460 nm and emission at 520 nm.

[0131] Calcium influx Calcium ion influx assays were performed with in-house CHO cells stably expressing both hCXCR2 and Gα16 proteins. 20,000 cells / well were plated in 384-well plates and cultured for 4-6 hours in growth medium supplemented with 1% FBS. The medium was removed and the cells were washed twice with HBSS buffer. 25 μL of freshly prepared loading solution containing calcium dye Fluo-4 Direct™ (#F10471; Invitrogen) was added to each well and incubated at 37°C for 30 minutes. The assay plate was equilibrated to room temperature before FLIPR detection. For agonist effects, 5 μL of ligand stock (in HBSS buffer) at different concentrations for IL8, Groα(CXCL1), and combinatorial antibodies were quickly mixed into each well containing dye-loaded cells and immediately recorded in a FLIPR reader (Tetra Multi-Mode Microplate Reader, Molecular Devices) at wavelengths of 494 nm (excitation) and 516 nm (emission). Final assay concentrations were 100, 25, 6.2, 1.6, 0.39, 0.098, 0.024, and 0.0061 nM for IL8 and Groα(CXCL1), and 3600, 900, 225, 56, 14, 3.5, 0.88, and 0.22 nM for abN48-IgG1 and abN48-2-IgG.

[0132] For inhibition studies of abN48-IgG1 and abN48-2-IgG1, 5 μL of HBSS stock containing different concentrations of abN48-IgG1 and abN48-2-IgG1 were first mixed into each corresponding well containing 25 μL of dye-loaded CHO cells and then equilibrated for 30 min at room temperature. Ca2+ influx signals were initiated by quickly mixing 5 μL HBSS stock of IL-8 (final concentration in assay 2.5 nM) or Groα(CXCL1) (final concentration in assay 7 nM) into each well on the FLIPR. The final concentrations of abN48-IgG1 and abN48-2-IgG1 in the assay were 3600 nM, 1200, 400, 130, 44, 15, 4.9, and 1.6 nM for IL8 inhibition, and 3600, 900, 225, 56, 14, 3.5, 0.88, and 0.22 nM for Groα (CXCL1) inhibition, respectively. 2+ The fluorescent signal of influx was recorded in real time as described above.

[0133] hCXCR2 Internalization Antibody-stimulated CXCR2 endocytosis was measured using an improved method utilizing the adaptor-toxin fusion protein AL2-PE38KDEL, which specifically binds to IgG and forms an immunotoxin complex (Hou, S.-C. et al,(2016).Scientific reports 6, 31878). The resulting immunotoxin complex induces cell death upon entry into cells. Briefly, AL2-PE38KDEL was added to the antibody solution at a 1:1 molar ratio and incubated at room temperature for 1 h to generate the immunotoxin complex. This immunotoxin was mixed with cells overexpressing hCXCR2 at different final concentrations and incubated at room temperature for 4 h. The resulting cells were reseeded in fresh medium and cultured at 37°C / 5% CO2 for 3 days. Cell viability was determined by measuring absorbance at 450 nm using WST-1 (#W201-12; Dojindo) according to the manufacturer's protocol.

[0134] Neutrophil chemotaxis Neutrophil migration was detected using a 24-well transwell chamber (#3422, Corning Costar) equipped with an 8 μm pore size membrane. Primary human neutrophils (#PBN-1F, MT-BIO) were cultured in chemotaxis medium (RPMI 1640 medium containing 0.5% BSA) at 5 × 10 5 Neutrophil samples were suspended at 1000 cells / ml. These neutrophil samples were incubated in chemotaxis medium with abN48-IgG1, abN48-2-IgG1, isotype antibody, and CXCR1 / 2 inhibitor navarixin (MK7123, #HY-10198, MedChemExpress) at different concentrations for 30 min at 37°C. The chemoattractant recombinant human IL8 (#Z03262-25, GenScript) was included in 600 μl chemotaxis medium in each lower chamber at a concentration of 10 nM. 250 μl of neutrophil suspension was placed in each upper chamber and incubated at 37°C for 60 min. After incubation, cells in the chemotaxis medium in the lower chamber were counted using a hemocytometer (#717810, BRAND).

[0135] Crystallization, X-ray data collection and structure determination Two peptides, pepN48 and pepN9-19, corresponding to residues 1-48 and 9-19 of CXCR2, respectively, were synthesized at Sangon Biotech. The purity and identity of the peptides were tested by HPLC and mass spectrometry. abN48 Fab was generated by papain (Sigma-Aldrich, 1:50 w / w ratio) cleavage of abN48-IgG1 (lambda type) in PBS buffer pH 7.4 at 4°C overnight. After cleavage, abN48 Fab was loaded onto a HiTrap lambda HP column (GE Healthcare) and eluted with 0.1 M sodium acetate, pH 3.0. The eluted fractions were pooled and immediately applied to a Superdex200 increase 10 / 300GL gel filtration column (GE Healthcare) equilibrated with a buffer containing 20 mM Tris pH 7.5 and 50 mM NaCl. The fractions containing purified abN48 Fab from the gel filtration column were then pooled and mixed with pepN9-19 (Sangon Biotech) at a molar ratio of 1:3. The mixture was incubated overnight at 4°C and then further concentrated. The complex of abN48-2 Fab and pepN9-19 was assembled following a similar protocol.

[0136] Crystals of the Fab antibody-peptide complex were obtained at 18 °C using the hanging drop, vapor diffusion method. Diffraction-quality crystals of abN48 Fab complexed to pepN9-19 were grown on siliconized cover clips by mixing 1 μL of protein solution (15 mg / mL) with 1 μL of reservoir solution (0.1 M HEPES sodium pH 7.5; 2% v / v polyethylene glycol 400; 2.0 M ammonium sulfate). Crystals of abN48-2 Fab complexed to pepN9-19 were obtained in 0.1 M HEPES pH 7.5; 25% w / v polyethylene glycol 3,350.

[0137] Crystals were flash-cooled in liquid nitrogen after adding 20% ​​glycerol as a cryoprotectant and used for data collection. Diffraction data were collected at beamline BL19U1 of the Shanghai Synchrotron Radiation Facility (SSRF) at a wavelength of 0.9789 Å and processed with the HKL3000 program. The structure of the complex of abN48 Fab and pepN9-19 was solved by molecular replacement using the Phaser program in PHENIX. The search model was a V-model constructed with SWISS-MODEL. H Domains and V of 4E10 Fab structure (PDBID: 4XCN, L chain) L The structure of the abN48-2 and pepN9-19 complex was similarly solved using the abN48-pepN9-19 complex structure as a molecular replacement search model. This initial model was further improved by cycles of manual building and refinement using COOT and Refmac5 in ccp4i. The quality of the final model was analyzed with MolProbity. A summary of data collection and refinement statistics is shown in Table S2. Figures were generated using PyMol (The PyMOL Molecular Graphics System, Version 2.1 Schrodinger, LLC). Electrostatic calculations were performed in PDB2PQR.

[0138] Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Amide hydrogen exchange of pepN48 alone was initiated by diluting 1 μL pepN48 at 50 μM into 19 μL DO buffer (25 mM Tris, pH 8.0, 150 mM NaCl, 1 mM TCEP) at 10 °C. At different time points (0 s, 10 s, 30 s, 60 s, 120 s), the labeling reaction was stopped by adding chilled quench buffer (400 mM KH2PO4 / K2PO4, pH 2.2, 50 mM TCEP) and immediately frozen in liquid nitrogen. For HDX-MS of pepN48 in the presence of abN48, 1 μL pepN48 at 50 μM was first mixed with 1 μL abN48 at 67 μM. The mixture was then labeled for 0, 10, 30, 60, or 120 seconds by adding 18 μL D2O buffer and then rapidly flash frozen. All frozen samples were stored at -80°C until analysis.

[0139] The thawed samples were immediately injected into an HPLC-MS (Agilent 1100) system with in-line peptide digestion and desalting. The desalted digests were analyzed using Hypersil Gold™. The HPLC system was extensively washed with blank injections between samples to minimize carryover. Peptide identification was performed by tandem MS / MS in orbi / orbi mode. All MS / MS spectra were analyzed using the MASCOT program and the final PSMs were filtered at 1% FDR. An initial analysis of peptide centroids was performed with HD-Examiner v1.3 (Sierra Analytics), after which all peptides were manually verified to confirm retention time, charge state, m / z range and the presence of overlapping peptides. Peptide coverage of pepN48 was found to be 100%, and the relative deuteration level (%D) of each peptide was automatically calculated by HD-Examiner assuming that fully deuterated samples retain 90%D at the current LC settings.

[0140] statistical analysis All statistical tests were performed with Graphpad Prism 7 software. Measurements were expressed as mean or mean ± standard deviation unless otherwise stated. P values ​​< 0.05 were considered significant.

[0141] Example 2. Selection of combinatorial antibodies that bind to human CXCR2 The human CXCR2 protein is a transmembrane protein with an N-terminus and three extracellular loops on the outside of the cell. The N-terminus and each of the three loops are involved in the binding of CXCR2 to IL8 (Ahuja, SK et al., (1996) The Journal of biological chemistry 271, 225-232; Katancik, JA, et al., (1997) Biochemical and Biophysical Research Communications 232, 663-668; Luo, ZW, et al., (1997) Protein Engineering 10, 1039-1045; Berkamp, ​​S., et al., (2017) Journal of Biomolecular Nmr 69, 111-121; Park, SH et al., (2017) Biophysical Journal 113, 2695-2705; Park, SH et al., (2011) J Mol Biol 414, 194-203; and Park, SH et al., (2012) Nature 491, 779-783).

[0142] Through sequence analysis, the inventors decided to target the extracellular N-terminal 48 amino acids as an epitope in panning and optimizing highly selective and potent combinatorial antibodies targeting IL8 signaling through CXCR2. A peptide (pepN48, Figure 1A) corresponding to the first 48 amino acids of the flexible N-terminus of human CXCR2 was synthesized (Chinese Peptide, Hangzhou). 11Phage panning was performed three times using the combinatorial library of and pepN48. Nine scFv sequences that showed high enrichment in the panning were subcloned into the pFuse expression vector. Expression and affinity binding of each scFv combinatorial antibody was confirmed by ELISA screening of cell supernatants in the presence of pepN48 peptide (Figure 5A). Further analysis of the supernatants using FACS showed that only one clone, H8(abN48), recognized membrane human CXCR2-mCherry on U2OS cells (Figure 5B). Next, the H8(abN48) scFv combinatorial antibody was overexpressed, purified to homogeneity, and subjected to SPR analysis on Biacore. The K of H8(abN48) with pepN48 was D The value is 2.6 × 10 -9 It was determined to be M (Figure 5C).

[0143] Example 3. Generation and affinity maturation of full-length IgG1 combinatorial antibodies To optimize the binding affinity of H8(abN48), the scFv antibody was first converted to a full-length combinatorial antibody in IgG1 format (Figure 6A), designated abN48-IgG1. D The value is 7.8 × 10 -10 The V of abN48-IgG1 was determined to be M (Figure 1B). The improved affinity of abN48-IgG1 compared to the scFv version suggests that affinity maturation by yeast display may be used to further optimize the Fab configuration. To maximize the chances of finding an optimal binder to the target epitope sequence, the V H The 11 CDR3 residues GYCSSTSCYDY were randomly mutated, and the resulting Fab sequence was displayed on the yeast surface using a pESC vector containing the GAL1-10 bidirectional promoter to express the heavy and light chains. 7A diverse combinatorial antibody library was generated. After four rounds of panning against pepN48, eight highly enriched sequences, abN48-1 to abN48-8, were selected from 100 positive clones and confirmed by sequencing analysis. Weblogo analysis revealed that the most frequent mutations in the enriched sequences were S5R and S7R, which constituted the sequence of a new combinatorial antibody, abN48-2 (Figure 5B). The eight sequences were subcloned into the pFuse vector and expressed in HEK293T cells. The binding affinity and homogeneity of the eight purified antibodies were determined by SPR and SEC-HPLC, respectively (Table 1 and Figure 7).

[0144] [Table 6]

[0145] All new constructs showed subnanomolar to picomolar binding affinity with pepN48, suggesting a common epitope sequence at the N-terminus of CXCR2. As shown in Figure 1B, the combinatorial antibody abN48-2-IgG1 showed picomolar binding affinity, with a K D The value is 7.2 × 10 -12 M was improved by 100-fold compared to abN48-IgG1. Furthermore, after incubation at 42°C for 5 days, abN48-2-IgG1 showed no detectable aggregation or degradation (Figure 7, top right).

[0146] [Table 7]

[0147] The interaction of abN48-IgG1 or ab48-2-IgG1 with pepN48 was further verified and mapped to residues 12–19 and 13–21, respectively, using HDX-MS techniques (Figure 1C).

[0148] Example 4. Species and subtypes of combinatorial antibodies that specifically bind to hCXCR2 To determine the species and subtype specificity of abN48-IgG1 and abN48-2-IgG1, gene constructs containing hCXCR1-mCherry, hCXCR2-mCherry, mCXCR2-mCherry, rCXCR2-mCherry, rbCXCR2-mCherry and mcCXCR2-mCherry were overexpressed in U2OS cells. Immunofluorescence confocal imaging of hCXCR1-mCherry, hCXCR2-mCherry and mCXCR2-mCherry overexpressing U2OS cells with abN48-IgG1 and abN48-2-IgG1 demonstrated that both antibodies specifically recognized surface hCXCR2 (Figure 2A). This high specificity was further confirmed by FACS analysis, showing that both abN48-IgG1 and abN48-2-IgG1 bound exclusively to CXCR2 in humans (hCXCR2) and the closely related macaque monkey (mcCXCR2), but not to CXCR1 or CXCR2 in mice, rats, or rabbits (Figure 2B).

[0149] Example 5. Combinatorial antibodies potently inhibited hCXCR2-mediated β-arrestin signaling and calcium influx To understand the cellular functions of hCXCR2-specific antibody binders, we investigated two independent signaling pathways through CXCR2: β-arrestin recruitment and cytoplasmic Ca 2+ The influx of Ca was assessed using Tango and FLIPR, respectively. β-arrestin recruitment is a G protein-independent intracellular event, whereas Ca 2+ The influx of Ca is induced by the activation of Gα proteins and IP3. 2+ from the endoplasmic reticulum (ER) into the cytoplasm, both of which are known to be directly associated with CXCR2 activation.

[0150] First, the agonistic effects of the natural ligands and the two selected combinatorial antibodies on β-arrestin recruitment were tested using the Tango assay. Only the two natural ligands, IL8 and GROα, induced β-arrestin recruitment, with apparent EC50 values ​​of 4.5 nM and 5.3 nM, respectively (Figure 3A). On the other hand, the two antibodies, abN48-IgG1 and abN48-2-IgG1, showed no activation of recruitment up to 100 nM, but completely inhibited recruitment induced by the natural ligands (EC50 values ​​of 2.8 nM and 0.90 nM for IL8, and 4.7 nM and 0.37 nM for GROα, respectively). 80 It is noteworthy that both abN48-IgG1 and abN48-2-IgG1 exhibited inverse agonist effects, and that both antibodies at 20 nM significantly suppressed endogenous β-arrestin recruitment induced by CXCR2 expression (Figure 8).

[0151] Next, we investigated the Ca concentration of IL8, GROα, and the combinatorial antibody. 2+ The natural ligands IL8 and GROα stimulated Ca influx in a dose-dependent manner. 2+ Activates the influx of EC 50 The EC values ​​were 0.42 nM and 1.7 nM, respectively (Figure 3C). Interestingly, the strong binding antibody, abN48-2-IgG1, showed partial agonist effects in the high concentration range of 10-1000 nM, with apparent EC 50 The EC value was 310 nM (Figure 3C). On the other hand, both abN48-IgG1 and abN48-2-IgG1 were significantly higher than those of the natural ligand (EC 90 Ca at levels 2+ Influx was inhibited in a dose-dependent manner, with IC 50 The values ​​were 190 nM and 44 nM, respectively, for IL8 and 1000 nM and 110 nM, respectively, for GROα (Fig. 3D).

[0152] The improved antagonism of CXCR2-mediated signaling by abN48-2-IgG1 compared with abN48-IgG1 is consistent with its improved binding affinity to the N-terminal epitope sequence of CXCR2.

[0153] Example 6. Combinatorial antibodies potently induced internalization of hCXCR2 It has also been shown that IL8 induces endocytic signaling of CXCR2 at 5-10 nM. To verify the effect of combinatorial antibody ligands on endocytosis of CXCR2, we examined CXCR2 internalization using U2OS cells overexpressing membrane hCXCR2 rather than FcR receptor. We used immunotoxin conjugates of abN48-IgG1 or abN48-2-IgG1 as molecular probes. As shown in Figure 3E, only immunotoxin conjugates of abN48-IgG1 and abN48-2-IgG1 showed dose-dependent cytotoxicity against U2OS cells overexpressing hCXCR2. Immunotoxin conjugates of unrelated combinatorial antibodies and U2OS cells not expressing hCXCR2 did not show detectable cytotoxicity under the experimental conditions. The activating effect of abN48-IgG1 or abN48-2-IgG1 was observed in the apparent EC 50 The value was highly sensitive at less than 0.1 nM.

[0154] Example 7. Monoclonal antibody abN48-2-IgG1 potently inhibited IL8-induced neutrophil chemotaxis Neutrophil chemotaxis occurs along the gradient of corresponding chemokines secreted at distant sites of acute injury or infection. CXCR2 is known to be expressed primarily on neutrophils, and its cognitive ligand, IL8, has been found to be a key regulator in many pathological processes, making the IL8-CXCR2 axis an important therapeutic intervention target. In this example, chemotaxis testing of an optimized combinatorial antibody, abN48-2-IgG1, was performed with primary human neutrophils. Validated neutrophils were negatively selected by immunomagnetic bead separation from whole blood collection and transfected with CD15 + , CD16 + , CD11b + , CD66b + (MT-Bio, Shanghai) was confirmed for purity.

[0155] First, we confirmed hCXCR2 membrane expression on human neutrophils by immunostaining with both abN48-IgG1 and abN48-2-IgG1 (Figure 4A). FACS analysis showed that the entire neutrophil cell population could be captured by abN48-IgG1 or abN48-2-IgG1, which was consistent with previous literature reports. Next, IL8-induced neutrophil chemotaxis was tested by transwell migration assay. The combinatorial antibody, abN48-2-IgG1, dose-dependently, highly potently, and completely inhibited neutrophil chemotaxis induced by the maximum IL8 concentration (10 nM) (Figure 4B). As shown in Figure 4B, abN48-2-IgG1 showed extremely potent inhibition of neutrophil chemotaxis, 75% at 1 nM and 120% at 20 nM. Meanwhile, the small molecule CXCR1 / CXCR2 dual inhibitor MK7123 showed 100% inhibition of neutrophil chemotaxis at 1 μM. The complete inhibition of both chemokine-dependent and -independent neutrophil migration by abN48-2-IgG1 may suggest the involvement of CXCR2 in the basal intrinsic migration of neutrophils.

[0156] Example 8. Interaction of abN48 antibody with the N-terminus of CXCR2 characterized by X-ray crystallography and mutagenesis Based on the HDX-MS results (Figure 1C), to identify the minimal interacting peptide sequence of pepN48 for crystallographic studies, in this example, a series of truncations from the N- and C-termini of the pepN48 peptide were designed and it was found that the minimal 9-19 amino acid (aa) peptide (pepN9-19, DSFEDFWKGED, SEQ ID NO:16) bound to the Fab forms of both abN48-IgG1 and abN48-2-IgG2 at a level comparable to pepN48, whereas pepN48 lacking the 9-19 aa (pepN48?9-19) was no longer recognized by the antibody (Figure 9).

[0157] We solved the crystal structures of abN48 and abN48-2 (Fab form) in complex with pepN9-19 to a final resolution of 2.8 Å (Fig. 1D, Table 3). Two abN48 / pep9-19 protein complexes (protomers) exist in one asymmetric unit (Fig. 10A). In one such protomer (Fig. 10B, left), the electron density for bound pepN9-19 was much better than in the other (Fig. 10B, right), so we focused our analysis on the former in this example.

[0158] [Table 8] *Stats for highest resolution shell in brackets.

[0159] In particular, in both abN48 / pepN9-19 protomers, FW14-15aa of pepN9-19 could be well traced from the electron density (Figure S10B). This indicates that these two residues play an important role in defining the binding between abN48 and pepN9-19. Indeed, the CDR loops of abN48 were positioned to form a large hydrophobic cavity on top of its variable region (Figure S11A and S11B). Residues F14 and W15 inserted their bulky aromatic side chains into this hydrophobic cavity and formed strong hydrophobic interactions with residues on all CDR loops except CDR2L of abN48 (Figure S11C). Specifically, W104 and F106 of CDR3L and W111 of CDR3H of abN48 form strong π-π stacking interactions with W15 of pepN9-19, which interacts with Y38 of the light chain frame region and W48 of the heavy chain frame region of abN48 (Figure S11C). Meanwhile, F14 of pepN9-19 forms strong π-π stacking interactions with Y36 of CDR1L and F48 and Y38 of the light chain frame region of abN48 (Figure S11C). In addition, W51 of CDR2H is hydrophobically packed against the side chain of K16 of pepN9-19, thereby also contributing to the high affinity interaction between abN48 and NT9-19 (Figure S11C). Therefore, the structure of abN48 / NT9-19 definitely indicates that F14 and W15 in the N-terminal region of CXCR2 are the important epitopes recognized by abN48 and abN48-2. Interestingly, both CDR2H and CDR3H of abN48 exist as antiparallel β-sheets, and CDR3H is even stabilized by a pair of α-strand disulfide bonds (C102-C107). Such a CDR loop conformation, although very rare, has been observed before.

[0160] For further validation, in this example, alanine-scanning site-directed mutagenesis was performed on the D13, F14, W15, and K16 residues at the N-terminus of hCXCR2. The mutants were fused with mCherry and expressed in U2OS cells, then confirmed by FACS and immunocytofluorescence (Figure 12). The results were consistent with the crystal analysis that D13, F14, and W15 are important epitopes for binding to antibody ligands. Therefore, W15 of CXCR2 is likely important for the interaction of IL8 with CXCR2, and abN48 can antagonize CXCR2 by competing with IL8 upon F14-W15 binding of CXCR2.

[0161] The present disclosure is not limited in scope by the specific embodiments described, which are intended as single illustrations of individual aspects of the present disclosure, and any compositions or methods that are functionally equivalent are also within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present disclosure without departing from the spirit and scope of the present disclosure. Therefore, the present disclosure is intended to cover the modifications and variations of the present disclosure, provided that they fall within the scope of the appended claims and their equivalents.

[0162] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. An antibody or an antigen-binding fragment thereof, which has specificity to human C-X-C motif chemokine receptor 2 (CXCR2) protein and comprises a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3, and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3, the VH CDR1 comprises the amino acid sequence of SEQ ID NO:1; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:2; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3 and 7-14; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:4; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:5; An antibody or antigen-binding fragment thereof, wherein the VL CDR3 comprises the amino acid sequence of SEQ ID NO:

6.

2. The antibody or antigen-binding fragment thereof of claim 1 , wherein the VHCDR3 comprises the amino acid sequence of SEQ ID NO:

3.

3. The antibody or antigen-binding fragment thereof of claim 1 , further comprising a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody is a human antibody.

5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the VH comprises an amino acid sequence of SEQ ID NO:17 or 18 or a peptide having at least 90% sequence identity to SEQ ID NO:17 or 18.

6. The antibody or antigen-binding fragment thereof of any one of claims 1 to 5, wherein the VH comprises an amino acid sequence of SEQ ID NO:18 or a peptide having at least 90% sequence identity to SEQ ID NO:

18.

7. The antibody or antigen-binding fragment thereof of claim 5 or 6, wherein the VL comprises an amino acid sequence of SEQ ID NO:19 or a peptide having at least 90% sequence identity to SEQ ID NO:

19.

8. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 and a pharma- ceutically acceptable carrier.

9. An isolated cell comprising one or more polynucleotides encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

10. An antibody or antigen-binding fragment thereof described in any one of claims 1 to 7 for treating cancer or an inflammatory disease in a patient in need of treatment thereof.

11. The antibody or antigen-binding fragment thereof of claim 10, wherein the cancer is selected from the group consisting of bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer and thyroid cancer.

12. The antibody or antigen-binding fragment thereof of claim 10, wherein the inflammatory disease is selected from the group consisting of Parkinson's disease, arthritis, rheumatoid arthritis, multiple sclerosis, psoriasis, psoriatic arthritis, Crohn's disease, inflammatory bowel disease, ulcerative colitis, lupus, systemic lupus erythematosus, juvenile rheumatoid arthritis, juvenile idiopathic arthritis, Grave's disease, Hashimoto's thyroiditis, Addison's disease, celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis, pernicious anemia, Sjogren's syndrome, type I diabetes, vasculitis, uveitis, atherosclerosis, and ankylosing spondylitis.

13. The antibody or antigen-binding fragment thereof of claim 10, wherein the inflammatory disease is caused by an infectious disease.

14. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 for the manufacture of a medicament for the treatment of cancer or an inflammatory disease.

15. The antibody or antigen-binding fragment thereof of any one of claims 1 to 7 for treating cancer in a patient in need thereof, said treatment comprising: (a) treating T cells in vitro with an antibody or antigen-binding fragment thereof; (b) administering the treated T cells to the patient. An antibody or antigen-binding fragment thereof comprising:

16. The antibody or antigen-binding fragment thereof described in claim 15, wherein the treatment further comprises isolating T cells from the patient prior to step (a).

17. The antibody or antigen-binding fragment thereof of claim 15, wherein the T cells are tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or a combination thereof.

18. 8. A method for detecting CXCR2 expression in a sample comprising contacting an antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 with the sample under conditions in which the antibody or antigen-binding fragment thereof binds to CXCR2, and detecting binding indicative of expression of CXCR2 in the sample.

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