Anti-CD157 antibodies, antigen binding fragments and compositions thereof, and methods of making and using same

Abstract

Compositions and methods for making and using anti-CD157 antibodies or antigen-binding fragments thereof (e.g., monoclonal antibodies, CD157-binding antibody fragments and derivatives) are described, as well as nucleic acids encoding such molecules, diagnostic reagents and kits including anti-CD157 antibodies or antigen-binding fragments thereof, and methods of making and using the same.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 390,602, filed on July 19, 2022, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Technical Field

[0003] The present technology relates in part to antibodies and antigen-binding fragments thereof that bind to cluster of differentiation 157 (i.e., CD157) and variants thereof, in particular monoclonal antibodies, antibody fragments, and antibody derivatives that specifically react to CD157 under physiological and / or in vitro conditions. Such antibodies and antigen-binding fragments thereof can be used for laboratory / research purposes (e.g., flow cytometry), and can be used to treat and / or prevent various diseases or conditions by delivering drugs or other compositions containing such antibodies and antigen-binding fragments thereof. Background Art

[0004] Bone marrow stromal cell antigen 1 (BST-1), also known as CD157, is a member of the immunoglobulin superfamily. CD157 is a cell surface molecule that supports pre-B cell growth and has increased expression on bone marrow stromal cell lines derived from patients with rheumatoid arthritis. The function of CD157 is associated with leukocyte adhesion to extracellular matrix (ECM) proteins and transendothelial extravasation, and can promote metastatic spread and epithelial-mesenchymal transition. However, currently available anti-CD157 antibodies and related methods have limited application in vitro and in vivo. Summary of the invention

[0005] In one aspect, the present disclosure includes an isolated antibody or antigen-binding fragment thereof that specifically binds to CD157, wherein the isolated antibody comprises:

[0006] a) a heavy chain variable region comprising:

[0007] (i) Heavy chain complementarity determining region 1 (CDRH1) having X1X2X3X4X5X6YX8X9X 10 (SEQ ID NO: 30), wherein X1 is G or no amino acid, X2 is F or no amino acid, X3 is S or no amino acid, X4 is L or no amino acid, X5 is T or no amino acid, X6 is S or N, X8 is H or D, X9 is V or M, and X 10 is S or A;

[0008] (ii) CDRH2, which has X1IX3X4X5GGSTX 10 YX 12 X 13 X 14 X 15 KX17 (SEQ ID NO: 31), wherein X1 is S or no amino acid, X3 is I or S, X4 is W or I, X5 is T or R, X6 is T or R, 10 A or Y, X 12 N or R, X 13 For S or D, X 14 For L or S, X 15 is L or V, and X 17 is S or G;

[0009] (iii) a CDRH3 having a sequence of X1X2X3X4X5X6X7X8FDY (SEQ ID NO: 32), wherein X1 is G or no amino acid, X2 is T or no amino acid, X3 is S or D, X4 is I or Y, X5 is T or Y, X6 is P or Y, X7 is T or D, and X8 is F or Y; and

[0010] b) a light chain variable region comprising:

[0011] (iv) light chain complementary determining region 1 (CDRL1), which has KX2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 (SEQ ID NO: 33), wherein X2 is R or A, X3 is S or G, X4 is T or R, X5 is G or N, X6 is N or I, X7 is F or N, X8 is G or S, X9 is N, S or Y, X 10 N or L, X 11 is Y or A, X 12 is V or no amino acid, and X 11 N or no amino acid;

[0012] (v) CDRL2 having a sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 34), wherein X1 is R or N, X2 is D or A, X3 is D or N, X4 is K or S, X5 is R or L, X6 is P or Q, and X7 is D or T; and

[0013] (vi) CDRL3 having a sequence of QX2YX4SX6X7X8X9 (SEQ ID NO: 35), wherein X2 is S or Q, X4 is S or N, X6 is G or W, X7 is I or T, X8 is V or N, and X9 is T or no amino acid.

[0014] In some embodiments, the antibody comprises: (1) a CDRH1 having a sequence of any one of SEQ ID NOs: 16, 17, and 18, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 16, 17, and 18; (2) a CDRH1 having a sequence of any one of SEQ ID NOs: 19 and 20, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: NO: 19 and 20 having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 19 and 20; and (3) a CDRH3 having a sequence of any one of SEQ ID NO: 21 and 22 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 21 and 22.

[0015] In some embodiments, the antibody comprises: (1) a CDRL1 having a sequence of any one of SEQ ID NOs: 23, 24, and 25, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 23, 24, and 25; (2) a CDRL1 having a sequence of any one of SEQ ID NOs: 26 and 27, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: NO:26 and 27 having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO:28 and 29; and (3) a CDRL3 having a sequence of any one of SEQ ID NO:28 and 29 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO:28 and 29.

[0016] In some embodiments, the antibody comprises: (1) a CDRH1 having a sequence of any one of SEQ ID NOs: 16, 17, and 18, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 16, 17, and 18; (2) a CDRH1 having a sequence of any one of SEQ ID NOs: 19 and 20, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: NO: 19 and 20 having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; (3) a CDRH3 having a sequence of any one of SEQ ID NO: 21 and 22 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 21 and 22; (4) a CDRH3 having a sequence of SEQ ID NO: NO: 23, 24 and 25, or a CDRL1 having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 23, 24 and 25; (5) a CDRL1 having a sequence of any one of SEQ ID NO: 26 and 27, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 23, 24 and 25; NO:26 and 27 having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO:28 and 29; and (6) a CDRL3 having a sequence of any one of SEQ ID NO:28 and 29 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO:28 and 29.

[0017] In some embodiments, the antibody comprises a CDRH1 having a sequence of SEQ ID NO: 16, a CDRH2 having a sequence of SEQ ID NO: 19, and a CDRH3 having a sequence of SEQ ID NO: 21, a CDRL1 having a sequence of SEQ ID NO: 23, a CDRL2 having a sequence of SEQ ID NO: 26, and a CDRL3 having a sequence of SEQ ID NO: 28.

[0018] In some embodiments, the antibody comprises a CDRH1 having a sequence of SEQ ID NO: 17, a CDRH2 having a sequence of SEQ ID NO: 19, and a CDRH3 having a sequence of SEQ ID NO: 21, a CDRL1 having a sequence of SEQ ID NO: 24, a CDRL2 having a sequence of SEQ ID NO: 26, and a CDRL3 having a sequence of SEQ ID NO: 28.

[0019] In some embodiments, the antibody comprises a CDRH1 having a sequence of SEQ ID NO: 18, a CDRH2 having a sequence of SEQ ID NO: 20, and a CDRH3 having a sequence of SEQ ID NO: 22, a CDRL1 having a sequence of SEQ ID NO: 25, a CDRL2 having a sequence of SEQ ID NO: 27, and a CDRL3 having a sequence of SEQ ID NO: 29.

[0020] In some embodiments, the heavy chain variable region comprises at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence set forth in any one of SEQ ID NOs: 1 and 2. In a specific embodiment, the heavy chain variable region comprises at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence set forth in SEQ ID NO: 1.

[0021] In some embodiments, the light chain variable region comprises at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence set forth in any one of SEQ ID NOs: 3, 4, and 5. In a specific embodiment, the light chain variable region comprises at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence set forth in SEQ ID NO: 4.

[0022] In some embodiments, the heavy chain variable region comprises at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence set forth in any one of SEQ ID NOs: 1 and 2, and the light chain variable region comprises at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence set forth in any one of SEQ ID NOs: 3, 4, and 5.

[0023] In some embodiments, the heavy chain variable region comprises at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence shown in SEQ ID NO:1, and the light chain variable region comprises at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence shown in SEQ ID NO:4.

[0024] In some embodiments, the antibody comprises an Fc polypeptide that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID NO:36.

[0025] In another aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to CD157, wherein the isolated antibody competes with an antibody described herein for binding to the CD157 receptor.

[0026] In some embodiments, the antibody binds to the same epitope as an antibody described herein.

[0027] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody comprises one or more human framework regions.

[0028] In some embodiments, the antibody is conjugated to a detectable marker or label. In some embodiments, the antibody is non-diffusively immobilized on a solid support.

[0029] In another aspect, the disclosure provides an isolated nucleic acid encoding an isolated antibody or antigen-binding fragment thereof described herein.

[0030] In another aspect, the present disclosure provides an isolated nucleic acid comprising a nucleotide sequence encoding a heavy chain variable region comprising at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the sequence shown in any one of SEQ ID NOs: 6-8.

[0031] In another aspect, the present disclosure provides an isolated nucleic acid comprising a nucleotide sequence encoding a light chain variable region comprising at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the sequence shown in any one of SEQ ID NOs: 9-11.

[0032] In another aspect, the disclosure provides an expression vector comprising a nucleic acid described herein.

[0033] In another aspect, the present disclosure provides an isolated host cell comprising an expression vector described herein.

[0034] In another aspect, the present disclosure provides a pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier.

[0035] In another aspect, the present disclosure provides a diagnostic reagent comprising the isolated antibody or antigen-binding fragment thereof described herein.

[0036] In another aspect, the present disclosure provides a kit comprising an isolated antibody or antigen-binding fragment thereof described herein or a diagnostic agent described herein.

[0037] In another aspect, the present disclosure provides a method for detecting CD157, comprising contacting a sample known or suspected to contain CD157 with the isolated antibody or antigen-binding fragment thereof described herein.

[0038] In another aspect, the present disclosure provides a method for detecting CD157, wherein the method comprises contacting a sample with an isolated antibody or antigen-binding fragment thereof described herein under conditions that allow the antibody to bind to CD157 on the sample, wherein the binding forms a receptor / antibody complex; and detecting the presence of the receptor / antibody complex, wherein the detection comprises the presence or absence of CD157 on the sample.

[0039] In another aspect, the present disclosure provides a method for treating or preventing a disease or condition associated with CD157, comprising contacting a sample from a subject (wherein the sample is known or suspected to contain CD157) with an isolated antibody or antigen-binding fragment thereof described herein; detecting the presence of a complex comprising CD157 and the antibody; wherein the presence of the complex indicates the presence of the disease or condition; and administering to the subject the isolated antibody or antigen-binding fragment thereof described herein.

[0040] In another aspect, the present disclosure provides a method for diagnosing a disease or condition, wherein the method comprises isolating a sample from a subject; incubating the sample with an isolated antibody or antigen-binding fragment thereof described herein for a period of time sufficient to generate a CD157:anti-CD157 complex; detecting the presence or absence of the CD157:anti-CD157 complex in the isolated tissue; and correlating the presence or abundance of CD157 with a target location in the tissue sample.

[0041] In any of the embodiments of the methods described herein, the method is performed in vitro.

[0042] In some embodiments of the methods described herein, detection comprises hybridization of a detectable moiety with an antibody or antigen-binding fragment thereof. In some embodiments, the detectable moiety comprises an oligonucleotide. In some embodiments, the detectable moiety comprises a fluorescent label. In some embodiments, detection comprises sequencing.

[0043] In some embodiments of the methods described herein, the sample comprises a cell. In some embodiments, the sample comprises a tissue sample.

[0044] Certain embodiments are further described in the following description, examples, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The accompanying drawings illustrate certain embodiments of the technology but are not limiting. For clarity and ease of illustration, the accompanying drawings are not drawn to scale, and in some cases, various aspects may be exaggerated or enlarged to facilitate understanding of specific embodiments.

[0046] Figure 1 Positive staining of the CD157-positive human monocyte U-937 cell line by all exemplified antibodies was shown, with AB2 showing enhanced specificity and staining intensity.

[0047] Figure 2A and 2B The results showed that compared with the commercially available antibody (CAb), the anti-CD157 antibody AB2 exhibited excellent specificity and reduced nonspecific staining of CD157-negative Jurkat cells ( Figure 2ASimilar staining characteristics were observed on the CD157-positive human monocytic cell line U-937 ( Figure 2B ).

[0048] Figures 3A-3C The specificity of the exemplary test antibodies is shown by expressing CD157 in granulocytes ( Figure 3C ) and monocytes ( Figure 3B ) and the absence of surface staining on lymphocytes that do not express CD157 ( Figure 3A ).

[0049] Figure 4 It is shown that all concentrations of AB2 tested were able to block CD157-mediated human monocyte adhesion to the extracellular matrix protein fibronectin, with optimal cell adhesion blockade observed at 1.26 μg (x-axis shows antibody concentration in ug / ml; y-axis shows cell adhesion normalized to an isotype control antibody).

[0050] Figure 5 Depicted is a significant enhancement in the ability of exemplary antibody AB2 to block CD157-mediated human monocyte adhesion to the extracellular matrix protein fibronectin compared to commercially available (CAb) and isotype control antibodies (P=0.0000116). (x-axis shows antibody concentration in ug / ml; y-axis shows cell adhesion normalized to isotype control antibody).

[0051] Figure 6 It is shown that the binding of the exemplary antibody AB2 to CD157 on human monocytes can induce signal transduction through CD157, as measured by induction of intracellular calcium signaling. This effect was absent when commercially available (CAb) and control antibodies were used. (The x-axis shows the antibody concentration in ug / ml; the y-axis shows the calcium signal intensity.)

[0052] Figure 7 Depicted is the ability of the exemplary antibody AB2 to induce polarization of human monocytes via CD157-induced signaling compared to commercially available (CAb) and control antibodies. AB2 binding to CD157 induces cell polarization and significantly increases F-actin (left). CD157 colocalizes with integrin β-1 (CD29) in different membrane domains. Cells treated with Cab and isotype control antibodies did not show this effect, as the cells remained unpolarized and the actin fibers remained disordered. DETAILED DESCRIPTION

[0053] Antibodies and antigen-binding fragments thereof that bind to CD157 are provided herein. For example, specific monoclonal antibodies to CD157 are described herein that provide excellent target specificity, signal-to-noise ratio, etc., compared to other reported anti-CD157 antibodies, as well as antigen-binding fragments of such antibodies that bind to CD157. Methods for producing anti-CD157 antibodies and antigen-binding fragments thereof having desired properties, including affinity and / or specificity for CD157 and / or its variants, are also provided herein.

[0054] The following description includes information that may be helpful in understanding the present technology. This is not an admission that any information provided herein or any publication explicitly or implicitly referenced herein is prior art or is particularly relevant to the presently claimed technology.

[0055] Antibody generation and characterization

[0056] Anti-CD157 antibodies and antigen binding fragments thereof provided herein can have strong binding affinity and / or specificity to CD157. In some embodiments, anti-CD157 antibodies and antigen binding fragments thereof can be chimeric antibodies. In some embodiments, anti-CD157 antibodies and antigen binding fragments thereof can be humanized antibodies. In some embodiments, anti-CD157 antibodies and antigen binding fragments thereof can be variant antibodies. For example, from the perspective of treatment, antibodies can have benefit characteristics. Determinations for determining the activity of anti-CD157 antibodies provided herein include, for example, cell-based ELISA (for example, to measure the cell specificity of antibodies) and cytotoxicity (for example, to measure the potential of mediating direct or indirect killing of target cells expressing CD157). In some cases, humanized antibodies or variant antibodies fail to induce an immunogenic response after administering a therapeutically effective amount of antibodies to human patients. In some cases, if an immunogenic response is induced, the response may cause the antibody to still provide therapeutic benefits for the patient being treated.

[0057] In some embodiments, the anti-CD157 antibodies and antigen-binding fragments thereof (e.g., humanized anti-CD157 antibodies) herein bind to the same epitope. In order to screen for antibodies that bind to an epitope on CD157 that binds to an antibody of interest (e.g., antibodies that block binding to CD157), a cross-blocking assay, such as Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988) can be performed. In some cases, epitope mapping can be performed, for example, as described in Champe et al., J. Biol. Chem. 270: 1388-1394 (1995), in Cunningham and Wells, Science 244: 1081-1085 (1989), or Davidson and Doranz, Immunology 143: 13-20 (2014), to determine whether the antibody binds to the target epitope.

[0058] The antibodies herein generally have a heavy chain variable domain comprising an amino acid sequence represented by the formula: FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4, wherein "FRH1-4" represents four heavy chain framework regions, and "CDRH1-3" represents three hypervariable regions of the heavy chain variable domain of an anti-CD157 antibody. FRH1-4 can be derived from a consensus sequence (e.g., the most common amino acids in a class, subclass, or subgroup of a human immunoglobulin heavy or light chain), or can be derived from a single human antibody framework region or a combination of different framework region sequences. Many human antibody framework region sequences have been compiled, for example, in Kabat et al. (1992) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTERES T, National Institutes of Health Publication No. 91-3242. In one embodiment, the heavy chain variable FR is provided by a consensus sequence of a human immunoglobulin subgroup as compiled by Kabat et al. (supra).

[0059] The human heavy chain variable FR sequence may have substitutions, for example, where a human FR residue is replaced by a corresponding non-human residue (when human and non-human sequences are aligned, "corresponding non-human residue" means a non-human residue with the same Kabat position number as the target human residue), but it is not necessary to replace it with a non-human residue. For example, replacement FR residues other than the corresponding non-human residue can be selected by phage display.

[0060] The antibody herein can have a light chain variable domain comprising an amino acid sequence represented by the formula: FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4, wherein "FRL1-4" represents four framework regions, and "CDRL1-3" represents three hypervariable regions of an anti-CD157 antibody light chain variable domain. FRL1-4 can be derived from a consensus sequence (e.g., the most common amino acid in a class, subclass, or subgroup of a human immunoglobulin heavy chain or light chain), or can be derived from a combination of a single human antibody framework region or different framework region sequences. In one embodiment, the light chain variable FR is provided by a consensus sequence of a human immunoglobulin subgroup as compiled by Kabat et al. (supra).

[0061] The human light chain variable FR sequence may have substitutions, for example, wherein the human FR residues are replaced by corresponding mouse residues, but it is not necessary to replace them with non-human residues. For example, the replacement residues except the corresponding non-human residues can be selected by phage display. The method for producing the humanized anti-CD157 antibody of interest herein will be described in more detail below.

[0062] Anti-CD157 antibodies and antigen-binding fragments thereof

[0063] Antibodies and antigen-binding fragments thereof that bind to CD157 are provided herein. Such antibodies and antigen-binding fragments thereof may include anti-CD157 antibodies, anti-CD157 antibody fragments (eg, antigen-binding fragments) and anti-CD157 antibody derivatives.

[0064] CD157 is the differentiation cluster nomenclature for bone marrow stromal cell antigen-1 (BST-1), which is a stromal cell line-derived glycosylphosphatidylinositol-anchored molecule that promotes the growth of pre-B cells. The deduced amino acid sequence shows 33% similarity to CD38. CD157 can be shed as a soluble protein, produced by protease cleavage of the membrane-bound form, or as an exosome-anchored protein. In addition to bone marrow stromal cells, vascular endothelial cells, and circulating endothelial cells, CD157 is also expressed in several other cell types and tissues of lymphoid and non-lymphoid origin (Yakymiv et al. 2019. Cells 8(12): 1580).

[0065] CD157 is a bifunctional receptor and a member of the ADP-ribosyl cyclase family of β-NAD +Metabolic exoenzyme. CD157 interacts with extracellular matrix components and regulates leukocyte infiltration through integrin-mediated signaling in inflammation. CD157 also regulates cell migration and is a marker for poor prognosis in epithelial ovarian cancer and pleural mesothelioma (Ferrero et al. 2017. Scientific Reports. 7: 15923). Other diseases associated with BST1 include paroxysmal nocturnal hemoglobinuria and hemoglobinuria.

[0066] In some embodiments, the antibody or antigen binding fragment thereof is isolated (e.g., separated from a component of its natural environment (e.g., an animal, a biological sample)). In some embodiments, the antibody or antigen binding fragment thereof is a humanized antibody or antigen binding fragment thereof. In some embodiments, the antibody or antigen binding fragment thereof is a derivative of a humanized antibody that binds to CD157. In some embodiments, the antibody or antigen binding fragment thereof binds to CD157 under laboratory conditions (e.g., binds to CD157 in vitro, binds to CD157 in a flow cytometry assay, binds to CD157 in an ELISA). In some embodiments, the antibody or antigen binding fragment thereof binds to CD157 under physiological conditions (e.g., binds to CD157 in a subject's cells).

[0067] Typically, any anti-CD157 antibody or its antigen binding fragment provided herein comprises at least one immunoglobulin heavy chain variable domain and at least one immunoglobulin light chain variable domain. In some embodiments, any anti-CD157 antibody or its antigen binding fragment herein comprises two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains. Typically, each immunoglobulin heavy chain variable domain of anti-CD157 antibody or its antigen binding fragment comprises the first, second and third heavy chain complementary determining region or "CDR" (e.g., CDRH1, CDRH2, CDRH3), and each immunoglobulin light chain variable domain of anti-CD157 antibody or its antigen binding fragment comprises the first, second and third light chain CDR (e.g., CDRL1, CDRL2, CDRL3).

[0068] CDRH1

[0069] In some embodiments, the CDRH1 of any anti-CD157 antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence selected from GFSLTSYHVS (SEQ ID NO: 16), SYHVS (SEQ ID NO: 17), and NYDMA (SEQ ID NO: 18).

[0070] In some embodiments, the CDRH1 of any anti-CD157 antibody or antigen-binding fragment thereof provided herein comprises X1X2X3X4X5X6YX8X9X 10 (SEQ ID NO: 30), wherein X1 is G or no amino acid; X2 is F or no amino acid; X3 is S or no amino acid; X4 is L or no amino acid; X5 is T or no amino acid; X6 is S or N; X8 is H or D; X9 is V or M; X 10 It is S or A.

[0071] CDRH2

[0072] In some embodiments, the CDRH2 of any anti-CD157 antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence selected from IIWTGGSTAYNSLLKS (SEQ ID NO: 19) and SISIRGGSTYYRDSVKG (SEQ ID NO: 20).

[0073] In some embodiments, the CDRH2 of any anti-CD157 antibody or antigen-binding fragment thereof provided herein comprises X1IX3X4X5GGSTX 10 YX 12 X 13 X 14 X 15 KX 17 (SEQ ID NO: 31), wherein X1 is S or no amino acid; X3 is I or S; X4 is W or I; X5 is T or R; 10 A or Y; X 12 N or R; X 13 S or D; X 14 L or S; X 15 is L or V; and X 17 It is S or G.

[0074] CDRH3

[0075] In some embodiments, the CDRH3 of any anti-CD157 antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence selected from the group consisting of SITPTFFDY (SEQ ID NO: 21) and GTDYYYDYFDY (SEQ ID NO: 22).

[0076] In some embodiments, the CDRH3 of any anti-CD157 antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence of X1X2X3X4X5X6X7X8FDY (SEQ ID NO: 32), wherein X1 is G or no amino acid; X2 is T or no amino acid; X3 is S or D; X4 is I or Y; X5 is T or Y; X6 is P or Y; X7 is T or D; and X8 is F or Y.

[0077] CDRL1

[0078] In some embodiments, the CDRL1 of any anti-CD157 antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence selected from KRSTGNFGNNYVN (SEQ ID NO: 23), KRSTGNFGSNYVN (SEQ ID NO: 24), and KAGRNINSYLA (SEQ ID NO: 25).

[0079] In some embodiments, the CDRL1 of any anti-CD157 antibody or antigen-binding fragment thereof provided herein comprises KX2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 (SEQ ID NO: 33), wherein X2 is R or A; X3 is S or G; X4 is T or R; X5 is G or N; X6 is N or I; X7 is F or N; X8 is G or S; X9 is N, S or Y; X 10 N or L; X 11 is Y or A; X 12 is V or no amino acid; and X 13 N or no amino acid.

[0080] CDRL2

[0081] In some embodiments, the CDRL2 of any anti-CD157 antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence selected from the group consisting of RDDKRPD (SEQ ID NO: 26) and NANSLQT (SEQ ID NO: 27).

[0082] In some embodiments, the CDRL2 of any anti-CD157 antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 34), wherein X1 is R or N; X2 is D or A; X3 is D or N; X4 is K or S; X5 is R or L; X6 is P or Q; and X7 is D or T.

[0083] CDRL3

[0084] In some embodiments, the CDRL3 of any anti-CD157 antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence selected from the group consisting of QSYSSGIV (SEQ ID NO: 28) and QQYNSWTNT (SEQ ID NO: 29).

[0085] In some embodiments, the CDRL3 of any anti-CD157 antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence of QX2YX4SX6X7X8X9 (SEQ ID NO: 35), wherein X2 is S or Q; X4 is S or N; X6 is G or W; X7 is I or T; X8 is V or N; and X9 is T or no amino acid.

[0086] CDR Group

[0087] In some embodiments, any anti-CD157 antibody or its antigen-binding fragment described herein comprises: an immunoglobulin heavy chain variable domain comprising a set of CDRs (i.e., CDRH1, CDRH2, CDRH3); and an immunoglobulin light chain variable domain comprising a set of CDRs (i.e., CDRL1, CDRL2, CDRL3). In some embodiments, the anti-CD157 antibody or its antigen-binding fragment herein comprises two immunoglobulin heavy chain variable domains, each of which comprises a set of CDRs (i.e., CDRH1, CDRH2, CDRH3); and two immunoglobulin light chain variable domains, each of which comprises a set of CDRs (i.e., CDRL1, CDRL2, CDRL3). The CDR group may comprise any combination of CDR amino acid sequences (i.e., CDRH1, CDRH2, CDRH3; and CDRL1, CDRL2, CDRL3) provided herein. In some embodiments, the immunoglobulin heavy chain variable domain comprises a set of CDRH1, CDRH2, and CDRH3 amino acid sequences, and the immunoglobulin light chain variable domain comprises a set of CDRL1, CDRL2, and CDRL3 amino acid sequences selected from Groups 1-16 provided in the table below.

[0088] Table 1. CDR groups

[0089]

[0090] In some embodiments, all CDRs are from the same group. For example, for an anti-CD157 antibody or antigen-binding fragment thereof comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains, each immunoglobulin heavy chain variable domain can comprise a pair of CDRH1, CDRH2 and CDRH3 amino acid sequences from Group 1, and each immunoglobulin light chain variable domain can comprise a pair of CDRL1, CDRL2 and CDRL3 amino acid sequences from Group 1.

[0091] In some embodiments, the CDRs are from different groups. For example, for an anti-CD157 antibody or antigen-binding fragment thereof comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains, each immunoglobulin heavy chain variable domain may comprise a pair of CDRH1, CDRH2 and CDRH3 amino acid sequences from group 1, and each immunoglobulin light chain variable domain may comprise a pair of CDRL1, CDRL2 and CDRL3 amino acid sequences from group 2. In another example, for an anti-CD157 antibody or antigen-binding fragment thereof comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains, one immunoglobulin heavy chain variable domain may comprise a pair of CDRH1, CDRH2 and CDRH3 amino acid sequences from group 2; one immunoglobulin light chain variable domain may comprise one of the CDRL1, CDRL2 and CDRL3 amino acid sequences from group 1; and another immunoglobulin light chain variable domain may comprise one of the CDRL1, CDRL2 and CDRL3 amino acid sequences from group 2.

[0092] VH

[0093] In some embodiments, any anti-CD157 antibody or antigen-binding fragment thereof provided herein may include a heavy chain variable domain (VH). In some embodiments, the heavy chain variable domain (VH) of any anti-CD157 antibody or antigen-binding fragment thereof provided herein includes a polypeptide having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence QVQLKESGPGLVQPSQTLSLTCTVSGFSLTSYHVSWVRQPPGKGLEWMGIIWTGGSTAYNSLLKSRLSISRDTSKSQVFLKMNSLQTEDTATYYCARSITPTFFDYWGQGVMVTVSS (SEQ ID NO: 1), for example, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to SEQ ID NO:1 has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of SEQ ID NO:1. In some embodiments, the VH comprises a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:1. In some embodiments, the VH comprises a polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO:1. In some embodiments, the VH comprises a polypeptide having 100% identity to the amino acid sequence of SEQ ID NO:1.

[0094] In some embodiments, the heavy chain variable domain (VH) of any anti-CD157 antibody or antigen-binding fragment thereof provided herein comprises a polypeptide having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence EVHLVESGGGLVQPGRSLKLSCAASGFTFSNYDMAWIRQAPAKGLEWVASISIRGGSTYYRDSVKGRFTVSRDNAKSTLYLQMDSLRSEDTATYYCVRGTDYYYDYFDYWGQGVMVTVSS (SEQ ID NO: 2), e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence EVHLVESGGGLVQPGRSLKLSCAASGFTFSNYDMAWIRQAPAKGLEWVASISIRGGSTYYRDSVKGRFTVSRDNAKSTLYLQMDSLRSEDTATYYCVRGTDYYYDYFDYWGQGVMVTVSS NO:2 amino acid sequence has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, the VH comprises a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:2. In some embodiments, the VH comprises a polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO:2. In some embodiments, the VH comprises a polypeptide having 100% identity to the amino acid sequence of SEQ ID NO:2.

[0095] In some embodiments, the VH of the anti-CD157 antibodies or antigen-binding fragments thereof provided herein comprises a polypeptide selected from SEQ ID NO: 1 and SEQ ID NO:2.

[0096] In some embodiments, the VH of any anti-CD157 antibody or antigen-binding fragment thereof provided herein further comprises a signal sequence. In some embodiments, the VH signal sequence of any anti-CD157 antibody or antigen-binding fragment thereof provided herein comprises the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the VH signal sequence of any anti-CD157 antibody or antigen-binding fragment thereof provided herein comprises the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the VH signal sequence of any anti-CD157 antibody or antigen-binding fragment thereof provided herein comprises the amino acid sequence shown in SEQ ID NO: 13.

[0097] V L

[0098] In some embodiments, any anti-CD157 antibody or antigen-binding fragment thereof provided herein may comprise a light chain variable domain (VL). In some embodiments, the light chain variable domain (VL) of any anti-CD157 antibody or antigen-binding fragment thereof provided herein comprises a polypeptide having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence QFVLTQPNSVSTNLGSTVKLSCKRSTGNFGNNYVNWYQQHEGRSPTTMIYRDDKRPDGVPDRFSGSIDRSSNSALLTISNVQTEDEADYFCQSYSSG IVFGGGTKLTVL (SEQ ID NO: 3), for example, a polypeptide having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to SEQ ID NO: NO:3 has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of NO:3. In some embodiments, the VL comprises a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:3. In some embodiments, the VL comprises a polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO:3. In some embodiments, the VL comprises a polypeptide having 100% identity to the amino acid sequence of SEQ ID NO:3.

[0099] In some embodiments, the light chain variable domain (VL) of any anti-CD157 antibody or antigen-binding fragment thereof provided herein comprises a polypeptide having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence QFVLTQPNSVSTNLGSTVKLSCKRSTGNFGSNYVNWYQQHEGRSPTTM IYRDDKRPDGVPDRFSGSIDRSSNSALLTINNVQTEDEADYFCQSYSSGIVFGGGTKLTVL (SEQ ID NO: 4), e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: NO:4 amino acid sequence has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, the VL comprises a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:4. In some embodiments, the VL comprises a polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO:4. In some embodiments, the VL comprises a polypeptide having 100% identity to the amino acid sequence of SEQ ID NO:4.

[0100] In some embodiments, the light chain variable domain (VL) of any anti-CD157 antibody or antigen-binding fragment thereof provided herein comprises a polypeptide having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence NIQMTQSPSLLSASVGDRVTLSCKAGRNINSYLAWYQQMLGEAPKLLIYNANSLQTGIPSRFSGSGSGTDYTLTISSLQPEDVATYFCQQYNSWTNTFGAGTKLELK (SEQ ID NO: 5), e ...% identity to the amino acid sequence NIQMTQSPSLLSASVGDRVTLSCKAGRNINSYLAWYQQMLGEAPKLLIYNANSLQTGIPSRFSGSGSGTDYTLTISSLQPEDVATYFCQQYNSWTNTFGAGTKLELK NO:5 amino acid sequence has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, the VL comprises a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:5. In some embodiments, the VL comprises a polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO:5. In some embodiments, the VL comprises a polypeptide having 100% identity to the amino acid sequence of SEQ ID NO:5.

[0101] In some embodiments, the VL of the anti-CD157 antibodies or antigen-binding fragments thereof provided herein comprises a polypeptide selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

[0102] In some embodiments, the VL of any anti-CD157 antibody or antigen-binding fragment thereof provided herein further comprises a signal sequence. In some embodiments, the VL signal sequence of any anti-CD157 antibody or antigen-binding fragment thereof provided herein comprises the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the VL signal sequence of any anti-CD157 antibody or antigen-binding fragment thereof provided herein comprises the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the VL signal sequence of any anti-CD157 antibody or antigen-binding fragment thereof provided herein comprises the amino acid sequence shown in SEQ ID NO: 15.

[0103] Fc

[0104] Anti-CD157 antibodies or their antigen-binding fragments provided herein may include a fragment crystallizable region (Fc region). The Fc region generally forms the tail of an antibody and may interact with certain components of certain cell surface receptors and complement systems. The Fc region may include, for example, two polypeptides, each of which is derived from the second (CH2) and third (CH3) constant domains of an antibody heavy chain.

[0105] The amino acid sequence of the wild-type CH2-CH3 portion of the Fc region is provided below (positioned the same as the EU index of Kabat et al. (1992) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, National Institutes of Health Publication No. 91-3242) (SEQ ID NO: 36). The CH2 portion is from amino acids 1-110 of SEQ ID NO: 36, and the CH3 portion is from amino acids 111-217 of SEQ ID NO: 36.

[0106] In some embodiments, the Fc region includes one or more modifications (e.g., one or more amino acid substitutions, insertions or deletions relative to a comparable wild-type Fc region). Antibodies and antigen-binding fragments thereof (variant agents) comprising modified Fc regions generally have an altered phenotype relative to agents comprising wild-type Fc regions. The variant agent phenotype may be manifested as an altered serum half-life, altered stability, altered cellular enzyme sensitivity, or altered effector function (e.g., as determined in NK-dependent or macrophage-dependent assays). Fc region modifications that alter effector function may include modifications that increase binding to activating receptors (e.g., FcγRIIA (CD16A)) and reduce binding to inhibitory receptors (e.g., FcγRIIB (CD32B)) (see, e.g., Stavenhagen, J Betal. (2007) Cancer Res. 57 (18): 8882-8890). Examples of human IgG1 Fc region variants with reduced binding to CD32B and / or increased binding to CD16A include F243L, R292P, Y300L, V305I and / or P396L substitutions. The amino acid positions correspond to the amino acid numbering of the CH2-CH3 domain provided above.

[0107] In some embodiments, the Fc region includes one or more modifications that reduce or eliminate Fc binding to Fc receptors. Such modifications may include amino acid substitutions at positions 234, 235, 265, and 297 (see, e.g., U.S. Patent No. 5,624,821, which is incorporated herein by reference). Example substitutions include one or more of L234A, L235A, D265A, and N297Q. The amino acid positions correspond to the amino acid numbers of the CH2-CH3 domains provided above.

[0108] In some embodiments, the Fc region comprises one or more modifications that alter (relative to a wild-type Fc region) the affinity ratio of the modified Fc region for an activating FcγR (e.g., FcγRIIA or FcγRIIIA) relative to an inhibitory FcγR (e.g., FcγRIIB):

[0109]

[0110] In the case where the modified Fc region has an affinity ratio greater than 1, the anti-CD157 antibodies or antigen-binding fragments thereof herein may have particular uses in providing therapeutic or prophylactic treatment of a disease, disorder or infection, or amelioration of a symptom thereof, where it is desired to enhance the efficacy of FcγR-mediated effector cell function (e.g., ADCC), such as cancer or infectious diseases. In the case where the modified Fc region has an affinity ratio less than 1, the anti-CD157 antibodies or antigen-binding fragments thereof herein may have particular uses in providing therapeutic or prophylactic treatment of a disease or disorder, or amelioration of a symptom thereof, where it is desired to reduce the efficacy of FcγR-mediated effector cell function, such as autoimmune or inflammatory disorders. Table 4 lists examples of single, double, triple, quadruple and quintuple amino acid substitutions with affinity ratios greater than 1 or less than 1 (see, e.g., PCT Publication Nos. WO04 / 063351; WO 06 / 088494; WO 07 / 024249; WO 06 / 113665; WO 07 / 021841; WO 07 / 106707; WO2008 / 140603, each of which is incorporated herein by reference). The amino acid positions correspond to the amino acid numbers of the CH2-CH3 domains provided above.

[0111] Table 4: Examples of single and multiple substitutions listed by affinity ratio

[0112]

[0113]

[0114] Antibodies and antigen-binding fragments thereof that competitively bind to the anti-CD157 antibodies and antigen-binding fragments thereof provided herein

[0115] Anti-CD157 antibodies and antigen binding fragments thereof are provided herein, which are competitively bound to or can be competitively bound to one or more anti-CD157 antibodies and antigen binding fragments thereof as described herein. Specifically, anti-CD157 antibodies and antigen binding fragments thereof are provided herein, which compete with or can compete with one or more anti-CD157 antibodies and antigen binding fragments thereof as described herein for binding to CD157. Such antibodies and antigen binding fragments thereof that compete with or can compete with anti-CD157 as described herein can be referred to as competitor antibodies and antigen binding fragments thereof. In some cases, when a competitor is bound to the same general region of CD157 with an anti-CD157 antibody or its antigen binding fragment as described herein (i.e., an extracellular region or a leucine-rich binding domain), it can be considered that the antibody or its antigen binding fragment (i.e., a competitor antibody or its antigen binding fragment) competes with CD157 for binding. In some cases, when a competitor binds to the exact same region of CD157 as an anti-CD157 antibody or antigen-binding fragment thereof described herein (e.g., the exact same peptide (linear epitope) or the exact same surface amino acid (conformational epitope)), it can be considered that the antibody or its antigen-binding fragment (i.e., competitor antibody or its antigen-binding fragment) competes for binding to CD157. In some cases, when a competitor binds to the same general region of CD157 as an anti-CD157 antibody or its antigen-binding fragment described herein (i.e., the extracellular region or the leucine-rich binding domain) under suitable assay conditions, it can be considered that the antibody or its antigen-binding fragment (i.e., competitor antibody or its antigen-binding fragment) can compete for binding to CD157. In some cases, when a competitor binds to the exact same region of CD157 as an anti-CD157 antibody or its antigen-binding fragment described herein (e.g., the exact same peptide (linear epitope) or the exact same surface amino acid (conformational epitope)) under suitable assay conditions, it can be considered that the antibody or its antigen-binding fragment (i.e., competitor antibody or its antigen-binding fragment) can compete for binding to CD157.

[0116] In some cases, when a competitor blocks the binding of one or more anti-CD157 antibodies or antigen-binding fragments thereof described herein to CD157, it can be considered that the antibody or its antigen-binding fragment (i.e., competitor antibody or antigen-binding fragment thereof) competes for binding to CD157. In some cases, when a competitor blocks the binding of one or more anti-CD157 antibodies or antigen-binding fragments thereof described herein to CD157 under suitable assay conditions, it can be considered that the antibody or its antigen-binding fragment (i.e., competitor antibody or antigen-binding fragment thereof) is able to compete for binding to CD157. Whether a competitor blocks the binding of one or more anti-CD157 antibodies or antigen-binding fragments thereof described herein to CD157 can be determined using a suitable competition assay or blocking assay (e.g., a blocking assay described in the Examples herein). Competitor antibodies or antigen-binding fragments thereof can block the binding of one or more anti-CD157 antibodies or antigen-binding fragments thereof described herein to CD157 by 50% or more in a competition or blocking assay, and conversely, one or more anti-CD157 antibodies or antigen-binding fragments thereof described herein can block the binding of competitor antibodies or antigen-binding fragments thereof to CD157 by about 50% or more in a competition or blocking assay. For example, an antibody or antigen-binding fragment thereof (i.e., a competitor antibody or antigen-binding fragment thereof) can block binding of one or more anti-CD157 antibodies or antigen-binding fragments thereof described herein to CD157 by about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in a competition or blocking assay, and conversely, one or more anti-CD157 antibodies or antigen-binding fragments thereof described herein can block binding of a competitor antibody or antigen-binding fragment thereof to CD157 by about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in a competition or blocking assay.

[0117] In some cases, when a competitor binds to CD157 with an affinity similar to one or more anti-CD157 antibodies or antigen-binding fragments thereof described herein, it can be considered that the antibody or its antigen-binding fragment (i.e., competitor antibody or its antigen-binding fragment) competes for binding to CD157. In some cases, when a competitor binds to CD157 with an affinity similar to one or more anti-CD157 antibodies or their antigen-binding fragments described herein under appropriate assay conditions, it can be considered that the antibody or its antigen-binding fragment (i.e., competitor antibody or its antigen-binding fragment) is able to compete for binding to CD157. In some embodiments, when a competitor binds to CD157 with an affinity of at least about 50% of the affinity of one or more anti-CD157 antibodies or their antigen-binding fragments described herein, it is considered that the antibody or its antigen-binding fragment (i.e., competitor antibody or its antigen-binding fragment) competes for binding to CD157. For example, when a competitor binds to CD157 with at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the affinity of one or more anti-CD157 antibodies or antigen-binding fragments thereof described herein, the antibody or antigen-binding fragment thereof (i.e., competitor antibody or antigen-binding fragment thereof) can be considered to compete for binding to CD157. The competing antibody or antigen-binding fragment thereof can comprise any of the features of the anti-CD157 antibodies or antigen-binding fragments thereof described herein.

[0118] Anti-CD157 antibodies or their Fabs are also provided herein, which are bound to or can be bound to the same epitope as one or more anti-CD157 antibodies or their Fabs as described herein. Specifically, anti-CD157 antibodies or their Fabs are provided herein, which compete with one or more anti-CD157 antibodies or their Fabs as described herein and bind to the same epitope on CD157. Such antibodies or their Fabs in conjunction with the same epitope can be referred to as epitope competitors. In some cases, epitope competitors can be bound to the exact same region of CD157 (e.g., the exact same peptide (linear epitope) or the exact same surface amino acid (conformational epitope)) with anti-CD157 antibodies or their Fabs as described herein. In some cases, epitope competitors block the combination of one or more anti-CD157 antibodies or their Fabs as described herein with CD157. Epitope competitors can block the binding of one or more anti-CD157 antibodies or antigen-binding fragments thereof described herein to CD157 by about 50% or more in a competition assay, and conversely, one or more anti-CD157 antibodies or antigen-binding fragments thereof described herein can block the binding of epitope competitors to CD157 by 50% or more in a competition assay. In some cases, epitope competitors bind to CD157 with an affinity similar to that of one or more anti-CD157 antibodies or antigen-binding fragments thereof described herein. In some embodiments, epitope competitors bind to CD157 with an affinity of at least about 50% of the affinity of one or more anti-CD157 antibodies or antigen-binding fragments thereof described herein. For example, epitope competitors can bind to CD157 with an affinity of at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the affinity of one or more anti-CD157 antibodies or antigen-binding fragments thereof described herein. An epitope competitor may comprise any of the features of an anti-CD157 antibody or antigen-binding fragment thereof described herein.

[0119] Antibody preparation

[0120] The following examples describe methods for generating anti-CD157 antibodies and their antigen binding fragments. In some embodiments, anti-CD157 antibodies or their antigen binding fragments are humanized antibodies or their antigen binding fragments. Humanized anti-CD157 antibodies can be prepared in, for example, genetically engineered (i.e., transgenic) mice (e.g., from Medarex), which can produce human antibodies that do not necessarily require CDR transplantation when presenting immunogens. These antibodies are fully human antibodies (100% human protein sequences) from animals (e.g., mice), in which non-human antibody genes are suppressed and replaced by human antibody gene expression. When presented to these genetically engineered mice or other animals that can produce the human framework of the relevant CDR, antibodies against CD157 can be generated.

[0121] In the case of generating variants of anti-CD157 antibodies, parent antibodies are prepared. The following sections will describe exemplary techniques for generating such non-human antibodies and parent antibodies.

[0122] Antigen preparation

[0123] The antigen used to generate the antibody can be, for example, a complete CD157 (particularly expressed in a cell) or a portion of CD157 (e.g., an N-terminal domain, a C-terminal domain, a cytoplasmic domain, an intracellular domain, a transmembrane domain, an extracellular domain, an ectodomain, a TIR domain, a leucine-rich domain, or a CD157 fragment containing a desired epitope). For those skilled in the art, other forms of antigens for generating antibodies are obvious.

[0124] Polyclonal antibodies

[0125] Polyclonal antibodies can be produced in animals (vertebrates or invertebrates, including mammals, birds, and fish, including cartilaginous fish) by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. Antibodies can be produced by using bifunctional or derivatizing agents (e.g., maleimidobenzoyl sulfosuccinimide ester (conjugated via cysteine ​​residues), N-hydroxysuccinimide (conjugated via lysine residues), glutaraldehyde, succinic anhydride, SOCl2 or R 1 N=C=NR, where R and R 1 It may be useful to conjugate the relevant antigen to a protein or other carrier that is immunogenic in the species to be immunized (e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor). Non-protein carriers (e.g., colloidal gold) can also be used for antibody production.

[0126] Animals are immunized against antigens, immunogenic conjugates or derivatives by combining, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with three volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, animals are boosted by subcutaneous injection of one-fifth to one-tenth of the original amount of peptide or conjugate in Freund's complete adjuvant at multiple sites. Seven to 14 days later, blood is drawn from the animals and serum antibody titers are determined. Animals are boosted until the titer is stable. Typically, animals are boosted with conjugates of the same antigen, but conjugated to different proteins and / or through different cross-linking agents. Conjugates can also be made as protein fusions in recombinant cell culture. In addition, aggregating agents (e.g., alum) are also suitable for enhancing immune responses.

[0127] Monoclonal antibodies

[0128] Monoclonal antibodies can be prepared using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or can be prepared by other methods, such as recombinant DNA methods (U.S. Pat. No. 4,816,567). In the hybridoma method, mice or other appropriate host animals, such as hamsters or macaques, are immunized as described above to induce lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes can also be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0129] The hybridoma cells thus prepared are inoculated and grown in an appropriate culture medium that may contain one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas will usually include hypoxanthine, aminopterin and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

[0130] Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among them, preferred myeloma cell lines are murine myeloma cell lines, such as SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection in Rockville, Maryland, USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0131] The production of monoclonal antibodies against the antigen in the culture medium in which the hybridoma cells are grown is measured. The binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation, in vitro binding assays (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)) or by flow cytometric analysis of cells expressing membrane antigens. The binding affinity of the monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107: 220 (1980).

[0132] After identifying hybridoma cells that produce antibodies of the desired specificity, affinity, and / or activity, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown as ascites tumors in animals. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites or serum by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0133] The DNA encoding the monoclonal antibody can be easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can be specifically bound to the genes encoding the heavy and light chains of the monoclonal antibodies). Alternatively, cDNA can be prepared from mRNA, and then the cDNA is subjected to DNA sequencing. Hybridoma cells are used as the preferred source of this genomic DNA or RNA for preparing cDNA. Once separated, DNA can be placed in expression vectors well known in the art and then transfected into host cells, such as Escherichia coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells or myeloma cells, which do not produce immunoglobulins in addition, to obtain the synthesis of monoclonal antibodies in recombinant host cells. The recombinant production of antibodies will be described in more detail below.

[0134] Humanization and amino acid sequence variants

[0135] General methods for antibody humanization are described in, e.g., U.S. Pat. Nos. 5861155, 6479284, 6407213, 6639055, 6500931, 5530101, 5585089, 5693761, 5693762, 6180370, 5714350, 6350861, 5777085, 5834597, 5882644, 5932448, 6013256, 6129914, 6210671, 6329511, 5225539, 6548640, and 5624821, each of which is incorporated herein by reference. In certain embodiments, it may be desirable to generate amino acid sequence variants of these humanized antibodies, particularly when these variants improve the binding affinity or other biological properties of the antibody.

[0136] By introducing the anti-CD157 antibody DNA with appropriate nucleotide changes or by peptide synthesis to prepare the amino acid sequence variants of anti-CD157 antibody. For example, such variants include, for example, the disappearance and / or insertion and / or replacement of residues in the anti-CD157 antibody amino acid sequence. Any combination of disappearance, insertion and replacement can be performed to obtain the final construct, as long as the final construct has the desired characteristics. Amino acid changes can also change the post-translational process of humanized or variant anti-CD157 antibodies, for example, change the number or position of glycosylation sites.

[0137] A method for identifying certain residues or regions of anti-CD157 antibodies as preferred positions for mutagenesis is called "alanine scanning mutagenesis", as described in Cunningham and Wells Science, 244: 1081-1085 (1989). Here, a residue or a group of target residues (e.g., charged residues such as arg, asp, his, lys and glu) are identified and replaced with neutral or negatively charged amino acids (most preferably alanine or polyalanine) to affect the interaction of the amino acid with the CD157 antigen. Then, the amino acid positions that show functional sensitivity to the substitution are refined by introducing further or other variants at the substitution site or introducing further or other variants for the substitution site. Therefore, although the site for introducing amino acid sequence variation is predetermined, the nature of the mutation itself does not need to be predetermined. For example, in order to analyze the performance of a mutation at a given site, alanine scanning or random mutagenesis is performed at the target codon or region, and the expressed anti-CD157 antibody variants are screened to obtain the desired activity. Amino acid sequence insertions include amino and / or carboxyl terminal fusions of polypeptides ranging in length from one residue to one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an N-terminal methionyl residue or an antibody fused to an epitope tag. Other insertion variants include fusions of enzymes or polypeptides that increase the serum half-life of the antibody to the N- or C-terminus of the antibody.

[0138] Another variant is an amino acid substitution variant. These variants have at least one amino acid residue removed from the antibody molecule and a different residue inserted in its place. The sites of greatest interest for substitution mutagenesis include hypervariable regions, but FR changes are also envisioned. Conservative substitutions are preferred, but more substantial changes may be introduced and the product screened. Examples of substitutions are listed below:

[0139] Examples of amino acid residue substitutions

[0140] A1a(A)val;leu;ile;val

[0141] Arg(R)lys;gln;asn;lys

[0142] Asn(N)gln;his;asp,lys;gln;arg

[0143] Asp(D)glu;asn

[0144] Cys(C)ser;ala

[0145] Gln(Q)asn;glu

[0146] Glu(E)asp;gln

[0147] Gly(G)ala

[0148] His(H)asn;gln;lys;arg

[0149] Ile(I)1eu; val; met; ala; leu; phe; norleucine

[0150] Leu(L)norleucine;ile;val;ile;met;ala;phe

[0151] Lys(K)arg;gln;asn

[0152] Met(M)1eu;phe;ile

[0153] Phe(F)1eu;val;ile;ala;tyr

[0154] Pro(P)ala

[0155] Ser(S)thr

[0156] Thr(T)ser

[0157] Trp(W)tyr;phe

[0158] Tyr(Y)trp;phe;thr;ser

[0159] Val(V)ile;leu;met;phe;ala;norleucine

[0160] Substantial changes in the biological properties of the antibody are achieved by selecting substitutions that have significant differences in maintaining (a) the structure of the polypeptide backbone in the area of ​​the substitution, e.g., as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into several groups based on common side chain properties:

[0161] (1) Hydrophobicity: norleucine, met, ala, val, leu, ile:

[0162] (2) Neutral hydrophilicity: Cys, Ser, Thr;

[0163] (3) Acidic: asp, glu;

[0164] (4) Basic: Asn, Gln, His, Lys, Arg;

[0165] (5) Residues that affect chain direction: gly, pro; and

[0166] (6) Aromaticity: trp, tyr, phe

[0167] Non-conservative substitutions will entail exchanging a member of one class for a member of another class.

[0168] Any cysteine ​​residues not involved in maintaining the correct conformation of the antibody may also be substituted to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​bonds may be added to the antibody to improve its stability (particularly when the antibody is an antibody fragment (eg, Fv fragment)).

[0169] A substitution variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., humanized or human antibody). Typically, the final variant selected for further development will have improved biological properties relative to the parent antibody that produced them. A convenient method for generating such substitution variants is to use phage display for affinity maturation. In short, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site.

[0170] The antibody variants thus generated are displayed as fusions of the M13 gene III products packaged in each particle from filamentous phage particles in a monovalent manner. Then, the biological activity (e.g., binding affinity) of the phage display variants is screened, as disclosed herein. In order to identify the modified candidate hypervariable region sites, alanine scanning mutagenesis can be performed to identify the hypervariable region residues that contribute significantly to antigen binding. Alternatively, or in addition, analyzing the crystal structure of the antigen-antibody complex may be beneficial to identify the contact points between the antibody and the antigen. According to the technology described in detail herein, such contact residues and adjacent residues are candidates for substitution. Once such variants are generated, the variant group is screened as described herein, and antibodies with excellent properties in one or more related assays can be selected for further development.

[0171] Another amino acid variant of an antibody changes the original glycosylation pattern of the antibody. The change means to make one of the multiple carbohydrate moieties found in the antibody missing, and / or to add one or more glycosylation sites that are not present in the antibody. The glycosylation of an antibody is usually N-connected and / or O-connected. N-connected refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (wherein X is any amino acid except proline) are the most common recognition sequences for the enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of any tripeptide sequence in a polypeptide produces potential glycosylation sites. O-connected glycosylation refers to the attachment of one of the carbohydrate N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0172] Adding glycosylation sites to antibodies can be conveniently accomplished by altering the amino acid sequence so that it contains one or more of the above tripeptide sequences (for N-linked glycosylation sites). Changes can also be made by adding or replacing one or more serine or threonine residues in the original antibody sequence (for O-linked glycosylation sites).

[0173] The nucleic acid molecules encoding the amino acid sequence variants of the anti-CD157 antibodies herein can be prepared by a variety of methods known in the art. These methods include but are not limited to separation from natural sources (with regard to naturally occurring amino acid sequence variants) or prepared by cassette mutagenesis of oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis and early prepared variants or non-variant versions of anti-CD157 antibodies.

[0174] Human Antibodies

[0175] As an optional humanization, human antibodies can be generated. For example, transgenic animals (e.g., mice) can be generated that are able to produce a full set of human antibodies in the absence of endogenous immunoglobulin production after immunization. For example, homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric mice and germline mutant mice results in complete inhibition of endogenous antibody production. Transferring the human germline immunoglobulin gene array into such germline mutant mice can result in the production of human antibodies after antigen attack (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann et al., Year in Immuno., 7: 33 (1993); and U.S. Patent Nos. 5,591,669, 5,589,369 and 5,545,807). Human antibodies can also be derived from phage display libraries (Hoogenboom et al., J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581-597 (1991); and U.S. Pat. Nos. 5,565,332 and 5,573,905). Human antibodies can also be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

[0176] Antigen-binding antibody fragment

[0177] In certain embodiments, the anti-CD157 antibody or its antigen-binding fragment is an antibody fragment that retains at least one desired activity (including antigen binding). Various techniques for producing antibody fragments have been developed. In some cases, these fragments are obtained by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). In some cases, these fragments are produced directly by recombinant host cells. For example, Fab'-SH fragments can be directly recovered from Escherichia coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). In some cases, F(ab')2 is formed using leucine zipper GCN4 to facilitate the assembly of F(ab')2 molecules. According to another approach, Fv, Fab or F(ab')2 fragments can be isolated directly from recombinant host cell culture.Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.

[0178] Multispecific antibodies and antigen-binding fragments thereof

[0179] In some embodiments, the anti-CD157 antibody or antigen-binding fragment thereof comprises a first binding moiety and a second binding moiety, wherein the first binding moiety specifically reacts with a first molecule (which is CD157) and the second binding moiety specifically reacts with a second molecule (which is a molecular species different from the first molecule). Such antibodies or antigen-binding fragments thereof may comprise a plurality of first binding moieties, a plurality of second binding moieties, or a plurality of first binding moieties and a plurality of second binding moieties. Preferably, the ratio of the first binding moiety to the second binding moiety is about 1:1, although it may range from about 1000:1 to about 1:1000, wherein the ratio may be measured in terms of valency.

[0180] In those embodiments where the first moiety is an antibody, the second binding moiety may also be an antibody. In some embodiments, the first and second moieties are connected by a linker moiety that may have a valency of two to hundreds or even thousands for attaching the first and second binding moieties by one or more different chemical reactions. Examples of bispecific antibodies include antibodies that react to two different epitopes; in some cases, one epitope is a CD157 epitope and the second epitope is located on an unrelated soluble molecule. In some embodiments, the bispecific antibody is reactive to an epitope on CD157 and reactive to epitopes on different molecules found on different cell surfaces.

[0181] The composition herein may also include a first antibody or antigen binding fragment thereof and a second antibody or antigen binding fragment thereof, wherein the first antibody or antigen binding fragment thereof comprises a first binding portion that specifically reacts with a first molecule (e.g., CD157), and the second antibody or antigen binding fragment thereof comprises a second binding portion that specifically reacts with a second molecule (which is a molecular species different from the first molecule). The first and / or second antibody or antigen binding fragment thereof may be an antibody. The ratio of the first antibody or antigen binding fragment thereof to the second antibody or antigen binding fragment thereof may range from about 1,000:1 to 1:1,000, although the preferred ratio is about 1:1. In some embodiments, it may be desirable to generate a multispecific (e.g., bispecific) anti-CD157 antibody having binding specificity for at least two different epitopes. Certain bispecific antibodies may bind to two different epitopes of CD157. Bispecific antibodies may be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0182] According to a method for preparing bispecific antibodies, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture.Preferred interfaces comprise at least a portion of the CH3 domains of antibody constant domains.In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced by larger side chains (e.g., tyrosine or tryptophan).By replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensation "cavity" identical or similar to the large side chain size is formed on the interface of the second antibody molecule.This provides a mechanism for improving the output of heterodimers relative to other unwanted final products (e.g., homodimers) (see, e.g., Wo96 / 27011 disclosed on September 6, 1996).

[0183] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one antibody in the heteroconjugate can be coupled to avidin and the other antibody can be coupled to biotin. Heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. No. 4,678,980, along with a number of cross-linking techniques.

[0184] Any suitable technology can be used to generate bispecific antibodies from antibody fragments. For example, chemical linkage can be used to prepare bispecific antibodies. In some methods, intact antibodies are proteolytically cleaved to generate F(ab')2 fragments (see, for example, Brennan et al., Science 229: 81 (1985), which is incorporated herein by reference). These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent the formation of intermolecular disulfides. Then, the generated Fab' fragments are converted into thionitrobenzoate (TNB) derivatives. Then, one of the Fab'-TNB derivatives is reconverted into Fab'-thiol by reduction with mercaptoethylamine and mixed with other Fab'-thiol derivatives in equimolar amounts to form bispecific antibodies. In a still further embodiment, Fab'-SH fragments directly recovered from E. coli can be chemically coupled in vitro to form bispecific antibodies (see, eg, Shalaby et al., J. Exp. Med. 175:217-225 (1992), incorporated herein by reference).

[0185] Any technique suitable for preparing and isolating bispecific antibody fragments directly from recombinant cell culture can be used. For example, leucine zippers have been used to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992), which is incorporated herein by reference). The leucine zipper peptides of Fos and Jun proteins are linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers are reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) provides an alternative mechanism for preparing bispecific antibody fragments. These fragments contain the heavy chain variable domain (V H ), which is linked to the light chain variable domain (V L ) is connected, and the linker is too short to allow pairing between the two domains on the same chain. H and V L The domain is forced to bind to another fragment of V L and V HThe domains are paired to form two antigen binding sites. Another strategy for preparing bispecific antibody fragments is through the use of single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol. 152: 5368 (1994), which is incorporated herein by reference). In some cases, bispecific antibodies can be "linear antibodies" produced as described in Zapata et al. Protein Eng. 8 (10): 1057-1062 (1995), which is incorporated herein by reference.

[0186] Antibodies with two or more valencies are contemplated herein. Antibodies (or polymers or polypeptides) in which each arm or fragment thereof comprises one or more binding sites are referred to herein as "multivalent" antibodies. For example, each Fab or fragment thereof of a "bivalent" antibody herein comprises two binding sites, while each Fab or fragment thereof of a "trivalent" polypeptide herein comprises three binding sites. In the multivalent polymers herein, the two or more binding sites of each Fab may bind to the same or different antigens. For example, the two or more binding sites in the multivalent polypeptide herein may be directed against the same antigen, such as against the same portion or epitope of the antigen, or against two or more identical or different portions or epitopes of the antigen; and / or may be directed against different antigens; or a combination thereof. Thus, the bivalent polypeptide herein, for example, may comprise two identical binding sites, may comprise a first binding site for a first portion or epitope of an antigen and a second binding site for the same portion or epitope of the antigen or for another portion or epitope of the antigen; or may comprise a first binding site for a first portion or epitope of an antigen and a second binding site for a different antigen. However, as will be clear from the above description, the technology herein is not limited thereto, as the multivalent polypeptides herein can comprise any number of binding sites for the same or different antigens. In one embodiment, the multivalent polypeptide comprises at least two ligand binding elements, one of which contains one or more CDR peptide sequences set forth herein. In another embodiment, the multivalent polypeptide comprises three ligand binding sites, each of which is independently selected from the CDR sequences disclosed herein.

[0187] In certain embodiments, at least one of the ligand binding elements binds CD157. In one embodiment, at least one of the ligand binding elements binds another target. In one embodiment, there are up to 10,000 binding elements in the multivalent binding molecule, and the ligand binding elements can be attached to the scaffold.

[0188] Each Fab or fragment thereof of the antibody (or polymer or polypeptide) herein contains at least two binding sites, wherein at least one first binding site is for a first antigen and the second binding site is for a second antigen different from the first antigen, which may also be referred to as "multi-specific". Thus, a "bispecific" polymer comprises at least one site for a first antigen and at least one second site for a second antigen, while a "trispecific" is a polymer comprising at least one (first) binding site for a first antigen, at least one further (second) binding site for a second antigen, and at least one further (third) binding site for a third antigen: and so on. Thus, in its simplest form, a bispecific polypeptide herein is a bivalent polypeptide (per Fab) of the technology provided herein. However, as will be clear from the above description, the technology herein is not limited thereto, as the multispecific polypeptide herein may comprise any number of binding sites for two or more different antigens.

[0189] Other Modifications

[0190] Other modifications of anti-CD157 antibodies or antigen-binding fragments thereof are contemplated. For example, the technology herein also relates to immunoconjugates comprising an antibody described herein (e.g., anti-CD157 antibody) conjugated to a cytotoxic agent (e.g., a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or a fragment thereof)) or a radioactive isotope (e.g., a radioconjugate) or a cytotoxic drug. Such conjugates are sometimes referred to as "antibody-drug conjugates" or "ADCs". Conjugates are prepared using various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyladipimide HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene)).

[0191] Anti-CD157 antibodies or antigen-binding fragments thereof disclosed herein can be formulated as immunoliposomes. Liposomes containing antibodies are prepared by methods known in the art, such as Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030 (1980); and described in U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556. For example, liposomes can be generated by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derived phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter with a determined pore size to obtain liposomes with a desired diameter. Fab' fragments of the antibodies provided herein can be conjugated to liposomes by a disulfide exchange reaction as described in Martin et al., J. Biol. Chem. 257:286-288 (1982).Another active ingredient is optionally contained within the liposome.

[0192] Enzyme or other polypeptides can be covalently bound to anti-CD157 antibodies or their antigen-binding fragments by techniques well known in the art (e.g., using heterobifunctional cross-linking reagents discussed above). In some embodiments, recombinant DNA techniques well known in the art can be used to construct a fusion protein comprising at least the antigen-binding region of an antibody provided herein connected to at least the functionally active portion of an enzyme (see, e.g., Neuberger et al., Nature 312: 604-608 (1984)).

[0193] In certain embodiments, for example, it may be desirable to use antibody fragments rather than complete antibodies to increase the permeability of target tissues and cells. In this case, it may be desirable to modify the antibody fragment to increase its serum half-life. This can be achieved, for example, by incorporating a salvage receptor binding epitope into the antibody fragment (e.g., by mutating the appropriate region in the antibody fragment, or by incorporating the epitope into a peptide tag, which is then fused to either end or the middle of the antibody fragment, e.g., by DNA or peptide synthesis; see, e.g., Wo96 / 32478, published on October 17, 1996).

[0194] Covalent modification of anti-CD157 antibodies or their antigen-binding fragments is also included in the scope of the present technology. For example, anti-CD157 antibodies can be modified by chemical synthesis or by enzymatic or chemical cleavage. Other types of covalent modifications of antibodies are introduced into molecules by reacting the targeted amino acid residues of the antibody with an organic derivatizing agent that can react with a selected side chain or N-terminal or C-terminal residue. Exemplary covalent modifications of polypeptides are described in U.S. Patent No. 5,534,615, which is specifically incorporated herein by reference. Preferred covalent modification types of antibodies include connecting the antibody to one of a variety of non-protein polymers (e.g., polyethylene glycol, polypropylene glycol or polyoxyalkylene) in the manner described in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

[0195] In some embodiments, any antibody or antigen fragment thereof disclosed herein is conjugated or hybridized with an oligonucleotide marker. In some embodiments, the oligonucleotide marker includes a sample barcode sequence, a binding site for a primer, and an anchor. In some embodiments, the oligonucleotide marker may be conjugated or hybridized with any detectable marker or marker disclosed herein. In some embodiments, the oligonucleotide marker is a polymer sequence. In some embodiments, the terms "oligonucleotide" and "polynucleotide" are used interchangeably and refer to a single-stranded nucleotide polymer having a length of about 2 to about 500 nucleotides. In some embodiments, any oligonucleotide marker described herein may be synthesized, enzymatically prepared (e.g., by polymerization) or prepared using a "split-pool" method. In some embodiments, any oligonucleotide marker described herein may include ribonucleotide monomers (i.e., oligoribonucleotides) and / or deoxyribonucleotide monomers (i.e., oligodeoxyribonucleotides). In some embodiments, any oligonucleotide marker described herein may include a combination of deoxyribonucleotide monomers and ribonucleotide monomers in an oligonucleotide (e.g., a random or ordered combination of deoxyribonucleotide monomers and ribonucleotide monomers). In some embodiments, the length of oligonucleotide labeling can be 4 to 10, 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400 or 400-500 nucleotides. In some embodiments, any oligonucleotide labeling as described herein can include (for example, covalently or non-covalently) one or more functional parts attached to another structure. In some embodiments, any oligonucleotide labeling as described herein can include one or more detectable labels (for example, radioisotopes or fluorophores). In some embodiments, anchor is a determined polymer, for example, a polynucleotide or oligonucleotide sequence, which is designed to hybridize with a complementary oligonucleotide sequence. In some embodiments, anchor is designed to generate a double-stranded construct oligonucleotide sequence. In some embodiments, the anchor is located at the 3' end of the construct oligonucleotide sequence. In other embodiments, the anchor is located at the 5' end of the construct oligonucleotide sequence. Each anchor is specific for its intended complementary sequence.

[0196] In some embodiments, the sample barcode sequence is a polymer, e.g., a polynucleotide, which, when a functional element, is specific to a single ligand. In some embodiments, the sample barcode sequence can be used to identify a specific cell or substrate, e.g., Drop-seq beads. In some embodiments, the sample barcode sequence can be formed by a defined sequence of DNA, RNA, modified bases, or a combination of these bases, and any other polymer defined above. In some embodiments, the sample barcode sequence is about 2 to 4 monomer components, e.g., nucleotide bases, in length. In other embodiments, the barcode is at least about 1 to 100 monomer components, e.g., nucleotides in length. Thus, in various embodiments, the barcode is comprised of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 99, 91, 92, 93, 94, 95, 96, 97, 98, 99 or up to 100 monomer components (e.g., nucleic acids). In some embodiments, the sample barcode sequence is a specific barcode that is unique relative to other barcodes.

[0197] In some of any embodiments, the sample barcode sequence can have a variety of different forms. For example, the sample barcode sequence can include a polynucleotide barcode, a random nucleic acid and / or amino acid sequence, and a synthetic nucleic acid and / or amino acid sequence. The sample barcode sequence can be attached to an analyte or other part or structure in a reversible or irreversible manner. For example, the sample barcode sequence can be added to a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before or during sequencing of the sample. The sample barcode sequence can be used to identify and / or quantify a single sequencing read (e.g., a barcode can be or can include a unique molecular identifier or "UMI").

[0198] The sample barcode sequence can spatially resolve molecular components found in a biological sample, e.g., at single-cell resolution (e.g., the barcode can be or can include a "spatial barcode"). In some embodiments, the barcode includes a UMI and a spatial barcode. In some embodiments, the barcode includes two or more sub-barcodes that together act as a single barcode. For example, a polynucleotide barcode can include two or more polynucleotide sequences (e.g., sub-barcodes) separated by one or more non-barcode sequences.

[0199] In some embodiments, the binding site of primer is the functional component of oligonucleotide, which itself is an oligonucleotide or polynucleotide sequence that is provided for the annealing site of oligonucleotide amplification.The binding site of primer can be formed by the polymer of the combination of DNA, RNA, PNA, modified base or these bases, or polyamide etc.In some embodiments, the length of the binding site of primer is about 10 such monomer components, for example, nucleotide base.In other embodiments, the length of the binding site of primer is at least about 5 to 100 monomer components, for example, nucleotide. Thus, in various embodiments, the binding site for a primer consists of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 99, 91, 92, 93, 94, 95, 96, 97, 98, 99 or up to 100 monomer components (e.g., nucleic acids). In certain embodiments, the binding site of the primer can be a universal sequence suitable as an annealing site for a variety of amplification techniques. Amplification techniques include, but are not limited to, DNA polymerase-based amplification systems, such as polymerase chain reaction (PCR), real-time PCR, loop-mediated isothermal amplification (LAMP, MALBAC), strand displacement amplification (SDA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), and polymerization by any number of DNA polymerases (e.g., T4 DNA polymerase, Sulfulobus DNA polymerase, Klenow DNA polymerase, Bst polymerase, Phi29 polymerase), as well as RNA polymerase-based amplification systems (e.g., T7-, T3-, and SP6-RNA polymerase amplification), nucleic acid sequence-based amplification (NASBA), self-directed sequence replication (3SR), rolling circle amplification (RCA), ligase chain reaction (LCR), helicase-dependent amplification (I), branched amplification method, and RNA-seq. The method for conjugating or hybridizing oligonucleotide labels can be carried out in the manner described in WO / 2018 / 144813, WO / 2016 / 018960, WO / 2018 / 089438, WO / 2014 / 182528, WO / 2018 / 026873, and WO / 2021 / 188838.

[0200] Nucleic acids, vectors, host cells and recombinant methods

[0201] The technology described herein also provides isolated nucleic acids encoding anti-CD157 antibodies or their antigen-binding fragments, vectors and host cells comprising the nucleic acids, and recombinant techniques for producing the antibodies or their antigen-binding fragments. The nucleic acids herein may include one or more subsequences, each of which is referred to as a polynucleotide.

[0202] Provided herein are nucleic acids (e.g., isolated nucleic acids) comprising nucleotide sequences encoding anti-CD157 antibodies or antigen-binding fragments thereof. In some embodiments, the nucleic acid encodes the immunoglobulin heavy chain variable domains of any anti-CD157 antibodies or antigen-binding fragments thereof provided herein. In some embodiments, the nucleic acid encodes the immunoglobulin light chain variable domains of any anti-CD157 antibodies or antigen-binding fragments thereof provided herein. In some embodiments, the nucleic acid encodes the immunoglobulin heavy chain variable domains and immunoglobulin light chain variable domains of the anti-CD157 antibodies or antigen-binding fragments thereof provided herein. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of any one of SEQ ID NOs: 6-11. The nucleic acid may comprise a nucleotide sequence encoding the amino acid sequence of any one of the immunoglobulin heavy chain variable domains of SEQ ID NOs: 6-8. The nucleic acid may comprise a nucleotide sequence encoding the amino acid sequence of any one of the immunoglobulin light chain variable domains of SEQ ID NOs: 9-11.

[0203] Provided herein are isolated nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable domain of any of the anti-CD157 antibodies or antigen-binding fragments thereof described herein, wherein the nucleotide sequence is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to any one of SEQ ID NOs: 6-8.

[0204] Provided herein are isolated nucleic acids comprising a nucleotide sequence encoding an immunoglobulin light chain variable domain of any of the anti-CD157 antibodies or antigen-binding fragments thereof described herein, wherein the nucleotide sequence is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs: 9-11.

[0205] Provided herein are isolated nucleic acids comprising nucleotide sequences encoding an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain of any anti-CD157 antibody or antigen-binding fragment thereof described herein, wherein the nucleotide sequence encoding the immunoglobulin heavy chain variable domain is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs: 6-8, and the nucleotide sequence encoding the immunoglobulin light chain variable domain is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs: 9-11.

[0206] Examples of VH nucleotide (NT) sequences associated with the antibodies described herein are provided below.

[0207] Table 2: Nucleotide sequences of VH

[0208]

[0209] Examples of VL nucleotide (NT) sequences associated with the antibodies described herein are provided below.

[0210] Table 3: Nucleic acid sequence of VL

[0211]

[0212] In some embodiments, any nucleic acid provided herein comprises a signal sequence.In some embodiments, any nucleic acid described herein does not comprise a signal sequence.

[0213] In order to recombinantly produce anti-CD157 antibodies or their antigen-binding fragments, nucleic acids encoding anti-CD157 antibodies or their antigen-binding fragments can be separated and inserted into replicable vectors for further cloning (DNA amplification) or expression. In some cases, anti-CD157 antibodies or their antigen-binding fragments can be produced by homologous recombination, for example, as described in U.S. Patent No. 5,204,244, which is specifically incorporated herein by reference. Conventional procedures can be used (for example, by using oligonucleotide probes that can specifically bind to genes encoding antibody heavy chains and light chains) to easily separate and sequence the DNA encoding anti-CD157 antibodies or their antigen-binding fragments. Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: signal sequence and origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences, for example, as described in U.S. Patent No. 5,534,615 issued on July 9, 1996, and it is specifically incorporated herein by reference.

[0214] Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryotes, yeast or higher eukaryotic cells. Prokaryotes suitable for this purpose include true bacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia (Escherichia, for example, Escherichia coli (E. coli)), Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella (Klebsiella), Proteus (Proteus), Salmonella (Salmonella, for example, Salmonella typhimurium (Salmonella typhimurium)), Serratia (Serratia, for example, Serratia marcescens (Serratia marcescans) and Shigella, as well as Bacilli (e.g., B. subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710 published on April 12, 1989), Pseudomonas (e.g., Pseudomonas aeruginosa) and Streptomyce. A preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains (e.g., E. coli B, E. coli X1776 (ATCC 31,537) and E. coli W3110 (ATCC 27,325)) are also suitable. These examples are illustrative only and not limiting.

[0215] In addition to prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are also suitable cloning or expression hosts for anti-CD157 antibody or antigen-binding fragment encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used of the lower eukaryotic host microorganisms. Many other genera, species and strains are generally available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesia (EP 244,234); Neurosporacrassa: Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger.

[0216] Host cells suitable for expressing anti-CD157 antibodies or their antigen-binding fragments (e.g., glycosylated anti-CD157 antibodies or their antigen-binding fragments) are derived from multicellular organisms. Examples of invertebrate cells include plants and insect cells. A variety of baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells from hosts (e.g., Spodoptera frugiperda (caterpillars), Aedes aegypti (mosquitoes), Aedes albopictus (mosquitoes), Drosophila melanogaster (fruit flies) and Bombyx mori (bombyx mori)). Various virus strains for transfection are publicly available, for example, the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and according to the present technology, these viruses can be used as viruses herein, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0217] Suitable host cells for expressing anti-CD157 antibodies or antigen-binding fragments thereof may also include vertebrate cells (eg, mammalian cells). Vertebrate cells may be propagated in culture (tissue culture). Examples of useful mammalian host cell lines include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 cells or subcloned 293 cells for suspension culture growth, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human hepatocytes (HepG2, HB8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cell line (Hep G2).

[0218] Host cells can be transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0219] Host cells used to produce the antibodies or antigen-binding fragments thereof herein can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle Medium ((DMEM), Sigma) are suitable for culturing host cells. In addition, Ham et al., Meth. Enz. 58: 44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or any medium described in U.S. Patent Reissue No. 30,985 can be used as a culture medium for host cells. Any of these media can be supplemented with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN TM ), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements known to those skilled in the art at appropriate concentrations may also be included. The culture conditions (e.g., temperature, pH, etc.) are those previously used for the host cell selected for expression and will be apparent to the skilled person.

[0220] When recombinant techniques are used, the antibody or its antigen-binding fragment can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the antibody is produced intracellularly, as a first step, the particulate debris (host cell or lysed debris) is removed, for example by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163-167 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, the cell paste is thawed for about 30 min in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. In the case where the antibody is secreted into the culture medium, the supernatant from such an expression system is usually first concentrated using a commercially available protein concentration filter (e.g., Amicon or Millipore Pellicon ultrafiltration device). Protease inhibitors (e.g., PMSF) can be included in any of the preceding steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

[0221] The antibody or antigen-binding fragment composition prepared from the cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify human heavy chain-based antibodies (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand is attached is most commonly agarose, but other matrices are also available. Mechanically stable matrices (e.g., controlled pore glass or poly(styrenedivinyl)benzene) achieve faster flow rates and shorter processing times than can be achieved with agarose. In the case of antibodies containing C H3 In the case of a domain, Bakerbond ABX.TM. resin (JTBaker, Phillipsburg, NJ) can be used for purification. Other protein purification techniques, such as ion exchange column fractionation, ethanol precipitation, reversed phase HPLC, silica chromatography, heparin SEPHAROSE TM Chromatography, anion or cation exchange resin chromatography (eg, polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0222] Following any preliminary purification steps, the mixture comprising the antibody or antigen-binding fragment thereof of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer having a pH of about 2.5-4.5 and can be performed at a low salt concentration (e.g., about 0-0.25 M salt).

[0223] Drug formulation, dosage and route of administration

[0224] The present technology provides anti-CD157 antibodies or antigen-binding fragments thereof and related compositions, which can be used, for example, to eliminate cells expressing CD157 from the body, and to identify and quantify the number of cells expressing CD157 in a tissue sample.

[0225] The therapeutic methods and compositions of the present technology may be referred to as "CD157-based" to indicate that these therapies can alter the relative or absolute numbers of undesirable or toxic CD157-expressing cells (e.g., lymphoma or autoimmune B lymphocytes).

[0226] For example, one way to control the amount of undesirable CD157 expressing cells in a patient is to provide a composition comprising one or more anti-CD157 antibodies to produce cytotoxic activity against CD157 expressing cells.

[0227] Anti-CD157 antibodies or their antigen-binding fragments can be formulated into pharmaceutical compositions for a variety of purposes, including treatment of disease, illness or physical trauma. Pharmaceutical compositions comprising one or more anti-CD157 antibodies or their antigen-binding fragments can be used to administer the pharmaceutical compositions herein to patients in need, and according to one embodiment of the technology, a kit comprising such a device is provided. Such devices and kits can be designed for conventional administration of the pharmaceutical compositions herein, including self-administration.

[0228] The antibodies or antigen-binding fragments thereof having the desired degree of purity can be prepared by mixing with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16 th edition, Osol, A. Ed. (1980)) to prepare therapeutic preparations of antibodies or antigen-binding fragments thereof in the form of lyophilized preparations or aqueous solutions for storage. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the doses and concentrations employed and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN®. TM 、PLURONICS TM or polyethylene glycol (PEG).

[0229] The formulations herein may also contain more than one active compound as required for the particular indication being treated, preferably those compounds having complementary activities and which do not adversely affect each other. Such molecules are suitably present in combination in amounts effective for the intended purpose.

[0230] The active ingredients can also be encapsulated in microcapsules, for example, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions prepared by coacervation techniques or interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules, respectively. Such techniques have been described in Remington's Pharmaceutical Sciences 16. th ed., Osol, A. Ed. (1980).

[0231] Preparations for in vivo administration are generally sterile. This can be achieved, for example, by filtration through sterile filtration membranes.

[0232] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody or antigen-binding fragment thereof in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or polyvinyl alcohol), polylactic acid (U.S. Pat. No. 3,773,919), L-glutamic acid and γ Copolymers of ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (e.g. Lupron (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid). Although polymers (e.g., ethylene-vinyl acetate and lactic acid-glycolic acid) are able to release molecules for more than 100 days, some hydrogels release proteins for shorter periods of time. When the encapsulated antibody or its antigen-binding fragment remains in the body for a longer time, it may denature or aggregate due to exposure to a humid environment at 37°C, thereby losing biological activity, and the immunogenicity may change. Depending on the mechanism involved, a rational stabilization strategy can be formulated. For example, if the aggregation mechanism is found to be the formation of intermolecular SS bonds through thiol-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling the moisture content, using appropriate additives, and developing specific polymer matrix compositions.

[0233] For therapeutic applications, the anti-CD157 antibodies or antigen-binding fragments thereof provided herein are administered to mammals (e.g., humans) in a pharmaceutically acceptable dosage form (e.g., the above dosage forms), including intravenous administration as a bolus or continuous infusion over a period of time, or administration to humans via intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical or inhalation routes.

[0234] For the prevention or treatment of disease, the appropriate dosage of the antibody or antigen-binding fragment thereof will depend on the type of disease to be treated (as defined above), the severity and course of the disease, whether the antibody is being administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

[0235] Depending on the type and severity of the disease, an antibody of about 1 μg / kg to about 50 mg / kg (e.g., 0.1-20 mg / kg) can be an initial candidate dose for administration to a patient, for example, by one or more separate administrations, or by continuous infusion. Depending on the above factors, a typical daily or weekly dose may be between about 1 μg / kg to about 20 mg / kg or more. For repeated administration over several days or longer, depending on the condition, the treatment is repeated until the desired disease symptom suppression occurs. However, other dosing regimens may also be useful. The progress of this therapy is easily monitored by conventional techniques and assays (including, for example, radiography). Detection methods for determining CD157 levels in body fluids or tissues using antibodies can be used to optimize patient exposure to therapeutic antibodies.

[0236] In some embodiments, the composition comprising the anti-CD157 antibody or its antigen binding fragment herein (e.g., mAb interfering with CD157 activity) is administered as a monotherapy, and in some embodiments, the composition comprising the anti-CD157 antibody or its antigen binding fragment is administered as part of a combination therapy. In some cases, the antibody or its antigen binding fragment can be continuously administered or co-administered with another antibody or its antigen binding fragment effective for these purposes (e.g., a chemotherapeutic drug for treating cancer or microbial infection) to improve the effectiveness of the antibody or its antigen binding fragment in preventing or treating the disease. In other cases, the anti-CD157 antibody or its antigen binding fragment can be used to enhance or increase the sensitivity of cells to chemotherapy, thereby achieving efficacy with lower doses and lower toxicity. In addition to administering a composition comprising an antibody or its antigen binding fragment that reduces the number of CD157 expressing cells, some combination therapies also include delivering a second treatment regimen selected from the group consisting of: administering a chemotherapeutic agent, radiotherapy, surgery, and any of the foregoing combinations.

[0237] Such other agents may be present in the composition administered or may be administered alone. In addition, anti-CD157 antibodies or antigen-binding fragments thereof may be appropriately administered continuously or in combination with other agents or modalities (e.g., chemotherapeutic agents or radiotherapy for treating cancer, infection, etc., or immunosuppressive drugs).

[0238] Research and diagnostic (including clinical diagnosis) uses of the anti-CD157 antibodies and antigen-binding fragments thereof provided herein

[0239] Provided herein are diagnostic reagents comprising anti-CD157 antibodies or antigen binding fragments thereof as described herein. For example, anti-CD157 antibodies or antigen binding fragments thereof provided herein can be used to detect and / or purify CD157, for example, CD157 expressed on cells from body fluids or in body fluids or tissues. Also provided herein are methods for detecting CD157. For example, the method may include contacting a sample (e.g., a biological sample known or suspected to contain CD157) with an anti-CD157 antibody or antigen binding fragment thereof provided herein, and if the sample contains CD157, then detecting CD157: anti-CD157 complexes. Also provided herein are reagents comprising anti-CD157 antibodies or antigen binding fragments thereof as described herein and methods for detecting CD157 for research purposes.

[0240] For example, anti-CD157 antibodies can contribute to the diagnostic assay of CD157, for example, detecting its presence in specific cells, tissues or body fluids. Such diagnostic methods can be used for diagnosis (for example, of a hyperproliferative disease or disorder). Therefore, clinical diagnostic uses as well as research uses are included herein.

[0241] In some embodiments, the anti-CD157 antibody or its antigen-binding fragment comprises a detectable marker or label. In some embodiments, the anti-CD157 antibody or its antigen-binding fragment is conjugated to a detectable marker or label. For example, for research and diagnostic applications, the anti-CD157 antibody or its antigen-binding fragment can be labeled with a detectable portion. There are many labels available, which are generally divided into the following categories:

[0242] (a) Radioisotopes, e.g. 35 S. 14 C. 125 I. 3 H and 131 I. For example, antibodies can be labeled with radioactive isotopes using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, NY, Pubs. (1991), and the radioactivity can be measured using scintillation counting.

[0243] (b) Fluorescent labels, such as rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lissamine, phycoerythrin, Texas Red and BrilliantViolet TMFor example, fluorescent labels can be conjugated to antibodies using techniques disclosed in Current Protocols in Immunology (supra). Fluorescence can be quantified using a flow cytometer, an imaging microscope, or a fluorimeter.

[0244] (c) A variety of enzyme-substrate labels are available, and U.S. Patent No. 4,275,149 provides a review of some of them. Enzymes generally catalyze chemical changes in chromogenic substrates, which can be measured using various techniques. For example, enzymes can catalyze color changes in substrates, which can be measured by spectrophotometry. Alternatively, enzymes can change the fluorescence or chemiluminescence of substrates. The techniques for quantitative fluorescence changes are described above. Chemiluminescent substrates produce electronic excitations through chemical reactions, which can then emit light that can be measured (e.g., using a chemiluminometer) or supply energy to a fluorescent receptor. Examples of enzyme labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, peroxidase (e.g., horseradish peroxidase (HRPO)), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies are described in O'Sullivan et al., Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. Van Vunakis), Academic press, New York, 73: 147-166 (1981).

[0245] Examples of enzyme-substrate combinations include, for example:

[0246] (i) horseradish peroxidase (HRP) with catalase as substrate, wherein catalase oxidizes a dye precursor (e.g., o-phenylenediamine (OPD) or 3,3′,5,5′-tetramethylbenzidine hydrochloride (TMB));

[0247] (ii) alkaline phosphatase (AP) with p-nitrophenyl phosphate as a chromogenic substrate; and (iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) or the fluorogenic substrate 4-methylumbelliferyl-β-D-galactosidase.

[0248] A variety of other enzyme-substrate combinations can be used (eg, U.S. Pat. Nos. 4,275,149 and 4,318,980, each of which is incorporated herein by reference).

[0249] In some cases, the label is indirectly conjugated to the antibody or its antigen-binding fragment. Those skilled in the art will understand various techniques for achieving this purpose. For example, the antibody can be conjugated to biotin, and any of the three major categories of labels mentioned above can be conjugated to avidin, and vice versa. Biotin selectively binds to avidin, so the label can be conjugated to the antibody in this indirect manner. Alternatively, in order to achieve indirect conjugation of the label to the antibody, the antibody can be conjugated to a small hapten (e.g., digoxin), and one of the above different types of labels can be conjugated to an anti-hapten antibody (e.g., anti-digoxin antibody). Therefore, indirect conjugation of the label to the antibody can be achieved.

[0250] In some embodiments, the anti-CD157 antibody or antigen-binding fragment thereof need not be labeled, and its presence can be detected, for example, using a labeled antibody that binds to the anti-CD157 antibody.

[0251] In some embodiments, the anti-CD157 antibody or its Fab herein is fixed on a solid support or substrate. In some embodiments, the anti-CD157 antibody or its Fab herein is fixed on a solid support in a non-diffusion manner (for example, the anti-CD157 antibody or its Fab does not break away from the solid support). The solid support or substrate can be any physically separable solid, and the anti-CD157 antibody or its Fab can be directly or indirectly attached thereto, including but not limited to: the surface provided by microarray and hole, and particles, such as beads (for example, paramagnetic beads, magnetic beads, microbeads, nanobeads), microparticles and nanoparticles. Solid supports can also include, for example, chips, columns, optical fibers, wipes, filters (e.g., planar filters), one or more capillaries, glass and modified or functionalized glass (e.g., controlled pore glass (CPG)), quartz, mica, diazotized membranes (paper or nylon), polyoxymethylene, cellulose, cellulose acetate, paper, ceramics, metals, metalloids, semiconductor materials, quantum dots, coated beads or particles, other chromatographic materials, magnetic particles; plastics (including acrylic resins, polystyrene, copolymers of styrene or other materials, polybutylene, polyurethane, TEFLON TM , polyethylene, polypropylene, polyamide, polyester, polyvinylidene fluoride (PVDF), etc.), polysaccharides, nylon or nitrocellulose, resins, silicon or silicon-based materials (including silicon, silica gel and modified silicon), Carbon, metal (e.g., steel, gold, silver, aluminum, silicon, and copper), inorganic glass, conductive polymers (including polymers such as polypyrrole and polyindole); microstructured or nanostructured surfaces, such as nucleic acid tile arrays, nanotubes, nanowires, or nanoparticles decorated surfaces; or porous surfaces or gels, such as methacrylates, acrylamides, sugar polymers, cellulose, silicates, or other fibers or chain polymers. In some embodiments, the solid support or substrate can be coated with a passive or chemically derived coating with any number of materials (including polymers such as dextran, acrylamide, gelatin, or agarose). Beads and / or particles can be free, or connected to another (e.g., sintered). In some embodiments, the solid support or substrate can be a collection of particles. In some embodiments, the particles can include silicon, and the silicon can include silicon dioxide. In some embodiments, the silicon can be porous, and in certain embodiments, the silicon can be non-porous. In some embodiments, the particles further include an agent that imparts paramagnetism to the particles. In certain embodiments, the reagent comprises a metal, and in certain embodiments, the reagent is a metal oxide (e.g., iron or iron oxide, wherein the iron oxide contains a mixture of Fe2+ and Fe3+). The anti-CD157 antibody or antigen-binding fragment thereof can be attached to a solid support by covalent bonds or non-covalent interactions, and can be attached to a solid support directly or indirectly (e.g., through an intermediary, such as a spacer molecule or biotin).

[0252] The antibodies or antigen-binding fragments thereof provided herein can be used in any known assay method, such as flow cytometry, immunohistochemistry, immunofluorescence, mass cytometry (e.g., Cytof instrument), competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

[0253] Flow cytometry and mass spectrometry flow cytometry analysis generally involve the use of a single primary antibody to specifically identify the presence of target molecules expressed on the surface of a single cell dispersion suspension. Dispersed cells are generally obtained from biological fluid samples (e.g., blood), but can also be obtained from single cell dispersions prepared from solid tissue samples (e.g., spleen or tumor biopsy). A primary antibody can be directly conjugated to a detectable portion, for example, a fluorophore (e.g., phycoerythrin) for flow cytometry or a heavy metal chelate for mass spectrometry flow cytometry. Alternatively, a primary antibody can be unlabeled, or labeled with an undetectable label (e.g., biotin), and then detected by a detectably labeled secondary antibody, which specifically identifies a primary antibody itself or a label on a primary antibody. Then, the labeled cells are analyzed in an instrument (e.g., flow cytometer, mass spectrometry flow cytometer, fluorescence microscope, or bright field optical microscope) capable of single cell detection to identify a single cell expressing a target identified by a primary antibody in a dispersed population or tissue sample. A detailed description of the technical basis and practical applications of flow cytometry principles can be found, for example, in Shapiro, Practical Flow Cytometry, 4 th Edition,Wiley,2003.

[0254] Sandwich assays involve the use of two antibodies, each of which can bind to different immunogenic parts or epitopes of the detected protein. In sandwich assays, the test sample analyte is bound to the first antibody fixed on a solid support, after which the second antibody is bound to the analyte, thereby forming an insoluble three-part complex. See, for example, U.S. Patent No. 4,376,110. The second antibody itself can be labeled with a detectable portion (direct sandwich assay), or an anti-immunoglobulin antibody labeled with a detectable portion can be used to measure (indirect sandwich assay). For example, a sandwich assay is an ELISA assay, in which case the detectable portion is an enzyme. In cell ELISA, antibodies that are first attached to a support and recognize different cell surface proteins can be used to attach a target cell population to a solid support. These primary antibodies capture cells to a support. Then, CD157 on the cell surface can be detected by adding anti-CD157 antibodies to the captured cells and detecting the amount of CD157 antibodies attached to the cells. In some cases, fixed and permeabilized cells can be used, in which case surface CD157 and intracellular CD157 can be detected.

[0255] For immunohistochemistry, blood or tissue samples may be fresh or frozen, or may be embedded in paraffin and fixed with a preservative such as formalin.

[0256] The antibodies or antigen-binding fragments thereof herein can also be used in in vivo diagnostic assays. Typically, antibodies are labeled with a radionuclide (e.g. 111 In, 99Tc, 14 C. 131 I. 125 I. 3 H. 32 P or 35 S), allowing the localization of bound target molecules using immunoscintigraphy.

[0257] Detection of CD157 in immune cells

[0258] Provided herein are antibodies or their antigen binding fragments and methods for detecting CD157 in immune cells.Detecting CD157 in immune cells can refer to detecting on the surface of immune cells (e.g., by surface staining) and / or inside immune cells (e.g., by intracellular staining).In some embodiments, antibodies or their antigen binding fragments and methods for detecting CD157 in heterogeneous immune cell populations are provided.Heterogeneous immune cell populations can include two or more types of immune cells.For example, heterogeneous immune cell populations can include two or more B cells, plasmacytoid dendritic cells (pDC), lymphocytes, leukocytes, T cells, monocytes, macrophages, neutrophils, myeloid dendritic cells (mDC), innate lymphocytes, mast cells, eosinophils, basophils, natural killer cells, etc.In some embodiments, heterogeneous immune cell populations include peripheral blood mononuclear cells (PBMC), which can include, for example, T cells, B cells, natural killer cells and monocytes.

[0259] Typically, cells are contacted with an anti-CD157 antibody or antigen-binding fragment thereof as described herein (e.g., in a flow cytometry assay described in the Examples; or in any suitable protein or cell detection assay). In some embodiments, an anti-CD157 antibody or antigen-binding fragment thereof as described herein detects significant levels of CD157 in certain immune cells. An anti-CD157 antibody or antigen-binding fragment thereof as described herein can detect significant levels of CD157 in certain immune cells, while no significant levels of CD157 are detected in other immune cells. The level of CD157 detection in certain immune cells can vary depending on certain factors (e.g., the type of detection assay, the type of detection reagent (e.g., the type of dye), antibody concentration, donor cell variability, etc.).

[0260] In some of any embodiments, any antibody or antigen-binding fragment thereof provided herein can be used to characterize single cells by measuring gene expression levels and cellular proteins. Such known single-cell sequencing platforms suitable for integration with the antibodies or antigen-binding fragments thereof described herein include Drop-seq methods, including but not limited to microfluidics, plate-based or microwell, Seq-WellT M Methods and adaptation of basic protocols and InDropTM method.

[0261] In another embodiment, a single cell sequencing platform suitable for integration with the antibodies or antigen-binding fragments thereof described herein is the 10x genomics single cell 3′ solution or single cell V(D)J solution, which can be run on a Chromium controller or a dedicated Chromium single cell controller. Other suitable sequencing methods include Wafergen iCell8 TM Methods, Microwell-seq methods, Fluidigm CI TM Methods and equivalent single cell products. Still other known sequencing solutions used with the antibodies or antigen-binding fragments thereof described herein include BD Resolve TM Single-cell analysis platform and ddSeq (from SureCellT M WTA 3′Library Prep Kit for the ddSEQ TM System, 2017, Pub. No. 1070-2016-014-B, Illumina Inc., Bio-Rad Laboratories, Inc.). In some other embodiments, the antibodies or antigen-binding fragments thereof described herein can be used in a combined index-based method (sci-RNA-seq TM SPLiT-seq TM Methods) and spatial transcriptomics, or equivalent spatially resolved sequencing methods. The methods and compositions described herein can also be used as an additional information layer for standard index sorting (FACS) and mRNA sequencing-based methods.

[0262] In some of any of the embodiments, any of the antibodies or antigen-binding fragments thereof described herein can be used to detect the presence, absence, or amount of various nucleic acids, proteins, targets, oligonucleotides, amplification products, and barcodes described herein.

[0263] In some of any embodiments, the detection comprises hybridization of a detectable moiety with an antibody or an antigen-binding fragment thereof. In some of any embodiments, the sample is contacted with a second antibody. In some of any embodiments, the second antibody is an antibody comprising a detectable moiety. In some of any embodiments, the detectable moiety comprises an oligonucleotide. In some of any embodiments, the detectable moiety comprises a fluorescent label. In some of any embodiments, the measurement comprises sequencing. In some of any embodiments, the detectable moiety comprises immunofluorescence. In some of any embodiments, the sample is a formalin-fixed paraffin-embedded sample. In some of any embodiments, the sample comprises a cell. In some of any embodiments, the sample comprises a tissue sample.

[0264] Detecting significant levels of CD157 can refer to a particular signal-to-noise ratio (S:N) (eg, a threshold or range) measured in a flow cytometry assay.

[0265] In some embodiments, significant levels of CD157 are detected in immune cells (eg, lymphocytes).

[0266] Kits incorporating the anti-CD157 antibodies or antigen-binding fragments thereof provided herein

[0267] The anti-CD157 antibody or its antigen-binding fragment herein can be provided in a kit, for example, a packaged combination of a predetermined amount of reagent and instructions for use (e.g., instructions for performing diagnostic assays; instructions for performing laboratory assays). In some embodiments, the kit is a diagnostic kit configured to detect CD157 in a sample (e.g., a biological sample). In the case where the anti-CD157 antibody or its antigen-binding fragment is labeled with a fluorophore, the kit may include an isotype negative control unrelated antibody of the same type to control the non-specific binding of the anti-CD157 antibody or its antigen-binding fragment. In the case where the anti-CD157 antibody or its antigen-binding fragment is labeled with an enzyme, the kit may include substrates and cofactors required for the enzyme (e.g., substrate precursors providing a detectable chromophore or fluorophore). Additional additives may be included, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), etc. The relative amounts of various reagents may vary widely to provide the concentration of the reagent in the solution, thereby substantially optimizing the sensitivity of the assay. In some cases, the reagent may be provided as a dry powder (e.g., lyophilized powder), including an auxiliary material that will provide a reagent solution with an appropriate concentration when dissolved.

[0268] Products

[0269] In another aspect of the present technology, there is provided an article containing materials that can be used to treat or diagnose the conditions described herein. The article may include a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container may be formed of a variety of materials (e.g., glass or plastic). The container may contain a composition effective for treating the condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The active anti-CD157 antibody or its antigen-binding fragment in the composition may be an anti-CD157 antibody. The label on or attached to the container indicates that the composition is used to treat or diagnose the selected condition. The article may further include a second container, which includes a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and dextrose solution; and may further include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0270] definition

[0271] "Acceptor human framework" generally refers to a framework comprising an amino acid sequence of a heavy chain variable domain (VH) framework or a light chain variable domain (VL) framework derived from a human immunoglobulin framework or a human consensus framework, as defined herein. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or may contain amino acid sequence changes. In some embodiments, the number of framework amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VH and / or VL acceptor human framework is identical in sequence to the VH and / or VL human immunoglobulin framework amino acid sequence or a human consensus framework amino acid sequence.

[0272] "Framework" or "FR" generally refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1; FR2; FR3; and FR4. Thus, HVR and FR sequences generally appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0273] "Human consensus framework" generally refers to a framework that represents the most frequently occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup such as Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols.1-3. In some embodiments, for VL, the subgroup is subgroup κI, such as Kabat et al. (supra). In some embodiments, for VH, the subgroup is subgroup III, such as Kabat et al. (supra).

[0274] The term "hypervariable region" or "HVR" generally refers to each region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops ("hypervariable loops"). Typically, a natural four-chain antibody comprises six HVRs: three in VH (H1, H2, H3), and three in VL (L1, L2, L3). HVRs generally comprise amino acid residues from hypervariable loops and / or "complementarity determining regions" (CDRs), the latter having the highest sequence variability and / or being involved in antigen recognition.

[0275] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using a number of well-known schemes, including those described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5 thEd. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme), MacCallum et al., J. Mol. Biol. 262: 732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745, ("Contact" numbering scheme), Martin et al., Proc. Natl. Acad. Sci., 86: 9268-9272 (1989) ("AbM" numbering scheme), Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," DevComp Immunol, 2003 Jan;27(1):55-77 ("IMGT" numbering scheme), and Honegger A and Plückthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun 8;309(3):657-70 ("Aho" numbering scheme).

[0276] The boundaries of a given CDR or FR can vary according to the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering of the Kabat and Chothia schemes is based on the most common antibody region sequence length, and insertion is represented by an inserted letter, for example, "30a", and deletions occur in some antibodies. These two schemes place certain insertions and deletions ("indels") at different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures, and is similar to the Chothia numbering scheme in many respects.

[0277] Table 5 below lists exemplary position boundaries for CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2 and CDRL3 identified by the Kabat, Chothia and Contact schemes, respectively. For CDRH1, the residue numbers are listed using the Kabat and Chothia numbering schemes. FRs are located between CDRs, e.g., FRH1 is located between CDRH1 and CDRH2, etc. It is noteworthy that because the Kabat numbering scheme shown places the insertion at H35A and H35B, the ends of the Chothia CDRH1 loop vary between H32 and H34 when numbered using the Kabat numbering convention shown, depending on the length of the loop.

[0278] Table 5

[0279] CDR Kabat Chothia AbM Contact <![CDATA[CDRH1 (Kabat number 1 )]]> H31--H35B H26--H32..34 H26--H35B H30--H35B <![CDATA[CDRH1 (Chothia number 2 )]]> H31--H35 H26--H32 H26--H35 H30--H35 CDRH2 H50--H65 H52--H56 H50--H58 H47--H58 CDRH3 H95--H102 H95--H102 H95--H102 H93--H101 CDRL1 L24--L34 L24--L34 L24--L34 L30--L36 CDRL2 L50--L56 L50--L56 L50--L56 L46--L55 CDRL3 L89--L97 L89--L97 L89--L97 L89--L96

[0280] 1-Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5 th Ed. Public Health Service, National Institutes of Health, Bethesda. MD

[0281] 2-Al-Lazikani et al., (1997) JMB 273,927-948

[0282] CDRs also contain "specificity determining residues" or "SDRs," which are residues that make contact with specific antigens. Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al. (supra).

[0283] The term "variable region" or "variable domain" generally refers to the domain of an antibody heavy chain or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, VH or VL domains from antibodies that bind to a particular antigen can be used to isolate antibodies that bind to the antigen to screen complementary VL or VH domain libraries, respectively. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0284] "Affinity" generally refers to the overall strength of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can often be expressed in terms of the dissociation constant (K). d ). Affinity can be measured by conventional methods known in the art (including those described herein). Specific illustrative and exemplary embodiments for measuring binding affinity are described elsewhere herein. In some cases, the antibodies herein bind to a target (e.g., CD157) with high affinity, e.g., K d The value does not exceed about 1×10 -7 M; preferably not more than about 1×10 -8 M; and preferably not more than about 5×10 -9 M.

[0285] An "affinity matured" antibody generally refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody that does not have such alterations. Preferably, such alterations result in an improvement in the affinity of the antibody for its target antigen.

[0286] The term "anti-CD157 antibody or antigen-binding fragment thereof" generally refers to a molecule that is or comprises one or more anti-CD157 antibodies, CD157-binding antibody fragments, or CD157-binding antibody derivatives.

[0287] The terms "anti-CD157 antibody" and "antibody that binds to CD157" generally refer to an antibody that can bind to CD157 with sufficient affinity and / or specificity so that the antibody can be used as a research tool, diagnostic agent, and / or therapeutic agent targeting CD157. In some embodiments, the degree of binding of an anti-CD157 antibody (or its antigen-binding fragment) to an unrelated non-CD157 protein is less than about 10% of the binding of the antibody to CD157, for example, as measured by radioimmunoassay (RIA) or Scatchard analysis or surface plasmon resonance (e.g., Biacore). In certain embodiments, the dissociation constant (kD) of the antibody that binds to CD157 is 0.1 μM, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM (e.g., 10 -7 M or less, e.g., 10 -7 M to 10 -13 M). In certain embodiments, the anti-CD157 antibody binds to a CD157 epitope that is conserved between CD157 of different species.

[0288] The term "antibody" as used herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, variable heavy chains (VL) that are capable of specifically binding to an antigen, and variable heavy chains (VL) that are capable of specifically binding to an antigen. H ) region, single-chain antibody fragments (including single-chain variable fragments (scFv)) and single-domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term covers genetically engineered and / or other modified forms of immunoglobulins, such as intracellular antibodies, peptide antibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heterologous conjugate antibodies, multispecific antibodies (e.g., bispecific antibodies), double antibodies, three antibodies and four antibodies, tandem double scFv, tandem three scFv. Unless otherwise indicated, the term "antibody" should be understood to cover its functional antibody fragments. The term also covers complete or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses IgM, IgE, IgA and IgD.

[0289] "Antibody derivatives" generally refer to molecules other than intact antibodies, which contain portions derived from intact antibodies (or antigen-binding fragments thereof) and bind to the antigen to which the intact antibody (or antigen-binding fragment thereof) binds. Examples of antibody derivatives include, but are not limited to, single-chain variable fragments (scFv), diabodies, triabodies, etc., aptamers, single-chain variable fragments, diabodies, triabodies, etc., containing multiple antigen-binding antibody fragments.

[0290] "Antibody fragments" or "antigen-binding antibody fragments" generally refer to molecules other than intact antibodies, which contain a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, and multispecific antibodies formed from antibody fragments.

[0291] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0292] The term "Fc region" generally refers to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0293] An "antibody that binds to the same epitope as a reference antibody" (e.g., an antibody that binds CD157) generally refers to an antibody that blocks binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks binding of the antibody to its antigen by 50% or more in a competition assay.

[0294] The term "chimeric" antibody generally refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0295] "Human antibody" generally refers to an antibody having an amino acid sequence corresponding to an antibody produced by a human or human cell or derived from a non-human source utilizing a human antibody library or other human antibody encoding sequences. This definition of a human antibody specifically excludes "humanized" antibodies comprising non-human antigen binding residues.

[0296] "Humanized" antibodies generally refer to chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, humanized antibodies will include substantially all of at least one and usually two variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of non-human antibodies, and all or substantially all of the FRs correspond to those of human antibodies. Humanized antibodies may optionally include at least a portion of the antibody constant region derived from human antibodies. In some embodiments, humanized antibodies (or their antigen-binding fragments or derivatives) include one or more amino acid substitutions (or deletions or insertions) at the desired position when compared with antibodies derived from the receptor framework region. In some such embodiments, the amino acid residues substituted (or inserted or deleted) at specific positions in people (or other) or other FRs correspond to the amino acid residues at the corresponding positions in the parent antibody (i.e., the non-human antibody from which CDR or HVR is derived). The "humanized form" (e.g., non-human antibody) of an antibody refers to an antibody that has undergone humanization.

[0297] The term "antibody drug conjugate" (ADC) or "immunoconjugate" generally refers to a specific class of antibody-drug conjugates. Here, an "antibody-drug conjugate" is an anti-CD157 antibody or antigen-binding fragment thereof (e.g., an anti-CD157 antibody or CD157 binding fragment or derivative) conjugated to one or more heterologous molecules (including but not limited to cytotoxic agents).

[0298] The term "cytotoxic agent" generally refers to a substance that inhibits or prevents cellular function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At 211 ,I 131 ,I 125 , Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, doxorubicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitors; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and various anti-tumor or anti-cancer agents disclosed below.

[0299] A "diagnostic agent" generally refers to a compound used to perform a diagnostic assay, eg, a target-specific antibody (or antigen binding thereof).

[0300] "Effector function" generally refers to biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0301] An "effective amount" of an antibody or antigen-binding fragment thereof (eg, a pharmaceutical agent) generally refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0302] The term "epitope" generally refers to a specific site on an antigen molecule to which an antibody binds.

[0303] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and generally refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and the daughter cells derived therefrom, regardless of the number of passages. The nucleic acid content of the daughter cells may not be exactly the same as that of the parent cell, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected in the initially transformed cell are included herein.

[0304] "Rabbit antibody" generally refers to an antibody having an amino acid sequence corresponding to an antibody produced by a rabbit or rabbit cells, or an antibody derived from a non-rabbit source utilizing a rabbit antibody library or other rabbit antibody encoding sequence.

[0305] "Immunoconjugate" generally refers to an antibody (or antigen-binding fragment or derivative thereof) conjugated to one or more heterologous molecules (including but not limited to cytotoxic agents). Immunoconjugate is equivalent to the term "antibody drug conjugate" (ADC).

[0306] An "individual" or "patient" or "subject" is typically a mammal. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0307] "Isolated" molecules (e.g., nucleic acids, antibodies) generally refer to molecules that have been separated from components of their original environment (e.g., the natural environment if naturally occurring, or a host cell if exogenously expressed), and are therefore altered from their original environment by human intervention (e.g., "by the hand"). In some embodiments, for example, antibodies are purified to a purity greater than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). An isolated nucleic acid may refer to a nucleic acid molecule contained in a cell that typically contains the nucleic acid molecule, but the nucleic acid molecule is present outside the chromosome or in a chromosomal location different from its natural chromosomal location. In some embodiments, an isolated nucleic acid may contain less non-nucleic acid components (e.g., proteins, lipids) than the amount of components present in the source sample. A composition comprising an isolated nucleic acid may be about 50% to greater than 99% free of non-nucleic acid components. A composition comprising an isolated nucleic acid may be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% free of non-nucleic acid components.

[0308] "Isolated nucleic acid encoding an anti-CD157 antibody" or "isolated polynucleotide encoding an anti-CD157 antibody" generally refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations in a recombinant host cell.

[0309] The term "CD157" generally refers to any natural, mature CD157 produced by the processing of CD157 precursor proteins in cells. Unless otherwise indicated, the term includes CD157 from any vertebrate source, including mammals, such as primates (e.g., humans and cynomolgus monkeys or rhesus monkeys) and rodents (e.g., mice and rats). The term also includes natural variants of CD157, such as splice variants or allelic variants. The exemplary amino acid sequence of human CD157 protein is shown in SEQ ID NO: 37.

[0310] The term "CD157 positive cell" generally refers to any cell expressing CD157 on its surface or intracellular membrane or organelle (e.g., endosome, endoplasmic reticulum, Golgi apparatus, lysosome, etc.). Some cells (e.g., immune cells (e.g., lymphocytes)) show upregulated CD157 expression.

[0311] The term "monoclonal antibody" generally refers to an antibody obtained from a substantially homogeneous antibody group, i.e., the single antibodies comprising the group are identical (e.g., assessed at the level of Ig heavy chain and / or light chain amino acid sequences) and / or bind to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or appearing during the production of monoclonal antibody preparations), which variants are generally present in very small amounts. Unlike polyclonal antibody preparations that generally include different antibodies for different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the modifier "monoclonal" represents the characteristics of an antibody obtained from a substantially homogeneous antibody group, and should not be interpreted as requiring antibodies to be produced by any particular method. For example, the monoclonal antibodies used according to the present technology can be made by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0312] The term "package insert" generally refers to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0313] "Percentage (%) of amino acid sequence identity" relative to a reference polypeptide sequence generally refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing spaces, if necessary, to achieve the maximum percentage of sequence identity, and any conservative substitutions are not considered part of the sequence identity. Alignment for determining percentage of amino acid sequence identity can be achieved in a variety of ways known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0314] The term "pharmaceutical composition" generally refers to a preparation that is in such a form that the biological activity of the active ingredients contained therein is effective, and that contains no additional components that are unacceptably toxic to a subject to which the preparation would be administered.

[0315] "Pharmaceutically acceptable carrier" generally refers to a component of a pharmaceutical preparation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0316] As used herein, "treatment" (and grammatical variants thereof, such as "treat" or "treating") generally refers to a clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed for prevention or during clinical pathology. The desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies herein are used to delay the development of the disease or slow the progression of the disease.

[0317] The term "vector" generally refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0318] Exemplary embodiments

[0319] Exemplary embodiments provided in accordance with the presently disclosed subject matter include:

[0320] 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to CD157, wherein the isolated antibody comprises:

[0321] a) a heavy chain variable region comprising:

[0322] (i) Heavy chain complementarity determining region 1 (CDRH1), which comprises X1X2X3X4X5X6YX8X9X 10 (SEQ ID NO: 30), wherein X1 is G or no amino acid, X2 is F or no amino acid, X3 is S or no amino acid, X4 is L or no amino acid, X5 is T or no amino acid, X6 is S or N, X8 is H or D, X9 is V or M, and X 10 is S or A;

[0323] (ii) Heavy chain complementarity determining region 2 (CDRH2), which comprises X1IX3X4X5GGSTX 10 YX 12 X 13 X 14 X 15 KX 17 (SEQ ID NO: 31), wherein X1 is S or no amino acid, X3 is I or S, X4 is W or I, X5 is T or R, X6 is T or R, 10 A or Y, X 12 N or R, X 13 For S or D, X 14For L or S, X 15 is L or V, and X 17 is S or G;

[0324] (iii) heavy chain complementarity determining region 3 (CDRH3), which comprises X1X2X3X4X s X6X7X8FDY (SEQ ID NO: 32), wherein X1 is G or no amino acid, X2 is T or no amino acid, X3 is S or D, X4 is I or Y, X5 is T or Y, X6 is P or Y, X7 is T or D, and X8 is F or Y: and

[0325] b) a light chain variable region comprising:

[0326] (iv) light chain complementary determining region 1 (CDRL1), which comprises KX2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 (SEQ ID NO: 33), wherein X2 is R or A, X3 is S or G, X4 is T or R, X5 is G or N, X6 is N or I, X7 is F or N, X8 is G or S, X9 is N, S or Y, X 10 N or L, X 11 is Y or A, X 12 is V or no amino acid, and X 11 N or no amino acid;

[0327] (v) a light chain complementary determining region 2 (CDRL2) comprising a sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 34), wherein X1 is R or N, X2 is D or A, X3 is D or N, X4 is K or S, X5 is R or L, X6 is P or Q, and X7 is D or T; and

[0328] (vi) a light chain complementary determining region 3 (CDRL3) comprising a sequence of QX2YX4SX6X7X8X9 (SEQ ID NO: 35), wherein X2 is S or Q, X4 is S or N, X6 is G or W, X7 is I or T, X8 is V or N, and X9 is T or no amino acid.

[0329] 2. The isolated antibody or antigen-binding fragment thereof of embodiment 1, wherein:

[0330] (1) a CDRH1 comprising the sequence set forth in any one of SEQ ID NOs: 16, 17, and 18, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 16, 17, and 18;

[0331] (2) the CDRH2 comprises the sequence set forth in any one of SEQ ID NOs: 19 and 20, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 19 and 20; and

[0332] (3) CDRH3 comprises the sequence set forth in any one of SEQ ID NOs:21 and 22, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs:21 and 22.

[0333] 3. The isolated antibody or antigen-binding fragment thereof of embodiment 1 or 2, wherein:

[0334] (1) CDRH1 comprises a sequence as shown in any one of SEQ ID NOs: 16, 17, and 18, or a sequence having an amino acid substitution relative to the sequence of any one of SEQ ID NOs: 16, 17, and 18;

[0335] (2) CDRH2 comprises the sequence shown in any one of SEQ ID NOs: 19 and 20, or a sequence having one, two or three amino acid substitutions relative to the sequence of any one of SEQ ID NOs: 19 and 20; and

[0336] (3) CDRH3 comprises the sequence shown in any one of SEQ ID NOs: 21 and 22, or a sequence having one or two amino acid substitutions relative to the sequence shown in any one of SEQ ID NOs: 21 and 22.

[0337] 4. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 3, wherein:

[0338] (1) CDRH1 comprises a sequence as shown in any one of SEQ ID NOs: 16, 17, and 18;

[0339] (2) CDRH2 comprises the sequence shown in any one of SEQ ID NOs: 19 and 20; and

[0340] (3) CDRH3 comprises the sequence shown in any one of SEQ ID NOs: 21 and 22.

[0341] 5. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 4, wherein CDRH1 comprises the sequence shown in SEQ ID NO: 16, CDRH2 comprises the sequence shown in SEQ ID NO: 19, and CDRH3 comprises the sequence shown in SEQ ID NO: 21.

[0342] 6. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 4, wherein CDRH1 comprises the sequence shown in SEQ ID NO: 17, CDRH2 comprises the sequence shown in SEQ ID NO: 19, and CDRH3 comprises the sequence shown in SEQ ID NO: 21.

[0343] 7. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 4, wherein CDRH1 comprises the sequence shown in SEQ ID NO: 18, CDRH2 comprises the sequence shown in SEQ ID NO: 20, and CDRH3 comprises the sequence shown in SEQ ID NO: 22.

[0344] 8. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 7, wherein:

[0345] (1) CDRL1 comprises the sequence set forth in any one of SEQ ID NOs: 23, 24 and 25, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 23, 24 and 25;

[0346] (2) CDRL2 comprises the sequence set forth in any one of SEQ ID NOs: 26 and 27, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 26 and 27; and

[0347] (3) CDRL3 comprises the sequence shown in any one of SEQ ID NOs: 28 and 29, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 28 and 29.

[0348] 9. The isolated antibody or antigen-binding fragment thereof of embodiment 1 or 8, wherein:

[0349] (1) CDRL1 comprises the sequence shown in any one of SEQ ID NOs: 23, 24, 25, or a sequence having one or two amino acid substitutions relative to the sequence of any one of SEQ ID NOs: 23, 24, 25;

[0350] (2) the CDRL2 comprises the sequence shown in any one of SEQ ID NOs: 26 and 27, or a sequence having an amino acid substitution relative to the sequence of any one of SEQ ID NOs: 26 and 27; and

[0351] (3) CDRL3 comprises the sequence shown in any one of SEQ ID NOs: 28 and 29, or a sequence having one or two amino acid substitutions relative to the sequence shown in any one of SEQ ID NOs: 28 and 29.

[0352] 10. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 9, wherein:

[0353] (1) CDRL1 comprises the sequence shown in any one of SEQ ID NOs: 23, 24, and 25;

[0354] (2) CDRL2 comprises the sequence shown in any one of SEQ ID NOs: 26 and 27; and

[0355] (3) CDRL3 comprises the sequence shown in any one of SEQ ID NOs: 28 and 29.

[0356] 11. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 10, wherein CDRL1 comprises the sequence shown in SEQ ID NO: 23 or 24, CDRL2 comprises the sequence shown in SEQ ID NO: 26, and CDRL3 comprises the sequence shown in SEQ ID NO: 28.

[0357] 12. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 10, wherein CDRL1 comprises the sequence shown in SEQ ID NO: 23, CDRL2 comprises the sequence shown in SEQ ID NO: 26, and CDRL3 comprises the sequence shown in SEQ ID NO: 28.

[0358] 13. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 10, wherein CDRL1 comprises the sequence shown in SEQ ID NO:24, CDRL2 comprises the sequence shown in SEQ ID NO:26, and CDRL3 comprises the sequence shown in SEQ ID NO:28.

[0359] 14. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 10, wherein CDRL1 comprises the sequence shown in SEQ ID NO: 25, CDRL2 comprises the sequence shown in SEQ ID NO: 27, and CDRL3 comprises the sequence shown in SEQ ID NO: 29.

[0360] 15. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 14, wherein:

[0361] (1) a CDRH1 comprising the sequence set forth in any one of SEQ ID NOs: 16, 17, and 18, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 16, 17, and 18;

[0362] (2) a CDRH2 comprising the sequence set forth in any one of SEQ ID NOs: 19 and 20, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 19 and 20;

[0363] (3) a CDRH3 comprising the sequence set forth in any one of SEQ ID NOs:21 and 22, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs:21 and 22;

[0364] (4) CDRL1 comprises the sequence set forth in any one of SEQ ID NOs: 23, 24, and 25, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 23, 24, and 25;

[0365] (5) CDRL2 comprises the sequence set forth in any one of SEQ ID NOs: 26 and 27, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 26 and 27; and

[0366] (6) CDRL3 comprises the sequence shown in any one of SEQ ID NOs: 28 and 29, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 28 and 29.

[0367] 16. The isolated antibody or antigen-binding fragment thereof of embodiments 1 to 15, wherein:

[0368] (1) CDRH1 comprises the sequence shown in any one of SEQ ID NOs: 16, 17 and 18, or a sequence having an amino acid substitution relative to the sequence of any one of SEQ ID NOs: 16, 17 and 18:

[0369] (2) CDRH2 comprises the sequence shown in any one of SEQ ID NOs: 19 and 20, or a sequence having one, two or three amino acid substitutions relative to the sequence of any one of SEQ ID NOs: 19 and 20:

[0370] (3) CDRH3 comprises the sequence shown in any one of SEQ ID NOs: 21 and 22, or a sequence having one or two amino acid substitutions relative to the sequence of any one of SEQ ID NOs: 21 and 22:

[0371] (4) CDRL1 comprises the sequence shown in any one of SEQ ID NOs: 23, 24 and 25, or a sequence having one or two amino acid substitutions relative to the sequence of any one of SEQ ID NOs: 23, 24 and 25:

[0372] (5) CDRL2 comprises the sequence shown in any one of SEQ ID NOs: 26 and 27, or a sequence having an amino acid substitution relative to the sequence of any one of SEQ ID NOs: 26 and 27; and

[0373] (6) CDRL3 comprises the sequence shown in any one of SEQ ID NOs: 28 and 29, or a sequence having one or two amino acid substitutions relative to the sequence shown in any one of SEQ ID NOs: 28 and 29.

[0374] 17. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 16, wherein:

[0375] (1) CDRH1 comprises a sequence shown in any one of SEQ ID NOs: 16, 17, and 18;

[0376] (2) CDRH2 comprises the sequence shown in any one of SEQ ID NOs: 19 and 20;

[0377] (3) CDRH3 comprises the sequence shown in any one of SEQ ID NOs: 21 and 22;

[0378] (4) CDRL1 comprises the sequence shown in any one of SEQ ID NOs: 23, 24, and 25;

[0379] (5) CDRL2 comprises the sequence shown in any one of SEQ ID NOs: 26 and 27; and

[0380] (6) CDRL3 comprises the sequence shown in any one of SEQ ID NOs: 28 and 29.

[0381] 18. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 17, wherein CDRH1 comprises the sequence shown in SEQ ID NO: 16, CDRH2 comprises the sequence shown in SEQ ID NO: 19, and CDRH3 comprises the sequence shown in SEQ ID NO: 21, CDRL1 comprises the sequence shown in SEQ ID NO: 23, CDRL2 comprises the sequence shown in SEQ ID NO: 26, and CDRL3 comprises the sequence shown in SEQ ID NO: 28.

[0382] 19. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 17, wherein CDRH1 comprises the sequence shown in SEQ ID NO: 17, CDRH2 comprises the sequence shown in SEQ ID NO: 19, and CDRH3 comprises the sequence shown in SEQ ID NO: 21, CDRL1 comprises the sequence shown in SEQ ID NO: 24, CDRL2 comprises the sequence shown in SEQ ID NO: 26, and CDRL3 comprises the sequence shown in SEQ ID NO: 28.

[0383] 20. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 17, wherein CDRH1 comprises the sequence shown in SEQ ID NO: 18, CDRH2 comprises the sequence shown in SEQ ID NO: 20, and CDRH3 comprises the sequence shown in SEQ ID NO: 22, CDRL1 comprises the sequence shown in SEQ ID NO: 25, CDRL2 comprises the sequence shown in SEQ ID NO: 27, and CDRL3 comprises the sequence shown in SEQ ID NO: 29.

[0384] 21. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 20, wherein the heavy chain variable region comprises at least 90% identity to the sequence shown in any one of SEQ ID NOs: 1 and 2.

[0385] 22. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 21, wherein the heavy chain variable region comprises at least 90% identity to the sequence shown in SEQ ID NO: 1.

[0386] 23. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 21, wherein the heavy chain variable region comprises at least 90% identity to the sequence of SEQ ID NO: 2.

[0387] 24. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 23, wherein the heavy chain variable region comprises the sequence of any one of SEQ ID NOs: 1 and 2.

[0388] 25. The isolated antibody or antigen-binding fragment thereof of embodiment 24, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 1.

[0389] 26. The isolated antibody or antigen-binding fragment thereof of embodiment 24, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 2.

[0390] 27. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 26, wherein the light chain variable region comprises at least 90% identity to the sequence shown in any one of SEQ ID NOs: 3, 4 and 5.

[0391] 28. The isolated antibody or antigen-binding fragment thereof of embodiment 27, wherein the light chain variable region comprises at least 90% identity to the sequence shown in SEQ ID NO:3.

[0392] 29. The isolated antibody or antigen-binding fragment thereof of embodiment 27, wherein the light chain variable region comprises at least 90% identity to the sequence shown in SEQ ID NO:4.

[0393] 30. The isolated antibody or antigen-binding fragment thereof of embodiment 27, wherein the light chain variable region comprises at least 90% identity to the sequence shown in SEQ ID NO:5.

[0394] 31. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 27, wherein the light chain variable region comprises the sequence shown in any one of SEQ ID NOs: 3, 4 and 5.

[0395] 32. The isolated antibody or antigen-binding fragment thereof of embodiment 31, wherein the light chain variable region comprises the sequence shown in SEQ ID NO:3.

[0396] 33. The isolated antibody or antigen-binding fragment thereof of embodiment 31, wherein the light chain variable region comprises the sequence shown in SEQ ID NO:4.

[0397] 34. The isolated antibody or antigen-binding fragment thereof of embodiment 31, wherein the light chain variable region comprises the sequence shown in SEQ ID NO:5.

[0398] 35. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 34, wherein the heavy chain variable region comprises at least 90% identity to the sequence shown in any one of SEO ID NOs: 1 and 2, and wherein the light chain variable region comprises at least 90% identity to the sequence shown in any one of SEQ ID NOs: 3, 4 and 5.

[0399] 36. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 35, wherein the heavy chain variable region comprises at least 90% identity to the sequence shown in SEQ ID NO: 1, and wherein the light chain variable region comprises at least 90% identity to the sequence shown in SEQ ID NO: 3.

[0400] 37. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 35, wherein the heavy chain variable region comprises at least 90% identity to the sequence shown in SEQ ID NO: 1, and wherein the light chain variable region comprises at least 90% identity to the sequence shown in SEQ ID NO: 4.

[0401] 38. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 35, wherein the heavy chain variable region comprises at least 90% identity to the sequence shown in SEQ ID NO: 2, and wherein the light chain variable region comprises at least 90% identity to the sequence shown in SEQ ID NO: 5.

[0402] 39. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 34, wherein the heavy chain variable region comprises the sequence shown in any one of SEQ ID NOs: 1 and 2, and wherein the light chain variable region comprises the sequence shown in any one of SEQ ID NOs: 3, 4 and 5.

[0403] 40. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 39, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 1, and wherein the light chain variable region comprises the sequence shown in SEQ ID NO: 3.

[0404] 41. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 39, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 1, and wherein the light chain variable region comprises the sequence shown in SEQ ID NO: 4.

[0405] 42. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 39, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 2, and wherein the light chain variable region comprises the sequence shown in SEQ ID NO: 5.

[0406] 43. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 42, wherein the heavy chain variable region further comprises a signal sequence.

[0407] 44. The isolated antibody or antigen-binding fragment thereof of claim 43, wherein the signal sequence comprises the sequence set forth in any one of SEQ ID NOs: 12 and 13.

[0408] 45. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 44, wherein the light chain variable region further comprises a signal sequence.

[0409] 46. ​​The isolated antibody or antigen-binding fragment thereof of claim 45, wherein the signal sequence comprises the sequence set forth in any one of SEQ ID NOs: 14 and 15.

[0410] 47. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 46, wherein the antibody comprises an Fc polypeptide having at least 90% identity to the sequence of SEQ ID NO: 36.

[0411] 48. An isolated antibody or antigen-binding fragment thereof that specifically binds to CD157, wherein the isolated antibody competes with the antibody of any one of embodiments 1 to 47 for binding to the CD157 receptor.

[0412] 49. The isolated antibody or antigen-binding fragment thereof of embodiment 48, wherein the antibody binds to the same epitope as the antibody of any one of embodiments 1 to 47.

[0413] 50. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 49, wherein the antibody is a monoclonal antibody.

[0414] 51. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 50, wherein the antibody is a humanized antibody.

[0415] 52. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 51, wherein the antibody comprises one or more human framework regions.

[0416] 53. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 52, wherein the antibody is conjugated to a detectable marker or label.

[0417] 54. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 53, wherein the antibody is non-diffusively immobilized on a solid support.

[0418] 55. An isolated nucleic acid encoding the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 54.

[0419] 56. An isolated nucleic acid comprising a nucleotide sequence encoding a heavy chain variable region comprising a sequence having at least 90% sequence identity to the sequence shown in any one of SEQ ID NOs: 6-8.

[0420] 57. The isolated nucleic acid of embodiment 56, wherein the heavy chain variable region comprises at least 90% sequence identity to the sequence shown in SEQ ID NO:6.

[0421] 58. The isolated nucleic acid of embodiment 56, wherein the heavy chain variable region comprises at least 90% sequence identity to the sequence shown in SEQ ID NO:7.

[0422] 59. The isolated nucleic acid of embodiment 56, wherein the heavy chain variable region comprises at least 90% sequence identity to the sequence shown in SEQ ID NO:8.

[0423] 60. An isolated nucleic acid comprising a nucleotide sequence encoding a heavy chain variable region comprising the sequence set forth in any one of SEQ ID NOs: 6-8.

[0424] 61. The isolated nucleic acid of embodiment 60, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO:6.

[0425] 62. The isolated nucleic acid of embodiment 60, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO:7.

[0426] 63. The isolated nucleic acid of embodiment 60, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO:8.

[0427] 64. An isolated nucleic acid comprising a nucleotide sequence encoding a light chain variable region, the light chain variable region comprising a sequence having at least 90% sequence identity to the sequence shown in any one of SEQ ID NOs: 9-11.

[0428] 65. The isolated nucleic acid of embodiment 64, wherein the light chain variable region comprises at least 90% sequence identity to the sequence shown in SEQ ID NO:9.

[0429] 66. The isolated nucleic acid of embodiment 64, wherein the light chain variable region comprises at least 90% sequence identity to the sequence shown in SEQ ID NO:10.

[0430] 67. The isolated nucleic acid of embodiment 64, wherein the light chain variable region comprises at least 90% sequence identity to the sequence shown in SEQ ID NO:11.

[0431] 68. An isolated nucleic acid comprising a nucleotide sequence encoding a light chain variable region, the light chain variable region comprising the sequence shown in any one of SEQ ID NOs: 9-11.

[0432] 69. The isolated nucleic acid of embodiment 68, wherein the light chain variable region comprises the sequence shown in SEQ ID NO:9.

[0433] 70. The isolated nucleic acid of embodiment 68, wherein the light chain variable region comprises the sequence shown in SEQ ID NO:10.

[0434] 71. The isolated nucleic acid of embodiment 68, wherein the light chain variable region comprises the sequence shown in SEQ ID NO:11.

[0435] 72. An expression vector comprising the nucleic acid of any one of embodiments 55 to 71.

[0436] 73. An isolated host cell comprising the expression vector of embodiment 72.

[0437] 74. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 54 and a pharmaceutically acceptable carrier.

[0438] 75. A diagnostic reagent comprising the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 54.

[0439] 76. A kit comprising the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 54 or the diagnostic agent of embodiment 75.

[0440] 77. A method for detecting CD157, comprising contacting a sample known or suspected to contain CD157 with the isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 54.

[0441] 78. A method for detecting CD157, comprising

[0442] a) contacting the sample with the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 54 under conditions such that the antibody binds to CD157 on the sample, wherein the binding forms a receptor / antibody complex; and

[0443] b) detecting the presence of the receptor / antibody complex,

[0444] The detection includes the presence or absence of CD157 on the sample.

[0445] 79. A method for treating or preventing a disease or condition associated with CD157 in a subject, comprising:

[0446] a) contacting a sample known or suspected to contain CD157 with the isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 54;

[0447] b) detecting the presence of a complex comprising CD157 and the antibody; wherein the presence of the complex indicates the presence of a disease or condition; and

[0448] c) administering the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 54 to a subject.

[0449] 80. A method of diagnosing a disease or condition, comprising:

[0450] a) isolating a sample from a subject;

[0451] b) incubating the sample with the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 54 for a period of time sufficient to generate a CD157:anti-CD157 complex;

[0452] c) detecting the presence or absence of a CD157:anti-CD157 complex in the isolated tissue; and

[0453] d) Correlating the presence or abundance of CD157 to a target location in a tissue sample.

[0454] 81. The method of embodiment 80, wherein an increase in CD157 in the target location of the tissue sample relative to a control level is indicative of a disease or condition in the subject.

[0455] 82. The method of any one of embodiments 77 to 81, wherein the method is performed in vitro.

[0456] 83. The method of any one of embodiments 77 to 81, wherein the method is performed in vivo.

[0457] 84. The method of any one of embodiments 77 to 83, wherein the detection comprises intracellular detection.

[0458] 85. The method of any one of embodiments 77 to 74, wherein the detection comprises detection on the surface of a cell.

[0459] 86. The method of any one of embodiments 77 to 85, wherein the detection comprises hybridization of the detectable moiety to the antibody or antigen-binding fragment thereof.

[0460] 87. The method of any one of embodiments 77 to 86, wherein the sample is contacted with a second antibody.

[0461] 88. The method of any one of embodiments 77 to 87, wherein the second antibody is an antibody comprising a detectable moiety.

[0462] 89. The method of any one of embodiments 77 to 88, wherein the detectable moiety comprises an oligonucleotide.

[0463] 90. The method of any one of embodiments 77 to 89, wherein the detectable moiety comprises a fluorescent label.

[0464] 91. The method of any one of embodiments 77 to 90, wherein measuring comprises sequencing.

[0465] 92. The method of any one of embodiments 77 to 91, wherein the detectable moiety comprises immunofluorescence.

[0466] 93. The method of any one of embodiments 77 to 92, wherein the sample is a formalin-fixed paraffin-embedded sample.

[0467] 94. The method of any one of embodiments 77 to 93, wherein the sample comprises cells.

[0468] 95. The method of any one of embodiments 77 to 94, wherein the sample comprises a tissue sample.

[0469] 96. The method of any one of embodiments 77 to 95, wherein the sample comprises immune cells.

[0470] 97. The method of embodiment 177, wherein the immune cell is selected from the group consisting of B cells, plasmacytoid dendritic cells (pDC), lymphocytes, leukocytes, T cells, monocytes, macrophages, neutrophils, myeloid dendritic cells (mDC), innate lymphocytes, mast cells, eosinophils, basophils, natural killer cells and peripheral blood mononuclear cells (PBMC).

[0471] 98. The method of any one of embodiments 77 to 97, wherein the sample comprises tissue or cells associated with a disease or disorder.

[0472] 99. The method of any one of embodiments 77 to 98, wherein the disease or disorder is cancer, an autoimmune disorder, an inflammatory disorder, or an infection.

[0473] 100. The method of embodiment 98 or 99, wherein the disease or condition is selected from non-viral cancer, virus-related cancer, cancer associated with HBV infection, cancer associated with Epstein-Barr virus (EBV) infection, cancer associated with polyoma virus infection, erythema nodosum leprosum (ENL), autoimmune diseases, autoimmune inflammation, autoimmune thyroid disease, B-cell lymphoma, T-cell lymphoma, acute myeloid leukemia, Hodgkin's disease, acute myeloid leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma cell leukemia, B-cell leukemia, myelodysplastic syndrome, solid phase cancer, herpes virus infection and / or rejection of transplanted tissue or organ.

[0474] 101. The antibody or antigen-binding fragment thereof of any one of embodiments 1 to 54, for use in a method of correlating the presence or abundance of CD157 with a target location in a tissue sample.

[0475] 102. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 54, for use in a method for detecting CD157 in a tissue sample.

[0476] 103. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 77-100, wherein the method comprises generating all or part of a nucleic acid molecule comprising the oligonucleotide sequence or its complement.

[0477] 104. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 54, for use in constructing a protein library.

[0478] 105. The isolated antibody or antigen-binding fragment thereof of embodiment 104, wherein the construction of the protein library comprises sequencing.

[0479] 106. The isolated antibody or antigen-binding fragment thereof of embodiment 104, wherein construction of the protein library comprises the use of flow cytometry.

[0480] Example

[0481] The examples presented below illustrate certain embodiments but do not limit the technology.

[0482] Example 1: Establishment and characterization of anti-CD157 hybridomas

[0483] Hybridomas secreting monoclonal antibodies reactive with CD157 expressed in vivo were prepared as described in this Example.

[0484] Common laboratory mouse strains (e.g., BALB / c or C57 / B16) or rats (e.g., Sprague Dawley) are immunized with CD157 immunogens. After successful immunization of mice, myeloma cells are fused with spleen and lymph node cells collected from animals are discharged using standard protocols to form hybridomas. After selecting successful fusion in HAT culture medium and cloning it to approximately one cell / well in microtiter plates, culture supernatant is detected for CD157 expressing cell transfectants (e.g., HEK293 or RBL) by flow cytometry. The wells with successful staining characteristics are subcultured in larger containers until there are enough cells to allow subclones. Cells transfected with CD157 are further characterized by flow cytometry to hybridoma subclone candidates. The clone selected as the best candidate will be further screened by flow cytometry for human blood cells divided into different subgroups (e.g., lymphocytes, monocytes, etc.) and for one or more cell lines generated by sick and / or infected human cells. The percentage of positive cells in each blood cell subset was quantified compared with the isotype control.

[0485] Exemplary clones were selected based on strong reactivity to CD157-expressing immune cells (eg, monocytes) but no significant reactivity to CD157-negative blood cell populations (eg, lymphocytes).

[0486] Example 2: Sequencing of anti-CD157 antibody variable regions

[0487] Cells from well-performing anti-CD157 hybridoma cell lines (as described in Example 1 above) were grown in standard mammalian tissue culture medium. Using a program based on RNeasy Mini Kit (Qiagen), total RNA was isolated from hybridoma cells expressing various clones of anti-CD157 monoclonal antibodies. This RNA was used to generate the first strand cDNA. Both light chain and heavy chain variable region cDNA were amplified by 5'-RACE technology. Positive clones were prepared by PCR, and then DNA sequencing was performed on multiple clones.

[0488] Table E1 and Table E2 below show the amino acid sequences of the individual variable domains (CDRs and framework regions, including CDR1, CDR2 and CDR3 regions) of the heavy and light chains of three different antibodies (clones), designated AB 1-3 (also referred to herein as Antibody 1-3 and Clone 1-3).

[0489] Table E1: CDR sequences and SEQ ID NOs of the VH and VL domains of representative anti-CD157 monoclonal antibodies

[0490] SEQ ID NO CDR Type AB (clone) number Amino acid sequence 16 CDRH1 AB1 GFSLTSYHVS 17 CDRH1 AB2 SYHVS 18 CDRH1 AB3 NYDMA 19 CDRH2 AB1 and AB2 IIWTGGSTAYNSLLKS 20 CDRH2 AB3 SISIRGGSTYYRDSVKG 21 CDRH3 AB1 and AB2 SITPTFFDY 22 CDRH3 AB3 GTDYYYDYFDY 23 CDRL1 AB1 KRSTGNFGNNYVN 24 CDRL1 AB2 KRSTGNFGSNYVN 25 CDRL1 AB3 KAGRNINSYLA 26 CDRL2 AB1 and AB2 RDDKRPD 27 CDRL2 AB3 QT 28 CDRL3 AB1 and AB2 QSYSSGIV 29 CDRL3 AB3 QQYNSWTNT

[0491] Table E2: Sequences and SEQ ID NOs of the VH and VL domains of representative anti-CD157 monoclonal antibodies

[0492]

[0493]

[0494] Example 3: Detection of cells expressing CD157

[0495] This example describes the ability of exemplary generated anti-CD157 antibodies to detect cells expressing CD157 by flow cytometry and immunohistochemistry.

[0496] In the first experiment, cells from a human promonocytic cell line (U-937; CRL-1539.2 TM ) cells were evaluated for exemplary anti-CD157 antibodies.

[0497] U-937 cells were grown in RPMI medium supplemented with 10% FBS in a T75 culture flask to a confluence of approximately 80%. Various concentrations of exemplary generated anti-CD157 antibodies were added, including 2, 1, 0.5, 0.25, 0.125, and 0.06 μg, and allowed to incubate for 15 minutes. The cells were then washed twice with FACS wash buffer and stained with anti-rat IgG-PE secondary antibody for 15 minutes. The cells were washed with FACS buffer and analyzed on a BD LSRII flow cytometer. Figure 1 As shown, all exemplary antibodies positively stained the CD157-positive human monocyte U-937 cell line, with AB2 showing enhanced specificity and staining intensity.

[0498] Furthermore, in CD157 positive human monocyte U-937 cells ( Figure 2B ) and CD157-negative human T lymphocyte immortalized cell line (Jurkat cells-ATCC; Figure 2A ) and compared to a commercially available antibody (CAb). Cells were grown similarly to above and various concentrations of the exemplary generated anti-CD157 antibody AB2 were added, including 1, 0.5, 0.25, and 0.125 μg, and allowed to incubate for 15 minutes. Cells were then washed and prepared as described above and analyzed on a BD LSRII flow cytometer. Figure 2A As shown, the exemplary tested anti-CD157 antibody AB2 exhibited superior specificity with reduced nonspecific staining of CD157-negative Jurkat cells compared to the commercial antibody (CAb). Similar staining characteristics were observed on the CD157-positive human monocytic cell line U-937 ( Figure 2B ).

[0499] Exemplary antibody evaluations were further performed on leukocytes (lymphocytes, monocytes, and granulocytes) isolated from healthy volunteer donors. Briefly, leukocytes in whole blood were stained with antibodies against human CD45, CD14, and CD15 and 1.5 μ FITC-conjugated AB2 or a commercial antibody control (CAb), followed by lysis of red blood cells. Lysed blood was washed twice with FACS wash buffer and then analyzed on a BD LSRII flow cytometer. Figures 3A-3C As shown, the exemplary tested antibodies expressed CD157 in granulocytes ( Figure 3C ) and monocytes ( Figure 3B ) showed positive staining, while lymphocytes that did not express CD157 ( Figure 3A ) without surface staining.

[0500] These results demonstrate that the exemplary generated anti-CD157 antibodies are able to specifically recognize cells expressing the cognate receptor CD157 with little to no non-specific binding, and that the exemplary AB2 exhibits higher specificity than the commercially available antibody (CAb).

[0501] Example 4: Monocyte adhesion function assay

[0502] This example describes a functional assay based on the ability of exemplary generated anti-CD157 antibodies to block monocyte adhesion.

[0503] Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll gradient and the cell concentration was adjusted to 1 million / ml in complete RMPI medium. In the first experiment, exemplary antibody AB2 was added to the cells at 2.25, 1.6, 1.26, 0.9 and 0.6 μg and incubated at room temperature for 15 minutes with occasional shaking.

[0504] The cells were then transferred to a 24-well plate coated with recombinant human fibronectin and subsequently incubated in an incubator at 37 degrees for 1 hour. Non-adherent cells were removed by washing 4 times with PBS (horizontally shaking). Fixation buffer was added for 30 minutes, followed by another wash in PBS. Bright field images were taken at 10× magnification (at least 3 random fields per well). The number of cells in each image was counted using the cell counter function in FIJI, and the mean and standard deviation were calculated using an Excel spreadsheet. The data shown were normalized to the isotype control wells for each antibody concentration. As Figure 4 As shown, all tested concentrations were able to block CD157-mediated adhesion of human monocytes to the extracellular matrix protein fibronectin, with the best cell adhesion blockade observed at 1.26 μg.

[0505] In a second experiment, exemplary antibody AB2 or commercially available (CAb) antibody was added to cells at 10, 5, 2.5, 1.25, 0.625 and 0.3125 μg and incubated at room temperature for 15 minutes with occasional shaking.

[0506] The cells were then transferred to an opaque 24-well plate coated with recombinant human fibronectin and subsequently incubated in an incubator at 37 degrees for 1 h. After incubation, Deep Blue Cell Viability Assay Reagent was added to the culture medium at a final concentration of 10% and allowed to incubate at 37 degrees for 3 hours. The number of viable cells was assessed by fluorescence using a SpectraMax M3 microplate reader (Molecular Devices) at an excitation wavelength of 530-570 nm and an emission wavelength of 590-620 nm. Means and standard deviations were calculated in Excel. The data shown were normalized to the isotype control wells for each antibody concentration. Figure 5As shown, AB2 exhibited an enhanced ability to block CD157-mediated adhesion of human monocytes to the extracellular matrix protein fibronectin compared to a commercially available (CAb) antibody.

[0507] Example 5: Assessment of calcium signaling and cell polarization

[0508] This example describes a functional assay based on the ability of exemplary generated anti-CD157 antibodies to induce calcium signaling and cell polarization.

[0509] Peripheral blood mononuclear cells (PBMC) were separated by Ficoll gradient and treated with CalceinAM at a concentration of 2 ug / ml for 30 minutes. Exemplary antibody AB2 or commercially available antibody (CAb) was added to the cells at various concentrations ranging from 0.625 to 10 μg.

[0510] The cells were then transferred to a 24-well plate coated with recombinant human fibronectin and then incubated in an incubator at 37 degrees for 1 hour. Wash three times with PBS (horizontally shaking) to remove non-adherent cells. Add fixation buffer for 30 minutes, followed by another wash in PBS. Calcein fluorescence was read at 488nm / 520nm on a Spectramax M3 microplate reader. The cells were then washed three times with PBS, and fresh RPMI medium containing a 1 / 10 dilution of Deep Blue cell viability reagent was added to the wells and incubated for 3 hours. After incubation, cell fluorescence was read on a Spectramax M3 microplate reader at an excitation wavelength of 530-570nm and an emission wavelength of 590-620nm. The ratio of calcein fluorescence to deep blue fluorescence was then calculated, and the calcein signal was normalized to the number of cells. As Figure 6 As shown, exemplary antibody AB2 was able to induce calcium signaling at all concentrations tested and to a greater extent than the commercially available antibody.

[0511] In separate experiments, the U937 myelomonocytic cell line was treated with 5ug / ml of exemplary antibody AB2, commercially available antibodies (CAb), or isotype controls in RPMI medium supplemented with 10% FBS for 15 minutes. The cells were then plated onto chamber slides coated with recombinant human fibronectin and incubated at 37C for 1 hour. The medium was removed and the cells were fixed with fixation buffer (BL Cat#420801) for 30 minutes. The cells were washed twice with 1× intracellular staining permeabilization wash buffer (BL Cat#421002) and then incubated with exemplary antibody AB2 (5ug / ml), Alexa-647 anti-CD29 (clone TS2 / 16, BioLegend, 5ug / ml), or Flash Phalloidin TMGreen 488 (BioLegend) was used to visualize F-actin (1 / 500) for 1 hour in 1× intracellular staining permeabilization wash buffer. After washing 3 times with 1× intracellular staining permeabilization wash buffer, the cells were stained with Alexa-555 anti-rat secondary antibody (BioLegend, 5ug / ml) for 1 hour. After washing 3 times with 1× intracellular staining permeabilization wash buffer, the cells were covered with PBS and imaged using an Olympus IX83 inverted fluorescence microscope, and the images were analyzed using Metamorph software.

[0512] like Figure 7 As shown, the exemplary antibody AB2 is able to polarize the cells, and filamentous actin increases significantly (left figure), and CD157 (but not CD29) accumulates at the tail foot. Most of the F-actin fibers are located at the other end of CD157, and CD157 and integrin β-1 are co-localized in different membrane domains. The control antibody and the commercially available antibody did not show this effect, and the cells were still not polarized, and the actin fibers remained disordered. Neither CD157 nor CD29 accumulated in different regions of the cell.

[0513] Example 6: Blocking assay

[0514] This example describes the ability of exemplary generated anti-CD157 antibodies to block the binding of other anti-CD157 antibodies.

[0515] For blocking studies, 100 μL of lysed whole blood was incubated with 10 μg of commercially available (CAb) antibody. After 15 minutes, FITC-conjugated AB2 was added and incubated for another 15 minutes. Cells were washed twice with cell staining buffer and acquired in an LSRII flow cytometer (BD Biosciences); data were analyzed using FlowJo software.

[0516] Calculate the raw MFI percentage by dividing the MFI of the sample blocked with Cab by the MFI of the sample stained with AB2 alone, as shown in Table E3. Subtract this value from 100 to get the blocking percentage. The formula is as follows:

[0517]

[0518] Table E3: FITC MFI and % Blocking of Commercial Clones

[0519]

[0520] AB1 was blocked by a reference commercial clone (data not shown), indicating that AB2 recognizes a different epitope.

[0521] Sequence Listing

[0522]

[0523]

[0524]

[0525]

[0526] The foregoing may be modified without departing from the basic aspects of the present technology. Although the present technology has been described in considerable detail with reference to one or more specific embodiments, those skilled in the art will recognize that modifications may be made to the embodiments specifically disclosed in this application, yet these modifications and improvements are within the scope and spirit of the present technology.

[0527] In the absence of any element not specifically disclosed herein, the technology described exemplarily herein can be appropriately put into practice.Therefore, for example, in each example herein, any one of the terms "comprising", "substantially consisting of ... consisting of" and "consisting of ... consisting of" can be replaced by any one of the other two terms.The terms and expressions adopted are used as descriptive terms rather than restrictive terms, and the use of these terms and expressions does not exclude any equivalents of the features shown and described or their parts, and various modifications can be made within the claimed technical scope.The term "a (a)" or "an (an)" can refer to one or more of the elements modified by it (for example, "reagent (areagent)" can mean one or more reagents), unless the context clearly describes one of the elements or multiple elements.As used herein, the term "about" refers to a numerical value within 10% of the basic parameter (i.e., plus or minus 10%), and the term "about" is used at the beginning of a string of numerical values ​​to modify each numerical value (i.e., "about 1, 2 and 3" refers to about 1, about 2 and about 3).For example, the weight of "about 100 grams" can include the weight between 90 grams and 110 grams. In addition, when a numerical list is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%), the list includes all intermediate values ​​and fractional values ​​thereof (e.g., 54%, 85.4%). Therefore, it should be understood that although the present technology has been specifically disclosed through representative embodiments and optional features, those skilled in the art may make modifications and variations to the concepts disclosed herein, and such modifications and variations are considered to be within the scope of the present technology.

Claims

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to CD157, wherein the isolated antibody comprises: a) a heavy chain variable region comprising: (i) a heavy chain complementarity determining region 1 (CDRH1) comprising a sequence as set forth in any one of SEQ ID NOs: 16, 17, and 18, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 16, 17, and 18; (ii) a heavy chain complementarity determining region 2 (CDRH2) comprising a sequence as set forth in any one of SEQ ID NOs: 19 and 20, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 19 and 20; (iii) a heavy chain complementarity determining region 3 (CDRH3) comprising a sequence as set forth in any one of SEQ ID NOs: 21 and 22, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 21 and 22; and b) a light chain variable region comprising: (iv) a light chain complementary determining region 1 (CDRL1) comprising a sequence as set forth in any one of SEQ ID NOs: 23, 24, and 25, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 23, 24, and 25; (v) a light chain complementary determining region 2 (CDRL2) comprising a sequence as set forth in any one of SEQ ID NOs: 26 and 27, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 26 and 27; and (vi) a light chain complementarity determining region 3 (CDRL3) comprising a sequence as set forth in any one of SEQ ID NOs: 28 and 29, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 28 and 29. 2 . The isolated antibody or antigen-binding fragment thereof according to claim 1 , wherein the heavy chain variable region comprises at least 90% identity to the sequence shown in any one of SEQ ID NOs: 1 and 2. 3 . The isolated antibody or antigen-binding fragment thereof according to claim 1 , wherein the light chain variable region comprises at least 90% identity to the sequence shown in any one of SEQ ID NOs: 3, 4 and 5. 4 .

4. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, further comprising an Fc polypeptide having at least 90% identity to the sequence of SEQ ID NO:

36.

5. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein the antibody is conjugated to a detectable marker or label.

6. An isolated nucleic acid encoding the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

7. An isolated nucleic acid comprising a nucleotide sequence encoding a heavy chain variable region comprising at least 90% sequence identity to the sequence set forth in any one of SEQ ID NOs: 6-8.

8. An isolated nucleic acid comprising a nucleotide sequence encoding a light chain variable region comprising at least 90% sequence identity to the sequence set forth in any one of SEQ ID NOs: 9-11.

9. An expression vector comprising the nucleic acid of any one of claims 1 to 8.

10. An isolated host cell comprising the expression vector of claim 9.

11. A composition comprising the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

12. A kit comprising the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

13. A method for detecting CD157, the method comprising contacting a sample known or suspected to contain CD157 with the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

14. A method for detecting CD157, comprising: a) contacting the sample with the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 12 under conditions such that the antibody binds to CD157 on the sample, wherein the binding forms a receptor / antibody complex; and b) detecting the presence of said receptor / antibody complex, The detection comprises the presence or absence of CD157 on the sample.

15. A method of diagnosing a disease or condition, comprising: a) isolating a sample from a subject; b) incubating the sample with the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 for a period of time sufficient to generate a CD157:anti-CD157 complex; c) detecting the presence or absence of the CD157:anti-CD157 complex in the isolated tissue; and d) Correlating the presence or abundance of CD157 to a target location in a tissue sample.

16. The method according to any one of claims 13 to 15, wherein the method is performed in vitro.

17. The method of any one of claims 26 to 30, wherein the detecting comprises hybridization of a detectable moiety to the antibody or antigen-binding fragment thereof.

18. The method of any one of claims 13 to 17, wherein the detectable moiety comprises a fluorescent label.

19. The method of any one of claims 13 to 18, wherein the sample comprises a tissue sample.

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