Antibodies that bind CD228

Abstract

The present disclosure provides antibodies, or antigen binding fragments thereof, that have specificity against CD228. Such antibodies or antigen-binding fragments may be used in a variety of pharmaceutical applications, e.g., as anticancer agents and / or immunomodulators. The present disclosure also relates to methods of making the antibodies or antigen-binding fragments described herein and compositions comprising such antibodies or antigen-binding fragments. The disclosure further relates to nucleic acid molecules encoding such antibodies or antigen binding fragments. In addition, the application discloses therapeutic and / or diagnostic uses of such antibodies or antigen binding fragments.

Description

I. Cross - Reference to Related Applications

[0001] This application claims the priority benefit of U.S. Provisional Application No. 63 / 408,605, filed on September 21, 2022, which is incorporated herein by reference in its entirety for any purpose. II. Introduction and Overview

[0002] Cluster of differentiation 228 or CD228 (also known as melanotransferrin, MELTF, p97, and MFI2) is a glycosylphosphatidylinositol (GPI) - anchored glycoprotein that belongs to the transferrin family of iron - binding proteins and was initially described as a carcinoembryonic protein that is highly expressed on malignant melanoma cells (Rose et al., Proc Natl Acad Sci USA, 1986).

[0003] CD228 is expressed in a variety of cancers, including melanoma, mesothelioma, thyroid cancer, lung cancer, liver cancer, pancreatic cancer, head and neck cancer, gastric cancer, colorectal cancer, urothelial cancer, breast cancer, and cervical cancer. Melanoma, also known as malignant melanoma, is a type of cancer that develops from melanocytes, which are pigment - containing cells. Melanoma is the most dangerous type of skin cancer. In 2015, 3.1 million people had active disease and melanoma caused 59,800 deaths. Surgery can be effective for early - stage melanoma, but may not be a treatment option for disease that has metastasized to distant organs. Spread of melanoma typically spreads to lymph nodes in the area and then elsewhere. Attempting to improve survival by surgically removing lymph nodes is associated with many complications and no overall survival benefit. Immunotherapy, chemotherapy, and radiotherapy have all been used, but are generally not curative, especially for advanced melanoma. When there are distant metastases, the cancer is generally considered incurable. The five - year survival rate for stage IV disease is 15 - 20%.

[0004] The present disclosure particularly provides novel antibodies that bind to CD228 and antigen - binding fragments thereof. III. Definitions

[0005] The following list defines terms, phrases, and abbreviations used throughout this specification. All terms listed and defined herein are intended to cover all grammatical forms.

[0006] As used herein, unless otherwise specified, "CD137" refers to human CD137 (huCD137). Human CD137 refers to the full-length protein, fragments thereof, or variants thereof as defined by UniProt Q07011. CD137 is also known as 4-1BB, tumor necrosis factor receptor superfamily member 9 (TNFRSF9), and is induced by lymphocyte activation (ILA). In certain specific embodiments, CD137 of a non-human species is used, such as cynomolgus monkey CD137 or mouse CD137.

[0007] As used herein, unless otherwise specified, "CD228" refers to human CD228. Human CD228 refers to the full-length protein, its mature form, its subtypes, fragments thereof, or variants thereof as defined by UniProt P08582. Human CD228 is encoded by the MELTF gene. CD228 is also known as melanotransferrin, MELTF, p97, and MFI2, and these terms may be used interchangeably herein. In certain specific embodiments, CD228 of a non-human species is used, such as cynomolgus monkey CD228 or mouse CD228.

[0008] As used herein, "binding affinity" describes the ability of a biomolecule of the present disclosure (such as a polypeptide or protein) (such as an antibody or an antigen-binding fragment thereof, a fusion protein, or any other peptide or protein) to bind to a selected target (and form a complex). Binding affinity is measured by a variety of methods known to those skilled in the art, including but not limited to fluorescence titration, enzyme-linked immunosorbent assay (ELISA)-based assays (including direct and competitive ELISA), calorimetry, such as isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). These methods are well recognized in the art and some examples of such methods are further described herein. Binding affinity is thus reported as the dissociation constant (K D ), half-maximal effective concentration (EC 50 ), or half-maximal inhibitory concentration (IC 50 ) values measured using such methods. Lower K D , EC 50 , or IC 50 values reflect better (higher) binding ability (affinity). Thus, the binding affinities of two biomolecules for a selected target can be measured and compared. When comparing the binding affinities of two biomolecules for a selected target, the terms "comparable to", "about the same", "substantially the same", or "substantially similar" mean that one biomolecule has a K D , EC 50 , or IC 50The binding affinity of a value that is the same as or similar to the binding affinity of another molecule within the experimental variability of the binding affinity measurement. Preferably, "comparable to", "about the same", "substantially the same", or "substantially similar" refer to values within 50% deviation from a given reference value, more preferably within 20% deviation, and most preferably within 10% deviation. The experimental variability of the binding affinity measurement depends on the specific method used and is known to those skilled in the art.

[0009] As used herein, the term "substantially" may also refer to a qualitative condition that exhibits all or nearly all of the range or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will appreciate that biological and chemical phenomena rarely, if ever, proceed to completion and / or continue to complete or achieve or avoid absolute results. Thus, the term "substantially" as used herein is intended to account for the potential lack of completeness inherent in many biological and chemical phenomena.

[0010] As used herein, the terms "detect", "detection", "detectable", or "detecting" are understood at both the quantitative and qualitative levels and in combinations thereof. Thus, it includes quantitative, semi - quantitative, and qualitative measurement results of the biomolecules of the present disclosure.

[0011] As used herein, "detectable affinity" generally refers to the binding ability between a biomolecule and its target, which is reported as a K D , EC 50 or IC 50 value and is at most about 10 -5 M or lower. A binding affinity reported as a K D , EC 50 or IC 50 value higher than 10 -5 M is generally no longer measurable by conventional methods such as ELISA and SPR and thus has secondary importance. Thus, "detectable affinity" may refer to a K -5 value of about 10 D M or lower as determined by ELISA or SPR, preferably SPR.

[0012] As used herein, "specific for", "specific binding", "specifically bind" or "binding specificity" in reference to a biomolecule refers to the ability of the biomolecule to distinguish a desired target (e.g., CD228) from one or more reference targets. It should be understood that such specificity is not an absolute property but a relative property and can be determined, for example, by SPR, Western blot, ELISA, fluorescence-activated cell sorting (FACS), radioimmunoassay (RIA), electrochemiluminescence (ECL), immunoradiometric assay (IRMA), immunohistochemistry (IHC), and peptide scanning assays.

[0013] When used herein in the context of a biomolecule such as an antibody, antigen-binding fragment, or fusion protein of the disclosure that binds to CD228, "specific for", "specific binding", "specifically bind" or "binding specificity" refers to the biomolecule binding to, reacting with, or being specific for CD228 as described herein, but not significantly binding to another protein. The term "another protein" includes any protein that is not CD228 and is not a protein closely related or homologous to CD228. However, the term "another protein" does not exclude CD228 from a species other than human and fragments and / or variants of CD228. The term "not significantly bind" means that the biomolecule of the disclosure binds to another protein with a lower binding affinity than to CD228, i.e., exhibits less than 30%, preferably 20%, more preferably 10%, and particularly preferably less than 9%, 8%, 7%, 6%, or 5% cross-reactivity. Whether a biomolecule specifically reacts as defined above can be readily tested, for example, by comparing the reaction of the biomolecule of the disclosure with CD228 and the reaction of the biomolecule with the other protein(s).

[0014] As used herein, the term "lipocalin" refers to a monomeric protein of about 18 - 20 kDa by weight that has a cylindrical β - sheet super - secondary structure region comprising multiple β - strands (preferably eight β - strands designated A through H), the β - strands being pairwise connected at one end by multiple (preferably four) loops, thereby comprising a ligand - binding pocket and defining the entrance to the ligand - binding pocket. Preferably, the loops that constitute the ligand - binding pocket as used in the present disclosure are the loops connecting the open ends of β - strands A and B, C and D, E and F, and G and H and are designated as loops AB, CD, EF, and GH. It is known that the diversity of such loops in the otherwise rigid lipocalin scaffold gives rise to a variety of different binding modes among lipocalin family members, each of which is capable of accommodating targets of different sizes, shapes, and chemical characteristics (reviewed, for example, in Skerra, Biochim Biophys Acta, 2000; Flower et al., Biochim Biophys Acta, 2000; Flower, Biochem J, 1996). It should be understood that the lipocalin family of proteins has evolved naturally to bind a wide range of ligands that share an unusually low level of overall sequence conservation (typically having less than 20% sequence identity), yet retain a highly conserved overall folding pattern. The correspondence between positions in various lipocalins is also well - known to those of skill in the art (see, for example, U.S. Patent No. 7,250,297). Proteins that fall within the definition of "lipocalin" as used herein include, but are not limited to, human lipocalins, including tear lipocalin (Tlc, Lcn1), lipocalin - 2 (Lcn2) or neutrophil gelatinase - associated lipocalin (NGAL), apolipoprotein D (ApoD), apolipoprotein M, alpha - 1 - acid glycoprotein 1, alpha - 1 - acid glycoprotein 2, alpha - 1 - microglobulin, complement component 8 gamma, retinol - binding protein (RBP), epididymal retinoic acid - binding protein, immunosuppressive glycoprotein (glycodelin), odorant - binding protein IIa, odorant - binding protein IIb, lipocalin - 15 (Lcn15), and prostaglandin D synthase.

[0015] As used herein, a "mutein", "mutated" entity (whether protein or nucleic acid), or "mutant" refers to an exchange, deletion, or insertion of one or more amino acids or nucleotides as compared to a naturally - occurring (wild - type) protein or nucleic acid. The term also includes fragments of the mutein proteins as described herein. In some embodiments, as described herein, lipocalin muteins (also referred to as The protein) has a cylindrical β-sheet super-secondary structure region containing eight β-strands, and the β-strands are pairwise connected by four loops at one end, thereby including a ligand-binding pocket and defining the entrance of the ligand-binding pocket, wherein at least one amino acid located within the four loops has been mutated compared to the native sequence lipocalin.

[0016] As used herein, the term "variant" refers to derivatives of modified proteins or polypeptides, for example, by substitution, deletion, insertion, and / or chemical modification of an amino acid sequence or a nucleotide sequence. In some embodiments, such mutations and / or chemical modifications do not reduce the function of the protein or peptide. Such substitutions can be conservative, that is, an amino acid residue is replaced with a chemically similar amino acid residue. Examples of conservative substitutions are replacements among members of the following groups: 1) alanine, serine, threonine, and valine; 2) aspartic acid, glutamic acid, glutamine, asparagine, and histidine; 3) arginine, lysine, glutamine, asparagine, and histidine; 4) isoleucine, leucine, methionine, valine, alanine, phenylalanine, threonine, and proline; and 5) isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Such variants include proteins or polypeptides in which one or more amino acids have been replaced by their respective D-stereoisomers, or by amino acids other than the 20 naturally occurring amino acids, such as ornithine, hydroxyproline, citrulline, homoserine, hydroxylysine, norvaline. Such variants also include, for example, proteins or polypeptides with one or more amino acid residues added or deleted at the N-terminus and / or C-terminus. Generally, a variant has at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or at least about 98% amino acid sequence identity with the native sequence protein or polypeptide. The variant preferably retains the biological activity of the protein or polypeptide from which it is derived, such as binding to the same target.

[0017] As used herein with respect to an antibody or an antigen-binding fragment thereof, the term "variant" refers to an antibody or an antigen-binding fragment thereof of the present disclosure, wherein the sequence has mutations, including substitutions, deletions, insertions, and / or chemical modifications. A variant of an antibody or an antigen-binding fragment thereof as described herein retains the biological activity of the antibody or an antigen-binding fragment thereof from which it is derived, such as binding to CD228. Generally, a variant of an antibody or an antigen-binding fragment thereof has at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or 98% amino acid sequence identity with the antibody or an antigen-binding fragment thereof from which it is derived.

[0018] As used herein, the term "sequence identity" or "identity" refers to the property of a sequence that measures its similarity or relationship. The term "sequence identity" or "identity" as used in the present disclosure refers to the percentage of paired identical residues relative to the number of residues in the longer of the two sequences after (homologous) alignment of the sequences of the proteins or polypeptides of the present disclosure with the sequence under discussion. Sequence identity is measured by dividing the number of identical amino acid residues by the total number of residues and multiplying the result by 100.

[0019] As used herein, the term "sequence homology" or "homology" has its ordinary meaning, and homologous amino acids include identical amino acids as well as amino acids that are considered to be conservatively substituted at equivalent positions in the linear amino acid sequence of the proteins or polypeptides of the present disclosure.

[0020] Those skilled in the art will recognize available computer programs for determining sequence homology or sequence identity using standard parameters, such as BLAST (Altschul et al., Nucleic Acids Res, 1997), BLAST2 (Altschul et al., J Mol Biol, 1990) and Smith-Waterman (Smith and Waterman, J Mol Biol, 1981). The percentage of sequence homology or sequence identity can be determined, for example, in the present application using the program BLASTP, version 2.2.5 (November 16, 2002) (Altschul et al., Nucleic Acids Res, 1997). In some embodiments, the percentage of homology is based on the alignment of the entire protein or polypeptide sequence including the propeptide sequence (matrix: BLOSUM 62; gap cost: 11.1; cutoff value set to 10 -3 ), preferably using the wild-type protein backbone as a reference in pairwise comparisons. It is calculated as the percentage of the number of "positive" (homologous amino acids) as indicated by the results in the BLASTP program output divided by the total number of amino acids selected by the program used for alignment.

[0021] A "gap" is a void in the alignment resulting from the addition or deletion of amino acids. Thus, two copies of an exactly identical sequence have 100% identity, but sequences that are less highly conserved and have deletions, additions, or substitutions may have a lower degree of sequence identity.

[0022] As used interchangeably herein, the terms “conjugate,” “conjugation,” “fuse,” “fusion,” or “linkage” refer to all forms of attachment of two or more subunits by covalent or non-covalent linkages, including but not limited to gene fusions, chemical conjugations, attachment via a linker or crosslinker, and non-covalent associative linkages.

[0023] As used herein, the term “fusion polypeptide” or “fusion protein” refers to a polypeptide or protein that comprises two or more subunits. In some embodiments, a fusion protein as described herein comprises two or more subunits, at least one of which is capable of specifically binding to CD228. Within the fusion protein, the subunits may be linked by covalent or non-covalent bonds. Preferably, the fusion protein is a translational fusion between two or more subunits. A translational fusion can be generated by engineering the coding sequence of one subunit in frame with the coding sequence of another subunit. Both subunits may be interspersed by a nucleotide sequence encoding a linker. However, the subunits of the fusion proteins of the present disclosure may also be linked by chemical conjugation. The subunits forming the fusion protein are typically attached to each other as follows: the C-terminus of one subunit to the N-terminus of another subunit, or the C-terminus of one subunit to the C-terminus of another subunit, or the N-terminus of one subunit to the N-terminus of another subunit, or the N-terminus of one subunit to the C-terminus of another subunit. The subunits of the fusion protein may be attached in any order and may include more than one of any of the constituent subunits. If one or more of the subunits is part of a protein (complex) consisting of more than one polypeptide chain, the term “fusion protein” may also refer to a protein that comprises the fusion sequence and all other polypeptide chains of the protein (complex). As an illustrative example, when a full-length immunoglobulin / antibody is fused to a lipocalin mutant protein via the heavy or light chain of the immunoglobulin / antibody, the term “fusion protein” may refer to a single polypeptide chain that comprises the lipocalin mutant protein and the heavy or light chain of the immunoglobulin / antibody. The term “fusion protein” may also refer to the entire immunoglobulin / antibody (both light and heavy chains) and the lipocalin mutant protein fused to one or both of its heavy and / or light chains.

[0024] As used herein, the term “subunit” of a fusion protein disclosed herein refers to a single protein or separate polypeptide chain that by itself can form a stable folded structure and can define a unique function that provides a binding motif for a target. In some embodiments, a preferred subunit of the present disclosure is an antibody, such as a full-length antibody or an antigen-binding domain / fragment thereof.

[0025] A “linker” that may be included in the fusion proteins of the present disclosure joins together two or more subunits of a fusion protein as described herein.

[0026] As used herein, the term "albumin" includes all mammalian albumins, such as human serum albumin or bovine serum albumin or rat serum albumin.

[0027] As used herein, the term "organic molecule" or "organic small molecule" refers to an organic molecule containing at least two carbon atoms, but preferably no more than 7 or 12 rotatable carbon-carbon bonds, having a molecular weight in the range of 100 to 2,000 daltons, preferably in the range of 100 to 1,000 daltons, and optionally including one or two metal atoms.

[0028] A "sample" is defined as a biological sample taken from any subject. Biological samples include, but are not limited to, blood, serum, urine, feces, semen, or tissue, including tumor tissue.

[0029] A "subject" is a vertebrate, preferably a mammal, more preferably a human. The term "mammal" is used herein to refer to any animal classified as a mammal, including but not limited to humans, domesticated animals and farm animals, as well as zoo animals, sport animals or pet animals, such as sheep, dogs, horses, cats, cows, rats, pigs, apes (such as cynomolgus monkeys), to name just a few illustrative examples. Preferably, the "mammal" used herein is a human.

[0030] An "effective amount" is an amount sufficient to produce a beneficial or desired result. The effective amount may be administered in one or more separate administrations or doses.

[0031] As used herein, "antibody" includes whole antibodies or any antigen-binding fragment thereof (i.e., "antigen-binding portion" or "antigen-binding domain") or single chains. The terms "antibody" and "immunoglobulin" are used interchangeably herein. A whole antibody refers to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable domain / region (V H or HCVR) and a heavy chain constant region (C H ). The heavy chain constant region contains three domains: C H1 , C H2 and C H3 . Each light chain contains a light chain variable domain / region (V L or LCVR) and a light chain constant region (C L ). The light chain constant region contains one domain C L . The VH region and the VL region can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each V H and V LIt contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with an antigen (such as CD228). The constant region of the antibody can optionally mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component of the classical complement system (C1q).

[0032] As used herein, an "antigen-binding fragment" (also referred to as an "antigen-binding domain") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (such as CD228). It has been shown that the antigen-binding function of an antibody can be carried out by fragments of the full-length antibody. Examples of binding fragments included within the term "antigen-binding fragment" of an antibody include (i) Fab fragments consisting of V H 、V L 、C L and C H1 domains; (ii) F(ab')2 containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fab' fragments consisting of V H 、V L 、C L and C H1 domains and the region between the C H1 and C H2 domains; (iv) Fd fragments consisting of V H and C H1 domains; (v) single-chain Fv fragments consisting of the V H and V L domains of a single arm of an antibody; (vi) dAb fragments consisting of V H domains (Ward et al., Nature, 1989); (vii) isolated complementarity-determining regions (CDRs) or combinations of two or more isolated CDRs, which may optionally be linked by a synthetic linker; (viii) "diabodies" containing V H and V L linked in the same polypeptide chain using a short linker (see, for example, patent documents EP 404,097; WO 93 / 11161; and Holliger et al., Proc Natl Acad Sci USA, 1993); and (ix) "domain antibody fragments" containing only V H or V L domains, where in some cases, two or more V H regions are covalently linked.

[0033] The antibody can be polyclonal or monoclonal; xenogeneic, allogeneic or syngeneic; or a modified form thereof (e.g., humanized, chimeric or multispecific). The antibody can also be a fully human antibody.

[0034] As used herein, "framework" or "FR" refers to the variable domain residues other than the hypervariable region (CDR) residues.

[0035] "Fragment crystallizable region" or "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, including the native sequence Fc region and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus according to the EU index numbering of Kabat (Johnson and Wu, Nucleic Acids Res, 2000). The C-terminal lysine of the Fc region (residue 447 according to the EU index of Kabat) can be removed, for example, during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies can include a population of antibodies in which all K447 residues have been removed, a population of antibodies in which no K447 residues have been removed, and a population of antibodies having a mixture of antibodies with and without K447 residues. Native sequence Fc regions in antibodies suitable for the present disclosure include human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.

[0036] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody.

[0037] As used herein, an "isolated antibody" refers to an antibody that is substantially free from its natural environment. For example, an isolated antibody is substantially free of cellular material and other proteins from the cell or tissue source from which it is derived. An "isolated antibody" further refers to an antibody that is substantially free of other antibodies having different antigen specificities. In the context of the present invention, an isolated antibody that specifically binds CD228 is substantially free of antibodies that specifically bind antigens other than CD228. However, an isolated antibody that specifically binds CD228 may have cross-reactivity with other antigens, such as CD228 molecules from other species.

[0038] As used herein, a "monoclonal antibody" refers to a preparation of antibody molecules consisting of a single molecule. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

[0039] As used herein, a "humanized antibody" refers to an antibody comprising CDRs from an antibody of a mammal other than a human and FR regions and constant regions from a human antibody or a human antibody-derived sequence. A humanized antibody can comprise a variable domain having a variable region amino acid sequence that, as evaluated using the IMGT DomainGapAlign tool, is more closely related to humans overall as described by Ehrenmann et al. (2010). Due to reduced antigenicity, humanized antibodies can be used as effective components in therapeutic agents. As used herein, the terms "therapeutic agent" or "therapeutically active agent" refer to pharmaceutically useful agents. A therapeutic agent can be any agent used for the prevention, amelioration, or treatment of a disease, physiological condition, symptom, or for its assessment or diagnosis.

[0040] As used herein, a "human antibody" includes an antibody having a variable region in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis or by in vivo somatic mutation). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto a human framework sequence.

[0041] As used herein, "antibody clone X" can also be referred to as "OMTX". For example, "antibody clone 30" can also be referred to as "OMT30". Antibody clone 8 can also be referred to as "EE03"; antibody clone 24 can also be referred to as "EB03"; antibody clone 30 can also be referred to as "OC04"; antibody clone 35 can also be referred to as "OB02"; and antibody clone 36 can also be referred to as "OE12".

[0042] As used herein, "AAFX" refers to a fusion protein comprising antibody clone X and a lipocalin mutant protein (lipocalin mutant protein I) having the amino acid sequence of SEQ ID NO: 40. For example, "AAF30" refers to a fusion protein comprising antibody clone 30 and lipocalin mutant protein I. The symbol "HC" in the name of the fusion protein refers to a lipocalin mutant protein conjugated to the antibody via the heavy chain of the antibody, and the symbol "LC" in the name of the fusion protein refers to a lipocalin mutant protein conjugated to the antibody via the light chain of the antibody. A fusion protein name containing a clone number before "HC" or "LC" refers to a fusion protein comprising an antibody clone and lipocalin mutant protein I conjugated to the antibody via the heavy chain or light chain of the antibody, respectively. For example, "30HC" refers to a fusion protein comprising antibody clone 30 and lipocalin mutant protein I conjugated thereto via the heavy chain of the antibody.

[0043] An antibody or fusion protein can also be represented by its heavy and light chain sequences (e.g., a fusion protein having the sequences of SEQ ID NO:80 and 76), one of which (e.g., SEQ ID NO:80) contains the sequence of the lipocalin mutant protein in the case of a fusion protein. IV. DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 A schematic diagram is provided showing the clustering of CD137 by bridging CD137-positive T cells with CD228-expressing tumor cells using the fusion proteins provided herein.

[0045] Figure 2A and Figure 2B Showing the CDR sequences of certain antibodies provided herein.

[0046] Figure 3 Showing the results of an ELISA in which an anti-CD228 antibody binds to recombinant human CD228.

[0047] Figures 4A - 4C Showing the binding of an anti-CD228 antibody to CD228-expressing cells as measured by fluorescence intensity. Figure 4D Showing the EC of each antibody against each cell line 50 value.

[0048] Figures 5A - 5E Showing the results of a biolayer interferometry (BLI) assay for the binding kinetics and affinity of an anti-CD228 antibody to CD228.

[0049] Figures 6A - 6C Showing the results of a binding assay of an anti-CD228 antibody to CD228 and certain other transferrin family members.

[0050] Figures 7A - 7B and Figures 8A - 8C Showing the results of a binding assay of an anti-CD228 antibody to human, murine, and cynomolgus macaque CD228.

[0051] Figure 9 Showing the results of a cross-competition assay among various anti-CD228 antibodies.

[0052] Figure 10 Showing the results of an antibody internalization assay using a CD228+ tumor cell line.

[0053] Figures 11A - 11E Showing the results of an antigen recall assay in which the fusion protein co-stimulates innate and adaptive immune cytokines from PBMCs in response to a viral peptide.

[0054] Figure 12 ,Figure 13 , Figure 14 and Figure 15 show the results of an antigen recall assay, wherein the fusion protein co-stimulates T cell and NK cell responses in response to viral peptides.

[0055] Figures 16A - 16E show the results of an antigen recall assay, wherein the fusion protein co-stimulates cytotoxic effector molecules and cytokines from PBMCs in response to viral peptides.

[0056] Figures 17A - 17B show the ability of a representative fusion protein to co-stimulate T cell activation in a CD228 target-dependent manner.

[0057] Figures 18A - 18C show the production of T cell cytokines in co-cultures with CD228-expressing tumor cell lines engineered with anti-CD3 scFv.

[0058] Figure 19 show CD8 T cell proliferation in co-cultures with CD228-expressing tumor cell lines engineered with anti-CD3 scFv, and Figure 20 show tumor cell killing in the co-cultures.

[0059] Figure 21 show the pharmacokinetics of the fusion protein in cynomolgus monkeys. Figures 22A - 22D , Figures 23A - 23C and Figures 24A - 24B show the in vivo activity of the fusion protein in a humanized xenograft model. V. DETAILED DESCRIPTION OF THE DISCLOSURE

[0060] As described herein, in one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds CD228. Such anti-CD228 antibodies or antigen-binding fragments thereof can be used as antibody therapeutics per se, conjugated with a therapeutic agent to produce an antibody-drug conjugate, included as part of a bispecific or multispecific antibody, or included as part of a fusion molecule. In certain embodiments, the antibody or antigen-binding fragment is incorporated into a fusion molecule that includes a binding domain that binds a target different from CD228. For example, as described in more detail below, in some embodiments, the antibody or antigen-binding fragment is incorporated into a fusion molecule that can bind both CD228 and CD137. The use of an anti-CD228 antibody or antigen-binding fragment as provided herein in a fusion protein (such as those that target both CD228 and CD137) can be used to promote CD137 clustering by bridging CD137-positive T cells with CD228-expressing tumor cells located in the tumor microenvironment, as exemplified Figure 1As shown. The anti-CD228 antibodies and antigen-binding fragments provided herein can be used to generate fusion proteins against other targets to achieve similar effects. In other aspects, the present disclosure provides methods and useful applications of the anti-CD228 antibodies and their antigen-binding fragments provided herein. The present disclosure also provides methods for making the CD228-binding antibodies or their antigen-binding fragments described herein and compositions comprising such proteins. The CD228-binding antibodies or their antigen-binding fragments and their compositions of the present disclosure can be used in methods for detecting CD228 in a sample or in methods for binding CD228 in a subject. Such antibodies or their antigen-binding fragments having these features related to the uses provided herein have not been previously described. A. Exemplary antibody or its antigen-binding fragment specific for CD228

[0061] In some embodiments, the antibody or its antigen-binding fragment that binds CD228 provided herein comprises: i) A heavy chain variable domain (VH) that comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:110, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:111, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:112, and a light chain variable domain (VL) that comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:116, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:117, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:118; ii) A VH that comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:113, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:114, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:115, and a VL that comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:119, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:120, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:121; iii) VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:130, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:131, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:132, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:136, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:137, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:138; iv) VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:133, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:134, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:135, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:139, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:140, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:141; v) VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:150, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:151, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:152, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:156, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:157, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:158; vi) VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:153, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:154, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:155, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:159, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:160, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:161; vii) VH, which comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:170, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:171, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:172, and VL, which comprises (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:176, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:177, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:178; viii) VH, which comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:173, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:174, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:175, and VL, which comprises (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:179, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:180, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:181; ix) VH, which comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:190, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:191, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:192, and VL, which comprises (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:196, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:197, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:198; x) VH, which comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:193, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:194, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:195, and VL, which comprises (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:199, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:200, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:201; xi) VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 210, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 211, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 212, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 216, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 217, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 218; xii) VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 213, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 214, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 215, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 219, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 220, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 221; xiii) VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 230, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 231, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 232, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 236, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 237, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 238; xiv) VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 233, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 234, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 235, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 239, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 240, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 241; xv) VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:250, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:251, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:252, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:256, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:257, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:258; xvi) VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:253, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:254, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:255, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:259, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:260, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:261; xvii) VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:270, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:271, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:272, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:276, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:277, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:278; or xviii) VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:273, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:274, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:275, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:279, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:280, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:281.

[0062] In some embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein comprises: i) VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:210, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:211, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:212, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:216, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:217, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:218; ii) VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:213, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:214, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:215, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:219, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:220, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:221; iii) VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:250, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:251, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:252, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:256, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:257, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:258; or iv) VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:253, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:254, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:255, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:259, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:260, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:261.

[0063] In some embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein comprises: a VH that comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:210, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:211, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:212, and a VL that comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:216, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:217, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:218; or a VH that comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:213, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:214, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:215, and a VL that comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:219, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:220, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:221.

[0064] In some embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein comprises: i) a VH that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:122; and a VL that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:124; ii) a VH that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:142; and a VL that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:144; iii) VH, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 162; and VL, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 164; iv) VH, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 182; and VL, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 184; v) VH, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 202; and VL, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 204; vi) VH, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 222; and VL, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 224; vii) VH, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 242; and VL, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 244; viii) VH, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 262; and VL, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 264; or ix) VH, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 282; and VL, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 284.

[0065] In some embodiments, an antibody or antigen-binding fragment thereof that binds CD228 provided herein comprises: a VH that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 222; and a VL that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 224; or a VH that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 262; and a VL that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 264.

[0066] In some embodiments, an antibody or antigen-binding fragment thereof that binds CD228 provided herein comprises: a VH that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 222; and a VL that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 224.

[0067] In some embodiments, an antibody or antigen-binding fragment thereof that binds CD228 provided herein comprises: i) a VH that comprises the amino acid sequence of SEQ ID NO: 122; and a VL that comprises the amino acid sequence of SEQ ID NO: 124; ii) a VH that comprises the amino acid sequence of SEQ ID NO: 142; and a VL that comprises the amino acid sequence of SEQ ID NO: 144; iii) VH, which comprises the amino acid sequence of SEQ ID NO:162; and VL, which comprises the amino acid sequence of SEQ ID NO:164; iv) VH, which comprises the amino acid sequence of SEQ ID NO:182; and VL, which comprises the amino acid sequence of SEQ ID NO:184; v) VH, which comprises the amino acid sequence of SEQ ID NO:202; and VL, which comprises the amino acid sequence of SEQ ID NO:204; vi) VH, which comprises the amino acid sequence of SEQ ID NO:222; and VL, which comprises the amino acid sequence of SEQ ID NO:224; vii) VH, which comprises the amino acid sequence of SEQ ID NO:242; and VL, which comprises the amino acid sequence of SEQ ID NO:244; viii) VH, which comprises the amino acid sequence of SEQ ID NO:262; and VL, which comprises the amino acid sequence of SEQ ID NO:264; or ix) VH, which comprises the amino acid sequence of SEQ ID NO:282; and VL, which comprises the amino acid sequence of SEQ ID NO:284.

[0068] In some embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein comprises: VH, which comprises the amino acid sequence of SEQ ID NO:222; and VL, which comprises the amino acid sequence of SEQ ID NO:224; or VH, which comprises the amino acid sequence of SEQ ID NO:262; and VL, which comprises the amino acid sequence of SEQ ID NO:264.

[0069] In some embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein comprises: VH, which comprises the amino acid sequence of SEQ ID NO:222; and VL, which comprises the amino acid sequence of SEQ ID NO:224.

[0070] In some embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein comprises: i) A heavy chain (HC) that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 126; and a light chain (LC) that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 128; ii) A HC that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 146; and a LC that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 148; iii) A HC that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 166; and a LC that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 168; iv) A HC that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 186; and a LC that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 188; v) An HC that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 206; and an LC that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 208; vi) An HC that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 226; and an LC that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 228; vii) An HC that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 246; and an LC that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 248; viii) An HC that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 266; and an LC that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 268; or ix) An HC that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 286; and an LC that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 288.

[0071] In some embodiments, an antibody or antigen-binding fragment thereof that binds CD228 provided herein comprises: an HC that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 226; and an LC that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 228; or an HC that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 266; and an LC that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 268.

[0072] In some embodiments, an antibody or antigen-binding fragment thereof that binds CD228 provided herein comprises: an HC that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 226; and an LC that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 228.

[0073] In some embodiments, the anti-CD228 antibodies or antigen-binding fragments thereof provided herein comprise: i) an HC that comprises the amino acid sequence of SEQ ID NO:126; and an LC that comprises the amino acid sequence of SEQ ID NO:128; ii) an HC that comprises the amino acid sequence of SEQ ID NO:146; and an LC that comprises the amino acid sequence of SEQ ID NO:148; iii) an HC that comprises the amino acid sequence of SEQ ID NO:166; and an LC that comprises the amino acid sequence of SEQ ID NO:168; iv) an HC that comprises the amino acid sequence of SEQ ID NO:186; and an LC that comprises the amino acid sequence of SEQ ID NO:188; v) an HC that comprises the amino acid sequence of SEQ ID NO:206; and an LC that comprises the amino acid sequence of SEQ ID NO:208; vi) an HC that comprises the amino acid sequence of SEQ ID NO:226; and an LC that comprises the amino acid sequence of SEQ ID NO:228; vii) an HC that comprises the amino acid sequence of SEQ ID NO:246; and an LC that comprises the amino acid sequence of SEQ ID NO:248; viii) an HC that comprises the amino acid sequence of SEQ ID NO:266; and an LC that comprises the amino acid sequence of SEQ ID NO:268; or ix) an HC that comprises the amino acid sequence of SEQ ID NO:286; and an LC that comprises the amino acid sequence of SEQ ID NO:288.

[0074] In some embodiments, the anti-CD228 antibodies or antigen-binding fragments thereof provided herein comprise: an HC that comprises the amino acid sequence of SEQ ID NO:226; and an LC that comprises the amino acid sequence of SEQ ID NO:228; or an HC that comprises the amino acid sequence of SEQ ID NO:266; and an LC that comprises the amino acid sequence of SEQ ID NO:268.

[0075] In some embodiments, the anti-CD228 antibodies or antigen-binding fragments thereof provided herein comprise: an HC that comprises the amino acid sequence of SEQ ID NO:226; and an LC that comprises the amino acid sequence of SEQ ID NO:228.

[0076] In some embodiments, the anti-CD228 antibodies provided herein are monoclonal antibodies. In some embodiments, the anti-CD228 antibodies provided herein are humanized or chimeric antibodies. In some embodiments, the anti-CD228 antibodies provided herein are IgG1, IgG2, IgG3, or IgG4 antibodies.

[0077] In some embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein binds to CD228 with a K D value of 175 nM or lower. In some embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein binds to CD228 with a K D value of 150 nM or lower. In some embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein binds to CD228 with a K D value of 100 nM or lower. In some embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein binds to CD228 with a K D value of 50 nM or lower. In some embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein binds to CD228 with a K D value of 75 nM or lower. In some embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein binds to CD228 with a K D value of 25 nM or lower. In some embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein binds to CD228 with a K D value of 20 nM or lower. In some embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein binds to CD228 with a K D value of 15 nM or lower. In some embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein binds to CD228 with a K D value of 10 nM or lower. In some embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein binds to CD228 with a K D value of 5 nM or lower. In some embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein binds to CD228 with a K D value of 4 nM or lower. In some embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein binds to CD228 with a K D value of 3 nM or lower. In some embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein binds to CD228 with a K DValues bind to CD228. In some embodiments, the anti-CD228 antibodies or antigen-binding fragments thereof provided herein bind to CD228 with a K D value that is 2 nM or lower. In some embodiments, the anti-CD228 antibodies or antigen-binding fragments thereof provided herein bind to CD228 with a K D value that is 1.5 nM or lower. In some embodiments, the anti-CD228 antibodies or antigen-binding fragments thereof provided herein bind to CD228 with a K D value that is 1 nM or lower.

[0078] In some embodiments, the anti-CD228 antibodies or antigen-binding fragments thereof provided herein bind to cynomolgus macaque CD228. In some such embodiments, the anti-CD228 antibodies or antigen-binding fragments thereof provided herein comprise: i) a VH that comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:170, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:171, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:172, and a VL that comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:176, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:177, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:178; ii) a VH that comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:173, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:174, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:175, and a VL that comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:179, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:180, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:181; iii) a VH that comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:210, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:211, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:212, and a VL that comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:216, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:217, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:218; iv) VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:213, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:214, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:215, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:219, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:220, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:221; v) VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:250, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:251, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:252, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:256, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:257, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:258; or vi) VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:253, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:254, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:255, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:259, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:260, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:261.

[0079] In some such embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein comprises the CDR sets as set forth above and: i) VH, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:182; and VL, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:184; ii) a VH that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 222; and a VL that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 224; or iii) a VH that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 262; and a VL that comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 264.

[0080] In some such embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein comprises: a VH comprising the amino acid sequence of SEQ ID NO: 182, and a VL comprising the amino acid sequence of SEQ ID NO: 184; a VH comprising the amino acid sequence of SEQ ID NO: 222, and a VL comprising the amino acid sequence of SEQ ID NO: 224; or a VH comprising the amino acid sequence of SEQ ID NO: 262, and a VL comprising the amino acid sequence of SEQ ID NO: 264.

[0081] In some embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein does not bind murine CD228, or binds murine CD228 with an affinity that is at least 100-fold lower than that for human CD228. In some embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein does not bind transferrin or lactotransferrin. In some such embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein comprises: i) VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 170, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 171, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 172, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; ii) VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 173, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 174, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 175, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 179, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 180, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 181; iii) VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 190, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 191, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 192, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 196, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 197, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 198; iv) VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 193, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 194, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 195, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 199, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 200, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 201; v) VH, which comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 210, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 211, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 212, and VL, which comprises (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 216, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 217, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 218; vi) VH, which comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 213, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 214, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 215, and VL, which comprises (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 219, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 220, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 221; vii) VH, which comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 230, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 231, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 232, and VL, which comprises (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 236, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 237, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 238; viii) VH, which comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 233, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 234, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 235, and VL, which comprises (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 239, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 240, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 241; ix) A VH that comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:250, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:251, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:252, and a VL that comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:256, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:257, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:258; x) A VH that comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:253, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:254, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:255, and a VL that comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:259, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:260, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:261; xi) A VH that comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:270, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:271, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:272, and a VL that comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:276, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:277, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:278; or xii) A VH that comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:273, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:274, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:275, and a VL that comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:279, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:280, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:281.

[0082] In some such embodiments, the anti-CD228 antibodies or antigen-binding fragments thereof provided herein comprise the CDR sets as set forth above and: i) VH, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 182; and VL, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 184; ii) VH, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 202; and VL, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 204; iii) VH, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 222; and VL, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 224; iv) VH, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 242; and VL, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 244; or v) VH, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:262; and VL, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:264; or vi) VH, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:282; and VL, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:284.

[0083] In some such embodiments, the anti-CD228 antibody or antigen-binding fragment thereof provided herein comprises: VH comprising the amino acid sequence of SEQ ID NO:182, and VL comprising the amino acid sequence of SEQ ID NO:184; VH comprising the amino acid sequence of SEQ ID NO:202, and VL comprising the amino acid sequence of SEQ ID NO:204; VH comprising the amino acid sequence of SEQ ID NO:222, and VL comprising the amino acid sequence of SEQ ID NO:224; VH comprising the amino acid sequence of SEQ ID NO:242, and VL comprising the amino acid sequence of SEQ ID NO:244; VH comprising the amino acid sequence of SEQ ID NO:262, and VL comprising the amino acid sequence of SEQ ID NO:264; or VH comprising the amino acid sequence of SEQ ID NO:282, and VL comprising the amino acid sequence of SEQ ID NO:284.

[0084] In some embodiments, an antibody or an antigen-binding fragment thereof that binds to CD228 is provided, wherein the antibody or the antigen-binding fragment thereof competes with any one of the antibodies or antigen-binding fragments thereof disclosed herein for binding to CD228. In some such embodiments, the antibody is a monoclonal antibody. In some such embodiments, the antibody is a humanized antibody or a chimeric antibody. In some such embodiments, the antibody is an IgG1, IgG2, IgG3 or IgG4 antibody. In some such embodiments, the antibody or the antigen-binding fragment thereof has a K of 175 nM or lower DThe value binds to CD228. In some such embodiments, the antibody or antigen-binding fragment thereof binds to cynomolgus monkey CD228. In some such embodiments, the antibody or antigen-binding fragment thereof does not bind to murine CD228, or binds to murine CD228 with an affinity that is at least 100-fold lower than that for human CD228. In some such embodiments, the antibody or antigen-binding fragment thereof does not bind to transferrin or lactotransferrin. In some such embodiments, the antibody or antigen-binding fragment thereof competes with an antibody or antigen-binding fragment thereof that comprises a VH comprising the amino acid sequence of SEQ ID NO:122 and a VL comprising the amino acid sequence of SEQ ID NO:124; a VH comprising the amino acid sequence of SEQ ID NO:142 and a VL comprising the amino acid sequence of SEQ ID NO:144; a VH comprising the amino acid sequence of SEQ ID NO:162 and a VL comprising the amino acid sequence of SEQ ID NO:164; a VH comprising the amino acid sequence of SEQ ID NO:182 and a VL comprising the amino acid sequence of SEQ ID NO:184; a VH comprising the amino acid sequence of SEQ ID NO:202 and a VL comprising the amino acid sequence of SEQ ID NO:204; a VH comprising the amino acid sequence of SEQ ID NO:222 and a VL comprising the amino acid sequence of SEQ ID NO:224; a VH comprising the amino acid sequence of SEQ ID NO:242 and a VL comprising the amino acid sequence of SEQ ID NO:244; a VH comprising the amino acid sequence of SEQ ID NO:262 and a VL comprising the amino acid sequence of SEQ ID NO:264; or a VH comprising the amino acid sequence of SEQ ID NO:282 and a VL comprising the amino acid sequence of SEQ ID NO:284 for binding to CD228. In some such embodiments, the antibody or antigen-binding fragment thereof competes with an antibody or antigen-binding fragment thereof that comprises a VH comprising the amino acid sequence of SEQ ID NO:202 and a VL comprising the amino acid sequence of SEQ ID NO:204; a VH comprising the amino acid sequence of SEQ ID NO:242 and a VL comprising the amino acid sequence of SEQ ID NO:244; or a VH comprising the amino acid sequence of SEQ ID NO:262 and a VL comprising the amino acid sequence of SEQ ID NO:264 for binding to CD228.In some such embodiments, the antibody or antigen-binding fragment thereof competes for binding to CD228 with an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence of SEQ ID NO:202 and a VL comprising the amino acid sequence of SEQ ID NO:204; or a VH comprising the amino acid sequence of SEQ ID NO:242 and a VL comprising the amino acid sequence of SEQ ID NO:244. In some such embodiments, competition for binding to CD228 is measured by flow cytometry using at least one labeled antibody or antigen-binding fragment thereof.

[0085] In some embodiments, the CDR sequences disclosed herein are defined according to the Kabat numbering scheme as described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD. In some embodiments, the CDR sequences disclosed herein are defined according to the IMGT method as described in Lefranc, M.-P., The Immunologist, 7, 132-136 (1999).

[0086] The antibody that specifically binds to CD228 comprised in the fusion proteins disclosed herein may comprise an Fc portion that allows for an extended in vivo half-life of the bispecific binding molecules disclosed herein. In some embodiments, such Fc portions are preferably of human origin, more preferably the human Fc portion of an IgG1 or IgG4 antibody, even more preferably an engineered human Fc portion of IgG1 or IgG4 having activating or silent effector functions. In some embodiments, silent effector functions may be more preferred as compared to activating effector functions. In some embodiments, such Fc portions are engineered to silence effector functions with mutations at positions 234 and / or 235 (EU index numbering according to Kabat) (Johnson and Wu, Nucleic Acids Res, 2000). In some embodiments, mutations may be introduced at positions F234 and L235 in the provided anti-CD228 antibody to silence effector functions. In other embodiments, mutations may be introduced at positions D265 and P329 in the provided anti-CD228 antibody to silence effector functions. The numbering of both sets of these potential mutations is according to the Kabat EU index (Shields et al., J Biol Chem, 2001). In some embodiments, the provided anti-CD228 antibody has an engineered IgG4 backbone carrying the mutations S228P, F234A and L235A.

[0087] A variety of techniques for generating antibodies and their antigen-binding fragments are well known in the art and are described, for example, in Altshuler et al. (2010). Thus, for example, polyclonal antibodies can be obtained from the blood of an animal after immunization with a mixture of an antigen and an additive and an adjuvant, and monoclonal antibodies can be generated by any technique that provides antibodies produced by a continuous cell line culture. Examples of such techniques are described, for example, in Harlow and Lane (1999), (1988), and include the hybridoma technique originally described by and Milstein, 1975, the trioma technique, the human B-cell hybridoma technique (see, for example, Li et al., Proc Natl Acad Sci USA, 2006; Kozbor and Roder, Immunol Today, 1983), and the EBV-hybridoma technique for generating human monoclonal antibodies (Cole et al., Cancer Res, 1984). In addition, recombinant antibodies can be obtained from monoclonal antibodies or can be prepared de novo using various display methods, such as phage, ribosome, mRNA, or cell display. In some embodiments, suitable systems for expressing recombinant (humanized) antibodies or fragments thereof can be selected from, for example, bacteria, yeast, insects, mammalian cell lines, or transgenic animals or plants (see, for example, U.S. Patent No. 6,080,560; Holliger and Hudson, Nat Biotechnol, 2005). In addition, the techniques described for generating single-chain antibodies (see especially U.S. Patent No. 4,946,778) can be adapted to generate single-chain antibodies specific for the targets of the present invention. Surface plasmon resonance as employed in the BIAcore system can be used to enhance the efficiency of phage antibodies. B. Immunoconjugates of Antibodies or Their Antigen-Binding Fragments

[0088] The antibodies or antigen-binding fragments thereof provided herein can be conjugated to a cytotoxic or cytostatic moiety (including its pharmaceutically compatible salts) to form an immunoconjugate, such as an antibody-drug conjugate (ADC). Particularly suitable moieties for conjugation to an antibody or antigen-binding fragment thereof are cytotoxic agents (e.g., chemotherapeutic agents), prodrug converting enzymes, radioisotopes or compounds, or toxins (collectively referred to as therapeutic agents). For example, an antibody or antigen-binding fragment thereof can be conjugated to a cytotoxic agent, such as a chemotherapeutic agent or toxin (e.g., a cytostatic or cytocidal agent, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin). Examples of useful classes of cytotoxic agents include, for example, DNA minor groove binders, DNA alkylating agents, and tubulin inhibitors. Exemplary cytotoxic agents include, for example, auristatins, camptothecin, calicheamicins, duocarmycins, etoposides, maytansinoids (e.g., DM1, DM2, DM3, DM4), taxanes, benzodiazepines (e.g., pyrrolo[1,4]benzodiazepines indolinobenzodiazepines (indolinobenzodiazepines) and oxazolidinobenzodiazepines (oxazolidinobenzodiazepines)) and vinca alkaloids.

[0089] In one embodiment, the antibody or antigen-binding fragment thereof is conjugated to a prodrug converting enzyme. The prodrug converting enzyme can be recombinantly fused to the antibody or antigen-binding fragment thereof or chemically conjugated thereto using known methods. Exemplary prodrug converting enzymes are carboxypeptidase G2, β-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, β-lactamase, β-glucosidase, nitroreductase, and carboxypeptidase A.

[0090] Techniques for conjugating therapeutic agents to antibodies or antigen-binding fragments thereof are well known. (See, e.g., Alley et al., Current Opinion in Chemical Biology 2010 14:1-9; Senter, Cancer J., 2008, 14(3):154-169.) A therapeutic agent can be conjugated in a manner that reduces its activity unless it is cleaved from the antibody or antigen-binding fragment thereof (e.g., by hydrolysis, by proteolytic degradation, or by a cleaving agent). In some aspects, the therapeutic agent is attached to the antibody or antigen-binding fragment thereof via a cleavable linker that is sensitive to cleavage in the intracellular environment of cancer cells expressing CD228 but not significantly sensitive to the extracellular environment, such that the immunoconjugate cleaves from the antibody or antigen-binding fragment thereof upon its internalization by cancer cells expressing CD228 (e.g., in endosomes, or, e.g., in the lysosomal environment or in the caveolear environment by virtue of pH sensitivity or protease sensitivity). In some aspects, the therapeutic agent can also be attached to the antibody or antigen-binding fragment thereof with a non-cleavable linker.

[0091] Generally, an immunoconjugate comprises a linker region between the therapeutic agent and the antibody or antigen-binding fragment thereof. The linker is typically cleavable under intracellular conditions such that cleavage of the linker releases the therapeutic agent from the antibody or antigen-binding fragment thereof in the intracellular environment (e.g., in lysosomes or endosomes or vesicles). The linker can be, for example, a peptidyl linker that is cleaved by intracellular peptidases or proteases (including lysosomal or endosomal proteases). Cleaving agents can include cathepsin B and D and plasmin (see, e.g., Dubowchik and Walker, Pharm. Therapeutics 83:67-123, 1999). Most typically, the peptidyl linker is cleavable by an enzyme present in cells expressing CD228. For example, a peptidyl linker cleavable by the thiol-dependent protease cathepsin-B, which is highly expressed in cancer tissue (e.g., a linker containing a Phe-Leu or Val-Cit peptide) can be used.

[0092] The cleavable linker can be pH-sensitive, i.e., sensitive to hydrolysis at certain pH values. Generally, pH-sensitive linkers can be hydrolyzed under acidic conditions. For example, acid-labile linkers that can be hydrolyzed in lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitylamides, orthoesters, acetals, ketals, etc.) can be used. (See, e.g., U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, Pharm. Therapeutics 83:67-123, 1999; Neville et al., Biol. Chem. 264:14653-14661, 1989.) Such linkers are relatively stable at neutral pH conditions, such as those in blood, but are unstable at pH values below 5.5 or 5.0 (approximate pH of lysosomes).

[0093] Other linkers can be cleaved under reducing conditions (e.g., disulfide linkers). Disulfide linkers include those formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene), SPDB, and SMPT. (See, e.g., Thorpe et al., Cancer Res. 47:5924-5931, 1987; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel ed., Oxford U.Press, 1987). See also U.S. Patent No. 4,880,935.)

[0094] The linker can also be a malonate linker (Johnson et al., Anticancer Res. 15:1387-93, 1995), a maleimidobenzoyl linker (Lau et al., Bioorg-Med-Chem. 3:1299-1304, 1995), or a 3'-N-amide analogue (Lau et al., Bioorg-Med-Chem. 3:1305-12, 1995).

[0095] In other embodiments, the linker is a non-cleavable linker, such as a maleimido-alkylene- or maleimido-aryl linker, which is directly attached to the therapeutic agent and is released by proteolytic degradation of the antibody or its antigen-binding fragment.

[0096] Generally, the linker is not significantly sensitive to the extracellular environment, which means that no more than about 20%, typically no more than about 15%, more typically no more than about 10%, and even more typically no more than about 5%, no more than about 3% or no more than about 1% of the linker in a sample of the immunoconjugate cleaves when the immunoconjugate is present in the extracellular environment (e.g., in plasma). Whether the linker is not significantly sensitive to the extracellular environment can be determined, for example, by independently incubating (a) the immunoconjugate (“immunoconjugate sample”) and (b) an equimolar amount of the unconjugated antibody or its antigen-binding fragment or therapeutic agent (“control sample”) with plasma for a predetermined time (e.g., 2, 4, 8, 16, or 24 hours), and then comparing the amount of the unconjugated antibody or its antigen-binding fragment or therapeutic agent present in the immunoconjugate sample as measured, for example, by high performance liquid chromatography with the amount present in the control sample.

[0097] The linker can also facilitate cellular internalization. The linker can facilitate cellular internalization when conjugated to a therapeutic agent (i.e., in the context of the linker-therapeutic agent moiety of an immunoconjugate or immunoconjugate derivative as described herein). Alternatively, the linker can facilitate cellular internalization when conjugated to a therapeutic agent and an antibody or its antigen-binding fragment (i.e., in the context of an immunoconjugate as described herein).

[0098] Exemplary immunoconjugates include auristatin-based conjugates, which means that the drug component is an auristatin drug. Auristatins bind to tubulin, which has been shown to interfere with microtubule dynamics as well as nuclear and cell division, and have anti-cancer activity. Generally, an auristatin-based antibody-drug conjugate comprises a linker between the auristatin drug and the antibody or its antigen-binding fragment. The linker can be, for example, a cleavable linker (e.g., a peptidyl linker) or a non-cleavable linker (e.g., a linker released by degradation of the antibody). The auristatin can be auristatin E or a derivative thereof. The auristatin can be, for example, an ester formed between auristatin E and a keto acid. For example, auristatin E can react with p-acetylbenzoic acid or benzoylvaleric acid to produce auristatin EB (AEB) and auristatin EVB (AEVB), respectively. Other typical auristatins include MMAF and MMAE. The synthesis and structure of exemplary auristatins are described in U.S. Patents or Publication Nos. 7,659,241, 7,498,298, 2009-0111756, 2009-0018086, and 7,968,687, each of which is incorporated herein by reference in its entirety and for all purposes.

[0099] Exemplary auristatin-based conjugates include the vcMMAE, vcMMAF, and mcMMAF immunoconjugates as shown below, where Ab is an anti-CD228 antibody or antigen-binding fragment thereof as described herein and val-cit represents the valine-citrulline dipeptide. The drug payload is represented by p, which is the number of drug-linker molecules per antibody or antigen-binding fragment thereof. Depending on the context, p can represent the average number of drug-linker molecules per antibody or antigen-binding fragment in a composition of antibodies or antigen-binding fragments thereof, which is also referred to as the average drug payload. In some embodiments, p ranges from 1 to 20 and is preferably from 1 to 8. In some embodiments, when p represents the average drug payload, p ranges from about 2 to about 5. In some embodiments, p is about 2, about 3, about 4, or about 5. The average drug number per antibody in a formulation can be characterized by conventional means such as mass spectrometry, HIC, ELISA assays, and HPLC. In some aspects, the anti-CD228 antibody or antigen-binding fragment thereof is attached to the drug-linker through a cysteine residue of the antibody or antigen-binding fragment. In some embodiments, the cysteine residue is a residue engineered into the antibody or antigen-binding fragment. In other aspects, the cysteine residue is an interchain disulfide cysteine residue. C. Exemplary Uses and Applications of Antibodies or Antigen-Binding Fragments Thereof That Are Specific for CD228, or Immunoconjugates of Antibodies or Antigen-Binding Fragments Thereof

[0100] In some aspects, the present disclosure provides diagnostic and / or analytical kits that comprise one or more antibodies, antigen-binding fragments thereof, or immunoconjugates of antibodies or antigen-binding fragments thereof according to the present disclosure.

[0101] In addition to their use in diagnostics, in yet another aspect, the present disclosure contemplates pharmaceutical compositions that comprise one or more antibodies, antigen-binding fragments thereof, or immunoconjugates of antibodies or antigen-binding fragments thereof of the present disclosure and a pharmaceutically acceptable excipient.

[0102] In addition, in some embodiments, the provided antibodies, antigen-binding fragments thereof, or immunoconjugates of the antibodies or antigen-binding fragments thereof can be used in therapy, such as as anti-tumor agents and / or anti-infective agents and / or immunomodulators. In some embodiments, the provided antibodies, antigen-binding fragments thereof, or immunoconjugates of the antibodies or antigen-binding fragments thereof can be used in the manufacture of a medicament (such as a medicament for treating cancer, including CD228-positive cancer). In some embodiments, the antibodies, antigen-binding fragments thereof, or immunoconjugates of the antibodies or antigen-binding fragments thereof of the present disclosure are contemplated for use in methods for preventing, ameliorating, or treating human diseases, such as cancer, including CD228-positive cancer. Accordingly, there is also provided a method for preventing, ameliorating, or treating a human disease, such as cancer, including CD228-positive cancer, in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of one or more antibodies, antigen-binding fragments thereof, or immunoconjugates of the antibodies or antigen-binding fragments thereof of the present disclosure, or one or more compositions comprising such antibodies, antigen-binding fragments thereof, or immunoconjugates of the antibodies or antigen-binding fragments thereof. In some embodiments, the cancer is CD228-positive cancer.

[0103] Examples of cancers that can be treated with the antibodies, antigen-binding fragments thereof, or immunoconjugates of the antibodies or antigen-binding fragments thereof of the present disclosure include lung cancer and melanoma (such as cutaneous or uveal malignant melanoma), pancreatic cancer, mesothelioma, colorectal cancer, thyroid cancer, breast cancer, cholangiocarcinoma, esophageal cancer, and head and neck cancer. In some embodiments, the cancer includes metastatic cancer.

[0104] In some embodiments, the fusion proteins of the present disclosure can simultaneously target tumor cells expressing CD228 and activate lymphocytes that activate the host immune system adjacent to such tumor cells. In some embodiments, the fusion proteins of the present disclosure can enhance the activity of targeted anti-tumor T cells, enhance anti-tumor immunity and / or have a direct inhibitory effect on tumor growth, thereby producing a synergistic anti-tumor result. In some embodiments, the fusion proteins of the present disclosure can activate the immune response in the tumor microenvironment. In some embodiments, the fusion proteins of the present disclosure can reduce the side effects, i.e., off-target toxicity, of effector lymphocytes on healthy cells, such as by locally inhibiting oncogene activity and / or inducing lymphocyte activation.

[0105] In some embodiments, the present disclosure encompasses using the fusion proteins of the present disclosure or compositions comprising the provided fusion proteins to induce a local lymphocyte response near CD228-positive tumor cells. Accordingly, in some embodiments, the present disclosure provides a method for inducing a local lymphocyte response near CD228-positive tumor cells, which comprises administering one or more fusion proteins of the present disclosure or one or more compositions comprising such fusion proteins. D. Generation of Exemplary Provided Antibodies or Antigen-Binding Fragments Thereof Specific for CD228

[0106] In some embodiments, the present disclosure provides nucleic acid molecules (e.g., DNA or RNA) comprising nucleotide sequences encoding the provided antibodies or antigen-binding fragments thereof. In some embodiments, the present disclosure encompasses vectors containing the provided nucleic acid molecules. In some embodiments, the present disclosure encompasses host cells containing the provided nucleic acid molecules or vectors. Because the degeneracy of the genetic code permits certain codons to be replaced by other codons that specify the same amino acid, the present disclosure is not limited to specific nucleic acid molecules encoding antibodies or antigen-binding fragments thereof as described herein, but rather encompasses all nucleic acid molecules comprising nucleotide sequences encoding functional antibodies or antigen-binding fragments thereof. In this regard, the present disclosure also relates to nucleotide sequences encoding the provided antibodies or antigen-binding fragments thereof.

[0107] A nucleic acid molecule is said to be "capable of expressing a nucleic acid molecule" or "capable of permitting expression of a nucleotide sequence" if the nucleic acid molecule, e.g., DNA, includes sequence elements that contain information regarding transcriptional and / or translational regulation and such sequences are "operably linked" to the nucleotide sequence encoding the protein. An operable linkage is a linkage in which the regulatory sequence elements and the sequence to be expressed are connected in such a way as to enable gene expression. The exact nature of the regulatory regions required for gene expression can vary between species, but generally, these regions include a promoter, which in prokaryotes contains both the promoter itself (i.e., the DNA element that directs the initiation of transcription) and a DNA element that will send a translation initiation signal when transcribed into RNA. Such promoter regions typically include 5' non-coding sequences involved in transcription and translation initiation, such as the -35 / -10 box and Shine-Dalgarno element in prokaryotes, or the TATA box, CAAT sequence, and 5' cap element in eukaryotes. These regions may also include enhancer or suppressor elements as well as translation signals and leader sequences for targeting the native protein to specific compartments of the host cell.

[0108] In addition, 3' non-coding sequences may contain regulatory elements involved in transcription termination, polyadenylation, etc. However, if these termination sequences do not function satisfactorily in a particular host cell, they may be replaced with signals that function in that cell.

[0109] Accordingly, the nucleic acid molecules of the present disclosure can be "operably linked" to one or more regulatory sequences, such as a promoter sequence, to permit expression of the nucleic acid molecule. In some embodiments, the nucleic acid molecules of the present disclosure include a promoter sequence and a transcription termination sequence. Suitable prokaryotic promoters are, for example, the tet promoter, the lacUV5 promoter, or the T7 promoter. Examples of promoters that can be used for expression in eukaryotic cells are the SV40 promoter or the CMV promoter.

[0110] In some embodiments, the provided nucleic acid molecule can also be part of a vector or any other kind of cloning vehicle (e.g., plasmid, phagemid, phage, baculovirus, cosmid, or artificial chromosome).

[0111] In some embodiments, the provided nucleic acid molecule can be contained in a phagemid. As used in this context, a phagemid vector refers to a vector encoding the intergenic region of a temperate phage, such as M13 or f1, or a functional part thereof fused to a cDNA of interest. For example, in some embodiments, after repeated infection of bacterial host cells with such provided phagemid vectors and appropriate helper phages (e.g., M13K07, VCS-M13, or R408), intact phage particles are produced, thereby enabling the encoded heterologous cDNA to be physically linked to its corresponding polypeptide displayed on the phage surface (Lowman, Annu Rev Biophys Biomol Struct, 1997; Rodi and Makowski, Curr Opin Biotechnol, 1999).

[0112] According to various embodiments, in addition to the regulatory sequences described above and the nucleic acid sequences encoding an antibody or an antigen-binding fragment thereof as described herein, the cloning vehicle can include replication and control sequences derived from a species compatible with the host cell for expression, as well as selectable markers that confer a selectable phenotype on the transformed or transfected cells. A large number of suitable cloning vectors are known in the art and commercially available.

[0113] In some embodiments, the present disclosure also relates to methods of producing the antibodies or antigen-binding fragments thereof of the present disclosure starting from nucleic acids encoding an antibody or an antigen-binding fragment thereof or any subunit thereof using genetic engineering methods. In some embodiments, the provided methods can be carried out in vivo, wherein the provided antibody or antigen-binding fragment thereof can be produced, for example, in a bacterial or eukaryotic host organism and then isolated from the host organism or its culture. The antibodies or antigen-binding fragments thereof of the present disclosure can also be produced in vitro, for example, using an in vitro translation system.

[0114] When producing an antibody or an antigen-binding fragment thereof in vivo, nucleic acids encoding such an antibody or an antigen-binding fragment thereof can be introduced into a suitable bacterial or eukaryotic host organism using recombinant DNA techniques well known in the art. In some embodiments, a DNA molecule encoding an antibody or an antigen-binding fragment thereof as described herein, and in particular a cloning vector containing the coding sequence of such an antibody or an antigen-binding fragment thereof, can be transformed into a host cell capable of expressing the gene. Transformation can be carried out using standard techniques. Accordingly, the present disclosure also relates to host cells containing the nucleic acid molecules as disclosed herein.

[0115] In some embodiments, the transformed host cells can be cultured under conditions suitable for expressing the nucleotide sequences encoding the antibodies or antigen-binding fragments thereof of the present disclosure. In some embodiments, the host cells can be prokaryotic, such as Escherichia coli (E. coli) or Bacillus subtilis; or eukaryotic, such as Saccharomyces cerevisiae, Pichia pastoris, SF9 or High5 insect cells, immortalized mammalian cell lines (such as HeLa cells or CHO cells), or primary mammalian cells.

[0116] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure can also be produced in the cytoplasm of a host cell, preferably E. coli. In this case, the provided antibodies or antigen-binding fragments thereof can be obtained directly in a soluble and folded state or recovered as inclusion bodies and subsequently renatured in vitro. Another option is to use a specific host strain that has an oxidative intracellular environment (which can thus allow the formation of disulfide bonds in the cytoplasm) (Venturi et al., J Mol Biol, 2002).

[0117] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure as described herein do not have to be generated or produced in whole or in part by using genetic engineering. Instead, such proteins can also be obtained by any of a number of conventional and well-known techniques, such as direct organic synthesis strategies, solid-phase assisted synthesis techniques, commercially available automated synthesizers, or by in vitro transcription and translation. For example, molecular modeling can be used to identify promising antibodies or antigen-binding fragments thereof, synthesize them in vitro, and explore their binding activity against the target of interest. Methods for solid-phase and / or solution-phase synthesis of proteins are well known in the art (see, for example, Bruckdorfer et al., Curr Pharm Biotechnol, 2004).

[0118] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure can be produced by in vitro transcription / translation using well-known methods known to those skilled in the art.

[0119] In some additional embodiments, the antibodies or antigen-binding fragments thereof as described herein can also be prepared by conventional recombinant techniques alone or in combination with conventional synthesis techniques.

[0120] Those skilled in the art will be aware of methods that can be used to prepare antibodies or antigen-binding fragments thereof for which the protein or nucleic acid sequences are contemplated by the present disclosure but not explicitly disclosed herein. As an overview, such modifications of amino acid sequences include, for example, site-directed mutagenesis at a single amino acid position to simplify subcloning of a protein gene or a portion thereof by incorporating cleavage sites for certain restriction enzymes. In addition, these mutations can be incorporated to further improve the affinity of the antibody or antigen-binding fragment thereof for its target (e.g., CD228). Further, if necessary, mutations can be introduced to modulate one or more characteristics of the protein to improve folding stability, serum stability, protein resistance, or water solubility or to reduce the tendency to aggregate.

[0121] After studying the following non-limiting examples and figures, additional objectives, advantages, and features of the present disclosure will become apparent to those skilled in the art. Accordingly, it should be understood that although the present disclosure is specifically disclosed by way of exemplary embodiments and optional features, those skilled in the art may adopt modifications and variations of the disclosure embodied herein and such modifications and variations are considered to be within the scope of the present disclosure. VI. Examples Example 1: Generation of Anti-CD228 Antibodies

[0122] A human immunoglobulin transgenic rat strain ( OMT, Inc.) was used to develop hybridoma cells expressing monoclonal antibodies. Comprising a chimeric human / rat IgH locus (containing 22 human VH, all human D and JH segments linked to the rat CH locus in their native configuration) and a fully human IgL locus (12 Vκ linked to Jκ-Cκ and 16 Vλ linked to Jλ-Cλ). See Osborn et al. (2013) J Immunol 190(4):1481-1490); WO 2014 / 093908. In response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic hypermutation to generate high-affinity IgG monoclonal antibodies.

[0123] The transgenic rats were immunized with recombinant human CD228 protein. Subcutaneous injections included 48.2 μg of recombinant protein in Freund's complete adjuvant on day 1 and 48 μg of recombinant protein in Freund's incomplete adjuvant on days 33, 81, and 127.

[0124] RPMI-7951 cells engineered to stably express human CD228 were used to monitor the presence of antibodies against human CD228 in the sera of transgenic rats by flow cytometry on days 33, 81, and 127. Transgenic rats with detectable immunoreactivity were boosted for five and seven days, and then spleens and lymph nodes were harvested. Boosting prior to organ harvest included an intravenous injection of 90 μg of recombinant human CD228 protein suspended in phosphate-buffered solution (PBS) and an intraperitoneal injection of 18 μg.

[0125] Spleen cells and lymphocytes that exhibited a B cell lineage and were specific for fluorescently labeled recombinant human CD228 were sorted as single cells into cell lysis buffer by fluorescence-activated cell sorting (FACS). RNA from individual B cells was reverse transcribed into cDNA and amplified from primer sets for known human variable regions. Amplicons underwent DNA purification for Sanger sequencing using appropriate sequencing primers, and the heavy and light chain sequences were then identified and annotated by IgBLAST.

[0126] The validated heavy and light chain sequences of the human variable regions identified from single B cell cloning and Sanger sequencing were selected for gene synthesis and cloned into an expression vector using traditional cloning techniques. The expression vector encoded the human IgG1, human κ, or human λ constant region, which was in-frame and downstream of the human variable region. Sanger sequencing verified the antibody expression construct.

[0127] To produce full-length antibodies, the heavy and light chain expression vectors were co-transfected into ExpiCHO cells according to the manufacturer's protocol (Life Technologies). Nine days after transfection, the cell culture supernatant was collected by centrifugation. 10% Triton-X (final concentration 0.1%) was added to the filtered culture supernatant and incubated overnight at 4 °C on an orbital shaker. Then, the supernatant was loaded onto a 5 mL HiTrap MabSelectSuRe column at 2.5 mL / min using an Avant 25 chromatography system. The column was then washed with: 5 column volumes (CV) of endo wash buffer (1× PBS, 0.1% Triton-X), 5 CV of high salt buffer (1× PBS, 0.5 M NaCl), and 7.5 CV of standard 1× PBS, pH 7.4. The protein was eluted with 1 CV of 20 mM citrate, pH 3, and the buffer was immediately exchanged to 1× PBS, pH 7.4 using a HiPrep Sephadex G-25 desalting column. Fractionation was determined by A280 UV monitoring parameters to allow optimal collection of the purified protein.

[0128] Figure 2A and 2B shown as by Kabat (Figure 2A ) and IMGT Figure 2B ), the CDR sequences of the antibody defined by Example 2: Binding of anti-CD228 antibody to recombinant human CD228

[0129] The binding of a humanized antibody specific for human melanotransferrin (CD228) to recombinant human CD228 (R&D Systems) was evaluated by ELISA. The antibody was titrated on plates coated with 1 μg / ml recombinant human CD228, detected with an HRP-labeled goat anti-human IgG secondary reagent (Thermo), and developed with a TMB substrate (Thermo). The OD450 values were read on a plate reader using SoftMax Pro software. The OD450 values of each experiment were transferred to GraphPad Prism 8 for plotting and analysis. The results are shown in Figure 3 . Example 3: Binding of anti-CD228 antibody to cells

[0130] The relative binding of a humanized antibody specific for human CD228 to cancer cell lines expressing CD228 was evaluated. The cells were incubated with titrations of an Alexa-647-labeled humanized antibody clone, washed, and the fluorescence intensity was evaluated by flow cytometry on an Attune NXT flow cytometer. The MFI values were transferred to GraphPad Prism 8 for plotting and analysis. The EC50 values were determined by non-linear regression. The numbers immediately following the cell line name reflect the estimated surface CD228 copy number as estimated using the QIFIKIT quantitative analysis kit (Agilent).

[0131] The results are shown as the raw mean fluorescence intensity (MFI) values in Figures 4A - 4C . Figure 4D Show the EC 50 values of each antibody against each cell line. Example 4: Binding profile of anti-CD228 antibody

[0132] By Biolayer Interferometry (BLI), using RED 384 (Sartorius) was used to determine the binding kinetics and affinity of human CD228 and C-terminal polyhistidine tag (R&D Systems). An anti-human antibody capture AHC (GE Healthcare) biosensor was used for the analysis. Then, 0.5 μg / mL of anti-CD228 antibodies OMT8, OMT24, OMT30, OMT35, and OMT36 (IgG) in HBS-EP+ buffer were captured on the chip surface by anti-human IgG-Fc antibody for 180 seconds. After each capture step, the biosensor was washed in HBS-EB+ blank. For the affinity assay, dilutions of recombinant huCD228 (100 nM, 40 nM, 16 nM, 6.4 nM, 2.6 nM, and 1.0 nM) or blank were prepared in HBS-EP+ buffer and applied to the biosensor. A binding assay was performed with a contact time of 300 seconds and a dissociation time of 1,200 seconds. All measurements were carried out at 25 °C. Fresh AHC biosensors were used for each analysis. With Satorius Data Analysis Software (v12.0) was used to evaluate the data, and the results are shown in Figures 5A - 5E this figure. A single reference was used, and a 1:1 binding model was used to fit the raw data. Example 5: Anti-CD228 antibodies lack cross-reactivity with transferrin and lactoferrin

[0133] The binding of humanized antibodies specific for human melanotransferrin (CD228) to the related transferrin family members lactoferrin and transferrin was evaluated by ELISA. Antibodies were titrated on plates coated with 1 μg / ml of recombinant human melanotransferrin (CD228), lactoferrin, and transferrin, detected with an HRP-labeled goat anti-human IgG secondary reagent (Thermo), developed with TMB substrate (Thermo), and the OD450 values were read on a plate reader using SoftMax Pro software. The OD450 values of each experiment were transferred to GraphPad Prism 8 for plotting and analysis. As Figures 6A - 6C shown in this figure, these results indicate that the anti-CD228 antibodies do not exhibit cross-reactive binding with other transferrin family members. Example 6: Cross-reactivity of anti-CD228 antibodies with non-human CD228

[0134] The CD228-negative human melanoma cell line RPMI-7951 was engineered to express cynomolgus macaque CD228 to test the binding of anti-CD228 antibody clones. 50,000 RPMI-7951 cells were incubated with titers of humanized antibody clones, washed with staining buffer to remove excess antibody, and incubated with 250 ng / mL of a monoclonal antibody (Thermo) specific for human IgG1 labeled with a fluorescent dye to detect bound antibody. As determined by measurement on an Attune NXT flow cytometer (Thermo), the results are shown as mean fluorescence intensity (MFI) in Figure 7A in.

[0135] The reactivity of CD228 antibody clones against recombinant his-tagged cynomolgus macaque CD228 was tested by ELISA. Plates were coated with 1 μg / ml of recombinant his-cynomolgus macaque CD228 in phosphate-buffered saline, blocked with 250 μL of Superblock blocking buffer (Thermo), the antibodies were titrated on the plates, bound antibodies were detected with HRP-labeled goat anti-human IgG (Sigma), developed with TMB substrate (Thermo), and the OD450 values were read on a plate reader using SoftmaxPro software. The results are shown in Figure 7B in.

[0136] The binding of humanized antibodies specific for human CD228 to recombinant cynomolgus macaque and murine CD228 was evaluated by ELISA. Antibodies were titrated on plates coated with 1 μg / ml of recombinant human, cynomolgus macaque, and murine CD228, detected with an HRP-labeled goat anti-human IgG secondary reagent (Thermo), developed with TMB substrate (Thermo), and the OD450 values were read on a plate reader using SoftMax Pro software. The OD450 values for each experiment are shown in Figures 8A - 8C in.

[0137] These results indicate that a subset of antibody clones (OMT30, OMT35, OMT24, OMT8, OMT36, and L235) are cross-reactive with cynomolgus macaque CD228 and have minimal cross-reactivity with murine CD228. Example 7: Anti-CD228 Antibody Cross-Competition Assay

[0138] Evaluate the competition of a fluorescent dye-labeled humanized monoclonal antibody specific for human CD228 with an unlabeled humanized monoclonal antibody for binding to HT-1080 tumor cell line and SK-MEL-5 tumor cell line to identify potential shared epitopes. At 4 °C, HT-1080 cells and SK-MEL-5 cells were pre-incubated with the unlabeled humanized antibody in 1 μg / ml staining buffer for 30 minutes, washed in staining buffer, and incubated with the A647-labeled version at 200 ng / mL. The bound fluorescent dye-labeled antibody was measured by flow cytometry on an Attune NXT flow cytometer. The results are shown as the percentage of the mean fluorescence intensity compared to that pre-incubated with control non-binding human IgG1 (Sigma) in Figure 9 . Clones with direct epitopes or steric competition showed low staining percentage values. These results indicate that antibody clones 28, 32, and 35 may share closely related epitopes, while clones 8, 11, 24, 30, and 36 may bind to unique CD228 epitopes. Example 8: Internalization of antibody clones on CD228+ tumor cell lines

[0139] Evaluate the relative internalization of humanized antibodies specific for human CD228 on melanoma and lung cancer cell lines SK-MEL-5 and Calu-1 expressing CD228, respectively. The cells were incubated with 2 μg / ml anti-CD228 antibody, washed three times to remove free antibody, and incubated at 37 °C, 5% CO2 for the indicated times. At each time point, the cells were fixed (BDCytofix) and stained for bound human IgG1 with a fluorescent dye-labeled anti-human IgG1 antibody (Invitrogen). At the end of the time course, the mean fluorescence intensity of the antibody bound to the cell surface was evaluated by FACS on an Attune NXT flow cytometer. The internalization results are shown as the percentage of MFI relative to the starting MFI at time point 0 in Figure 10 . These results show that there is a range of internalization rates among the antibody clones, with antibody clone 35 internalizing at the fastest rate. Example 9: Expression and analysis of representative fusion proteins

[0140] A representative antibody-lipocalin mutant protein fusion protein is generated by fusing a CD228-specific antibody and a CD137-specific lipocalin mutant protein (such as the lipocalin mutant protein of SEQ ID NO: 40) via a linker (such as the unstructured (G4S)3 linker of SEQ ID NO: 13) to simultaneously engage CD228 and CD137. Two different exemplary CD228-specific antibodies are used. The first CD228-specific antibody has a heavy chain provided by SEQ ID NO: 75 (or comprising the heavy chain variable domain of SEQ ID NO: 70, or comprising the heavy chain CDRs (HCDR1, HCDR2, HCDR3) of SEQ ID NO: 58-60), and has a light chain provided by SEQ ID NO: 76 (or comprising the light chain variable domain of SEQ ID NO: 71, or comprising the light chain CDRs (LCDR1, LCDR2, LCDR3) of SEQ ID NO: 61-63). The second CD228-specific antibody has a heavy chain provided by SEQ ID NO: 78 (or comprising the heavy chain variable domain of SEQ ID NO: 72, or comprising the heavy chain CDRs (HCDR1, HCDR2, HCDR3) of SEQ ID NO: 64-66), and has a light chain provided by SEQ ID NO: 79 (or comprising the light chain variable domain of SEQ ID NO: 73, or comprising the light chain CDRs (LCDR1, LCDR2, LCDR3) of SEQ ID NO: 67-69). Exemplary fusion proteins of SEQ ID NO: 80 and 76, SEQ ID NO: 82 and 79, SEQ ID NO: 75 and 81, and SEQ ID NO: 78 and 83 are bivalent for CD137, wherein the CD137-specific lipocalin mutant protein is fused to the C-terminus of each heavy chain or each light chain.

[0141] The CD228-specific antibodies and all antibody-lipocalin mutant protein fusion proteins described in this example have an engineered IgG4 backbone that contains an S228P mutation to minimize IgG4 half-antibody exchange in vitro and in vivo (Silva et al., J Biol Chem, 2015). Additional mutations in the IgG4 backbone may also be present in all antibodies described herein, including any one or more of the mutations F234A, L235A, M428L, N434S, M252Y, S254T, and T256E. The F234A and L235A mutations can be introduced to reduce ADCC and ADCP (Glaesner et al., Diabetes Metab Res Rev, 2010). The M428L and N434S mutations or the M252Y, S254T, and T256E mutations can be introduced for extended serum half-life (Dall'Acqua et al., J Biol Chem, 2006; Zalevsky et al., Nat Biotechnol, 2010). All antibodies are expressed without a carboxyl-terminal lysine to avoid heterogeneity.

[0142] Constructs of exemplary fusion proteins are generated by gene synthesis and cloned into mammalian expression vectors. They are then transiently expressed in suspension-adapted CHO-K1 cells. Example 10: Evaluation of PBMC Responses to Viral Peptides in the Presence of Tumor Cells with High or No CD228 Expression

[0143] Assays were performed to evaluate the ability of the fusion proteins to co-stimulate innate and adaptive immune cytokines from PBMCs in a CD228 target-dependent manner in response to viral peptides. PBMCs isolated from healthy donors were co-incubated 10:1 with engineered CD228+ or wild-type (CD228-) RPMI-7951 tumor cell lines in RPMI with 10% FCS and provided with viral peptides (CEF peptides) from CMV, EBV, and influenza virus. Bispecific fusion proteins and controls were titrated into the assays and the changes in IFN-γ, TNF-α, IL-5, IL-12 (p70), and CXCL10 (IP-10) were measured at the end of 4 days of stimulation at 37 °C, 5% CO2. The T cell (IFN-γ and TNF-α) and myeloid cell (IL-12 and CXCL10) responses of the supernatants were evaluated by multiplex cytokine array. Data are shown as fold change of cytokines relative to untreated control wells in Figures 11A - 11EIn the middle. Before cytokine measurement, pooled samples were processed from triplicate test articles. These results showed that, compared to antibodies, the fusion proteins (such as AAF30(HC)) co-stimulated the production of various innate and adaptive immune cytokines from PBMCs in a CD228 target-dependent and dose-dependent manner in response to viral peptides. Example 11: Evaluation of PBMC cellular responses to viral peptides in the presence of CD228+ tumor cells

[0144] To evaluate the activity of the bispecific fusion protein in an antigen recall assay, PBMCs (Bloodworks Northwest) cryopreserved from healthy donors were thawed in pre-warmed RPMI 10% FCS, washed, and labeled at room temperature with 1.5 mL of 10 nM CFSE in PBS 3% FCS. To quench the labeling reaction, the cells were washed 2 times with 12 mL of RPMI 10% FCS. The cells were counted and mixed with the CD228-expressing cell line RPMI-7951 CD228 (ATCC) (engineered to express human CD228) at a ratio of 10:1 in complete RPMI (10% FCS, 1×Glutamax, 1×MEM NEAA, 1× sodium pyruvate, 1× penicillin / streptomycin (Gibco TM )) and distributed in non-adherent 96-well round bottom plates (Sbio), 1.5×10 5 cells / well. CEF peptide was added to a final concentration of 100 ng / mL, and the bispecific fusion protein and control were added in equimolar titrations in triplicate. The assay was incubated at 37 °C, 5% CO2 for 5 days. Figure 12 、 Figure 13 and Figure 14 Show representative examples of the calculation of CD8 T cell, NK cell, and CD8 T cell / Treg ratios from an antigen recall assay co-cultured with the CD228-engineered RPMI-7951 cell line, respectively. Similarly, Figure 15 Shows a representative example of the calculation of NK cell and CD8 T cell / Treg ratios from an antigen recall assay. These results showed that, compared to antibodies, the fusion proteins (such as AAF30(HC)) co-stimulated the proliferation / division of CD8+ T cells and NK cells in a CD228 target-dependent and dose-dependent manner in response to viral peptides. Example 12: Evaluation of PBMC cytokine responses to viral peptides in the presence of CD228+ tumor cells

[0145] To evaluate the activity of the bispecific fusion protein in an antigen recall assay, PBMCs (Bloodworks Northwest) cryopreserved from healthy donors were thawed, washed in pre-warmed RPMI 10% FCS, and labeled at room temperature with 1.5 mL of 10 nM CFSE in PBS 3% FCS. To quench the labeling reaction, the cells were washed 2 times with 12 mL of RPMI 10% FCS. The cells were counted and mixed with the CD228-expressing cell line RPMI-7951 CD228 (ATCC) (engineered to express human CD228), CALU-1 cells (ATCC), or H3677 cells (Seagen) at a ratio of 10:1 in complete RPMI (10% FCS, 1×Glutamax, 1×MEM NEAA, 1×sodium pyruvate, 1×penicillin / streptomycin (Gibco TM )) and distributed in non-adherent 96-well round bottom plates (Sbio), 1.5×10 5 cells / well. CEF peptide was added to a final concentration of 100 ng / mL, and the bispecific fusion protein and controls were added in equimolar titrations in triplicate. The assay was incubated at 37 °C, 5% CO2 for 5 days. At the end of the assay, the plates were centrifuged and the supernatants were collected for cytokine evaluation. Cytokines were measured using MAP Human CD8+T CellMagnetic Bead Panel Premixed 17Plex and read on the system. Figures 16A - 16E The mean representative fold change of cytokines in the three CD228-expressing cell lines is shown. The raw data was exported and analyzed in Microsoft Excel. These data highlight the consistent changes in cytotoxic effector molecules and various cytokines during antigen recall in the presence of the bispecific fusion protein. Example 13: Evaluation of T cell activation in the presence of tumor cells with high CD228 expression or no CD228 expression

[0146] Assays were performed to evaluate the requirement for CD228 expression on cancer cells for primary T cell co-stimulation with the fusion protein. Healthy donor primary T cells were incubated in anti-CD3 coated plates (0.25 μg / ml in 50 μL PBS, overnight) to drive T cell receptor stimulation, and CD228-positive Calu-1 cells or CD228-negative SK-BR-3 cells were added to cultures with titrations of the fusion protein. T cells were mixed with tumor cells at a final ratio of 10:1 in RPMI with 10% FCS. At the end of the three-day stimulation at 37 °C, 5% CO2, supernatants were collected and measured for the cytokine IL-2. Figures 17A - 17B IL-2 secretion in response to co-stimulation with the fusion protein and urelumab is shown. These results show that the fusion protein (e.g., AAF35 (HC)) co-stimulates T cell activation in a CD228 target-dependent and dose-dependent manner. Example 14: Evaluation of cytokines of T cells generated in co-cultures with CD228-expressing tumor cell lines engineered with anti-CD3 scFv

[0147] Experiments were conducted to evaluate the effect of the fusion protein on cytokine and soluble 4-1BB (sCD137) production by T cells receiving direct T cell receptor stimulation from CD228-expressing tumor cells. Healthy donor PBMCs were co-cultured with CD228+ CALU-1 tumor cells (10:1, in RPMI 10% FCS), which were engineered to express surface anti-CD3 scFv to initiate direct T cell receptor engagement by the tumor cells. Titrations of the fusion protein or a control were added in triplicate to 96-well round bottom plates. At the end of the three-day stimulation at 37 °C, 5% CO2, supernatants were collected and soluble cytokines IL-2, IL-13, and sCD137 were measured by multiplex array. Samples were pooled from triplicate test article treatments prior to cytokine measurement. Results are shown in Figures 18A - 18C which demonstrate that the fusion protein increases cytokines from direct T cell-tumor cell interactions. Example 15: Evaluation of CD8 T cell proliferation in co-cultures with CD228-expressing tumor cell lines engineered with anti-CD3 scFv

[0148] Experiments were conducted to evaluate the effect of the bispecific fusion protein on cytotoxic T cells receiving direct T cell receptor stimulation from tumor cells expressing CD228. Healthy donor PBMCs were CFSE-labeled and co-cultured with CD228+ CALU-1 tumor cells (10:1), which were engineered to express surface anti-CD3 scFv to trigger T cell receptor engagement. Titers of the bispecific fusion protein or a control were added in triplicate to 96-well round bottom plates. After incubation at 37 °C, 5% CO2 for 72 hours, the plates were washed and stained with a live dead viability dye and antibodies specific for CD3, CD4, CD8, and CD228. Live CD8 T cell proliferation and CD228+ tumor cells in the plates were evaluated by an Attune TM NxT flow cytometer. Figure 19 Shows the proportion of CD8 T cells with diluted CFSE relative to wells without added treatment. Figure 20 Shows the percentage of remaining viable tumor cells relative to wells without added treatment, which reflects tumor cell killing. These results show that in the presence of CD228, the fusion protein (e.g., AAF30 (HC)) co-stimulates CD8+ T cell proliferation and kills tumor cells in a dose-dependent manner compared to the antibody. Example 16: Pharmacokinetics of the fusion protein in cynomolgus monkeys

[0149] Compare the pharmacokinetic differences of the bispecific heavy chain fusion proteins 30HC and 35HC (SEQ ID NO:80 and 76 and SEQ ID NO:82 and 79, respectively) in cynomolgus monkeys. Animals received a single dose of the bispecific fusion at a dose of 1 mg / kg or 6 mg / kg. As Figure 21 shown, the bispecific fusion proteins exhibited similar pharmacokinetic profiles with slightly different profiles after day 14, which could be attributed to anti-drug antibody responses. Example 17: Evaluate the in vivo activity of the fusion protein in a humanized xenograft model

[0150] To evaluate the in vivo activity of the bispecific fusion protein, a humanized xenograft model using the CD228+ melanoma cell lines CALU-1 (ATCC) and H3677 (SGEN, in-house) was employed. Tumor cells in 25% (Corning) were implanted into immunodeficient NSG mice and monitored until the tumors reached 100 mm 3The average volume, at which time healthy donor peripheral blood mononuclear cells (PBMCs) were adoptively transferred via tail vein injection to provide a source of human T cells. Subsequently, mice were dosed with equimolar amounts of bispecific fusion protein (10 mg / kg), antibody (non-bispecific antibody, anti-PD-1 and / or anti-4-1BB), or control (8 mg / kg) at 5-day intervals, and tumor growth was monitored. The growth curves in the entire Calu-1 study are shown in Figure 22A and the final tumor volumes at the end of the study, i.e., day 80, are shown in Figure 22B The growth curves in the entire H3677 study are shown in Figure 22C and the final tumor volumes at the end of the study, i.e., day 22, are shown in Figure 22D The statistical data provided are the results of Tukey analysis. These results show that, compared to the antibody, the fusion protein (e.g., AAF30 (HC)) reduces the growth of CD228+ tumor cells in vivo.

[0151] To evaluate the intratumoral PD effects in the humanized CD228+ Calu-1 xenograft model from animals treated with bispecific fusions, tumors were collected and processed into single-cell suspensions for staining of tumor and immune cell populations and analysis by flow cytometry. Figure 23A Shows the ratio of CD8+ T cells to tumor cells as determined by dividing the count of CD8+ T cells by the count of CD228+ non-immune cells. Figure 23B Shows the change in the CD8+ / CD4+ T cell ratio. Figure 23C Shows the proportion of CD8+ T cells in active degranulation as measured by surface expression of CD107a at the end of the study. The dashed lines in the figures indicate the CD8+ / CD4+ T cell ratio ( Figure 23B ) or the percentage of CD8 T cells expressing CD107a ( Figure 23C ) in samples of the remaining PBMCs (i.e., non-tumor models) from the same donor. These data demonstrate that the expansion and activation of cytotoxic CD8+ T cells are enhanced after treatment with bispecific fusion protein, which is superior to the control. The statistical data provided are the results of Tukey analysis.

[0152] To evaluate the intratumoral PD effects in the humanized CD228+ Calu-1 xenograft model alone from animals treated with bispecific fusions, tumors were collected and processed into single-cell suspensions for staining of tumor and immune cell populations and analysis by flow cytometry. Figure 24A Shows the ratio of CD8+ T cells to CD4+ T cells. Figure 24BShow the changes in intracellular TCF1 expression. These data indicate that, as identified by the expression of the transcription factor TCF1 after treatment with a bispecific fusion protein targeting 4-1BB, cytotoxic CD8+ T cells enhance the expansion and differentiation into cells with more stem cell-like properties, which indicates greater anti-tumor potential. The statistical data provided are the results of Tukey analysis.

[0153] The embodiments illustratively described herein can be suitably implemented in the absence of any one or more elements or one or more limitations not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," etc. should be understood broadly and without limitation. Additionally, the terms and expressions used herein are used as terms of description and not of limitation, and there is no intention, in using such terms and expressions, of excluding any equivalents of the features shown and described or portions thereof, but it should be recognized that various modifications can be made within the scope of the claimed invention. Accordingly, it should be understood that although the embodiments of the present invention have been specifically disclosed by preferred embodiments and optional features, those skilled in the art can adopt their equivalents, modifications, and variations, and such equivalents, modifications, and variations are considered to be within the scope of the present invention. All patents, patent applications, textbooks, and (peer-reviewed) publications described herein are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. Further, when the definition or use of a term in a reference (which is incorporated herein by reference) is inconsistent with or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply. The various items falling within the broader generic and subgeneric groupings disclosed herein also form part of the present invention. This includes the general description of the present invention with the removal of any subject matter restrictive conditions or negative limitations from the subordination, whether or not the excluded material is specifically recited herein. Additionally, when describing features in terms of a Markush group, those skilled in the art should recognize that the disclosure also hereby describes the invention in terms of any individual member or subgroup of members of the Markush group. Additional embodiments will become apparent from the appended claims. VII. Sequence Listing

[0154] Exemplary Sequence Listing:

Claims

1. An antibody or antigen-binding fragment thereof that binds to CD228, wherein the antibody or antigen-binding fragment thereof comprises: A heavy-chain variable domain (VH) that comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 110, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 111, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 112, and a light-chain variable domain (VL) that comprises (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 116, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 118; VH that comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 113, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 114, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 115, and VL that comprises (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 119, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 120, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 121; VH that comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 130, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 131, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 132, and VL that comprises (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 136, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 137, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 138; VH that comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 133, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 134, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 135, and VL that comprises (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 139, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 140, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 141; VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:150, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:151, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:152, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:156, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:157, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:158; VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:153, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:154, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:155, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:159, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:160, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:161; VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:170, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:171, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:172, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:176, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:177, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:178; VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:173, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:174, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:175, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:179, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:180, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:181; VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:190, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:191, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:192, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:196, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:197, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:198; VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:193, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:194, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:195, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:199, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:200, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:201; VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:210, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:211, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:212, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:216, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:217, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:218; VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:213, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:214, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:215, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:219, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:220, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:221; VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:230, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:231, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:232, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:236, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:237, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:238; VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:233, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:234, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:235, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:239, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:240, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:241; VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:250, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:251, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:252, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:256, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:257, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:258; VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:253, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:254, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:255, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:259, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:260, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:261; VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:270, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:271, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:272, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:276, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:277, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:278; or VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:273, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:274, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:275, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:279, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:280, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

281.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises: VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:210, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:211, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:212, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:216, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:217, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:218; VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:213, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:214, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:215, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:219, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:220, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

221. VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:250, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:251, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:252, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:256, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:257, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:258; or VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:253, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:254, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:255, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:259, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:260, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

261.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises: VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:210, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:211, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:212, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:216, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:217, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:218; or VH, which comprises (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:213, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:214, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:215, and VL, which comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:219, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:220, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

221.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises: VH, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:122; and VL, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:124; VH, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 142; and VL, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 144; VH, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 162; and VL, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 164; VH, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 182; and VL, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 184; VH, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 202; and VL, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 204; VH, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 222; and VL, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 224; VH, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 242; and VL, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 244; VH, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 262; and VL, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 264; or VH, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 282; and VL, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

284.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1, 2 and 4, wherein the antibody or antigen-binding fragment thereof comprises: VH, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 222; and VL, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 224; or VH, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 262; and VL, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

264.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof comprises: VH, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 222; and VL, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

224.

7. The antibody or antigen-binding fragment thereof according to claim 1 or 4, wherein the antibody or antigen-binding fragment thereof comprises: VH, which comprises the amino acid sequence of SEQ ID NO: 122; and VL, which comprises the amino acid sequence of SEQ ID NO: 124; VH, which comprises the amino acid sequence of SEQ ID NO: 142; and VL, which comprises the amino acid sequence of SEQ ID NO: 144; VH, which comprises the amino acid sequence of SEQ ID NO: 162; and VL, which comprises the amino acid sequence of SEQ ID NO: 164; VH, which comprises the amino acid sequence of SEQ ID NO: 182; and VL, which comprises the amino acid sequence of SEQ ID NO: 184; VH, which comprises the amino acid sequence of SEQ ID NO: 202; and VL, which comprises the amino acid sequence of SEQ ID NO: 204; VH, which comprises the amino acid sequence of SEQ ID NO: 222; and VL, which comprises the amino acid sequence of SEQ ID NO: 224; VH, which comprises the amino acid sequence of SEQ ID NO: 242; and VL, which comprises the amino acid sequence of SEQ ID NO: 244; VH, which comprises the amino acid sequence of SEQ ID NO: 262; and VL, which comprises the amino acid sequence of SEQ ID NO: 264; or VH, which comprises the amino acid sequence of SEQ ID NO: 282; and VL, which comprises the amino acid sequence of SEQ ID NO:

284.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1, 2, 4, 5 and 7, wherein the antibody or antigen-binding fragment thereof comprises: VH, which comprises the amino acid sequence of SEQ ID NO: 222; and VL, which comprises the amino acid sequence of SEQ ID NO: 224; or VH, which comprises the amino acid sequence of SEQ ID NO: 262; and VL, which comprises the amino acid sequence of SEQ ID NO:

264.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment thereof comprises: VH, which comprises the amino acid sequence of SEQ ID NO: 222; and VL, which comprises the amino acid sequence of SEQ ID NO:

224.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1, 4 and 7, wherein the antibody or antigen-binding fragment thereof comprises: Heavy chain (HC), which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 126; and light chain (LC), which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 128; HC, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 146; and LC, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 148; HC, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 166; and LC, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 168; HC, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 186; and LC, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 188; HC, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 206; and LC, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 208; HC, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 226; and LC, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 228; HC, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 246; and LC, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 248; HC, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 266; and LC, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 268; or HC, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 286; and LC, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

288.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1, 2, 4, 5, 7, 8, and 10, wherein the antibody or antigen-binding fragment thereof comprises: HC, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 226; and LC, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 228; or HC, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 266; and LC, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

268.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the antibody or antigen-binding fragment thereof comprises: HC, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 226; and LC, which comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

228.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1, 4, 7, and 10, wherein the antibody or antigen-binding fragment thereof comprises: HC, which comprises the amino acid sequence of SEQ ID NO:126; and LC, which comprises the amino acid sequence of SEQ ID NO:128; HC, which comprises the amino acid sequence of SEQ ID NO:146; and LC, which comprises the amino acid sequence of SEQ ID NO:148; HC, which comprises the amino acid sequence of SEQ ID NO:166; and LC, which comprises the amino acid sequence of SEQ ID NO:168; HC, which comprises the amino acid sequence of SEQ ID NO:186; and LC, which comprises the amino acid sequence of SEQ ID NO:188; HC, which comprises the amino acid sequence of SEQ ID NO:206; and LC, which comprises the amino acid sequence of SEQ ID NO:208; HC, which comprises the amino acid sequence of SEQ ID NO:226; and LC, which comprises the amino acid sequence of SEQ ID NO:228; HC, which comprises the amino acid sequence of SEQ ID NO:246; and LC, which comprises the amino acid sequence of SEQ ID NO:248; HC, which comprises the amino acid sequence of SEQ ID NO:266; and LC, which comprises the amino acid sequence of SEQ ID NO:268; or HC, which comprises the amino acid sequence of SEQ ID NO:286; and LC, which comprises the amino acid sequence of SEQ ID NO:

288.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1, 2, 4, 5, 7, 8, 10, 11 and 13, wherein the antibody or antigen-binding fragment thereof comprises: HC, which comprises the amino acid sequence of SEQ ID NO:226; and LC, which comprises the amino acid sequence of SEQ ID NO:228; or HC, which comprises the amino acid sequence of SEQ ID NO:266; and LC, which comprises the amino acid sequence of SEQ ID NO:

268.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, wherein the antibody or antigen-binding fragment thereof comprises: HC, which comprises the amino acid sequence of SEQ ID NO:226; and LC, which comprises the amino acid sequence of SEQ ID NO:

228.

16. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein the antibody is a monoclonal antibody.

17. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, wherein the antibody is a humanized antibody or a chimeric antibody.

18. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, wherein the antibody is an IgG1, IgG2, IgG3 or IgG4 antibody.

19. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, wherein the antibody or antigen-binding fragment thereof binds to CD228 with a K D value of 175 nM or less.

20. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 19, wherein the antibody or antigen-binding fragment thereof binds cynomolgus macaque CD228.

21. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, wherein the antibody or antigen-binding fragment thereof does not bind to murine CD228, or binds to murine CD228 with an affinity at least 100-fold lower than that for human CD228.

22. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, wherein the antibody or antigen-binding fragment thereof does not bind to transferrin or lactotransferrin.

23. An antibody or antigen-binding fragment thereof that binds to CD228, wherein the antibody or antigen-binding fragment thereof competes with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 22 for binding to CD228.

24. The antibody or antigen-binding fragment thereof according to claim 23, wherein the antibody is a monoclonal antibody.

25. The antibody or antigen-binding fragment thereof according to claim 23 or 24, wherein the antibody is a humanized antibody or a chimeric antibody.

26. The antibody or antigen-binding fragment thereof according to any one of claims 23 to 25, wherein the antibody is an IgG1, IgG2, IgG3 or IgG4 antibody.

27. The antibody or antigen-binding fragment thereof according to any one of claims 23 to 26, wherein the antibody or antigen-binding fragment thereof binds to CD228 with a K D value of 175 nM or less.

28. The antibody or antigen-binding fragment thereof according to any one of claims 23 to 27, wherein the antibody or antigen-binding fragment thereof binds to cynomolgus macaque CD228.

29. The antibody or antigen-binding fragment thereof according to any one of claims 23 to 28, wherein the antibody or antigen-binding fragment thereof does not bind to murine CD228, or binds to murine CD228 with an affinity at least 100-fold lower than that for human CD228.

30. The antibody or antigen-binding fragment thereof according to any one of claims 23 to 29, wherein the antibody or antigen-binding fragment thereof does not bind to transferrin or lactotransferrin.

31. The antibody or antigen-binding fragment thereof according to any one of claims 23 to 30, wherein the antibody or antigen-binding fragment thereof competes with the antibody or antigen-binding fragment thereof comprising: VH, which comprises the amino acid sequence of SEQ ID NO: 202; and VL, which comprises the amino acid sequence of SEQ ID NO: 204; VH, which comprises the amino acid sequence of SEQ ID NO: 242; and VL, which comprises the amino acid sequence of SEQ ID NO: 244; or VH, which comprises the amino acid sequence of SEQ ID NO: 262; and VL, which comprises the amino acid sequence of SEQ ID NO:

264.

32. The antibody or antigen-binding fragment thereof according to claim 31, wherein the antibody or antigen-binding fragment thereof competes with the antibody or antigen-binding fragment thereof comprising: VH, which comprises the amino acid sequence of SEQ ID NO: 202; and VL, which comprises the amino acid sequence of SEQ ID NO: 204; or VH, which comprises the amino acid sequence of SEQ ID NO: 242; and VL, which comprises the amino acid sequence of SEQ ID NO:

244.

33. The antibody or antigen-binding fragment thereof according to any one of claims 23 to 32, wherein competition for binding to CD228 is measured by flow cytometry using at least one labeled antibody or antigen-binding fragment thereof.

34. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 33.

35. The isolated nucleic acid molecule according to claim 34, wherein the nucleic acid molecule is operably linked to regulatory sequences to permit expression of the nucleic acid molecule.

36. A vector comprising the nucleic acid molecule according to claim 34 or claim 35.

37. A host cell comprising the nucleic acid molecule according to claim 34 or claim 35 or the vector according to claim 36.

38. A host cell that expresses the antibody or antigen-binding fragment thereof according to any one of claims 1 to 33.

39. The host cell according to claim 37 or claim 38, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

40. A method of producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 33, the method comprising incubating the host cell according to any one of claims 37 to 39 under conditions suitable for producing the antibody or antigen-binding fragment thereof.

41. The method according to claim 40, further comprising isolating the antibody or antigen-binding fragment thereof.

42. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 33 and a pharmaceutically acceptable excipient.

43. An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 33 and a cytotoxic agent.

44. The immunoconjugate according to claim 43, wherein the cytotoxic agent is an auristatin.

45. The immunoconjugate according to claim 44, wherein the cytotoxic agent is monomethyl auristatin E (MMAE).

46. The immunoconjugate according to claim 44, wherein the cytotoxic agent is monomethyl auristatin F (MMAF).

47. A method of treating cancer, comprising administering to a subject having cancer the antibody or antigen-binding fragment thereof according to any one of claims 1 to 33, the pharmaceutical composition according to claim 42, or the immunoconjugate according to any one of claims 43 to 46.

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