Antibodies targeting integrin beta-2

Abstract

Provided herein are antibodies that specifically target integrin beta-2 and compositions comprising such antibodies for therapeutic and diagnostic applications. The antibody comprises an integrin beta-2 binding structural domain, the integrin beta-2 binding structural domain comprises a heavy chain variable region, and the heavy chain variable region comprises an HCDR1 sequence containing ISYYM, an HCDR2 sequence containing SISSSSGYTY and an HCDR3 sequence containing GAM. And a light chain variable region, the light chain variable region comprising an LCDR1 sequence containing SVSSA, an LCDR2 sequence containing SASSLYS, and an LCDR3 sequence containing FSSGSWAPI.

Description

Technical Field

[0001] Embodiments disclosed herein relate to antibody therapeutics, and in particular to antibodies that specifically target integrin beta-2. Background Art

[0002] Given the lack of characterized cancer-specific surface markers, the development of safe and effective immunotherapeutics has proven difficult. It has been hypothesized that cancer-specific surface protein conformations may in fact be widespread, given abnormalities in tumor signaling, metabolism, or cellular microenvironment communication, all of which largely involve membrane proteins. In particular, integrin beta-2 may be a promising immunotherapeutic target, being widely expressed in cell lines and patient tumors.

[0003] Therefore, new antibodies that specifically target integrin beta-2 are needed to further develop immunotherapeutic and diagnostic agents targeting cancer. Summary of the invention

[0004] Phage display selections were used to identify anti-integrin beta-2 antibodies that could be used for diagnostic and therapeutic purposes.

[0005] In some embodiments, a method is provided in which the K of the peptide is less than about 10 nM. D Anti-integrin beta-2 antibody.

[0006] In some embodiments, the antibody comprises an anti-integrin beta-2 binding domain having at least one, at least two, or three CDRs of a variable domain sequence of SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the anti-integrin beta-2 binding domain comprises a HCDR3 of SEQ ID NO: 2 and a LCDR3 of SEQ ID NO: 3. In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 2 and LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 3.

[0007] In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 2, wherein one of the CDRs comprises a substitution relative to the corresponding CDR shown in SEQ ID NO: 2. In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 2, wherein two of the CDRs comprise a substitution relative to the corresponding CDR shown in SEQ ID NO: 2. In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 2, wherein all three CDRs comprise a substitution relative to the corresponding CDR sequences shown in SEQ ID NO: 2.

[0008] In some embodiments, the anti-integrin beta-2 binding domain comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 3, wherein one of the CDRs comprises a substitution relative to the corresponding CDR shown in SEQ ID NO: 3. In some embodiments, the anti-integrin beta-2 binding domain comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 3, wherein two of the CDRs comprise a substitution relative to the corresponding CDR shown in SEQ ID NO: 3. In some embodiments, the anti-integrin beta-2 binding domain comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 3, wherein all three CDRs comprise a substitution relative to the corresponding CDR sequences shown in SEQ ID NO: 3.

[0009] In some embodiments, the anti-integrin beta-2 binding domain comprises a variable region having at least 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the variable region sequence of SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the variable domain comprises a substitution, insertion or deletion in the framework of the variable region as shown in SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the anti-integrin beta-2 binding domain of the present disclosure comprises a heavy chain variable region comprising the HCD1, HCDR2 and HCDR3 sequences of SEQ ID NO: 2 and having at least 95% identity to SEQ ID NO: 2; and a light chain variable region comprising the LCD1, LCDR2, LCDR3 sequences of SEQ ID NO: 3 and having at least 95% identity to SEQ ID NO: 3.

[0010] Other aspects and features will become apparent to those of ordinary skill in the art upon reading the following description of certain exemplary embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The accompanying drawings included herein are used to illustrate various examples of the products, methods and devices of the present specification. In the accompanying drawings:

[0012] Figure 1 is a diagram of the phage-fab display selection strategy used to develop anti-integrin beta-2 antibodies;

[0013] Figures 2A-2D is a representative ELISA profile of phage-selected anti-integrin β-2 antibody candidates;

[0014] Figure 3 Yes Display Figures 2A-2D Table of complementarity determining regions (CDRs) of anti-integrin beta-2 antibody candidates shown in .

[0015] Figures 4A-4H is an ELISA graph showing integrin β-2 binding specificity of antibodies obtained from phage display selection;

[0016] Figures 5A-5R is a representative bio-layer interferometry plot showing the binding kinetics of antibodies to integrin-2 heterodimers; and

[0017] Figure 6 Is from Figures 5A-5R A table showing the binding affinity of antibodies against integrin-2 heterodimers is shown in the biolayer interferometry plots. DETAILED DESCRIPTION

[0018] Various material compositions will be described below to provide examples of each claimed embodiment. The embodiments described below do not limit any claimed embodiment, and any claimed embodiment may encompass compositions different from those described below. The claimed embodiments are not limited to compositions having all the features of any of the following compositions or features common to a plurality or all of the following compositions.

[0019] As used herein, the term "about" refers to the usual error range of the corresponding value that is readily known to those skilled in the art. D and IC 50 Values, ±20%, ±10%, or ±5% are within the intended meaning of the values.

[0020] As used herein, the term integrin beta-2 or Integrin beta-2 (also known as CD18, LAD, LCAMB, LFA-1, MAC-1, MF17, MFI7 or integrin subunit beta 2) refers to a polypeptide encoded by the ITGB2 gene (chr21:44,885,949-44,931,989 (GRCH38 / hg38)), which is cytogenetically mapped to human chromosome 21q22.3 by HGNC, Entrez Gene and Ensembl (genomic coordinates (GRCh38 / hg38 assembly December 2013:)), and plays a role in cell adhesion, cell surface-mediated sequencing and immune response. An illustrative human integrin beta-2 protein sequence encoded by the human ITGB2 gene (P05107-1) can be obtained under Uniprot number P05107 and is provided as SEQ ID NO: 1. Integrin beta-2 can bind to many alpha chains and thus can form multiple heterodimers, but also exists in a soluble ligand-bound form. Defects in Itgb2 expression can lead to adhesion defects of circulating leukocytes in the human body, reducing the ability of the immune system to defend against foreign invaders. Illustrative integrin beta-2 heterodimers include, for example, integrin ITGAL / ITGB2, which is a receptor for ICAM1, ICAM2, ICAM3 and ICAM4, and is also a receptor for the secreted form of the ubiquitin-like protein ISG15; integrin ITGAM / ITGB2 and ITGAX / ITGB2, which are receptors for the iC3b fragment of the third complement component and fibrinogen; integrin ITGAX / ITGB2, which recognizes the sequence GPR in the fibrinogen alpha chain, integrin ITGAM / ITGB2, which recognizes the P1 and P2 peptides of the fibrinogen gamma chain, and is also a receptor for factor X; and integrin ITGAD / ITGB2, which is a receptor for ICAM3 and VCAM1.

[0021] The terms "anti-integrin beta-2 antibody," "integrin beta-2 specific antibody," "integrin beta-2 antibody," and "anti-integrin beta-2" are used synonymously herein to refer to an antibody that specifically binds to integrin beta-2. An illustrative human integrin beta-2 sequence is provided in SEQ ID NO: 1.

[0022] As used herein, an "anti-integrin beta-2 binding domain" refers to a V domain comprising an anti-integrin beta-2 antibody as described herein. H and V L The antigen binding domain of the region binds to integrin beta-2.

[0023] The anti-integrin beta-2 antibodies of the present disclosure bind to integrin beta-2. The active state of integrin beta-2 is an extended-open conformation (see, e.g., Nishida et al., Immunity 25:583-94, 2006; Li et al., EMBO J. 36:629-45, 2017). The ligand affinity of the active conformation (extended-open) is increased by 4,000 times compared to the other two states (bent-closed, inactive; and extended-closed (intermediate)) (Li et al., 2017, supra). Integrin activation occurs through various cell surface receptors upon cell stimulation. Cell stimulation triggers an inside-out signaling pathway that ultimately recruits cytoplasmic factors such as talin and kindlin to the NPxY motif of the cytoplasmic tail of the beta chain of integrin, which causes the cytoplasmic tail of the integrin subunit to separate and convert the integrin to an active (extended-open) conformation.

[0024] The term "antibody" as used herein refers to a polypeptide or fragment thereof comprising a framework region encoded by an immunoglobulin gene, which specifically binds and recognizes an antigen, such as integrin beta-2. Typically, the "variable region" contains the antigen binding region of the antibody (or its functional equivalent), and is important in terms of specificity and affinity of binding. The term "antibody" as used herein thus encompasses antigen binding fragments, such as antigen binding domains or other antigen binding fragments. Antigen binding fragments can be produced by modifying whole antibodies, or using recombinant DNA methods (e.g., single-chain Fv formats).

[0025] Illustrative immunoglobulin (antibody) structural units include tetramers. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The term variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains, respectively.

[0026] As used herein, a "V region" refers to an antibody, e.g., an antibody, variable region domain that comprises segments of framework 1, CDR1, framework 2, CDR2, and framework 3, including segments of CDR3 and framework 4 that are added to the V segment as a result of rearrangement of V region genes during B cell differentiation.

[0027] As used herein, "complementarity determining region (CDR)" refers to the three hypervariable regions that interrupt the four "framework" regions of the variable domain. The CDRs are the major contributors to epitope binding to an antigen. The CDRs are referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus.

[0028] The amino acid sequences of the CDR and framework regions can be determined using various definitions known in the art, such as Kabat, Chothia, the international ImMunoGeneTics database (IMGT) and AbM (see, e.g., Johnson et al., supra; Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992, structural repertoire of the human VH segments J. Mol. Biol. 227, 799-817; Al-Lazikani et al., J. Mol. Biol 1997, 273(4)).The definition of antigen binding sites is also described in: Ruiz et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219-221 (2000); and Lefranc, M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan 1; 29(1): 207-9 (2001); MacCallum et al., Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262(5), 732-745 (1996); and Martin et al., Proc. Natl Acad. Sci. USA, 86, 9268-9272 (1989); Martin et al., Methods Enzymol., 203, 121-153, (1991); Pedersen et al., Immunomethods, 1, 126, (1992); and Rees et al., In Sternberg MJE (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172 1996). Reference to CDRs as determined by Kabat numbering is based on, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institute of Health, Bethesda, MD (1991)). Chothia CDRs are determined as defined by Chothia (see, for example, Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0029] As used herein, "isotype" is a class of antibodies defined by the constant region of the heavy chain. The antibodies described herein can be any isotype of the isotype class. Immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes. Light chains are classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the isotype classes IgG, IgM, IgA, IgD, and IgE, respectively. In some embodiments, IgG is IgG1, IgG2, IgG3, or IgG4.

[0030] Antibodies can exist as intact immunoglobulins or as any of a number of well-characterized fragments that contain specific antigen binding activity. Such fragments can be produced by digestion with various peptidases. Pepsin digests antibodies below the disulfide bonds in the hinge region to produce F(ab)'2, which is a dimer of Fab, which itself is linked to V by disulfide bonds. H -C H 1 connected light chain. F(ab)'2 can be reduced under mild conditions to destroy the disulfide bond in the hinge region, thereby converting the F(ab)'2 dimer into a Fab' monomer. Fab' monomer is basically a Fab with a hinge region portion (see Fundamental Immunology (Paul ed., 3rd edition, 1993). Although various antibody fragments are defined from the perspective of digestion of intact antibodies, it will be appreciated by the skilled artisan that such fragments can be synthesized de novo by chemical methods or by using recombinant DNA methods.

[0031] The antibody or antigen-binding molecule of the present invention further includes one or more immunoglobulin chains, which are chemically conjugated to other proteins or expressed as fusion proteins with other proteins. It also includes bispecific antibodies. Bispecific or bifunctional antibodies are artificial hybrid antibodies with two different heavy chain / light chain pairs and two different binding sites. Other antigen-binding fragments or antibody portions of the present invention include single-chain variable fragments (scFv), bivalent scFv (bi-antibodies), bispecific scFv antibodies (wherein the antibody molecule recognizes two different epitopes), single binding domains (dAbs) and mini-antibodies. The term "antibody" additionally encompasses bispecific and multispecific antibodies and any other monovalent, bivalent or multivalent antibody forms.

[0032] The various antibodies or antigen-binding fragments described herein can be produced by enzymatic or chemical modification of intact antibodies, or synthesized de novo using recombinant DNA methods (e.g., single-chain Fv), or identified using yeast or phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554, 1990; Boder et al., (2000) Proc. Natl. Acad. Sci. USA 97:10701). For example, miniantibodies can be produced using methods described in the art (e.g., Vaughan and Sollazzo, Comb Chem High Throughput Screen. 4:417-30 2001). Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or connection of Fab' fragments. See, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992). Single-chain antibodies can be identified using phage display libraries, yeast display or ribosome display libraries, gene shuffling libraries. Such libraries can be constructed from synthetic, semisynthetic or natural and immunologically active sources.

[0033] "Monoclonal antibody" refers to a clonal preparation of antibody with a single binding specificity and affinity for a given epitope on an antigen.

[0034] A "chimeric antibody" is an antibody molecule in which (a) the constant region or a portion thereof is altered, replaced or exchanged so that the antigen binding site (variable region, CDR or a portion thereof) is linked to a constant region of a different or altered class, effector function and / or species, or to an entirely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug, etc.) that confers new properties to the chimeric antibody; or (b) the variable region or a portion thereof is altered, replaced or exchanged with a variable region having a different or altered antigenic specificity (e.g., CDR and framework regions from a different species).

[0035] "Humanized" antibodies are antibodies that retain the reactivity of non-human antibodies but have lower immunogenicity in humans. This can be achieved, for example, by retaining non-human CDR regions and replacing the remainder of the antibody with their human counterparts. In one embodiment, some, most, or all of the amino acids outside the CDR domains are replaced by amino acids corresponding to the human immunoglobulin germline, while the amino acids in one or more CDR regions remain unchanged. In some embodiments, one or more CDR residues can be changed, for example, to provide a sequence closer to the germline or to replace residues that may hinder production.

[0036] The term "specific binding" or "specific targeting" refers to a molecule (e.g., an antibody or antibody fragment) that binds to a target with at least 2 times greater affinity than a non-target compound, such as at least 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 25 times, 50 times, or 100 times greater affinity. For example, an antibody that specifically binds to integrin beta-2 typically binds to integrin beta-2 with at least 2 times greater affinity than a non-integrin beta-2 target, or in the case of an antibody that specifically binds to active integrin beta-2, binds to active integrin beta-2 with at least 2 times greater affinity than an inactive form of integrin beta-2. In some embodiments, an antibody binds to an inactive integrin beta-2 with a K of at least 100 times greater than its affinity for inactive integrin beta-2. D Binds to active integrin beta-2.

[0037] "Epitope" or "antigenic determinant" refers to the site on the antigen to which an antibody binds. An epitope can be formed by continuous amino acids or by non-continuous amino acids juxtaposed by the tertiary folding of the protein. Epitopes formed by continuous amino acids are usually retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are usually lost when treated with denaturing solvents. In a unique spatial conformation, an epitope usually includes at least 3, and more usually at least 5 or 8-10 amino acids. Methods for determining the spatial conformation of an epitope include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, edited by Glenn E. Morris (1996).

[0038] As used herein, the term "valency" refers to the number of different binding sites of an antibody for an antigen. A monovalent antibody contains one binding site for an antigen. A multivalent antibody contains multiple binding sites.

[0039] The words "protein", "peptide" and "polypeptide" are used interchangeably to refer to an amino acid polymer or a group of two or more interacting or bound amino acid polymers. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymers.

[0040] As used herein, "flexible linker" refers to an amino acid sequence that connects domains to provide a certain degree of motion or interaction. Such linkers are usually composed of small non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids, but may also include polar amino acids such as Lys and Glu, for example to improve solubility. The small size of these amino acids provides flexibility and enables the connected functional domains to have mobility. Incorporation of Ser or Thr can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, and thus reduce unfavorable interactions between the linker and the protein portion. In some embodiments, the flexible linker is mainly composed of stretches of Gly and Ser residues ("GS" linker). The most widely used example of a flexible linker has (Gly-Gly-Gly-Gly-Ser) n By adjusting the copy number "n", the length of the GS linker can be adjusted to achieve appropriate separation of functional domains and / or maintain necessary domain interactions. In addition to the GS linker, many other flexible linkers have been designed for recombinant protein expression.

[0041] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those amino acids encoded by the genetic code, as well as those amino acids that are subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure (e.g., an alpha carbon bound to a hydrogen, carboxyl group, amino group, and R group) as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfoxide, methionine methyl thiocyanate. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid but function similarly to naturally occurring amino acids.

[0042] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides may be referred to by their commonly accepted single-letter codes.

[0043] "Conservatively modified variants" are applicable to amino acids and nucleic acid sequences. With respect to a specific nucleic acid sequence, conservatively modified variants refer to those nucleic acids encoding the same or substantially the same amino acid sequence, or when the nucleic acid does not encode an amino acid sequence, it refers to substantially the same or associated, such as a natural continuous sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For example, codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at each position where alanine is specified by a codon, the codon can be changed to another of the corresponding codons without changing the encoded polypeptide. Such nucleic acid variations are "silent variations", which are a type of conservatively modified variations. Each nucleic acid sequence encoding a polypeptide herein also describes the silent variations of nucleic acids. The skilled person will recognize that, in some cases, each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to produce functionally identical molecules. Therefore, the silent variations of nucleic acids encoding polypeptides are implicit in the sequence with respect to the expression product rather than with respect to the actual probe sequence.

[0044] As used herein, "substitution" refers to the substitution of amino acids so that the charge, hydrophobicity and / or size of the side chain are maintained. Illustrative groups of amino acids that can be substituted for each other include (i) positively charged amino acids Lys, Arg and His; (ii) negatively charged amino acids Glu and Asp; (iii) aromatic amino acids Phe, Tyr and Trp; (iv) nitrogen ring amino acids His and Trp; (v) large aliphatic non-polar amino acids Val, Leu and Ile; (vi) slightly polar amino acids Met and Cys; (vii) small side chain amino acids Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gln and Pro; (viii) aliphatic amino acids Val, Leu, Ile, Met and Cys; and (ix) small hydroxy amino acids Ser and Thr. References to the charge of amino acids in this paragraph refer to the charge at physiological pH.

[0045] The terms "nucleic acid" and "polynucleotide" are used interchangeably and, as used herein, refer to the sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. In specific embodiments, nucleotides refer to ribonucleotides, deoxynucleotides, or modified forms of either type of nucleotide, and combinations thereof. The term also includes, but is not limited to, single-stranded and double-stranded forms of DNA. In addition, a polynucleotide, such as a cDNA or mRNA, may include either or both of naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide bonds. As will be readily appreciated by those skilled in the art, nucleic acid molecules, such as oligonucleotide probes or primers, may be chemically or biochemically modified, or may contain non-natural or derived nucleotide bases. Such modifications include, for example, labeling, methylation, substitution of one or more naturally occurring nucleotides with analogs, nucleotide modifications such as uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), pendant moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralens, etc.), chelators, alkylating agents, and modified bonds (e.g., α-anomeric nucleic acids, etc.). The above terms are also intended to include any topological conformation, including single-stranded, double-stranded, partially double-helix (duplex), triple-helix (triplex), hairpin, circular, and locked conformations. Unless otherwise indicated, reference to a nucleic acid sequence encompasses its complement. Thus, reference to a nucleic acid molecule with a specific sequence should be understood to encompass its complementary chain and its complementary sequence. The term also includes codon-optimized nucleic acids encoding the same polypeptide sequence.

[0046] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is connected. The term includes vectors that are self-replicating nucleic acid structures and vectors that are incorporated into the genome of a host cell into which it is introduced. "Vector" as used herein refers to a recombinant construct in which a target nucleic acid sequence is inserted into a vector. Certain vectors are capable of directing the expression of nucleic acids to which they are operably connected. Such vectors are referred to herein as "expression vectors."

[0047] In the context of two or more nucleic acids or two or more polypeptides, the term "identical" or "percent identity" refers to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotides or amino acids (i.e., about 60% identity over a specified region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity when compared and aligned over a comparison window or specified region for maximum correspondence) as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters or by manual alignment and visual inspection. See, for example, the NCBI website ncbi.nlm.nih.gov / BLAST. Such sequences are subsequently referred to as being "substantially identical". This definition also refers to or can be applied to the complement of a nucleotide test sequence. This definition also includes sequences with deletions and / or additions, as well as sequences with substitutions. As described below, the algorithm can take into account gaps, etc. Typically, identity exists over a region that encompasses an antibody epitope, or over a sequence that is at least about 25 amino acids or nucleotides in length, or over a region that is 50-100 amino acids or nucleotides in length, or over the entire length of a reference sequence.

[0048] When used in the context of determining a given amino acid residue in a polypeptide sequence, the terms "corresponding to," "determined with reference to," or "numbered with reference to," refer to the position of the residue of the designated reference sequence when the given amino acid sequence is maximally aligned and compared to the reference sequence. Thus, for example, when optimally aligned with SEQ ID NO: 1, the amino acid residue in a variable domain polypeptide "corresponds to" the amino acid in the variable domain polypeptide of SEQ ID NO: 1 when the residue is aligned with the amino acid in SEQ ID NO: 1. A polypeptide aligned to a reference sequence need not be the same length as the reference sequence.

[0049] The term "recombinant" when used with respect to, for example, a cell or a nucleic acid, protein or vector, means that the cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein, or by the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes that are not present in the native (non-recombinant) form of the cell, or expresses native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all.

[0050] The term "heterologous" when used with reference to a portion of a nucleic acid means that the nucleic acid comprises two or more subsequences that do not exist in the same relationship to each other in nature. For example, nucleic acids are typically recombinantly produced, having two or more sequences from unrelated genes arranged to produce a new functional nucleic acid, such as a promoter from one source and a coding region from another source. Similarly, a heterologous protein means that the protein comprises two or more subsequences that do not exist in the same relationship to each other in nature (e.g., a fusion protein).

[0051] The term "isolated" when applied to a nucleic acid or protein means that the nucleic acid or protein is substantially free of other cellular components associated with it in the native state. It is preferably in a homogeneous state. It can be a dry product or an aqueous solution. Purity and homogeneity are usually determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. Proteins that are the main species present in a preparation are substantially purified. In particular, the isolated gene is separated from the open reading frame that flanks the gene and encodes proteins other than the target gene. The term "purified" means that the nucleic acid or protein basically produces one band in an electrophoretic gel. In particular, this means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.

[0052] "Subject," "patient," "individual," and similar terms are used interchangeably and, unless otherwise indicated, refer to mammals, such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammalian species. The term does not necessarily mean that the subject has been diagnosed with a particular disease, but generally refers to an individual under medical supervision. A patient can be an individual for whom treatment is sought, monitoring, adjustment or modification of an existing treatment regimen, etc.

[0053] "Cancer," "tumor," "transformed," and similar terms include precancerous cells, neoplastic cells, transformed cells, and cancer cells, and may refer to solid tumors or non-solid cancers. Cancer includes both benign and malignant tumors (abnormal growths). The term "cancer" may thus refer to carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, solid cancers, and lymphomas, among others. Examples of different types of cancer include, but are not limited to, lung cancer (e.g., non-small cell lung cancer or NSCLC), ovarian cancer, prostate cancer, colorectal cancer, liver cancer, and prostate cancer. Cancer (i.e., hepatocarcinoma), kidney cancer (i.e., renal cell carcinoma), bladder cancer, breast cancer, thyroid cancer, pleural cancer, pancreatic cancer, uterine cancer, cervical cancer, testicular cancer, anal cancer, pancreatic cancer, bile duct cancer, gastrointestinal carcinoid tumors, esophageal cancer, gallbladder cancer, appendix cancer, small intestine cancer, stomach (stomach) cancer, central nervous system cancer, skin cancer, choriocarcinoma; head and neck cancer, blood cancer, osteogenic sarcoma, fibrosarcoma, neuroblastoma, glioma, melanoma, B-cell lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, small cell lymphoma, large cell lymphoma, monocytic leukemia, myeloid leukemia, acute lymphocytic leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and multiple myeloma. In some embodiments, the antibody compositions and methods described herein can be used to treat cancer.

[0054] The terms "chimeric antigen receptor" and "CAR" used interchangeably herein refer to artificial multi-module molecules that can trigger or inhibit immune cell activation, which generally but not exclusively include an extracellular domain (e.g., a ligand / antigen binding domain), a transmembrane domain, and one or more intracellular signaling domains. The term CAR is not particularly limited to CAR molecules, but also includes CAR variants. CAR variants include split CAR, wherein the extracellular portion (e.g., ligand binding portion) and the intracellular portion (e.g., intracellular signaling portion) of CAR are present on two separate molecules. CAR variants also include ON-switch CAR, which is a conditionally activated CAR, for example, including split CAR, wherein the conditional heterodimerization of the two parts of the split CAR is pharmacologically controlled. CAR variants also include bispecific CAR, which includes a secondary CAR binding domain that can enhance or inhibit the activity of a primary CAR. CAR variants also include inhibitory chimeric antigen receptors (iCAR), which can be used, for example, as a component of a bispecific CAR system, wherein the binding of the secondary CAR binding domain results in inhibition of primary CAR activation. CAR molecules and their derivatives (i.e., CAR variants) are described in, for example, PCT Application No. US2014 / 016527; Fedorov et al., Sci Transl Med (2013); 5(215):215ra172; Glienke et al., Front Pharmacol (2015) 6:21; Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151-5; Riddell et al., Cancer J (2014) 20(2):141-4; Pegram et al., Cancer J (2014) 20(2):127-33; Cheadle et al., Immunol Rev (2014) 257(1):91-106; Barrett et al., Annu Rev Med (2014) 65:333-47; Sadelain et al., Cancer Discov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; the disclosures of which are incorporated herein by reference in their entirety.

[0055] The term "immune cell" as used herein generally includes white blood cells (leukocytes) derived from hematopoietic stem cells (HSC) produced in the bone marrow."Immune cells" include, for example, lymphocytes (T cells, B cells, natural killer (NK) cells) and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells).

[0056] "T cells" include all types of immune cells that express CD3, including T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), T regulatory cells (Tregs), and gamma-delta T cells.

[0057] "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells and neutrophils, which are capable of mediating a cytotoxic response. Phage display selection of anti-integrin β-2 antibodies

[0058] Reference Figure 1 , wherein a diagram of a phage display selection strategy for developing anti-integrin beta-2 antibodies is shown. A previously described Fab-phage display platform based on fully human framework sequences (Persson et al., J. Mol. Biol. 425:803–811, 2013) was used to perform selections against recombinant integrin beta-2, including integrin beta-2 / integrin alpha-M (R and D 4047-AM, antibodies #7061, #7062, #7063, #7064, #7065), integrin-beta2 / integrin alpha-L (R and D 3868-AV, antibodies #7060, #7341), and integrin-beta2 / integrin alpha-X (R and D5755-AX, antibodies #7055, #7056, #7057) recombinant heterodimeric protein complexes.

[0059] Briefly, integrin beta-2 recombinant protein complexes were immobilized on Maxisorp immunoplates (ThermoFisher, 12-565-135) and used for positive binding selection using library phage pools that were first exposed to neutravidin-coated wells to deplete nonspecific binders. After four rounds of binding selection, clonal phage were prepared and evaluated by phage ELISA and sequencing as previously described (Persson et al., supra) and summarized below.

[0060] From about 10 10 In the initial library diversity of 10 binders, we identified Figures 2A-2D and Figure 3 The 10 initial Fab hits shown in . were identified using biolayer interferometry (BLI) (see Figures 5A-5R and Figure 6 ) and nonspecific ELISA (see Figures 4A-4H) further validated that seven of the Fab hits (#7055, #7056, #7057, #7060, #7061, #7062, #7063, #7064, #7065, #7341) had binding affinity to integrin beta-2 in the low nM range and lacked binding to unrelated proteins. These Fabs were cloned into a human IgG1 backbone and purified after recombinant expression in mammalian cells (e.g., Expi93 human embryonic kidney cells or Chinese hamster ovary cells). Antibody production

[0061] Anti-integrin beta-2 antibodies were produced using the human Expi293 expression system (Thermofisher). Expi293 cells were transiently transfected with construct DNA in a volume of 2 mL using FectoPro transfection reagent (Polyplus Transfection, 101000014). After a 5-day expression period, antibodies were purified using rProteinA Sepharaso (GE Healthcare) and stored in phosphate buffer (50 mM NaH2PO4, 75 mM Na2HPO4, 100 mM H3PO4, 154 mM NaCl). Biolayer interferometry (BLI) binding assay

[0062] The binding of human integrin beta-2 antibodies was tested against three different integrin beta-2 complexes, including integrin beta-2 / integrin alpha-M (R and D4047-AM), integrin beta-2 / integrin alpha-X (R and D 5755-AX), and integrin beta-2 / integrin alpha-L (R and D3868-AV). To determine the binding kinetic parameters of the antibodies, BLI experiments were performed on an OctetHTX instrument (Sartorius) at 1000 rpm and 25°C. All proteins were diluted in assay buffer (PBS, 1% BSA, 0.05% Tween 20). Test antibodies and control antibodies were first captured on the AHQ biosensor at a concentration of 2 μg / ml to achieve a binding signal of 0.8-1.3 nm. Unoccupied Fc binding sites on the antibody-coated sensor were subsequently quenched by 20 μg / mL of Fc protein. After equilibration with assay buffer, the biosensor was then immersed in wells containing 5-fold serial dilutions of integrin-β2 complexes for 600 seconds (association phase), followed by transfer back to assay buffer for another 600 seconds (dissociation phase). Assay buffer alone was used as a negative control. The binding response data were reference subtracted and globally fitted with a 1:1 binding model using ForteBio's Octet Systems software 9.0. ELISA The ELISA protocol for assessing the interaction of antibodies with unrelated macromolecules was adapted from Meirsch et al. (J Vis Exp. 2015 Jan 17;(95):51492). Antigens tested included integrin AL / B2 (50 μg / mL, R&D Systems 3868-AV-050), integrin AX / B2 (50 μg / mL, R&D Systems 5755-AX-050), integrin AM / B2 (50 μg / mL, R&D Systems 4047-AM-050), histidine-tagged Sumo domain (100 μg / mL, recombinant), biotinylated Robo domain (100 μg / mL, recombinant), neutravidin (100 μg / mL, Pierce TPPI31000) or all other integrins (50 μg / mL R&D Systems). In addition, each antibody was tested for binding to empty wells (BSA only control) and wells containing goat anti-human Fc antibody (positive control, 1 μg / ml, Jackson 109-005-098). Antigen was coated in 384-well Maxisorp plates at 30 μL / well and incubated overnight at 4°C. The plates were blocked with 0.5% bovine serum albumin (BSA) for 1 hour at room temperature and washed with PBS+0.05% Tween20. Phage-Fab was added and allowed to bind for 60 minutes at room temperature. The plates were washed with PBS+0.05% Tween20, and the binding was detected with anti-M13 HRP antibody (1:5000, Sinobiological 11973-MM05T-H) and developed with TMB substrate (KPL (Mandel) KP-50-76-03). Statistical analysis

[0063] Unless otherwise stated, all statistical analyses were performed using GraphPad Prism. Data are expressed as ± mean, and p values ​​< 0.05 were considered statistically significant. Anti-integrin beta-2 antibody

[0064] Provided herein are anti-integrin beta-2 antibodies useful for diagnostic and therapeutic purposes.

[0065] In some embodiments, the anti-integrin beta-2 antibodies of the present disclosure have a K of less than about 10 nM. D .

[0066] In some embodiments, the anti-integrin beta-2 binding domains of the present disclosure have at least one, at least two, or three CDRs of the variable domain sequence of SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the anti-integrin beta-2 binding domains of the present disclosure comprise HCDR3 of SEQ ID NO: 2 and LCDR3 of SEQ ID NO: 3. In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 2 and LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 3.

[0067] In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 2, wherein one of the CDRs comprises a substitution relative to the corresponding CDR shown in SEQ ID NO: 2. In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 2, wherein two of the CDRs comprise a substitution relative to the corresponding CDR shown in SEQ ID NO: 2. In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 2, wherein all three CDRs comprise a substitution relative to the corresponding CDR sequences shown in SEQ ID NO: 2.

[0068] In some embodiments, the anti-integrin beta-2 binding domain comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 3, wherein one of the CDRs comprises a substitution relative to the corresponding CDR shown in SEQ ID NO: 3. In some embodiments, the anti-integrin beta-2 binding domain comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 3, wherein two of the CDRs comprise a substitution relative to the corresponding CDR shown in SEQ ID NO: 3. In some embodiments, the anti-integrin beta-2 binding domain comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 3, wherein all three CDRs comprise a substitution relative to the corresponding CDR sequences shown in SEQ ID NO: 3.

[0069] In some embodiments, the anti-integrin beta-2 binding domain of the present disclosure comprises a variable region having at least 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the variable region sequence of SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the variable domain comprises a substitution, insertion or deletion in the framework of the variable region as shown in SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the anti-integrin beta-2 binding domain of the present disclosure comprises a heavy chain variable region comprising the HCD1, HCDR2 and HCDR3 sequences of SEQ ID NO: 2 and having at least 95% identity to SEQ ID NO: 2; and a light chain variable region comprising the LCD1, LCDR2, LCDR3 sequences of SEQ ID NO: 3 and having at least 95% identity to SEQ ID NO: 3.

[0070] In some embodiments, the anti-integrin beta-2 binding domains of the present disclosure have at least one, at least two, or three CDRs of the variable domain sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the anti-integrin beta-2 binding domains of the present disclosure comprise HCDR3 of SEQ ID NO: 4 and LCDR3 of SEQ ID NO: 5. In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 4 and LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 5.

[0071] In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 4, wherein one of the CDRs comprises a substitution relative to the corresponding CDR shown in SEQ ID NO: 4. In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 4, wherein two of the CDRs comprise substitutions relative to the corresponding CDRs shown in SEQ ID NO: 4. In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 4, wherein all three CDRs comprise substitutions relative to the corresponding CDR sequences shown in SEQ ID NO: 4.

[0072] In some embodiments, the anti-integrin beta-2 binding domain comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 5, wherein one of the CDRs comprises a substitution relative to the corresponding CDR shown in SEQ ID NO: 5. In some embodiments, the anti-integrin beta-2 binding domain comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 5, wherein two of the CDRs comprise a substitution relative to the corresponding CDR shown in SEQ ID NO: 5. In some embodiments, the anti-integrin beta-2 binding domain comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 5, wherein all three CDRs comprise a substitution relative to the corresponding CDR sequences shown in SEQ ID NO: 5.

[0073] In some embodiments, the anti-integrin beta-2 binding domain of the present disclosure comprises a variable region having at least 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the variable region sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the variable domain comprises a substitution, insertion or deletion in the framework of the variable region as shown in SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the anti-integrin beta-2 binding domain of the present disclosure comprises a heavy chain variable region comprising the HCD1, HCDR2 and HCDR3 sequences of SEQ ID NO: 4 and having at least 95% identity to SEQ ID NO: 4; and a light chain variable region comprising the LCD1, LCDR2, LCDR3 sequences of SEQ ID NO: 5 and having at least 95% identity to SEQ ID NO: 5.

[0074] In some embodiments, the anti-integrin beta-2 binding domains of the present disclosure have at least one, at least two, or three CDRs of the variable domain sequence of SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the anti-integrin beta-2 binding domains of the present disclosure comprise HCDR3 of SEQ ID NO: 6 and LCDR3 of SEQ ID NO: 7. In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 6 and LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 7.

[0075] In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 6, wherein one of the CDRs comprises a substitution relative to the corresponding CDR shown in SEQ ID NO: 6. In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 6, wherein two of the CDRs comprise a substitution relative to the corresponding CDR shown in SEQ ID NO: 6. In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 6, wherein all three CDRs comprise a substitution relative to the corresponding CDR sequences shown in SEQ ID NO: 6.

[0076] In some embodiments, the anti-integrin beta-2 binding domain comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 7, wherein one of the CDRs comprises a substitution relative to the corresponding CDR shown in SEQ ID NO: 7. In some embodiments, the anti-integrin beta-2 binding domain comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 7, wherein two of the CDRs comprise a substitution relative to the corresponding CDR shown in SEQ ID NO: 7. In some embodiments, the anti-integrin beta-2 binding domain comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 7, wherein all three CDRs comprise a substitution relative to the corresponding CDR sequences shown in SEQ ID NO: 7.

[0077] In some embodiments, the anti-integrin beta-2 binding domain of the present disclosure comprises a variable region having at least 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the variable region sequence of SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the variable domain comprises a substitution, insertion or deletion in the framework of the variable region as shown in SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the anti-integrin beta-2 binding domain of the present disclosure comprises a heavy chain variable region comprising the HCD1, HCDR2 and HCDR3 sequences of SEQ ID NO: 6 and having at least 95% identity to SEQ ID NO: 6; and a light chain variable region comprising the LCD1, LCDR2, LCDR3 sequences of SEQ ID NO: 7 and having at least 95% identity to SEQ ID NO: 7.

[0078] In some embodiments, the anti-integrin beta-2 binding domains of the present disclosure have at least one, at least two, or three CDRs of the variable domain sequence of SEQ ID NO: 8 or SEQ ID NO: 9. In some embodiments, the anti-integrin beta-2 binding domains of the present disclosure comprise HCDR3 of SEQ ID NO: 8 and LCDR3 of SEQ ID NO: 9. In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 8 and LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 9.

[0079] In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 8, wherein one of the CDRs comprises a substitution relative to the corresponding CDR shown in SEQ ID NO: 8. In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 8, wherein two of the CDRs comprise a substitution relative to the corresponding CDR shown in SEQ ID NO: 8. In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 8, wherein all three CDRs comprise a substitution relative to the corresponding CDR sequences shown in SEQ ID NO: 8.

[0080] In some embodiments, the anti-integrin beta-2 binding domain comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 9, wherein one of the CDRs comprises a substitution relative to the corresponding CDR shown in SEQ ID NO: 9. In some embodiments, the anti-integrin beta-2 binding domain comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 9, wherein two of the CDRs comprise a substitution relative to the corresponding CDR shown in SEQ ID NO: 9. In some embodiments, the anti-integrin beta-2 binding domain comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 9, wherein all three CDRs comprise a substitution relative to the corresponding CDR sequences shown in SEQ ID NO: 9.

[0082] In some embodiments, the anti-integrin beta-2 binding domain of the present disclosure comprises a variable region having at least 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the variable region sequence of SEQ ID NO: 8 or SEQ ID NO: 9. In some embodiments, the variable domain comprises a substitution, insertion or deletion in the framework of the variable region as shown in SEQ ID NO: 8 or SEQ ID NO: 9. In some embodiments, the anti-integrin beta-2 binding domain of the present disclosure comprises a heavy chain variable region comprising the HCD1, HCDR2 and HCDR3 sequences of SEQ ID NO: 8 and having at least 95% identity to SEQ ID NO: 8; and a light chain variable region comprising the LCD1, LCDR2, LCDR3 sequences of SEQ ID NO: 9 and having at least 95% identity to SEQ ID NO: 9.

[0082] In some embodiments, the anti-integrin beta-2 binding domains of the present disclosure have at least one, at least two, or three CDRs of the variable domain sequence of SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the anti-integrin beta-2 binding domains of the present disclosure comprise HCDR3 of SEQ ID NO: 10 and LCDR3 of SEQ ID NO: 11. In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 10 and LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 11.

[0083] In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 10, wherein one of the CDRs comprises a substitution relative to the corresponding CDR shown in SEQ ID NO: 10. In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 10, wherein two of the CDRs comprise substitutions relative to the corresponding CDRs shown in SEQ ID NO: 10. In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 10, wherein all three CDRs comprise substitutions relative to the corresponding CDR sequences shown in SEQ ID NO: 10.

[0084] In some embodiments, the anti-integrin beta-2 binding domain comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 11, wherein one of the CDRs comprises a substitution relative to the corresponding CDR shown in SEQ ID NO: 11. In some embodiments, the anti-integrin beta-2 binding domain comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 11, wherein two of the CDRs comprise a substitution relative to the corresponding CDR shown in SEQ ID NO: 11. In some embodiments, the anti-integrin beta-2 binding domain comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 11, wherein all three CDRs comprise a substitution relative to the corresponding CDR sequences shown in SEQ ID NO: 11.

[0085] In some embodiments, an anti-integrin beta-2 binding domain of the present disclosure comprises a variable region having at least 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the variable region sequence of SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the variable domain comprises a substitution, insertion, or deletion in the framework of the variable region as shown in SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, an anti-integrin beta-2 binding domain of the present disclosure comprises a heavy chain variable region comprising the HCD1, HCDR2, and HCDR3 sequences of SEQ ID NO: 10 and having at least 95% identity to SEQ ID NO: 10; and a light chain variable region comprising the LCD1, LCDR2, LCDR3 sequences of SEQ ID NO: 11 and having at least 95% identity to SEQ ID NO: 11.

[0086] In some embodiments, the anti-integrin beta-2 binding domains of the present disclosure have at least one, at least two, or three CDRs of the variable domain sequence of SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the anti-integrin beta-2 binding domains of the present disclosure comprise HCDR3 of SEQ ID NO: 12 and LCDR3 of SEQ ID NO: 13. In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 12 and LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 13.

[0087] In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 12, wherein one of the CDRs comprises a substitution relative to the corresponding CDR shown in SEQ ID NO: 12. In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 12, wherein two of the CDRs comprise a substitution relative to the corresponding CDR shown in SEQ ID NO: 12. In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 12, wherein all three CDRs comprise a substitution relative to the corresponding CDR sequences shown in SEQ ID NO: 12.

[0088] In some embodiments, the anti-integrin beta-2 binding domain comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 13, wherein one of the CDRs comprises a substitution relative to the corresponding CDR shown in SEQ ID NO: 13. In some embodiments, the anti-integrin beta-2 binding domain comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 13, wherein two of the CDRs comprise a substitution relative to the corresponding CDR shown in SEQ ID NO: 13. In some embodiments, the anti-integrin beta-2 binding domain comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 13, wherein all three CDRs comprise a substitution relative to the corresponding CDR sequences shown in SEQ ID NO: 13.

[0089] In some embodiments, an anti-integrin beta-2 binding domain of the present disclosure comprises a variable region having at least 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the variable region sequence of SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the variable domain comprises a substitution, insertion, or deletion in the framework of the variable region as shown in SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, an anti-integrin beta-2 binding domain of the present disclosure comprises a heavy chain variable region comprising the HCD1, HCDR2, and HCDR3 sequences of SEQ ID NO: 12 and having at least 95% identity to SEQ ID NO: 12; and a light chain variable region comprising the LCD1, LCDR2, LCDR3 sequences of SEQ ID NO: 13 and having at least 95% identity to SEQ ID NO: 13.

[0090] In some embodiments, the anti-integrin beta-2 binding domains of the present disclosure have at least one, at least two, or three CDRs of the variable domain sequence of SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the anti-integrin beta-2 binding domains of the present disclosure comprise HCDR3 of SEQ ID NO: 14 and LCDR3 of SEQ ID NO: 15. In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 14 and LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 15.

[0091] In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 14, wherein one of the CDRs comprises a substitution relative to the corresponding CDR shown in SEQ ID NO: 14. In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 14, wherein two of the CDRs comprise substitutions relative to the corresponding CDRs shown in SEQ ID NO: 14. In some embodiments, the anti-integrin beta-2 binding domain comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 14, wherein all three CDRs comprise substitutions relative to the corresponding CDR sequences shown in SEQ ID NO: 14.

[0092] In some embodiments, the anti-integrin beta-2 binding domain comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 15, wherein one of the CDRs comprises a substitution relative to the corresponding CDR shown in SEQ ID NO: 15. In some embodiments, the anti-integrin beta-2 binding domain comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 15, wherein two of the CDRs comprise a substitution relative to the corresponding CDR shown in SEQ ID NO: 15. In some embodiments, the anti-integrin beta-2 binding domain comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 15, wherein all three CDRs comprise a substitution relative to the corresponding CDR sequences shown in SEQ ID NO: 15.

[0093] In some embodiments, an anti-integrin beta-2 binding domain of the present disclosure comprises a variable region having at least 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the variable region sequence of SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the variable domain comprises a substitution, insertion, or deletion in the framework of the variable region as shown in SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, an anti-integrin beta-2 binding domain of the present disclosure comprises a heavy chain variable region comprising the HCD1, HCDR2, and HCDR3 sequences of SEQ ID NO: 14 and having at least 95% identity to SEQ ID NO: 14; and a light chain variable region comprising the LCD1, LCDR2, LCDR3 sequences of SEQ ID NO: 15 and having at least 95% identity to SEQ ID NO: 15. Antibody format

[0094] The anti-integrin beta-2 antibodies of the present disclosure may be incorporated into bivalent antibodies or multivalent antibodies that bind to the same or different antigens. In some embodiments, the anti-integrin beta-2 antibodies of the present disclosure may be incorporated into bispecific antibodies or multispecific antibodies that bind to antigens at different epitopes or bind to different antigens. In some embodiments, such antibodies may comprise an Fc region. In some embodiments, the anti-integrin beta-2 antibodies of the present disclosure may exist as an antigen binding domain of a larger molecule, for example, as an antigen binding domain of a chimeric antigen receptor or a synthetic Notch receptor. Nucleic acid encoding CAR and vector

[0095] Any method can be used to genetically modify effector cells such as T cells or NK cells to express CARs containing anti-integrin beta-2 antibodies of the present disclosure. Non-limiting examples of methods for genetically engineering immune cells include, but are not limited to, retroviral or lentiviral mediated transduction. Other viral delivery systems include adenovirus, adeno-associated virus, herpes simplex virus vectors, poxvirus vectors, alphavirus vectors, poliovirus vectors, and other positive and negative strand RNA viruses, viroids, and mimic viruses or parts thereof. The method of transduction includes direct co-culture of cells with production cells, such as by Bregni et al., Blood 80: 1418-1422 (1992), or cultured with a separate viral supernatant or a concentrated carrier stock solution containing or not containing appropriate growth factors and polycations, such as by Xu et al., Exp. Hemat. 22: 223-230 (1994); and Hughes, et al. J. Clin. Invest. 89: 1817 (1992).

[0096] In some embodiments, genetic modification is performed using a transposase-based system for gene integration, CRISPR / Cas-mediated gene integration, TALEN or zinc finger nuclease integration technology. For example, CRISPR / Cas-mediated gene integration can be used to introduce CAR or synthetic Notch receptors into immune effector cells, which can then be selected and amplified for administration to patients. Antibody conjugates

[0097] In another aspect, the anti-integrin beta-2 antibodies of the present disclosure can be conjugated or linked directly or indirectly to a therapeutic and / or imaging / detectable moiety. For example, in some embodiments, the antibodies of the present disclosure or antigen binding regions comprising the antibodies of the present disclosure can be conjugated to an agent, including but not limited to a detectable marker, a cytotoxic agent, an imaging agent, a therapeutic agent, or an oligonucleotide. Methods for conjugating or linking antibodies or antigen binding regions comprising antibodies to a desired molecular moiety are well known in the art. The moiety can be covalently linked to the antibody or linked to the antibody by a non-covalent bond.

[0098] In some embodiments, an anti-integrin beta-2 antibody of the disclosure, or an antigen binding domain comprising an anti-integrin beta-2 antibody of the disclosure, is conjugated to a cytotoxic moiety or other moiety that inhibits cell proliferation.

[0099] In some embodiments, an anti-integrin beta-2 antibody of the present disclosure is conjugated to a cytotoxic agent, including, but not limited to, for example, ricin A chain, doxorubicin, daunomycin, maytansinoids, taxol, ethidium bromide, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, dihydroxyanthraquinone, methotrexact, actinomycin, diphtheria toxin, exotoxin A from Pseudomonas, Pseudomonas exotoxin 40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, restrictocin, cobra venom factor, ribonuclease, engineered Shiga toxin, phenomycin, enomycin, curicin, crotonin, calicheamicin, Saponaria officinalis, officinalis inhibitors, glucocorticoids, auristatins, chlortetracycline, yttrium, bismuth, combrestatin, duocarmycins, dolastatin, cc1065, or cisplatin. In some embodiments, the antibody can be linked to an agent such as an enzyme inhibitor, a proliferation inhibitor, a cleavage agent, a DNA or RNA synthesis inhibitor, a membrane permeability regulator, a DNA metabolite, a dichloroethyl sulfide derivative, a protein production inhibitor, a ribosome inhibitor, or an apoptosis inducer.

[0100] In some embodiments, the anti-integrin beta-2 antibodies of the present disclosure or antigen binding domains comprising the anti-integrin beta-2 antibodies of the present disclosure may be linked to a radionuclide, an iron-related compound, a dye, a fluorescent agent, or an imaging agent. In some embodiments, the antibodies may be linked to agents such as, but not limited to, metals; metal chelators; lanthanides; lanthanide chelators; radioactive metals; radioactive metal chelators; positron emitting nuclei; microbubbles (for ultrasound); liposomes; molecules microencapsulated in liposomes or nanospheres; single crystalline iron oxide nanocompounds; magnetic resonance imaging contrast agents; light absorbing, reflecting and / or scattering agents; colloidal particles; fluorophores, such as near-infrared fluorophores.

[0101] Although the above description provides examples of one or more antibodies and nucleic acids, it should be understood that other compositions of matter may also be within the scope of the claims as interpreted by one skilled in the art. Peptide sequence: SEQ ID NO:1 Uniprot P05107-1 amino acid sequence SEQ ID NO: 2 Antibody-7065 heavy chain variable region; CDRs are underlined SEQ ID NO:3 Antibody-7065 light chain variable region; CDRs are underlined SEQ ID NO:4 Antibody-7060 heavy chain variable region; CDRs are underlined SEQ ID NO:5 Antibody-7060 light chain variable region; CDRs are underlined SEQ ID NO:6 Antibody-7062 heavy chain variable region; CDRs are underlined SEQ ID NO:7 Antibody-7062 light chain variable region; CDRs are underlined SEQ ID NO:8 Antibody-7063 heavy chain variable region; CDRs are underlined SEQ ID NO: 9 Antibody-7063 light chain variable region; CDRs are underlined SEQ ID NO: 10 Antibody-7064 heavy chain variable region; CDRs are underlined SEQ ID NO: 11 Antibody-7064 light chain variable region; CDRs are underlined SEQ ID NO: 12 Antibody-7056 heavy chain variable region; CDRs are underlined SEQ ID NO: 13 Antibody-7056 light chain variable region; CDRs are underlined SEQ ID NO: 14 Antibody-7341 heavy chain variable region; CDRs are underlined SEQ ID NO: 15 Antibody-7341 light chain variable region; CDRs are underlined

Claims

1. An antibody that specifically binds to integrin beta-2, wherein the antibody comprises an integrin beta-2 binding domain, wherein the integrin beta-2 binding domain comprises: Heavy chain variable region (V H ), which comprises a HCDR1 sequence comprising ISYYYM, a HCDR2 sequence comprising SISSSSGYTY, and a HCDR3 sequence comprising GAM; and Light chain variable region (V L ), which comprises a LCDR1 sequence containing SVSSA, a LCDR2 sequence containing SASSLYS, and a LCDR3 sequence containing FSSGSWAPI.

2. The antibody of claim 1, wherein said V H comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 2; and / or Wherein V L Comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:

3.

3. The antibody of claim 1, wherein said V H Comprising the amino acid sequence SEQ ID NO: 2; and / or Wherein V L Comprising the amino acid sequence SEQ ID NO:

3.

4. The antibody of any one of claims 1-3, wherein the antibody is a single chain variable fragment (scFv).

5. The antibody of claim 4, wherein said V L In the V H N-terminus.

6. The antibody of claim 4, wherein said V H In the V L N-terminus.

7. The antibody of any one of claims 5-6, wherein the V H and the V L Separated by flexible joints.

8. The antibody of claim 7, wherein the flexible linker comprises Gly-Ser.

9. The antibody of claim 8, wherein the flexible linker comprises one or more Gly-Ser sequences.

10. A bispecific or multispecific antibody comprising the antibody of any one of claims 1 to 9.

11. A chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises the antibody of any one of claims 1-9.

12. An immune effector cell comprising the CAR of claim 11.

13. The immune effector cell of claim 12, wherein the cell is a T cell or a NK cell.

14. A polynucleotide encoding the antibody of any one of claims 1-11.

15. A vector comprising the polynucleotide of claim 14.

16. An immune effector cell comprising the vector of claim 15.

17. Encoding antibody V H and / or V L A nucleic acid, wherein the V H comprising a HCDR1 sequence comprising ISYYYM, a HCDR2 sequence comprising SISSSSGYTY, and a HCDR3 sequence comprising GAM; and Wherein V L It comprises a LCDR1 sequence containing SVSSA, a LCDR2 sequence containing SASSLYS, and a LCDR3 sequence containing FSSGSWAPI.

18. The nucleic acid of claim 17, wherein said V H comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 2; and / or Wherein V L Comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:

3.

19. The nucleic acid of claim 17, wherein said V H Comprising the amino acid sequence SEQ ID NO: 2; and / or Wherein V L Comprising the amino acid sequence SEQ ID NO:

3.

20. A vector comprising the nucleic acid of any one of claims 17 to 19.

21. A host cell comprising the vector of claim 20.

22. An antibody that specifically binds to integrin beta-2, wherein the antibody comprises an integrin beta-2 binding domain, wherein the integrin beta-2 binding domain comprises: Heavy chain variable region (V H ), which comprises a HCDR1 sequence comprising LSYSSM, a HCDR2 sequence comprising YIYPSYGYTY, and a HCDR3 sequence comprising WSPGSGWAF; and Light chain variable region (V L ), which comprises a LCDR1 sequence containing SVSSA, a LCDR2 sequence containing SASSLYS, and a LCDR3 sequence containing YHGSLI.

23. An antibody that specifically binds to integrin beta-2, wherein the antibody comprises an integrin beta-2 binding domain, wherein the integrin beta-2 binding domain comprises: Heavy chain variable region (V H ), which comprises a HCDR1 sequence comprising LYYYSM, a HCDR2 sequence comprising YIYPYYGYTS, and a HCDR3 sequence comprising TVRGSKKPYFSGWAM; and Light chain variable region (V L ), which comprises a LCDR1 sequence containing SVSSA, a LCDR2 sequence containing SASSLYS, and a LCDR3 sequence containing WGAWGPLI.

24. An antibody that specifically binds to integrin beta-2, wherein the antibody comprises an integrin beta-2 binding domain, wherein the integrin beta-2 binding domain comprises: Heavy chain variable region (V H ), which comprises a HCDR1 sequence comprising LSYYYM, a HCDR2 sequence comprising SISSYYGYTS, and a HCDR3 sequence comprising GAL; and Light chain variable region (V L ), which comprises a LCDR1 sequence containing SVSSA, a LCDR2 sequence containing SASSLYS, and a LCDR3 sequence containing FYGGYSLI.

25. An antibody that specifically binds to integrin beta-2, wherein the antibody comprises an integrin beta-2 binding domain, wherein the integrin beta-2 binding domain comprises: Heavy chain variable region (V H ), which comprises a HCDR1 sequence comprising ISSYSI, a HCDR2 sequence comprising SIYSYYGYTS, and a HCDR3 sequence comprising YWGYPYAM; and Light chain variable region (V L ), which comprises a LCDR1 sequence containing SVSSA, a LCDR2 sequence containing SASSLYS, and a LCDR3 sequence containing YYYAASLF.

26. An antibody that specifically binds to integrin beta-2, wherein the antibody comprises an integrin beta-2 binding domain, wherein the integrin beta-2 binding domain comprises: Heavy chain variable region (V H ), which comprises a HCDR1 sequence comprising LSYSYM, a HCDR2 sequence comprising SIYSYYSSTS, and a HCDR3 sequence comprising SYHYSYYAGL; and Light chain variable region (V L ), which comprises a LCDR1 sequence containing SVSSA, a LCDR2 sequence containing SASSLYS, and a LCDR3 sequence containing WYFLI.

27. An antibody that specifically binds to integrin beta-2, wherein the antibody comprises an integrin beta-2 binding domain, wherein the integrin beta-2 binding domain comprises: Heavy chain variable region (V H ), which comprises a HCDR1 sequence comprising LSYSSM, a HCDR2 sequence comprising YIYSSSGYTY, and a HCDR3 sequence comprising WGWYAHAGM; and Light chain variable region (V L ), which comprises a LCDR1 sequence containing SVSSA, a LCDR2 sequence containing SASSLYS, and a LCDR3 sequence containing WVHGLI.

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