Agnonistic antibodies targeting JAML

Abstract

Antibodies or antigen-binding fragments that bind to junctional adhesion molecule-like (JAML) proteins or fragments thereof are provided herein. In one embodiment, the antibody or antigen-binding fragment is a humanized antibody or its antigen-binding fragment. In several embodiments, the antibody or antigen-binding fragment comprises, consists of, or essentially consists of heavy chain complementarity-determining regions 1-3 (CDRH1-3) CDRH1, CDRH2, CDRH3, and light chain complementarity-determining regions 1-3 (CDRH1-3) 1, CDRH2, and CDRH3, each selected from Table 5.

Description

[Technical Field]

[0001] Cross-reference of related applications This application asserts the interests under Section 119(e) of U.S. Provisional Application No. 63 / 460,835, filed on April 20, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Incorporating sequence lists by reference This application has a sequence listing submitted electronically in XML format, the entirety of which is incorporated herein by reference. The XML copy, created on April 5, 2024, is named 116639-2660.SL.xml and has a size of 202,202 bytes.

[0003] This disclosure generally relates to agonist antibodies that specifically target and bind to junctional adhesion molecule-like proteins (JAMLs). [Background technology]

[0004] The following background explanation of this technology is provided solely as an aid to understanding this technology and is not intended to describe or constitute prior art. Junctional adhesion molecule-like proteins (JAMLs) function as costimulatory molecules in γδT cells, influencing tissue homeostasis and repair. JAMLs were initially identified as a major costimulatory molecule in epithelial γδT cells, and their activation by their ligands, coxsackievirus and adenovirus receptors (CXADRs), expressed by epithelial cells, has been shown to be important for tissue homeostasis and wound repair (Verdino et al., Science. 2010 Sep 3;329(5996):1210-4, Witherden et al., Science. 2010 Sep 3;329(5996):1205-10). Although JAML has low overall sequence identity with the co-stimulatory molecule CD28 (approximately 11%), their intracellular signaling motifs are substantially similar, and upon ligation, they recruit phosphatidylinositol-3-OH-kinase (PI3K), leading to cell activation, proliferation, and cytokine production (Verdino et al., 2010, Witherden et al., 2010). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Verdino et al.,Science.2010 Sep 3;329(5996):1210-4 [Non-Patent Document 2] Witherden et al.,Science.2010 Sep 3;329(5996):1205-10 [Overview of the project] [Means for solving the problem]

[0006] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to a junctional adhesion molecule-like (JAML) protein or a fragment thereof. In some embodiments, the antibody or antigen-binding fragment comprises heavy chain complementarity-determining regions 1-3 (CDRH1-3) and light chain complementarity-determining regions 1-3 (CDRL1-3) selected from a single row in Table 2, or each of their equivalents. In another embodiment, the antibody or antigen-binding fragment comprises, consists of, or essentially consists of, heavy chain variable regions (HC) and light chain variable regions (LC) selected from a single row in Table 1, or each of their equivalents.

[0007] In one embodiment, the antibody or antigen-binding fragment is a humanized antibody or its antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment includes, consists of, or essentially consists of, heavy chain complementarity-determining regions 1-3 (CDRH1-3) CDRH1, CDRH2, CDRH3, light chain complementarity-determining regions 1-3 (CDRH1-3) 1, CDRH2, and CDRH3, each selected from Table 5. In some other embodiments, the antibody or its antigen-binding fragment includes, consists of, or essentially consists of, heavy chain complementarity-determining regions 1-3 (CDRH1-3) and light chain complementarity-determining regions 1-3 (CDRL1-3), or their respective equivalents, selected from a single row in Table 5. In yet another embodiment, the antibody or its antigen-binding fragment includes, consists of, or essentially consists of, HC or their respective equivalents, selected from SEQ ID NOs. 135-138, and LC or their respective equivalents, selected from SEQ ID NOs. 139-142. In some embodiments, the HC equivalent holds the corresponding CDRH1-3 selected from a single row in Table 5, or the LC equivalent holds the corresponding CDRL selected from a single row in Table 5. In some embodiments, the antibody or its antigen-binding fragment contains, consists of, or essentially consists of HC and LC, or their equivalents, selected from a single row in Table 3. In some embodiments, the HC equivalent holds the corresponding CDRH1-3 selected from a single row in Table 5, and the LC equivalent holds the corresponding CDRL selected from a single row in Table 5.

[0008] In some embodiments, the antibody is a monoclonal antibody or a fragment thereof, such as an antigen-binding fragment thereof. In some embodiments, the antibody comprises a constant region selected from the group consisting of an IgA constant region, an IgD constant region, an IgE constant region, an IgG constant region, or an IgM constant region. In some embodiments, the constant region comprises, consists of, or consists essentially of an IgG1 constant region. In some embodiments, the antigen-binding fragment comprises, consists of, or consists essentially of Fab, F(ab’)2, Fab’, scFv, or Fv. In some embodiments, the antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of an Fc region comprising one or more mutations selected from G237D, P238D, H268D, P271G, and A330R compared to the wild-type Fc region of the antibody.

[0009] In some embodiments, the antibodies or antigen-binding fragments described herein comprise a detectable label or a purification label.

[0010] In some embodiments, an equivalent of the HC or LC of a single row in Table 1 comprises, consists of, or consists essentially of a polypeptide having at least 80%, or at least 85%, or at least 90%, or at least 95% amino acid identity to the polypeptide, or an equivalent to the amino acid sequence hybridizes under high stringency conditions to a complement of the polynucleotide encoding the amino acid sequence and optionally comprises a polypeptide encoded by a polynucleotide that retains the reference or HC or LC CDRH1-3 or CDRL1-3, respectively. In one embodiment, the equivalent encodes a related HC and / or LC having at least 90% or greater binding affinity to the JAML polypeptide as the reference or parental HC and / or LC.

[0011] In one embodiment, the antibody or antigen-binding fragment includes modifications. In several embodiments, the modifications are selected from the group consisting of PEGylation, PEG mimicry, polysialylation, HESation, or glycosylation.

[0012] In some embodiments, isolated polynucleotides encoding antibody or antigen-binding fragments are provided herein. In some embodiments, the isolated polynucleotides contain, consist of, or essentially consist of any one of the polynucleotide sequences from SEQ ID NOs. 143-150, or their respective equivalents. In some embodiments, the equivalents encode HC and LC selected from a single row in Table 3.

[0013] In some embodiments, polynucleotides are operably ligated to promoter and / or enhancer elements. In some embodiments, the polynucleotides include, consist of, or essentially consist of, a detectable label of a purified label. In some embodiments, vectors comprising, consisting of, or essentially consisting of, polynucleotides are provided herein. In some embodiments, the vectors include, consist of, or essentially consist of, heterologous promoter sequences. In yet another embodiment, isolated host cells comprising, consisting of, or essentially consisting of, polynucleotides or vectors are provided herein.

[0014] In one embodiment, the Specified Information provides a composition comprising, comprising, or essentially comprising, a carrier and one or more of the following: an antibody or antigen-binding fragment, a polynucleotide encoding an antibody or antigen-binding fragment, a vector, or a host cell. In some embodiments, the carrier comprises, comprises, or essentially comprises a pharmaceutically acceptable carrier.

[0015] In another embodiment, this specification provides a kit comprising, or essentially comprising, one or more of the following: an antibody or antigen-binding fragment, a polynucleotide encoding an antibody or antigen-binding fragment, a vector, or a host cell, each as described herein. In some embodiments, the kit further comprises instructions for use.

[0016] In one embodiment, a method for producing an antibody or antigen-binding fragment is provided herein. In some embodiments, the method comprises, or essentially comprises, culturing a host cell containing a polynucleotide encoding the antibody or the antigen-binding fragment under conditions for the expression of the polynucleotide encoding the antibody or the antigen-binding fragment. In some embodiments, the method further comprises, or essentially comprises, isolating the antibody or antigen-binding fragment. Alternatively, the antibody or fragment can be produced using each sequence by chemical means, such as the use of a polypeptide synthesizer.

[0017] In yet another aspect of this disclosure, a method for conjugating a JAML protein or a fragment thereof is provided herein. In some aspects, the method comprises, or essentially comprises, contacting a JAML protein or a fragment thereof with an antibody or antigen-binding fragment thereof under conditions preferred for the conjugation of the antibody or antigen-binding fragment to the JAML protein or fragment, and optionally, isolating the antibody or antigen-binding fragment conjugated to the JAML protein. These conditions are known in the art and are briefly described herein. [Brief explanation of the drawing]

[0018] [Figure 1]The alignment of three selected humanized heavy chain sequences compared to the consensus and parent heavy chain amino acid sequences is shown. "HC_hu(IGHV1 3)BM", "HC_hu(IGHV1 18)BM", and "HC_hu(IGHV1 46)BM" were selected as the three humanized VH sequences. The figure discloses sequence numbers 205, 20, 206, 172, 207, 174, 208, and 176, respectively, in order of appearance. [Figure 2] The alignment of three selected humanized light chain sequences compared to the consensus and parent light chain amino acid sequences is shown. "LC_hu(IGKV2 28)BM", "LC_hu(IGKV2 30)BM", and "LC_hu(IGKV2D 30)BM" were selected as the three humanized VK sequences. The figure discloses sequence numbers 177, 55, 209, 173, 210, 175, 211, and 177, respectively, in order of appearance. [Figure 3] Interim results of kinetic data for selected humanized antibodies with heavy and light chain pairs are shown. [Figure 4] Qualitative binding evaluation data for all IgG antibodies and HC and LC combinations are shown. [Figure 5] The sensorograms and kinetic data for parental and LC1 antibodies in combinations of humanized antibodies HC1, HC2, and HC3 from Session 2 are shown. [Figure 6] The sensorograms and kinetic data for parental and LC1 antibodies in combinations of humanized antibodies HC1, HC2, and HC3 from Session 3 are shown. [Figure 7] The data shows the activation markers after 24 hours of stimulation (0.5 μg / mL aCD3+ co-stimulation). [Figure 8] The proliferation data after 72 hours of stimulation (co-stimulation with 0.5 μg / mL aCD3 + 2.5 μg / mL) is shown. [Figure 9] The activation markers after 24 hours of stimulation (0.5 μg / mL aCD3+ co-stimulation) are shown. [Figure 10] The activation markers after 24 hours of stimulation (0.5 μg / mL aCD3+ co-stimulation) are shown. [Figure 11] This shows proliferation data after 72 hours of stimulation and pAKT levels after 24 hours of stimulation. [Figure 12] This shows the mouse activation markers after 24 hours of stimulation (0.5 μg / mL aCD3+ co-stimulation). [Figure 13] This shows the mouse activation markers after 72 hours of stimulation (0.5 μg / mL aCD3+ co-stimulation). [Figure 14] This shows the activation marker 24 hours after stimulation. It was evaluated in two separate experiments and represents one initial activation marker (CD69). [Modes for carrying out the invention]

[0019] Embodiments provided herein are described in full below. However, aspects of this disclosure may be embodied in different forms and should not be construed as being limited to the embodiments described herein. Rather, these embodiments are provided to make this disclosure thorough and complete and to fully convey the scope of this disclosure to those skilled in the art. The terms used in this description are for the purpose of describing specific embodiments only and are not intended to limit the invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which this disclosure pertains. All nucleotide sequences provided herein are presented in the 5' to 3' direction. Any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this disclosure, but particularly non-limiting exemplary methods, apparatus, and materials are described herein. All technical and patent publications cited herein are incorporated herein by reference in their entirety. Nothing herein should be construed as acknowledging that this disclosure does not have prior rights to any prior disclosure.

[0021] Unless otherwise indicated, the implementation of this disclosure will involve conventional techniques within the scope of the art, including tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA. For example, Sambrook and Russell eds, (2001) Molecular Cloning: A Laboratory Manual, 3rd edition, the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology, the series Methods in Enzymology (Academic Press, Inc., NY), MacPherson et al. (1991) PCR 1:A Practical Approach (IRL Press at Oxford University Press), MacPherson et al. al. (1995) PCR 2: A Practical Approach, Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual, Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition, Gait ed. (1984) Oligonucleotide Synthesis, U.S. Patent No. 4,683,195, Hames and Higgins eds.(1984)Nucleic Acid Hybridization, Anderson (1999) Nucleic Acid Hybridization, Hames and Higgins eds. (1984) Transcription and Translation, Immobilized Cells and Enzymes (IRL Press (1986)), Perbal (1984) A Practical Guide to Molecular Cloning, Miller and Calos eds, (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory), Makrides ed.See (2003) Gene Transfer and Expression in Mammalian Cells, Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London), and Herzenberg et al. eds. (1996) Weir's Handbook of Experimental Immunology.

[0022] The terms used herein are for the purpose of describing specific embodiments and are not intended to limit this disclosure. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety.

[0023] The implementation of this technology will, unless otherwise specified, utilize conventional techniques within the scope of the art, including tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA.

[0024] Unless otherwise indicated by the context, the various features of the Disclosure described herein are particularly intended to be used in any combination. Furthermore, the Disclosure also intends that in some embodiments, any feature or combination of features described herein may be excluded or omitted. For illustrative purposes, where the Disclosure herein states that a complex includes components A, B, and C, it is specifically intended that any one of A, B, or C, or any combination thereof, may be omitted or excluded individually or in any combination.

[0025] Unless otherwise expressly indicated, all specific embodiments, features, and terms are intended to include both the listed embodiments, features, or terms and their biological equivalents.

[0026] All numerical notations, including ranges, such as pH, temperature, time, concentration, and molecular weight, are approximations that vary (+) or (-) by increments of 1.0 or 0.1, or alternatively by variations of + / - 15%, 10%, 5%, or 2%, as needed, and such ranges are included. It should be understood that all numerical specifications are preceded by the term “approximately,” although this is not always explicitly stated. Furthermore, it should be understood that the reagents described herein are merely illustrative, and such equivalents are known in the art, although this is not always explicitly stated.

[0027] Throughout this disclosure, various publications, patents, and published patent specifications may be referenced by specific citations or by Arabic numerals. These publications, patents, and published patent specifications are incorporated herein by whole reference in order to provide a more complete explanation of the current technology relating to this disclosure.

[0028] definition Where used in this disclosure and the accompanying claims, the singular forms "a," "an," and "the" are intended to also include the plural forms unless the context explicitly indicates otherwise.

[0029] Where used herein, the term “comprising” is intended to mean that a composition and method includes the enumerated elements but does not exclude other elements. Where used herein, the transitional phrase “essentially consisting of” (and grammatical variations thereof) is to be interpreted as including the enumerated materials or steps and those that do not materially affect the basic and novel features of the enumerated embodiments. Accordingly, where used herein, the term “essentially consisting of” should not be interpreted as equivalent to “including.” “Consists of” is to mean excluding trace elements of other components and anything beyond substantial method steps for administering the compositions disclosed herein. The embodiments defined by each of these transitional terms are within the scope of this disclosure.

[0030] As used herein, the term “about” means, when referring to a measurable value such as a quantity or concentration, to include variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified quantity.

[0031] As used herein, “increased,” “decreased,” “high,” “low,” or any grammatical variation thereof, refer to variations of about 90%, 80%, 50%, 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the reference composition, polypeptide, protein, etc.

[0032] Where used to describe any selection of components, ranges, dosage forms, etc. disclosed herein, terms such as “acceptable,” “effective,” or “sufficient” are intended to indicate that such components, ranges, dosage forms, etc. are suitable for the purposes of disclosure.

[0033] As used herein, "and / or" means and encompasses any and all possible combinations with one or more of the related enumerated items, as well as the absence of any combination if interpreted as an alternative ("or").

[0034] A polynucleotide or polypeptide equivalent (hereinafter referred to as "reference") shares at least 50% (or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%) identity with respect to the reference (compared to when a comparison program such as BLAST is run with default parameters), and when referring to a polynucleotide, it codes for the same polypeptide or a similar polypeptide encoded by the reference, or an equivalent of the polypeptide encoded by the reference.

[0035] Sequence alignment is performed between the test sequence and the reference sequence to reach the position or segment of the test sequence that corresponds to (or is equivalent to) an amino acid / nucleotide residue or segment of the reference sequence in the reference sequence. The aligned positions or segments are determined to be equivalents.

[0036] The term "analog" refers to an equivalent having one or more modified amino acids and one or more amino acids substituted with other amino acids. Such modifications include conjugation with molecules (e.g., small molecules, cytotoxic molecules, linkers, pH-sensitive linkers, and / or thiol linkers), sialylation, polysialylation, O-glycosylation, N-glycosylation, myristoylation, palmitoylation, isoprenylation or prenylation, glipyatyon, lipoylation, phosphopantetheinylation, ethanolamine phosphoglycerol attachment, diphthamide formation, hypsination, acylation, acetylation, formylation, alkylation, methylation, amidation, citrullination, deamidation, eliminylation, ISG formation, and SUMOylation. This may include, but is not limited to, ubiquitination, nedylation, pupation, biotinylation, carbamylation, oxidation, pegylation, glycation, carbamylation, carbonylation, spontaneous isopeptide bond formation, butyrylation, gammacarboxylation, malonylation, hydroxylation, iodation, nucleotide addition, formation of phosphate esters (O-bonds) or phosphoramides (N-bonds), phosphorylation, adenylation, uridilylation, propionylation, pyroglutamate formation, S-glutathionylation, S-nitrosylation, S-sulfenylation, S-sulfinylation, S-sulfonylation, succinylation, and / or sulfation. The term "albumin equivalent" includes, essentially consists of, or further consists of, polypeptides that are known in the art or described herein, which can be expressed in reasonable amounts and still retain or improve certain albumin properties, including binding of albumin fragments to FcRn receptors.

[0037] The term "affinity tag" refers to a polypeptide that can be included within a fusion protein to enable detection and / or purification of the fusion protein from the cellular environment by using a ligand that can bind to the affinity tag, i.e., has affinity. The ligand may be, but is not limited to, an antibody, a resin, or a complementary polypeptide. Affinity tags may include small peptides, generally peptides with a length of about 4 to 16 amino acids, or larger polypeptides. Commonly used affinity tags include, among others, polyarginine, FLAG, V5, polyhistidine, c-Myc, Strep II, maltose-binding protein (MBP), N-utilizing protein A (NusA), thioredoxin (Trx), and glutathione S-transferase (GST) (see, for example, the GST Gene Fusion System Handbook - Sigma-Aldrich). In one embodiment, the affinity tag is a polyhistidine tag, e.g., the His6 tag (SEQ ID NO: 159). By including an affinity tag in the fusion protein or polypeptide, the fusion protein can be purified from the cellular environment by affinity purification using an affinity medium that can tightly and specifically bind to the affinity tag. The affinity medium may contain, for example, a metal-charged resin or ligand covalently bonded to a stationary phase (matrix) such as agarose or metal beads. For example, a polyhistidine-tagged fusion protein or polypeptide (also called a His-tagged fusion protein) may contain Ni 2+ or Co 2+ The GST-tagged fusion proteins can be recovered by immobilized metal ion chromatography using a resin loaded with the GST, or they may be captured using an anti-FLAG affinity gel, or they may be captured using glutathione crosslinked to a solid support such as agarose.

[0038] As used herein, the terms “purification,” “purifying,” or “separation” refer to the process of isolating one or more polypeptides from a complex mixture, such as a cell lysate or a mixture of polypeptides. Purification, separation, or isolation does not need to be complete; that is, some components of the complex mixture may remain in one or more polypeptides after the purification process. However, the product of purification should be concentrated with respect to one or more polypeptides relative to the complex mixture before purification, and a substantial portion of the other components initially present in the complex mixture should be removed by the purification process.

[0039] As used herein, the term “cells” may optionally refer to either prokaryotic or eukaryotic cells obtained from the subject or a commercially available source.

[0040] "Eukaryotic cells" include all living organisms except Monera. They can be easily distinguished by their membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms in which cells are organized into complex structures by an inner membrane and cytoskeleton. The most characteristic membrane-bound structure is the nucleus. Unless otherwise specified, the term "host" includes eukaryotic hosts, such as yeast, higher plants, insects, and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include monkeys, cattle, pigs, mice, rats, birds, reptiles, and humans, such as HEK293 cells, Chinese hamster ovaries (CHO), and 293T cells.

[0041] Prokaryotic cells typically lack a nucleus or any other membrane-bound organelle and are divided into two domains: bacteria and archaea. In addition to chromosomal DNA, these cells can also contain genetic information in circular loops called episomes. Bacterial cells are very small, roughly the size of animal mitochondria (about 1-2 μm in diameter and 10 μm in length). Prokaryotic cells are characterized by three main shapes: rod-shaped, spherical, and helical. Instead of undergoing the complex replication process of eukaryotes, bacterial cells divide by binary fission. Examples include, but are not limited to, Bacillus, E. coli, and Salmonella.

[0042] The term “coding” as applied to nucleic acid sequences refers to a polynucleotide said to “code” a polypeptide, which, in its natural state or when manipulated by methods well known to those skilled in the art, is capable of being transcribed and / or translated to produce mRNA of a polypeptide and / or fragment thereof. The antisense strand is the complementary strand of such nucleic acid, from which the coding sequence can be inferred.

[0043] As used herein, “expression” refers to the process by which polynucleotides are transcribed into mRNA, and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. If the polynucleotides are derived from genomic DNA, expression may include the splicing of mRNA in eukaryotic cells.

[0044] As used herein, the term “isolated” or its grammatical variation refers to a molecule substantially free of other material, or to a biological or cellular material.

[0045] As used herein, the term “functional” may be used to describe any modification of any molecule, biological material, or cellular material intended to achieve a specific, designated effect.

[0046] As used herein, the terms “nucleic acid sequence” and “polynucleotide” are used interchangeably to refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Therefore, these terms include, but are not limited to, polymers containing single-, double-, or multi-stranded DNA or RNA, genomic DNA, complementary DNA (cDNA), DNA-RNA hybrids, or purine and pyrimidine bases, or other natural, chemically or biochemically modified unnatural, or derivatized nucleotide bases. In certain embodiments, a polynucleotide includes and / or codes for messenger RNA (mRNA), short hairpin RNA, and / or small hairpin RNA. In one embodiment, a polynucleotide is either mRNA or codes for mRNA. In certain embodiments, a polynucleotide is double-stranded (ds)DNA, such as engineered dsDNA or double-stranded cDNA synthesized from single-stranded RNA.

[0047] The terms “polynucleotide” and “oligonucleotide” are used interchangeably and refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides, ribonucleotides, or their analogues. Polynucleotides can have any three-dimensional structure and may perform any known or unknown function. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogues. Modifications to the nucleotide structure, if present, may be conjugated before or after the assembly of the polynucleotide. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeling components. This term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or requested, any embodiment disclosed herein that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or expected to make up the double-stranded form.

[0048] A polynucleotide consists of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and, if the polynucleotide is RNA, uracil (U) instead of thymine. Therefore, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be entered into a database on a computer with a central processing unit and used in bioinformatics applications such as functional genomics and homology searches.

[0049] As used herein with respect to nucleic acids such as DNA or RNA, the terms “isolated” and “recombinant” refer to molecules and polypeptides, respectively, that have been isolated from other DNA or RNA present in the natural source of a macromolecule. The term “isolated or recombinant nucleic acid” means that it includes nucleic acid fragments that do not exist naturally as fragments and are not found in nature. The term “isolated” is also used to refer to polynucleotides, polypeptides, and proteins, meaning that it includes both polypeptides and recombinant polypeptides that have been isolated and purified from other cellular proteins. In other embodiments, the term “isolated or recombinant” means isolated from cells or other components, including cells, tissues, polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof, which normally associate in nature. For example, isolated cells are cells isolated from tissues or cells of a different phenotype or genotype. Isolated polynucleotides are isolated from 3' and 5' consecutive nucleotides that normally associate in their natural or natural environment, for example, on chromosomes. As will be apparent to those skilled in the art, polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof that exist unnaturally do not require “isolation” to distinguish them from their naturally occurring counterparts.

[0050] As used herein, the terms “manipulated,” “synthetic,” “recombinant,” and “unnaturally occurring” are interchangeable and refer to intentional human manipulation, e.g., modification from its naturally occurring form and / or optimization of its sequence.

[0051] The terms “equivalent” or “biological equivalent” are used interchangeably when referring to specific molecules, biological materials, or cellular materials, and are intended to have minimal homology while still maintaining the desired structure or functionality (e.g., having similar functional activity). When referring to an equivalent or biological equivalent of a reference polypeptide, protein, or polynucleotide, it should be understood that the equivalent or biological equivalent has an enumerated structural relationship to the reference polypeptide, protein, or polynucleotide, and has equivalent or substantially equivalent biological activity. For example, non-limiting examples of equivalent polypeptides, proteins, or polynucleotides include polypeptides, proteins, or polynucleotides that have at least 60%, or alternatively at least 65%, or alternatively at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% or 97% identity with respect to them, or with respect to the reference polypeptide, polynucleotide, or protein sequence over the length of the reference polypeptide, polynucleotide, or protein sequence, respectively. Alternatively, in one embodiment, the equivalent polypeptide is encoded by a polynucleotide or its complement that hybridizes to a polynucleotide encoding such a reference polypeptide sequence under high stringency conditions and has substantially equivalent or equivalent biological activity. High stringency conditions are described herein and incorporated herein by reference. Alternatively, the equivalent is a polypeptide encoded by a polynucleotide or its complement that has at least 70%, or alternatively at least 75%, or alternatively 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or at least 97% sequence identity over the length of a polynucleotide equivalent to the reference polynucleotide, e.g., a wild-type polynucleotide. Such an equivalent polypeptide has the same or similar biological activity as the reference polypeptide.

[0052] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 80%, 85%, 90%, or 95%) of “sequence identity” with respect to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same when comparing the two sequences over the length of the reference polynucleotide or polypeptide. Alignment and homology percentages or sequence identity can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987), Supplement 30, section 7.7.18, Table 7.7.1. In certain embodiments, default parameters are used for alignment. A non-exclusive exemplary alignment program is BLAST, which uses default parameters. Other exemplary programs include BLASTN and BLASTP, which use the following default parameters: Genetic code=Standard;Filter=None;Strand=Both;Cutoff=60;Expected=10;Matrix=BLOSUM62;Description=50 sequences;Sorting=HIGH SCORE;Database=Non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS Translation+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following internet address:ncbi.nlm.nih.gov / cgi-bin / BLAST. Sequence identity and percent identity can be determined by incorporating them into clustalW (available at web address:https: / / www.genome.jp / tools / clustalw, last accessed: March 25, 2024).

[0053] "Homologousness," "identity," or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Homologousness can be determined by comparing the positions in each sequence that can be aligned for comparison purposes. Molecules are homologous at a position if the positions in the comparison sequences are occupied by the same base or amino acid. The degree of homology between sequences correlates to the number of matching or homologous positions shared by the sequences. "Unrelated" or "non-homologous" sequences share less than 40% identity, or alternatively less than 25% identity, with one of the sequences of this disclosure. "Homologousness," "identity," or "similarity" can also refer to two nucleic acid molecules that hybridize under stringent conditions.

[0054] As used herein, the term “at least 90% identical” means that two sequences (polynucleotides or polypeptides) being compared are about 90% to about 100% identical. It also includes at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100% identical.

[0055] As used herein, the terms “retain,” “similar,” and “identical” are used interchangeably when describing the activity or functional activity of polynucleotides, proteins, and / or peptides, referring to the functional activity of at least about 20% (including, but not limited to, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or about 100%) of the activity of the reference protein, polynucleotide, and / or peptide.

[0056] Without explicit enumeration, unless otherwise intended, whereever this disclosure relates to a polypeptide, protein, polynucleotide, or antibody, it should be inferred that such equivalents or bioequivalents are intended within the scope of this disclosure. Where used herein, the term “its bioequivalent” is intended to be synonymous with “its equivalent” when referring to a reference protein, antibody, fragment, polypeptide, or nucleic acid, and is intended to have minimal homology while still maintaining the desired structure or functionality. Unless specifically enumerated herein, any polynucleotide, polypeptide, or protein described herein is also intended to include its equivalents. In another aspect, where referring to an antibody or fragment thereof, its equivalent competes for the binding of the antibody or antigen-binding fragment to its antigen under a competitive ELISA assay.

[0057] Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonds between the bases of nucleotide residues. Hydrogen bonds can arise through Watson-Crick base pairing, Hoogsteen bonds, or any other sequence-specific mode. The complex may consist of two strands forming a double-stranded structure, three or more strands forming a multi-stranded complex, a single self-hybridization strand, or any combination thereof. Hybridization reactions may constitute a step in a broader process, such as initiating a PCR reaction or enzymatic cleavage of polynucleotides by ribozymes.

[0058] Examples of stringent hybridization conditions include incubation temperatures of approximately 25°C to 37°C, hybridization buffer concentrations of approximately 6×SSC to 10×SSC, formamide concentrations of approximately 0% to 25%, and washing solutions of approximately 4×SSC to 8×SSC. Examples of moderate hybridization conditions include incubation temperatures of approximately 40°C to 50°C, buffer concentrations of approximately 9×SSC to 2×SSC, formamide concentrations of approximately 30% to 50%, and washing solutions of approximately 5×SSC to 2×SSC. Examples of highly stringent conditions include incubation temperatures of approximately 55°C to 68°C, buffer concentrations of approximately 1×SSC to 0.1×SSC, formamide concentrations of approximately 55% to 75%, and washing solutions of approximately 1×SSC, 0.1×SSC, or deionized water. Generally, the hybridization incubation time is 5 minutes to 24 hours, accompanied by one, two, or more washing steps, with a washing incubation time of approximately 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be used.

[0059] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably and in their broadest sense to refer to compounds of two or more subunits of amino acids, amino acid analogs, or peptide mimetics. Subunits may be linked by peptide bonds. In other embodiments, subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may make up a protein or peptide sequence. As used herein, the term “amino acid” refers to any natural and / or unnatural or synthetic amino acid, including glycine, as well as both D and L optical isomers, amino acid analogs, and peptide mimetics.

[0060] As used herein, a contiguous amino acid sequence refers to a sequence having at least two amino acids. However, it should be noted that a contiguous amino acid sequence of a first and second part does not restrict the amino acid sequence to such that the first part is directly conjugated to the second part. The first part can also be joined to the second part via a third part, such as a linkage, and thus form a single contiguous amino acid sequence.

[0061] The polynucleotides disclosed herein may be delivered to cells or tissues using gene delivery vehicles. As used herein, “gene delivery,” “gene transfer,” “mRNA-based delivery,” and “transduction” are terms referring to the introduction of exogenous polynucleotides (sometimes referred to as “transgenes”) into host cells, regardless of the method used for introduction. Such methods include a variety of well-known techniques such as vector-mediated gene transfer (e.g., viral infection / transfection, or by various other protein-based or lipid-based gene delivery complexes, including, for example, protamine complexes, lipid nanoparticles, polymer nanoparticles, lipid-polymer hybrid nanoparticles, and inorganic nanoparticles, or combinations thereof), as well as techniques that facilitate the delivery of “naked” polynucleotides (such as electroporation, “gene gun” delivery, and various other techniques used for the introduction of polynucleotides). The introduced polynucleotide may be unmodified or may contain one or more modifications. For example, a modified mRNA may include ARCA capping, enzymatic polyadenylation (SEQ ID NO: 160) adding a tail of 100–250 adenosine residues, and substitution of one or both of the cytidines with 5-methylcytidine and / or substitution of uridine with pseudouridine. The introduced polynucleotide may be maintained stably or transiently in the host cell. Stable maintenance typically requires the introduced polynucleotide to contain a host cell-compatible origin of replication or to be incorporated into an extrachromosomal replicon (e.g., a plasmid) or a host cell replicon such as a nuclear or mitochondrial chromosome. As is known in the art and described herein, several vectors are known to be capable of mediating the transfer of genes into mammalian cells.

[0062] A plasmid is an extrachromosomal DNA molecule distinct from chromosomal DNA that can replicate independently of chromosomal DNA. Often, it is round and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a microbial population, typically offering a selective advantage under given environmental conditions. A plasmid may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively, the proteins produced may act as toxins under similar circumstances.

[0063] The "plasmids" used in genetic engineering are called "plasmid vectors." Many plasmids for such use are commercially available. The gene to be replicated is inserted into a multi-cloning site (MCS, or polylinker), which is a short region containing a copy of the plasmid containing the gene that makes cells resistant to a particular antibiotic, and several commonly used restriction sites (allowing for the easy insertion of DNA fragments at this location). Another primary use of plasmids is to produce large quantities of protein. In this case, researchers grow bacteria containing a plasmid that has the gene of interest. It is also possible to induce the bacteria to produce large quantities of protein from the inserted gene, just as bacteria produce protein to confer their antibiotic resistance.

[0064] "Yeast artificial chromosomes" or "YACs" refer to vectors used to clone large DNA fragments (over 100kb, up to 3000kb). These are artificially constructed chromosomes containing telomere origin sequences, centromere origin sequences, and replication origin sequences necessary for replication and storage within yeast cells. They are constructed using early circular plasmids, which are linearized using restriction enzymes, and then DNA ligases can be used to add the desired sequence or gene into the linear molecule using adherent ends. Yeast expression vectors such as YACs, YIp (yeast integration plasmids), and YEp (yeast episome plasmids) are very useful because yeast itself is a eukaryotic cell, allowing for the acquisition of eukaryotic protein products with post-translational modifications. However, YACs have been found to be less stable than BACs and can result in chimeric effects.

[0065] A "viral vector" is defined as a recombinantly produced virus or viral particle containing polynucleotides that is delivered to a host cell either in vivo, ex vivo, or in vitro.

[0066] Examples of viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors, herpes simplex virus vectors, and alphavirus vectors.

[0067] As used herein, the term “animal” refers to living multicellular vertebrate organisms, and this category includes, for example, mammals and birds. The term “mammal” includes both humans and non-human mammals, such as dogs, cattle, cats, monkeys, horses, or mice.

[0068] In one embodiment, the "subject" or "patient" to whom a therapy such as anti-JMAL therapy or a combination of anti-JMAL therapy and an immune checkpoint inhibitor is administered is preferably a mammal such as a non-primate (e.g., cattle, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys or humans). The subject or patient may be a human, such as an adult patient or a pediatric patient.

[0069] An "effective dose" refers to the amount of a drug, or a combination of two or more drugs, that is sufficient to produce such treatment for a disease when administered to a mammal or other subject. The effective dose will vary depending on the drug(s), the disease and its severity, as well as the age, weight, etc., of the subject being treated.

[0070] As used herein, biological samples or specimens may be obtained from subjects, cell lines, or cultured cells or tissues. Exemplary specimens include cell samples, tissue samples, tumor biopsies, blood and other fluid specimens of biological origin (but not limited to, ocular fluid (aqueous humor and vitreous humor), peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper's fluid or preejaculatory fluid, female ejaculate, sweat, tears, cystic fluid, pleural and peritoneal fluid, pericardial fluid, ascites, lymph, atherosclerotic fluid, chyle, bile, interstitial fluid, menstrual fluid, pus, sebum, vomit, vaginal secretions / flushing, synovial fluid, mucosal secretions, fecal water, pancreatic juice, sinus lavage fluid, bronchopulmonary aspirate, blastocyst cavity fluid. Examples include, but are not limited to, umbilical cord blood (fluid), or other similar samples. In some cases, the sample is a tumor / cancer biopsy.

[0071] A "solid tumor" is typically an abnormal mass of tissue that does not contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell type that forms them. Examples of solid tumors include sarcomas, carcinomas, and lymphomas. Solid tumors can be localized or metastatic.

[0072] In certain embodiments, the terms “disease,” “disorder,” and “condition” are used interchangeably herein to refer to cancer, a diagnosed cancerous condition, or a suspected cancerous condition.

[0073] As used herein, “cancer” is a disease condition characterized by the presence of cells exhibiting abnormal, uncontrolled replication in the subject and may be used interchangeably with the term “tumor.” In some embodiments, cancer is leukemia or lymphoma. “Cells associated with cancer” refers to cells of the subject exhibiting abnormal, uncontrolled replication. In certain embodiments, cancer is acute myeloid leukemia or acute lymphoblastic leukemia. As used herein, “leukemia” is a cancer of the blood or bone marrow characterized by an abnormal increase in immature white blood cells. A particular condition of acute myeloid leukemia (AML) (also known as acute myelogenous leukemia or acute myeloblastic leukemia) is a cancer of blood cells of myeloid origin, characterized by the rapid growth of abnormal myeloid cells that accumulate in the bone marrow and interfere with the production of normal blood cells. Acute lymphoblastic leukemia (ALL) (also known as acute lymphoblastic leukemia or acute lymphoblastic leukemia) is a specific condition of leukocyte cancer characterized by the overproduction and accumulation of malignant immature white blood cells (lymphoblasts) resulting in a lack of normal, healthy blood cells. As used herein, “lymphoma” is a cancer of the blood characterized by the development of hematological malignancies and enlarged lymph nodes, fever, excessive sweating, involuntary weight loss, itching, and persistent fatigue.

[0074] As used herein, “cancer” is a disease condition characterized by the presence of cells exhibiting abnormal, uncontrolled replication in the subject and may be used interchangeably with the term “tumor.” In some embodiments, cancer is leukemia or lymphoma. “Cells associated with cancer” refers to cells of the subject exhibiting abnormal, uncontrolled replication. In certain embodiments, cancer is acute myeloid leukemia or acute lymphoblastic leukemia. As used herein, “leukemia” is a cancer of the blood or bone marrow characterized by an abnormal increase in immature white blood cells. A particular condition of acute myeloid leukemia (AML) (also known as acute myelogenous leukemia or acute myeloblastic leukemia) is a cancer of blood cells of myeloid origin, characterized by the rapid growth of abnormal myeloid cells that accumulate in the bone marrow and interfere with the production of normal blood cells. Acute lymphoblastic leukemia (ALL) (also known as acute lymphoblastic leukemia or acute lymphoblastic leukemia) is a specific condition of leukocyte cancer characterized by the overproduction and accumulation of malignant immature white blood cells (lymphoblasts) resulting in a lack of normal, healthy blood cells. As used herein, “lymphoma” is a cancer of the blood characterized by the development of hematological malignancies and enlarged lymph nodes, fever, excessive sweating, involuntary weight loss, itching, and persistent fatigue.

[0075] "Cancer," also referred to herein as "tumor," is medically known as the uncontrolled division of abnormal cells in a part of the body, whether benign or malignant. In one embodiment, cancer refers to a broad group of diseases characterized by malignant neoplasms, unregulated cell division and growth, and invasion into nearby parts of the body. Non-exclusive examples of cancer include carcinomas, sarcomas, leukemias, and lymphomas, e.g., colon cancer, colorectal cancer, rectal cancer, stomach cancer, esophageal cancer, head and neck cancer, breast cancer, brain cancer, lung cancer, stomach cancer, liver cancer, gallbladder cancer, or pancreatic cancer. In one embodiment, the term "cancer" refers to solid tumors, which are usually masses of abnormal tissue that do not contain cysts or fluid areas, including but not limited to sarcomas, carcinomas, and certain lymphomas (such as non-Hodgkin lymphomas). In another embodiment, the term "cancer" refers to liquid cancers, which are cancers that reside in bodily fluids (such as blood and bone marrow), e.g., leukemia (a cancer of the blood) and certain lymphomas.

[0076] In addition or alternatively, cancer may refer to local cancer (an invasive malignant cancer that is confined entirely to the organ or tissue in which it originated), metastatic cancer (a cancer that spreads from its site of origin to another part of the body), non-metastatic cancer, primary cancer (a term used to describe the first cancer a subject experiences), secondary cancer (a metastasis from a primary cancer or a secondary cancer unrelated to the original cancer), advanced cancer, unresectable cancer, or recurrent cancer. As used herein, advanced cancer refers to cancer that has progressed after receiving one or more first-line, second-line, or third-line therapies.

[0077] As used herein, the term “extracellular matrix” (ECM) refers to a three-dimensional network of extracellular macromolecules, such as collagen, enzymes, and glycoproteins, that provide structural and biochemical support to surrounding cells. It is an essential component of the tumor microenvironment. While cancer development and progression are associated with increased adhesion and crosslinking of the ECM, the chemical and physical signals induced by the ECM are necessary for the proliferation and invasion of cancer cells. In one embodiment, the cancer ECM includes pericellular or tissue cells.

[0078] In certain embodiments, the terms “disease,” “disorder,” and “condition” are used interchangeably herein to refer to cancer, a diagnosed cancerous condition, or a suspected cancerous condition.

[0079] As used herein, the term “detectable marker” means at least one marker that can directly or indirectly generate a detectable signal. A non-exclusive list of markers includes, for example, enzymes that generate a detectable signal by colorimetric analysis, fluorescence, or luminescence, such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose-6-phosphate, dehydrogenase, chromophores such as fluorescence and luminescence dyes, groups having electron density detectable by electron microscopy, or groups detectable by electrical properties such as conductivity, amperometry, voltameometry, impedance, etc., where the molecule is large enough to induce a detectable change in its physical and / or chemical properties, and such detection is by optical methods such as diffraction, surface plasmon resonance, surface fluctuations, or changes in contact angle, or by atomic force spectroscopy, tunneling effect, or 32 P, 35 S, 89 Zr, or 125 This can be achieved by physical methods such as radioactive molecules like I.

[0080] As used herein, the term “purification marker” refers to at least one marker useful for purification or identification. A non-exclusive list of markers includes His, lacZ, GST, maltose-binding protein, NusA, BCCP, c-myc, CaM, FLAG, GFP, YFP, cherry, thioredoxin, poly(NANP), V5, Snap, HA, chitin-binding protein, Softag1, Softag3, Strep, or S-protein. Suitable direct or indirect fluorescent markers include FLAG, GFP, YFP, RFP, dTomato, cherry, Cy3, Cy5, Cy5.5, Cy7, DNP, AMCA, biotin, digoxigenin, Tamra, Texas Red, rhodamine, Alexa fluor, FITC, TRITC, or any other fluorescent dye or hapten.

[0081] As used herein, “immunophenotyping” refers to the analysis of heterogeneous populations of cells for the purpose of identifying the presence and proportion of different populations in a sample. Antibodies are used to identify cells by detecting specific antigens (referred to as markers) expressed by these cells. In one embodiment, a cell sample is characterized by immunophenotyping using techniques such as flow cytometry. In an alternative embodiment, characterization of the various cell types present in a cell sample (such as T cells, B cells, and their subsets) may be performed using any preferred methodology such as reverse transcriptase polymerase chain reaction (RT-PCR) or immunocytochemistry (IHC).

[0082] The terms “first-line,” “second-line,” “third-line,” “fourth-line,” and “fifth-line” refer to the order of treatments a patient receives. A first-line therapy regimen is the initial treatment given, while second-line or third-line therapies are given after the first-line or second-line therapy, respectively. The National Cancer Institute defines first-line therapy as “the initial treatment for the disease or condition.” In cancer patients, first-line treatment can be surgery, chemotherapy, radiation therapy, or a combination of these therapies. First-line therapy is also referred to by those skilled in the art as “first-line therapy and first-line treatment.” Typically, a patient is given a subsequent chemotherapy regimen because they did not show a positive clinical or nonclinical response to first-line therapy, or because first-line therapy was discontinued.

[0083] As used herein, the term “T cell” refers to a type of lymphocyte that matures in the thymus. T cells play a vital role in cell-mediated immunity and are distinguished from other lymphocytes, such as B cells, by the presence of T cell receptors on their cell surface. T cells may be isolated or obtained from commercially available sources. “T cells” include T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells, T regulatory cells (Treg), and tissue-resident memory T cells (T). RMThis includes all CD3-expressing immune cell types, including stem T cells and gamma-delta T cells. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells, and neutrophils, which can mediate cytotoxic responses. Non-exclusive examples of commercially available T cell lines include BCL2(AAA)Jurkat (ATCC® CRL-2902®), BCL2(S70A)Jurkat (ATCC® CRL-2900®), BCL2(S87A)Jurkat (ATCC® CRL-2901®), BCL2 Jurkat (ATCC® CRL-2899®), Neo Jurkat (ATCC® CRL-2898®), and the TALL-104 cytotoxic human T cell line (ATCC#CRL-11386).Further examples include mature T cell lines such as Deglis, EBT-8, HPB-MLp-W, HUT 78, HUT 102, Karpas 384, Ki 225, My-La, Se-Ax, SKW-3, SMZ-1, and T34, as well as immature T cell lines such as ALL-SIL, Be13, CCRF-CEM, CML-T1, DND-41, DU.528, EU-9, HD-Mar, HPB-ALL, H-SB2, HT-1, JK-T1, Jurkat, Karpas 45, KE-37, KOPT-K1, K-T1, L-KAW, Loucy, MAT, MOLT-1, MOLT 3, MOLT-4, MOLT 13, MOLT-16, MT-1, MT-ALL, P12 / Ichikawa, Peer, PER0117, PER-255, PF-382, PFI-285, RPMI-8402, ST-4, SUP-T1~T14, TALL-1, TALL-101, T ALL-103 / 2, TALL-104, TALL-105, TALL-106, TALL-107, TALL-197, TK-6, TLBR-1, -2, -3, and -4, CCRF-HSB-2 (CCL-120.1), J.RT3-T3.5 (ATCC TIB-153), J45.01 (ATCC CRL-1990), J.CaM1.6 (ATCC CRL-2063), RS4;11 (ATCC Examples of cutaneous T-cell lymphoma cell lines include, but are not limited to, CRL-1873, CCRF-CEM (ATCC CRM-CCL-119), and cutaneous T-cell lymphoma cell lines such as HuT78 (ATCC CRM-TIB-161), MJ[G11] (ATCC CRL-8294), and HuT102 (ATCC TIB-162). Null leukemia cell lines, including but not limited to REH, NALL-1, KM-3, and L92-221, are another commercially available immune cell source, as are cell lines derived from other leukemias and lymphomas such as K562 erythroleukemia, THP-1 monocytic leukemia, U937 lymphoma, HEL erythroleukemia, HL60 leukemia, HMC-1 leukemia, KG-1 leukemia, and U266 myeloma.Non-exclusive exemplary sources of such commercially available cell lines include the American Type Culture Collection or ATCC (http: / / www.atcc.org / ) and the German Collection of Microorganisms and Cell Cultures (https: / / www.dsmz.de / ).

[0084] The "frequency" of cells expressing any one specific molecule, biomarker, or antigen refers to the likelihood or proportion of cells expressing that molecule, biomarker, or antigen compared to the general population of T cells.

[0085] As used herein, the terms “antibody,” “antibodies,” and “immunoglobulin” include the whole antibody and any antigen-binding fragment or single-chain thereof. Therefore, the term “antibody” includes any protein or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule. The terms “antibody,” “antibodies,” and “immunoglobulin” also include, but are not limited to, Fab, Fab', F(ab)2, Fv, scFv, dsFv, Fd fragment, dAb, VH, VL, VhH, and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies, and kappabodies; multispecific antibody fragments formed from antibody fragments, as well as any isotype of immunoglobulin, including one or more isolated ones, and antibody fragments that retain specific binding to antigens. Such examples include, but are not limited to, the complementarity-determining region (CDR) of the heavy or light chain, or its ligand-binding portion, the variable region of the heavy or light chain, the constant region of the heavy or light chain, the framework (FR) region, or any portion thereof, at least one portion of a binding protein, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and a non-antibody protein. The variable regions of the heavy and light chains of an immunoglobulin molecule contain binding domains that interact with the antigen. The constant region of an antibody (Ab) can mediate the binding of the immunoglobulin to host tissue. The term "anti" when used before a protein name (e.g., anti-JAML) refers to a monoclonal or polyclonal antibody that binds to a particular protein and / or has affinity for a particular protein.

[0086] Antibodies can be polyclonal, monoclonal, multispecific (e.g., bispecific antibodies), humanized, and their respective antibody fragments, as long as they exhibit the desired biological activity. Antibodies can be isolated from any suitable biological source, such as mice, rats, sheep, and dogs, or produced by recombinant DNA.

[0087] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of an intact antibody. In some embodiments, the term "antibody fragment" refers to at least one portion of an intact antibody or a recombinant variant thereof and refers to the antigen-determining variable region of an intact antibody that is sufficient to confer recognition and specific binding of the antibody fragment to a target such as an antigen. Examples of antibody fragments include Fab, Fab’, F(ab’)2, Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies such as sdAb (V L or V H either), camelid VHH domains, and bispecific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region, as well as isolated CDRs or other epitope-binding fragments of an antibody, but are not limited thereto. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies). "Fab" means a monovalent antigen-binding fragment of an immunoglobulin composed of a portion of the light and heavy chains. F(ab’)2 means a bivalent antigen-binding fragment of an immunoglobulin containing both light chains and a portion of both heavy chains.

[0088] As used herein, the term "Fv fragment" or "variable domain fragment" refers to the VH and VL domains of an antibody that specifically bind to an antigen, and both domains together form an Fv fragment. In some embodiments, the Fv fragment is the V H and V LThis refers to antibody fragments containing domains, which are present in a single polypeptide chain. Generally, Fv fragment polypeptides allow for the formation of scFv. H Domain polypeptide and V L It further includes a polypeptide linker between the domain polypeptide and the polypeptide.

[0089] The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are sequentially linked via a short flexible polypeptide linker and can be expressed as a single polypeptide chain, and the scFv retains the specificity of the intact antibody from which it is derived. Where used herein, unless otherwise specified, scFv refers to, for example, the N-terminus and C-terminus of the polypeptide in either order. L and V H It may have a variable region, and scFv is V L -Linker-V H It may include, or V H -Linker-V L It may include.

[0090] As used herein, the term “antibody heavy chain” refers to the larger of the two polypeptide chains present in all antibody molecules in their naturally occurring three-dimensional structure.

[0091] As used herein, the term “antibody light chain” refers to the smaller of the two polypeptide chains present in all antibody molecules in their naturally occurring three-dimensional structure. Kappa (κ) and lambda (λ) light chains refer to the two main antibody light chain isotypes.

[0092] As used herein, the term “synthetic antibody” refers to an antibody produced using recombinant DNA technology, such as an antibody expressed by a bacteriophage. The term should also be interpreted as meaning an antibody produced by the synthesis of an antibody-encoding DNA molecule, in which the DNA molecule expresses an antibody protein, or an amino acid sequence that identifies an antibody, where the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence technologies that are available and well known in the art.

[0093] As used herein, the term “antibody variant” includes synthetic and manipulated forms of antibodies that are modified to not exist in nature, such as antibodies containing at least two heavy chain portions but not two complete heavy chains (e.g., domain deletion antibodies or minibodies), polyspecific forms of antibodies modified to bind to two or more different antigens or different epitopes on a single antigen (e.g., bispecific, tripspecific, etc.), and heavy chain molecules conjugated to scFv molecules. Furthermore, the term “antibody variant” also includes polyvalent forms of antibodies (e.g., trivalent, tetravalent, etc., antibodies that bind to three, four, or more copies of the same antigen).

[0094] As used herein, the terms “antigen” or “Ag” are defined as molecules that induce an immune response. This immune response may involve antibody production, activation of specific immune cells, or both. Those skilled in the art will understand that virtually any macromolecule, including any protein or peptide, can function as an antigen. Furthermore, antigens may originate from recombinant DNA or genomic DNA. Those skilled in the art will understand that any DNA contains nucleotide sequences or partial nucleotide sequences that encode a protein that induces an immune response, and therefore encodes an “antigen” as the term is used herein. Furthermore, those skilled in the art will understand that antigens do not need to be encoded by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention involves, but is not limited to, the use of partial nucleotide sequences of two or more genes, and that these nucleotide sequences are arranged in various combinations to induce a desired immune response. Furthermore, those skilled in the art will understand that antigens do not need to be encoded by a “gene” at all. It is readily apparent that antigens can be generated and synthesized, or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or other biological fluids.

[0095] The term “bispecific antibody” refers to an antibody that can simultaneously bind to two different receptors, epitopes, or antigens. The bispecific antibodies of this disclosure may target and bind to antigens on the same cell or on different cells. In some embodiments, the bispecific antibody binds to JAML and a second molecule on T cells. JAML may be expressed on T cells. In some embodiments, the second molecule is expressed on the same T cells. In one embodiment, the bispecific antibody disclosed in the claims increases target specificity to JAML-expressing T cells while limiting undesirable off-target activity. In some embodiments, the bispecificity binds to JAML on or on T cells or JAML-expressing T cells and modulates its expression or activity.

[0096] In some other embodiments described herein, bispecific antibodies bind to JAML and tumor or cancer antigens (including, but not limited to, tumor-associated antigens or tumor-specific antigens) expressed by tumor or cancer cells. The bispecific antibodies may simultaneously bind to and activate JAML-expressing T cells while also binding to tumor or cancer antigens. The activated T cells can then target tumor or cancer cells expressing the antigens. In some embodiments, the antigens are either overexpressed or specifically expressed by tumor or cancer cells. Therefore, the bispecific antibodies of this disclosure may be configured to bind to overexpressed or specifically expressed tumor or cancer antigens, including tumor-associated or tumor-specific antigens, which are identifiable markers of tumor or cancer cells, rather than to off-target cells and undesirable binding to the antigens.

[0097] As used herein, “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous antibody population. Each monoclonal antibody is highly specific because it is directed to a single determinant on an antigen. The antibody may be detectably labeled, for example, with a radioisotope, an enzyme that produces a detectable product, or a fluorescent protein. The antibody may also be further conjugated to other parts, such as members of a specific binding pair, for example, biotin (a member of the biotin-avidin specific binding pair). The antibody may also be conjugated to a solid support, including, but not limited to, a polystyrene plate or beads.

[0098] Monoclonal antibodies can be produced using hybridoma techniques or recombinant DNA methods known in the art. Hybridomas are cells produced in the laboratory from the fusion of antibody-producing lymphocytes with non-antibody-producing cancer cells, typically myeloma or lymphoma. Hybridomas proliferate and produce serial samples of a particular monoclonal antibody. Alternative techniques for generating or selecting antibodies include in vitro exposure of lymphocytes to the antigen of interest and screening of antibody display libraries in cells, phages, or similar systems.

[0099] As used herein, the term “human antibody” is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies disclosed herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo). However, as used herein, the term “human antibody” is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, is transplanted onto a human framework sequence. Therefore, as used herein, the term “human antibody” refers to proteins (e.g., CDR, framework, C). L , C H Domain (for example, C H1 , C H2 , C H3This refers to antibodies whose substantially all parts (VL, VH) are substantially non-immunogenic in humans, possessing only minor sequence changes or mutations. Similarly, antibodies designated for primates (monkeys, baboons, chimpanzees, etc.), rodents (mice, rats, rabbits, guinea pigs, hamsters, etc.), and other mammals designate such species, subgenus, genus, subfamily, and family-specific antibodies. Furthermore, chimeric antibodies include any combination of the above. Such changes or mutations, at will, retain or reduce immunogenicity in humans or other species compared to unmodified antibodies. For this reason, human antibodies are distinct from chimeric antibodies or humanized antibodies. It has been noted that human antibodies can be produced by non-human animals or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and / or light chain) genes. Furthermore, if a human antibody is a single-chain antibody, it may contain linker peptides not found in naturally occurring human antibodies. For example, Fv may contain linker peptides, such as 2 to about 8 glycine or other amino acid residues, that connect the variable regions of the heavy chain and the variable regions of the light chain. Such linker peptides are considered to be of human origin.

[0100] "Effector functions" derived from the interaction between the antibody Fc region and a specific Fc receptor include, but are not limited to, FcyR-mediated effector functions such as Clq binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, ADCC, and antibody-dependent cell-mediated phagocytosis (ADCP), as well as downregulation of cell surface receptors (e.g., B cell receptors; BCRs). Such effector functions generally require the Fc region to be combined with an antigen-binding domain (e.g., an antibody variable domain).

[0101] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of immunoglobulins. FcRs that bind to IgG antibodies include the FcyR family of receptors (including allelic variants and alternatively spliced ​​forms of these receptors). The FcyR family consists of three activating receptors (FcyRI, FcyRIII, and FcyRIV in mice; FcyRIA, FcyRIIA, and FcyRIIIA in humans) and one inhibitory receptor (FcyRIIb, or the equivalent FcyRIIB). Table 1 summarizes the various characteristics of human FcyRs. Most congenital effector cell types co-express one or more activating FcyRs and inhibitory FcyRIIb, while natural killer (NK) cells selectively express one activating Fc receptor (FcyRIII in mice, FcyRIIIA in humans) but do not express inhibitory FcyRIIb in mice or humans. Human IgG1 binds to most human Fc receptors and is considered equivalent to mouse IgG2a in terms of the types of activated Fc receptors it binds to.

[0102] The "Fc region" (fragment crystallizable region), "Fc domain," or "Fc" refers to the C-terminal region of an antibody's heavy chain that mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complementation system. Thus, the Fc region includes the constant region of the antibody excluding the first constant region immunoglobulin domain (e.g., CHI or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region includes the CH2 and CH3 constant domains in each of the two heavy chains of the antibody, while the IgM and IgE Fc regions include three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. In the case of IgG, the Fc region includes the immunoglobulin domains Cy2 and Cy3, as well as the hinge between Cyl and Cyl. While the boundaries of the Fc region of immunoglobulin heavy chains can vary, the human IgG heavy chain Fc region is typically defined as extending from the amino acid residue at position C226 or P230 (or the amino acid between these two) to the carboxyl terminus of the heavy chain, with numbering following the EU index as found in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD; see also Figures 3c-3f of U.S. Patent Application Publication No. 2008 / 0248028. The CH2 domain of the human IgG Fc region extends from approximately amino acid 231 to approximately amino acid 340 of Sequence ID No. 139, while the CH3 domain is located C-terminal to the CH2 domain within the Fc region, i.e., from approximately amino acid 341 to approximately amino acid 447 (including C-terminal lysine) of IgG. As used herein, the Fc region may be a native sequence Fc containing any allotype variant, or a variant Fc (e.g., a non-naturally occurring Fc). Fc may also refer to this region in isolation, or in the context of a protein polypeptide containing Fc, such as a “binding protein containing an Fc region” (e.g., an antibody or immunoadhesian), also referred to as an “Fc fusion protein.”

[0103] As used herein, a human antibody “derived” from a specific germline sequence if the antibody is obtained from a system that uses a human immunoglobulin sequence, for example, by immunizing transgenic mice possessing a human immunoglobulin gene, or by screening a human immunoglobulin gene library. A human antibody “derived” from a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody with the amino acid sequence of the human germline immunoglobulin. A selected human antibody typically contains amino acid residues that are at least 90% identical in amino acid sequence to the amino acid sequence encoded by the human germline immunoglobulin gene, and that identify the human antibody as human when compared with the germline immunoglobulin amino acid sequence of another species (e.g., mouse germline sequence). In certain particular cases, a human antibody may be at least 95%, or even more than 96%, 97%, 98%, or 99%, identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, human antibodies derived from a specific human germline sequence differ from the amino acid sequence encoded by the human germline immunoglobulin gene by 10 or fewer amino acids. In certain cases, human antibodies may differ from the amino acid sequence encoded by the germline immunoglobulin gene by 5 or fewer amino acids, or even 4, 3, 2, or 1 amino acid.

[0104] A "human monoclonal antibody" refers to a single antibody exhibiting binding specificity, possessing variable and constant regions derived from a human germline immunoglobulin sequence. This term also intends to refer to recombinant human antibodies. Methods for producing these antibodies are described herein.

[0105] In one embodiment, the antibodies used herein may be recombinant antibodies. As used herein, the term “recombinant human antibody” includes all human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies isolated from animals (e.g., mice) or hybridomas prepared therefrom that are transgenic or transchromosomal to human immunoglobulin genes, antibodies isolated from transformed host cells that express antibodies, such as transfectomas, antibodies isolated from recombinant combinatorial human antibody libraries, and antibodies prepared, expressed, produced or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, if transgenic animals for the human Ig sequence are used, in vivo somatic mutagenesis), and therefore the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but may not be naturally present in the human antibody germline repertoire in vivo. Methods for producing these antibodies are described herein.

[0106] As used herein, a chimeric antibody is typically an antibody in which light and heavy chain genes are constructed from antibody variable and constant region genes belonging to different species through genetic engineering.

[0107] As used herein, the terms “humanized antibody” or “humanized immunoglobulin” refer to a human / non-human chimeric antibody containing the minimum sequence derived from a non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the variable region of the recipient are replaced by residues from the variable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and capabilities. A humanized antibody may contain residues not found in the recipient antibody or donor antibody. A humanized antibody may also optionally include at least a portion of a human immunoglobulin that is a non-human antibody containing one or more amino acids in the framework region, constant region, or CDR, typically substituted with correspondingly positioned amino acids from the human antibody, in the immunoglobulin constant region (Fc). In general, humanized antibodies are expected to produce a reduced immune response in the human host compared to the non-humanized version of the same antibody. Humanized antibodies may have conserved amino acid substitutions that do not substantially affect antigen binding or other antibody functions. Examples of conservative substitutions include glycine-alanine, valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, serine-threonine, and asparagine-glutamine. The antibodies of this disclosure may bind to an antigen or molecule. In some embodiments, the antigen or molecule is expressed in target T cells, tumor cells, or tissues.

[0108] As used herein, the terms “polyclonal antibody” or “polyclonal antibody composition” refer to preparations of antibodies derived from different B cell lines. These are mixtures of immunoglobulin molecules secreted in response to specific antigens, each recognizing a different epitope.

[0109] As used herein, the term “antibody derivative” includes a full-length antibody or a fragment of an antibody in which one or more amino acids are chemically modified, for example, by alkylation, pegylation, acylation, esterification, or amide formation, to link the antibody to a second molecule. This includes, but is not limited to, pegylated antibodies, cysteine ​​pegylated antibodies, and their variants.

[0110] Complementarity-determining regions (CDRs) are parts of the variable regions of antibodies or T cell receptors produced by B cells and T cells, respectively, which bind to their specific antigens (also called epitopes). In certain embodiments, the terms “variable region” and “variable domain” are used interchangeably and refer to polypeptides of the light or heavy chain of an antibody in which the sequence of amino acid residues differs significantly from one antibody to another, determining the three-dimensional structure of the combination site that confers the antibody's specificity to a particular antigen. In further embodiments, the variable region is approximately 90 to 200 amino acid lengths, but is not limited to approximately 100 amino acid lengths, or alternatively approximately 110 amino acid lengths, or alternatively approximately 120 amino acid lengths, or alternatively approximately 130 amino acid lengths, or alternatively approximately 140 amino acid lengths, or alternatively approximately 150 amino acid lengths, or alternatively approximately 160 amino acid lengths, or alternatively approximately 170 amino acid lengths, or alternatively approximately 180 amino acid lengths, or alternatively approximately 190 amino acid lengths. In certain embodiments, as used herein, the variable region of an amino acid sequence refers to a variable region consisting of the first approximately 100 amino acids, or alternatively approximately 110 amino acids, or alternatively approximately 120 amino acids, or alternatively approximately 130 amino acids, or alternatively approximately 140 amino acids, or alternatively approximately 150 amino acid sequences (including or excluding signal peptides, where applicable).

[0111] A set of CDRs constitutes a paratope, also called an antigen-binding site, which is part of an antibody that recognizes and binds to an antigen. There are three CDRs (CDR1, CDR2, and CDR3) positioned discontinuously and optionally from the amino terminus to the carboxyl terminus on the amino acid sequence of the variable region of the antigen receptor, such as the heavy or light chain. As used herein, CDRn refers to a CDRn within or derived from the immunoglobulin chain, where n is selected from 1 to 3. In one embodiment, CDRLn refers to a CDRn within or derived from the light chain, where n is selected from 1 to 3, while CDRHn refers to a CDRn within or derived from the heavy chain, where n is selected from 1 to 3. In certain embodiments, a framework region (FR) refers to a portion of the variable region that is not a CDR. In certain embodiments, FRn refers to an FR within or derived from the heavy or light chain, where n is selected from 1 to 4. In certain embodiments, the variable regions include, essentially consist of, or even consist of, the following (optionally, in the order provided, and further optionally, from the amino terminus to the carboxyl terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0112] The variable region and / or CDR of an antibody or its fragment can be determined by a person skilled in the art, for example, using publicly available or commercially available tools. Non-exclusive examples of such tools include IgBlast (accessible from www.ncbi.nlm.nih.gov / igblast / ), Scaligner (available from drugdesigntech at www.scaligner.com / ), IMGT rules and / or tools (see, e.g., www.imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition.html, also accessible from www.imgt.org / ), Chothia Canonical Assignment (accessible from www.bioinf.org.uk / abs / chothia.html), Antigen receptor Numbering And Receptor Classification (ANARCI, accessible from opig.stats.ox.ac.uk / webapps / newsabdab / sabpred / anarci / ), and Kabat numbering method / scheme (e.g., Kabat, EA, et al., (1991) Sequences of Proteins of Immunological Examples include *Interest*, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242, or the Paratome web server (accessible from www.ofranlab.org / paratome / ; see Vered Kunik, et al, *Nucleic Acids Research*, Volume 40, Issue W1, 1 July 2012, Pages W521-W524).

[0113] Junctional adhesion molecule-like proteins (JAMLs) are human protein-coding genes (also known as AMICA1 and Gm638) that encode transmembrane proteins of the plasma membrane of leukocytes. JAMLs can regulate leukocyte migration and activation through interactions with the plasma membrane receptor coxsackievirus and adenovirus receptor CXADR, which are found on adjacent epithelial and endothelial cells. The interaction between JAML and CXADRs can mediate the activation of gamma-delta T cells present in epithelium. Upon binding to epithelial CXADRs, JAMLs can induce downstream cell signaling events in gamma-delta T cells via PI3 kinase and MAP kinase, resulting in T cell proliferation and production of cytokines and growth factors that stimulate epithelial tissue repair. JAMLs can also regulate leukocyte migration within epithelial and endothelial tissues through adhesion interactions with epithelial and endothelial CXADRs. Furthermore, the binding of JAMLs to their endogenous ligands, coxsackievirus and adenovirus receptors (CXADRs), provides a co-stimulus that leads to cell proliferation and the production of cytokines and growth factors. The protein sequence of JAML is publicly available at https: / / www.uniprot.org / uniprot / Q86YT9 (accessed October 28, 2021) and is reproduced below. [ka]

[0114] Generally, agonist antibodies have the ability to bind to and activate a target receptor in a manner that mimics the activity of a ligand. Agonist anti-junctional adhesion molecule-like protein (JAML) antibodies (anti-JAML antibodies) are intended to recognize and bind to the JAML protein, and are antibodies, antigen-binding fragments, derivatives, or other modifications described herein. The agonizing antibodies described herein can bind to JAML to increase, enhance, upregulate, and / or otherwise modulate the activity of the JAML receptor and / or JAML-expressing cells. Such activity may include proliferation and cellular signaling activity of cells expressing the JAML receptor. The agonist antibodies of this disclosure target JAML and bind specifically to JAML. In some embodiments, the agonist antibody binds to the receptor in a manner that mimics the binding of a physiological ligand resulting in antibody-mediated agonism. In some embodiments, treatment with an agonist anti-JAML antibody significantly inhibits tumor growth, which may be mediated through stimulation of tumor-infiltrating CD8+ T cells. Immunotherapy utilizing agonist antibodies, particularly those targeting trimer receptors such as 4-1BB or CD40, requires antibody crosslinking via Fcg receptors expressed on APCs for efficient T cell activation. Depending on the immunoglobulin (IgG) antibody subclass, they bind to Fcg receptors with different affinities and can therefore be crosslinked. (Li et al., 2011 Science. 2011 Aug 19;333(6045):1030-4, Nimmerjahn et al., 2005 Science 310 1510-1512, Claus et al., 2019 Sci Transl Med. 2019 Jun 12;11(496):eaav5989). Therefore, antibody subclass, as well as the availability, type, and expression level of Fcg receptors on APCs, are important determinants of the therapeutic efficacy of immunotherapy. However, the agonist activity of antibodies targeting costimulatory receptors depends on various factors and does not always require Fc crosslinking. Such factors include, but are not limited to, antibody affinity, Fc modifications such as glycotechnology or point mutations, antibody subclasses, and antigen expression.

[0115] "Immune response" broadly refers to the antigen-specific response of lymphocytes to foreign substances. The terms "immunogen" and "immunogenicity" refer to molecules that have the ability to induce an immune response. Not all immunogens are antigens, but not all antigens are immunogenic. The immune responses disclosed herein can be humoral (via antibody activity) or cell-mediated (via T cell activation). The response may occur in vivo or in vitro. Those skilled in the art will understand that a variety of macromolecules, including proteins, nucleic acids, fatty acids, lipids, lipopolysaccharides, and polysaccharides, may be immunogenic. Those skilled in the art will further understand that nucleic acids that encode molecules capable of inducing an immune response necessarily encode immunogens. Those skilled in the art will further understand that immunogens are not limited to full-length molecules and may include partial molecules.

[0116] As used herein, the terms “induction of an immune response” or “modulation of an immune response” are terms well understood in the art and refer to an increase or decrease in the immune response to an antigen (or epitope) (i.e., a T cell or antibody response) of at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 100-fold, at least about 500-fold, or at least about 1000-fold or more, which can be detected or measured by various methods known in the art. For example, the frequency or activity of antigen-specific T cells can be measured by several methods, including but not limited to flow cytometry, RNA sequencing, or in vitro assays.

[0117] As used herein, the term “modulation of activity” refers to an increase or decrease in the activity of a particular population of T cells involved in an immune response. Modulation of activity may be achieved by administering a drug containing an antibody that targets and binds to a particular T cell receptor in order to activate a population of T cells expressing that molecule. Modulation may occur when T cells are associated with a costimulatory ligand, an agonist antibody, or a cytokine. In some embodiments, modulation of activity may involve the administration of a drug that targets a molecule on T cells. In some embodiments, the molecule is JAML, and the drug is an antibody that targets JAML and thus activates JAML-expressing T cells.

[0118] "Immunotherapy" refers to a type of cancer treatment that uses a patient's own immune system to fight cancer, and includes, but is not limited to, physical interventions, chemicals, biomolecules or particles, cells, tissues or organs, or any combination thereof, that enhance, activate or initiate a patient's immune response against cancer. Non-exclusive examples of immunotherapies include antibodies, immunomodulators, checkpoint inhibitors, antisense oligonucleotides (ASOs), RNA interference (RNAi), clustered regular-spacing short palindromic repeat (CRISPR) systems, viral vectors, anti-cancer cell therapies (e.g., transplantation of optionally amplified and / or activated anti-cancer immune cells in vivo, or administration of immune cells expressing chimeric antigen receptors (CARs)), CAR therapies, and cancer vaccines.

[0119] As used herein, immune checkpoints refer to regulators and / or modulators of the immune system (such as immune responses, antitumor immune responses, nascent antitumor immune responses, antitumor immune cell responses, antitumor T cell responses, and / or antigen recognition by T cell receptors in the course of an immune response). Their interactions activate either inhibition or activation of immune signaling pathways. Thus, checkpoints may comprise one of two signals: stimulative immune checkpoints that stimulate an immune response, and inhibitory immune checkpoints that inhibit an immune response. In some embodiments, immune checkpoints are important for self-tolerance, which prevents the immune system from indiscriminately attacking cells. However, some cancers can protect themselves from attack by stimulating immune checkpoint targets. In some embodiments, immune checkpoints are located on T cells, antigen-presenting cells (APCs), and / or tumor cells.

[0120] As used herein, the terms “recombinant host cell,” “recombinant cell,” “engineered host cell,” or “engineered cell” mean a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to specific target cells but also to the offspring of such cells. Such offspring may not be identical to the parent cells in practice, as certain modifications may occur in later generations due to mutation or environmental influences, but they are still included within the scope of the terms “host cell” or “cell” as used herein. A host cell includes the offspring of a single host cell, which may not necessarily be exactly identical (morphologically or in total DNA complement) to the original parent cell due to natural, accidental, or intentional mutations and / or changes. A host cell includes a cell transfected or infected in vivo or in vitro with a recombinant vector, expression vector, or nucleic acid encoding an antibody of this disclosure. A host cell containing a recombinant vector, expression vector, or nucleic acid encoding an antibody disclosed herein may also be referred to as a “recombinant host cell,” “engineered host cell,” or “engineered cell.”

[0121] As used herein, the term “host cell” refers to a cell that may be used in a process for purifying an immunogenic protein or recombinant antibody in accordance with this disclosure. Such a host cell expresses the protein of interest (the antibody disclosed herein). Host cells may also be called protein-expressing cells. “Host cell” refers not only to a specific target cell but also to the offspring or potential offspring of such a cell. Such offspring may not be identical to the parent cell in practice because certain modifications may occur in later generations due to either mutation or environmental influences, but they are still included within the scope of the term as used herein.

[0122] The host cells described herein may be, but are not limited to, prokaryotic cells, eukaryotic cells, archaea, bacterial cells, insect cells, yeast, mammalian cells, and / or plant cells. The bacteria assumed to be host cells may be Gram-negative or Gram-positive, for example, Escherichia coli, Erwinia sp., Klebsellia sp., Lactobacillus sp., or Bacillus subtilis. In some embodiments, the host cell is a yeast cell. In those embodiments, the yeast host cell is selected from the group consisting of Saccharomyces cerevisiae, Hansenula polymorpha, and Pichia pastoris.

[0123] "Composition" means a combination of an activator and another compound or composition, either inactive (e.g., a detectable drug or label), active, such as an adjuvant, diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, etc., and includes a pharmaceutically acceptable carrier. The carrier also includes pharmaceutically acceptable excipients and additives, proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides, derivatized sugars such as alditol, aldonic acid, esterified sugars, and polysaccharides or sugar polymers), which may be present alone or in combination, and may be present alone or in combination in amounts of 1 to 99.99% by weight or volume. Exemplary protein excipients include serum albumins such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, and casein. Representative amino acid / antibody components that can also function in buffering capacity include alanine, arginine, glycine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, and aspartame. Carbohydrate excipients are also intended to be within the scope of this technology, and examples include monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, and sorbose; disaccharides such as lactose, sucrose, trehalose, and cellobiose; polysaccharides such as raffinose, melegitose, maltodextrin, dextran, and starch; and argitols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), and myo-inositol, but are not limited to these. A "pharmaceutical composition" is intended to include a combination of an active polypeptide, polynucleotide, or antibody with an inactive or active carrier, such as a solid support, making the composition suitable for in vitro, in vivo, or ex vivo diagnostic or therapeutic use.

[0124] "Pharmacologically acceptable carriers" refers to any diluent, excipient, or carrier that may be used in the compositions disclosed herein. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffers such as phosphates, saturated vegetable fatty acids such as glycine, sorbic acid, potassium sorbate, and protamine sulfate, water, salts, or partial glyceride mixtures of electrolytes, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin. Suitable pharmaceutical carriers are listed in Remington's Pharmaceutical Sciences, Mack Publishing Company, the standard reference literature in this field. These can be selected with respect to the intended dosage form, i.e., oral tablets, capsules, elixirs, syrups, etc., and are consistent with conventional pharmaceutical practices.

[0125] "Administration" can be completed in a single dose, either continuously or intermittently, throughout the course of treatment. Methods for determining the most effective means of administration and dosage are known to those skilled in the art and vary depending on the composition used in the therapy, the purpose of the therapy, the target cells to be treated, and the subject being treated. Single or multiple administrations can be performed at dose levels and patterns selected by the treating physician. Suitable dosage formulations and methods for administering the drug are known in the art. The route of administration can also be determined, and methods for determining the most effective route of administration are known to those skilled in the art and vary depending on the composition used in the therapy, the purpose of the therapy, the health status or disease stage of the subject being treated, and the target cells or tissues. Non-limiting examples of routes of administration include oral administration, intranasal administration, injection, and topical application.

[0126] The agents of this disclosure can be administered for therapeutic purposes by any preferred route of administration. It will also be understood that the optimal route of administration may vary depending on the recipient's condition and age, as well as the disease being treated.

[0127] The term "effective dose" refers to the amount sufficient to achieve the desired effect. In the context of therapeutic or prophylactic use, the effective dose will depend on the type and severity of the condition in question, as well as the characteristics of the individual subject, such as general health, age, sex, weight, and tolerance to the pharmaceutical composition. With respect to immunogenic compositions, in some embodiments, the effective dose will depend, in addition to the factors mentioned above, on the intended use, the degree of immunogenicity of the particular antigenic compound, and the health / responsiveness of the subject's immune system. Those skilled in the art will be able to determine the appropriate amount according to these and other factors.

[0128] For in vitro applications, in some embodiments, the effective dose will depend on the size and nature of the application in question. It will also depend on the nature and sensitivity of the in vitro target, as well as the method of use. Those skilled in the art will be able to determine the effective dose based on these and other considerations. Depending on the embodiment, the effective dose may consist of one or more doses of the composition.

[0129] As used herein, “concurrent use” means administering two compounds of the compositions herein in a single identical pharmaceutically acceptable form, or in two different pharmaceutically acceptable forms simultaneously.

[0130] As used herein, “separate use” means administering two compounds of the composition provided herein simultaneously in different pharmaceutically acceptable forms.

[0131] As used herein, “sequential use” refers to the sequential administration of two compounds of the composition according to this disclosure, each in a different pharmaceutical form.

[0132] The term "adjuvant" therapy refers to administering a therapy or chemotherapy regimen to a patient in addition to primary or initial treatment, such as after surgical removal of a tumor. Adjuvant therapy is typically performed to minimize or prevent the likelihood of cancer recurrence. Alternatively, "neoadjuvant" therapy typically refers to the administration of a preoperative therapy or chemotherapy regimen that attempts to shrink the tumor before surgical intervention to minimize the extent of tissue removed during the procedure. In addition or alternatively, the potential of such adjuvant therapy (i.e., sensitizing the subject to the primary therapy) may help the subject reach one or more clinical endpoints of cancer treatment.

[0133] The term “tissue” is used herein to mean any tissue of a living or dead organism, or any tissue derived from or designed to mimic a living or dead organism. Tissues may be healthy, diseased, and / or have genetic mutations. Biological tissues can include any single tissue (e.g., an assembly of cells that can be interconnected), or a group of tissues that make up an organ of an organism, or a part or region of the body. Tissues may contain homogeneous cellular material or may be complex structures, such as those found in regions of the body, including the chest, which may include, for example, lung tissue, skeletal tissue, and / or muscle tissue. Exemplary tissues include, but are not limited to, those derived from the liver, lungs, thyroid gland, skin, pancreas, blood vessels, bladder, kidneys, brain, bile ducts, duodenum, abdominal aorta, iliac veins, heart, and intestines, and any combination thereof.

[0134] As used herein, “treating” or “curing” a disease in a subject means (1) preventing the development of symptoms or disease in a subject that is predisposed to the disease or has not yet shown symptoms; (2) inhibiting or preventing the onset of the disease; or (3) restoring or causing regression of the disease or symptoms of the disease. As understood in the art, “treatment” is an approach to obtain a beneficial or desired outcome, including clinical outcomes. For the purposes of this art, beneficial or desired outcomes, whether detectable or undetectable, include, but are not limited to, one or more of the following: relief or recovery of one or more symptoms; reduction of the degree of a condition (including disease); a stable (i.e., non-worsening) stage of a condition (including disease); delay or slowing of a condition (including disease); progression, recovery, or relief of a condition (including disease); a stage, and remission (partial or complete), whether detectable or undetectable. In one embodiment, the term “treatment” excludes prevention.

[0135] The compositions used in accordance with this disclosure may be packaged in dosing unit form to facilitate administration and ensure uniformity of dosage. The terms “unit dose” or “dosage” refer to physically distinct units suitable for use in a subject, each unit containing a predetermined amount of the composition calculated to produce a desired response in relation to its administration (i.e., appropriate route and regimen). Depending on both the number of treatments and the unit dose, the amount administered depends on the desired outcome and / or protection. The exact amount of the composition also depends on the physician’s judgment and is unique to each individual. Factors influencing the dose include the subject’s physical and clinical condition, the route of administration, the intended goal of the treatment (symptom relief versus cure), as well as the potency, stability, and toxicity of the particular composition. When formulated, the solution may be administered in a form compatible with the dosing formulation and in a therapeutically or prophylactically effective amount. The formulation is readily administered in various dosage forms, such as the types of injectable solutions described herein.

[0136] As used herein, the term “single-chain antibody” refers to an antibody formed by recombinant DNA techniques in which immunoglobulin heavy and light chain fragments are linked to an Fv region via an engineered range of amino acids. Various methods for generating single-chain antibodies are known, including those described in U.S. Patent No. 4,694,778, Bird, Science 242:423-442 (1988), Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988), Ward et al., Nature 334:54454 (1989), and Skerra et al., Science 242:1038-1041 (1988).

[0137] As used herein, the term “specifically binding” means, with respect to an antibody, an antibody or its binding fragment (e.g., Fv fragment or scFv) that recognizes a specific antigen but substantially does not recognize or bind to other molecules in the sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, such cross-reactivity itself does not change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allele forms of the antigen. However, such cross-reactivity itself does not change the classification of the antibody as specific. In some examples, the terms “specific binding” or “specifically binding” can be used to refer to the interaction between an antibody, protein, chimeric antigen receptor, or peptide and a second chemical species, where the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) for the chemical species, for example, a chimeric antigen receptor generally recognizes and binds to a specific protein structure rather than a protein. If the antibody is specific to epitope "A", the presence of molecules containing epitope A (or free, unlabeled A) in a reaction involving labeled "A" and the antibody reduces the amount of labeled A bound to the antibody.

[0138] Forms for implementing this disclosure In some embodiments, the antibodies or antibody fragments disclosed herein include heavy chain complementarity-determining regions 1-3 (CDRH1-3) and light chain complementarity-determining regions 1-3 (CDRL1-3) selected from Table 2 or Table 5. In some embodiments, the combinations of CDRH1-3 and CDRL1-3 are selected from a single row in Table 2 or Table 5, or their respective equivalents.

[0139] In some embodiments, the antibodies or antibody fragments disclosed herein include, or essentially consist of, or consist of, a heavy chain variable sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity to the heavy chain variable amino acid sequences of Table 1 or Table 3, and / or a light chain variable sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity to the light chain variable amino acid sequences of Table 1 or Table 3. In further embodiments, the equivalent has the identity percentages described, but retains 100% identity for each CDR region of the reference antibody or fragment.

[0140] In some embodiments, the Fv fragment further comprises a linker domain for generating a single-stranded variable fragment (scFv). The scFv may be obtained by linking the VH and VL domains by a linker in a single polypeptide. In one embodiment, the linker domain is operably linked to the heavy-chain variable domain and the light-chain variable domain. In some embodiments, the light-chain and heavy-chain variable regions are sequentially linked via a short flexible polypeptide linker and can be expressed as a single-stranded polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. In some embodiments, the linker may have a length of 10 to 25 amino acid residues, more preferably 15 to 20 amino acid residues, or most preferably 18 to 20 amino acid residues.

[0141] In one embodiment, the flexible polypeptide linker includes, but is not limited to, (Gly4 Ser)4 (SEQ ID NO: 162) or (Gly4Ser)3 (SEQ ID NO: 163). In another embodiment, the linker includes multiple repeats of (Gly2Ser), (GlySer), or (Gly3Ser) (SEQ ID NO: 164). This may include a 15-mer (G4S)3 (SEQ ID NO: 163), an 18-mer GGSSRSSSSGGGGSGGGG (SEQ ID NO: 165), or a 20-mer (G4S)4 (SEQ ID NO: 162). In some other embodiments, the linker may contain the peptide sequences GSTSGSGKPGSGEGSTKG (SEQ ID NO: 166) (L1), KESGSVSSEQLAQFRSLD (SEQ ID NO: 167) (L2), EGKSSGSGSESKST (SEQ ID NO: 168) (L3), (GGGGS)3 (SEQ ID NO: 163) (L4), and GSAGSAAGSGEF (SEQ ID NO: 169) (L5). See Navabi P et al. Designing and generating a single-chain fragment variable (scFv) antibody against IL2Rα (CD25): An in silico and in vitro study. Iran J Basic Med Sci. 2021 Mar;24(3):360-368.

[0142] While the linker described above is provided, those skilled in the art will understand that the scFv fragment of this disclosure may utilize any conventional linker that can be used to connect the VH and VL domains so as to maintain the binding activity of the variable domain.

[0143] Humanized antibodies JAML-specific humanized antibodies are disclosed herein, as shown in the experimental examples section provided below. Specific examples of humanized antibodies include the following heavy and light chain sequences.

[0144] Humanized IgG1 parental E10 sequence-HC MDPKGSLSWRILLFLSLAFELSYGEVQLQESGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWIGEIYPRSGNTYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFC ARERYYGSSYAFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 135) Signal peptide MDPKGSLSWRILLFLSLAFELSYG (Sequence ID 170) Variable region EVQLQESGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWIGEIYPRSGNTYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARERYYGSSYAFDYWGQGTTLTVSS(Sequence ID 20) [Table 6]

[0145] Humanized IgG1 parental E10 sequence-LC METDTLLLWVLLLWVPGSTGDIVLTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 139) Signal peptide METDTLLLWVLLLWVPGSTG (Sequence ID 171) Variable region DIVLTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK (Sequence ID 55) [Table 7]

[0146] Hu E10 HC1 MDPKGSLSWRILLFLSLAFELSYGQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGMSWVRQAPGQRLEWMGEIYPRSGNTYYSQKFQGRVTITADTSASTAYMELSSLRSEDTAVYYC ARERYYGSSYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 136) Signal peptide MDPKGSLSWRILLFLSLAFELSYG (Sequence ID 170) Variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGMSWVRQAPGQRLEWMGEIYPRSGNTYYSQKFQGRVTITADTSASTAYMELSSLRSEDTAVYYCARERYYGSSYAFDYWGQGTLVTVSS(Sequence ID 172) [Table 8]

[0147] DHu E10 LC1 METDTLLLWVLLLWVPGSTGDIVMTQSPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 140) Signal peptide METDTLLLWVLLLWVPGSTG (Sequence ID 171) Variable region DIVMTQSPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIK (Sequence ID 173) [Table 9]

[0148] Hu E10 HC2 MDPKGSLSWRILLFLSLAFELSYGQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGEIYPRSGNTYYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYC ARERYYGSSYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 137) Signal peptide MDPKGSLSWRILLFLSLAFELSYG (Sequence ID 170) Variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGEIYPRSGNTYYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARERYYGSSYAFDYWGQGTLVTVSS(Sequence ID 174) [Table 10]

[0149] Hu E10 LC2 METDTLLLWVLLLWVPGSTGDVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 141) Signal peptide METDTLLLWVLLLWVPGSTG (Sequence ID 171) Variable region DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKLEIK (Sequence ID 175) [Table 11]

[0150] Hu E10 HC3 MDPKGSLSWRILLFLSLAFELSYGQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGMSWVRQAPGQGLEWMGEIYPRSGNTYYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYC ARERYYGSSYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 138) Signal peptide MDPKGSLSWRILLFLSLAFELSYG (Sequence ID 170) Variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGMSWVRQAPGQGLEWMGEIYPRSGNTYYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARERYYGSSYAFDYWGQGTLVTVSS(Sequence ID 176) [Table 12]

[0151] Hu E10 LC3 METDTLLLWVLLLWVPGSTGDVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLNWFQQRPGQSPRLLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 142) Signal peptide METDTLLLWVLLLWVPGSTG (Sequence ID 171) Variable region DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLNWFQQRPGQSPRLLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIK (Sequence ID 177) [Table 13]

[0152] Fc mutation In some embodiments, an antibody or fragment thereof, or an encoding nucleic acid, is conjugated or bound to a label or therapeutic agent. In one embodiment, the antibody is an IgG antibody conjugated to a label and / or conjugated to a therapeutic agent. In some embodiments, the antibody is IgG. In some embodiments, the antibody is a recombinant IgG antibody. In some embodiments, the antibody is an antibody fragment containing a mutated Fc moiety to alter (eliminate or enhance) the FcR interaction. In one embodiment, the mutated Fc increases the half-life of the antibody disclosed herein and / or increases the therapeutic efficacy. In one embodiment, the mutated Fc moiety includes LALA, LALA PG, N297, GASD / ALIE, DHS, YTE, or LS mutations. In some embodiments, the Fc moiety is modified to alter, eliminate, or enhance the FcR interaction, such as enzymatic or chemical addition or removal, or expression of glycans in cell lines manipulated with a defined glycosylation pattern. In some embodiments, the antibody contains a YTE mutation.

[0153] This disclosure also aims at isotype modification. Different functionalities can be achieved by modifying the Fc region to have different isotypes. For example, modification to IgG1 can increase antibody-dependent cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve valency.

[0154] Alternatively or in addition, it may be useful to combine amino acid modifications with one or more further amino acid modifications that alter the complementarity-dependent cytotoxicity (CDC) function of the Fc region of C1q-binding and / or IL-23p19-binding molecules. A binding polypeptide of particular interest may bind to C1q and exhibit complementarity-dependent cytotoxicity. Existing C1q-binding polypeptides, optionally further possessing CDC-mediating ability, may be modified to enhance one or both of these activities. Amino acid modifications that alter C1q and / or modify its complementarity-dependent cytotoxicity are described in WO2000 / 0042072.

[0155] For example, the Fc region of an antibody can be designed with altered effector function by modifying C1q binding and / or FcγR binding, thereby changing CDC activity and / or ADCC activity. “Effector function” refers to involvement in activating or reducing biological activity (e.g., in a target). Examples of effector functions, but not limited to, include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and downregulation of cell surface receptors (e.g., B cell receptors; BCRs). Such effector functions may require the Fc region to be combined with a binding domain (e.g., an antibody-variable domain) and can be evaluated using various assays (e.g., Fc binding assays, ADCC assays, CDC assays, etc.).

[0156] For example, variant Fc regions of antibodies can be generated that have improved C1q binding and improved FcγRIII binding (e.g., having both improved ADCC activity and improved CDC activity). Alternatively, if it is desired that effector function be reduced or eliminated, the variant Fc region can be manipulated with reduced CDC activity and / or reduced ADCC activity. In other embodiments, only one of these activities may be increased, and optionally, the other activity may be reduced (e.g., to generate an Fc region variant that has improved ADCC activity but reduced CDC activity (and vice versa).

[0157] Fc mutations can also be introduced and manipulated to alter their interaction with the neonatal Fc receptor (FcRn) and improve their pharmacokinetic properties. A set of human Fc variants with improved binding to FcRn has been described (Shields et al., (2001). High resolution mapping of the binding site on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and design of IgG1 variants with improved binding to the FcγR, (J. Biol. Chem. 276:6591-6604). Several methods are known that can result in increased half-life, including amino acid modifications that can be generated by techniques including alanine scanning mutagenesis, random mutagenesis, and screening, to evaluate binding to the neonatal Fc receptor (FcRn) and / or in vivo behavior (Kuo and Aveson, (2011)). One of the amino acid mutations to be mutated may be selected using computational strategies followed by mutagenesis.

[0158] In some embodiments, the antibody or antibody fragment further includes a label. In some embodiments, the antibody is a chimeric antibody, a bispecific antibody, a humanized antibody, a human antibody, or a fully human antibody.

[0159] In some embodiments, the anti-JAML antibody of the present invention has an Fc that binds to FcyRIIb or has enhanced binding to FcyRIIb, which can provide enhanced agonism. See, for example, WO2012 / 087928, Li & Ravetch (2011) Science 333:1030, Wilson et al. (2011) Cancer Cell 19:101, and White et al. (2011) J.Immunol. 187:1754. The variable regions described herein may be linked to an Fc variant that enhances affinity to the inhibitory receptor FcyRIIb, for example, to enhance apoptosis induction or adjuvant activity. See Li & Ravetch (2012) Proc. Nat I Acad. Sci (USA) 109:10966, U.S. Patent Application Publication No. 2014 / 0010812. Such variants may provide antibodies with immunomodulatory activity associated with FcyRIIb+ cells, including, for example, B cells and monocytes. In some embodiments, the Fc variant provides selectively enhanced affinity for FcyRIIb compared to one or more activating receptors. Such variants may also exhibit enhanced FcK-mediated crosslinking, resulting in enhanced therapeutic efficacy. Modifications for altering binding to FcyRIb include one or more modifications at positions selected from the group consisting of 234, 235, 236, 237, 238, 239, 266, 267, 268, 271, 325, 326, 327, 328, 330, and 332. In some embodiments, the Fc region includes one or more mutations selected from G237D, P238D, H268D, P271G, and A330R.

[0160] In other embodiments, exemplary substitutions for enhancing FcyRIIb affinity include, but are not limited to, 234D, 234E, 234F, 234W, 235D, 235F, 235R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, and 332E of SEQ ID NO: 152. Exemplary substitutions include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y. Other Fc variants for enhancing binding to FcyRIIb include 235Y-267E, 236D-267E, 239D-268D, 239D-267E, 267E-268D, 267E-268E, and 267E-328F. Specifically, the S267E, G236D, S239D, L328F, and I332E variants of human IgG1 (including the S267E-L328F bivariate) are particularly useful for specifically enhancing affinity to inhibitory FcyRIIb receptors. Chu et al. (2008) Mol.Immunol. 45:3926, U.S. Patent Application Publication No. 2006 / 024298, WO2012 / 087928.

[0161] Among these, modifications that improve binding selectivity to FcγRIIb compared to FcγRIIa(R type) are preferred, and modifications that improve binding selectivity to FcγRIIb compared to FcγRIIa(H type) are even more preferred. A preferred example of amino acid substitution modification is SEQ ID NO: 152 A modification that replaces Gly (ranked 237th) with Trp. A modification that replaces Gly (ranked 237th) with Phe. A modification that replaces Pro (ranked 238th) with Phe. A modification that replaces Asn (ranked 325th) with Met. A modification that replaces Ser (ranked 267th) with Ile. A modification to replace Leu (ranked 328th) with Asp. A modification that replaces Ser, ranked 267th, with Val. A modification that replaces Leu (ranked 328th) with Trp. A modification that replaces Ser (ranked 267th) with Gln. A modification that replaces Ser (ranked 267th) with Met. A modification that replaces Gly (ranked 236th) with Asp. A modification that replaces Ala, ranked 327th, with Asn. A modification that replaces Asn (ranked 325th) with Ser. A modification that replaces Leu (ranked 235th) with Tyr. A modification that replaces Val (ranked 266th) with Met. A modification that replaces Leu (ranked 328th) with Tyr. A modification that replaces Leu (ranked 235th) with Trp. A modification that replaces Leu (ranked 235th) with Phe. A modification that replaces Ser (ranked 239th) with Gly. A modification that replaces Ala at position 327 with Glu. A modification that replaces Ala (ranked 327th) with Gly. A modification that replaces Pro (ranked 238th) with Leu. A modification that replaces Ser (ranked 239th) with Leu. A modification that replaces Leu (ranked 328th) with Thr. A modification that replaces Leu (ranked 328th) with Ser. A modification that replaces Leu (ranked 328th) with Met. A modification that replaces Pro (ranked 331st) with Trp. A modification that replaces Pro (ranked 331st) with Tyr. A modification that replaces Pro (ranked 331st) with Phe. A modification that replaces Ala (ranked 327th) with Asp. A modification that replaces Leu (ranked 328th) with Phe. A modification that replaces Pro (ranked 271st) with Leu. A modification that replaces Ser at position 267 with Glu. A modification that replaces Leu (ranked 328th) with Ala. A modification that replaces Leu (ranked 328th) with Ile. A modification that replaces Leu (ranked 328th) with Gln. A modification that replaces Leu (ranked 328th) with Val. A modification that replaces Lys (ranked 326th) with Trp. A modification that replaces Lys (ranked 334th) with Arg. A modification that replaces His (ranked 268th) with Gly. A modification to replace His (ranked 268th) with Asn. A modification that replaces Ser at rank 324 with Val. A modification that replaces Val, ranked 266th, with Leu. A modification that replaces Pro (ranked 271st) with Gly. A modification that replaces Ile (ranked 332nd) with Phe. A modification that replaces Ser at rank 324 with Ile. A modification to replace the 333rd ranked "glu" with "Pro". A modification to replace Tyr at rank 300 with Asp. A modification to replace Ser (ranked 337th) with Asp. A modification that replaces Tyr at rank 300 with Gln. A modification that replaces Thr (ranked 335th) with Asp. A modification that replaces Ser (ranked 239th) with Asn. A modification that replaces Lys (ranked 326th) with Leu. A modification that replaces Lys (ranked 326th) with Ile. A modification that replaces Ser at position 239 with Glu. A modification that replaces Lys (ranked 326th) with Phe. A modification that replaces Lys (ranked 326th) with Val. A modification that replaces Lys (ranked 326th) with Tyr. A modification to replace Ser (ranked 267th) with Asp. A modification to replace Lys (ranked 326th) with Pro. A modification that replaces Lys (ranked 326th) with His. A modification that replaces Lys (ranked 334th) with Ala. A modification that replaces Lys (ranked 334th) with Trp. A modification that replaces His (ranked 268th) with Gln. A modification that replaces Lys (ranked 326th) with Gln. A modification that replaces Lys at position 326 with Glu. A modification that replaces Lys (ranked 326th) with Met. A modification that replaces Val, ranked 266th, with Ile. A modification that replaces Lys at position 334 with Glu. A modification that replaces Tyr at position 300 with Glu. A modification that replaces Lys (ranked 334th) with Met. A modification that replaces Lys (ranked 334th) with Val. A modification that replaces Lys (ranked 334th) with Thr. A modification that replaces Lys (ranked 334th) with Ser. A modification that replaces Lys (ranked 334th) with His. A modification that replaces Lys (ranked 334th) with Phe. A modification that replaces Lys (ranked 334th) with Gln. A modification to replace Lys (ranked 334th) with Pro. A modification that replaces Lys (ranked 334th) with Tyr. A modification that replaces Lys (ranked 334th) with Ile. A modification that replaces Gln (ranked 295th) with Leu. A modification that replaces Lys (ranked 334th) with Leu. A modification to replace Lys (ranked 334th) with Asn. A modification that replaces His (ranked 268th) with Ala. A modification to replace Ser, ranked 239th, with Asp. A modification that replaces Ser, ranked 267th, with Ala. A modification that replaces Leu (ranked 234th) with Trp. A modification that replaces Leu (ranked 234th) with Tyr. A modification that replaces Gly, ranked 237th, with Ala. A modification that replaces Gly (ranked 237th) with Asp. A modification that replaces Gly (ranked 237th) with Glu. A modification that replaces Gly (ranked 237th) with Leu. A modification that replaces Gly (ranked 237th) with Met. A modification that replaces Gly, ranked 237th, with Tyr. A modification that replaces Ala (ranked 330th) with Lys. A modification that replaces Ala (ranked 330th) with Arg. A modification that replaces Glu at position 233 with Asp. A modification to replace His (ranked 268th) with Asp. A modification that replaces His at position 268 with Glu. A modification to replace Lys (ranked 326th) with Asp. Modification to replace Lys (ranked 326th) with Ser (EU numbering), Modification to replace Lys (ranked 326th) with Thr (EU numbering), Modification to replace Val (ranked 323rd) with Ile (EU numbering), Modification to replace Val (ranked 323rd) with Leu (EU numbering), A modification that replaces Val (ranked 323rd) with Met. A modification that replaces Tyr (ranked 296th) with Asp. A modification that replaces Lys (ranked 326th) with Ala. A modification to replace Lys at rank 326 with Asn, and One modification involves replacing Ala, ranked 330th, with Met.

[0162] Furthermore, an example of a preferred amino acid substitution among these modifications is the substitution of Gly at position 237 with Trp. A modification that replaces Gly (ranked 237th) with Phe. A modification that replaces Ser, ranked 267th, with Val. A modification that replaces Ser (ranked 267th) with Gln. A modification to replace His (ranked 268th) with Asn. A modification that replaces Pro (ranked 271st) with Gly. A modification that replaces Lys (ranked 326th) with Leu. A modification that replaces Lys (ranked 326th) with Gln. A modification that replaces Lys at position 326 with Glu. A modification that replaces Lys (ranked 326th) with Met. A modification to replace Ser, ranked 239th, with Asp. A modification that replaces Ser, ranked 267th, with Ala. A modification that replaces Leu (ranked 234th) with Trp. A modification that replaces Leu (ranked 234th) with Tyr. A modification that replaces Gly, ranked 237th, with Ala. A modification that replaces Gly (ranked 237th) with Asp. A modification that replaces Gly (ranked 237th) with Glu. A modification that replaces Gly (ranked 237th) with Leu. A modification that replaces Gly (ranked 237th) with Met. A modification that replaces Gly, ranked 237th, with Tyr. A modification that replaces Ala (ranked 330th) with Lys. A modification that replaces Ala (ranked 330th) with Arg. A modification that replaces Glu at position 233 with Asp. A modification to replace His (ranked 268th) with Asp. A modification that replaces His at position 268 with Glu. A modification to replace Lys (ranked 326th) with Asp. A modification that replaces Lys (ranked 326th) with Ser. A modification that replaces Lys (ranked 326th) with Thr. A modification that replaces Val, ranked 323rd, with Ile. A modification that replaces Val, ranked 323rd, with Leu. A modification that replaces Val (ranked 323rd) with Met. A modification that replaces Tyr (ranked 296th) with Asp. A modification that replaces Lys (ranked 326th) with Ala. A modification to replace Lys at rank 326 with Asn, and One modification involves replacing Ala, ranked 330th, with Met.

[0163] Enhanced specificity for FcyRIIb (distinguishing from FcyRIIaRi3i) may be obtained by adding P238D substitutions and other mutations (Mimoto et al. (2013) Protein.Eng.Des.& Selection 26:589, WO2012 / 1152410), as well as V262E and V264E (Yu et al. (2013) J.Am.Chem.Soc.135:9723, and WO2014 / 184545).

[0164] In certain embodiments, antibodies are modified to increase their biological half-life. Various approaches are possible. For example, this may be done by increasing the binding affinity of the Fc region to FcRn. In one embodiment, the antibody is modified within the CHI or CL region to contain a salvage receptor-binding epitope obtained from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Patents No. 5,869,046 and 6,121,022 by Presta et al. Other exemplary Fc variants that increase binding to FcRn and / or improve pharmacokinetic properties include substitutions at positions 259, 308, and 434, including, for example, 2591, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M. Other variants that increase Fc binding to FcRn include 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al, (2004), J. Biol. Chem. 279(8):6213-6216, Hinton et al. (2006) Journal of Immunology 176:346-356), 256A, 272A, 305A, 307A, 311A, 312A, 378Q, 380A, 382A, 434A (Shields et al, (2001) Journal of Biology Examples include Chemistry, 276(9):6591-6604), 252F, 252Y, 252W, 254T, 256Q, 256E, 256D, 433R, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H (Dall'Acqua et al. (2002) Journal of Immunology, 169:5171-5180, Dall'Acqua et al., (2006) Journal of Biological Chemistry 281:23514-23524). See U.S. Patent No. 8,367,805.

[0165] Modification of certain conserved residues in IgG Fc(1253, H310, Q311, H433, N434), such as the N434A variant (Yeung et al. (2009) J.Immunol. 182:7663), has been proposed as a way to increase FcRn affinity and thus increase the half-life of circulating antibodies. WO98 / 023289. Combinations of Fc variants including M428L and N434S have been shown to increase FcRn binding and increase serum half-life by up to 5 times. Zalevsky et al. (2010) Nat.Biotechnol. 28:157. Combinations of Fc variants including T307A, E380A, and N434A modifications also extend the half-life of IgG1 antibodies. Petkova et al. (2006) Int.Immunol. 18:1759. In addition, combinations of Fc variants, including the M252Y-M428L, M428L-N434H, M428L-N434F, M428L-N434Y, M428L-N434A, M428L-N434M, and M428L-N434S variants, have also been shown to extend the half-life. WO2009 / 086320.

[0166] Furthermore, combinations of Fc variants including M252Y, S254T, and T256E increase the half-life by nearly four times. Dall'Acqua et al. (2006) J Biol. Chem. 281:23514. Related IgGl modifications (M252Y-S254T-T256E-H433K-N434F) resulting in increased FcRn affinity but reduced pH dependence have been used to construct IgGl constructs ("MST-HN Abdeg") for use as competitors to prevent other antibodies from binding to FcRn, resulting in increased clearance of other antibodies, either endogenous IgG (e.g., autoimmune setting) or another exogenous (therapeutic) mAb. Vaccaro et al. (2005) Nat. Biotechnol. 23:1283, WO2006 / 130834. Other modifications to increase FcRn binding are described in Yeung et al (2010) J.Immunol. 182:7663-7671, 6,277,375, 6,821,505, WO97 / 34631, WO2002 / 060919.

[0167] In certain embodiments, hybrid IgG isotypes may be used to increase FcRn binding and potentially increase half-life. For example, an IgGl / IgG3 hybrid variant may be constructed by substituting the IgGl positions in the CH2 and / or CH3 regions with amino acids from IgG3 at different positions for the two isotypes. Thus, hybrid variant IgG antibodies may be constructed containing one or more substitutions, e.g., 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F. In other embodiments described herein, an IgGl / IgG2 hybrid variant may be constructed by substituting the IgG2 positions in the CH2 and / or CH3 regions with amino acids from IgGl at different positions for the two isotypes. Therefore, hybrid variant IgG antibodies may be constructed containing one or more substitutions, for example, one or more of the following amino acid substitutions: 233E, 234L, 235L, -236G (referring to a glycine insertion at position 236), and 327A. See U.S. Patent No. 8,629,113. Hybrids of IgG1 / IgG2 / IgG4 sequences have been generated to intentionally increase serum half-life and improve expression (U.S. Patent No. 7,867,491) (Sequence ID 18 as specified herein).

[0168] The serum half-life of the antibody of the present invention can also be increased by pegylation. Pegylation of an antibody can increase its biological (e.g., serum) half-life, for example. To pegylate an antibody, the antibody or a fragment thereof is reacted with a polyethylene glycol (PEG) reagent, such as a reactive ester or aldehyde derivative of PEG, typically under conditions in which one or more PEG groups bind to the antibody or antibody fragment. Preferably, pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG used to derivatize other proteins, such as mono(CI-CIO)alkoxy or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylation of proteins are known in the art and can be applied to the antibodies described herein. For example, see EP0154316 by Nishimura et al. and EP0401384 by Ishikawa et al.

[0169] Alternatively, in some situations, it may be desirable to decrease rather than increase the half-life of the antibody of the present invention. Modifications to the Fc of human IgG1, such as 1253A (Hornick et al. (2000) J. Nucl. Med. 41:355) and H435A / R, 1253A, or H310A (Kim et al. (2000) Eur. J. Immunol. 29:2819), can reduce FcRn binding and thus decrease the half-life (increase clearance) for use in situations where rapid clearance is desirable, such as medical imaging. See also Kenanova et al. (2005) Cancer Res. 65:622. Another means of enhancing clearance is to format the antigen-binding domain of the present invention as an antibody fragment lacking the ability to bind to FcRn, e.g., a Fab fragment. Such modifications can shorten the circulating half-life of the antibody from several weeks to several hours. Next, the half-life of the antibody fragment can be fine-tuned (increased) as needed using selective PEGylation of the antibody fragment. Chapman et al. (1999) Nat. Biotechnol. 17:780. The half-life of the antibody fragment may also be increased by fusing it with human serum albumin, for example, in a fusion protein construct. Yeh et al. (1992) Proc. Nat 7 Acad. Sci. (USA) 89:1904. Alternatively, a bispecific antibody may be constructed using a first antigen-binding domain of the present invention and a second antigen-binding domain that binds to human serum albumin (HSA). See International Patent Application Publication No. 2009 / 127691 and the patent references cited therein. Alternatively, the half-life can be increased by adding a specialized polypeptide sequence to the antibody fragment, for example, the "CTEN" polypeptide sequence. Schellenberger et al. (2009) Nat. Biotechnol. 27:1186, International Patent Application Publication No. 2010 / 091122. Additional Fc variant

[0170] When using the constant domain of IgG4, it is generally preferable to include the substitution S228P, which mimics the hinge sequence of IgGl and thereby stabilizes the IgG4 molecule, for example, by reducing Fab arm exchange between the therapeutic antibody and endogenous IgG4 in patients undergoing treatment. Labrijn et al. (2009) Nat. Biotechnol. 27:767, Reddy et al. (2000) J. Immunol. 164:1925. Potential protease cleavage sites in the hinge of the IgGl construct can be eliminated by D221G and K222S modifications, increasing antibody stability. See WO2014 / 043344.

[0171] The affinity and binding properties of an Fc variant to its ligand (Fc receptor) can be determined by various in vitro assay methods (biochemical or immunological assays) known in the art, including but not limited to equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetic methods (e.g., BIACORE® SPR analysis), as well as other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize labeling on one or more of the components being examined and / or employ various detection methods, including but not limited to chromogenic, fluorescent, luminescent, or isotopic labeling. A detailed description of binding affinity and kinetics can be found in Paul, WE, ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.

[0172] In further embodiments, antibody glycosylation is modified to increase or decrease effector function. For example, by mutating the conserved asparagine residue at position 297 (e.g., N297A), non-glycosylated antibodies lacking all effector function can be produced, thus eliminating complement and FcyRI binding. See also Bolt et al. (1993) Eur. J. Immunol. 23:403. Tao & Morrison (1989) J. Immunol. 143:2595 (removing glycosylation at position 297 using N297Q in IgG1).

[0173] Non-glycosylated antibodies generally lack effector function, but mutations can be introduced to restore this function. Non-glycosylated antibodies, such as those resulting from the N297A / C / D / or H mutation, or antibodies produced in non-glycosylated protein systems (e.g., E. coli), can be further mutated to restore FcyR binding (e.g., S298G and / or T299A / G / or H (WO2009 / 079242), or E382V and M428I (Jung et al. (2010) Proc. Nat I Acad. Sci. (USA) 107:604)).

[0174] The anti-inflammatory properties of IgG constructs can also be modified by altering the α2,6 sialyl content of the carbohydrate chain attached to Asn297 in the Fc region using glycotechnology, with increased proportion of the α2,6 sialylated form enhancing the anti-inflammatory effect. See Nimmerjahn et al. (2008) Ann. Rev. Immunol. 26:513. Conversely, a decrease in the proportion of antibodies with α2,6 sialylated carbohydrates may be useful when anti-inflammatory properties are undesirable. Methods for modifying the α2,6 sialylated content of antibodies, for example, by selective purification of the α2,6 sialylated form or by enzymatic modification, are provided in U.S. Patent Application Publication 2008 / 0206246. In other embodiments, the amino acid sequence of the Fc region may be modified to mimic the effect of α2,6 sialylation, for example, by including the F241 A modification. WO2013 / 095966.

[0175] antibody derivative One aspect of this disclosure provides “derivatives” of antibodies or antigen fragments described herein or disclosed in Table 4. The term “derivative” refers to an antibody or antigen-binding fragment that binds immunospecifically to an antigen but involves one, two, three, four, five, or more amino acid substitutions, additions, deletions, or modifications to the “parent” (or wild-type) molecule. Such amino acid substitutions or additions may introduce naturally occurring (i.e., DNA-encoded) or unnaturally occurring amino acid residues. The term “derivative” also encompasses, for example, variants having a modified CH1, hinge, CH2, CH3, or CH4 region, and forming antibodies such as those having a variant Fc region with enhanced or impaired effector or binding characteristics. The term "derivative" also includes non-amino acid modifications, such as glycosylation (e.g., modified mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-glycorneuraminic acid, etc.), acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, and amino acids that can be linked to cell ligands or other proteins.

[0176] In some embodiments, modified carbohydrate modifications modulate one or more of the following: antibody solubilization, facilitating subcellular transport, and antibody secretion, promoting antibody assembly, structural integrity, and antibody-mediated effector function. In certain embodiments, modified carbohydrate modifications enhance antibody-mediated effector function compared to antibodies lacking carbohydrate modifications. Carbohydrate modifications leading to altered antibody-mediated effector function are well known in the art. See, for example, Shields et al., J. Biool. Chem. 277(30):26733-26740 (2002) and Davies J. et al., Biotechnology & Bioengineering 74(4):288-294 (2001). Methods for altering carbohydrate content are also known to those skilled in the art. For example, see Wallick et al. J.Exp.Med.168(3):1099-1109(1988), Tao et al. J.Immunol.143(8):2595-2601(1989), Routledge et al.,Transplantation 60(8):847-53(1995), Elliott et al.,Nature Biotechnol.21:414-21(2003), and Shield et al. J.Biol.Chem.277(30):26733-26740(2002).

[0177] Derivative antibodies or antibody fragments may be generated in an engineered sequence or glycosylated state to confer a favorable level of activity in antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), or antibody-dependent complement deposition (ADCD) function, as measured by bead-based or cell-based assays, or by in vivo studies in animal models.

[0178] Derivative antibodies or antibody fragments may be modified by chemical modifications using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formulation, and metabolic synthesis of tunicamycin. In one embodiment, the antibody derivative has similar or identical function to the parent antibody. In another embodiment, the antibody derivative exhibits altered activity compared to the parent antibody. For example, the derivative antibody (or fragment thereof) may be able to bind more strongly to its epitope than the parent antibody, or may be more resistant to proteolysis.

[0179] Antibody conjugate In some embodiments, the antibody or antibody fragment of this disclosure may be conjugated to at least one drug to form an antibody conjugate. It is conventional to conjugate, covalently bond, or complex at least one desired molecule or part to an antibody molecule in order to increase its efficacy as a diagnostic or therapeutic agent. Such molecules or parts may, but are not limited to, at least one effector or reporter molecule. The effector molecule includes a molecule having a desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules conjugated to an antibody include toxins, antitumor agents, therapeutic enzymes, radionuclides, antivirals, chelators, cytokines, growth factors, and oligonucleotides or polynucleotides. In contrast, a reporter molecule is defined as any part that can be detected using an assay. Non-limiting examples of reporter molecules conjugated to an antibody include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles, or ligands, e.g., biotin.

[0180] Labeled antibody Antibody conjugates are generally preferred for use as diagnostic agents. Antibody diagnostic agents are generally classified into two classes: those for use in in vitro diagnostics such as various immunoassays, and those for use with in vivo diagnostic protocols commonly known as “antibody-directed imaging.” Many suitable imaging agents are known in the art, as are methods for attaching them to antibodies. See, for example, U.S. Patents 5,021,236, 4,938,948, and 4,472,509. The imaging portions used can be paramagnetic ions, radioisotopes, fluorochromes, NMR-detectable substances, and X-ray imaging agents. Examples of paramagnetic ions include chromium(III), manganese(II), iron(III), iron(II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), and / or erbium(III), with gadolinium being particularly preferred. Ions useful in other contexts such as X-ray imaging include, but are not limited to, lanthanum(III), gold(III), lead(II), and especially bismuth(III).

[0181] For radioisotopes used in therapeutic and / or diagnostic applications, examples include astatine 211, 14 carbon, 51 chromium, 36 chlorine, 57 cobalt, 58 cobalt, copper 67, 152 Eu, gallium 67, 3 hydrogen, iodine 123, iodine 125, iodine 131, indium 111, 59 iron, 32 phosphorus, rhenium 186, rhenium 188, 75 selenium, 35 sulfur, technicium 99m, and / or yttrium 90. 125I is often preferred for use in certain embodiments, and technicium 99m and / or indium 111 are also often preferred due to their suitability for low-energy and long-range detection. The radiolabeled monoclonal antibodies of this disclosure can be produced by methods well known in the art. For example, monoclonal antibodies can be iodized by contact with sodium iodide and / or potassium iodide, as well as a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent such as lactoperoxidase. Monoclonal antibodies according to this disclosure can be labeled with technetium-99m by a ligand exchange process, for example, by reducing pertechnate with a tin solution, chelating the reduced technetium onto a Sephadex column, and applying the antibody to this column. Alternatively, direct labeling techniques may be used, for example, by incubating pertechnate, a reducing agent such as SNC12, a buffer such as a sodium phthalate-potassium phthalate solution, and the antibody. Intermediate functional groups often used to conjugate radioisotopes present in antibodies as metal ions are diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0182] Fluorescent labels intended for use as conjugates include Alexa350, Alexa430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green488, Oregon Green500, Oregon Green514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and / or Texas Red.

[0183] The additional types of antibodies contemplated in this disclosure are primarily intended for in vitro use, and the antibodies are linked to secondary ligands and / or enzymes (enzyme tags) that produce a colored product upon contact with a chromogenic substrate. Examples of preferred enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase, or glucose oxidase. Preferred secondary ligands are biotin and avidin, as well as streptavidin compounds. The use of such labels is well known to those skilled in the art and is described, for example, in U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241.

[0184] Another known method for site-specific attachment of molecules to antibodies involves the reaction of the antibody with a hapten-based affinity label. Essentially, the hapten-based affinity label reacts with amino acids at the antigen-binding site, thereby disrupting this site and blocking the specific antigenic reaction. However, this may not always be advantageous as it results in the loss of antigen binding due to the antibody conjugate.

[0185] Molecules containing azide groups may be used to form covalent bonds to proteins via reactive nitrene intermediates generated by low-intensity ultraviolet light (Potter and Haley, Methods Enzymol. 1983;91:613-33). In particular, 2- and 8-azide analogs of purine nucleotides have been used as site-specific photoprobes for identifying nucleotide-binding proteins in crude cell extracts. 2- and 8-azide nucleotides have also been used to map nucleotide-binding domains of purified proteins and may be used as antibody conjugates.

[0186] Several methods for attaching or conjugating antibodies to their conjugate moieties are known in the art. Some attachment methods involve the use of metal chelate complexes with organic chelating agents such as diethylenetriaminepentaacetic anhydride (DTPA), ethylenetriaminetetraacetic acid, N-chloro-p-toluenesulfonamide, and / or tetrachloro-3α-6α-diphenylglycyryl-3 attached to the antibody (U.S. Patents 4,472,509 and 4,938,948). Monoclonal antibodies may also be reacted with enzymes in the presence of coupling agents such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with isothiocyanates. In U.S. Patent No. 4,938,948, imaging of breast tumors is achieved using a monoclonal antibody, and the detectable imaging portion is conjugated to the antibody using a linker such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.

[0187] In other embodiments, derivatization of immunoglobulins is contemplated by selectively introducing sulfhydryl groups into the Fc region of the immunoglobulin and using reaction conditions that do not modify the antibody binding site. Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity, and sensitivity (U.S. Patent No. 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, where the reporter or effector molecule is conjugated to carbohydrate residues within the Fc region, has also been disclosed in the literature (O’Shannessy et al., 1987. Methods. 99:153-191). This approach has been reported to produce diagnostically and therapeutically promising antibodies currently under clinical evaluation.

[0188] constant region of the antibody In one aspect, the technology provides an antibody or an antigen-binding fragment thereof comprising the heavy chain immunoglobulin variable domain (V H ) and the light chain immunoglobulin variable domain (V L ) disclosed herein, and the Fc domain of any isotype, such as, but not limited to, IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, and IgY. Non-limiting examples of constant region sequences include the following:

[0189] The constant region of the antibody can also vary. For example, antibodies having the Fc region of any isotype: IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), or IgM are provided. Non-limiting examples of constant region sequences include the following:

[0190] Human IgD constant region, Uniprot: P01880 (SEQ ID NO: 151) APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK, and its equivalents.

[0191] Human IgG1 constant region, Uniprot:P01857 (SEQ ID NO: 152) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK and its equivalents.

[0192] Human IgG2 constant region, Uniprot:P01859 (SEQ ID NO: 153) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and its equivalents.

[0193] Human IgG3 constant region, Uniprot:P01860 (SEQ ID NO: 154) ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK, and its equivalents.

[0194] Human IgM constant region, Uniprot:P01871 (SEQ ID NO: 155) GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSV FVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSF ASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY, and its equivalents.

[0195] Human IgG4 constant region, Uniprot:P01861 (SEQ ID NO: 156) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK, and its equivalents.

[0196] Human IgA1 constant region, Uniprot:P01876 (SEQ ID NO: 157) ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY, and its equivalents.

[0197] Human IgA2 constant region, Uniprot:P01877 (SEQ ID NO: 158) ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY, and its equivalents.

[0198] Human Ig kappa constant region, Uniprot:P01834 (SEQ ID NO: 214) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, and its equivalents.

[0199] nucleic acid In another aspect, the Disclosure provides isolated nucleic acid molecules comprising, essentially comprising, or comprising nucleotide sequences encoding CDRs, heavy chains, light chains, scFVs, antibodies, or antibody fragments disclosed herein, which are optionally detectably labeled.

[0200] In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a heavy chain variable sequence containing an amino acid sequence selected from SEQ ID NOs: 1-26 or 135-138. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a heavy chain variable sequence containing an amino acid sequence disclosed in Table 1 or Table 3. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a heavy chain variable sequence containing an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequences disclosed in Table 1 or Table 3. In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the nucleic acid sequences disclosed in Table 4.

[0201] In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a light chain variable sequence containing an amino acid sequence selected from SEQ ID NOs. 27-60 or 139-142. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a light chain variable sequence containing an amino acid sequence disclosed in Table 1 or Table 3. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a light chain variable sequence containing an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequences disclosed in Table 1 or Table 3. In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the nucleic acid sequences disclosed in Table 4.

[0202] In one embodiment, the Disclosure provides an isolated polynucleotide comprising a nucleic acid sequence encoding the heavy chain variable region of an antibody or antibody fragment disclosed herein and / or a nucleic acid sequence encoding the light chain variable region of an antibody or antibody fragment disclosed herein. In some embodiments, the isolated polynucleotide comprises a nucleic acid sequence encoding the heavy chain variable region of an antibody or antibody fragment disclosed in Table 1 or Table 3, and / or a nucleic acid sequence encoding the light chain variable region of an antibody or antibody fragment disclosed in Table 1 or Table 3. In some embodiments, the polynucleotide comprises the sequences provided in Table 3. In some embodiments, the isolated polynucleotides include nucleic acid sequences having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with nucleic acid sequences encoding amino acid sequences of the heavy chain variable region of an antibody or antibody fragment disclosed in Table 1, and / or nucleic acid sequences having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with nucleic acid sequences encoding amino acid sequences of the light chain variable region of an antibody or antibody fragment disclosed in Table 1 or Table 3.

[0203] In some embodiments, the nucleic acid includes a nucleotide sequence encoding a heavy chain complementarity determination region (CDRH1, CDRH2, and / or CDRH3). In some embodiments, the nucleic acid includes a nucleotide sequence encoding a heavy chain complementarity determination region that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the heavy chain complementarity determination region amino acid sequence. Combinations of CDRH1-3 may be selected from Table 2 or Table 5.

[0204] In some embodiments, the nucleic acid includes a nucleotide sequence encoding a light chain complementarity-determining region (CDRL1, CDRL2, and / or CDRL3). In some embodiments, the nucleic acid includes a nucleotide sequence encoding a light chain complementarity-determining region that has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the light chain complementarity-determining region amino acid sequence. Combinations of CDRL1-3 may be selected from Table 2 or Table 5.

[0205] The polynucleotide may be DNA or RNA, and may be operably ligated to expression elements for transcription, translation, or replication of the polynucleotide. Such elements may include, for example, promoter and enhancer elements, as are known in the art. The polynucleotide can be used for the recombinant production of polynucleotides or antibodies and fragments thereof, as disclosed herein.

[0206] vector In another aspect, the disclosure provides vectors comprising isolated nucleic acid molecules comprising, essentially consisting of, or comprising a nucleotide sequence encoding an antibody or antibody fragment disclosed herein. Retrovirus-derived vectors, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for the long-term, stable integration of the transgene and its proliferation in daughter cells. Lentiviral vectors have an additional advantage over onchoretrovirus-derived vectors, such as mouse leukemia virus, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the additional advantage of low immunogenicity.

[0207] In one embodiment, the vector containing the nucleic acid encoding the antibody or antibody fragment disclosed herein is DNA, RNA, plasmid, adenovirus vector, lentivirus vector, or retrovirus vector. The retrovirus vector may also be, for example, a gamma-retrovirus vector. The gamma-retrovirus vector may include, for example, a promoter, a packaging signal (w), a primer binding site (PBS), one or more (e.g., two) long-terminal repeats (LTRs), and the desired transgene (e.g., a gene encoding the antibody or antibody fragment disclosed herein). The gamma-retrovirus vector may lack viral structural genes such as gag, pol, and env. Exemplary gamma-retrovirus vectors include mouse leukemia virus (MLV), splenic focal-forming virus (SFFV), and myeloproliferative sarcoma virus (MPSV), and vectors derived therefrom. Other gamma-retrovirus vectors are described, for example, in Tobias Maetzig et al., Viruses 3(6):677-713 (2011).

[0208] In another embodiment, the vector containing the nucleic acid encoding the antibody or antibody fragment disclosed herein is an adenovirus vector (A5 / 35). In another embodiment, the expression of the nucleic acid encoding the antibody or antibody fragment disclosed herein can be achieved using transposons such as sleeping beauty, CRISPR, CAS9, and zinc finger nucleases. See, for example, June et al. Nature Reviews Immunology 9(10):704-716 (2009). In short, the expression of the natural or synthetic nucleic acid encoding the antibody or antibody fragment disclosed herein is typically achieved by operably ligating the nucleic acid encoding the antibody or antibody fragment polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector may be suitable for eukaryotic replication and incorporation. Typical cloning vectors contain transcription and translation termination factors, start sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence. The expression vectors disclosed herein may also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. For example, see U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466.

[0209] Nucleic acids can be cloned into several types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. Further, expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. See, for example, WO2001 / 096584, WO2001 / 029058, and U.S. Patent No. 6,326,193.

[0210] Several virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered either in vivo or ex vivo to the cells of interest. Several retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0211] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located 30–110 bp upstream of the initiation site, although some promoters have been shown to also contain functional elements downstream of the initiation site. The spacing between promoter elements is often flexible so that promoter function is preserved when elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to be able to activate transcription either cooperatively or independently. Vectors may also contain signal sequences to facilitate secretion, polyadenylation signals, and transcription termination factors (e.g., from the bovine growth hormone (BGH) gene), elements that enable episomal replication and replication in prokaryotes (e.g., from SV40 and ColE1 or others known in the art), and / or elements that enable selection (e.g., ampicillin resistance genes and / or zeosin markers).

[0212] To evaluate the expression of an antibody or antibody fragment, an expression vector introduced into a cell may contain either a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells subjected to viral vector-mediated transfection or infection. In other embodiments, the selectable marker may be supported on a separate DNA fragment and used in a simultaneous transfection procedure. Both the selectable marker and reporter genes can be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes such as Neo.

[0213] Reporter genes are used to identify potentially transfected cells and evaluate the functionality of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue, and whose expression manifests as a polypeptide with several readily detectable properties, such as enzymatic activity. The expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cell. Suitable reporter genes may include those encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and may be prepared using known techniques or commercially available. Generally, a construct with a minimum 5' adjacent region exhibiting the highest level of reporter gene expression is identified as a promoter. Such promoter regions may be ligated to the reporter gene and used to evaluate drugs for their ability to regulate promoter-driven transcription.

[0214] Method for producing antibodies Manipulated cells Aspects of this disclosure provide engineered cells comprising isolated nucleic acid molecules comprising, essentially comprising, or comprising nucleotide sequences encoding the antibodies or antibody fragments disclosed herein, or vectors comprising isolated nucleic acid molecules comprising nucleotide sequences encoding the antibodies or antibody fragments disclosed herein. In some embodiments, the engineered cells produce the antibodies or antibody fragments disclosed herein. In some embodiments, the antibodies or antibody fragments disclosed herein bind to a JAML protein. In some embodiments, the host cell or engineered cell comprises the vector disclosed herein. In one embodiment, the cell is a prokaryotic cell. In another embodiment, it is a eukaryotic cell, such as HEK293 cells.

[0215] Antibody production One aspect of the present disclosure provides a method for producing an antibody or antibody fragment, comprising culturing manipulated cells as disclosed herein and isolating an antibody or antibody fragment from the cultured cells, or essentially consisting of the same, or consisting of the same. A "monoclonal antibody" can also be isolated from a phage antibody library using techniques described, for example, Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0216] Methods for producing monoclonal antibodies generally begin along the same line as methods for preparing polyclonal antibodies. The first step in both of these methods is the immunization of a suitable host, or identification of a target with immunity resulting from a previous natural infection, or vaccination with an approved or experimental vaccine. As is well known in the art, a given composition for immunization may differ in its immunogenicity. Therefore, it is often necessary to enhance the host's immune system, as can be achieved by conjugating a peptide or polypeptide immunogen onto a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin, or rabbit serum albumin can also be used as carriers. Means for conjugating polypeptides onto carrier proteins are well known in the art and include glutaraldehyde, m-maleimidebencyl-N-hydroxysuccinimide ester, carbodiimide, and bis-biazoted benzidine. As is well known in the art, the immunogenicity of certain immunogen compositions can be enhanced by the use of nonspecific stimulants of the immune response known as adjuvants. Exemplary and preferred adjuvants in animals include complete Freund's adjuvants (nonspecific stimulants of the immune response containing dead Mycobacterium tuberculosis), incomplete Freund's adjuvants, and aluminum hydroxide adjuvants, while in humans, examples include alum, CpG, MFP59, and combinations of immunostimulatory molecules ("adjuvant systems" such as AS01 or AS03). Additional experimental forms of inoculation to induce JAML-specific B cells are possible, including nanoparticle vaccines or genetically coded antigens delivered as DNA or RNA genes via physical delivery systems (e.g., on lipid nanoparticles or gold nanoparticle gun beads) and delivered by needles, gene guns, or percutaneous electroporation devices.The antigen gene can also be carried so as to be encoded by a replicable viral vector such as an adenovirus, adeno-associated virus, poxvirus, herpesvirus, or alphavirus replicon, or by a deficient viral vector, or alternatively, by a virus-like particle.

[0217] In the case of human antibodies against natural pathogens, a preferred approach is to identify subjects exposed to the pathogen, for example, subjects diagnosed with a disease, or subjects vaccinated to generate protective immunity against the pathogen or to test the safety or efficacy of an experimental vaccine. Circulating anti-pathogen antibodies can be detected, and then antibodies encoding or producing B cells may be obtained from antibody-positive subjects.

[0218] The amount of immunogen composition used to produce polyclonal antibodies varies depending on the properties of the immunogen and the animals used for immunization. Various routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous, and intraperitoneal). Polyclonal antibody production can be monitored by sampling the blood of immunized animals at various points after immunization. A second booster injection may be given. The boosting and titration process is repeated until a suitable titer is achieved. When the desired level of immunogenicity is obtained, the immunized animals can be bled, and the serum can be isolated and stored, and / or the animals can be used to produce monoclonal antibodies.

[0219] Monoclonal antibodies produced by either means may be further purified, if necessary, by filtration, centrifugation, and various chromatographic methods such as FPLC or affinity chromatography. Monoclonal antibody fragments of this disclosure can be obtained from purified monoclonal antibodies by methods including digestion with enzymes such as pepsin or papain, and / or cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments incorporated in this disclosure can be synthesized using an automated peptide synthesizer.

[0220] Furthermore, it is intended that monoclonal antibodies can be generated using molecular cloning approaches. Single B cells identified as responding to infection or vaccination can be physically sorted using paramagnetic bead selection or flow cytometry sorting for plasmablasts or activated B cell markers, or memory B cells labeled with the antigen of interest. RNA can then be isolated from the single cells, and the antibody gene can be amplified by RT-PCR. Various single-cell RNA-seq methods are available for obtaining antibody variable genes from single cells. Alternatively, antigen-specific bulk sorted populations of cells can be separated into microvesicles, and matched heavy and light chain variable genes can be recovered from single cells using physical binding of heavy and light chain amplicons or common barcoding of heavy and light chain genes from vesicles. Matched heavy and light chain genes from single cells can also be obtained from a population of antigen-specific B cells by treating the cells with RT-PCR primers and cell-permeable nanoparticles having barcodes for marking transcripts with one barcode per cell. Antibody variable genes can also be isolated by RNA extraction of antibody genes obtained from hybridoma strains and RT-PCR, and cloned into immunoglobulin expression vectors. Alternatively, combinatorial immunoglobulin phagemide libraries are prepared from RNA isolated from cell lines, and phagemides expressing the appropriate antibodies are selected by panning using viral antigens. The advantages of this approach over conventional hybridoma techniques are that it can produce and screen approximately 104 times the number of antibodies in a single round, and novel specificities are generated by the combination of H and L chains, further increasing the chances of finding the appropriate antibody.

[0221] Other U.S. patents teaching antibody production useful for this disclosure include U.S. Patent No. 5,565,332 describing the production of chimeric antibodies using a combinatorial approach, U.S. Patent No. 4,816,567 describing recombinant immunoglobulin preparations, and U.S. Patent No. 4,867,973 describing antibody-therapeutic conjugates (each incorporated herein by reference).

[0222] In various embodiments, the sequence of a specified antibody may be manipulated for various reasons, such as improved expression, improved cross-reactivity, or reduced off-target binding. Modified antibodies can be prepared by any technique known to those skilled in the art, including expression by standard molecular biological techniques or chemical synthesis of polypeptides. Methods for recombinant expression are discussed elsewhere in this specification. The following is a general consideration of relevant target techniques for antibody engineering.

[0223] Recombinant full-length IgG antibodies can be produced by subcloning heavy and light chain Fv DNA from a cloning vector into an IgG plasmid vector and transfecting 293 (e.g., Freestyle) cells or CHO cells, collecting the antibodies, and purifying them from the 293 or CHO cell supernatant. Other suitable host cell lines include bacteria such as E. coli, insect cells (S2, Sf9, Sf29, High Five), plant cells (e.g., tobacco, with or without manipulation for human-like glycans), algae, or various non-human transgenic contexts such as mice, rats, goats, or cattle.

[0224] For both the purpose of subsequent antibody purification and host immunization, the expression of antibody-coding nucleic acids is also planned. The antibody-coding sequence can be RNA, such as native RNA or modified RNA. Modified RNA is intended to have certain chemical modifications that confer increased stability and lower immunogenicity to mRNA, thereby facilitating the expression of therapeutically important proteins. For example, N1-methyl-psoidouridine (N1mΨ) is superior in terms of translational ability to several other nucleoside modifications and their combinations. In addition to turning off immune / eIF2α phosphorylation-dependent inhibition of translation, incorporated N1mΨ nucleotides dramatically alter the dynamics of the translation process by increasing ribosome pausing and density of mRNA. Increased ribosome attachment of modified mRNA makes them more tolerant to initiation by preferring either ribosome reuse or novel ribosome recruitment on the same mRNA. Such modifications can be used to enhance antibody expression in vivo after RNA inoculation. RNA, whether natural or modified, may be delivered as naked RNA or in a delivery vehicle such as lipid nanoparticles.

[0225] Antibodies can be produced using DNA that encodes antibodies. The DNA is contained in an expression cassette that includes an active promoter in the host cell in which it is designed. The expression cassette is advantageously contained in a replicable vector such as a conventional plasmid or minivector. Vectors include and are intended to include viral vectors such as poxviruses, adenoviruses, herpesviruses, adeno-associated viruses, and lentiviruses. Replicons encoding antibody genes, such as alphavirus replicons based on VEE virus or Sindbis virus, are also intended. Delivery of such vectors can be carried out by needle via intramuscular, subcutaneous, or intradermal pathways, or by percutaneous electroporation if in vivo expression is desired.

[0226] Recombinant Antibody Expression. As described above, antibodies of the Technology can be produced by the application of recombinant DNA technology. Recombinant polynucleotides construct antibodies of the Technology and typically include an expression control sequence operably linked to the coding sequence of the antibody chain, including a naturally related or heterologous promoter region. Thus, another aspect of the Technology includes a vector containing one or more nucleic acid sequences encoding antibodies of the Technology. For the recombinant expression of one or more polypeptides of the Technology, nucleic acids containing all or part of the nucleotide sequences encoding the antibodies are inserted into a suitable cloning vector or expression vector (i.e., a vector containing the elements necessary for the transcription and translation of the inserted polypeptide coding sequence) by recombinant DNA techniques well known in the Art and detailed below. Methods for producing diverse vector populations are described in U.S. Patents 6,291,160 and 6,680,192 by Lerner et al.

[0227] Generally, expression vectors useful in recombinant DNA techniques are often in plasmid form. In this disclosure, “plasmid” and “vector” can be used interchangeably, as plasmids are the most commonly used form of vectors. However, this technique is intended to include other forms of expression vectors that are not technically plasmids, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses), which perform equivalent functions. Such viral vectors enable infection of a target and expression of a construct in that target. In some embodiments, the expression regulatory sequence is a eukaryotic promoter system in the vector that can transform or transfect eukaryotic host cells. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of antibody-encoding nucleotide sequences, and for the collection and purification of antibodies, e.g., cross-reactive antibodies. See generally US2002 / 0199213. These expression vectors are typically replicable within the host organism, either as episomes or as an integral part of host chromosomal DNA. Generally, expression vectors contain a selection marker, such as ampicillin resistance or hygromycin resistance, to enable the detection of cells transformed with a desired DNA sequence. Vectors can also encode signal peptides, such as peptide triases, useful for directing the secretion of extracellular antibody fragments. See U.S. Patent No. 5,576,195.

[0228] The recombinant expression vector of this technology comprises a nucleic acid encoding a protein having the binding properties of the antibody and its fragments disclosed herein, in a form suitable for nucleic acid expression in a host cell, meaning that the recombinant expression vector includes one or more regulatory sequences selected based on the host cell used for expression, which are operably ligated to the nucleic acid sequence to be expressed. In a recombinant expression vector, “operably ligated” is intended to mean that the target nucleotide sequence is ligated to the regulatory sequence(s) in a manner that enables the expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system, or in the host cell if the vector is introduced into a host cell). The term “regulatory sequence” is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct the constitutive expression of nucleotide sequences in many types of host cells and those that direct the expression of nucleotide sequences only in specific host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will understand that the design of expression vectors can depend on factors such as the selection of host cells to be transformed and the desired polypeptide expression level. Typical regulatory sequences useful as promoters for recombinant polypeptide expression (i.e., antibodies or fragments thereof) include, but are not limited to, the promoter of 3-phosphoglycerate kinase and other glycosphagocytes. Inducible yeast promoters include, among others, those from alcohol dehydrogenase, isocytochrome C, and enzymes involved in maltose and galactose utilization. In one embodiment, the polynucleotide encoding the antibody of this technology is operably ligated to the ara B promoter and is expressible in host cells. See U.S. Patent No. 5,028,530.The expression vector of this technology can be introduced into host cells to produce polypeptides or peptides, including fusion polypeptides encoded by the nucleic acids described herein.

[0229] Another aspect of this technology relates to antibody-expressing host cells containing nucleic acids encoding one or more antibodies or fragments thereof. Recombinant expression vectors of this technology can be designed for antibody expression in prokaryotic or eukaryotic cells. For example, antibodies can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), fungal cells, such as yeast, yeast cells, or mammalian cells. Suitable host cells are further discussed in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, recombinant expression vectors can be transcribed and translated in vitro, for example, using a T7 promoter control sequence and T7 polymerase. Methods useful for the preparation and screening of polypeptides with predetermined properties, such as antibodies, via the expression of stochastically generated polynucleotide sequences have been previously described. See U.S. Patent Nos. 5,763,192, 5,723,323, 5,814,476, 5,817,483, 5,824,514, 5,976,862, 6,492,107, and 6,569,641.

[0230] Polypeptide expression in prokaryotes is often performed in E. coli using vectors containing constitutive or inducible promoters that direct the expression of either fusion or non-fusion polypeptides. Fusion vectors add several amino acids to the polypeptide they encode, usually to the amino terminus of the recombinant polypeptide. Such fusion vectors typically serve three purposes: (i) to increase the expression of the recombinant polypeptide, (ii) to increase the solubility of the recombinant polypeptide, and (iii) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, in fusion expression vectors, proteolytic cleavage sites are introduced at the junction between the fusion site and the recombinant polypeptide to allow for the separation of the recombinant polypeptide from the fusion site after the purification of the fusion polypeptide. Such enzymes, and their homorecognition sequences, include factor Xa, thrombin, and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.), and pRIT5 (Pharmacia, Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose E-binding polypeptide, or polypeptide A to the target recombinant polypeptide, respectively.

[0231] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89). Methods for targeted assembly of different active peptide or protein domains to obtain multifunctional polypeptides via polypeptide fusion are described by Pack et al., U.S. Patent Nos. 6,294,353 and 6,692,935. One strategy to maximize recombinant polypeptide expression in E. coli, e.g., anti-L1-CAM antibody, is to express the polypeptide in a host bacterium in which the ability to proteolytically cleave the recombinant polypeptide is impaired. For example, see Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to modify the nucleic acid sequence of the expression vector so that each individual codon of each amino acid is preferentially utilized by the expression host, e.g., E. coli (see Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such modification of nucleic acid sequences in this technique can be performed using standard DNA synthesis techniques.

[0232] In another embodiment, the antibody expression vector is a yeast expression vector. Examples of vectors for expression in the yeast Saccharomyces cerevisiae include pYepSec1 (Baldari, et al., 1987. EMBO J.6:229-234), pMFa (Kurjan and Herskowitz, Cell 30:933-943, 1982), pJRY88 (Schultz et al., Gene 54:113-123, 1987), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp, San Diego, Calif.). Alternatively, anti-L1-CAM antibodies can be expressed in insect cells using a baculovirus expression vector. Examples of baculovirus vectors that can be used for the expression of polypeptides, such as anti-L1-CAM antibodies, in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., Mol. Cell. Biol. 3:2156-2165, 1983) and the pVL series (Lucklow and Summers, 1989, Virology 170:31-39).

[0233] In yet another embodiment, the nucleic acid encoding the antibody or a fragment of the Technology is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include, but are not limited to, pCDM8 (Seed, Nature 329:840, 1987) and pMT2PC (Kaufman, et al., EMBO J.6:187-195, 1987). When used in mammalian cells, the regulatory function of the expression vector is often provided by a viral regulatory element. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells useful for expressing the anti-L1-CAM antibody of the Technology, see, for example, Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0234] In another embodiment, recombinant mammalian expression vectors can direct nucleic acid expression in specific cell types (e.g., tissue-specific regulatory elements). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include albumin promoters (liver-specific; Pinkert, et al., Genes Dev. 1:268-277, 1987), lymphocyte-specific promoters (Calame and Eaton, Adv. Immunol. 43:235-275, 1988), T cell receptor promoters (Winoto and Baltimore, EMBO J. 8:729-733, 1989), and immunoglobulins (Banerji, et al., 1983. Cell 33:729-740, Queen and Baltimore, Cell 33:741-748, 1983), neuron-specific promoters (e.g., neuronal filament promoters; Byrne and Ruddle, Proc. Natl. Acad. Sci. USA 86:5473-5477, 1989), and pancreas-specific promoters (Edlund, et al.) Examples include mammary gland-specific promoters (e.g., whey promoter; U.S. Patent No. 4,873,316 and European Patent Publication No. 264,166). Developmentally regulated promoters, such as the mouse hox promoter (Kessel and Gruss, Science 249:374-379, 1990) and the α-fetal protein promoter (Campes and Tilghman, Genes Dev.3:537-546, 1989), are also included.

[0235] Another aspect of the present invention relates to host cells into which the recombinant expression vector of the present technology has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to specific target cells but also to the offspring or potential offspring of such cells. Such offspring may not be identical to the parent cells in practice because certain modifications may occur in later generations due to either mutation or environmental influences, but they are still included within the scope of the present terminology as used herein.

[0236] The host cell can be any prokaryotic or eukaryotic cell. For example, an antibody or fragment thereof can be expressed in bacterial cells such as E. coli, insect cells, yeast, or mammalian cells. Mammalian cells are suitable hosts for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes To Clones, (VCH Publishers, NY, 1987). Several suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, including Chinese hamster ovary (CHO) cell lines, various COS cell lines, HeLa cells, L cells, and myeloma cell lines. In some embodiments, the cells are non-human. Expression vectors for these cells may include expression regulatory sequences, e.g., origin of replication, promoter, enhancer, and necessary processing information sites, e.g., ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. See Queen et al., Immunol. Rev. 89:49, 1986. Exemplary regulatory sequences include promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, and bovine papillomavirus. Co et al., J Immunol. 148:1149, 1992. Other suitable host cells are known to those skilled in the art.

[0237] Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of techniques recognized in the art for introducing foreign nucleic acids (e.g., DNA) into host cells, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran mediated transfection, lipofection, electroporation, microparticle guns, or virus-based transfection. Other methods used to transform mammalian cells include the use of polyblens, protoplast fusion, liposomes, electroporation, and microinjection methods (generally, Sambrook et al., Molecular Cloning). Preferred methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals. Vectors containing the target DNA segment can be transferred to host cells by known methods, depending on the type of cell host.

[0238] For stable transfection of mammalian cells, it is known that only a small number of cells can incorporate foreign DNA into their genome, depending on the expression vector and transfection technique used. To identify and select these incorporates, genes encoding selectable markers (e.g., antibiotic resistance) are generally introduced into host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs such as G418, hygromycin, and methotrexate. Nucleic acids encoding selectable markers can be introduced into host cells on the same vector as those encoding antibodies or fragments thereof, or on separate vectors. Cells stably transfected with the introduced nucleic acids can be identified by drug selection (e.g., cells incorporating the selectable marker gene survive while other cells die).

[0239] Host cells containing the antibody or fragment thereof of the present technology, such as prokaryotic or eukaryotic host cells in culture, can be used to produce (i.e., express) the recombinant antibody or fragment thereof. In one embodiment, the method comprises culturing host cells (in which a recombinant expression vector encoding the antibody or fragment thereof has been introduced) in a suitable medium so that the antibody is produced. In another embodiment, the method further comprises the step of isolating the antibody from the medium or host cells. Once expressed, the antibody, e.g., a collection of the antibody or antibody-related polypeptide, is purified from the culture medium and host cells. The antibody can be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, etc. In one embodiment, the antibody is produced in a host organism by the method of Boss et al., U.S. Patent No. 4,816,397. Typically, the antibody chain is expressed in a signal sequence and therefore released into the culture medium. However, if the antibody chain is not spontaneously secreted by the host cells, the antibody chain can be released by treatment with a neutral detergent. The purification of recombinant polypeptides is well known in the art and includes ammonium sulfate precipitation, affinity chromatography, column chromatography, ion exchange purification, and gel electrophoresis (see, in general, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0240] Polynucleotides encoding antibodies, such as antibody-coding sequences, can be incorporated into transgenes for introduction into the genome of transgenic animals and subsequent expression in the milk of transgenic animals. See, for example, U.S. Patents 5,741,957, 5,304,489, and 5,849,992. Suitable transgenes include light and / or heavy chain coding sequences operably linked to promoters and enhancers from mammary gland-specific genes such as casein or β-lactoglobulin. For the production of transgenic animals, the transgene can be microinjected into fertilized oocytes or incorporated into the genome of embryonic stem cells, the nuclei of such cells being transferred to enucleated oocytes.

[0241] Single-chain antibodies. In one embodiment, the antibodies of this technology are single-chain antibodies. According to this technology, the technique can be adapted to produce single-chain antibodies specific to the JAML protein. Examples of techniques that can be used to produce single-chain Fv and antibodies of this technology include those described in U.S. Patents 4,946,778 and 5,258,498, Huston et al., Methods in Enzymology, 203:46-88, 1991, Shu, L. et al., Proc. Natl. Acad. Sci. USA, 90:7995-7999, 1993, and Skerra et al., Science 240:1038-1040, 1988.

[0242] Chimeric antibody. In one embodiment, the antibody of this technology is a chimeric antibody. In one embodiment of this technology, the donor and acceptor antibodies are monoclonal antibodies from different species. For example, the acceptor antibody is a mouse antibody (to minimize its antigenicity in the mouse animal model).

[0243] Recombinant antibodies, such as chimeric monoclonal antibodies containing both human and non-human portions, can be produced using standard recombinant DNA techniques and are within the scope of this technology. Chimeric antibodies can be used for several applications, including in vivo use of antibodies of this technology in human or non-human animals, and use of these agents in in vitro detection assays. Such chimeric monoclonal antibodies can be produced by recombinant DNA techniques known in the art.Such useful methods include, for example, International Application No. PCT / US86 / 02269, U.S. Patent No. 5,225,539, European Patent No. 184187, European Patent No. 171496, European Patent No. 173494, PCT International Publication No. WO86 / 01533, U.S. Patent No. 4,816,567, U.S. Patent No. 5,225,539, European Patent No. 125023, Better, et al., 1988. Science 240:1041-1043, Liu, et al., 1987. Proc. Natl. Acad. Sci. USA 84:3439-3443, Liu, et al., 1987. J. Immunol. 139:3521-3526, Sun, et al. al.,1987.Proc.Natl.Acad.Sci.USA 84:214-218, Nishimura,et al.,1987.Cancer Res.47:999-1005,Wood,et al.,1985.Nature 314:446-449,Shaw,et al.,1988.J.Natl.Cancer Inst.80:1553-1559, Morrison(1985)Science 229:1202-1207, Oi,et al.(1986)BioTechniques 4:214;Jones,et al.,1986.Nature 321:552-525,Verhoeyan,et al.,1988.Science 239:1534;Morrison,Science Examples of methods include, but are not limited to, those described in 229:1202,1985, Oi et al., BioTechniques 4:214,1986, Gillies et al., J.Immunol.Methods, 125:191-202,1989, U.S. Patent No. 5,807,715, and Beidler, et al., 1988. J.Immunol. 141:4053-4060.For example, antibodies can be used to produce chimerices using a variety of techniques, including CDR grafting (EP 0 239 400, WO91 / 09967, U.S. Patent Nos. 5,530,101, 5,585,089, 5,859,205, 6,248,516, EP460167), veneering, or resurfacing (EP 0 592 106, EP 0 519 596, Padlan EA, Molecular Immunology, 28:489-498, 1991, Studnicka et al., Protein Engineering 7:805-814, 1994, Roguska et al., PNAS 91:969-973, 1994), and chain shuffling (U.S. Patent No. 5,565,332).In one embodiment, a cDNA encoding a monoclonal antibody is digested with a restriction enzyme specifically selected to remove the sequence encoding the Fc constant region, and an equivalent portion of the cDNA encoding a different Fc constant region is substituted (Robinson et al., PCT / US86 / 02269, Akira et al., European Patent Application Nos. 184,187, Taniguchi, European Patent Application No. 171,496, Morrison et al., European Patent Application No. 173,494, Neuberger et al., WO86 / 01533, Cabilly et al., U.S. Patent No. 4,816,567, Cabilly et al., European Patent Application No. 125,023, Better et al. (1988) Science 240:1041-1043, Liu et al. (1987) Proc. Natl. Acad. Sci. USA See 84:3439-3443, Liu et al. (1987) J Immunol 139:3521-3526, Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218, Nishimura et al. (1987) Cancer Res 47:999-1005, Wood et al. (1985) Nature 314:446-449, and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559, U.S. Patent Nos. 6,180,370, 6,300,064, 6,696,248, 6,706,484, and 6,828,422.

[0244] CDR antibody. Generally, the donor and acceptor antibodies used to produce the antibody are monoclonal antibodies from different species, and typically, the acceptor antibody is a non-human antibody (to minimize its antigenicity in non-human animals). The graft is a single V of the acceptor antibody. H or V L It may be a single CDR (or part of a single CDR) within, or V H and V LIt can be one or both of the CDRs (or a portion thereof). Often, all three CDRs within all variable domains of the acceptor antibody are replaced with the corresponding donor CDRs, but only as many replacements as necessary to allow proper binding of the resulting CDR-transplanted antibody. Methods for generating CDR-transplanted antibodies are taught by Queen et al., U.S. Patents 5,585,089, 5,693,761, 5,693,762, and Winter U.S. 5,225,539, as well as EP0682040. H and V L Useful methods for preparing polypeptides are taught by Winter et al., U.S. Patents Nos. 4,816,397, 6,291,158, 6,291,159, 6,291,161, 6,545,142, EP0368684, EP0451216, and EP0120694.

[0245] After selecting suitable framework region candidates from the same family and / or the same family members, either or both of the heavy chain and light chain variable regions are produced by transplanting the CDR from the original species into the hybrid framework region. With respect to any of the above embodiments, the assembly of hybrid antibodies or hybrid antibody fragments having the hybrid variable chain region can be achieved using conventional methods known to those skilled in the art. For example, the DNA sequences encoding the hybrid variable domains described herein (i.e., the framework based on the target species and the CDR from the origin species) can be produced by oligonucleotide synthesis and / or PCR. The nucleic acid encoding the CDR region can also be isolated from the origin species antibody using a suitable restriction enzyme and ligated into the target species framework by ligation with a suitable ligation enzyme. Alternatively, the framework region of the variable chain of the origin species antibody can be modified by site-directed mutagenesis.

[0246] Since hybrids are constructed from multiple candidate choices corresponding to each framework domain, there are many combinations of sequences that can be constructed according to the principles described herein. Therefore, it is possible to assemble a library of hybrids having members with different combinations of individual framework domains. Such a library can be an electronic database collection of sequences or a physical collection of hybrids.

[0247] This process typically does not modify the FR of the acceptor antibody adjacent to the transplanted CDR. However, those skilled in the art can improve the antigen-binding affinity of the resulting CDR-transplanted antibody by replacing certain residues of a given FR to make the FR more similar to the corresponding FR of the donor antibody. Preferred sites for substitution include amino acid residues adjacent to the CDR or amino acid residues that can interact with the CDR (see, e.g., US5,585,089, particularly columns 12-16). Techniques for making these modifications are known in the art. In particular, if the resulting FR matches the human consensus FR at that site, or is at least 90% identical to such consensus FR, doing so may not significantly increase the antigenicity of the resulting modified anti-CDR-transplanted antibody compared to the same antibody with a complete human FR.

[0248] The rapid availability of antibodies produced using the same host cells and cell culture processes as the final cGMP manufacturing process can shorten the timeline of process development programs. Lonza has developed a common method using pooled transfectants grown in CDACF medium to rapidly produce small amounts (up to 50 g) of antibody in CHO cells. While slightly slower than a true transient response system, the advantages include higher product concentrations, as well as the use of the same host and process as the production cell line. An example of GS-CHO pool growth and productivity expressing model antibodies in a disposable bioreactor: In disposable bag bioreactor cultures (5 L working volume) operated in supply batch mode, a harvest antibody concentration of 2 g / L was achieved within 9 weeks of transfection.

[0249] Antibody molecules include, for example, fragments produced by proteolytic cleavage of monoclonal antibodies (e.g., F(ab'), F(ab')2), or single-chain immunoglobulins that can be produced via recombinant means. F(ab') antibody derivatives are monovalent, while F(ab')2 antibody derivatives are divalent. In one embodiment, such fragments can form a “chimeric” binding molecule with each other or with other antibody fragments or receptor ligands. Importantly, such a chimeric molecule may contain substituents that can bind to different epitopes of the same molecule.

[0250] In the relevant embodiments, the antibody is a derivative of the disclosed antibody, for example, an antibody containing the same CDR sequence as that in the disclosed antibody (e.g., a chimeric or CDR-implanted antibody). Alternatively, modifications such as introducing a conservative change into the antibody molecule may be desired. When making such modifications, the hydrophobic-hydrophilic index of amino acids may be taken into consideration. The importance of the hydrophobic-hydrophilic amino acid index in conferring interactive biological function to proteins is generally understood in the art (Kyte and Doolittle, 1982). It is recognized that the relative hydrophobic-hydrophilic properties of amino acids contribute to the resulting secondary structure of the protein, which in turn defines the interaction between the protein and other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0251] It is also understood in the art that similar amino acid substitutions can be effectively carried out based on hydrophilicity. U.S. Patent No. 4,554,101, incorporated herein by reference, states that the maximum local mean hydrophilicity of a protein correlates with the biological properties of the protein, such that it is governed by the hydrophilicity of its adjacent amino acids. As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to amino acid residues: Basic amino acids: Arginine (+3.0), Lysine (+3.0), and Histidine (-0.5); Acidic amino acids: Aspartic acid (+3.0±1), Glutamic acid (+3.0±1), Asparagine (+0.2), and Glutamine (+0.2); Hydrophilic, nonionic amino acids: Serine (+0.3), Asparagine (+0.2), Glutamine (+0.2) and threonine (-0.4); sulfur-containing amino acids: cysteine ​​(-1.0) and methionine (-1.3); hydrophobic, non-aromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5±1), aluanine (-0.5), and glycine (0); hydrophobic, aromatic amino acids: tryptophan (-3.4), phenylalanine (-2.5), and tyrosine (-2.3).

[0252] It is understood that amino acids can be replaced with other amino acids having similar hydrophilicity to produce biologically or immunologically modified proteins. In such modifications, substitutions of amino acids with hydrophilicity values ​​of ±2 are preferred, those of ±1 are particularly preferred, and those of ±0.5 are even more preferred.

[0253] Amino acid substitutions are generally based on the relative similarity of the amino acid side chains of the substituents, such as their hydrophobicity, hydrophilicity, charge, and size. Exemplary substitutions considering the various characteristics mentioned above are well known to those skilled in the art, and these include arginine and lysine, glutamic acid and aspartic acid, serine and threonine, glutamine and asparagine, as well as valine, leucine, and isoleucine.

[0254] Antibodies according to this disclosure may, in the first example, be defined by their binding specificity. A person skilled in the art can determine whether such an antibody falls within the scope of the claims herein by evaluating the binding specificity / affinity of a given antibody using techniques well known to those skilled in the art. For example, the epitope to which a given antibody binds may consist of a single continuous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acids located within an antigen molecule (e.g., a linear epitope within a domain). Alternatively, the epitope may consist of a plurality of discontinuous amino acids (or amino acid sequences) located within an antigen molecule (e.g., a structural epitope).

[0255] Using various techniques known to those skilled in the art, it is possible to determine whether an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, for example, conventional cross-blocking assays, such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Cross-blocking can be measured by various binding assays such as ELISA, biolayer interferometry, or surface plasmon resonance. Other methods include alanine scanning mutation analysis, peptide blot analysis (Reineke, Methods Mol. Biol. 248:443-63, 2004), peptide cleavage analysis, high-resolution electron microscopy using single-particle reconstruction, cryoEM, or tomography, crystallographic studies, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer Prot. Sci. 9:487-496, 2000). Another method that can be used to identify amino acids within polypeptides that antibodies interact with is hydrogen / deuterium exchange, detected by mass spectrometry. Generally speaking, hydrogen / deuterium exchange involves deuterizing the protein of interest and then binding the antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, where the exchangeable protons within the amino acids protected by the antibody complex undergo reverse exchange from deuterium to hydrogen at a slower rate than the exchangeable protons within amino acids that are not part of the interface. As a result, the amino acids that form part of the protein / antibody interface can retain deuterium and therefore exhibit a relatively higher mass compared to amino acids not included in the interface. After antibody dissociation, the target protein is subjected to protease cleavage and mass spectrometry to reveal the deuterium-labeled residues corresponding to the specific amino acids that the antibody interacts with. See, for example, Ehring, Analytical Biochemistry 267:252-259 (1999) and Engen and Smith, Anal. Chem. 73:256A-265A (2001).

[0256] The term "epitope" refers to a site on an antigen to which B and / or T cells respond. B cell epitopes can be formed from both consecutive amino acids or discontinuous amino acids juxtaposed by tertiary protein folding. Epitopes formed from consecutive amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three, more commonly at least five, or eight to ten amino acids within their unique spatial structure.

[0257] Modification-assisted profiling (MAP), also known as antigen-based antibody profiling (ASAP), is a method for classifying a number of monoclonal antibodies (mAbs) directed to the same antigen based on the similarity of their binding profiles to chemically or enzymatically modified antigen surfaces (see U.S. Patent Publication 2004 / 0101920, which is incorporated herein by reference in its entirety). Each category may reflect a unique epitope that is either distinctly different from or partially overlapping with the epitopes represented by another category. This technique allows for rapid filtering of genetically identical antibodies, and as a result, characterization can focus on genetically distinct antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce mAbs with desired characteristics. Using MAP, the antibodies of this disclosure may be classified into groups of antibodies that bind to different epitopes.

[0258] This disclosure includes antibodies that may bind to the same epitope or a portion of an epitope. Similarly, this disclosure also includes antibodies that compete with any of the specific exemplary antibodies described herein for binding to a target or a fragment thereof. Using common methods known in the art, it is readily possible to determine whether an antibody binds to the same epitope as a reference antibody or competes for binding to it. For example, to determine whether a test antibody binds to the same epitope as the reference, the reference antibody is bound to the target under saturated conditions. The ability of the test antibody to bind to the target molecule is then evaluated. If the test antibody can bind to the target molecule after saturated binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope than the reference antibody. On the other hand, if the test antibody cannot bind to the target molecule after saturated binding with the reference antibody, the test antibody may be bound to the same epitope to which the reference antibody binds.

[0259] To determine whether an antibody competes for binding to the reference anti-JAML antibody, the binding methodology described above is performed in two directions: In the first direction, the reference antibody is bound to the JAML antigen under saturated conditions, followed by evaluation of the binding of the test antibody to the JAML molecule. In the second direction, the test antibody is bound to the JAML antigen molecule under saturated conditions, followed by evaluation of the binding of the reference antibody to the JAML molecule. In both directions, if only the first (saturated) antibody is capable of binding to JAML, it is concluded that the test antibody and the reference antibody compete for binding to JAML. As will be understood by those skilled in the art, an antibody competing for binding to a reference antibody does not necessarily need to bind to the same epitope as the reference antibody, but can sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0260] If two antibodies each competitively inhibit (block) the binding of other antibodies to an antigen, then the two antibodies will bind to the same or overlapping epitopes. That is, as measured in a competitive binding assay, a 1, 5, 10, 20, or 100-fold excess of one antibody will inhibit the binding of the other antibody by at least 50%, but preferably 75%, 90%, or even 99% (see, for example, Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, if essentially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other, then the two antibodies will have the same epitope. If several amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other, then the two antibodies will have overlapping epitopes.

[0261] Next, further conventional experiments (e.g., peptide mutation and binding analysis) can be performed to confirm whether the observed deletion of the test antibody's binding is indeed due to binding to the same epitope as the reference antibody, or whether steric blockage (or another phenomenon) is the cause of the observed deletion of binding. These types of experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. Structural studies using EM or crystallography can also indicate whether two antibodies competing for binding recognize the same epitope.

[0262] In another embodiment, monoclonal antibodies having clonal pairing CDRs from heavy and light chains are provided, as shown in Table 1 or Table 3. The combination of heavy and light chain CDRs may be selected from Table 2 or Table 5. Such antibodies may be produced by clones disclosed in the Examples section using the methods described herein.

[0263] In another embodiment, antibodies may be defined by their variable sequences, which include additional “framework” regions. Furthermore, antibody sequences may, at their discretion, differ from these sequences using methods discussed in more detail below. For example, a nucleic acid sequence may (a) have its variable region separated from the constant domains of the light and heavy chains; (b) be different from those described above, without affecting the residues it encodes; (c) be different from those described above by a given percentage of homology, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%; or (d) be low-salt, such as being supplied by about 0.02 M to about 0.15 M NaCl at a temperature of about 50°C to about 70°C. (e) The amino acids may differ from those described above by their ability to hybridize under high stringency conditions, such as and / or high temperature conditions; (f) The amino acids may differ from those described above by a given percentage of homology, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%; or (f) The amino acids may differ from those described above by enabling conservative substitutions (discussed below). Each of the foregoing applies to nucleic acid sequences and amino acid sequences. In addition, various publications describe methods for obtaining physiologically active molecules, whose half-lives are modified either by introducing an FcRn-binding polypeptide into the molecule, or by fusing it with an antibody that has conserved FcRn-binding affinity but significantly reduced affinity to other Fc receptors, or by fusing it with an antibody that is fused to the antibody's FcRn-binding domain (see, for example, Kontermann (2009)).

[0264] derivatized antibodies Derivatized antibodies may be used to modify the half-life (e.g., serum half-life) of a parent antibody in mammals, particularly humans. Such modifications may result in a half-life of more than 15 days, preferably more than 20 days, more than 25 days, more than 30 days, more than 35 days, more than 40 days, more than 45 days, more than 2 months, more than 3 months, more than 4 months, or more than 5 months. An increase in the half-life of the antibody or fragment of the present disclosure in mammals, preferably humans, results in a higher serum titer of the antibody or antibody fragment in mammals, and therefore reduces the frequency of administration of the antibody or antibody fragment and / or the concentration of the antibody or antibody fragment administered. Antibodies or fragments having an increased in vivo half-life can be produced by techniques known to those skilled in the art. For example, an antibody or fragment with an increased in vivo half-life can be produced by modifying (e.g., substituting, deleting, or adding) amino acid residues identified as being involved in the interaction between the Fc domain and the FcRn receptor.

[0265] Beltramello et al. (2010) previously reported a modification that neutralizes mAbs by generating a variant in which the leucine residues at positions 1.3 and 1.2 of the CH2 domain (according to IMGT unique numbering of the C-domain) are replaced with alanine residues, as this tends to enhance dengue virus infection. This modification, also known as the "LALA" mutation, eliminates antibody binding to FcγRI, FcγRII, and FcγRIIIa, as described by Hessell et al. (2007). The variant and unmodified recombinant mAbs were compared for their ability to neutralize and enhance infection by four dengue virus serotypes. The LALA variant retained the same neutralizing activity as the unmodified mAb but did not enhance activity at all. Therefore, the LALA mutation of this nature is intended in the context of the antibody of this disclosure.

[0266] Modified glycosylation Specific embodiments of this disclosure are isolated monoclonal antibodies, or antigen-binding fragments thereof, containing a substantially homogeneous glycan that does not contain sialic acid, galactose, or fucose. The monoclonal antibody comprises a heavy-chain variable region and a light-chain variable region, both of which may be attached to the heavy-chain or light-chain constant region, respectively. The aforementioned substantially homogeneous glycan may be covalently bound to the heavy-chain constant region.

[0267] Another embodiment of the present disclosure comprises a monoclonal antibody having a novel Fc glycosylation pattern. The isolated monoclonal antibody, or its antigen-binding fragment, is present in a substantially homogeneous composition represented by GNGN or G1 / G2 glycoform. Fc glycosylation plays a crucial role in the antiviral and anticancer properties of therapeutic monoclonal antibodies. The present disclosure is consistent with recent studies showing increased antilentiviral cell-mediated viral inhibition of fucose-free anti-HIV monoclonal antibodies in vitro. This embodiment of the present disclosure, having a homogeneous glycan lacking core fucose, has shown that protection against specific viruses increased by more than a twofold factor. Elimination of core fucose dramatically improves the ADCC activity of monoclonal antibodies mediated by natural killer (NK) cells, but appears to have the opposite effect on ADCC activity in polymorphonuclear cells (PMNs).

[0268] Isolated monoclonal antibodies or their antigen-binding fragments containing a substantially homogeneous composition represented by GNGN or G1 / G2 glycoforms show increased binding affinity to Fc gamma RI and Fc gamma RIII compared to the same antibodies containing G0, G1F, G2F, GNF, GNGNF, or GNGNFX-containing glycoforms, while not containing a substantially homogeneous GNGN glycoform. In one embodiment of this disclosure, the antibody is 1 × 10⁶ -8 K below M D Fc gamma RI having, and 1 × 10 -7 K below M D It dissociates from Fc gamma RIII, which has [a certain characteristic].

[0269] Glycosylation of the Fc region is typically either N-linked or O-linked. "N-linked" refers to the attachment of the asparagine residue of the carbohydrate moiety to the side chain. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. The recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side-chain peptide sequence are asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. Therefore, the presence of either of these peptide sequences in the polypeptide creates a potential glycosylation site.

[0270] The glycosylation pattern may be modified, for example, by deleting one or more glycosylation sites found in the polypeptide and / or by adding one or more glycosylation sites that are not present in the polypeptide. Addition of glycosylation sites to the Fc region of the antibody is conveniently achieved by modifying the amino acid sequence to contain one or more of the tripeptide sequences described above (for the N-linked glycosylation site). An exemplary glycosylation variant has an amino acid substitution at the heavy chain residue Asn297. Modification can also be carried out by adding or substituting one or more serine or threonine residues to the original polypeptide sequence (for the O-linked glycosylation site). In addition, the change from Asn297 to Ala can remove one of the glycosylation sites.

[0271] In certain embodiments, the antibody is expressed in cells expressing beta(1,4)-N-acetylglucosaminyltransferase III (GnT III), and as a result, GnT III adds GlcNAc to the IL-23p19 antibody. Methods for producing the antibody in such a manner are provided in WO / 9954342, WO2003 / 011878, U.S. Patent Publication No. 2003 / 0003097A1, and Umana et al., Nature Biotechnology, 17:176-180 (1999). Cell lines can be modified using genome editing techniques such as clustered, regularly spaced, short-palindrical repeats (CRISPR) to enhance, reduce, or eliminate certain post-translational modifications, such as glycosylation. For example, CRISPR technology can be used to eliminate genes encoding glycosylation enzymes in HEK293 or CHO cells used to express recombinant monoclonal antibodies.

[0272] It is possible to eliminate protein sequence burdens in monoclonal antibodies. For example, the manipulability and safety of antibody variable gene sequences obtained from human B cells can be improved by manipulating them. Potential protein sequence burdens can be identified by searching for sequence motifs associated with sites containing: 1) unpaired Cys residues, 2) N-linked glycosylation, 3) Asn deamidation, 4) Asp isomerization, 5) SYE cleavage, 6) Met oxidation, 7) Trp oxidation, 8) N-terminal glutamate, 9) integrin binding, 10) CD11c / CD18 binding, or 11) fragmentation. Such motifs can be eliminated by modifying the synthetic gene of the cDNA encoding the recombinant antibody.

[0273] Efforts in protein engineering in the field of therapeutic antibody development have clearly demonstrated that certain sequences or residues are associated with differences in solubility (Fernandez-Escamilla et al., Nature Biotech., 22(10), 1302-1306 (2004), Chennamsetty et al., PNAS 106:(29), 11937-11942 (2009), Voynov et al., Biocon. Chem., 21:(2), 385-392, (2010). Evidence from solubility-altering mutations in the literature shows that several hydrophilic residues, such as aspartic acid, glutamic acid, and serine, contribute significantly more favorably to protein solubility than other hydrophilic residues, such as asparagine, glutamine, threonine, lysine, and arginine).

[0274] stability Antibodies can be manipulated for enhanced biophysical properties. Antibodies can be unfolded using high temperatures, and their relative stability can be determined using their mean apparent melting temperature. Differential scanning calorimetry (DSC) measures the heat capacity (Cp) of a molecule (the heat required for heating per degree) as a function of temperature. DSC can be used to study the thermal stability of antibodies. DSC data of mAbs are particularly interesting because they can isolate the unfolding of individual domains within the mAb structure, sometimes producing up to three peaks in the thermogram (from the unfolding of the Fab, CH2, and CH3 domains). Typically, unfolding of the Fab domain produces the strongest peak. DSC profiles and the relative stability of the Fc portion reveal characteristic differences among human IgG1, IgG2, IgG3, and IgG4 subclasses (Garber and Demarest, Biochem. Biophys. Res. Commun. 355, 751-757, 2007). Furthermore, the average apparent melting temperature can be determined using circular dichroism (CD) performed with a CD spectrometer. Far-ultraviolet CD spectra are measured for antibodies in the 200–260 nm range with 0.5 nm increments. The final spectrum can be determined as the average of 20 accumulations. Residual ellipticity values ​​can be calculated after background subtraction. Thermal unfolding of the antibody (0.1 mg / mL) can be monitored at 235 nm with heating rates of 25–95°C and 1°C / min. Dynamic light scattering (DLS) can be used to assess the tendency towards aggregation. DLS is used to characterize the sizes of various particles, including proteins. If the size of the system is not dispersed, the average effective diameter of the particles can be determined. This measurement varies depending on the particle core size, surface structure size, and particle concentration. Since DLS essentially measures variations in scattered light intensity due to the particles, the diffusion coefficient of the particles can be determined. DLS software for commercially available DLS instruments displays particle populations of different diameters. Stability studies can be conveniently performed using DLS.DLS measurement of a sample can indicate whether particles aggregate over time or with temperature changes by determining whether the hydrodynamic radius of the particles increases. When particles aggregate, a larger collection of particles with a larger radius can be observed. Temperature-dependent stability can be analyzed by controlling the temperature in situ. Capillary electrophoresis (CE) techniques include a proven methodology for determining antibody stability characteristics. Using the iCE approach, antibody protein charge variants resulting from deamidation, C-terminal lysine, sialylation, oxidation, glycosylation, and any other modifications to the protein that may result in changes in the protein's pI can be separated. Each expressed antibody protein can be evaluated by high-throughput free-solution isoelectric focusing (IEF) (cIEF) in a capillary column using the Protein Simple Maurice instrument. Full-row UV absorption detection can be performed every 30 seconds for real-time monitoring of molecules focused on isoelectric point (pI). This approach combines the high resolution of conventional gel IEF with the quantification and automation advantages of column-based separations, while eliminating the need for a recruitment step. This technique provides reproducible quantitative analysis of the identity, purity, and heterogeneity profiles of expressed antibodies. The results identify antibody charge heterogeneity and molecular sizing with detection sensitivity down to 0.7 μg / mL in both absorbance and innate fluorescence detection modes.

[0275] solubility The intrinsic solubility score of an antibody sequence can be determined. The intrinsic solubility score can be calculated using CamSol Intrinsic (Sormanni et al., J Mol Biol 427, 478-490, 2015). The solubility score can be calculated by evaluating the amino acids in HCDR3 (heavy chain CDR3; CDRH3) of each antibody fragment, such as scFv or Fv fragments, via an online program. Alternatively, solubility can be determined using laboratory techniques. Various techniques exist, including adding lyophilized protein to a solution until the solution is saturated and reaches its solubility limit, or concentration by ultrafiltration in a microconcentrator with a suitable molecular weight cutoff. The simplest method is the induction of amorphous precipitates, which measure protein solubility using a method involving protein precipitation with ammonium sulfate (Trevino et al., J Mol Biol, 366:449-460, 2007). Ammonium sulfate precipitation provides rapid and accurate information regarding relative solubility values. Ammonium sulfate precipitation yields a precipitate solution with clearly defined aqueous and solid phases and requires relatively small amounts of protein. Solubility measurements performed using amorphous precipitation induction with ammonium sulfate can also be easily carried out at different pH values. Protein solubility is highly dependent on pH, and pH is considered the most important exogenous factor influencing solubility.

[0276] Self-reactive While it is generally believed that autoreactive clones should be eliminated during ontogeny through negative selection, it has become clear that many naturally occurring human antibodies with autoreactive properties remain in the adult maturation repertoire, and that autoreactivity may enhance the antiviral function of many antibodies against pathogens. It has been noted that CDRH3 (heavy chain CDR3; CDRH3) loops in antibodies during early B cell development are often positively charged and exhibit autoreactive patterns (Wardemann et al., Science 301, 1374-1377, 2003). Autoreactivity of a given antibody can be tested by evaluating the level of binding to human-derived cells in microscopic examination (using adherent Hela or HEp-2 epithelial cells) and flow cytometry cell surface staining (using suspension Jurkat T cells and 293 S human embryonic kidney cells). Autoreactivity can also be investigated using evaluation of binding to tissues in tissue arrays.

[0277] Preferred residues ("human similarity"). Deep sequencing of the B cell repertoire of human B cells from blood donors has been widely performed in many recent studies. Sequence information on a significant portion of the human antibody repertoire facilitates the statistical evaluation of antibody sequence features common to healthy humans. Using knowledge of antibody sequence features in the Human Recombinant Antibody Variable Gene Reference Database, the degree of position-specificity of "human similarity" (HL) of antibody sequences can be estimated. HL has been shown to be useful in the development of antibodies for clinical use, such as therapeutic antibodies or antibodies as vaccines. The goal is to increase the human similarity of antibodies to reduce potential adverse effects and anti-antibody immune responses that can significantly reduce the efficacy of antibody drugs or induce serious health effects. By evaluating the antibody features of a combined antibody repertoire of approximately 400 million sequences from three healthy human blood donors, a novel "relative human similarity" (rHL) score focusing on the hypervariable regions of antibodies can be created. The rHL score allows for easy distinction between human (positive score) and non-human sequences (negative score). Antibodies can be engineered to eliminate residues that are not common in the human repertoire.

[0278] composition Another aspect of this disclosure provides a composition comprising, essentially consisting of, one or more antibodies or antibody fragments disclosed herein, isolated nucleic acids or vectors described herein. In some embodiments, the composition is a pharmaceutically acceptable composition and may optionally contain other therapeutic agents for combination therapy.

[0279] This disclosure provides pharmaceutical compositions comprising, or essentially consisting of, anti-JAML antibodies and antigens for generating them. Such compositions comprise a prophylactic or therapeutically effective amount of the antibody or a fragment thereof, or a peptide immunogen, and a pharmaceutically acceptable carrier. In certain embodiments, the term “pharmaceutically acceptable” means approved by a federal or state regulatory authority for use in animals, more specifically in humans, or listed in the United States Pharmacopeia or other generally accepted pharmacopoeias. The term “carrier” refers to a diluent, excipient, or vehicle administered with the therapeutic agent. Such pharmaceutical carriers may be sterile liquids, e.g., water and oils, and may be of petroleum, animal, plant, or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, the particular carrier is water. Saline solutions, as well as aqueous dextrose and glycerol solutions, can also be used as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, and ethanol.

[0280] If desired, the composition may also contain small amounts of wetting or emulsifying agents or pH buffers. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations may contain standard pharmaceutical-grade carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Examples of suitable pharmaceuticals are listed in "Remington's Pharmaceutical Sciences." Such compositions contain, together with a suitable amount of carrier, a prophylactic or therapeutically effective amount of antibody or fragment thereof, preferably in a purified form, to provide a form for appropriate administration to a patient. The formulations must be suitable for modes of administration that can be oral, intravenous, oral, systemic, intranasal, intra-arterial, intrabuccal, intranasal, ocular, spray, injection, infusion, bronchial inhalation, inhalation, inhalation, rectal, transdermal, rectal, vaginal, topical, or delivered by mechanical ventilation.

[0281] Generally, the components of the compositions of this disclosure are supplied either separately as dry, lyophilized powder or water-free concentrate in unit dosage forms, for example, in airtight containers such as ampoules or sachets indicating the amount of activator, or as a mixture. When the compositions are administered by infusion, they can be dispensed using infusion bottles containing sterile pharmaceutical-grade water or saline. When the compositions are administered by injection, ampoules of sterile water or saline for injection can be provided so that the components can be mixed before administration.

[0282] The composition may further include additional agents for enhancing the antibody response or treatment.

[0283] This disclosure includes antibodies or antigen-binding fragments thereof that bind to junctional adhesion molecule-like (JAML) proteins or fragments thereof. In some embodiments, the antibody or antigen-binding fragment comprises, consists of, or essentially comprises heavy chain complementarity-determining regions 1-3 (CDRH1-3) and light chain complementarity-determining regions 1-3 (CDRL1-3), or their equivalents, selected from a single row in Table 2 or Table 5. In one embodiment, the antibody or antigen-binding fragment comprises, consists of, or essentially comprises heavy chain variable regions (HC) and light chain variable regions (LC), or their equivalents, selected from a single row in Table 1 or Table 3.

[0284] In some embodiments, the antibody comprises, consists of, or essentially comprises a monoclonal antibody or a fragment thereof. In some embodiments, the antibody comprises, consists of, or essentially comprises a constant region selected from the group consisting of an IgA constant region, an IgD constant region, an IgE constant region, an IgG constant region, or an IgM constant region. In some embodiments, the constant region comprises, consists of, or essentially comprises an IgG1 constant region.

[0285] In some embodiments, the antigen-binding fragment comprises, consists of, or essentially comprises Fab, F(ab')2, Fab', scFv, or Fv. In some embodiments, the antibody or antigen-binding fragment comprises, consists of, or essentially comprises a detectable label or purified label.

[0286] In some embodiments, the equivalent comprises, consists of, or substantially comprises a polypeptide having at least 80% amino acid identity to the polypeptide, or the equivalent to an amino acid sequence comprises a polypeptide encoded by a polynucleotide that hybridizes under conditions of high stringency to the complement of the polynucleotide encoding the amino acid sequence.

[0287] In one embodiment, the antibody or antigen-binding fragment includes modifications. In several embodiments, the modifications include, consist of, or essentially consist of PEGylation, PEG mimicry, polysialation, HESation, or glycosylation.

[0288] In some embodiments, isolated polynucleotides encoding antibody or antigen-binding fragments are provided herein. In some embodiments, the polynucleotides are operably ligated to promoter and / or enhancer elements. In some embodiments, the polynucleotides include, consist of, or essentially consist of, a detectable label with a purified label. In some embodiments, vectors containing, consisting of, or essentially consisting of, a polynucleotide are provided herein. In some embodiments, the vectors include, consist of, or essentially consist of, a heterologous promoter sequence. In yet another embodiment, isolated host cells containing, consisting of, or essentially consisting of, a polynucleotide or vector are provided herein.

[0289] In one embodiment, the Specified Information provides a composition comprising, or essentially comprising, a carrier and an antibody or antigen-binding fragment, a polynucleotide encoding the antibody or antigen-binding fragment, a vector, or a host cell. In some embodiments, the carrier comprises, or essentially comprises, a pharmaceutically acceptable carrier.

[0290] In another embodiment, this specification provides a kit comprising, or essentially comprising, an antibody or antigen-binding fragment, a polynucleotide encoding the antibody or antigen-binding fragment, a vector, or a host cell. In some embodiments, the kit comprises, or essentially comprises, instructions for use.

[0291] method Methods for producing antibodies or antigen-binding fragments are provided herein. In some embodiments, the method comprises, or essentially comprises, culturing host cells containing a polynucleotide encoding an antibody or an antigen-binding fragment under conditions for the expression of the antibody or the antigen-binding fragment. In some embodiments, the method further comprises, or essentially comprises, isolating the antibody or antigen-binding fragment.

[0292] In yet another aspect of this disclosure, methods for conjugating JAML proteins or fragments thereof are provided herein. In some aspects, the methods include, or essentially consist of, contacting a JAML protein with an antibody or its antigen-binding fragment under conditions favorable for the conjugation of the antibody or its antigen-binding fragment, and optionally, isolating the antibody or its antigen-binding fragment conjugated to the JAML protein. The disclosed methods are useful for in vitro or in vivo detection of JAML proteins, if optionally the antibody or its antigen-binding fragment is detectably labeled. Cells may be animal or mammalian, such as humans. They may be from patient biopsies or from cultured cell lines, such as commercially available cell lines (e.g., available from the American Type Culture Collection).

[0293] When performed in vitro or in vivo in animal models, the composition is useful for assaying agonists or antagonists that mediate the JAML pathway (e.g., leading to cell activation, proliferation, and / or cytokine production) when the JAML protein is bound to an antibody, agonist, or antagonist. As will be understood by those skilled in the art, one or more controls should be assayed simultaneously or sequentially and can be used for comparative purposes.

[0294] The Disclosure also provides a method for activating or agonizing the JAML pathway, which includes, essentially consists of, or further comprises, administering a composition of the Disclosure to a subject to thereby activate or agonize the JAML pathway in the subject.

[0295] Further methods are provided for treating subjects requiring treatment of tumors or cancer, which include, essentially consist of, or further consist of, administering the compositions of the present disclosure to a subject to treat cancer in the subject. The subject may be an animal, mammal, or human patient.

[0296] In some embodiments, compositions comprising the antibodies disclosed herein are administered for the treatment of cancer in a subject. In some embodiments of this disclosure, cancer or tumor is cancer of at least one of the following organs: the circulatory system, respiratory system, gastrointestinal system, genitourinary system, live, bone, nervous system, reproductive system, hematological system, oral cavity, skin, and connective tissue and soft tissue, retroperitoneum and peritoneum, eye, intraocular melanoma, and adnexa, breast, head and / or neck, anal region, thyroid gland, parathyroid gland, adrenal colon cancer, pancreatic cancer, and other endocrine glands and associated structures, and other tissues including lymph nodes. Cancer may be a solid tumor, or alternatively, cancer may be a liquid cancer. Cancer may be primary cancer or metastatic cancer and / or cancer selected from carcinoma, sarcoma, myeloma, leukemia, or lymphoma, testicular cancer, brain cancer, metastatic or recurrent cancer, non-small cell lung cancer (NSCLC) and / or head and neck squamous cell carcinoma (HNSCC). In addition, cancer of tissue selected from the epithelium, head, neck, lungs, prostate, colon, breast, testes, bones, lymphatic system, blood, endometrium, uterus, ovaries, pancreas, esophagus, liver, skin, kidneys, adrenal glands, and brain. Cancer may be from the group of lymphoma, leukemia, breast cancer, early triple-negative breast cancer, endometrial cancer, uterine cancer, ovarian cancer, testicular cancer, lung cancer, prostate cancer, colon cancer, rectal cancer, pancreatic cancer, esophageal cancer, liver cancer, melanoma, or other skin cancers, ovarian cancer, kidney cancer, adrenal cancer, non-small cell lung cancer (NSCLC) and / or head and neck squamous cell carcinoma (HNSCC), and / or brain cancer or brain tumors. It may be any stage (primary or metastatic) or recurrent tumor or cancer or neoplasm.

[0297] experiment material and method Immunization. Five C57BL / 6J mice (Jackson Laboratories) were immunized intraperitoneally with 100 μg of recombinant protein (human JAML-Sinobiological) in 100 μl of ddH2O mixed with 100 μl of alum (prime immunization), and then immunized again 4 weeks later (booster immunization). After 7 days, three mice were euthanized by CO2 and neck dislocation, and their spleens and femurs were collected. Plasma cells from these organs were purified (Stemcell Technologies), pooled, resuspended in 15 mL of plasma B cell medium (Berkeley Lights), and loaded onto chips (Beacon instrument) according to the manufacturer's instructions. Plasma cells secreting antibodies against the target antigen (human JAML) were identified on the Beacon (specific workflow described in MAN-08133).

[0298] Opto Plasma B Discovery 4.0 workflow. Purified plasma cells were loaded onto a 23k Optoselect chip, isolated as single cells, and placed into Nanopens via optoelectronic positioning (OEP). Cells were then assayed for secretion of antigen-specific (extracellular domain of human JAML) IgG antibody by loading the chip with biotinylated human JAML and fluorescently labeled anti-mouse IgG antibody conjugated to streptavidin-coated beads (slide 6 of pptx document). Subsequently, the beads and antibody were washed away, and mRNA capture beads were loaded onto the chip and into each nanopen containing (antigen-specific) plasma cells. Cells were then lysed, and cDNA was synthesized via reverse transcription. cDNA conjugated to barcoded BCR beads was exported to a 96-well plate with up to 12 barcoded cDNA / well for downstream processing (NGS sequencing and antibody reexpression) ("unpenned"). Metadata summarized in the Excel sheet "OptoSeq BCR summary file".

[0299] Amplification and NGS sequencing of barcoded cDNA. The exported cDNA was amplified and purified according to the manufacturer's instructions (MAN-08137). Subsequently, the cDNA was quantified via the PicoGreen assay (a highly sensitive method for quantifying DNA using fluorescent nucleic acid staining), and their fragment sizes were determined using a bioanalyzer. A portion of this cDNA was used for the re-expression of the antibody described in MAN-08148. After determining the fragment sizes with the bioanalyzer, the applicant performed BCR amplification using the provided barcoded BCR forward primers, quantified the BCR amplification product via PicoGreen, and then pooled the amplicons in a single tube. The amplicons were then bead-purified and quantified. The applicant then performed library tagging using the Nextera XT DNA Library Preparation Kit, followed by PCR library indexing. Finally, the library was purified, quantified, analyzed with a fragment analyzer, and then sequenced with NovaSeq. Subsequently, the generated fastq files were analyzed with PrineSeq BCR software (Berkeley Lights). Hc and Lc sequences derived from the NGS sequencing workflow are summarized in the Excel sheet "Pipeline_output_summary".

[0300] Antibody re-expression from the Opto Plasma B discovery 4.0 workflow. The applicant first performed Hc and Lc domain amplification from cDNA (as described in step 3), quantified the cDNA concentration via PicoGreen, and quantified the Hc and Lc amplicon sizes via gel electrophoresis. The applicant then generated heavy and light chain constructs and again quantified the amplicon concentrations and their respective sizes via gel electrophoresis. The applicant then combined the respective Hc and Lc constructs and transfected HEK293 T cells, growing the cells at 37°C for 4–5 days. Subsequently, the applicant collected the supernatant and tested the antigen specificity of the generated antibodies via enzyme-linked immunosorbent assay (ELISA).

[0301] Sequencing of Hc and Lc chains, and production of monoclonal antibodies. Considering that the applicant obtained only a small fraction of the Hc sequences (51 / 287) from 287 exported proprietary cDNAs from the NGS sequencing step, the applicant further performed a deconvolution assay (ELISA assay) for antigen-specific antibodies from wells A3-A10 from the Beacon export plate (summarized in the Excel sheet, tab "Antibody re-expression") by performing nested PCR (MAN-08148) of the cDNA cloned into expression vectors. The final PCR products were used for Gibson cloning of the Hc (MsHC Koz IgG pHCMV R1 / Xho vector and Lc MsKLC Koz IgG pHCMV R1 / Xho vector)-mouse IgG1 backbone. Subsequently, the Hc and Lc (kappa light chain) plasmids were used for bacterial transformation (DH5a competent cells). Bacteria were grown at 37°C for 24 hours in SOC medium containing kanamycin, allowing only bacteria that successfully incorporated the plasmid containing the kanamycin resistance gene to grow. Using a multichannel, 6 μl of cells were streaked onto kanamycin plates to generate single colonies, which were grown at 37°C for 20 hours and then stored at 4°C. Plasmid DNA from the bulk transformations was then purified (QIAprep 96 Turbo kit) and sent for Sanger sequencing. CHO cells were then transfected using plasmid DNA from the bulk transformations to produce antibodies. ELISA was performed using the CHO cell culture supernatant to test antigen specificity (recognition of human JAML). Sanger sequencing revealed impurity sequences for several Hc and Lc plasmids. Eight single colonies were seeded (bacterial growth) for each of the Hc and Lc plasmids from the previous step into a new S block. Bacteria from the individual colonies were grown at 37C for 20 hours, and the plasmid DNA was purified (using the QIAprep 96 Turbo kit). The purified plasmids were sent for Sanger sequencing. The remaining DNA was stored at 4C.For several Hc and Lc plasmids, the applicant obtained diverse sequences from eight individual colonies and suggested that some antibodies derived from MAN-08148 were polyclonal. To generate true monoclonal antibodies, the applicant utilized Hc plasmids from individual colonies and paired them with their respective Lc plasmids from the same antibody clone for subsequent CHO transfection. As before, the applicant used the CHO supernatant to identify hJAML-specific antibodies (monoclonal).

[0302] Humanization of JAML-specific antibodies Affinity measurement using Octet The kinetics of recombinant humanized variants and parental chimeras were analyzed using biolayer interferometry. A very slow dissociation rate of 1 E 05 s 1 was observed using standard assay conditions and biosensor regeneration, which exceeded the detection limit of the instrument. In addition, an unstable (e.g., dissociative) baseline was observed in at least one reference well, which may result in poor line fit or erroneous kinetic rate. Processed sensor gram with reference subtraction from one Octet session was included. Based on the current results, the humanized variants appeared to bind to huJAML in the Octet assay compared to the parental chimeras. Two additional sessions were performed to assess whether alternative conditions yielded improved results. The first session was performed to evaluate qualitative "yes / binding of all IgG and 1000 nM JAML analytes" with reference subtraction. The second session run included kinetic analysis of up to four antibodies that showed qualitative binding in session 1, which were tested for huJAML HIS binding using non-regeneration conditions. Sensor regeneration is common and typically used under standard conditions, and did not appear to impair IgG reloading, but regeneration was excluded to minimize potential variables.

[0303] Humanity assessment of HC The humanity scores for parental and humanized antibodies are shown in the table below. Based on this method, a score of 90 or higher indicates a human-like heavy chain framework. For fully variable-length regions, a VH cutoff score of 80 is recommended. [Table 14]

[0304] Humanity assessment of LC The humanity scores for parental and humanized antibodies are shown in the table below. Based on this method, a score of 90 or higher indicates humanity of the kappa light chain framework. For fully variable-length regions, a Vκis cutoff score of 86 is recommended. [Table 15]

[0305] Antibody production yield Humanized variants and (1) parental chimeras were transiently produced using 0.01 L of TunaCHO® and an extended 14-day process, and purified by protein A chromatography. Yields are summarized in the table below: [Table 16]

[0306] Octet method The ligand was diluted with assay buffer to prepare a 5 μg / mL solution for the assay. The huJAML-HIS protein was reconstituted in water to prepare a 0.25 mg / mL analyte solution according to the manufacturer's COA. Buffer exchange (1×) of the resulting JAML protein solution was performed by dialysfiltration using AmiconUltra Centricon, with 100% recovery expected. A serial dilution series of huJAML was prepared, starting at 1000 nM and diluting 1:3 to produce seven concentrations (i.e., 1000, 333, 111, 37, 12.4, 4.1, and 1.4 nM). Binding experiments were performed in an Octet HTX at 25°C.

[0307] 8.4 Ab parental chimeras and (9) humanized 8.4 Ab variant hIgG were captured on an AHC sensor. The loaded sensor was immersed in serial dilutions of huJAML-HIS protein. A reference sample well (buffer) was used as a reference for subtraction during data processing. Rate constants were calculated using a monovalent (1:1) binding model.

[0308] The binding curves for analyte concentrations at 12.4 nM and 1.4 nM were excluded from the analysis due to low binding signals. [Table 17]

[0309] result Scout experiments showed that the parental chimera and three humanized variants paired with LC1 associated with JAML. Subsequent assays of these four IgGs against a dilution series of the JAML analyte again showed that HC2+LC1 and HC3+LC1 did not exhibit repeatable and titrable binding to JAML. Importantly, the parental chimera and humanized Hu8.4 Ab HC1+LC1 showed binding to JAML with intermediate-range nM affinity.

[0310] Baseline instability improved while the buffer effect persisted, and was most pronounced in the reference-subtracted 1000 nM binding curve. Even with additional buffer exchanges for the JAML protein between runs, the highly sensitive Octet assay continued to exhibit some degree of anomalous behavior in JAML. Affinity determination of HC1+LC1 and parental chimeras may require additional research to eliminate inter-experimental variability, including the use of the more sensitive assay method SPR and testing of other assay buffer formulations (Figure 3). [Table 18]

[0311] Qualitative binding evaluation of SR-24951 to all IgGs. Humanized variants and (1) parental chimeras were loaded into AHC sensors at 5 μg / mL, and the binding rate was evaluated with 1000 nM JAML in assay buffer. Sensorgrams and results are summarized in the table below. (3) Humanized variants (Hu8.4 Ab HC1+LC1, HC2+LC1, and HC3+LC1) and parental chimeras (8.4 Ab parent) showed a JAML binding response greater than 0.05 nm. Subsequent kinetic analysis of these (4) test samples from Session 2 was performed using Octet (BLI). (Figure 4) [Table 19]

[0312] K d Summary of Results (Session 2) The test samples were assayed for binding to human JAML His protein using Octet (BLI), and their affinity (K D The values ​​will be reported on the following slides along with the summary table and sensorgrams below. A 1:1 coupled model was used for all four test samples. K D This was calculated using the ratio k dis / ka.

[0313] Ab parent and Hu8.4 Ab HC1+LC1 have nanomolar affinity (K D ) was shown.

[0314] Dissociation exceeding the detection limit (K DIS 1<10⁻⁵) was observed for Hu8.4 Ab HC1+LC1.

[0315] The Hu8.4 Ab HC2+LC1 and Hu8.4 Ab HC3+LC1 response signals were undetectable (nd, response <0.05). (Figure 5) [Table 20]

[0316] Octet BLI method Prior to kinetic analysis, buffer exchange of the analyte to the assay buffer was performed using a MilliPoreAmicon column, 30 kDa MWCO. Binding experiments were performed in Octet HTX at 25°C. 8.4 Ab parent chimera (1) and humanized 8.4 Ab variant (3) (5 μg / mL) were loaded into the AHC sensor. The loaded sensor was immersed in a serial dilution of the protein sample (1000 nM start, 1:3 dilution, 7 points) for 300 seconds, followed by dissociation in the assay buffer for 600 seconds. The reference sample well (buffer) was used for data analysis. For all four test samples, the rate constant was first calculated using a monovalent (1:1) binding model.

[0317] The binding curves for analyte concentrations at 12.35 nM, 1.37 nM, and 0.15 nM were excluded from sensor gram and analysis due to low binding signals. [Table 21] [Table 22]

[0318] Humanization Process Part 2 (Session 3) Previous binding evaluations were performed on Greiner One Bio plates (catalog #781900). Through a series of multiple BLI experiments at Curia, Curia observed more stable baseline wells on Sartorius Octet plates (catalog #18 5080), and continued binding testing of four antibodies (8.4 Ab parent, Hu8.4 Ab HC1+ LC1, Hu8.4 Ab HC2+ LC1, and Hu8.4 Ab HC3+ LC1) to the human JAML His protein using Sartorius plates under standard Octet conditions.

[0319] Affinity (K D The values ​​will be reported in subsequent slides along with the summary table and sensorgram below. 1:1 curve-fitting model used for velocity calculation. K D is, KDIS / K A The calculation was performed using the ratio.

[0320] Ab parent and humanized variant 3 have equivalent nanomolar affinity (K D ) was shown. [Table 23]

[0321] Octet method Binding experiments were performed using Octet HTX at 25°C. 8.4 Ab parent chimera (1) and humanized 8.4 Ab variant (3) (5 ug / mL) were loaded into the AHC sensor. The loaded sensor was immersed in a serial dilution of the protein sample (1000 nM start, 1:3 dilution, 7 points) for 300 seconds, followed by dissociation in assay buffer for 600 seconds. The reference sample well (buffer) was used for data analysis. For all four test samples, the rate constant was first calculated using a monovalent (1:1) binding model.

[0322] The binding curves for analyte concentrations at 12.35 nM, 1.37 nM, and 0.15 nM were excluded from sensor gram and analysis due to low binding signals. [Table 24]

[0323] Sensorograms and speed charts Sensorograms and speed tables are shown in the table and Figure 6 below. [Table 25] *Poor fit, R^2<0.9)

[0324] Agonist immunotherapy Agonist immunotherapy often targets costimulatory molecules such as 4-1BB, ICOS, or OX-40. The involvement of inhibitory FcγRIIB is important for the in vivo efficacy of such mouse and human agonist immunotherapy antibodies because it mimics the involvement of multimeric endogenous ligands (i.e., most TNF receptors such as CD40, OX-40, or 4-1BB are trimers and therefore require multimeric ligands for proper activation). Thus, antibodies with enhanced affinity for FcγRIIB (such as IgG1) or antibodies with Fc-targeting mutations that increase affinity for FcγRIIB exhibit increased antitumor effects.

[0325] In mouse models, the expression of the human Fcγ receptor is necessary to faithfully and confidently test the agonist activity of such Fc-optimized human antibodies. Therefore, we will perform a detailed analysis of Fc-optimized humanized antibody variants using syngeneic tumor models such as the MC38 model of colon cancer (Schroers et.al. Front Immunol. 2023 Mar 8;14:1102282.doi:10.3389 / fimmu.2023.1102282.PMID:36969213;PMCID:PMC10030996), and the B16F10 model (available at https: / / www.atcc.org / products / crl-6475) of melanoma, lung cancer, and head and neck cancer in humanized FcγR mice. Compared to parental antibodies (with intermediate FcγRIIB binding) or Fc-silencing IgG1 antibodies lacking detectable FcγR binding, Fc-optimized antibodies exhibit substantially enhanced antitumor effects (due to increased affinity for FcγRIIB).

[0326] Equivalents While this disclosure has been described in conjunction with the embodiments described above, it should be understood that the foregoing description and examples are illustrative and not intended to limit the scope of this disclosure. Other aspects, advantages, and modifications within the scope of this disclosure will be apparent to those skilled in the art to whom this disclosure relates.

[0327] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this disclosure pertains. All nucleotide sequences provided herein are presented in the 5' to 3' direction.

[0328] The embodiments described herein as illustrative can also be suitably implemented without any elements, limitations, or restrictions not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing” should be interpreted broadly and indefinitely. Furthermore, while the terms and expressions used herein are for illustrative purposes only and not limiting, and there is no intention to exclude any equivalent or part thereof of the illustrated and described features, it is recognized that various modifications are possible within the scope of this disclosure.

[0329] Therefore, although this disclosure has been specifically disclosed through specific embodiments, it should be understood that optional features, modifications, improvements, and variations of the embodiments disclosed herein may be reused by those skilled in the art, and such modifications, improvements, and variations are considered to be within the scope of this disclosure. The materials, methods, and examples provided herein are representative of specific embodiments, are illustrative, and are not intended as limitations on the scope of this disclosure.

[0330] The scope of this disclosure has been described broadly and comprehensively herein. Each of the narrower species and subgenera belonging to the comprehensive disclosure also forms part of this disclosure. This includes the comprehensive specification with provisos or negative limitations that remove any subject matter from its genus, regardless of whether the removed material is specifically enumerated herein.

[0331] In addition, if any feature or aspect of the present disclosure is described in terms of the Markush group, a person skilled in the art will recognize that embodiments of the present disclosure may thereby be described in terms of any individual member or subgroup of a member of the Markush group.

[0332] All publications, patent applications, patents, and other references described herein are expressly incorporated by reference to the same extent that each is incorporated by reference individually. In case of any conflict, this specification, including definitions, shall prevail. In some aspects, publications are referred to by Arabic numerals. A complete citation of these publications is provided below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 2-1] [Table 2-2] Table 2-3 Table 2-4 Table 2-5 Table 2-6 Table 3-1 Table 3-2 Table 4-1 Table 4-2 Table 4-3 Table 4-4 Table 4-5 Table 5-1 Table 5-2

Claims

1. An antibody or its antigen-binding fragment that binds to a junctional adhesion molecule-like (JAML) protein or a fragment thereof.

2. The antibody or antigen-binding fragment according to claim 1, comprising heavy chain complementarity-determining regions 1-3 (CDRH1-3) and light chain complementarity-determining regions 1-3 (CDRL1-3), or equivalents thereof, selected from a single row in Table 2.

3. The antibody or antigen-binding fragment according to claim 1, comprising a heavy chain variable region (HC) and a light chain variable region (LC), or equivalents thereof, selected from a single row in Table 1, wherein optionally, the equivalent of the HC holds corresponding CDRH1-3 selected from a single row in Table 2, or the equivalent of the LC holds corresponding CDRL1-3 selected from a single row in Table 2.

4. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment is a humanized antibody or its antigen-binding fragment.

5. The antibody or antigen-binding fragment according to claim 4, comprising heavy chain complementarity-determining regions (CDRH) 1, CDRH2, CDRH3, light chain complementarity-determining regions (CDRL) 1, CDRL2, and CDRL3, each selected from Table 5.

6. The antibody or antigen-binding fragment according to claim 4, comprising heavy chain complementarity-determining regions 1-3 (CDRH1-3) and light chain complementarity-determining regions 1-3 (CDRL1-3), or equivalents thereof, selected from a single row in Table 5.

7. The antibody or antigen-binding fragment according to claim 4, comprising an HC or equivalent selected from SEQ ID NOs. 135 to 138 and an LC or equivalent selected from SEQ ID NOs. 139 to 142, wherein optionally, the equivalent of the HC holds a corresponding CDRH1 to 3 selected from a single row in Table 5, or the equivalent of the LC holds a corresponding CDRL selected from a single row in Table 5.

8. The antibody or antigen-binding fragment according to claim 4, comprising HC and LC or equivalents selected from a single row in Table 3, wherein optionally the equivalent of HC holds corresponding CDRH1-3 selected from a single row in Table 5, and the equivalent of LC holds corresponding CDRL selected from a single row in Table 5.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or 5 to 8, wherein the antibody is a monoclonal antibody or a fragment thereof.

10. The antibody or its antigen-binding fragment according to any one of claims 1 to 9, wherein the antibody further comprises a constant region selected from the group consisting of an IgA constant region, an IgD constant region, an IgE constant region, an IgG constant region, or an IgM constant region.

11. The antibody or antigen-binding fragment according to claim 10, wherein the constant region is the IgG1 constant region.

12. The antigen-binding fragment is Fab, F(ab') ２ An antigen-binding fragment according to any one of claims 1 to 11, selected from the group consisting of Fab', scFv, or Fv.

13. The antibody or antigen-binding fragment according to any one of claims 2 to 12, wherein the equivalent comprises a polypeptide having at least 80% amino acid identity with respect to the polypeptide, or the equivalent to an amino acid sequence comprises a polypeptide encoded by a polynucleotide that hybridizes under conditions of high stringency with respect to the complement of the polynucleotide encoding the amino acid sequence.

14. The antibody or antigen-binding fragment according to any one of claims 1 to 13, wherein the antibody or fragment thereof is modified, and optionally the modification is selected from the group consisting of PEGylation, PEG mimicry, polysialation, HESization, or glycosylation.

15. The antibody or antigen-binding fragment according to any one of claims 1 to 14, further comprising an Fc region containing one or more mutations selected from G237D, P238D, H268D, P271G, and A330R.

16. Isolated polynucleotides encoding an antibody or antigen-binding fragment according to any one of claims 1 to 15, and optionally operably linked to a promoter and / or enhancer element.

17. An isolated polynucleotide encoding the antibody or its antigen-binding fragment according to claim 4, comprising any one polynucleotide sequence from SEQ ID NOs. 143 to 150, or an equivalent thereof, wherein the equivalent optionally encodes HC and LC selected from a single row in Table 3.

18. A vector comprising a polynucleotide according to claim 16 or claim 17, and optionally a heterologous promoter sequence.

19. An isolated host cell comprising an isolated polynucleotide according to claim 16 or claim 17 and / or a vector according to claim 18.

20. A method for producing an antibody or an antigen-binding fragment according to any one of claims 1 to 15, comprising culturing a host cell containing a polynucleotide encoding the antibody or the antigen-binding fragment under conditions for the expression of the antibody or the antigen-binding fragment, and optionally isolating the antibody or the antigen-binding fragment.

21. An antibody or antigen-binding fragment according to any one of claims 1 to 15, further comprising a detectable label, affinity tag, or purification label.

22. The polynucleotide according to claim 16 or 17, further comprising a detectable label or a purified label.

23. A method for binding to a JAML protein or a fragment thereof, comprising: contacting the JAML protein with an antibody or antigen-binding fragment according to any one of claims 1 to 15 or 21 under conditions favorable for binding to the antibody or antigen-binding fragment thereof; and optionally isolating the antibody or antigen-binding fragment bound to the JAML protein.

24. A composition comprising a carrier, one or more of the antibodies or antigen-binding fragments described in any one of claims 1 to 15 or 21, the polynucleotide described in any one of claims 16, 17, or 22, the vector described in claim 18, and the host cells described in claim 19, wherein optionally the carrier is a pharmaceutically acceptable carrier.

25. A kit comprising one or more of the antibodies or antigen-binding fragments according to any one of claims 1 to 15 or 21, the polynucleotide according to any one of claims 16, 17, or 22, the vector according to claim 18, and the host cells according to claim 19, optionally, and instructions for use.

Images