Antibodies that bind to CD228

Abstract

The present disclosure provides antibodies or antigen-binding fragments thereof specific for CD228. Such antibodies or antigen-binding fragments can be used in a number of pharmaceutical applications, for example, as anti-cancer and / or immunomodulatory agents. 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 present disclosure further relates to nucleic acid molecules encoding such antibodies or antigen-binding fragments. Additionally, the present application discloses therapeutic and / or diagnostic uses of such antibodies or antigen-binding fragments.

Description

Related Applications

[0001] I. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 408,605, filed September 21, 2022, which is incorporated herein by reference in its entirety for all purposes. [Background technology]

[0002] II. Introduction and Overview Cluster of differentiation 228 or CD228 (also known as melanotransferrin, MELTF, p97, and MFI2) is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein belonging to the transferrin family of iron-binding proteins, and was first described as an oncofetal protein highly expressed in 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, pigment-containing cells. It is the most dangerous type of skin cancer. In 2015, 3.1 million people had active disease, and 59,800 died from melanoma. While surgery can be effective for early-stage melanoma, it may not be a treatment option for disease that has metastasized to distant organs. Metastatic melanoma often spreads to lymph nodes in the area before spreading to other sites. Surgical removal of lymph nodes has attempted to improve survival, but has been associated with numerous complications and no benefit to overall survival. Immunotherapy, chemotherapy, and radiation therapy have all been used but are often incurable, especially for late-stage melanoma. Distant metastasis generally renders the cancer incurable. The five-year survival rate for stage IV disease is 15-20%. Summary of the Invention

[0004]

[0004] The present disclosure provides, inter alia, novel antibodies and antigen-binding fragments thereof that bind to CD228. III. Definition

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

[0005]

[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 embodiments, non-human CD137, such as cynomolgus monkey CD137 or mouse CD137, is used.

[0006]

[0007] As used herein, unless otherwise specified, "CD228" refers to human CD228. Human CD228 refers to the full-length protein, its mature form, its isoform, its fragment, or its variant 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 embodiments, non-human CD228, such as cynomolgus monkey CD228 or mouse CD228, is used.

[0007]

[0008] As used herein, "binding affinity" refers to the ability of a biomolecule (e.g., a polypeptide or protein) of the present disclosure (e.g., an antibody or antigen-binding fragment thereof, a fusion protein, or any other peptide or protein) to bind to (and form a complex with) a selected target. Binding affinity is measured by several methods known to those skilled in the art, including, but not limited to, fluorescence titration, enzyme-linked immunosorbent assay (ELISA)-based assays (including direct ELISA and competitive ELISA), calorimetric methods such as isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). These methods are well established in the art, and some examples of such methods are further described herein. Binding affinity is thereby determined by the dissociation constant (K ) measured using such methods. D ), half-maximum effective concentration (EC 50 ), or half-maximal inhibitory concentration (IC 50 ) value is reported as K D , E.C. 50 , or IC 50 A lower value of K indicates better (higher) binding ability (affinity). Thus, the binding affinity of two biomolecules for a selected target can be measured and compared. When comparing the binding affinity 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 that is identical or similar to that of another molecule, within the experimental variability of binding affinity measurements. D , E.C. 50 , or IC 50 "Comparable to," "about the same," "substantially the same," or "substantially similar" refers to a value that is within 50% deviation, more preferably within 20%, and most preferably within 10% deviation from a given reference value. Experimental variability in binding affinity measurements depends on the particular method used and is known to those skilled in the art.

[0008]

[0009] As used herein, the term "substantially" can also refer to the qualitative condition of exhibiting a total degree or near total degree of a desired characteristic or property. Those skilled in the biological arts will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or perfection, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of perfection inherent in many biological and chemical phenomena.

[0009]

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

[0010]

[0011] As used herein, "detectable affinity" generally refers to a D , E.C. 50 , or IC 50 The binding affinity between a biomolecule and its target, as reported by a value of up to about 10 -5 This means that the value is less than or equal to M. 10 -5 K is higher than M D , E.C. 50 , or IC 50 The binding affinity reported in values ​​is generally no longer measurable by common methods such as ELISA and SPR and is therefore of little importance. Therefore, a "detectable affinity" is defined as a value of about 10 as determined by ELISA or SPR, preferably SPR. -5 K below M D It may point to a value.

[0011]

[0012] As used herein, "specific for," "specific binding," "specifically binds," or "binding specificity" refers to the ability of a biomolecule to distinguish between a desired target (e.g., CD228) and one or more reference targets. It is understood that such specificity is a relative, rather than absolute, 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.

[0012]

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

[0013]

[0014] As used herein, the term "lipocalin" refers to a monomeric protein weighing approximately 18-20 kDa, comprising multiple β-strands (preferably eight β-strands designated A-H) connected in pairs at one end by multiple (preferably four) loops, thereby constituting a ligand-binding pocket, and having a cylindrical β-pleated sheet supersecondary structure region that defines the entrance to the ligand-binding pocket. Preferably, the loops constituting the ligand-binding pocket used in this disclosure are those connecting the open ends of β-strands A and B, C and D, E and F, and G and H, designated loops AB, CD, EF, and GH. The diversity of these loops in the otherwise rigid lipocalin scaffold allows for a variety of different binding modes among members of the lipocalin family, allowing for targets of various sizes, shapes, and chemical characteristics to be accommodated (e.g., as reviewed in Skerra, Biochim Biophys Acta, 2000; Flower et al., Biochim Biophys Acta, 2000; Flower, Biochem J, 1996). Lipocalin family proteins have naturally evolved to bind to a wide range of ligands, and although they share an unusually low level of overall sequence conservation (often less than 20% sequence identity), their overall folding patterns are highly conserved. The positional correspondences in various lipocalins are also well known to those skilled in the art (see, e.g., U.S. Patent No. 7,250,297). Proteins within the definition of "lipocalin," as used herein, include, but are not limited to, tear lipocalin (Tlc, Lcn1), lipocalin-2 (Lcn2) or neutrophil gelatinase-associated lipocalin (NGAL), apolipoprotein D (ApoD), apolipoprotein M, α1-acid glycoprotein 1, α1-acid glycoprotein 2, α1-microglobulin, complement component 8γ, retinol-binding protein (RBP), epididymal retinoic acid-binding protein, glycodelin, odorant-binding protein IIa, odorant-binding protein IIb, lipocalin-15 (Lcn15), and prostaglandin D synthase.

[0014]

[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 compared to a naturally occurring (wild-type) protein or nucleic acid. The terms also include fragments of the muteins described herein. In some embodiments, the lipocalin muteins described herein (also referred to as Anticalin® proteins) comprise eight beta strands connected in pairs at one end by four loops, thereby constituting a ligand-binding pocket, and have a cylindrical beta-pleated sheet supersecondary structure region that defines the entrance to the ligand-binding pocket, wherein at least one amino acid located within the four loops is mutated compared to native sequence lipocalin.

[0015]

[0016] As used herein, the term "variant" refers to a derivative of a protein or polypeptide, including mutations, for example, by substitution, deletion, insertion, and / or chemical modification of the amino acid or nucleotide sequence. In some embodiments, such mutations and / or chemical modifications do not reduce the functionality of the protein or peptide. Such substitutions may be conservative, i.e., an amino acid residue is replaced with a chemically similar amino acid residue. Examples of conservative substitutions include substitutions between 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 are substituted 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, etc. Such variants also include proteins or polypeptides in which one or more amino acid residues are added or deleted, for example, at the N-terminus and / or C-terminus. Generally, variants have at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or at least about 98% amino acid sequence identity with a native sequence protein or polypeptide. Variants preferably retain the biological activity of the protein or polypeptide from which they are derived, e.g., biological activity of binding to the same target.

[0016]

[0017] The term "variant," as used herein with respect to an antibody or antigen-binding fragment thereof, refers to an antibody or antigen-binding fragment thereof of the present disclosure in which the sequence has mutations, including substitutions, deletions, insertions, and / or chemical modifications. A variant of an antibody or antigen-binding fragment thereof described herein retains the biological activity of the antibody or antigen-binding fragment thereof from which it is derived, e.g., the biological activity of binding to CD228. Generally, a variant of an antibody or 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 antigen-binding fragment thereof from which it is derived.

[0017]

[0018] As used herein, the term "sequence identity" or "identity" refers to a property of sequences that measures their similarity or relationship. As used in this disclosure, the term "sequence identity" or "identity" refers to the percentage of a pair of identical residues relative to the number of residues in the longer of the two sequences after (homologous) alignment of a protein or polypeptide of the disclosure with the sequence in question. Sequence identity is measured by dividing the number of identical amino acid residues by the total number of residues and multiplying the product by 100.

[0018]

[0019] As used herein, the term "sequence homology" or "homology" has its ordinary meaning, where homologous amino acids include identical amino acids and amino acids that are considered conservative substitutions at equivalent positions in the linear amino acid sequence of a protein or polypeptide of the present disclosure.

[0019]

[0020] Those skilled in the art will be aware of available computer programs, 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), for determining sequence homology or sequence identity using standard parameters. Percentage of sequence homology or sequence identity may be determined herein, for example, 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 determined based on an alignment of the entire protein or polypeptide sequence, including the propeptide sequence, preferably using the wild-type protein scaffold as a reference in pairwise comparisons (matrix: BLOSUM 62; gap cost: 11.1; cutoff value: 10 -3 It is calculated as a percentage of the number of "positives" (homologous amino acids) resulting in the BLASTP program output divided by the total number of amino acids selected by the program for the alignment.

[0020]

[0021] "Gaps" are spaces in an alignment that result from the addition or deletion of amino acids. Thus, two copies of the exact same sequence will have 100% identity, but less well-conserved sequences with deletions, additions, or substitutions may have a lower degree of sequence identity.

[0021]

[0022] As used interchangeably herein, the terms "conjugate," "conjugation," "fuse," "fusion," or "linked" refer to the joining of two or more subunits to one another through all forms of covalent or non-covalent bonding, including, but not limited to, genetic fusion, chemical conjugation, bonding via a linker or cross-linking agent, and non-covalent association.

[0022]

[0023] The terms "fusion polypeptide" or "fusion protein," as used herein, refer to a polypeptide or protein comprising two or more subunits. In some embodiments, the fusion proteins described herein comprise two or more subunits, at least one of which is capable of specifically binding to CD228. Within the fusion protein, the subunits can be linked by a covalent or non-covalent bond. Preferably, the fusion protein is a translational fusion between two or more subunits. A translational fusion can be generated by genetically engineering the coding sequence of one subunit in the reading frame with the coding sequence of an additional subunit. A nucleotide sequence encoding a linker can be interposed between the subunits. However, the subunits of the fusion protein of the present disclosure can also be linked by chemical conjugation. The subunits forming the fusion protein are typically linked 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 N-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 a fusion protein may be linked in any order and may contain more than one of any of the constituent subunits. When one or more subunits are part of a protein (complex) consisting of more than one polypeptide chain, the term "fusion protein" can refer to the protein containing 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 mutein via the heavy or light chain of the immunoglobulin / antibody, the term "fusion protein" can refer to a single polypeptide chain containing the lipocalin mutein and the heavy or light chain of the immunoglobulin / antibody. The term "fusion protein" can refer to the entire immunoglobulin / antibody (both light and heavy chains) and the lipocalin mutein fused to one or both of the heavy and / or light chains.

[0023]

[0024] As used herein, the term "subunit" of the fusion proteins disclosed herein refers to a single protein or separate polypeptide chain that can form a stable folded structure by itself and 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, e.g., a full-length antibody, or an antigen-binding domain / fragment thereof.

[0024]

[0025] A "linker," which may be constituted by the fusion proteins of the present disclosure, joins together two or more subunits of the fusion proteins 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.

[0025]

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

[0026]

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

[0027]

[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, domestic and farm animals, as well as zoo, sport, or pet animals, such as sheep, dogs, horses, cats, cows, rats, pigs, and apes, such as cynomolgus monkeys, to name a few illustrative examples. Preferably, a "mammal" as used herein is a human.

[0028]

[0030] An "effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more individual administrations or doses.

[0031] As used herein, "antibody" includes whole antibodies or any antigen-binding fragments thereof (i.e., "antigen-binding portion" or "antigen-binding domain") or single chains. The terms "antibody" and "immunoglobulin" can be and 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 heavy chain constant region (C H The heavy chain constant region is composed of C H1 , C H2 and C H3 Each light chain consists of a light chain variable domain / region (V L or LCVR) and the light chain constant region (C L The light chain constant region consists of one domain, C L The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen (e.g., 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 (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0029]

[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 (e.g., CD228). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding domain" of an antibody include: (i) V H , V L , C L and C H1 (ii) a Fab fragment consisting of two Fab fragments linked by a disulfide bridge in the hinge region; (iii) a V H , V L , C L and C H1 Domain and C H1 Domain and C H2 (iv) Fab' fragment consisting of the region between the V H and C H1 (v) a single arm V of an antibody; H and V L (vi) a single-chain Fv fragment consisting of a V H (vii) isolated complementarity-determining regions (CDRs) or combinations of two or more isolated CDRs that can optionally be joined by synthetic linkers; (viii) V domains connected within the same polypeptide chain using short linkers. H and V L (ix) "diabodies" including V H or V L "Domain antibody fragments" (in some cases, two or more V H domains are covalently bonded together).

[0030]

[0033] Antibodies can be polyclonal or monoclonal; xenogeneic, allogeneic, or syngenic; or modified forms thereof (e.g., humanized, chimeric, or multispecific). Antibodies can also be fully human.

[0031]

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

[0035] "Fragment crystallizable region" or "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions 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 usually defined to extend from the amino acid residue at Cys226, or from Pro230, to the carboxyl terminus, as numbered according to the EU index 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 antibody production or purification or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies can include antibody populations in which all K447 residues have been removed, antibody populations in which the K447 residue has not been removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Native sequence Fc regions suitable for use in the antibodies of the disclosure include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0032]

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

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

[0033]

[0038] As used herein, "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition, which display a single binding specificity and affinity for a particular epitope.

[0034]

[0039] As used herein, a "humanized antibody" refers to an antibody consisting of CDRs from an antibody derived from a non-human mammal and a human antibody or FR and constant regions derived from a human antibody. A humanized antibody may contain variable domains that, when analyzed as a whole, have a variable region amino acid sequence closer to that of humans than to that of other species, as assessed using the DomainGapAlign tool of the Immunogenetics Information System (IMGT) described in Ehrenmann et al. (2010). Because humanized antibodies have reduced antigenicity, they may be useful as active components of therapeutic agents. The terms "therapeutic agent" or "therapeutically active agent" as used herein refer to a therapeutically useful agent. A therapeutic agent may be any agent for the prevention, amelioration, or treatment of, or the evaluation or diagnosis of, a disease, physiological condition, or symptom.

[0035]

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

[0036]

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

[0037]

[0042] As used herein, "AAFX" refers to a fusion protein containing antibody clone X and a lipocalin mutein (lipocalin mutein I) having the amino acid sequence of SEQ ID NO: 40. For example, "AAF30" refers to a fusion protein containing antibody clone 30 and lipocalin mutein I. The designation "HC" in the name of a fusion protein indicates that the lipocalin mutein therein is conjugated to the antibody via the antibody's heavy chain, and the designation "LC" in the name of a fusion protein indicates that the lipocalin mutein therein is conjugated to the antibody via the antibody's light chain. A fusion protein name followed by "HC" or "LC" after the clone number refers to a fusion protein containing that antibody clone and lipocalin mutein I conjugated via the antibody's heavy chain or light chain, respectively. For example, "30HC" refers to a fusion protein containing antibody clone 30 and lipocalin mutein I conjugated thereto via the antibody's heavy chain.

[0038]

[0043] The antibody or fusion protein may also be designated by the sequences of its heavy and light chains (e.g., a fusion protein having the sequences of SEQ ID NOs: 80 and 76), one of which (e.g., SEQ ID NO: 80) contains the sequence of a lipocalin mutein in the case of the fusion protein.

[0039] IV. Figure Description [Brief explanation of the drawings]

[0040] [Figure 1]

[0044] FIG. 1 provides a schematic diagram showing CD137 clustering by cross-linking CD137-positive T cells with CD228-expressing tumor cells using the fusion proteins provided herein. [Figure 2A]

[0045] 2A and 2B are tables showing the CDR sequences of certain antibodies provided herein. [Figure 2B] 2A and 2B are tables showing the CDR sequences of certain antibodies provided herein. [Figure 3]

[0046] FIG. 1 shows the results of an ELISA demonstrating that anti-CD228 antibodies bind to recombinant human CD228. [Figure 4-1]

[0047] Figures 4A-4C show the binding of anti-CD228 antibodies to CD228-expressing cells as measured by fluorescence intensity, and Figure 4D is a table showing the EC50 values ​​of each antibody for each cell line. [Figure 4-2] Same as above. [Figure 5A]

[0048] 5A-5E show the results of a biolayer inferometry (BLI) assay of the binding kinetics and affinity of anti-CD228 antibodies to CD228. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 5D] Same as above. [Figure 5E] Same as above. [Figure 6A]

[0049] 6A-6C show the results of binding assays of anti-CD228 antibodies to CD228 and certain other transferrin family members. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 7A]

[0050] 7A to 7B and 8A to 8C show the results of binding assays of anti-CD228 antibodies to human, mouse, and cynomolgus monkey CD228. [Figure 7B] Same as above. [Figure 8A] 7A to 7B and 8A to 8C show the results of binding assays of anti-CD228 antibodies to human, mouse, and cynomolgus monkey CD228. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 9]

[0051] 1 is a table showing the results of a cross-competition assay between various anti-CD228 antibodies. [Figure 10]

[0052] FIG. 1 shows the results of an antibody internalization assay using a CD228+ tumor cell line. [Figure 11A]

[0053] 11A-11E show the results of an antigen recall assay in which fusion proteins costimulate innate and adaptive immune cytokines from PBMCs in response to viral peptides. [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 11D] Same as above. [Figure 11E] Same as above. [Figure 12]

[0054] 12, 13, 14 and 15 show the results of antigen recall assays in which fusion proteins costimulate T cell and NK cell responses in response to viral peptides. [Figure 13] 12, 13, 14 and 15 show the results of antigen recall assays in which fusion proteins costimulate T cell and NK cell responses in response to viral peptides. [Figure 14] 12, 13, 14 and 15 show the results of antigen recall assays in which fusion proteins costimulate T cell and NK cell responses in response to viral peptides. [Figure 15] 12, 13, 14 and 15 show the results of antigen recall assays in which fusion proteins costimulate T cell and NK cell responses in response to viral peptides. [Figure 16A]

[0055] 16A-16E show the results of an antigen recall assay in which fusion proteins costimulate cytotoxic effector molecules and cytokines from PBMCs in response to viral peptides. [Figure 16B] Same as above. [Figure 16C] Same as above. [Figure 16D] Same as above. [Figure 16E] Same as above. [Figure 17A]

[0056] 17A-17B show the ability of representative fusion proteins to costimulate T cell activation in a CD228 target-dependent manner. [Figure 17B] Same as above. [Figure 18A]

[0057] 18A-18C show T cell cytokine production in co-culture with anti-CD3 scFv engineered CD228-expressing tumor cell lines. [Figure 18B] Same as above. [Figure 18C] Same as above. [Figure 19]

[0058] FIG. 1 shows the proliferation of CD8 T cells in co-culture with anti-CD3 scFv-engineered CD228-expressing tumor cell lines. [Figure 20] FIG. 20 shows tumor cell killing in co-culture. [Figure 21]

[0059] FIG. 1 is a diagram and table showing the pharmacokinetics of fusion proteins in cynomolgus monkeys. [Figure 22A] 22A-22D, 23A-23C, and 24A-24B show the in vivo activity of the fusion proteins in a humanized xenograft model. [Figure 22B] 22A-22D, 23A-23C, and 24A-24B show the in vivo activity of the fusion proteins in a humanized xenograft model. [Figure 22C] 22A-22D, 23A-23C, and 24A-24B show the in vivo activity of the fusion proteins in a humanized xenograft model. [Figure 22D] 22A-22D, 23A-23C, and 24A-24B show the in vivo activity of the fusion proteins in a humanized xenograft model. [Figure 23A] 22A-22D, 23A-23C, and 24A-24B show the in vivo activity of the fusion proteins in a humanized xenograft model. [Figure 23B]22A-22D, 23A-23C, and 24A-24B show the in vivo activity of the fusion proteins in a humanized xenograft model. [Figure 23C] 22A-22D, 23A-23C, and 24A-24B show the in vivo activity of the fusion proteins in a humanized xenograft model. [Figure 24] 22A-22D, 23A-23C, and 24A-24B show the in vivo activity of the fusion proteins in a humanized xenograft model. DETAILED DESCRIPTION OF THE INVENTION

[0041] 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 to CD228. Such anti-CD228 antibodies or antigen-binding fragments thereof can be used as antibody therapeutics themselves, conjugated to therapeutic agents to generate antibody-drug conjugates, included as part of bispecific or multispecific antibodies, or included as part of fusion molecules. In certain embodiments, the antibody or antigen-binding fragment is incorporated into a fusion molecule containing a binding domain that binds to 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 protein that can bind to both CD228 and CD137. The use of the anti-CD228 antibodies or antigen-binding fragments provided herein in fusion proteins, such as those that target both CD228 and CD137, can be utilized to promote CD137 clustering by crosslinking CD137-positive T cells to CD228-expressing tumor cells located in the tumor microenvironment, as illustratively shown in FIG. 1. The anti-CD228 antibodies and antigen-binding fragments provided herein can be used to generate fusion proteins directed to other targets to achieve similar effects. In other aspects, the present disclosure also provides methods and useful applications for the anti-CD28 antibodies and antigen-binding fragments provided herein. The present disclosure also provides methods for producing the CD228-binding antibodies or antigen-binding fragments thereof described herein, and compositions comprising such proteins. The CD228-binding antibodies or antigen-binding fragments thereof of the present disclosure, and compositions thereof, can be used in methods for detecting CD228 in a sample and in methods for binding CD228 in a subject. Such antibodies or antigen-binding fragments having the characteristics associated with the uses provided by the present disclosure have not previously been described.

[0042] A. Exemplary Antibodies or Antigen-Binding Fragments Specific for CD228

[0061] In some embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein comprise: i) a heavy chain variable domain (VH) comprising (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) comprising (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 comprising (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 comprising (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) a VH comprising (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 a VL comprising (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) a VH comprising (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 a VL comprising (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) a VH comprising (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 a VL comprising (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) a VH comprising (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 a VL comprising (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) a VH comprising (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 comprising (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; viii) a VH comprising (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 comprising (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; ix) a VH comprising (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 a VL comprising (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; x) a VH comprising (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 a VL comprising (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; xi) a VH comprising (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 comprising (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) a VH comprising (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 comprising (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) a VH comprising (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 a VL comprising (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) a VH comprising (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 a VL comprising (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) a VH comprising (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 comprising (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) a VH comprising (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 comprising (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) a VH comprising (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 comprising (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) A VH comprising (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 273, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 274, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 275, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 279, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 280, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 281.

[0043]

[0062] In some embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein comprise: i) a VH comprising (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 comprising (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) a VH comprising (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 comprising (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) a VH comprising (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 comprising (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) A VH comprising (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 253, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 254, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 255, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 259, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 260, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 261.

[0044]

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

[0045]

[0064] In some embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein comprise: i) a VH comprising 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 comprising 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 comprising 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 comprising 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) a VH comprising 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 a VL comprising 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) a VH comprising 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 a VL comprising 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) a VH comprising 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 a VL comprising 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) a VH comprising 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 comprising 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) a VH comprising 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: 242, and a VL comprising 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: 244; viii) a VH comprising 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 comprising 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 ix) a VH comprising 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 a VL comprising 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.

[0046]

[0065] In some embodiments, the antibodies or antigen-binding fragments thereof that bind CD228 provided herein comprise a VH comprising 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 VH comprising 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 comprising 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 comprising 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.

[0047]

[0066] In some embodiments, an antibody or antigen-binding fragment thereof that binds to CD228 provided herein comprises a VH comprising 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 comprising 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.

[0048]

[0067] In some embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein comprise: i) 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; ii) 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; iii) 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; iv) 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; v) 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; vi) 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; vii) 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; viii) 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 ix) 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.

[0049]

[0068] In some embodiments, an antibody or antigen-binding fragment thereof that binds to CD228 provided herein comprises 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.

[0050]

[0069] In some embodiments, an antibody or antigen-binding fragment thereof that binds to CD228 provided herein comprises 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.

[0051]

[0070] In some embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein comprise: i) a heavy chain (HC) comprising 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) comprising 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) an HC comprising 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 an LC comprising 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) an HC comprising 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 an LC comprising 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) an HC comprising 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 an LC comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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.

[0052]

[0071] In some embodiments, the antibodies or antigen-binding fragments thereof that bind CD228 provided herein comprise an HC comprising 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 HC comprising 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 LC comprising 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 comprising 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.

[0053]

[0072] In some embodiments, an antibody or antigen-binding fragment thereof that binds to CD228 provided herein comprises an HC comprising 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 comprising 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.

[0054]

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

[0055]

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

[0056]

[0075] In some embodiments, an antibody or antigen-binding fragment thereof that binds to CD228 provided herein comprises an HC comprising the amino acid sequence of SEQ ID NO:226, and an LC comprising the amino acid sequence of SEQ ID NO:228.

[0057]

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

[0058]

[0077] In some embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein have a K D In some embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein bind to CD228 with a K value of 150 nM or less. D In some embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein bind to CD228 with a K value of 100 nM or less. D In some embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein bind to CD228 with a K value of 50 nM or less. D In some embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein bind to CD228 with a K value of 75 nM or less. DIn some embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein bind to CD228 with a K value of 25 nM or less. D In some embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein bind to CD228 with a K value of 20 nM or less. D In some embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein bind to CD228 with a K value of 15 nM or less. D In some embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein bind to CD228 with a K value of 10 nM or less. D In some embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein bind to CD228 with a K value of 5 nM or less. D In some embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein bind to CD228 with a K value of 4 nM or less. D In some embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein bind to CD228 with a K value of 3 nM or less. D In some embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein bind to CD228 with a K value of 2.5 nM or less. D In some embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein bind to CD228 with a K value of 2 nM or less. D In some embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein bind to CD228 with a K value of 1.5 nM or less. D In some embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein bind to CD228 with a K value of 1 nM or less. D Binds to CD228 at high levels.

[0059]

[0078] In some embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein bind to cynomolgus monkey CD228. In some such embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein comprise: i) a VH comprising (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 comprising (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 comprising (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 comprising (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 comprising (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 comprising (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) a VH comprising (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 comprising (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) a VH comprising (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 comprising (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) A VH comprising (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 253, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 254, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 255, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 259, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 260, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 261.

[0060] In some such embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein comprise a set of CDRs as described above, as well as: i) a VH comprising 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 a VL comprising 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 comprising 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 comprising 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 comprising 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 comprising 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. Includes.

[0061] In some such embodiments, an antibody or antigen-binding fragment thereof that binds to CD228 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.

[0062]

[0079] In some embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein do not bind to mouse CD228 or bind to mouse CD228 with an affinity that is at least 100-fold reduced compared to human CD228. In some embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein do not bind to transferrin or lactotransferrin. In some such embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein include: i) a VH comprising (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 comprising (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 comprising (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 comprising (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 comprising (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 a VL comprising (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) a VH comprising (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 a VL comprising (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) a VH comprising (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 comprising (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; vi) a VH comprising (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 comprising (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; vii) a VH comprising (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 a VL comprising (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; viii) a VH comprising (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 a VL comprising (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; ix) a VH comprising (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 comprising (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 comprising (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 comprising (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 comprising (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 comprising (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 comprising (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 273, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 274, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 275, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 279, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 280, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 281.

[0063] In some such embodiments, the antibodies or antigen-binding fragments thereof that bind to CD228 provided herein comprise a set of CDRs as described above, as well as: i) a VH comprising 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 a VL comprising 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 comprising 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 a VL comprising 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; iii) a VH comprising 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 comprising 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; iv) a VH comprising 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: 242; and a VL comprising 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: 244; or v) a VH comprising 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 comprising 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) a VH comprising 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 a VL comprising 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. Includes.

[0064] In some such embodiments, an antibody or antigen-binding fragment thereof that binds to CD228 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: 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.

[0065]

[0080] In some embodiments, an antibody or antigen-binding fragment thereof that binds to CD228 is provided, wherein the antibody or antigen-binding fragment thereof competes for binding to CD228 with any one of the antibodies or antigen-binding fragments disclosed herein. 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 antigen-binding fragment thereof has a K DIn some such embodiments, the antibody or antigen-binding fragment thereof binds to CD228 with an affinity that is at least 100-fold reduced compared to human CD228. In some such embodiments, the antibody or antigen-binding fragment thereof does not bind to mouse CD228 or binds to mouse CD228 with an affinity that is at least 100-fold reduced compared to 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 for binding to CD228 with an antibody or antigen-binding fragment thereof comprising 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. 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; 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.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.

[0066]

[0081] 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 ed. 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).

[0067]

[0082] The specific binding antibody to CD228 contained in the fusion protein of the present disclosure may comprise an Fc portion that allows for extended in vivo half-life of the bispecific binding molecule of the present disclosure. In some embodiments, such an Fc portion is preferably of human origin, more preferably a human Fc portion of an IgG1 or IgG4 antibody, and even more preferably an engineered human Fc portion of an IgG1 or IgG4 antibody with activating or silencing effector function. In some embodiments, silencing effector function may be preferred over activating effector function. In some embodiments, such an Fc portion is engineered to silence effector function by mutation(s) at positions 234 and / or 235, numbered according to the EU index of Kabat (Johnson and Wu, Nucleic Acids Res, 2000). In some embodiments, mutations at positions F234 and L235 of the provided anti-CD228 antibody may be introduced to silence effector function. In other embodiments, mutations at positions D265 and P329 of the provided anti-CD228 antibodies may be introduced to silence effector function. The numbering of both sets of these potential mutations is according to the EU index of Kabat (Shields et al., J Biol Chem, 2001). In some embodiments, the provided CD228 antibodies have an engineered IgG4 backbone with the mutations S228P, F234A, and L235A.

[0068]

[0083] Various techniques for producing antibodies and antigen-binding fragments thereof 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 animal blood after immunization with an antigen mixed with additives and adjuvants, and monoclonal antibodies can be produced by any technique that provides antibodies produced by continuous cell line culture. Examples of such techniques are described, for example, in Harlow and Lane (1999), (1988), and include the hybridoma technique first described by Kohler 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 producing human monoclonal antibodies (Cole et al., Cancer Res, 1984). Furthermore, recombinant antibodies can be derived from monoclonal antibodies or 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, insect, mammalian cell lines, or transgenic animals or plants (see, e.g., U.S. Pat. No. 6,080,560; Holliger and Hudson, Nat Biotechnol, 2005). Furthermore, techniques described for the production of single-chain antibodies (see, in particular, U.S. Pat. No. 4,946,778) can be adapted to produce single-chain antibodies specific to 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.

[0069] B. Immunoconjugates of Antibodies or Antigen-Binding Fragments Thereof

[0084] The antibodies or antigen-binding fragments thereof provided herein can be conjugated to cytotoxic or cytostatic moieties (including pharmaceutically compatible salts thereof) to form immunoconjugates, such as antibody-drug conjugates (ADCs). Particularly suitable moieties for conjugation to antibodies or antigen-binding fragments thereof are cytotoxic agents (e.g., chemotherapeutic agents), prodrug-converting enzymes, radioisotopes or compounds, or toxins (these moieties are collectively referred to as therapeutic agents). For example, antibodies or antigen-binding fragments thereof can be conjugated to cytotoxic agents such as chemotherapeutic agents or toxins (e.g., cytostatic or cytocidal agents, e.g., 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, camptothecins, calicheamicins, duocarmycins, etoposide, maytansinoids (e.g., DM1, DM2, DM3, DM4), taxanes, benzodiazepines (e.g., pyrrolo[1,4]benzodiazepines, indolinobenzodiazepines, and oxazolidinobenzodiazepines), and vinca alkaloids.

[0070]

[0085] In one embodiment, the antibody or antigen-binding fragment thereof is conjugated to a prodrug-converting enzyme. The prodrug-converting enzyme may 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.

[0071]

[0086] 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.) The therapeutic agent can be conjugated in a way that reduces its activity unless it is cleaved from the antibody or antigen-binding fragment thereof (e.g., by hydrolysis, proteolysis, or a cleaving agent). In some embodiments, the therapeutic agent is attached to the antibody or antigen-binding fragment thereof by a cleavable linker that is sensitive to cleavage in the intracellular environment of the CD228-expressing cancer cell, but is substantially insensitive to the extracellular environment, such that the immunoconjugate is cleaved from the antibody or antigen-binding fragment thereof when internalized by the CD228-expressing cancer cell (e.g., within an endosome, or within a lysosomal or caveolar environment, e.g., due to pH sensitivity or protease sensitivity). In some aspects, the therapeutic agent may be attached to the antibody or antigen-binding fragment thereof by a non-cleavable linker.

[0072]

[0087] Typically, immunoconjugates contain a linker region between the therapeutic agent and the antibody or antigen-binding fragment thereof. The linker is generally 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. The linker can be, for example, a peptide linker cleaved by intracellular peptidase or protease enzymes (including lysosomal or endosomal proteases). Cleaving agents can include cathepsin B and D and plasmin (e.g., Dubowchik and Walker, Pharm. Therapeutics 83:67-123, 1999). Most typical is a peptidyl linker that 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 cancerous tissues, can be used (e.g., a linker containing Phe-Leu or Val-Cit).

[0073]

[0088] The cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a certain pH value. Typically, the pH-sensitive linker is hydrolyzable under acidic conditions. For example, an acid-labile linker (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, etc.) that is hydrolyzable in lysosomes can be used. (See, for example, 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 under neutral pH conditions, such as in blood, but are unstable below pH 5.5 or 5.0, which is the approximate pH of lysosomes.

[0074]

[0089] Other linkers are cleavable under reducing conditions (e.g., disulfide linkers), including those that can be 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-alpha-methyl-alpha-(2-pyridyl-dithio)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.S. Press, 1987); see also U.S. Pat. No. 4,880,935).

[0075]

[0090] The linker can also be a malonic acid 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 analog (Lau et al., Bioorg-Med-Chem. 3:1305-12, 1995).

[0076]

[0091] In other embodiments, the linker is a non-cleavable linker, such as a maleimide-alkylene-linker or a maleimide-aryl linker, that is directly attached to the therapeutic agent and released by proteolysis of the antibody or antigen-binding fragment thereof.

[0077]

[0092] Typically, the linker is substantially insensitive to the extracellular environment, meaning that no more than about 20%, typically no more than 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 linkers in a sample of the immunoconjugate are cleaved when the immunoconjugate is present in an extracellular environment (e.g., in plasma). Whether a linker is substantially insensitive to the extracellular environment can be determined, for example, by incubating both (a) the immunoconjugate (the "immunoconjugate sample") and (b) an equimolar amount of an unconjugated antibody or antigen-binding fragment thereof or therapeutic agent (the "control sample") independently with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours), and then comparing the amount of unconjugated antibody or antigen-binding fragment thereof or therapeutic agent present in the immunoconjugate sample with the amount present in the control sample, as measured, for example, by high performance liquid chromatography.

[0078]

[0093] The linker may also facilitate internalization into a cell. The linker can facilitate internalization into a cell when conjugated to a therapeutic agent (i.e., in the context of the linker-therapeutic agent portion of an immunoconjugate or immunoconjugate derivative described herein). Alternatively, the linker can facilitate internalization into a cell when conjugated to both a therapeutic agent and an antibody or antigen-binding fragment thereof (i.e., in the context of an immunoconjugate described herein).

[0079]

[0094] Exemplary immunoconjugates include auristatin-based conjugates, meaning that the drug component is an auristatin drug. Auristatins bind to tubulin, disrupt microtubule dynamics and nuclear and cell division, and have been shown to have anti-cancer activity. Typically, auristatin-based antibody-drug conjugates include 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 antibody degradation). 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 be reacted with paraacetylbenzoic acid or benzoylvaleric acid to produce auristatin EB (AEB) and auristatin EVB (AEVB), respectively. Other exemplary auristatins include MMAF and MMAE. The synthesis and structure of exemplary auristatins are described in U.S. Patent or Publication Nos. 7,659,241, 7,498,298, 2009-0111756, 2009-0018086, and 7,968,687, each of which is incorporated by reference in its entirety for all purposes.

[0080]

[0095] Exemplary auristatin-based conjugates include vcMMAE, vcMMAF, and mcMMAF immunoconjugates, as shown below, where Ab is an anti-CD228 antibody or antigen-binding fragment thereof described herein, and val-cit represents a valine-citrulline dipeptide. Drug loading is represented by p, 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 thereof in a composition of antibodies or antigen-binding fragments, also referred to as average drug loading. In some embodiments, p is in the range of 1 to 20, preferably 1 to 8. In some embodiments, when p represents average drug loading, p is in the range of about 2 to about 5. In some embodiments, p is about 2, about 3, about 4, or about 5. The average number of drugs per antibody in a preparation can be characterized by conventional means, such as mass spectrometry, HIC, ELISA assay, and HPLC. In some aspects, the anti-CD228 antibody or antigen-binding fragment thereof is linked to the drug-linker via a cysteine ​​residue in the antibody or antigen-binding fragment thereof. In some embodiments, the cysteine ​​residue is engineered into the antibody or antigen-binding fragment thereof. In other aspects, the cysteine ​​residue is an intrachain disulfide cysteine ​​residue.

[0081] [ka]

[0082] C. Exemplary Uses and Applications of Antibodies or Antigen-Binding Fragments Thereof Specific for CD228, or Immunoconjugates of the Antibodies or Antigen-Binding Fragments Thereof

[0096] In some aspects, the present disclosure provides diagnostic and / or analytical kits comprising one or more antibodies, antigen-binding fragments thereof, or immunoconjugates of antibodies or antigen-binding fragments thereof according to the present disclosure.

[0083]

[0097] In addition to uses in diagnosis, in yet another aspect, the present disclosure contemplates pharmaceutical compositions comprising one or more antibodies, antigen-binding fragments thereof, or immunoconjugates of the antibodies or antigen-binding fragments thereof of the present disclosure and a pharmaceutically acceptable excipient.

[0084]

[0098] Furthermore, 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, for example, as anti-tumor 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 medicaments, for example, medicaments for the treatment of cancer, including CD228-positive cancer. In some embodiments, it is envisioned that the antibodies, antigen-binding fragments thereof, or immunoconjugates of the antibodies or antigen-binding fragments thereof of the present disclosure are used in methods for the prevention, amelioration, or treatment of human diseases, such as cancer, including CD228-positive cancer. Thus, also provided are methods of preventing, ameliorating, or treating a human disease, such as cancer, including CD228-positive cancer, in a subject in need thereof, comprising 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 a CD228-positive cancer.

[0085]

[0099] Examples of cancers that can be treated using 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, e.g., cutaneous or intraocular malignant melanoma, pancreatic cancer, mesothelioma, colorectal cancer, thyroid cancer, breast cancer, bile duct cancer, esophageal cancer, and head and neck cancer. In some embodiments, the cancer includes metastatic cancer.

[0086]

[0100] In some embodiments, the fusion proteins of the present disclosure can simultaneously target CD228-expressing tumor cells and activate lymphocytes of the host immune system adjacent to such tumor cells. In some embodiments, the fusion proteins of the present disclosure can increase targeted anti-tumor T cell activity, enhance anti-tumor immunity, and / or have a direct inhibitory effect on tumor growth, thereby producing synergistic anti-tumor results. In some embodiments, the fusion proteins of the present disclosure can activate immune responses in the tumor microenvironment. In some embodiments, the fusion proteins of the present disclosure can reduce the side effects of effector lymphocytes on healthy cells, i.e., off-target toxicity, for example, by locally inhibiting oncogene activity and / or inducing lymphocyte activation.

[0087]

[0101] In some embodiments, the present disclosure encompasses the use of fusion proteins of the present disclosure, or compositions comprising the provided fusion proteins, to induce localized lymphocyte responses in the vicinity of CD228-positive tumor cells. Thus, in some embodiments, the present disclosure provides methods of inducing a localized lymphocyte response in the vicinity of CD228-positive tumor cells, comprising applying one or more fusion proteins of the present disclosure, or one or more compositions comprising such fusion proteins.

[0088] D. Exemplary embodiments provide for the generation of antibodies or antigen-binding fragments thereof specific for CD228

[0102] In some embodiments, the present disclosure provides nucleic acid molecules (e.g., DNA or RNA) comprising a nucleotide sequence 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 the substitution of certain codons with other codons that specify the same amino acid, the present disclosure is not limited to the particular nucleic acid molecules encoding the antibodies, or antigen-binding fragments thereof, described herein, but rather encompasses all nucleic acid molecules comprising a nucleotide sequence encoding a functional antibody, or antigen-binding fragment thereof. In this regard, the present disclosure also relates to nucleotide sequences encoding the provided antibodies, or antigen-binding fragments thereof.

[0089]

[0103] Nucleic acid molecules, such as DNA, contain sequence elements containing information for transcriptional and / or translational regulation. When such sequences are "operably linked" to a nucleotide sequence encoding a protein, they are said to "permit expression of the nucleic acid molecule" or "permit expression of the nucleotide sequence." An operable linkage is one in which the regulatory sequence elements and the sequence to be expressed are connected in a manner that allows gene expression. While the exact nature of the regulatory regions required for gene expression can vary between species, these regions generally include a promoter, which in prokaryotes contains both the promoter itself, i.e., the DNA element that directs the initiation of transcription, and the DNA element that, once transcribed into RNA, signals the initiation of translation. Such promoter regions usually include 5' non-coding sequences involved in initiation of transcription and translation, such as the -35 / -10 box and Shine-Dalgarno element in prokaryotes, and the TATA box, CAAT sequence, and 5'-capping element in eukaryotes. These regions may also contain enhancer or repressor elements, as well as translational signals and leader sequences for targeting the native protein to specific compartments of the host cell.

[0090]

[0104] In addition, the 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 substituted with signals that are functional in that cell.

[0091]

[0105] Therefore, the nucleic acid molecule of the present disclosure can be "operably linked" to one or more regulatory sequences, such as a promoter sequence, to allow the expression of the nucleic acid molecule. In some embodiments, the nucleic acid molecule of the present disclosure includes a promoter sequence and a transcription termination sequence. Suitable prokaryotic promoters include, for example, the tet promoter, the lacUV5 promoter, or the T7 promoter. Examples of promoters useful for expression in eukaryotic cells include the SV40 promoter or the CMV promoter.

[0092]

[0106] In some embodiments, the provided nucleic acid molecules can also be part of a vector or any other type of cloning vehicle, such as a plasmid, phagemid, phage, baculovirus, cosmid, or artificial chromosome.

[0093]

[0107] In some embodiments, the provided nucleic acid molecule may be contained in a phagemid.In this context, a phagemid vector refers to a vector encoding the intergenic region of a temperate phage such as M13 or f1, or its functional portion fused to a target cDNA.For example, in some embodiments, after hyperinfecting bacterial host cells with such a provided phagemid vector and suitable helper phage (for example, M13K07, VCS-M13 or R408), intact phage particles are produced, thereby allowing the coded heterologous cDNA to be physically linked to the corresponding polypeptide displayed on the phage surface (Lowman, Annu Rev Biophys Biomol Struct, 1997; Rodi and Makowski, Curr Opin Biotechnol, 1999).

[0094]

[0108] According to various embodiments, the cloning vehicle may contain, in addition to the regulatory sequences described above and nucleic acid sequences encoding the antibodies, or antigen-binding fragments thereof, described herein, replication and control sequences derived from a species compatible with the host cell used for expression, and a selectable marker that confers a selectable phenotype on transformed or transfected cells. Many suitable cloning vectors are known in the art and are commercially available.

[0095]

[0109] The present disclosure also relates to methods for producing the antibodies or antigen-binding fragments thereof of the present disclosure, in some embodiments, using genetic engineering methods, starting with a nucleic acid encoding an antibody, or antigen-binding fragment thereof, or any subunit(s) thereof. In some embodiments, the provided methods can be performed in vivo, and the provided antibodies or antigen-binding fragments thereof can be produced, for example, in a bacterial or eukaryotic host organism, and then isolated from the host organism or a culture thereof. 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.

[0096]

[0110] For in vivo production of antibodies or antigen-binding fragments thereof, nucleic acids encoding such antibodies or antigen-binding fragments thereof may be introduced into suitable bacterial or eukaryotic host organisms using recombinant DNA techniques well known in the art. In some embodiments, DNA molecules encoding the antibodies or antigen-binding fragments thereof described herein, particularly cloning vectors containing the coding sequences of such antibodies or antigen-binding fragments thereof, can be transformed into host cells capable of expressing the genes. Transformation can be carried out using standard techniques. Thus, the present disclosure also covers host cells containing the nucleic acid molecules disclosed herein.

[0097]

[0111] In some embodiments, transformed host cells can be cultured under conditions suitable for expression of nucleotide sequences encoding antibodies, or antigen-binding fragments thereof, of the present disclosure. In some embodiments, host cells are prokaryotic cells such as Escherichia coli (E. coli) or Bacillus subtilis, or eukaryotic cells such as Saccharomyces cerevisiae, Pichia pastoris, SF9 or High5 insect cells, immortalized mammalian cell lines (e.g., HeLa cells or CHO cells), or primary mammalian cells.

[0098]

[0112] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure can be produced in the cytoplasm of a host cell, preferably E. coli. In this case, the provided antibody or antigen-binding fragment thereof can be directly obtained in a soluble, folded state or recovered in the form of inclusion bodies and then re-denatured in vitro. Another option is to use a specific host strain with an oxidative intracellular environment, which can allow disulfide bond formation in the cytoplasm (Venturi et al., J Mol Biol, 2002).

[0099]

[0113] In some embodiments, the antibodies of the present disclosure, or antigen-binding fragments thereof, described herein, need not necessarily be generated or produced, in whole or in part, by genetic engineering. Rather, such proteins can be obtained by any of a number of conventional, well-known techniques, such as straightforward organic synthesis strategies, solid-phase-assisted synthesis techniques, commercially available automated synthesizers, or in vitro transcription and translation. For example, promising antibodies or antigen-binding fragments thereof can be identified using molecular modeling, where they are synthesized in vitro and investigated for binding activity to the target(s) of interest. Methods for solid-phase and / or solution-phase synthesis of proteins are well known in the art (see, e.g., Bruckdorfer et al., Curr Pharm Biotechnol, 2004).

[0100]

[0114] In some embodiments, antibodies, or antigen-binding fragments thereof, of the present disclosure may be produced by in vitro transcription / translation using well-established methods known to those of skill in the art.

[0115] In some further embodiments, the antibodies, or antigen-binding fragments thereof, described herein can also be prepared using conventional recombinant techniques alone or in combination with conventional synthetic techniques.

[0101]

[0116] Those skilled in the art will understand useful methods for preparing antibodies or antigen-binding fragments thereof contemplated by the present disclosure, even though the protein or nucleic acid sequences are not explicitly disclosed herein. Generally, such amino acid sequence modifications include, for example, directed mutagenesis of a single amino acid position to simplify subcloning of a protein gene or portion thereof by incorporating a specific restriction enzyme cleavage site. These mutations can also be incorporated to further improve the affinity of the antibody or antigen-binding fragment thereof for its target(s) (e.g., CD228). Furthermore, mutations can be introduced, as needed, to modulate one or more properties of the protein, such as improving folding stability, serum stability, protein resistance, or water solubility, or reducing aggregation tendency.

[0102]

[0117] Further objects, advantages, and features of the present disclosure will become apparent to those skilled in the art upon examination of the following examples and accompanying drawings, which are not intended to be limiting. Thus, while the present disclosure has been specifically disclosed by exemplary embodiments and optional features, it should be understood that modifications and variations of the disclosure embodied therein disclosed herein may be resorted to by those skilled in the art, and such modifications and variations are considered to be within the scope of the present disclosure.

[0103] VI. Working Examples [Example]

[0104]

[0118] Example 1: Generation of anti-CD228 antibodies

[0119] A human immunoglobulin transgenic rat strain (OmniRat®; OMT, Inc.) was used to develop monoclonal antibody-expressing hybridoma cells. OmniRat® contains a chimeric human / rat IgH locus (containing 22 human VHs, with all human D and JH segments linked in their natural configuration to the rat CH locus) together with a fully human IgL locus (12 Vκs linked to Jκ-Cκs and 16 Vλs linked to Jλ-Cλs). See Osborn et al. (2013) J Immunol 190(4):1481-1490; WO2014 / 093908. In response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high-affinity IgG monoclonal antibodies.

[0105]

[0120] Transgenic rats were immunized with recombinant human CD228 protein. Subcutaneous injections included 48.2 μg of recombinant protein in complete Freund's adjuvant on day 1 and 48 μg of recombinant protein in incomplete Freund's adjuvant on days 33, 81, and 127.

[0106]

[0121] The presence of antibodies directed against human CD228 in the serum of transgenic rats was monitored by flow cytometry on days 33, 81, and 127 using RPMI-7951 cells engineered to stably express human CD228. Transgenic rats with detectable immune responses were boosted 5 and 7 days before harvesting the spleen and lymph nodes. The boosts before organ harvest included intravenous injection of 90 μg and intraperitoneal injection of 18 μg of recombinant human CD228 protein suspended in phosphate-buffered saline (PBS).

[0107]

[0122] Spleen cells and lymphocytes showing B cell lineage and specificity for fluorescently labeled recombinant human CD228 were sorted as single cells into cell lysis buffer by flow-activated cell sorting (FACS). RNA from single B cells was reverse transcribed into cDNA and amplified using primer sets against known human variable regions. Amplicons were purified for Sanger sequencing with appropriate sequencing primers, and heavy and light chain sequences were subsequently identified and annotated using IgBLAST.

[0108]

[0123] Highly productive heavy and light chain sequences of human variable regions identified from single B cell cloning and Sanger sequencing were selected for gene synthesis and cloned into expression vectors using conventional cloning techniques. The expression vectors encoded human IgG1, kappa, or lambda constant regions, which were in frame with and downstream of the human variable regions. Antibody expression constructs were verified by Sanger sequencing.

[0109]

[0124] To generate full-length antibodies, heavy and light chain expression vectors were co-transfected into ExpiCHO cells according to the manufacturer's protocol (Life Technologies). Nine days after transfection, cell culture supernatants were collected by centrifugation. The filtered culture supernatants were spiked with 10% Triton-X (final concentration 0.1%) and left overnight at 4°C on a shaking platform. The supernatants were loaded onto a 5mL HiTrap MabSelectSuRe column at 2.5mL / min using an AKTA Avant 25 chromatography system. The column was then washed with 5 column volumes (CV) of end-wash buffer (1x PBS, 0.1% Triton-X), 5CV of high-salt buffer (1x PBS, 0.5M NaCl), and 7.5CV of standard 1x PBS (pH 7.4). Protein was eluted with 1 CV of 20 mM citrate (pH 3) and immediately buffer exchanged into 1× PBS (pH 7.4) using a HiPrep Sephadex G-25 Desalting column. Fractions were determined by A280 UV watch parameters to allow optimal collection of purified protein.

[0110]

[0125] Figures 2A and 2B show the CDR sequences of antibodies as defined by Kabat (Figure 2A) and IMGT (Figure 2B). [Example]

[0111]

[0126] Example 2: Binding of anti-CD228 antibodies to recombinant human CD228

[0127] Humanized antibodies specific for human melanotransferrin (CD228) were evaluated for binding to recombinant human CD228 (R&D Systems) by ELISA. Antibodies were titrated on plates coated with 1 μg / ml recombinant human CD228, detected with HRP-conjugated goat anti-human IgG secondary reagent (Thermo), and developed with TMB substrate (Thermo). OD450 values ​​were read on a plate reader using SoftMax Pro software. OD450 values ​​from each experiment were transferred to GraphPad Prism 8 for plotting and analysis. The results are shown in Figure 3. [Example]

[0112]

[0128] Example 3: Binding of anti-CD228 antibodies to cells

[0129] Humanized antibodies specific for human CD228 were evaluated for relative binding to CD228-expressing cancer cell lines. Cells were incubated with titrations of Alexa-647-labeled humanized antibody clones, washed, and fluorescence intensity was assessed by flow cytometry on an Attune NXT flow cytometer. MFI values ​​were transferred to GraphPad Prism 8 for plotting and analysis. EC50 values ​​were determined by nonlinear regression. Numbers next to cell line names reflect surface CD228 copy numbers estimated using the QIFIKIT quantitative analysis kit (Agilent).

[0113]

[0130] The results are shown in Figures 4A-4C as raw mean fluorescence intensity (MFI) values. Figure 4D shows the EC values ​​of each antibody for each cell line. 50 Indicates the value. [Example]

[0114]

[0131] Example 4: Binding Profiles of Anti-CD228 Antibodies

[0132] The binding kinetics and affinity of human CD228 bearing a C-terminal polyhistidine tag (R&D Systems) were determined by biolayer interferometry (BLI) using the Octet® RED384 system (Sartorius). An anti-human antibody capture AHC (GE Healthcare) biosensor was used for the analysis. Anti-CD228 antibodies OMT8, OMT24, OMT30, OMT35, and OMT36 (IgG) were then applied to the chip surface at 0.5 μg / mL in HBS-EP+ buffer for 180 seconds, followed by capture with an anti-human IgG-Fc antibody. After each capture step, the biosensor was washed with HBS-EB+ blank. For affinity determination, dilutions of recombinant huCD228 (100 nM, 40 nM, 16 nM, 6.4 nM, 2.6 nM, and 1.0 nM) or a blank were prepared in HBS-EP+ buffer and applied to the biosensor. Binding assays were performed with a contact time of 300 seconds and a dissociation time of 1,200 seconds. All measurements were performed at 25°C. Fresh AHC biosensors were used for each analysis. Data were evaluated using Satorius Octet® Data Analysis Software (v12.0), and the results are shown in Figures 5A-5E. A single reference was used and the raw data were fitted using a 1:1 binding model. [Example]

[0115]

[0133] Example 5: Lack of cross-reactivity of anti-CD228 antibodies to transferrin and lactotransferrin

[0134] Humanized antibodies specific for human melanotransferrin (CD228) were evaluated by ELISA for binding to lactotransferrin and transferrin, related transferrin family members. The antibodies were titrated on plates coated with 1 μg / mL recombinant human melanotransferrin (CD228), lactotransferrin, or transferrin, and detected with HRP-conjugated goat anti-human IgG secondary reagent (Thermo) developed with TMB substrate (Thermo). OD450 values ​​were read using a plate reader equipped with SoftMax Pro software. OD450 values ​​from each experiment were imported into GraphPad Prism 8 for plotting and analysis. These results, shown in Figures 6A-6C, demonstrate no cross-reactive binding of the anti-CD228 antibody to other transferrin family members. [Example]

[0116]

[0135] Example 6: Cross-reactivity of anti-CD228 antibodies to non-human CD228

[0136] The CD228-negative human melanoma cell line RPMI-7951 was engineered to express cynomolgus monkey CD228 to test the binding of anti-CD228 antibody clones. 50,000 RPMI-7951 cells were incubated with titrations of the humanized antibody clones, washed with staining buffer to remove excess antibody, and incubated with 250 ng / mL of a fluorochrome-labeled monoclonal antibody against human IgG1 (Thermo) to detect bound antibody. Results are shown in Figure 7A as mean fluorescence intensity (MFI) determined by measurement on an Attune NXT flow cytometer (Thermo).

[0117]

[0137] The reactivity of CD228 antibody clones to recombinant his-tagged cynomolgus CD228 was tested by ELISA. Plates were coated with 1 μg / ml of recombinant his-cynomolgus CD228 in phosphate-buffered saline, blocked with 250 μL of Superblock Blocking Buffer (Thermo). The antibody was titrated across the plate, and bound antibody was detected with HRP-conjugated goat anti-human IgG (Sigma) and developed with TMB substrate (Thermo). OD450 values ​​were read using a plate reader equipped with SoftmaxPro software. The results are shown in Figure 7B.

[0118]

[0138] Humanized antibodies specific for human CD228 were evaluated for binding to recombinant cynomolgus monkey and mouse CD228 by ELISA. Antibodies were titrated on plates coated with 1 μg / mL recombinant human, cynomolgus monkey, and mouse CD228, detected with HRP-conjugated goat anti-human IgG secondary reagent (Thermo), developed with TMB substrate (Thermo), and OD450 values ​​were read using a plate reader equipped with SoftMax Pro software. OD450 values ​​for each experiment are shown in Figures 8A-8C.

[0119]

[0139] These results showed that a subset of antibody clones (OMT30, OMT35, OMT24, OMT8, OMT36, and L235) were cross-reactive with cynomolgus monkey CD228 and showed minimal cross-reactivity with mouse CD228. [Example]

[0120]

[0140] Example 7: Anti-CD228 antibody cross-competition assay

[0141] Fluorochrome-labeled humanized monoclonal antibodies specific for human CD228 were evaluated for binding to HT-1080 and SK-MEL-5 tumor cell lines in competition with unlabeled humanized antibodies to identify potential shared epitopes. HT-1080 and SK-MEL-5 cells were preincubated with 1 μg / mL of unlabeled humanized antibody in staining buffer for 30 minutes at 4°C, washed with staining buffer, and then incubated with 200 ng / mL of the A647-labeled version. Bound fluorochrome-labeled antibody was determined by flow cytometry using an Attune NXT flow cytometer. Results are shown in Figure 9 as the percent mean fluorescence intensity compared to preincubation with a control nonbinding human IgG1 (Sigma). Clones with direct epitopes or steric competition show low staining percentage values. These results indicate that antibody clones 28, 32, and 35 share closely related epitopes, while clones 8, 11, 24, 30, and 36 likely bind to unique CD228 epitopes. [Example]

[0121]

[0142] Example 8: Internalization of antibody clones in CD228+ tumor cell lines

[0143] Humanized antibodies specific for human CD228 were evaluated for relative internalization in the melanoma cell line SK-MEL-5 and lung cancer cell line Calu-1, which express CD228, respectively. Cells were incubated with 2 μg / ml anti-CD228 antibody, washed three times to remove free antibody, and incubated at 37°C in 5% CO2 for the indicated times. At each time point, cells were fixed (BD Cytofix) and stained for bound human IgG1 with a fluorochrome-conjugated anti-human IgG1 antibody (Invitrogen). At the end of the time course, cells were assessed by FACS for the mean fluorescence intensity of surface-bound antibody on an Attune NXT flow cytometer. Internalization results are shown in Figure 10 as a percentage of the MFI relative to the starting MFI at time point 0. These results demonstrate a range of internalization rates among the antibody clones, with antibody clone 35 internalizing at the fastest rate. [Example]

[0122]

[0144] Example 9: Expression and analysis of representative fusion proteins

[0145] Representative antibody-lipocalin mutein fusion proteins were generated by fusing a CD228-specific antibody and a CD137-specific lipocalin mutein, such as the lipocalin mutein of SEQ ID NO: 40, to each other via a linker, such as the unstructured (G4S)3 linker of SEQ ID NO: 13, and simultaneously engaging CD228 and CD137. Two different exemplary CD228-specific antibodies were used. The first CD228-specific antibody had 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 NOs: 58-60) and 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 NOs: 61-63). The second CD228-specific antibody had 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 NOs: 64-66) and 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 NOs: 67-69). Exemplary fusion proteins of SEQ ID NOs: 80 and 76, SEQ ID NOs: 82 and 79, SEQ ID NOs: 75 and 81, and SEQ ID NOs: 78 and 83 were bivalent for CD137 with a CD137-specific lipocalin mutein fused to the C-terminus of each heavy or light chain.

[0123]

[0146] The CD228-specific antibody and all antibody lipocalin mutein fusion proteins described in this example had an engineered IgG4 backbone containing the 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, including any one or more of the mutations F234A, L235A, M428L, N434S, M252Y, S254T, and T256E, may be present in all antibodies described herein. The F234A and L235A mutations may 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 to extend serum half-life (Dall'Acqua et al., J Biol Chem, 2006; Zalevsky et al., Nat Biotechnol, 2010). All antibodies were expressed without a carboxy-terminal lysine to avoid heterogeneity.

[0124]

[0147] Exemplary fusion protein constructs were generated by gene synthesis and cloned into mammalian expression vectors, after which they were transiently expressed in suspension-adapted CHO-K1 cells. [Example]

[0125]

[0148] Example 10: Evaluation of PBMC responses to viral peptides in the presence of tumor cells with or without high CD228 expression

[0149] Assays were performed to assess the ability of the fusion proteins to costimulate innate and adaptive immune cytokines from PBMCs in response to viral peptides in a CD228 target-dependent manner. PBMCs isolated from healthy donors were co-incubated at a 10:1 ratio with engineered CD228+ or wild-type (CD228-) RPMI-7951 tumor cell lines in RPMI containing 10% FCS and presented with viral peptides from CMV, EBV, and influenza viruses (CEF peptides). The bispecific fusion proteins and controls were titrated into the assay, and 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 in 5% CO2. Supernatants were evaluated for T cell (IFN-γ and TNF-α) and myeloid cell (IL-12 and CXCL10) responses using Luminex® multiplex cytokine arrays. Data are presented in Figures 11A-11E as fold changes in cytokines relative to untreated control wells. Samples were pooled from triplicate test article treatments prior to cytokine measurement. These results demonstrate that, compared to antibodies, fusion proteins (e.g., AAF30(HC)) costimulate various innate and adaptive immune cytokines from PBMCs in response to viral peptides in a CD228 target- and dose-dependent manner. [Example]

[0126]

[0150] Example 11: Evaluation of PBMC cellular responses to viral peptides in the presence of CD228+ tumor cells

[0151] To assess the activity of the bispecific fusion proteins in antigen recall assays, cryopreserved PBMCs (Bloodworks Northwest) from healthy donors were thawed in prewarmed RPMI 10% FCS, washed, and labeled with 1.5 mL of 10 nM CFSE in PBS 3% FCS at room temperature. To quench the labeling reaction, cells were washed twice with 12 mL of RPMI 10% FCS. Cells were counted and cultured in RPMI-complete (10% FCS, 1x Glutamax, 1x MEM NEAA, 1x sodium pyruvate, 1x penicillin / streptomycin (Gibco™)) containing the CD228-expressing cell line RPMI-7951. CD228 (ATCC) (engineered to express human CD228) at a 10:1 ratio and plated in a non-adherent 96-well round-bottom plate (Sbio) at 1.5 × 10 5 Cells were distributed at 100 cells / well. CEF peptide was added to a final concentration of 100 ng / mL, and the fusion protein bispecific and control were added in triplicate at equimolar titrations. Assays were incubated for 5 days at 37°C with 5% CO2. Figures 12, 13, and 14 show representative examples of CD8+ T cells, NK cells, and CD8+ T cell / Treg ratios calculated from antigen recall assays in cocultures with CD228-engineered RPMI-7951 cell lines, respectively. Similarly, Figure 15 shows representative examples of NK cell and CD8+ T cell / Treg ratios calculated from antigen recall assays. These results demonstrate that, compared with antibodies, fusion proteins (e.g., AAF30(HC)) costimulate the proliferation / division of CD8+ T cells and NK cells in response to viral peptides in a CD228 target- and dose-dependent manner. [Example]

[0127]

[0152] Example 12: Evaluation of PBMC cytokine responses to viral peptides in the presence of CD228+ tumor cells

[0153] To assess the activity of the bispecific fusion proteins in antigen recall assays, cryopreserved PBMCs (Bloodworks Northwest) from healthy donors were thawed in prewarmed RPMI 10% FCS, washed, and labeled with 1.5 mL of 10 nM CFSE in PBS 3% FCS at room temperature. To quench the labeling reaction, cells were washed twice with 12 mL of RPMI 10% FCS. Cells were counted and cultured in RPMI-complete (10% FCS, 1x Glutamax, 1x MEM NEAA, 1x sodium pyruvate, 1x penicillin / streptomycin (Gibco™)) containing the CD228-expressing cell line RPMI-7951. CD228 (ATCC) (engineered to express human CD228), CALU-1 cells (ATCC), or H3677 cells (Seagen) were mixed at a 10:1 ratio and plated in a non-adherent 96-well round-bottom plate (Sbio) at 1.5 × 10 5 Cells were distributed at 100 cells / well. CEF peptide was added to a final concentration of 100 ng / mL, and the fusion protein bispecific and control were added in triplicate in equimolar titrations. Assays were incubated for 5 days at 37°C with 5% CO2. After assay completion, plates were spun down and supernatants were collected for cytokine assessment. Cytokines were measured using MILLIPLEX® MAP Human CD8+ T Cell Magnetic Bead Panel Premixed 17 Plex and read on a Luminex® MAGPIX® system. Figures 16A-16E show representative mean fold changes in cytokines across three CD228-expressing cell lines. Raw Luminex® data were exported and analyzed in Microsoft Excel. These data highlight the consistent changes in cytotoxic effector molecules and various cytokines upon antigen recall in the presence of the bispecific fusion protein. [Example]

[0128]

[0154] Example 13: Evaluation of T cell activation in the presence of tumor cells with or without high CD228 expression

[0155] Assays were performed to evaluate the requirement for CD228 expression by cancer cells for costimulation of primary T cells with the fusion protein. Primary T cells from healthy donors were incubated in anti-CD3-coated plates (0.25 μg / ml in 50 μL of PBS, overnight) to stimulate the T cell receptor, and CD228-positive Calu-1 cells or CD228-negative SK-BR-3 cells were added to the cultures titrated with the fusion protein. T cells were mixed with tumor cells at a final ratio of 10:1 in RPMI containing 10% FCS. At the end of 3 days of stimulation at 37°C and 5% CO2, supernatants were collected and the cytokine IL-2 was measured. Figures 17A-17B show IL-2 secretion in response to costimulation with the fusion protein and urelumab. These results demonstrate that the fusion protein (e.g., AAF35(HC)) costimulates T cell activation in a CD228 target- and dose-dependent manner. [Example]

[0129]

[0156] Example 14: Evaluation of T cell cytokines produced in co-culture with anti-CD3 scFv engineered CD228-expressing tumor cell lines

[0157] Experiments were conducted to evaluate the effect of the fusion protein on cytokine and soluble 4-1BB (sCD137) production from T cells directly stimulated with CD228-expressing tumor cells. PBMCs from healthy donors were cocultured with CD228+ CALU-1 tumor cells (10:1 in RPMI 10% FCS) engineered to express surface anti-CD3 scFv to induce direct T cell receptor engagement by tumor cells. Titrations of fusion protein or control were added in triplicate to 96-well round-bottom plates. At the end of 3 days of stimulation at 37°C and 5% CO2, supernatants were collected and the soluble cytokines IL-2, IL-13, and sCD137 were measured by Luminex® multiplex array. Samples were pooled from triplicate test article treatments before cytokine measurement. The results are shown in Figures 18A-18C and demonstrate that the fusion protein increases cytokines from direct T cell-tumor cell interaction. [Example]

[0130]

[0158] Example 15: Evaluation of CD8 T cell proliferation in co-culture with anti-CD3 scFv engineered CD228-expressing tumor cell lines

[0159] Experiments were performed to evaluate the effect of fusion protein bispecifics on cytotoxic T cells stimulated directly by CD228-expressing tumor cells. PBMCs from healthy donors were cocultured (10:1) with CFSE-labeled CD228+ CALU-1 tumor cells engineered to express surface anti-CD3 scFv to induce T cell receptor engagement. Titrations of bispecific fusion proteins or controls were added in triplicate to 96-well round-bottom plates. After 72 hours of incubation at 37°C in 5% CO2, plates were washed and stained with live / dead viability dye and antibodies specific for CD3, CD4, and CD8, as well as CD228. Plates were assessed for proliferation of live CD8+ T cells and CD228+ tumor cells using an Attune™ NxT flow cytometer. Figure 19 shows the percentage of CD8+ T cells containing diluted CFSE compared to untreated wells. Figure 20 shows the percent of viable tumor cells remaining compared to untreated wells, reflecting tumor cell killing. These results demonstrate that in the presence of CD228, fusion proteins (e.g., AAF30(HC)) dose-dependently costimulate CD8+ T cell proliferation and tumor cell killing compared to antibodies. [Example]

[0131]

[0160] Example 16: Pharmacokinetics of fusion proteins in cynomolgus monkeys

[0161] The heavy chain fusion protein bispecifics 30HC and 35HC (SEQ ID NOs: 80 and 76, and SEQ ID NOs: 82 and 79, respectively) were compared for differences in pharmacokinetics in cynomolgus monkeys. Animals received a single dose of the fusion bispecific at a dose of 1 mg / kg or 6 mg / kg. As shown in Figure 21, the bispecific fusion proteins exhibited similar pharmacokinetic profiles, with slight differences after day 14, which may be due to anti-drug antibody responses. [Example]

[0132]

[0162] Example 17: Evaluation of in vivo activity of fusion proteins in a humanized xenograft model

[0163] To evaluate the activity of the bispecific fusion protein in vivo, a humanized xenograft model was performed using the CD228+ melanoma cell lines CALU-1 (ATCC) and H3677 (SGEN, Japan). Tumor cells were implanted into immunodeficient NSG mice in 25% Matrigel® (Corning), and tumors grew to an average volume of 100 mm. 3 The tumor growth curves were monitored until a 100% confidence interval (CI) was reached, at which point peripheral blood mononuclear cells (PBMCs) from healthy donors were adoptively transferred via tail vein injection to serve as a source of human T cells. Mice were then administered equimolar amounts of the bispecific fusion protein (10 mg / kg), antibody (non-bispecific antibody, anti-PD-1, and / or anti-4-1BB), or control (8 mg / kg) every 5 days, and tumor growth was monitored. The growth curve for Calu-1 over the study is shown in Figure 22A, and the final tumor volume at the end of the study on day 80 is shown in Figure 22B. The growth curve for H3677 over the study is shown in Figure 22C, and the final tumor volume at the end of the study on day 22 is shown in Figure 22D. Statistics are based on Tukey's analysis. These results demonstrate that fusion proteins (e.g., AAF30(HC)) compared with antibodies reduce the growth of CD228+ tumor cells in vivo.

[0133]

[0164] To evaluate intratumoral PD effects from animals treated with the bispecific fusion in a humanized CD228+ Calu-1 xenograft model, tumors were harvested, processed into single-cell suspensions, and tumor and immune cell populations were stained and analyzed by flow cytometry. Figure 23A shows the ratio of CD8+ T cells to tumor cells, determined by dividing the number of CD8+ T cells by the number of CD228+ non-immune cells. Figure 23B shows the change in the CD8+ / CD4+ T cell ratio. Figure 23C shows the percentage of actively degranulating CD8+ T cells, as determined 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 CD107a-expressing CD8+ T cells (Figure 23C) in resting PBMC (i.e., non-tumor model) samples from the same donor. These data demonstrate that treatment with the bispecific fusion protein enhanced the expansion and activation of cytotoxic CD8+ T cells compared to controls. Statistics are from Tukey's analysis.

[0134]

[0165] To evaluate intratumoral PD efficacy from animals treated with the bispecific fusion in separate humanized CD228+ Calu-1 xenograft models, tumors were harvested, processed into single-cell suspensions, and staining and flow cytometry of tumor and immune cell populations were performed. Figure 24A shows the ratio of CD8+ T cells to CD4+ T cells. Figure 24B shows the change in intracellular TCF1 expression. These data demonstrate that treatment with the bispecific fusion protein targeting 4-1BB promoted the expansion and differentiation of cytotoxic CD8+ T cells, transforming them into cells with stem cell-like properties that exhibited greater antitumor potential, as determined by the expression of the transcription factor TCF1. Statistics provided are the results of Tukey's analysis.

[0135]

[0166] The embodiments illustratively described herein may suitably be practiced in the absence of any element(s), limitation(ies) not specifically disclosed herein. Thus, for example, terms such as "comprising," "including," and "containing" are intended to be interpreted expansively and without limitation. Furthermore, the terms and phrases employed herein are used as terms of description and not of limitation, and the use of such terms and phrases is not intended to exclude equivalents of the illustrated and described features or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed invention. Thus, while the present embodiments have been specifically disclosed in terms of preferred embodiments and optional features, it should be understood that those skilled in the art may resort to equivalents, modifications, and variations thereof, and that 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 mentioned 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. Furthermore, if a definition or use of a term in a reference incorporated herein by reference contradicts or contradicts 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. Each narrower species and subgeneric group falling within the generic disclosure also forms part of the present invention. This includes the generic description of the invention with a provisos or negative limitation removing any subject matter from the genus, regardless of whether the excluded material is specifically described herein. Furthermore, where features are described in terms of a Markush group, one skilled in the art will recognize that the disclosure is thereby also described in terms of any individual member or subgroup of members of the Markush group. Further embodiments will become apparent from the claims that follow.

[0136] VIII. SEQUENCE LISTING

[0167] Table of exemplary sequences:

[0137]

Table 1-1

[0138]

Table 1-2

[0139]

Table 1-3

[0140]

Table 1-4

[0141]

Table 1-5

[0142]

Table 1-6

[0143]

Table 1-7

[0144]

Table 1-8

[0145]

Table 1-9

[0146]

Table 1-10

[0147]

Table 1-11

[0148]

Table 1-12

[0149]

Table 1-13

[0150]

Table 1-14

[0151]

Table 1-15

[0152]

Table 1-16

[0153]

Table 1-17

[0154]

Table 1-18

[0155]

Table 1-19

[0156]

Table 1-20

[0157]

Table 1-21

[0158]

Table 1-22

[0159]

Table 1-23

[0160]

Table 1-24

[0161]

Table 1-25

[0162]

Table 1-26

[0163]

Table 1-27

[0164]

Table 1-28

[0165]

Table 1-29

[0166]

Table 1-30

[0167]

Table 1-31

[0168]

Table 1-32

Claims

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

281. An antibody or antigen-binding fragment thereof comprising:

2. (a) a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO:210, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO:211, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO:212, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO:216, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO:217, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO:218; (a) a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO:213, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO:214, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO:215, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO:219, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO:220, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO:221; (a) a VH comprising 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 comprising (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 (a) a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 253, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 254, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 255; and (d) a VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 259, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 260, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO:

261. The antibody or antigen-binding fragment thereof of claim 1, comprising:

3. (a) a VH comprising 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 comprising (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) a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 213, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 214, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 215; and (d) a VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 219, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 220, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO:

221. The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising:

4. a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 122, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 124; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 142, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 144; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 162, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 164; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 182, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 184; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 202, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 204; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 222, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 224; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 242, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 244; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 262, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 264; or A VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 282, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

284. The antibody or antigen-binding fragment thereof of claim 1, comprising:

5. a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 222, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 224; or A VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 262, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

264. The antibody or antigen-binding fragment thereof according to any one of claims 1, 2 and 4, comprising:

6. A VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 222, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

224. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, comprising:

7. 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 VH comprising the amino acid sequence of SEQ ID NO: 282, and VL comprising the amino acid sequence of SEQ ID NO: 284 The antibody or antigen-binding fragment thereof of claim 1 or 4, comprising:

8. 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 VH comprising the amino acid sequence of SEQ ID NO: 262, and VL comprising the amino acid sequence of SEQ ID NO: 264 The antibody or antigen-binding fragment thereof of any one of claims 1, 2, 4, 5 and 7, comprising:

9. VH comprising the amino acid sequence of SEQ ID NO: 222, and VL comprising the amino acid sequence of SEQ ID NO: 224 The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, comprising:

10. a heavy chain (HC) comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 126, and a light chain (LC) comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 128; an HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 146, and an LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 148; an HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 166, and an LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 168; an HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 186, and an LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 188; an HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 206, and an LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 208; an HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 226, and an LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 228; an HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:246, and an LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:248; an HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 266, and an LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 268; or HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 286, and LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

288. The antibody or antigen-binding fragment thereof of any one of claims 1, 4 and 7, comprising:

11. an HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 226, and an LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 228; or HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 266, and LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

268.

11. The antibody or antigen-binding fragment thereof of claim 1, comprising:

12. HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 226, and LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

228.

12. The antibody or antigen-binding fragment thereof of claim 1 , comprising:

13. an HC comprising the amino acid sequence of SEQ ID NO: 126, and an LC comprising the amino acid sequence of SEQ ID NO: 128; an HC comprising the amino acid sequence of SEQ ID NO: 146, and an LC comprising the amino acid sequence of SEQ ID NO: 148; an HC comprising the amino acid sequence of SEQ ID NO: 166, and an LC comprising the amino acid sequence of SEQ ID NO: 168; an HC comprising the amino acid sequence of SEQ ID NO: 186, and an LC comprising the amino acid sequence of SEQ ID NO: 188; an HC comprising the amino acid sequence of SEQ ID NO: 206, and an LC comprising the amino acid sequence of SEQ ID NO: 208; an HC comprising the amino acid sequence of SEQ ID NO: 226, and an LC comprising the amino acid sequence of SEQ ID NO: 228; an HC comprising the amino acid sequence of SEQ ID NO: 246, and an LC comprising the amino acid sequence of SEQ ID NO: 248; an HC comprising the amino acid sequence of SEQ ID NO: 266, and an LC comprising the amino acid sequence of SEQ ID NO: 268; or HC comprising the amino acid sequence of SEQ ID NO: 286, and LC comprising the amino acid sequence of SEQ ID NO: 288 The antibody or antigen-binding fragment thereof of any one of claims 1, 4, 7 and 10, comprising:

14. an HC comprising the amino acid sequence of SEQ ID NO: 226, and an LC comprising the amino acid sequence of SEQ ID NO: 228; or HC comprising the amino acid sequence of SEQ ID NO: 266, and LC comprising the amino acid sequence of SEQ ID NO: 268 14. The antibody or antigen-binding fragment thereof of any one of claims 1, 2, 4, 5, 7, 8, 10, 11 and 13, comprising:

15. HC comprising the amino acid sequence of SEQ ID NO: 226, and LC comprising the amino acid sequence of SEQ ID NO:

228.

15. The antibody or antigen-binding fragment thereof of any one of claims 1 to 14, comprising:

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. 17. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a humanized antibody or a chimeric antibody.

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

19. K<175 nM Ｄ 19. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, which binds to CD228 at a value of 1:

1.

20. An antibody or antigen-binding fragment thereof described in any one of claims 1 to 19, which binds to cynomolgus monkey CD228.

21. An antibody or antigen-binding fragment thereof described in any one of claims 1 to 20, which does not bind to mouse CD228 or binds to mouse CD228 with an affinity that is 100-fold or less reduced compared to human CD228.

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

23. An antibody or antigen-binding fragment thereof that binds to CD228 and competes for binding to CD228 with an antibody or antigen-binding fragment thereof described in any one of claims 1 to 22.

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

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

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

27. K<175 nM Ｄ 27. The antibody or antigen-binding fragment thereof of any one of claims 23 to 26, which binds to CD228 at a value of 1:

1.

28. An antibody or antigen-binding fragment thereof described in any one of claims 23 to 27, which binds to cynomolgus monkey CD228.

29. An antibody or antigen-binding fragment thereof according to any one of claims 23 to 28, which does not bind to mouse CD228 or binds to mouse CD228 with an affinity that is 100-fold or less reduced compared to human CD228.

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

31. 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 VH comprising the amino acid sequence of SEQ ID NO: 262, and VL comprising the amino acid sequence of SEQ ID NO: 264 The antibody or antigen-binding fragment thereof of any one of claims 23 to 30, which competes for binding to CD228 with an antibody or antigen-binding fragment thereof comprising:

32. 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 VH comprising the amino acid sequence of SEQ ID NO: 242, and VL comprising the amino acid sequence of SEQ ID NO: 244 32. The antibody or antigen-binding fragment thereof of claim 31 , which competes for binding to CD228 with an antibody or antigen-binding fragment thereof comprising:

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

35. 35. The isolated nucleic acid molecule of claim 34, operably linked to a regulatory sequence to allow expression of the nucleic acid molecule.

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

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

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

39. 39. A host cell according to claim 37 or claim 38, which is a prokaryotic or eukaryotic cell.

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

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

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

43. 34. An immunoconjugate comprising an antibody or antigen-binding fragment thereof described in any one of claims 1 to 33 and a cytotoxic agent.

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

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

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

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

Images