Anti-CD228 antibodies and antibody-drug conjugates

Abstract

Novel anti-CD228 antibodies and antibody drug conjugates, and methods of using such anti-CD228 antibodies and antibody drug conjugates to treat cancer, are provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application Nos. 62 / 801,590, filed February 5, 2019, 62 / 824,923, filed March 27, 2019, 62 / 879,660, filed July 29, 2019, 62 / 882,016, filed August 2, 2019, and 62 / 934,424, filed November 12, 2019, the contents of which are incorporated herein by reference in their entireties.

[0002] Submission of sequence listing as an ASCII text file The contents of the following submission, as an ASCII text file, are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 761682001340SEQLIST.TXT, Date Recorded: January 21, 2020, Size: 28KB).

[0003] The present invention relates to novel anti-CD228 antibodies and antibody-drug conjugates, and methods of treating cancer using such anti-CD228 antibodies and antibody-drug conjugates. [Background technology]

[0004] CD228, also known as melanotransferrin, MELTF, p97, and MF12, is a glycosylphosphatidylinositol-linked glycoprotein that was first identified as a 97-kDa cell surface marker for malignant melanoma cells. CD228 is overexpressed in the majority of clinical melanoma isolates and has also been observed in many human carcinomas. CD228 has been shown to be expressed in a variety of cancers. CD228 belongs to the transferrin family of iron-binding proteins.

[0005] 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 is effective for early-stage melanoma, it may not be a treatment option for disease that has metastasized to distant organs. Melanoma often spreads to the lymph nodes in the area before spreading to other sites. Attempts to improve survival by surgically removing lymph nodes have been associated with numerous complications but have not shown any benefit in overall survival. Immunotherapy, chemotherapy, and radiation therapy have all been used but are often incurable, especially for late-stage melanoma. When distant metastasis is present, the cancer is generally considered incurable. The 5-year survival rate for stage IV melanoma is 15-20%. Therefore, improved melanoma treatment is needed.

[0006] All references cited herein, including patent applications, patent publications, and scientific literature, are hereby incorporated by reference in their entirety as if each individual reference was specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0007] Provided herein is an isolated anti-CD228 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 and the light chain variable region comprises: (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6 In some embodiments, the antibody is humanized.

[0008] Also provided herein is a humanized anti-CD228 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO:7, with the proviso that position H27 is occupied by D, position H30 is occupied by T, position H47 is occupied by Y, position H71 is occupied by R, and position H78 is occupied by Y; and a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO:8, with the proviso that position L2 is occupied by F, position L36 is occupied by Y, and position L46 is occupied by L. In some embodiments, position L28 is occupied by D.

[0009] Also provided herein is a humanized anti-CD228 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the three Kabat CDRs of SEQ ID NO: 7, in which position H27 is occupied by D, position H30 is occupied by T, position H47 is occupied by Y, position H71 is occupied by R, and position H78 is occupied by Y, and a light chain variable region comprising the three Kabat CDRs of SEQ ID NO: 8, in which position L2 is occupied by F, position L36 is occupied by Y, and position L46 is occupied by L.

[0010] In some of the embodiments herein, the heavy chain variable region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 8. In some of the embodiments herein, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8. In some of the embodiments herein, the antibody or antigen-binding fragment is an antigen-binding fragment. In some of the embodiments herein, the antibody or antigen-binding fragment is a full-length antibody.

[0011] Also provided herein are antibody-drug conjugates comprising the antibody or antigen-binding fragment provided herein conjugated to a cytotoxic or cytostatic drug. In some embodiments, the linker is an MDpr-PEG(12)-gluc linker. In some of the embodiments herein, the cytotoxic or cytostatic drug is monomethyl auristatin. In some embodiments, the monomethyl auristatin is monomethyl auristatin E (MMAE). In some of the embodiments herein, the linker is attached to monomethyl auristatin E to form a compound having the structure:

[0012] [ka] where Ab is antibody hL49, n is 12, and R PR is hydrogen and R 21 is CH3, and p is a number from 1 to 16. In some of the embodiments herein, the antibody drug conjugate is hL49-MDpr-PEG(12)-gluc-MMAE.

[0013] Also provided herein are nucleic acids encoding the heavy chain variable regions and / or light chain variable regions of the antibodies described herein. Also provided herein are vectors comprising the nucleic acids provided herein. Also provided herein are host cells comprising the nucleic acids provided herein.

[0014] Also provided herein is a method of producing an anti-CD228 antibody or antigen-binding fragment thereof provided herein, comprising culturing a host cell provided herein under conditions suitable for production of the anti-CD228 antibody or antigen-binding fragment thereof.

[0015] Also provided herein are methods for producing an anti-CD228 antibody drug conjugate provided herein, the method comprising culturing a host cell provided herein under conditions suitable for production of the anti-CD228 antibody, isolating the anti-CD228 antibody produced from the host cell, and conjugating the anti-CD228 antibody to a cytotoxic or cytostatic drug.

[0016] Also provided herein are methods of treating cancer in a subject, comprising administering to the subject an antibody or antigen-binding fragment provided herein, or an antibody-drug conjugate provided herein. In some embodiments, the cancer is selected from the group consisting of melanoma, pancreatic cancer, mesothelioma, colorectal cancer, lung cancer, thyroid cancer, breast cancer, cholangiocarcinoma, esophageal cancer, and head and neck cancer. In some embodiments, the subject is human.

[0017] Also provided herein are kits that include (a) an antibody or antigen-binding fragment provided herein, or an antibody drug conjugate provided herein, and (b) instructions for using the antibody or antigen-binding fragment, or antibody drug conjugate, according to the methods provided herein.

[0018] Also provided herein are pharmaceutical compositions comprising an antibody or antigen-binding fragment provided herein, or an antibody-drug conjugate provided herein, and one or more agents selected from the group consisting of physiologically acceptable carriers, diluents, excipients, and adjuvants.

[0019] The patent or application file will contain at least one drawing executed in color. Copies of this patent or patent application publication and the color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0020] [Figure 1]FIG. 1 shows analysis of CD228 protein expression by IHC in melanoma cancer patient samples. [Figure 2] FIG. 1 shows analysis of CD228 protein expression by IHC in mesothelioma cancer patient samples. [Figure 3] FIG. 1 shows analysis of CD228 protein expression by IHC in colorectal cancer patient samples. [Figure 4] Figure 1 shows analysis of CD228 protein expression by IHC in breast cancer patient samples (top panel shows analysis of CD228 protein expression by IHC in triple-negative breast cancer patient samples; bottom panel shows analysis of CD228 protein expression by IHC in Her2-HR+ breast cancer patient samples). [Figure 5] FIG. 1 shows analysis of CD228 protein expression by IHC in pancreatic cancer patient samples. [Figure 6] 1 shows analysis of CD228 protein expression by IHC in non-small cell lung cancer patient samples. The top panel shows analysis of CD228 protein expression by IHC in squamous cell NSCLC cancer patient samples. The bottom panel shows analysis of CD228 protein expression by IHC in adenocarcinoma NSCLC cancer patient samples. [Figure 7] FIG. 1 shows a comparison of the percentage of patient samples positive for CD228 expression as determined by IHC and by RNA levels as reported by The Cancer Genome Atlas for various tumor types. [Figure 8] FIG. 1 shows an alignment of the heavy chain variable region amino acid sequence of the parent murine anti-CD228 monoclonal antibody (referred to as Mu L49 vH (SEQ ID NO: 21)) with the human acceptor sequence (referred to as Hu IGHV4-59 / HJ4 (SEQ ID NO: 23)) and humanized versions of the L49 antibody (referred to as hvHA (SEQ ID NO: 7), hvHB (SEQ ID NO: 24), and hvHC (SEQ ID NO: 25)). CDR positions are designated using both the Kabat and IMGT numbering schemes. [Figure 9]FIG. 1 shows an alignment of the heavy chain variable region amino acid sequences of the humanized version of the L49 antibody (designated hvHA (SEQ ID NO: 7), hvHB (SEQ ID NO: 24), and hvHC (SEQ ID NO: 25)). CDR positions are designated using both the Kabat and IMGT numbering schemes. [Figure 10] Figure 1 shows an alignment of the light chain variable region amino acid sequence of the parent murine anti-CD228 monoclonal antibody (referred to as Mu L49 vL (SEQ ID NO: 31)) with the human acceptor sequence (referred to as Hu IGKV2-30 / KJ2 (SEQ ID NO: 32)) and humanized versions of the L49 antibody (referred to as hvLA (SEQ ID NO: 33), hvLB (SEQ ID NO: 34), and hvLC (SEQ ID NO: 35)). CDR positions are designated using both the Kabat and IMGT numbering schemes. [Figure 11]

[0023] Figure 1 shows an alignment of the light chain variable region amino acid sequences of the humanized versions of the L49 antibody (designated hvLA (SEQ ID NO: 33), hvLB (SEQ ID NO: 34), and hvLC (SEQ ID NO: 35)). CDR positions are designated using both the Kabat and IMGT numbering schemes. [Figure 12] 12A-12F show the results of competitive binding studies of recombinant humanized anti-CD228 antibodies, the parental murine antibody (designated mL49), and the chimeric antibody (cL49ec). [Figure 13] FIG. 1 shows the results of a saturation binding study on recombinant humanized anti-CD228 antibodies. [Figure 14] Figures 14A-14C show the percentage of viable cells over time in A2058, A375, and Colo-853 cell lines treated with hL49-MC-val-cit-PAB-MMAE (4), hL49-MP-gluc-MMAE (4), and hL49-MP-gluc-MMAE (8). [Figure 15]FIG. 1 shows A2058 tumor volume over time for untreated mice and mice treated with 3 mg / kg of hL49-HALC, hL49-HALC-Auristatin T (8), hL49-HALC-Lipophilic MMAF (8), hL49-HALC-Tublysin M (8), and hL49-HALC-MDpr-PEG(12)-gluc-MMAE (8). [Figure 16] FIG. 1 shows A2058 tumor volume over time for untreated mice and mice treated with 6 mg / kg IgG-MDpr-gluc-MMAE (2), 6 mg / kg hL49ec-MDpr-gluc-MMAE (2), 3 mg / kg hL49ec-MDpr-gluc-MMAE (2), 1 mg / kg hL49ec-MDpr-gluc-MMAE (2), 3 mg / kg IgG-MDpr-gluc-MMAE (4), 3 mg / kg hL49-MDpr-gluc-MMAE (4), and 3 mg / kg hL49-MDpr-gluc-MMAE (8). [Figure 17] FIG. 1 shows A2058 tumor volume over time for untreated mice and mice treated with 1 mg / kg hL49-MC-val-cit-PAB-MMAE (4), 3 mg / kg hL49-MC-val-cit-PAB-MMAE (4), 1 mg / kg hL49-MDpr-gluc-MMAE (8), and 3 mg / kg hL49-MDpr-gluc-MMAE (8). [Figure 18] FIG. 1 shows Colo-853 tumor volume over time for untreated mice and mice treated with 1 mg / kg hL49-MC-val-cit-PAB-MMAE (4), 3 mg / kg hL49-MC-val-cit-PAB-MMAE (4), 1 mg / kg hL49-MDpr-gluc-MMAE (8), and 3 mg / kg hL49-MDpr-gluc-MMAE (8). [Figure 19]FIG. 1 shows A2058 tumor volume over time for untreated mice and mice treated with 3 mg / kg IgG-MDpr-PEG(12)-gluc-MMAE(8), 3 mg / kg IgG-Tubulysin M(8), 1 mg / kg or 3 mg / kg hL49-Tubulysin M(8), or 1 mg / kg or 3 mg / kg hL49-MDpr-PEG(12)-gluc-MMAE(8). [Figure 20] FIG. 1 shows SK-MEL-5 tumor volume over time for untreated mice and mice treated with 3 mg / kg IgG-MDpr-PEG(12)-gluc-MMAE(8), 3 mg / kg IgG-Tubulysin M(8), 0.3 mg / kg, 1 mg / kg, or 3 mg / kg hL49-Tubulysin M(8), or 0.3 mg / kg, 1 mg / kg, or 3 mg / kg hL49-MDpr-PEG(12)-gluc-MMAE(8). [Figure 21] FIG. 1 shows IGR-37 tumor volume over time for untreated mice and mice treated with 1 mg / kg or 3 mg / kg of hL49-Tubulysin M(8), or 1 mg / kg or 3 mg / kg of hL49-MDpr-PEG(12)-gluc-MMAE(8). [Figure 22] FIG. 1 shows Colo-853 tumor volume over time for untreated mice and mice treated with 0.3, 1 mg / kg, or 3 mg / kg of hL49-MDpr-PEG(12)-gluc-MMAE(8), or 3 mg / kg of IgG-MDpr-PEG(12)-gluc-MMAE(8). [Figure 23] FIG. 1 shows LU0697 squamous NSCL PDX model tumor volume over time for untreated mice and mice treated with 1 mg / kg or 3 mg / kg hL49-MDpr-PEG(12)-gluc-MMAE(8), or 3 mg / kg IgG-MDpr-PEG(12)-gluc-MMAE(8). [Figure 24]FIG. 1 shows LU0697 adenocarcinoma NSCL PDX model tumor volume over time for untreated mice and mice treated with 1 mg / kg or 3 mg / kg hL49-MDpr-PEG(12)-gluc-MMAE(8), or 3 mg / kg IgG-MDpr-PEG(12)-gluc-MMAE(8). [Figure 25] FIG. 1 shows MDA-MB-231 TNBC tumor volume over time for untreated mice and mice treated with 0.5 mg / kg or 1 mg / kg hL49-MDpr-PEG(12)-gluc-MMAE(8), or 0.5 mg / kg or 1 mg / kg IgG-MDpr-PEG(12)-gluc-MMAE(8). [Figure 26] FIG. 1 shows HPAF-II tumor volume over time for untreated mice and mice treated with 3 mg / kg IgG hL49-MDpr-PEG(12)-gluc-MMAE(8) or 0.3 mg / kg, 1 mg / kg, or 3 mg / kg hL49-MDpr-PEG(12)-gluc-MMAE(8). [Figure 27] FIG. 1 shows the percent change in tumor volume in response to treatment with hL49-MDpr-PEG(12)-gluc-MMAE(8) in 22 different mouse PDX models of triple-negative breast cancer. [Figure 28] Figures 28A-28B show the % specific lysis (ADCC activity) of hL49 and another CD228 antibody, cL235, alone or conjugated to MDpr-PEG(12)-gluc-MMAE for two patients. [Figure 29] Figure 29A shows the plasma concentration of ADC over time in nude mice, and Figure 29B shows the plasma concentration of ADC over time in rats. [Figure 30] Figure 30A shows A2058 tumor volume over time for untreated mice and mice treated with various CD228 antibodies, and Figure 30B shows the percentage of animals with tumors that increased less than four-fold over time for each treatment condition. [Figure 31]31A-31B show the rate of conjugate cleavage over time in A375 and Colo-853 cells. [Figure 32] FIG. 1 shows that CD228 is recruited to the cell surface over time by comparing the rate of conjugate cleavage over time in cells treated with fluorescently labeled hL49 antibody using either a pulsed or continuous treatment of the labeled antibody. [Figure 33] 33A-33B show the mean fluorescence intensity per cell over time in cells incubated with fluorescently labeled hL49 antibody in the presence or absence of cycloheximide, an inhibitor of protein synthesis. [Figure 34] 34A-34F show the binding of various anti-CD228 antibodies to CD228 at pH values ​​ranging from 4 to 7.4. [Figure 35] 35A-35B show the ability of antibodies with similar binding affinities to internalize and catabolize drugs. [Figure 36A] FIG. 1 shows that a single dose of hL49-MDpr-PEG(12)-gluc-MMAE(8) has antitumor activity in patient-derived tumor (PDX) models. [Figure 36B] FIG. 1 shows that a single dose of hL49-MDpr-PEG(12)-gluc-MMAE(8) has antitumor activity in patient-derived tumor (PDX) models. DETAILED DESCRIPTION OF THE INVENTION

[0021] I. Definition In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless expressly stated otherwise herein, each of the following terms shall have the following meaning. Additional definitions are set forth throughout this application.

[0022] The term "and / or," as used herein, should be interpreted as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or" used in a phrase herein, for example, "A and / or B," is intended to include "A and B," "A or B," "A" (alone) and "B" (alone). Similarly, the term "and / or" used in a phrase, for example, "A, B, and / or C," is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0023] It will be understood that aspects and embodiments of the invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0025] Units, prefixes, and symbols are denoted in the format accepted by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of this disclosure, but may be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0026] The terms "CD228," "p97," "melanotransferrin," "MELTF," and "MF12" are used interchangeably herein and, unless otherwise specified, include any variants, isoforms, and species homologs of human CD228 that are generally expressed by or on cells transfected with the CD228 gene.

[0027] The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all interconnected by disulfide bonds. The structure of immunoglobulins is well characterized. See, e.g., Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)). Briefly, each heavy chain typically comprises a heavy chain variable region (referred to herein as V H or VH) and a heavy chain constant region (C H The heavy chain constant region is typically composed of C H 1. C H 2, and C H The heavy chains are generally interconnected via disulfide bonds in the so-called "hinge region." Each light chain typically contains a light chain variable region (referred to herein as V L or VL) and the light chain constant region (C L The light chain constant region is typically composed of one domain, C LThe CL may be of the κ (kappa) or λ (lambda) isotype. The terms "constant domain" and "constant region" are used interchangeably herein. Immunoglobulins can be derived from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the antibody class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region genes.

[0028] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable regions of the heavy and light chains (V, respectively) of a native antibody H and V L ) are further subdivided into regions of hypervariability (or hypervariable regions, which may be in the form of sequence- and / or structurally-defined loops), also called complementarity-determining regions (CDRs), which may be interspersed with more conserved regions called framework regions (FRs). The terms "complementarity-determining region" and "CDR," which are synonymous with "hypervariable region" or "HVR," are known in the art to refer to non-contiguous sequences of amino acids within an antibody variable region that confer antigen specificity and / or binding affinity. Generally, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, CDR-H3), and each light chain variable region has three CDRs (CDR-L1, CDR-L2, CDR-L3). The terms "framework region" and "FR" are known in the art to refer to the non-CDR portions of heavy and light chain variable regions. Generally, each full-length heavy chain variable region has four FRs (FR-H1, FR-H2, FR-H3, and FR-H4), and each full-length light chain variable region has four FRs (FR-L1, FR-L2, FR-L3, and FR-L4). H and V LWithin the FR, the three CDRs and four FRs are typically arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mot. Biol., 195, 901-917 (1987)).

[0029] The term "antibody" (Ab), in the context of the present invention, refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either, that has the ability to specifically bind to an antigen under typical physiological conditions with a half-life of a significant period, e.g., at least about 30 minutes, at least about 45 minutes, at least about 1 hour (h), at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours (h), about 24 hours or more, about 48 hours or more, about 3 days, 4 days, 5 days, 6 days, 7 days or more, etc., or any other relevant, functionally defined period (e.g., a period sufficient to induce, promote, enhance, and / or modulate a physiological response associated with binding of the antibody to the antigen and / or a period sufficient for the antibody to recruit effector activity). The variable regions of the heavy and light chains of an immunoglobulin molecule contain the binding domains that interact with the antigen. The constant regions of antibodies (Abs) can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system, such as C1q, the first component of the classical pathway of complement activation. Antibodies can also be bispecific antibodies, diabodies, multispecific antibodies, or similar molecules.

[0030] The term "monoclonal antibody," as used herein, refers to a preparation of recombinantly produced antibody molecules with a single primary amino acid sequence. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to an antibody displaying a single binding specificity which has variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced by hybridomas comprising B cells obtained from a transgenic or transchromosomal non-human animal, e.g., a transgenic mouse, whose genome comprises human heavy chain and light chain transgenes fused to an immortalized cell.

[0031] An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to CD228 is substantially free of antibodies that specifically bind to antigens other than CD228). However, an isolated antibody that specifically binds to CD228 may have cross-reactivity to other antigens, such as CD228 molecules from different species. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals. In one embodiment, an isolated antibody comprises an antibody conjugate linked to another agent (e.g., a small molecule drug). In some embodiments, an isolated anti-CD228 antibody comprises a conjugate of an anti-CD228 antibody with a small molecule drug (e.g., MMAE or MMAF).

[0032] A "human antibody" (HuMAb) refers to an antibody having a variable region in which both the FRs and CDRs 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. The terms "human antibody" and "fully human antibody" are used interchangeably.

[0033] The term "humanized antibody," as used herein, refers to a genetically engineered non-human antibody comprising a human antibody constant domain and a non-human variable domain that has been modified to contain a high level of sequence homology to the human variable domain. This can be achieved by grafting the six non-human antibody complementarity-determining regions (CDRs), which together form the antigen-binding site, onto homologous human acceptor framework regions (FRs) (see WO 92 / 22653 and EP 0629240). Substitution of framework residues from the parent antibody (i.e., non-human antibody) into human framework regions (back mutations) may be required to fully reconstitute the binding affinity and specificity of the parent antibody. Structural homology modeling can be useful in identifying amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody can comprise primarily human framework regions, including non-human CDR sequences, optionally one or more amino acid back mutations to non-human amino acid sequences, and a fully human constant region. Optionally, further amino acid modifications, not necessarily back mutations, can be applied to obtain humanized antibodies with favorable properties, such as affinity and biochemical properties.

[0034] The term "chimeric antibody," as used herein, refers to an antibody whose variable region originates from a non-human species (e.g., rodent) and whose constant region originates from a different species, e.g., human. Chimeric antibodies can be produced by antibody engineering. "Antibody engineering" is a term commonly used for the modification of different types of antibodies and is a method well known to those skilled in the art. In particular, chimeric antibodies can be produced using standard DNA techniques as described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Chapter 15. In this manner, chimeric antibodies can be genetically or enzymatically engineered recombinant antibodies. Producing chimeric antibodies is within the knowledge of those skilled in the art; therefore, chimeric antibodies of the present invention can be produced by methods other than those described herein. To reduce antibody immunogenicity, chimeric monoclonal antibodies have been developed for therapeutic applications. They typically contain non-human (e.g., murine) variable regions specific for the antigen of interest and human constant antibody heavy and light chain domains. The term "variable region" or "variable domain" when used in the context of a chimeric antibody refers to the region comprising the CDR and framework regions of both the heavy and light chains of an immunoglobulin.

[0035] An "anti-antigen antibody" refers to an antibody that binds to an antigen. For example, an anti-CD228 antibody is an antibody that binds to the antigen CD228.

[0036] An "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to the antigen bound by the whole antibody. Examples of antibody fragments (e.g., antigen-binding fragments) include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, the name reflecting its ability to readily crystallize. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0037] "Percent sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. For example, the percent sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (alternatively, it can be expressed as a given amino acid sequence A having or containing a certain percent sequence identity to, with, or relative to a given amino acid sequence B) can be expressed as follows: 100 x fraction X / Y where X is the number of amino acid residues scored as perfect identity to the sequences in the alignment of A and B by the program, and Y is the total number of amino acid residues in B. It is understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % sequence identity of A to B will not be equal to the % sequence identity of B to A.

[0038] As used herein, the terms "binding," "bind," or "specifically bind" in the context of antibody binding to a predetermined antigen typically refer to a binding activity of approximately 10, as determined, for example, by BioLayer Interferometry (BLI) technology on an Octet HTX instrument using the antibody as the ligand and the antigen as the analyte. -6 M or less, e.g. 10 -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 M or less, about 10 -10 M or less, or even about 10 -11 M or less, where the antibody binds with an affinity corresponding to a KD of binding to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). D at least 10 times lower, such as at least 100 times lower, such as at least 1,000 times lower, such as at least 10,000 times lower, such as at least 100,000 times lower, D binds to a given antigen with an affinity corresponding to the K D The lower the amount of antibody D Therefore, the K D If the K of binding to the antigen is very low, D is the K for nonspecific antigen binding D The amount may be at least 10,000 times lower (ie, the antibody is highly specific).

[0039] The term “K D " (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. As used herein, affinity and K Dis an inverse correlation, i.e., higher affinity corresponds to lower K D A lower affinity is intended to refer to a higher K D is intended to refer to.

[0040] The term "ADC" refers to an antibody drug conjugate, which in the context of the present invention refers to an anti-CD228 antibody linked to a drug moiety (e.g., MMAE or MMAF), as described in this application.

[0041] The abbreviations "vc" and "val-cit" refer to the dipeptide valine-citrulline.

[0042] The abbreviation "PAB" stands for self-immolative spacer: [ka]

[0043] The abbreviation "MC" stands for the stretcher maleimidocaproyl: [ka]

[0044] The abbreviation "MP" stands for the stretcher maleimidopropionyl: [ka]

[0045] "Cancer" refers to a broad group of diverse diseases characterized by the uncontrolled growth of abnormal cells in the body. "Cancer" or "cancerous tissue" can include tumors. Uncontrolled cell division and growth leads to the formation of malignant tumors, which can invade neighboring tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream. After metastasis, the distant tumor is said to "originate" from the pre-metastatic tumor.

[0046] The term "antibody-dependent cellular cytotoxicity" or ADCC is a mechanism for inducing cell death that depends on the interaction of antibody-coated target cells with immune cells (also called effector cells) that possess lytic activity. Such effector cells include natural killer cells, monocytes / macrophages, and neutrophils. Effector cells bind via their antigen-binding site to the Fc effector domain(s) of Ig bound to the target cell. Killing of the antibody-coated target cell occurs as a result of effector cell activity.

[0047] The term "antibody-dependent cellular phagocytosis" or ADCP refers to the process by which antibody-coated cells are wholly or partially internalized by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the Fc effector domain(s) of Ig.

[0048] The term "complement-dependent cytotoxicity" or CDC refers to a mechanism for inducing cell death in which the Fc effector domain(s) of a target-bound antibody activate a series of enzymatic reactions that result in the formation of pores in the target cell membrane. Typically, antigen-antibody complexes, e.g., on antibody-coated target cells, bind and activate the complement component C1q, which in turn activates the complement cascade leading to target cell death. Complement activation can also result in the deposition of complement components on the target cell surface, which promotes ADCC through binding of complement receptors (e.g., CR3) on leukocytes.

[0049] "Cytostatic effect" refers to the inhibition of cell proliferation. "Cytostatic drug" refers to an agent that has a cytostatic effect on cells, thereby inhibiting the growth and / or expansion of a specific subset of cells. The cytostatic drug may be conjugated to an antibody or administered in combination with an antibody.

[0050] "Treatment" or "therapy" of a subject means any type of intervention or process performed on a subject or administration of an active agent to a subject for the purpose of reversing, alleviating, ameliorating, inhibiting, delaying, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, conditions, or biochemical manifestations associated with a disease. In some embodiments, the disease is cancer.

[0051] A "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents (e.g., mice, rats, and guinea pigs). In some embodiments, the subject is a human. The terms "subject" and "patient" and "individual" are used interchangeably herein.

[0052] An "effective amount" or "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent is any amount of drug that, when used alone or in combination with another therapeutic agent, protects a subject from developing a disease or promotes regression of the disease as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of dysfunction or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to the skilled practitioner, for example, by assaying the activity of the agent in human subjects in clinical trials, in animal model systems that are predictive of efficacy in humans, or in in vitro assays.

[0053] By way of example with respect to tumor treatment, a therapeutically effective amount of an anti-cancer agent inhibits cell proliferation or tumor growth by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% in treated subjects (e.g., one or more treated subjects) compared to untreated subjects (e.g., one or more untreated subjects). In some embodiments, a therapeutically effective amount of an anti-cancer agent inhibits cell proliferation or tumor growth by 100% in treated subjects (e.g., one or more treated subjects) relative to untreated subjects (e.g., one or more untreated subjects).

[0054] In other embodiments of the present disclosure, tumor regression can be observed and continued for a period of at least about 20 days, at least about 30 days, at least about 40 days, at least about 50 days, or at least about 60 days.

[0055] A therapeutically effective amount of a drug (e.g., an anti-CD228 antibody drug conjugate) includes a "prophylactically effective amount," which is any amount of drug that, when administered alone or in combination with an anti-cancer agent to a subject at risk of developing cancer (e.g., a subject with a pre-malignant condition) or a subject at risk of suffering from cancer recurrence, inhibits the development or recurrence of cancer. In some embodiments, a prophylactically effective amount completely prevents the development or recurrence of cancer. "Inhibiting" the development or recurrence of cancer means either reducing the likelihood of cancer development or recurrence, or completely preventing the development or recurrence of cancer.

[0056] As used herein, a "subtherapeutic dose" means a dose of a therapeutic compound (e.g., an anti-CD228 antibody drug conjugate) that is lower than the usual or typical dose when the therapeutic compound is administered alone to treat a proliferative disease (e.g., cancer).

[0057] "Immune-related response pattern" refers to a clinical response pattern often observed in cancer patients treated with immunotherapeutic agents that produce antitumor effects by inducing cancer-specific immune responses or by modifying natural immune processes. This response pattern is characterized by an initial increase in tumor burden or the appearance of new lesions, which in conventional chemotherapeutic agent evaluations would be classified as disease progression and would be synonymous with drug failure, followed by a beneficial therapeutic effect. Therefore, proper evaluation of immunotherapeutic agents may require longitudinal monitoring of the effects of these agents on the target disease.

[0058] By way of example, an "anti-cancer agent" promotes the regression of cancer in a subject. In some embodiments, a therapeutically effective amount of a drug promotes the regression of cancer to the point where the cancer disappears. "Promoting cancer regression" means that administering an effective amount of a drug, alone or in combination with an anti-cancer agent, results in a decrease in tumor growth or size, tumor necrosis, a decrease in the severity of at least one disease symptom, an increase in the frequency and duration of disease-free periods, or prevention of dysfunction or disability due to disease affliction. Additionally, the terms "effective" and "efficacy," with respect to treatment, include both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a drug to promote the regression of cancer in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ, and / or organismal level resulting from the administration of a drug.

[0059] "Dured response" refers to a sustained effect on the reduction of tumor growth after treatment has stopped. For example, the tumor size may remain the same or smaller compared to the size at the beginning of the administration period. In some embodiments, the sustained response has a duration at least as long as the duration of treatment, or at least 1.5, 2.0, 2.5, or 3.0 times longer than the duration of treatment.

[0060] As used herein, "complete response" or "CR" means the disappearance of all target lesions; "partial response" or "PR" means at least a 30% reduction in the sum of the longest diameters (SLD) of target lesions, based on baseline SLD; and "stable disease" or "SD" means neither a sufficient shrinkage of target lesions to qualify for PR nor a sufficient increase, based on the smallest SLD since the start of treatment, to qualify for PD.

[0061] As used herein, "progression-free survival" or "PFS" means the length of time during and after treatment during which the disease being treated (e.g., cancer) does not worsen. Progression-free survival can include periods during which a patient experiences a complete or partial response, as well as periods during which a patient experiences stable disease.

[0062] As used herein, "overall response rate" or "ORR" means the sum of the complete response (CR) rate and the partial response (PR) rate.

[0063] As used herein, "overall survival" or "OS" refers to the percentage of individuals in a group who are likely to be alive after a specified period of time.

[0064] The phrase "pharmaceutically acceptable" indicates that a substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or with the mammal being treated therewith.

[0065] The phrase "pharmaceutically acceptable salt," as used herein, means a pharmaceutically acceptable organic or inorganic salt of a compound of the present invention. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate "mesylate", ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., 4,4'-methylene-bis-(2-hydroxy-3-naphthoate)), alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. Pharmaceutically acceptable salts can also include those containing another molecule, such as an acetate ion, a succinate ion, or other counter ion. The counter ion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt can have more than one charged atom in its structure. If multiple charged atoms are part of the pharmaceutically acceptable salt, it can have multiple counter ions. Thus, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counter ions.

[0066] "Administering" or "administration" refers to the physical introduction of a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration for anti-CD228 antibody-drug conjugates include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion (preferably intravenous). The phrase "parenteral administration," as used herein, refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. Therapeutic agents can be administered via non-parenteral routes or orally. Other non-parenteral routes include topical, epidermal, or mucosal routes of administration, such as intranasal, vaginal, rectal, sublingual, or topical routes of administration. Administration can also be, for example, single, multiple, and / or over one or more extended periods of time.

[0067] The terms "baseline" or "baseline value," as used interchangeably herein, can refer to the measurement or characterization of a symptom (symptom) before administration of a treatment (e.g., an anti-CD228 antibody drug conjugate described herein) or at the start of administration of a treatment. The baseline value can be compared to a reference value to determine the alleviation or amelioration of symptoms of a CD228-associated disease (e.g., cancer) contemplated herein. The terms "reference" or "reference value," as used interchangeably herein, can refer to the measurement or characterization of a symptom after administration of a treatment (e.g., an anti-CD228 antibody drug conjugate as described). The reference value can be measured one or more times during a dosing regimen or treatment cycle or at the completion of a dosing regimen or treatment cycle. A "reference value" can be an absolute value, a relative value, a value with upper and / or lower limits, a range of values, an average value, a median value, a mean value, or a value compared to a baseline value.

[0068] Similarly, a "baseline value" can be an absolute value, a relative value, a value with upper and / or lower limits, a range of values, an average value, a median value, a mean value, or a value compared to a reference value. Reference and / or baseline values ​​can be obtained from one individual, from two different individuals, or from a group of individuals (e.g., a group of 2, 3, 4, 5, or more individuals).

[0069] The term "monotherapy" as used herein means that the anti-CD228 antibody-drug conjugate is the only anti-cancer agent administered to a subject during a treatment cycle. However, other therapeutic agents may be administered to the subject. For example, anti-inflammatory agents or other agents administered to a subject with cancer to treat symptoms associated with cancer, such as inflammation, pain, weight loss, and general fatigue, rather than the underlying cancer itself, may be administered during monotherapy.

[0070] An "adverse event" (AE), as used herein, is any unfavorable and generally unintended or undesired sign (including abnormal laboratory findings), symptom, or disease associated with the use of a medical treatment. A medical treatment may have one or more associated AEs, each of which may have the same or different levels of severity. Reference to methods that can "modify adverse events" refers to a treatment regimen that reduces the incidence and / or severity of one or more AEs associated with the use of a different treatment regimen.

[0071] As used herein, a "serious adverse event" or "SAE" refers to an adverse event that meets one of the following criteria: -Fatal or life-threatening ("life-threatening" as used in the definition of serious adverse events refers to an event in which the patient was at risk of death at the time of the event, not to an event that would have been more severe and hypothetically fatal). -Resulting in persistent or significant disability / incapacity. -Congenital anomalies / birth defects. -A medically significant event, defined as an event that puts the patient at risk or that may require medical or surgical intervention to prevent one of the outcomes listed above. The determination of whether an AE is "medically significant" requires the exercise of medical and scientific judgment. -Requires hospitalization or extension of existing hospitalization, except for: 1) routine treatment or monitoring of an underlying disease that is not accompanied by a worsening of the condition; 2) elective or pre-planned treatment for a pre-existing condition that is unrelated to the indication under study and that has not worsened since the informed consent was signed; 3) social reasons and respite care when the patient's general condition has not worsened.

[0072] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to mean "one or more" of any described or listed members.

[0073] The terms "about" or "consisting essentially of" mean a value or composition that is within an acceptable error range for a particular value or composition, as determined by one of ordinary skill in the art, which will depend on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "consisting essentially of" can mean within one or more standard deviations, according to practice in the art. Alternatively, "about" or "consisting essentially of" can mean a range of up to 20%. Moreover, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to five times the value. When a particular value or composition is given in this application and claims, unless otherwise specified, the meaning of "about" or "consisting essentially of" should be presumed to be within an acceptable error range for that particular value or composition.

[0074] Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to that value or parameter per se. For example, a description that refers to "about X" includes and describes "X."

[0075] As used herein, any concentration range, percentage range, ratio range, or integer range, unless otherwise indicated, should be understood to include any integer value within the stated range and, where appropriate, fractions thereof (such as tenths and hundredths of integers).

[0076] Various aspects of the disclosure are described in further detail in the subsections below.

[0077] II. General The present invention provides antibodies that specifically bind to CD228. The present invention is based, in part, on the discovery that antibody-drug conjugates targeting CD228, such as PEGylated MMAE antibody-drug conjugates, are particularly effective at killing CD228+ expressing cells. CD228 has been shown to be expressed in a variety of cancers, including melanoma, thyroid cancer, lung cancer, liver cancer, pancreatic cancer, head and neck cancer, gastric cancer, colorectal cancer, urothelial cancer, breast cancer, and cervical cancer.

[0078] III. Target molecule Unless otherwise specified, CD228 refers to human CD228. An exemplary human protein sequence has been assigned UniProt ID NO. P08582.

[0079] IV. Antibodies of the Invention The present invention provides antibodies, e.g., humanized antibodies, derived from the murine antibody L49, a murine immunoglobulin G1 (IgG1) monoclonal antibody against CD228, obtained from BALB / c mice immunized with lung cancer and melanoma cell lines (Siemers et al., 1997, Bioconjug. Chem. 8:510-9).

[0080] The binding affinity (i.e., dissociation constant, K) of the humanized form of the murine L49 antibody D Preferably, the binding affinity of the humanized L49 antibody is within 5-fold or 2-fold of the binding affinity of the murine antibody L49 for human CD228. Similar to the murine antibody from which it is derived, the humanized L49 antibody specifically binds to human CD228. These antibodies bind to both native forms of CD228 and CD228 recombinantly expressed, for example, from Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells. Preferred humanized L49 antibodies have affinities for human CD228 that are equal to or greater than (i.e., greater than beyond the margin of measurement error) that of L49 (e.g., 1.1-5-fold, 1.1-3-fold, 1.5-3-fold, 1.7-2.3-fold, or 1.7-2.1-fold greater than L49, or approximately 2-fold greater). Preferred humanized L49 antibodies bind to the same epitope and / or compete with murine L49 for binding to human CD228.

[0081] Preferred antibodies of the invention are those that inhibit cancer (e.g., cell proliferation, metastasis, and / or organism lethality) as demonstrated in cancer cells grown in culture, in animal models, or in clinical trials. Animal models can be generated by implanting human tumor cell lines expressing CD228 into appropriate immunodeficient rodent strains, such as thymic nude mice or SCID mice. These tumor cell lines can be established in immunodeficient rodent hosts as solid tumors by subcutaneous injection or as disseminated tumors by intravenous injection.

[0082] Once established in the host, these tumor models can be applied to evaluate the therapeutic efficacy of anti-CD228 antibodies or conjugated forms thereof, as described in the Examples.

[0083] Generally, anti-CD228 antibodies and / or anti-CD228 antibody-drug conjugates of the present disclosure bind to CD228, e.g., human CD228, and exert cytostatic and cytotoxic effects on malignant cells (e.g., cancer cells). Anti-CD228 antibodies of the present disclosure are preferably monoclonal and may be multispecific, human, humanized, or chimeric antibodies, single-chain antibodies, Fab fragments, F(ab') fragments, fragments produced by an Fab expression library, and CD228-binding fragments of any of the above. In some embodiments, anti-CD228 antibodies of the present disclosure specifically bind to CD228. Immunoglobulin molecules of the present disclosure may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule.

[0084] In certain embodiments of the present disclosure, the anti-CD228 antibody is an antigen-binding fragment (e.g., a human antigen-binding fragment) as described herein, including, but not limited to, Fab, Fab' and F(ab'), Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), and V L or V H Antigen-binding fragments include fragments comprising any of the variable domains. Antigen-binding fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with all or a portion of the following: hinge region, CH1, CH2, CH3, and CL domains. Antigen-binding fragments comprising any combination of the variable region(s) and the hinge region, CH1, CH2, CH3, and CL domains are also included in the present disclosure. In some embodiments, the anti-CD228 antibody or antigen-binding fragment thereof is human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken.

[0085] The anti-CD228 antibodies of the present disclosure may be monospecific, bispecific, trispecific, or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of CD228, or may be specific for both CD228 and a heterologous protein. See, e.g., PCT Publication Nos. WO 93 / 17715; WO 92 / 08802; WO 91 / 00360; WO 92 / 05793; Tutt, et al., 1991, J. Immunol. 147:60-69; U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., 1992, J. Immunol. 148:1547-1553.

[0086] The anti-CD228 antibodies of the present disclosure can be described or specified in terms of the particular CDRs they contain.The precise amino acid sequence boundaries of a given CDR or FR can be determined using the methods described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003 Jan;27(1):55-77 ("IMGT" numbering scheme); Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun 8;309(3):657-70 ("Aho" numbering scheme); and Martin et al., "Modeling antibody hypervariable loops: a combined algorithm," PNAS, 1989, 86(23):9268-9272 ("AbM" numbering scheme).The boundaries of a given CDR may vary depending on the scheme used for identification. In some embodiments, the "CDR" or "complementarity determining region" of a given antibody or region thereof (e.g., its variable region), or individual designated CDRs (e.g., CDR-H1, CDR-H2, CDR-H3), should be understood to encompass the CDRs defined (or specified) by any of the aforementioned schemes. For example, a particular CDR (e.g., CDR-H3) may be included within a given V. H Area or V L When a region is described as containing the amino acid sequence of a corresponding CDR in its amino acid sequence, it is understood that such CDR has the sequence of the corresponding CDR (e.g., CDR-H3) within the variable region as defined by any of the aforementioned schemes. Schemes for identifying a particular CDR or CDRs may also be designated, such as CDRs defined by the Kabat, Chothia, AbM, or IMGT methods.

[0087] The CDR sequences of the anti-CD228 antibodies and anti-CD228 antibody-drug conjugates described herein follow the Kabat numbering scheme as set forth in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD.

[0088]

[0013] In one aspect, provided herein is an anti-CD228 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and / or the light chain variable region comprises (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, wherein the CDRs of the anti-CD228 antibody are defined according to the Kabat numbering scheme.

[0089] The anti-CD228 antibodies described herein can comprise any suitable framework variable domain sequence, provided that the antibody retains the ability to bind to CD228 (e.g., human CD228). As used herein, heavy chain framework regions are described as "HC-FR1-FR4," and light chain framework regions are described as "LC-FR1-FR4." In some embodiments, anti-CD228 antibodies comprise heavy chain variable domain framework sequences of SEQ ID NOs: 9, 10, 11, and 12 (HC-FR1, HC-FR2, HC-FR3, and HC-FR4, respectively). In some embodiments, anti-CD228 antibodies comprise light chain variable domain framework sequences of SEQ ID NOs: 13, 14, 15, and 16 (LC-FR1, LC-FR2, LC-FR3, and LC-FR4, respectively).

[0090] In some embodiments of the anti-CD228 antibodies described herein, the heavy chain variable domain comprises the amino acid sequence of QVQLQESGPGLVKPSETLSLTCTVSGDSITSGYWNWIRQPPGKGLEYIGYISDSGITYYNPSLKSRVTISRDTSKNQYSLKLSSVTAADTAVYYCARRTLATYYAMDYWGQGTLVTVSS (SEQ ID NO: 7), and the light chain variable domain comprises the amino acid sequence of DFVMTQSPLSLPVTLGQPASISCRASQSLVHSDGNTYLHWYQQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPPTFGQGTKLEIK (SEQ ID NO: 8).

[0091] In some embodiments of the anti-CD228 antibodies described herein, the heavy chain CDR sequences comprise: (a) CDR-H1 (SGYWN (SEQ ID NO: 1)); (b) CDR-H2 (YISDSGITYYNPSLKS (SEQ ID NO: 2)); and (c) CDR-H3 (RTLATYYAMDY (SEQ ID NO: 3)).

[0092] In some embodiments of the anti-CD228 antibodies described herein, the heavy chain FR sequence comprises: (a) HC-FR1 (QVQLQESGPGLVKPSETLSLTCTVSGDSIT (SEQ ID NO: 9)); (b) HC-FR2(WIRQPPGKGLEYIG (SEQ ID NO: 10)); (c) HC-FR3 (RVTISRDTSKNQYSLKLSSVTAADTAVYYCAR (SEQ ID NO: 11)); and (d) HC-FR4 (WGQGTLVTVSS (SEQ ID NO: 12)).

[0093] In some embodiments of the anti-CD228 antibodies described herein, the light chain CDR sequences comprise: (a) CDR-L1 (RASQSLVHSDGNTYLH (SEQ ID NO: 4)); (b) CDR-L2 (RVSNRFS (SEQ ID NO: 5)); and (c) CDR-L3 (SQSTHVPPT (SEQ ID NO: 6)).

[0094] In some embodiments of the anti-CD228 antibodies described herein, the light chain FR sequence comprises: (a) LC-FR1 (DFVMTQSPLSLPVTLGQPASISC (SEQ ID NO: 13)); (b) LC-FR2 (WYQQRPGQSPRLLIY (SEQ ID NO: 14)); (c) LC-FR3 (GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC (SEQ ID NO: 15)); and (d) LC-FR4 (FGQGTKLEIK (SEQ ID NO: 16)).

[0095] In some embodiments, provided herein are anti-CD228 antibodies and / or anti-CD228 antibody drug conjugates that bind to CD228 (e.g., human CD228), wherein the antibody or antibody drug conjugate comprises a heavy chain variable region and a light chain variable region, wherein the antibody comprises: (a) a heavy chain variable domain comprising: (1) HC-FR1 comprising the amino acid sequence of SEQ ID NO: 9; (2) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (3) HC-FR2 comprising the amino acid sequence of SEQ ID NO: 10; (4) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (5) HC-FR3 comprising the amino acid sequence of SEQ ID NO: 11; (6) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and (7) HC-FR4 comprising the amino acid sequence of SEQ ID NO: 12; and / or (b) a light chain variable domain comprising: (1) LC-FR1 comprising the amino acid sequence of SEQ ID NO: 13; (2) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (3) LC-FR2 comprising the amino acid sequence of SEQ ID NO: 14; (4) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; (5) LC-FR3 comprising the amino acid sequence of SEQ ID NO: 15; (6) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6; and (7) LC-FR4 comprising the amino acid sequence of SEQ ID NO: 16.

[0096] In one aspect, provided herein is an anti-CD228 antibody, and / or an anti-CD228 antibody-drug conjugate, comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 7, or comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the N-terminal glutamine of the heavy chain variable domain cyclizes to form pyroglutamic acid. In one aspect, provided herein is an anti-CD228 antibody comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the N-terminal glutamine of the heavy chain variable domain cyclizes to form pyroglutamic acid.

[0097] In some embodiments, provided herein are anti-CD228 antibodies and / or anti-CD228 antibody-drug conjugates comprising a heavy chain variable domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the N-terminal glutamine of the heavy chain variable domain cyclizes to form pyroglutamic acid. In certain embodiments, a heavy chain variable domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 7 contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence and retains the ability to bind to CD228 (e.g., human CD228). In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 7. In certain embodiments, the substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) occur in regions outside the CDRs (i.e., in the FRs). In some embodiments, an anti-CD228 antibody comprises the heavy chain variable domain sequence of SEQ ID NO: 7 (including post-translationally modified versions of that sequence). In some embodiments, the N-terminal glutamine of the heavy chain variable domain cyclizes to form pyroglutamic acid. In certain embodiments, the heavy chain variable domain comprises one, two, or three CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3.

[0098] In some embodiments, provided herein are anti-CD228 antibodies and / or anti-CD228 antibody drug conjugates comprising a light chain variable domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the light chain variable domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 8 contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence and retains the ability to bind to CD228 (e.g., human CD228). In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 8. In certain embodiments, the substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) occur in regions outside the CDRs (i.e., in the FRs). In some embodiments, the anti-CD228 antibody comprises a light chain variable domain sequence of SEQ ID NO: 8 (including post-translationally modified versions of that sequence). In certain embodiments, the light chain variable domain comprises one, two, or three CDRs selected from the following: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0099] In some embodiments, the anti-CD228 antibody and / or anti-CD228 antibody drug conjugate comprises a heavy chain variable domain according to any of the embodiments given above, and a light chain variable domain according to any of the embodiments given above. In one embodiment, the antibody comprises the heavy chain variable domain sequence of SEQ ID NO: 7 and the light chain variable domain sequence of SEQ ID NO: 8 (including post-translationally modified versions of those sequences). In some embodiments, the N-terminal glutamine of the heavy chain variable domain cyclizes to form pyroglutamic acid.

[0100] In some embodiments, the anti-CD228 antibody and / or anti-CD228 antibody drug conjugate comprises (i) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and (ii) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein the CDRs of the anti-CD228 antibody are defined by the Kabat numbering scheme.

[0101] In some embodiments, the anti-CD228 antibody and / or anti-CD228 antibody drug conjugate comprises (i) an amino acid sequence having at least 85% sequence identity to a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, and (ii) an amino acid sequence having at least 85% sequence identity to a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the N-terminal glutamine of the heavy chain variable domain cyclizes to form pyroglutamic acid.

[0102] In some embodiments, the anti-CD228 antibody and / or the anti-CD228 antibody of the anti-CD228 antibody drug conjugate is a monoclonal antibody.

[0103] Anti-CD228 antibodies of the invention may also be described or specified in terms of their binding affinity to CD228 (e.g., human CD228). Preferred binding affinities include those greater than 5×10 -2 M, 10 -2 M, 5 x 10 -3 M, 10 -3 M, 5 x 10 -4 M, 10 -4 M, 5 x 10 -5 M, 10 -5 M, 5 x 10 -6 M, 10 -6 M, 5 x 10 -7 M, 10 -7 M, 5 x 10 -8 M, 10 -8 M, 5 x 10 -9 M, 10 -9 M, 5 x 10 -10 M, 10-10 M, 5 x 10 -11 M, 10 -11 M, 5 x 10 -12 M, 10 -12 M, 5 x 10 -13 M, 10 -13 M, 5 x 10 -14 M, 10 -14 M, 5 x 10 -15 M or 10 -15 A dissociation constant or K less than M D Examples include those having the following structure.

[0104] In some embodiments, the binding of the anti-CD228 antibodies of the invention is pH dependent, such that the antibodies exhibit differential binding across a pH gradient. In some embodiments, the anti-CD228 antibodies exhibit maximal binding between about pH 5.5 and about pH 6.3. In some embodiments, the anti-CD228 antibodies exhibit maximal binding at about pH 5.6. In some embodiments, the anti-CD228 antibodies exhibit maximal binding at about pH 6.3. In some embodiments, the anti-CD228 antibodies exhibit minimal binding at a pH of about 5.1 or below.

[0105] There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each with a heavy chain designated α, δ, ε, γ, and μ. The γ and α classes are further divided into subclasses; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. IgG1 antibodies can exist in multiple polymorphic variants called allotypes (reviewed in Jefferis and Lefranc 2009. mAbs, Vol. 1, Issue 4, 1-7), any of which are suitable for use in some embodiments herein. Common allotypic variants in the human population are those designated by the letters a, f, n, z, or combinations thereof. In any of the embodiments herein, the antibody can comprise a heavy chain Fc region comprising a human IgG Fc region. In a further embodiment, the human IgG Fc comprises human IgG1.

[0106] In some embodiments, the anti-CD228 antibody and / or anti-CD228 antibody drug conjugate comprises a heavy chain variable domain according to any of the embodiments provided above and a light chain variable domain according to any of the embodiments provided above. In one embodiment, the antibody comprises a heavy chain variable domain described in any of the embodiments provided above. It comprises a heavy chain constant region comprising the amino acid sequence of LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 17), and a light chain constant region comprising the amino acid sequence of TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 18) (including post-translational modifications of these sequences).In another embodiment, the antibody is ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV The heavy chain constant region comprises the amino acid sequence SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 19), and the light chain constant region comprises the amino acid sequence TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 18), including post-translational modifications of these sequences. SEQ ID NO: 19 contains a serine-to-cysteine ​​substitution at amino acid position 239 of the human IgG1 isotype. The presence of the additional cysteine ​​residue allows interchain disulfide bond formation. Such interchain disulfide bond formation causes steric hindrance, thereby reducing the affinity of the Fc region-FcγR binding interaction. Cysteine ​​residues introduced into or adjacent to the Fc region of the IgG constant region can also serve as sites for conjugation to therapeutic agents (i.e., coupling of cytotoxic drugs using thiol-specific reagents such as maleimide derivatives of the drug). The presence of the therapeutic agent causes steric hindrance, thereby further reducing the affinity of the Fc region-FcγR binding interaction. Other substitutions at any of positions 234, 235, 236, and / or 237 reduce affinity for Fcγ receptors, particularly the FcγRI receptor (see, e.g., US 6,624,821, US 5,624,821).

[0107] In some embodiments, the anti-CD228 antibody of the anti-CD228 antibody or antibody drug conjugate is the humanized antibody hL49 HALC. hL49 HALC comprises the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8. In some embodiments, the N-terminal glutamine of the heavy chain variable domain is cyclized to form pyroglutamic acid. In some embodiments, the anti-CD228 antibody of the anti-CD228 antibody or antibody drug conjugate is the humanized antibody hL49. hL49 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8, a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 17, and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 18.

[0108] Antibodies also include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody, provided that the covalent attachment does not prevent the antibody from binding to CD228 or from exerting a cytostatic or cytotoxic effect on HD cells. For example, but not limited to, antibody derivatives include antibodies that have been modified by, e.g., glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of a number of chemical modifications can be made by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, derivatives can contain one or more non-classical amino acids.

[0109] humanized antibodies Humanized antibodies are genetically engineered antibodies in which CDRs from a non-human "donor" antibody are grafted onto human "acceptor" antibody sequences (see, e.g., Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539; Carter, US 6,407,213; Adair, US 5,859,205; and Foote, US 6,881,557). The acceptor antibody sequences can be, for example, mature human antibody sequences, composites of such sequences, consensus sequences of human antibody sequences, or germline region sequences. Preferred acceptor sequences for the heavy chain are germline V H Exon V H l-2 (also referred to as HV1-2 in the literature) (Shin et al., 1991, EMBO J. 10:3641-3645), and the hinge region (J H ) for Exon J H-6 (Mattila et al., 1995, Eur. J. Immunol. 25:2578-2582). For the light chain, the preferred acceptor sequence is exon VK2-30 (also referred to in the literature as KV2-30), and for the hinge region, exon JK-4 (Hieter et al., 1982, J. Biol. Chem. 257:1516-1522). Thus, a humanized antibody is an antibody that is derived entirely or substantially from a donor antibody and variable region framework sequences and constant regions, with some or all of the CDRs, if present, derived entirely or substantially from human antibody sequences. Similarly, a humanized heavy chain is derived entirely or substantially from a donor antibody heavy chain and heavy chain variable region framework sequences and heavy chain constant regions, with at least one, two, and usually all three CDRs, if present, derived substantially from human heavy chain variable region framework and constant region sequences. Similarly, a humanized light chain is derived entirely or substantially from a donor antibody light chain and light chain variable region framework sequence and light chain constant region, with at least one, two, and usually all three CDRs, if present, derived substantially from human light chain variable region framework and constant region sequences. Other than nanobodies and dAbs, humanized antibodies comprise a humanized heavy chain and a humanized light chain. CDRs in a humanized antibody are derived substantially from corresponding CDRs in a non-human antibody when at least 60%, 85%, 90%, 95%, or 100% of the corresponding residues (as defined by Kabat) are identical between the respective CDRs. The variable region framework sequence of an antibody chain or the constant region of an antibody chain is derived substantially from a human variable region framework sequence or human constant region, respectively, when at least 85%, 90%, 95%, or 100% of the corresponding residues as defined by Kabat are identical.

[0110] Humanized antibodies often incorporate all six CDRs (preferably as defined by Kabat) from a murine antibody, although they can also be made with fewer than all CDRs (e.g., at least three, four, or five) from the murine antibody (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al., Journal of Immunology, 164:1432-1441, 2000).

[0111] Certain amino acids from the human variable region framework residues may be selected for substitution based on their possible effects on CDR conformation and / or binding to antigen, studied by modeling, by examining the properties of the amino acid at a particular position, or by empirical observation of the effects of substituting or mutagenes- ing specific amino acids.

[0112] For example, when an amino acid differs between a murine variable region framework residue and a selected human variable region framework residue, the human framework amino acid can be substituted with the equivalent framework amino acid from the murine antibody, where the amino acid is reasonably predicted to be: (1) Direct non-covalent binding to antigens; (2) adjacent to the CDR region, (3) otherwise interacts with the CDR regions (e.g., is within about 6 Å of the CDR regions), or (4) Mediates the interaction between heavy and light chains.

[0113] One aspect of the present invention provides a humanized form of the murine antibody L49. One such humanized variant of the murine antibody L49 is designated HALC. HALC comprises a mature heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7 and a mature light chain variable region comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, the N-terminal glutamine of the heavy chain variable domain is cyclized to form pyroglutamic acid. Humanized antibodies of the present invention include variants of the HALC humanized antibody in which the humanized heavy chain mature variable region exhibits at least 90%, 95%, or 99% sequence identity to SEQ ID NO:7 and the humanized light chain mature variable region exhibits at least 90%, 95%, or 99% sequence identity to SEQ ID NO:8. Preferably, such antibodies retain some or all of the backmutations of HALC. That is, at least one, two, three, four, or preferably all five of the heavy chain positions H27, H30, H47, H71, and H78 are occupied by D, T, Y, R, and Y, respectively. Similarly, position L36 is preferably occupied by Y, and position L46 is preferably occupied by L. In some embodiments, position L2 is preferably occupied by F. In some embodiments, the CDR regions of such humanized antibodies are identical or substantially identical to the CDR regions of the murine donor antibody. In a preferred embodiment, light chain CDR1 position L28 is occupied by D. CDR regions can be defined by any conventional definition (e.g., Chothia), but are preferably as defined by Kabat. In one embodiment, the humanized antibody comprises a heavy chain comprising the three CDRs of SEQ ID NO:7 and a variable region framework having at least 95% identity to the variable region framework of SEQ ID NO:7. In another embodiment, the humanized antibody comprises a light chain comprising the three CDRs of SEQ ID NO:8 and a variable region framework having at least 95% identity to the variable region framework of SEQ ID NO:8.In a further embodiment, the humanized antibody comprises a heavy chain comprising three CDRs of SEQ ID NO:7 and a variable region framework at least 95% identical to the variable region framework of SEQ ID NO:7, and a light chain comprising three CDRs of SEQ ID NO:8 and a variable region framework at least 95% identical to the variable region framework of SEQ ID NO:8. In one embodiment, the humanized antibody comprises a heavy chain comprising three CDRs of SEQ ID NO:7 and a variable region framework at least 98% identical to the variable region framework of SEQ ID NO:7. In another embodiment, the humanized antibody comprises a light chain comprising three CDRs of SEQ ID NO:8 and a variable region framework at least 98% identical to the variable region framework of SEQ ID NO:8. In a further embodiment, the humanized antibody comprises a heavy chain comprising three CDRs of SEQ ID NO:7 and a variable region framework at least 98% identical to the variable region framework of SEQ ID NO:7, and a light chain comprising three CDRs of SEQ ID NO:8 and a variable region framework at least 98% identical to the variable region framework of SEQ ID NO:8. In one embodiment, the humanized antibody comprises a heavy chain comprising the three CDRs of SEQ ID NO: 7 and a variable region framework at least 99% identical to the variable region framework of SEQ ID NO: 7. In another embodiment, the humanized antibody comprises a light chain comprising the three CDRs of SEQ ID NO: 8 and a variable region framework at least 99% identical to the variable region framework of SEQ ID NO: 8. In a further embodiment, the humanized antibody comprises a heavy chain comprising the three CDRs of SEQ ID NO: 7 and a variable region framework at least 99% identical to the variable region framework of SEQ ID NO: 7, and a light chain comprising the three CDRs of SEQ ID NO: 8 and a variable region framework at least 99% identical to the variable region framework of SEQ ID NO: 8.

[0114] The humanized antibody HALC contains an asparagine-to-aspartic acid substitution at amino acid position L28, which is in the light chain CDR1, compared to the murine antibody L49. This substitution eliminates the deamidation observed in the humanized L49 variant HALB and also limits isomerization. In some embodiments, the light chain variable region of any of the antibodies described herein lacks this substitution at position L28. In some embodiments of the antibodies described herein, the light chain variable region comprises the amino acid sequence DFVMTQSPLSLPVTLGQPASISCRASQSLVHSNGNTYLHWYQQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPPTFGQGTKLEIK (SEQ ID NO: 20). In some embodiments, the humanized antibody is HALB, which comprises a heavy chain variable region comprising SEQ ID NO: 7 and a light chain variable region comprising SEQ ID NO: 20.

[0115] To the extent that the humanized antibody exhibits any variation from the exemplified HALC-humanized antibody, one possibility for such additional variation is additional backmutations in the variable region frameworks, however, such additional backmutations are not preferred as they generally do not improve affinity and may increase the risk of immunogenicity by introducing more mouse residues.

[0116] Another possible modification is to replace certain residues in the CDRs of a murine antibody with corresponding residues from human CDR sequences, typically from the CDRs of the human acceptor sequence used to design the exemplary humanized antibody. In some antibodies, only a portion of the CDRs, i.e., a subset of CDR residues required for binding, called SDRs, are required to retain binding in the humanized antibody. CDR residues that do not contact antigen and are not in the SDRs can be identified from regions of the Kabat CDRs outside the Chothia hypervariable loops based on previous studies (Chothia, J. Mol. Biol. 196:901, 1987), by molecular modeling, and / or experimentally, or as described in Gonzales et al., Mol. Immunol. 41:863 (2004) (e.g., residues H60-H65 in CDR H2 are often not required). In such humanized antibodies, where one or more donor CDR residues are absent or the entire donor CDR is deleted, the amino acid occupying that position may be the amino acid occupying the corresponding position (according to Kabat numbering) in the acceptor antibody sequence. The number of such acceptor to donor amino acid substitutions in the CDRs to include reflects a balance of competing considerations. Such substitutions may be advantageous for reducing the number of mouse amino acids in the humanized antibody and thus reducing potential immunogenicity. However, substitutions may also result in changes in affinity, and it is preferable to avoid significant decreases in affinity. The positions of substitutions within the CDRs and the amino acids to be substituted can also be selected empirically.

[0117] Although not preferred, other amino acid substitutions can be made, for example, in framework residues that do not contact the CDRs, or even in residues within the CDRs that contact some potential CDRs. The substitutions made in the variant humanized sequence are often conservative with respect to the HALC amino acid that is replaced. Preferably, substitutions (whether conservative or not) to HALC do not significantly affect the binding affinity or potency of the humanized mAb, i.e., its ability to bind to human CD228 and inhibit cancer cell proliferation.

[0118] Variants typically differ from the heavy and light chain mature variable regions of HALC by a small number of substitutions, deletions, or insertions (e.g., typically no more than 1, 2, 3, 5, or 10 in either the light chain mature variable region or the heavy chain mature variable region, or both).

[0119] Selection of constant regions The heavy and light chain variable regions of a humanized antibody can be linked to at least a portion of a human constant region. The choice of constant region depends, in part, on whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis, and / or complement-dependent cytotoxicity are desired. For example, human isotypes IgG1 and IgG3 have strong complement-dependent cytotoxicity, human isotype IgG2 has weak complement-dependent cytotoxicity, and human IgG4 lacks complement-dependent cytotoxicity. Human IgG1 and IgG3 also induce stronger cell-mediated effector functions than human IgG2 and IgG4. The light chain constant region can be lambda or kappa. Antibodies can be expressed as tetramers containing two light chains and two heavy chains, as separate heavy and light chains, as Fab, Fab', F(ab')2, and Fv, or as single-chain antibodies in which the heavy and light chain variable domains are linked via a spacer.

[0120] Human constant regions exhibit allotypic and isoallotypic diversity among different individuals, i.e., the constant region can differ in different individuals at one or more polymorphic positions. Isoallotypes differ from allotypes in that sera that recognize an isoallotype bind to non-polymorphic regions of one or more other isotypes.

[0121] One or several amino acids at the amino or carboxy termini of the light and / or heavy chains, such as the C-terminal lysine of the heavy chain, can be deleted or derivatized in a proportion or all of the molecules. Substitutions can be made in the constant regions to reduce or increase effector functions such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006) or to extend half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004).

[0122] Exemplary substitutions include amino acid substitutions of native amino acids with cysteine ​​residues introduced at amino acid positions 234, 235, 237, 239, 267, 298, 299, 326, 330, or 332, preferably the S239C mutation in the human IgG1 isotype (US 20100158909). The presence of the additional cysteine ​​residue allows interchain disulfide bond formation. Such interchain disulfide bond formation can cause steric hindrance, which can reduce the affinity of the Fc region-FcγR binding interaction. The cysteine ​​residue(s) introduced at or adjacent to the Fc region of the IgG constant region can also serve as a site for conjugation with a therapeutic agent (i.e., attachment of a cytotoxic drug using a thiol-specific reagent, such as a maleimide derivative of the drug). The presence of the therapeutic agent can cause steric hindrance, which can further reduce the affinity of the Fc region-FcγR binding interaction. Other substitutions at any of positions 234, 235, 236, and / or 237 result in reduced affinity for Fcγ receptors, particularly the FcγRI receptor (see, e.g., US 6,624,821, US 5,624,821).

[0123] The in vivo half-life of an antibody can also affect its effector function. Increasing or decreasing the half-life of an antibody can alter its therapeutic activity. FcRn is a receptor structurally similar to MHC class I antigens that noncovalently associate with β2-microglobulin. FcRn regulates the catabolism of IgG and its transcytosis across tissues (Ghetie and Ward, 2000, Annu. Rev. Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res. 25:97-113). IgG-FcRn interaction occurs at pH 6.0 (the pH of intracellular vesicles) but not at pH 7.4 (the pH of blood). This interaction allows IgG to be recycled back into the circulation (Ghetie and Ward, 2000, Ann. Rev. Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res. 25:97-113). The region on human IgG1 involved in FcRn binding has been mapped (Shields et al., 2001, J. Biol. Chem. 276:6591-604). Alanine substitutions at positions Pro238, Thr256, Thr307, Gln311, Asp312, Glu380, Glu382, or Asn434 of human IgG1 enhance FcRn binding (Shields et al., 2001, J. Biol. Chem. 276:6591-604). IgG1 molecules containing these substitutions have a longer serum half-life. As a result, these modified IgG1 molecules are able to perform their effector functions and therefore exert their therapeutic effect for a longer period of time compared to unmodified IgG1. Other exemplary substitutions to increase binding to FcRn include Gln at position 250 and / or Leu at position 428. EU numbering is used for all positions in the constant region.

[0124] The oligosaccharide covalently attached to the conserved Asn297 is responsible for the ability of the Fc region of IgG to bind to FcγR (Lund et al., 1996, J. Immunol. 157:4963-69; Wright and Morrison, 1997, Trends Biotechnol. 15:26-32). Engineering this glycoform on IgG can significantly improve IgG-mediated ADCC. Adding bisecting N-acetylglucosamine modifications to this glycoform (Umana et al, 1999, Nat. Biotechnol. 17:176-180; Davies et al, 2001, Biotech. Bioeng. 74:288-94) or removing fucose from this glycoform (Shields et al, 2002, J. Biol. Chem. 277:26733-40; Shinkawa et al, 2003, J. Biol. Chem. 278:6591-604; Niwa et al., 2004, Cancer Res. 64:2127-33) are two examples of IgG Fc engineering that improves binding between IgG Fc and FcγR, thereby enhancing Ig-mediated ADCC activity. In some embodiments, the anti-CD228 antibody of the anti-CD228 antibody or antibody drug conjugate described herein has a glycan attached to the conserved Asn297 residue in the constant region, where the numbering of amino acid residues in the constant region is according to the EU index as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991). In some embodiments, the glycan is biantennary. In some embodiments, the glycan is core-fucosylated. In some embodiments, the glycan has zero terminal galactose residues. In some embodiments, the glycan is biantennary and core-fucosylated.In some embodiments, the glycan is biantennary and has a zero terminal galactose residue. In some embodiments, the glycan is core-fucosylated and has a zero terminal galactose residue. In some embodiments, the glycan is biantennary, core-fucosylated and has a zero terminal galactose residue. In some embodiments, in the population of anti-CD228 antibodies of the anti-CD228 antibodies or antibody-drug conjugates described herein, the conserved Asn297 residue of the constant region (numbering of amino acid residues in the constant region is according to the EU index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) is predominantly occupied by glycans that are biantennary, core-fucosylated and have a zero terminal galactose residue.

[0125] Systematic substitution of solvent-exposed amino acids in the human IgG1 Fc region has generated IgG variants with altered FcγR binding affinity (Shields et al., 2001, J. Biol. Chem. 276:6591-604). Compared to the parent IgG1, a subset of these variants involving substitutions of Ala at Thr256 / Ser298, Ser298 / Glu333, Ser298 / Lys334, or Ser298 / Glu333 / Lys334 exhibits both increased FcγR binding affinity and ADCC activity (Shields et al., 2001, J. Biol. Chem. 276:6591-604; Okazaki et al., 2004, J. Mol. Biol. 336:1239-49).

[0126] The complement fixation activity (both C1q binding and CDC activity) of antibodies can be improved by substitutions at Lys326 and Glu333 (Idusogie et al., 2001, J. Immunol. 166:2571-2575). The same substitutions in the human IgG2 backbone can convert antibody isotypes that bind poorly to C1q and have severely lacking complement activation activity into those that can both bind to C1q and mediate CDC (Idusogie et al., 2001, J. Immunol. 166:2571-75). Several other methods have also been applied to improve the complement fixation activity of antibodies. For example, grafting the 18-amino acid carboxyl-terminal tail of IgM onto the carboxyl terminus of IgG enhances their CDC activity. This is observed even with IgG4, which normally has no detectable CDC activity (Smith et al., 1995, J. Immunol. 154:2226-36). Furthermore, substitution of Ser444, located near the carboxy terminus of the IgG1 heavy chain, with Cys induced tail-to-tail dimerization of IgG1, which increased CDC activity by 200-fold compared to monomeric IgG1 (Shopes et al., 1992, J. Immunol. 148:2918-22). Additionally, construction of bispecific diabodies with specificity for C1q also confers CDC activity (Kontermann et al., 1997, Nat. Biotech. 15:629-31).

[0127] Complement activity can be reduced by mutating at least one of amino acid residues 318, 320, and 322 of the heavy chain to a residue with a different side chain, such as Ala. Substituting other alkyl-substituted non-ionic residues, such as Gly, Ile, Leu, or Val, or aromatic non-polar residues, such as Phe, Tyr, Trp, and Pro, in place of any of the three residues also reduces or abolishes C1q binding. Ser, Thr, Cys, and Met can be used at residues 320 and 322, rather than residue 318, to reduce or abolish C1q binding activity.

[0128] Substitution of residue 318 (Glu) with a polar residue can alter C1q binding activity without abolishing it. Substitution of residue 297 (Asn) with Ala eliminates lytic activity but only slightly reduces affinity for C1q (approximately three-fold weakening). This modification destroys the glycosylation site and abolishes the presence of carbohydrates required for complement activation. Any other substitution at this site also destroys the glycosylation site. Mutations D270A, K322A, P329A, and P311S, and any combination thereof, also reduce C1q binding (see WO 06 / 036291).

[0129] Reference to a human constant region includes a constant region with any naturally occurring allotype or any permutation (permutation) of residues occupying polymorphic positions in the naturally occurring allotypes, and there may be up to 1, 2, 5, or 10 mutations, such as those listed above, relative to a naturally occurring human constant region to reduce Fc gamma receptor binding or increase binding to FcRn.

[0130] In some embodiments, the anti-CD228 antibodies and / or anti-CD228 antibody drug conjugates described herein comprise a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the anti-CD228 antibodies and / or anti-CD228 antibody drug conjugates described herein comprise a light chain constant region comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-CD228 antibodies and / or anti-CD228 antibody drug conjugates described herein comprise a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 17 and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-CD228 antibodies and / or anti-CD228 antibody drug conjugates described herein comprise a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-CD228 antibodies and / or anti-CD228 antibody drug conjugates described herein comprise a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 19 and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 18.

[0131] V. Expression of Recombinant Antibodies Humanized antibodies can typically be produced by recombinant expression. Recombinant polynucleotide constructs typically contain expression control sequences operably linked to the coding sequences of the antibody chains, including naturally associated or heterologous promoter regions. Preferably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into an appropriate host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequences, and the recovery and purification of cross-reacting antibodies.

[0132] Mammalian cells are preferred hosts for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). Several suitable host cell lines capable of screening intact heterologous proteins have been developed in the art, including CHO cell lines (e.g., DG44), various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myelomas (including Sp2 / 0 and NS0). Preferably, the cells are non-human. Expression vectors for these cells may include expression control sequences such as an origin of replication, a promoter, an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and a transcription termination sequence. Preferred expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0133] Once expressed, antibodies can be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, etc. (See generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0134] VI. Nucleic acids The present invention further provides nucleic acids encoding any of the above-described humanized heavy and light chains. Typically, the nucleic acid also encodes a signal peptide fused to the mature heavy and light chains. The coding sequence on the nucleic acid may be in operative linkage with regulatory sequences to ensure expression of the coding sequence, such as a promoter, enhancer, ribosome binding site, or transcription termination signal. The nucleic acids encoding the heavy and light chains may occur in isolated form or may be cloned into one or more vectors. The nucleic acids may be synthesized, for example, by solid-state synthesis or PCR of overlapping oligonucleotides. The nucleic acids encoding the heavy and light chains may be linked, for example, as a single contiguous nucleic acid within an expression vector, or may be separate, for example, each cloned into its own expression vector.

[0135] In some embodiments, also provided herein are nucleic acids encoding the anti-CD228 antibodies or antigen-binding fragments thereof described herein. Further provided herein are vectors comprising nucleic acids encoding the anti-CD228 antibodies or antigen-binding fragments thereof described herein. Further provided herein are host cells expressing nucleic acids encoding the anti-CD228 antibodies or antigen-binding fragments thereof described herein. Further provided herein are host cells comprising vectors comprising nucleic acids encoding the anti-CD228 antibodies or antigen-binding fragments thereof described herein.

[0136] The anti-CD228 antibodies described herein can be prepared by well-known recombinant techniques using well-known expression vector systems and host cells. In one embodiment, the antibodies are prepared in CHO cells using the GS expression vector system as disclosed in De la Cruz Edmunds et al., 2006, Molecular Biotechnology 34; 179-190, EP 216846, U.S. Pat. No. 5,981,216, WO 87 / 04462, EP 323997, U.S. Pat. No. 5,591,639, U.S. Pat. No. 5,658,759, EP 338841, U.S. Pat. No. 5,879,936, and U.S. Pat. No. 5,891,693.

[0137] The monoclonal anti-CD228 antibodies described herein may be produced by the hybridoma method first described, for example, by Kohler et al., Nature, 256, 495 (1975), or by recombinant DNA methods. Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature, 352, 624-628 (1991) and Marks et al., J Mol Biol., 222(3):581-597 (1991). Monoclonal antibodies may be obtained from any suitable source. Thus, for example, monoclonal antibodies may be obtained from hybridomas prepared from mouse splenic B cells obtained from mice immunized with the antigen of interest, e.g., cells expressing the antigen on their surface, or in the form of a nucleic acid encoding the antigen of interest. Monoclonal antibodies can also be obtained from hybridomas obtained from antibody-expressing cells of immunized humans or non-human mammals such as rats, dogs, and primates.

[0138] VII. Antibody-Drug Conjugates Anti-CD228 antibodies may be conjugated to a cytotoxic or cytostatic moiety (including pharmaceutically compatible salts thereof) to form an antibody-drug conjugate (ADC). Particularly suitable moieties for conjugation to antibodies are cytotoxic drugs (e.g., chemotherapeutic drugs), prodrug-converting enzymes, radioisotopes or compounds, or toxins (these moieties are collectively referred to as therapeutic agents). For example, anti-CD228 antibodies may be conjugated to a cytotoxic drug, such as a chemotherapeutic drug, or a toxin (e.g., a cytostatic or cytocidal drug, e.g., abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin).

[0139] The anti-CD228 antibody can be conjugated to a prodrug-converting enzyme. The prodrug-converting enzyme can be recombinantly fused to the antibody or chemically conjugated to the antibody using known methods. Exemplary prodrug-converting enzymes are carboxypeptidase G2, beta-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, beta-lactamase, beta-glucosidase, nitroreductase, and carboxypeptidase A.

[0140] Techniques for conjugating therapeutic agents to proteins, particularly antibodies, are well known. (For example, Arnon et al, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," Monoclonal Antibodies And Cancer Therapy (Reisfeld et al., ed., Alan R. Liss, Inc., 1985); Hellstrom et al, "Antibodies For Drug Delivery," Controlled Drug Delivery (Robinson et al., ed., Marcel Dekker, Inc., 2nd ed. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," Monoclonal Antibodies '84: Biological And Clinical Applications (ed. Pinchera et al., 1985); "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy," Monoclonal Antibodies For Cancer Detection And Therapy (ed. Baldwin et al., Academic Press, 1985); Thorpe et al. 1982, Immunol. Rev. 62:119-58; see also PCT Publication WO 89 / 12624).

[0141] The therapeutic agent can be conjugated in a manner that reduces its activity unless it is cleaved from the antibody (e.g., by hydrolysis, antibody degradation, or a cleaving agent). Such a therapeutic agent is attached to the antibody by a cleavable linker that is susceptible to cleavage in the intracellular environment of a CD228-expressing cancer cell, but is substantially insensitive to the extracellular environment, such that the conjugate is cleaved from the antibody when internalized by the CD228-expressing cancer cell (e.g., in an endosomal environment, or, e.g., by pH- or protease-sensitivity, in a lysosomal environment, or in a caveolear environment).

[0142] Typically, ADCs include a linker region between the therapeutic agent and the anti-CD228 antibody. As described above, typically, the linker can be cleavable under intracellular conditions such that cleavage of the linker releases the therapeutic agent from the antibody in the intracellular environment (e.g., within a lysosome, endosome, or caveolea). The linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including a lysosomal or endosomal protease. Typically, the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleavage agents can include cathepsins B and D and plasmin (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Most typical are peptidyl linkers that are cleavable by enzymes present in CD228-expressing cells. 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 comprising a Phe-Leu or Gly-Phe-Leu-Gly peptide (SEQ ID NO: 30)). Other such linkers are described, for example, in U.S. Pat. No. 6,214,345. In certain embodiments, the peptide linker cleavable by an intracellular protease includes a Val-Cit linker or a Phe-Lys dipeptide (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with a Val-Cit linker). The advantage of using intracellular proteolytic release of a therapeutic agent is that the agent is usually weakened when conjugated, and the serum stability of the conjugate is usually high.

[0143] The cleavable linker may be pH-sensitive, i.e., sensitive to hydrolysis at a specific 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 acid 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, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions, such as in blood, but are unstable at pH levels lower than 5.5 to 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (such as a thioether attached to a therapeutic agent via an acylhydrazone bond (see, eg, US Pat. No. 5,622,929)).

[0144] Other linkers are cleavable under reducing conditions (eg, disulfide linkers). Disulfide linkers include 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), linkers that can be made using SPDB and SMPT (see, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel ed., Oxford U. Press, 1987; see also U.S. Pat. No. 4,880,935).

[0145] The linker may be a malonic acid linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12). The linker may also be a malonic acid linker (Johnson et al, 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al, 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al, 1995, Bioorg-Med-Chem. 3(10):1305-12).

[0146] The linker can also be a non-cleavable linker, such as a maleimide-alkylene or maleimide-aryl linker that is directly attached to a therapeutic agent (e.g., a drug). The active drug linker is released upon degradation of the antibody.

[0147] Typically, the linkers are substantially insensitive to the extracellular environment, meaning that no more than about 20%, typically no more than about 15%, more typically no more than about 10%, and even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the linkers in a sample of the ADC are cleaved when the ADC is present in an extracellular environment (e.g., in plasma).

[0148] Whether a linker is substantially insensitive to the extracellular environment can be determined, for example, by incubating both (a) the ADC ("ADC sample") and (b) an equimolar amount of unconjugated antibody or therapeutic agent ("control sample") separately 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 therapeutic agent present in the ADC sample to that present in the control sample, as measured, for example, by high performance liquid chromatography.

[0149] The linker may also facilitate cellular internalization. The linker may facilitate cellular internalization when conjugated to a therapeutic agent (i.e., in the context of the linker-therapeutic agent portion of an ADC or ADC derivative described herein). Alternatively, the linker may facilitate cellular internalization when conjugated to both a therapeutic agent and an anti-CD228 antibody (i.e., in the context of an ADC described herein).

[0150] An anti-CD228 antibody can be conjugated to a linker through a heteroatom of the antibody. These heteroatoms can be naturally present on the antibody or can be introduced into the antibody. In some embodiments, an anti-CD228 antibody is conjugated to a linker through the nitrogen atom of a lysine residue. In other embodiments, an anti-CD228 antibody is conjugated to a linker through the sulfur atom of a cysteine ​​residue. The cysteine ​​residue can be natural or engineered into the antibody. Methods for conjugating antibodies to linkers and drug linkers through lysine and cysteine ​​residues are known in the art.

[0151] Exemplary antibody-drug conjugates include auristatin-based antibody-drug conjugates (i.e., the drug component is an auristatin drug). Auristatins have been shown to bind to tubulin and interfere with microtubule dynamics as well as nuclear and cell division, and have anti-cancer activity. Typically, auristatin-based antibody-drug conjugates include a linker between the auristatin drug and the anti-CD228 antibody. The linker can be, for example, a cleavable linker (e.g., a peptidyl linker, a carbohydrate linker) or a non-cleavable linker (e.g., a linker that is released by degradation of the antibody). Auristatins include auristatin T, MMAF, and MMAE. The synthesis and structures of exemplary auristatins are described in U.S. Publication Nos. 7,659,241, 7,498,298, 2009-0111756, 2009-0018086, and 7,968,687, each of which is incorporated herein by reference in its entirety and for all purposes.

[0152] Other exemplary antibody drug conjugates include maytansinoid antibody drug conjugates (i.e., the drug moiety is a maytansinoid drug), and benzodiazepine antibody drug conjugates (i.e., the drug moiety is a benzodiazepine (e.g., pyrrolo[1,4]benzodiazepine dimers (PDB dimers), indolinobenzodiazepine dimers, and oxazolidinobenzodiazepine dimers)).

[0153] In some embodiments, the PBD dimer for use in the present invention is represented by Formula I. The preferred stereochemistry of the PBD dimer is as shown in Formula Ia: [ka] or a pharmaceutical salt, solvate, or solvate of the salt, wherein the subscript n is 1 or 3.

[0154] Solvates of formula (I) and (Ia) are typically formed by adding water or an alcohol solvent across the imine functional groups of one or both PBD monomers to form carbinolamine(s) and / or carbinolamine ethers. For example, at the N10-C11 positions, there may be an imine (N=C), a carbinolamine (NH-CH(OH)), or a carbinolamine ether (NH-CH(OMe)), as represented by formulas I' and Ia' below: [ka] wherein: (a)R 10 is H and R 11 is OH or OR A where R A is saturated C 1-4 alkyl (preferably methyl); or (b)R 10 and R 11 form a nitrogen-carbon double bond between the nitrogen atom and the carbon atom to which they are attached; or (c)R 10 One of them is H and the other is R 11 is OH or OR A where R A is saturated C 1-4 alkyl (preferably methyl); and R 10 the other and R 11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are attached.

[0155] The PBD dimer of Formula I or Ia (or a pharmaceutical salt, solvate, or solvate of a salt thereof) is typically linked to an antibody via a linker unit, LU. The linker unit acts to release the PBD dimer of Formula I or Ia (or a pharmaceutical salt, solvate, or solvate of a salt thereof) at a target site (e.g., inside a cancer cell). PBD drug-linker compounds for use in the present invention are represented below by Formula II (preferred stereochemistry is shown in IIa), where LU is the linker unit. The linker unit can be, for example, a cleavable peptide linker unit (e.g., a linker comprising a valine-alanine peptide) or a cleavable disulfide linker unit: [ka] or a pharmaceutical salt, solvate, or solvate of the salt, wherein the subscript n is 1 or 3.

[0156] Preferred PBD drug-linker compounds for use in the present invention are represented by Formula III below: [ka] or a pharmaceutical salt, solvate, or solvate of a salt, wherein the subscript n is 1 or 3 and the subscript m is an integer from 2 to 5.

[0157] The PBD drug-linker is conjugated to an anti-CD228 antibody to generate a CD228-targeted antibody drug conjugate. For example, the antibody can be conjugated with a drug linker of Formula II or Formula III. Exemplary CD228 (C2248)-targeted antibody drug conjugates are shown below in Formulas IV, IVa, and IVb: [ka] or a pharmaceutical salt, solvate, or solvate of a salt, wherein the subscript n is 1 or 3, the subscript m is an integer from 2 to 5, and the subscript p is 1 to 4.

[0158] Exemplary drug linkers include MMAE drug linkers. The inventors have found that incorporating a polyethylene glycol polymer as a side chain into a cleavable β-glucuronide MMAE drug linker results in antibody drug conjugates with reduced plasma clearance and increased anti-tumor activity in xenograft models when compared to non-PEGylated controls. Thus, a particularly useful drug linker for attaching the antibodies of the invention is represented by the following formula V: [ka] or a pharmaceutically acceptable salt thereof.

[0159] A preferred stereochemistry for such a drug linker is shown below in Formula Va: [ka] or a pharmaceutically acceptable salt thereof, wherein, with respect to Formulas V and Va, Z represents an organic moiety having a reactive site capable of reacting with a functional group on an antibody to form a covalent bond therewith; n is in the range of 8 to 36, most preferably in the range of 8 to 14 (most preferably 12); R 21 is a capping unit of a polyethylene glycol moiety, preferably -CH3 or -CH2CH2CO2H.

[0160] Preferred Z moieties are maleimide-containing moieties. Particularly preferred Z moieties are the following drug-linkers: [ka] or a pharmaceutically acceptable salt thereof.

[0161] The preferred stereochemistry for such drug linkers is as follows: [ka] or a pharmaceutically acceptable salt thereof, wherein, for Formulas VI, VIa, VII, and VIIa, n is in the range of 8 to 36, most preferably in the range of 8 to 14 (most preferably 12); R PR is hydrogen or a protecting group, e.g., an acid-labile protecting group, e.g., BOC; R 21 is a capping unit of a polyethylene glycol moiety, preferably -CH3 or -CH2CH2CO2H.

[0162] As mentioned above, R PR can be hydrogen or a protecting group. As used herein, a protecting group refers to a group that selectively blocks a reactive site of a multifunctional compound, either temporarily or permanently. A protecting group is suitable if it can be removed under the reaction conditions necessary to effect a desired chemical transformation elsewhere in the molecule, and, if desired, during purification of the newly formed molecule, to prevent or avoid undesired side reactions or premature loss of the protecting group, and under conditions that do not adversely affect the structural or stereochemical integrity of the newly formed molecule. Suitable amine protecting groups include acid-labile nitrogen protecting groups, including those provided by Isidro-Llobel et al. "Amino acid-protecting groups" Chem. Rev. (2009) 109: 2455-2504. Typically, acid-labile nitrogen protecting groups convert a primary or secondary amino group to its corresponding carbamate and include t-butyl, allyl, and benzyl carbamates.

[0163] As mentioned above, R 21 is the capping unit of the polyethylene glycol moiety. As will be appreciated by those skilled in the art, the polyethylene glycol unit can be end-capped with a wide variety of organic moieties, typically those that are relatively unreactive. Alkyl and substituted alkyl groups are preferred.

[0164] Generally, 1 to 16 drug linkers are attached to each antibody.

[0165] When referring to a CD228-targeting antibody-drug conjugate, the subscript p represents the drug loading, which may represent the number of drug linker molecules attached to an individual antibody molecule, in which case it is an integer value, or it represents the average drug loading, in which case it may be an integer or non-integer value, but is typically a non-integer value. Average drug loading represents the average number of drug linker molecules per antibody in a population. Often, but not always, when referring to antibodies, e.g., monoclonal antibodies, reference is made to a population of antibody molecules. In a composition comprising a population of antibody-drug conjugate molecules, average drug loading is an important quality attribute because it determines the amount of drug that can be delivered to target cells. The percentage of unconjugated antibody molecules in the composition is included in the average drug loading value.

[0166] In preferred embodiments of the present invention, when referring to a composition comprising a population of antibody drug conjugate compounds, the average drug loading is 1 to about 16, preferably about 2 to about 14, and more preferably about 2 to about 10. For PBD-antibody drug conjugates, such as those exemplified herein, a particularly preferred average drug loading is about 2. In some embodiments, the actual drug loading of individual antibody molecules in a population of antibody drug conjugate compounds is 1 to 4, 1 to 3, or 1 to 2, with a predominant drug loading of 2. In preferred embodiments, an average drug loading of 2 is achieved via site-specific conjugation techniques (e.g., an engineered cysteine ​​introduced into an antibody comprising position 239 according to the EU index numbering system).

[0167] For MMAE-PEGylated ADCs, such as those exemplified herein, a particularly preferred average drug loading is about 8. In exemplary embodiments, the drug linker is conjugated to the cysteine ​​residue of the reduced interchain disulfide. In some aspects, the actual drug loading of individual antibody molecules in a population of antibody-drug conjugate compounds is 1-10 (or 6-10, or 6-8), with a predominant drug loading of 8. For example, higher drug loadings can be obtained when the drug linker is conjugated to an introduced cysteine ​​residue (such as a cysteine ​​residue introduced at position 239 according to the EU index) in addition to the interchain disulfide.

[0168] Exemplary ADCs include: [ka] TIFF0007754715000015.tif156170TIFF0007754715000016.tif187170, or a pharmaceutically acceptable salt thereof, wherein n is in the range of 8 to 36, most preferably in the range of 8 to 14 (most preferably 12); PR is hydrogen or a protecting group, e.g., an acid-labile protecting group, e.g., BOC; R 21 is a capping unit of a polyethylene glycol moiety, preferably -CH3 or -CH2CH2CO2H; Ab represents an anti-CD228 antibody; p represents an integer ranging from 1 to 16, preferably 1 to 14, 6 to 12, 6 to 10, or 8 to 10, when referring to an individual antibody molecule, or an average drug loading of about 4 or about 6 to about 14, preferably about 8, when referring to a population of antibody molecules.

[0169] As noted above, the PEG (polyethylene glycol) portion of the drug linker can range from 8 to 36 units, although PEGs with 12 ethylene oxide units have been found to be particularly preferred. It has been found that longer PEG chains may result in slower clearance, while shorter PEG chains may result in reduced activity. Thus, in all of the above embodiments, the subscript n is preferably 8 to 14, 8 to 12, 10 to 12, or 10 to 14, and most preferably 12.

[0170] Polydisperse PEGs, monodisperse PEGs, and individual PEGs can be used to prepare the PEGylated antibody-drug conjugates of the present invention. Polydisperse PEGs are a heterogeneous mixture of sizes and molecular weights, while monodisperse PEGs are typically purified from a heterogeneous mixture and therefore provide a single chain length and molecular weight. A preferred PEG unit is an individual PEG, which is a compound synthesized stepwise and does not undergo a polymerization process. An individual PEG provides a single molecule with a defined and specific chain length. Similar to the subscript "p," when referring to a population of antibody-drug conjugates, the value of the subscript "n" can be an average number and can be an integer or a non-integer.

[0171] In a preferred embodiment, covalent attachment of the antibody to the drug linker is achieved through a sulfhydryl functional group on the antibody, which interacts with a maleimide functional group on the drug linker to form a thio-substituted succinimide. The sulfhydryl functional group can be present on the ligand unit in the ligand's native state, e.g., a naturally occurring residue (interchain disulfide residue), or can be introduced into the ligand via chemical modification, or by biological alteration, or a combination of the two. It is understood that the antibody-substituted succinimide can exist in hydrolyzed form(s). For example, in a preferred embodiment, the ADC, when conjugated to an antibody, [ka] or when attached to an antibody, [ka] where the wavy line indicates the connection to the remainder of the drug-linker.

[0172] Useful classes of cytotoxic agents for conjugation to anti-CD228 antibodies include, for example, antitubulin agents, DNA minor groove binders, DNA replication inhibitors, chemotherapy sensitizers, etc. Examples of other classes of cytotoxic agents include anthracyclines, auristatins, camptothecins, duocarmycins, etoposide, maytansinoids, and vinca alkaloids. Some examples of cytotoxic agents include auristatins (e.g., auristatin T, auristatin E, AFP, monomethylauristatin F (MMAF), lipophilic monomethylauristatin F, monomethylauristatin E (MMAE)), DNA minor groove binding agents (e.g., enediynes and lexitropsins), duocarmycins, taxanes (e.g., paclitaxel and docetaxel), vinca alkaloids, nicotinamide phosphoribosyltransferase inhibitors (NAMPTi), tubulysin M, doxorubicin, morpholinodoxorubicin, and cyanomorpholinodoxorubicin.

[0173] The cytotoxic agent can be a chemotherapeutic agent, such as doxorubicin, paclitaxel, melphalan, a vinca alkaloid, methotrexate, mitomycin C, or etoposide. The drug can also be a CC-1065 analog, a calicheamicin, a maytansine, an analog of dolastatin 10, rhizoxin, or palytoxin.

[0174] The cytotoxic drug may be an auristatin. The auristatin may be an auristatin E derivative, such as an ester formed between auristatin E and a keto acid. For example, auristatin E may be reacted with paraacetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other exemplary auristatins include auristatin T, AFP, MMAF, and MMAE. The synthesis and structures of various auristatins are described, for example, in US 2005-0238649 and US 2006-0074008.

[0175] The cytotoxic agent can be a DNA minor groove binding agent. (See, e.g., U.S. Patent No. 6,130,237.) For example, the minor groove binding agent can be a CBI compound or an enediyne (e.g., calicheamicin).

[0176] The cytotoxic or cytostatic agent can be an antitubulin agent. Examples of antitubulin agents include taxanes (e.g., Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik), vinca alkaloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine), and auristatins (e.g., auristatin E, AFP, MMAF, MMAE, AEB, and AEVB). Examples of auristatins are shown in Formulas III-XIII below. Other suitable antitubulin agents include, for example, baccatin derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine and colcemid, estramustine, cryptophycin, cemadotin, maytansinoids, combretastatins, discodermolide, and eleutherobin.

[0177] The cytotoxic agent can be a maytansinoid, another group of antitubulin agents (e.g., DM1, DM2, DM3, DM4). For example, the maytansinoid can be maytansine or a maytansine containing a drug linker, such as DM-1 or DM-4 (ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res.).

[0178] In some embodiments, the anti-CD228 antibodies of the invention are conjugated to monomethyl auristatin E via a MDpr-PEG(12)-gluc linker and have the structure:

[0179] [ka] (wherein n is in the range of 8 to 36, most preferably in the range of 8 to 14 (most preferably 12), and R PR is hydrogen or a protecting group, for example an acid labile protecting group, for example BOC, and R 21 is a capping unit of a polyethylene glycol moiety, preferably -CH3 or -CH2CH2CO2H; Ab represents an anti-CD228 antibody; and p is an integer ranging from 1 to 16, preferably 1 to 14, 6 to 12, 6 to 10, or 8 to 10, when referring to an individual antibody molecule or an average drug loading of about 4 to about 14 or about 6 to about 14, and preferably about 8 when referring to a population of antibody molecules. or a pharmaceutically acceptable salt thereof. In some embodiments, the anti-CD228 is hL49, and the resulting antibody drug conjugate is hL49-Mdpr-PEG(12)-gluc-MMAE. hL49-Mdpr-PEG(12)-gluc-MMAE is also referred to as hL49-5088. The term hL49-5088(8) refers to hL49-5088 with an average drug loading of about 8 drug-linkers per antibody.

[0180] VIII. Therapeutic applications (therapeutic uses) The antibodies of the present invention, alone or as anti-CD228 antibody-drug conjugates thereof, can be used to treat cancer in a subject. Some such cancers exhibit detectable levels of CD228, measured either at the protein level (e.g., by immunoassay using one of the exemplified antibodies) or at the mRNA level. Some such cancers exhibit elevated CD228 levels compared to noncancerous tissue of the same type, preferably from the same patient. Exemplary levels of CD228 on cancer cells suitable for treatment are 5,000 to 500,000 CD228 molecules per cell, although higher or lower levels may be treated. Optionally, CD228 levels in the cancer are measured before treatment is administered. In some embodiments, the subject has previously been treated with one or more therapeutic agents and has not responded to treatment, and the one or more therapeutic agents are not antibodies, antigen-binding fragments, or antibody-drug conjugates. In some embodiments, the subject has previously been treated with one or more therapeutic agents and has relapsed after treatment, and the one or more therapeutic agents are not antibodies, antigen-binding fragments, or antibody-drug conjugates. In some embodiments, the subject has been previously treated with one or more therapeutic agents and experienced disease progression during treatment, wherein the one or more therapeutic agents are not an antibody, antigen-binding fragment, or antibody-drug conjugate. In some embodiments, the cancer is an advanced cancer. In some embodiments, the advanced cancer is stage 3 or stage 4 cancer. In some embodiments, the advanced cancer is metastatic cancer. In some embodiments, the cancer is a recurrent cancer. In some embodiments, the subject has been pretreated with standard therapy for the cancer and has failed the pretreatment. In some embodiments, the subject is a human.

[0181] Examples of cancers associated with CD228 expression and suitable for treatment include melanoma and other carcinomas, such as pancreatic cancer, lung cancer, including non-small lung cancer, thyroid cancer, esophageal cancer, head and neck cancer, breast cancer, including triple-negative breast cancer, colorectal cancer, mesothelioma, and cholangiocarcinoma. In some embodiments, the antibody or antibody-drug conjugate of the invention is used in a method for treating melanoma in a subject. In some embodiments, the melanoma is cutaneous melanoma. In some embodiments, the cutaneous melanoma is selected from the group consisting of superficial spreading melanoma, nodular melanoma, acral lentiginous melanoma, lentiginous maligna melanoma, and desmoplastic melanoma. In some embodiments, the cutaneous melanoma is superficial spreading melanoma. In some embodiments, the cutaneous melanoma is nodular melanoma. In some embodiments, the cutaneous melanoma is acral lentiginous melanoma. In some embodiments, the acral lentiginous melanoma is subungual melanoma. In some embodiments, the cutaneous melanoma is lentiginous maligna melanoma. In some embodiments, the cutaneous melanoma is desmoplastic melanoma. In some embodiments, the subject has been previously treated with a PD-1 or PD-L1 inhibitor for the cutaneous melanoma. In some embodiments, the subject has been previously treated with a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is selected from the group consisting of nivolumab (OPDIVO®, BMS-936558, or MDX-1106), pembrolizumab (KEYTRUDA®, MK-3475), pidilizumab (CT-011), and cemiplimab (REGN2810). In some embodiments, the subject has been previously treated with a PD-L1 inhibitor. In some embodiments, the PD-L1 inhibitor is selected from the group consisting of atezolizumab (TECENTRIQ®, MPDL3280A), avelumab (BAVENCIO®), durvalumab, and BMS-936559. In some embodiments, the melanoma is subcutaneous melanoma. In some embodiments, the subcutaneous melanoma is ocular melanoma or mucosal melanoma. In some embodiments, the melanoma is non-cutaneous melanoma. In some embodiments, the antibody or antibody-drug conjugate of the invention is used in a method for treating pancreatic cancer in a subject. In some embodiments, the pancreatic cancer is an exocrine cancer or a neuroendocrine cancer.In some embodiments, the pancreatic cancer is an exocrine cancer. In some embodiments, the pancreatic exocrine cancer is selected from the group consisting of pancreatic adenocarcinoma, acinar cell carcinoma, cystadenocarcinoma, pancreatoblastoma, adenosquamous carcinoma, signet ring cell carcinoma, hepatoid carcinoma, colloid carcinoma, undifferentiated carcinoma, and pancreatic mucinous cystic neoplasm. In some embodiments, the subject has received one or more prior lines of therapy for the exocrine pancreatic cancer. In some embodiments, the subject has received one prior line of therapy for the exocrine pancreatic cancer. In some embodiments, the subject has received more than one prior line of therapy for the exocrine pancreatic cancer. In some embodiments, the pancreatic cancer is pancreatic adenocarcinoma. In some embodiments, the pancreatic adenocarcinoma is pancreatic ductal adenocarcinoma. In some embodiments, the pancreatic cancer is acinar cell carcinoma. In some embodiments, the pancreatic cancer is cystadenocarcinoma. In some embodiments, the pancreatic cancer is pancreatoblastoma. In some embodiments, the pancreatic cancer is adenosquamous carcinoma. In some embodiments, the pancreatic cancer is signet ring cell carcinoma. In some embodiments, the pancreatic cancer is hepatoid carcinoma. In some embodiments, the pancreatic cancer is colloid carcinoma. In some embodiments, the pancreatic cancer is undifferentiated carcinoma. In some embodiments, the pancreatic cancer is pancreatic mucinous cystic neoplasm. In some embodiments, the pancreatic cancer is neuroendocrine carcinoma. In some embodiments, the antibody or antibody-drug conjugate of the invention is used in a method of treating lung cancer in a subject. In some embodiments, the antibody or antibody-drug conjugate of the invention is used in a method of treating non-small cell lung cancer in a subject. In some embodiments, the non-small cell lung cancer has a mutated form of epidermal growth factor receptor (EGFR). In some embodiments, the non-small cell lung cancer has wild-type EGFR. In some embodiments, the subject has been previously treated with a platinum-based therapy for the non-small cell lung cancer. In some embodiments, the platinum-based therapy is selected from the group consisting of carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin. In some embodiments, the platinum-based therapy is carboplatin. In some embodiments, the platinum-based therapy is cisplatin.In some embodiments, the platinum-based therapy is oxaliplatin. In some embodiments, the platinum-based therapy is nedaplatin. In some embodiments, the platinum-based therapy is triplatin tetranitrate. In some embodiments, the platinum-based therapy is phenanthriplatin. In some embodiments, the platinum-based therapy is picoplatin. In some embodiments, the platinum-based therapy is satraplatin. In some embodiments, the subject has been previously treated with a PD-1 or PD-L1 inhibitor for non-small cell lung cancer. In some embodiments, the subject has been previously treated with a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is selected from the group consisting of nivolumab (OPDIVO®, BMS-936558 or MDX-1106), pembrolizumab (KEYTRUDA®, MK-3475), pidilizumab (CT-011), and cemiplimab (REGN2810). In some embodiments, the subject has been previously treated with a PD-L1 inhibitor. In some embodiments, the PD-L1 inhibitor is selected from the group consisting of atezolizumab (TECENTRIQ®, MPDL3280A), avelumab (BAVENCIO®), durvalumab, and BMS-936559. In some embodiments, the subject has been previously treated for non-small cell lung cancer with a platinum-based therapy and a PD-1 or PD-L1 inhibitor. In some embodiments, the antibody or antibody drug conjugate of the invention is used in a method of treating thyroid cancer in a subject. In some embodiments, the antibody or antibody drug conjugate of the invention is used in a method of treating esophageal cancer in a subject. In some embodiments, the antibody or antibody drug conjugate of the invention is used in a method of treating head and neck cancer in a subject. In some embodiments, the antibody or antibody drug conjugate of the invention is used in a method of treating breast cancer in a subject. In some embodiments, the breast cancer is selected from the group consisting of HER2-positive, HER2-negative, estrogen receptor (ER)-positive, ER-negative, progesterone receptor (PR)-positive, PR-negative, and triple-negative breast cancer. In some embodiments, the breast cancer is a HER2-positive breast cancer. In some embodiments, the breast cancer is a HER2-negative breast cancer.In some embodiments, the subject has received one or more prior lines of therapy for HER2-negative breast cancer. In some embodiments, the one or more prior lines of therapy include treatment with a taxane. In some embodiments, the taxane is selected from the group consisting of paclitaxel, docetaxel, and cabazitaxel. In some embodiments, the taxane is paclitaxel. In some embodiments, the taxane is docetaxel. In some embodiments, the taxane is cabazitaxel. In some embodiments, the subject with HER2-negative breast cancer is hormone receptor positive. In some embodiments, the subject with HER2-negative hormone receptor-positive breast cancer has been previously treated with a CDK4 / 6 inhibitor. In some embodiments, the subject with HER2-negative hormone receptor-positive breast cancer has been previously treated with hormone-directed therapy. In some embodiments, the breast cancer is ER-positive breast cancer. In some embodiments, the breast cancer is ER-negative breast cancer. In some embodiments, the breast cancer is PR-positive breast cancer. In some embodiments, the breast cancer is PR-negative breast cancer. In some embodiments, the antibody or antibody-drug conjugate of the invention is used in a method for treating triple-negative breast cancer in a subject. Triple-negative breast cancer is a term of art for cancer that lacks detectable estrogen and progesterone receptors and lacks overexpression of HER2 / neu. In some embodiments, the antibody or antibody-drug conjugate of the invention is used in a method for treating colorectal cancer in a subject. In some embodiments, the colorectal cancer is selected from the group consisting of colorectal adenocarcinoma, gastrointestinal stromal tumor, primary colorectal lymphoma, gastrointestinal carcinoid tumor, and leiomyosarcoma. In some embodiments, the colorectal cancer is colorectal adenocarcinoma. In some embodiments, the colorectal cancer is gastrointestinal stromal tumor. In some embodiments, the colorectal cancer is primary colorectal lymphoma. In some embodiments, the colorectal cancer is gastrointestinal carcinoid tumor. In some embodiments, the colorectal cancer is leiomyosarcoma. In some embodiments, the subject has received two or more prior lines of therapy for colorectal cancer. In some embodiments, the subject has received two prior lines of treatment for colorectal cancer.In some embodiments, the subject has received more than two prior lines of therapy for colorectal cancer. In some embodiments, the antibody or antibody-drug conjugate of the invention is used in a method for treating mesothelioma in a subject. In some embodiments, the mesothelioma is selected from the group consisting of pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, and testicular mesothelioma. In some embodiments, the mesothelioma is pleural mesothelioma. In some embodiments, the subject has received prior platinum-based therapy for pleural mesothelioma. In some embodiments, the platinum-based therapy is selected from the group consisting of carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin. In some embodiments, the platinum-based therapy is carboplatin. In some embodiments, the platinum-based therapy is cisplatin. In some embodiments, the platinum-based therapy is oxaliplatin. In some embodiments, the platinum-based therapy is nedaplatin. In some embodiments, the platinum-based therapy is triplatin tetranitrate. In some embodiments, the platinum-based therapy is phenanthriplatin. In some embodiments, the platinum-based therapy is picoplatin. In some embodiments, the platinum-based therapy is satraplatin. In some embodiments, the subject has been previously treated with pemetrexed for pleural mesothelioma. In some embodiments, the mesothelioma is peritoneal mesothelioma. In some embodiments, the mesothelioma is pericardial mesothelioma. In some embodiments, the mesothelioma is testicular mesothelioma. In some embodiments, the antibodies or antibody-drug conjugates of the invention are used in methods of treating cholangiocarcinoma. This treatment can be applied to patients with primary or metastatic tumors of these types. This treatment can also be applied to patients who are refractory to conventional treatments or who have relapsed after responding to such treatments. In some embodiments, the subject is a human.

[0182] The antibodies of the present invention, e.g., humanized antibodies, alone or as conjugates thereof, are administered in an effective dosing regimen, meaning a dosage, route of administration, and frequency of administration that delays onset, reduces severity, inhibits further progression, and / or ameliorates at least one sign or symptom of cancer. If the patient already has cancer, the dosing regimen may be referred to as a therapeutically effective regimen. If the patient is at elevated risk for cancer compared to the general population but has not yet experienced symptoms, the dosing regimen may be referred to as a prophylactically effective regimen. In some cases, a therapeutic or prophylactic effect may be observed in an individual patient compared to historical controls or previous experience in the same patient. In other examples, therapeutic or prophylactic efficacy may be demonstrated in a population of treated patients compared to a control population of untreated patients in preclinical or clinical trials.

[0183] Exemplary dosages of monoclonal antibodies are 0.1 mg / kg to 50 mg / kg, more typically 1 mg / kg to 30 mg / kg, 1 mg / kg to 20 mg / kg, 1 mg / kg to 15 mg / kg, 1 mg / kg to 12 mg / kg, or 1 mg / kg to 10 mg / kg, 1 or 2 mg / kg to 30 mg / kg, 2 mg / kg to 20 mg / kg, 2 mg / kg to 15 mg / kg, 2 mg / kg to 12 mg / kg, or 2 mg / kg to 10 mg / kg, or 3 mg / kg to 30 mg / kg, 3 mg / kg to 20 mg / kg, 3 mg / kg to 15 mg / kg, 3 mg / kg to 12 mg / kg, or 3 mg / kg to 10 mg / kg based on the patient's body weight. Exemplary dosages for monoclonal antibodies or antibody-drug conjugates are 1 mg / kg to 7.5 mg / kg, or 2 mg / kg to 7.5 mg / kg, or 3 mg / kg to 7.5 mg / kg, or 0.1 to 20, or 0.5 to 5 mg / kg body weight (e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg), or 10 to 1500 or 200 to 1500 mg as a fixed dose based on the patient's body weight. In some methods, patients are administered a dosage of at least 1.5 mg / kg, at least 2 mg / kg, or at least 3 mg / kg, administered one or more times every three weeks. Dosage depends on, among other factors, the frequency of administration, the patient's condition, and response to prior therapy, if any, whether the treatment is prophylactic or therapeutic, and whether the disease is acute or chronic.

[0184] Administration can be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Administration can also be directly localized, for example, within a tumor. Administration into the systemic circulation via intravenous or subcutaneous administration is preferred. Intravenous administration can be by infusion over a period of time, such as 30 to 90 minutes, or by a single bolus injection.

[0185] The frequency of administration depends, inter alia, on the half-life of the antibody or conjugate in the circulation, the patient's condition, and the route of administration. The frequency can be daily, weekly, monthly, every three months, or at irregular intervals depending on changes in the patient's condition or the progression of the cancer being treated. An example of the frequency of intravenous administration is, for example, twice a week to four times a year over a course of treatment, although more frequent or less frequent administrations are also possible. Another example of the frequency of intravenous administration is, for example, once a week to three times every four weeks over a course of treatment, although more frequent or less frequent administrations are also possible. For subcutaneous administration, the frequency of administration can be, for example, daily to monthly, although more frequent or less frequent administrations are also possible.

[0186] The number of doses will depend on the characteristics of the cancer (e.g., acute or chronic) and the response of the disease to treatment. For acute disease or acute exacerbations of chronic disease, 1 to 10 doses are often sufficient. For acute disease or acute exacerbations of chronic disease, a single bolus dose, or optionally divided doses, may be sufficient. Treatment can be repeated for recurrence of acute disease or acute exacerbations. For chronic disease, the antibody can be administered at regular intervals, e.g., weekly, biweekly, monthly, every three months, or every six months, for at least one, five, or ten years, or for the life of the patient.

[0187] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., single-dose dosage form). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, excipients, additives, or adjuvants. The formulation will depend on the route of administration chosen. For injection, antibodies can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to reduce discomfort at the injection site). Solutions can contain formulating agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, antibodies can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use. The antibody concentration in liquid formulations can be, for example, 1-100 mg / ml, e.g., 10 mg / ml.

[0188] Treatment with the antibodies of the invention can be combined with chemotherapy, radiation, stem cell therapy, surgery, or other treatments effective against the disease being treated. Other useful classes of drugs that can be administered with antibodies and antibody drug conjugates to CD228 as described herein include, for example, antibodies to other receptors expressed on cancer cells, antitubulin agents (e.g., auristatins), DNA minor groove binding agents, DNA replication inhibitors, alkylating agents (e.g., platinum complexes, such as cisplatin, mono(platinum), bis(platinum), and trinuclear platinum complexes, and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluoropyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, pre-forming compounds, purine antimetabolites, puromycins, radiosensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, and the like.

[0189] Treatment with an anti-CD228 antibody or antibody drug conjugate (optionally in combination with any of the other agents or regimens described above, either alone or as an antibody drug conjugate) can increase the median progression-free survival or overall survival of patients with tumors (e.g., melanoma, pancreatic cancer, non-small cell lung cancer, thyroid cancer, head and neck cancer, triple-negative breast cancer, colorectal cancer, mesothelioma, cholangiocarcinoma), particularly in relapsed or refractory cases, by at least 30% or 40%, preferably 50%, 60% to 70%, or even 100% or more, compared to the same treatment (e.g., chemotherapy) but without the anti-CD228 antibody (alone or as a conjugate). Additionally or alternatively, a treatment (e.g., standard chemotherapy) comprising an anti-CD228 antibody (alone or as a conjugate) can increase the complete response rate, partial response rate, or objective response rate (complete plus partial) in tumor-bearing patients by at least 30% or 40%, preferably 50%, 60% to 70%, or even 100%, compared to the same treatment (e.g., chemotherapy) but without the anti-CD228 antibody (alone or as a conjugate).

[0190] Typically, in a clinical trial (e.g., a Phase II, Phase II / III, or Phase III trial), the increase in median progression-free survival and / or response rate for patients treated with standard of care plus an anti-CD228 antibody (alone or as a conjugate) relative to a control group of patients receiving standard of care alone (or plus a placebo) is statistically significant, e.g., at the p = 0.05 or 0.01, or even 0.001 level. Complete response and partial response rates are determined by objective criteria routinely used in cancer clinical trials, e.g., as described or approved by the National Cancer Institute and / or the Food and Drug Administration.

[0191] IX. Products or Kits In another aspect, an article of manufacture or kit is provided that includes an anti-CD228 antibody or anti-CD228 antibody drug conjugate described herein. The article of manufacture or kit can further include instructions for using the anti-CD228 antibody or anti-CD228 antibody drug conjugate described herein in the methods of the invention. Thus, in certain embodiments, the article of manufacture or kit includes instructions for using the anti-CD228 antibody or anti-CD228 antibody drug conjugate described herein in a method for treating cancer (e.g., melanoma and other carcinomas, including pancreatic cancer, non-small cell lung cancer, thyroid cancer, head and neck cancer, breast cancer (such as triple-negative breast cancer), colorectal cancer, mesothelioma, or cholangiocarcinoma) in a subject, comprising administering to the subject an effective amount of an anti-CD228 antibody or anti-CD228 antibody drug conjugate described herein. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is thyroid cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the breast cancer is triple-negative breast cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is mesothelioma. In some embodiments, the cancer is cholangiocarcinoma. In some embodiments, the subject is a human.

[0192] The product or kit may further comprise a container. Suitable containers include, for example, bottles, vials (e.g., dual-chamber vials), syringes (such as single- or dual-chamber syringes), and test tubes. In some embodiments, the container is a vial. The container can be made of various materials, such as glass or plastic. The container holds the formulation.

[0193] The article of manufacture or kit can further include a label or package insert, which can be on or associated with the container and provide instructions for reconstitution and / or use of the formulation. The label or package insert can further indicate that the formulation is useful for or intended for subcutaneous administration, intravenous administration, or other mode of administration for the treatment of cancer in a subject (e.g., melanoma and other carcinomas, e.g., pancreatic cancer, non-small cell lung cancer, thyroid cancer, head and neck cancer, breast cancer (such as triple-negative breast cancer), colorectal cancer, mesothelioma, or cholangiocarcinoma). The container holding the formulation can be a single-use or multi-use vial, which allows for repeated administration of the reconstituted formulation. The article of manufacture or kit can further include a second container containing a suitable diluent. The article of manufacture or kit can further include other materials desirable from a commercial, therapeutic, and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0194] The articles of manufacture or kits herein optionally further comprise a container containing a second medicament, wherein the anti-CD228 antibody or anti-CD228 antibody drug conjugate is a first medicament, and the articles of manufacture or kits further comprise instructions on the label or package insert for treating a subject with an effective amount of the second medicament. In some embodiments, the second medicament is for eliminating or reducing the severity of one or more adverse events.

[0195] In some embodiments, the anti-CD228 antibody or anti-CD228 antibody-drug conjugate is present in a container as a lyophilized powder. In some embodiments, the lyophilized powder is packaged in a sealed container, such as a vial, ampule, or sachet, indicating the quantity of active agent. If the medicament is administered by injection, an ampule of sterile water for injection or saline, for example, can optionally be provided as part of the kit so that the components can be mixed prior to administration. Such kits can optionally further include one or more of a variety of conventional pharmaceutical components, such as, for example, a container with one or more pharmaceutically acceptable carriers, additional containers, etc., as would be readily apparent to one of skill in the art. Printed instructions, such as a package insert or label, indicating the amounts of components to be administered, administration guidelines, and / or mixing guidelines for the components can also be included in the kit.

[0196] X. Other Uses The anti-CD228 antibodies described herein, e.g., humanized anti-CD228 antibodies, can be used to detect CD228 in clinical diagnostic or therapeutic or research contexts. Expression of CD228 in a cancer indicates that the cancer is amenable to treatment with the antibodies of the present invention. The antibodies can also be sold as research reagents for studies related to the detection of CD228-bearing cells and their responses to various stimuli. For such uses, the monoclonal antibodies can be labeled with fluorescent molecules, spin-labeled molecules, enzymes, or radioisotypes and provided in the form of kits containing all the necessary reagents to assay for CD228. The antibodies described herein can be used to detect CD228 protein expression and determine whether a cancer is amenable to treatment with a CD228 ADC. As an example, hL49(HALC) can be used to detect CD228 expression in melanoma cells, pancreatic cancer cells, non-small cell lung cancer cells, thyroid cancer cells, and head and neck cancer cells. The antibodies can also be used to purify CD228, for example, by affinity chromatography.

[0197] All patent applications, websites, other publications, accession numbers, etc., listed above or below, are herein incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. Where different versions of a sequence are associated with accession numbers from different times, the version associated with the accession number as of the effective filing date of this application is meant. The effective filing date means the earlier of the filing date or, if applicable, the filing date of the priority application that references that accession number. Similarly, where different versions of a publication, website, etc. are published at different times, the most recently published version as of the effective filing date of this application is meant unless otherwise specified. Any feature, step, element, embodiment, or aspect of the present invention can be used in combination with any other, unless specifically stated otherwise. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications can be practiced within the scope of the appended claims.

[0198] XI. Variable Domain Sequences For each of the variable region sequences below, the CDRs according to the Kabat numbering scheme are underlined and those according to the IMGT numbering scheme are shown in bold and italics.

[0199] Mouse L49 vH [ka]

[0200] Mu IGHV3-8 vH [ka]

[0201] Hu IGHV4-59 / HJ4 [ka]

[0202] hvHA

change

[0203] hhB

change

[0204] hv

change

[0205] L49

change

[0206] Mu IGKV1-110 vL

change

[0207] Hu IGKV2-30 / KJ2

change

[0208] hhL

change

[0209] hvL

change

[0210] LC

change

[0211] XII. Illustrative Embodiments Among the embodiments provided herein are the following: 1. An isolated anti-CD228 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 and the light chain variable region comprises: (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6 An antibody or antigen-binding fragment thereof comprising: 2. The antibody or antigen-binding fragment of embodiment 1, wherein the antibody is humanized. 3. A humanized anti-CD228 antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:7, with the proviso that position H27 is occupied by D, position H30 is occupied by T, position H47 is occupied by Y, position H71 is occupied by R, and position H78 is occupied by Y; and a light chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:8, with the proviso that position L2 is occupied by F, position L36 is occupied by Y, and position L46 is occupied by L. 4. The antibody or antigen-binding fragment of embodiment 3, further provided that position L28 is occupied by D. 5. A humanized anti-CD228 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the three Kabat CDRs of SEQ ID NO: 7, in which position H27 is occupied by D, position H30 is occupied by T, position H47 is occupied by Y, position H71 is occupied by R, and position H78 is occupied by Y, and a light chain variable region comprising the three Kabat CDRs of SEQ ID NO: 8, in which position L2 is occupied by F, position L36 is occupied by Y, and position L46 is occupied by L. 6. The antibody or antigen-binding fragment of any of embodiments 1 to 5, wherein the heavy chain variable region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 8. 7. The antibody or antigen-binding fragment of any of embodiments 1 to 5, wherein the heavy chain variable region comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 8. 8. The antibody or antigen-binding fragment of any of embodiments 1 to 5, wherein the heavy chain variable region comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 8. 9. The antibody or antigen-binding fragment of any of embodiments 1 to 5, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8. 10. The antibody or antigen-binding fragment of any of embodiments 1 to 9, which is an antigen-binding fragment. 11. The antibody or antigen-binding fragment of embodiment 10, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fab'-SH, Fv, diabody, linear antibody, and single-chain antibody fragment. 12. The antibody or antigen-binding fragment of any of embodiments 1 to 9, which is a full-length antibody. 13. The antibody or antigen-binding fragment of embodiment 12, wherein the heavy chain variable region is fused to a heavy chain constant region and the light chain variable region is fused to a light chain constant region. 14. The antibody or antigen-binding fragment of embodiment 13, wherein the heavy chain constant region is of the IgG1 isotype. 15. The antibody or antigen-binding fragment of embodiment 13 or 14, wherein the heavy chain constant region has an amino acid sequence comprising SEQ ID NO: 17, and the light chain constant region has an amino acid sequence comprising SEQ ID NO: 18. 16. The antibody or antigen-binding fragment of embodiment 13 or 14, wherein the heavy chain constant region is a mutated form of a native human constant region that has reduced binding to Fc gamma receptors compared to the native human constant region. 17. The antibody or antigen-binding fragment of embodiment 13 or 14, wherein the heavy chain constant region has an amino acid sequence comprising SEQ ID NO: 19 (S239C), and the light chain constant region has an amino acid sequence comprising SEQ ID NO: 18. 18. An antibody drug conjugate comprising the antibody or antigen-binding fragment of any of embodiments 1 to 17, conjugated to a cytotoxic or cytostatic drug. 19. The antibody drug conjugate of embodiment 18, wherein the antibody or antigen-binding fragment is conjugated to the cytotoxic or cytostatic drug via a linker. 20. The antibody drug conjugate of embodiment 19, wherein the linker is an MDpr-PEG(12)-gluc linker. 21. The antibody drug conjugate of any of embodiments 18-20, wherein the cytotoxic or cytostatic agent is monomethyl auristatin. 22. The antibody drug conjugate of embodiment 21, wherein the monomethyl auristatin is monomethyl auristatin E (MMAE). 23. A linker is attached to monomethyl auristatin E to form a compound having the structure: [ka] where Ab is antibody hL49, n is 12, and R PR is hydrogen and R 21 is CH3, and p is a number from 1 to 16. 23. The antibody drug conjugate of embodiment 22, wherein the antibody drug conjugate comprises: 24. The antibody drug conjugate of embodiment 23, wherein the average value of p in the population of antibody drug conjugates is about 8. 25. The antibody drug conjugate of any of embodiments 18 to 24, wherein the antibody drug conjugate is hL49-MDpr-PEG(12)-gluc-MMAE.

[0212] 26. A nucleic acid encoding the heavy chain variable region and / or the light chain variable region according to any one of embodiments 1 to 17. 27. A vector comprising the nucleic acid of embodiment 26. 28. The vector of embodiment 27, which is an expression vector. 29. A host cell comprising a nucleic acid according to embodiment 26. 30. The host cell of embodiment 29, which is a Chinese hamster ovary (CHO) cell. 31. A method for producing an anti-CD228 antibody or antigen-binding fragment thereof, comprising culturing a host cell of embodiment 29 or embodiment 30 under conditions suitable for producing the anti-CD228 antibody or antigen-binding fragment thereof. 32. The method of embodiment 31, further comprising isolating the anti-CD228 antibody or antigen-binding fragment thereof produced by the host cell.

[0213] 33. A method for producing an anti-CD228 antibody drug conjugate, comprising culturing a host cell of embodiment 29 or embodiment 30 under conditions suitable for producing an anti-CD228 antibody, isolating the anti-CD228 antibody produced from the host cell, and conjugating the anti-CD228 antibody to a cytotoxic or cytostatic drug. 34. The method of embodiment 33, wherein the anti-CD228 antibody is conjugated to the cytotoxic or cytostatic drug via a linker. 35. The method of embodiment 34, wherein the linker is an MDpr-PEG(12)-gluc linker. 36. The method of any of embodiments 33-35, wherein the cytotoxic or cytostatic agent is monomethyl auristatin. 37. The method of embodiment 36, wherein the monomethyl auristatin is monomethyl auristatin E (MMAE). 38. A linker is attached to monomethyl auristatin E to form a compound having the structure: [ka] where Ab is antibody hL49, n is 12, and R PR is hydrogen and R 21 is CH3, and p is a number from 1 to 16. 38. The method of embodiment 37, wherein the antibody drug conjugate is formed having the formula: 39. The method of embodiment 38, wherein the average value of p in the population of antibody drug conjugates is about 8. 40. The method of any of embodiments 33-39, wherein the antibody drug conjugate is hL49-MDpr-PEG(12)-gluc-MMAE.

[0214] 41. A method for treating cancer in a subject, comprising administering to the subject an antibody or antigen-binding fragment of any of embodiments 1-17, or an antibody-drug conjugate of any of embodiments 18-25. 42. The method of embodiment 41, wherein the subject has previously been treated with one or more therapeutic agents and has not responded to the treatment, and the one or more therapeutic agents are not said antibody, antigen-binding fragment, or antibody-drug conjugate. 43. The method of embodiment 41, wherein the subject has previously been treated with one or more therapeutic agents and has relapsed after treatment, and the one or more therapeutic agents are not said antibody, antigen-binding fragment, or antibody-drug conjugate. 44. The method of embodiment 41, wherein the subject has previously been treated with one or more therapeutic agents and experienced disease progression during treatment, and the one or more therapeutic agents are not said antibody, antigen-binding fragment, or antibody-drug conjugate. 45. The method of any one of embodiments 41-44, wherein the cancer is an advanced stage cancer. 46. ​​The method of embodiment 45, wherein the advanced cancer is stage 3 or stage 4 cancer. 47. The method of embodiment 45 or 46, wherein the advanced cancer is metastatic cancer. 48. The method of any one of embodiments 41-47, wherein the cancer is a recurrent cancer. 49. The method of any of embodiments 41-48, wherein the cancer is unresectable. 50. The method of any one of embodiments 41-49, wherein the subject has been pretreated with standard therapy for cancer and has failed the pretreatment. 51. The method of any of embodiments 41-50, wherein the cancer is selected from the group consisting of melanoma, pancreatic cancer, mesothelioma, colorectal cancer, lung cancer, thyroid cancer, breast cancer, cholangiocarcinoma, esophageal cancer, and head and neck cancer. 52. The method of embodiment 51, wherein the cancer is melanoma. 53. The method of embodiment 52, wherein the melanoma is cutaneous melanoma. 54. The method of embodiment 53, wherein the cutaneous melanoma is selected from the group consisting of superficial spreading melanoma, nodular melanoma, acral lentiginous melanoma, lentigo maligna melanoma, and desmoplastic melanoma. 55. The method of embodiment 54, wherein the acral lentiginous melanoma is subungual melanoma. 56. The method of any of embodiments 53-55, wherein the subject has undergone prior treatment with an inhibitor of PD-1 or PD-L1. 57. The method of embodiment 56, wherein the subject has undergone prior treatment with a PD-1 inhibitor. 58. The method of embodiment 52, wherein the melanoma is subcutaneous melanoma. 59. The method of embodiment 58, wherein the subcutaneous melanoma is ocular melanoma or mucosal melanoma. 60. The method of embodiment 52, wherein the melanoma is non-cutaneous melanoma. 61. The method of embodiment 51, wherein the cancer is mesothelioma. 62. The method of embodiment 61, wherein the mesothelioma is selected from the group consisting of pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, and testicular mesothelioma. 63. The method of embodiment 62, wherein the mesothelioma is pleural mesothelioma. 64. The method of embodiment 63, wherein the subject has been pretreated with a platinum-based therapy. 65. The method of embodiment 64, wherein the platinum-based therapy is cisplatin. 66. The method of any of embodiments 63-65, wherein the subject has been pretreated with pemetrexed. 67. The method of embodiment 51, wherein the lung cancer is non-small cell lung cancer. 68. The method of embodiment 67, wherein the non-small cell lung cancer has a mutated form of epidermal growth factor receptor (EGFR). 69. The method of embodiment 67, wherein the non-small cell lung cancer has wild-type EGFR. 70. The method of embodiment 69, wherein the subject has been pretreated with a platinum-based therapy. 71. The method of embodiment 69 or 70, wherein the subject has undergone prior treatment with an inhibitor of PD-1 or PD-L1. 72. The method of embodiment 71, wherein the subject has undergone prior treatment with an inhibitor of PD-1. 73. The method of embodiment 51, wherein the breast cancer is selected from the group consisting of HER2-positive, HER2-negative, estrogen receptor (ER)-positive, ER-negative, progesterone receptor (PR)-positive, PR-negative, and triple-negative breast cancer. 74. The method of embodiment 73, wherein the breast cancer is HER2-negative breast cancer. 75. The method of embodiment 74, wherein the subject has received one or more prior lines of treatment for HER2-negative breast cancer. 76. The method of embodiment 75, wherein one or more prior lines of treatment include treatment with a taxane. 77. The method of embodiment 75 or 76, wherein the subject is hormone receptor positive. 78. The method of embodiment 77, wherein the subject has undergone prior treatment with an inhibitor of CDK4 / 6. 79. The method of embodiment 77 or 78, wherein the subject has been pre-treated with hormone-directed therapy. 80. The method of embodiment 51, wherein the colorectal cancer is selected from the group consisting of colorectal adenocarcinoma, gastrointestinal stromal tumor, primary colorectal lymphoma, gastrointestinal carcinoid tumor, and leiomyosarcoma. 81. The method of embodiment 80, wherein the subject has undergone two or more prior lines of treatment for colorectal cancer. 82. The method of embodiment 51, wherein the pancreatic cancer is an exocrine cancer or a neuroendocrine cancer. 83. The method of embodiment 82, wherein the exocrine cancer is selected from the group consisting of pancreatic adenocarcinoma, acinar cell carcinoma, cystadenocarcinoma, pancreatoblastoma, adenosquamous carcinoma, signet ring cell carcinoma, hepatoid carcinoma, colloid carcinoma, undifferentiated carcinoma, and pancreatic mucinous cystic neoplasm. 84. The method of embodiment 83, wherein the pancreatic adenocarcinoma is pancreatic ductal adenocarcinoma. 85. The method of embodiment 83 or 84, wherein the subject has undergone one or more prior lines of treatment for pancreatic cancer. 86. The method of any of embodiments 41-85, wherein the antibody or antigen-binding fragment or antibody drug conjugate is in a pharmaceutical composition comprising the antibody or antigen-binding fragment or antibody drug conjugate and a pharmaceutically acceptable carrier. 87. The method of any one of embodiments 41-86, wherein the subject is a human.

[0215] 88. (a) an antibody or antigen-binding fragment according to any one of embodiments 1 to 17, or an antibody-drug conjugate according to any one of embodiments 18 to 25, and (b) Instructions for using the antibody or antigen-binding fragment or antibody-drug conjugate according to the method of any one of embodiments 41 to 87. Kit including:

[0216] 89. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any of embodiments 1-17, or the antibody-drug conjugate of any of embodiments 18-25, and one or more agents selected from the group consisting of physiologically acceptable carriers, diluents, excipients and adjuvants.

[0217] 90. The antibody or antigen-binding fragment of any of embodiments 1 to 17, or the antibody-drug conjugate of any of embodiments 18 to 25, for use in treating cancer in a subject. 91. The antibody or antigen-binding fragment of embodiment 90, wherein the subject has previously been treated with one or more therapeutic agents and has not responded to the treatment, and the one or more therapeutic agents are not the antibody, antigen-binding fragment, or antibody-drug conjugate. 92. The antibody or antigen-binding fragment of embodiment 90, wherein the subject has previously been treated with one or more therapeutic agents and has relapsed after treatment, and the one or more therapeutic agents are not the antibody, antigen-binding fragment, or antibody-drug conjugate. 93. The antibody or antigen-binding fragment of embodiment 90, wherein the subject has previously been treated with one or more therapeutic agents and experienced disease progression during treatment, and the one or more therapeutic agents are not the antibody, antigen-binding fragment, or antibody-drug conjugate. 94. The antibody or antigen-binding fragment of any of embodiments 90-93, wherein the cancer is an advanced stage cancer. 95. The antibody or antigen-binding fragment of embodiment 94, wherein the advanced cancer is stage 3 or stage 4 cancer. 96. The antibody or antigen-binding fragment of embodiment 94 or 95, wherein the advanced cancer is metastatic cancer. 97. The antibody or antigen-binding fragment of any of embodiments 90-96, wherein the cancer is a recurrent cancer. 98. The antibody or antigen-binding fragment of any of embodiments 90-97, wherein the cancer is unresectable. 99. The antibody or antigen-binding fragment of any of embodiments 90-98, wherein the subject has been pretreated with standard therapy for cancer and has failed the pretreatment. 100. The antibody or antigen-binding fragment of any of embodiments 90-99, wherein the cancer is selected from the group consisting of melanoma, pancreatic cancer, mesothelioma, colorectal cancer, lung cancer, thyroid cancer, breast cancer, cholangiocarcinoma, esophageal cancer, and head and neck cancer. 101. The antibody or antigen-binding fragment of embodiment 100, wherein the cancer is melanoma. 102. The antibody or antigen-binding fragment of embodiment 101, wherein the melanoma is cutaneous melanoma. 103. The antibody or antigen-binding fragment of embodiment 102, wherein the cutaneous melanoma is selected from the group consisting of superficial spreading melanoma, nodular melanoma, acral lentiginous melanoma, lentigo maligna melanoma, and desmoplastic melanoma. 104. The antibody or antigen-binding fragment of embodiment 103, wherein the acral lentiginous melanoma is subungual melanoma. 105. The antibody or antigen-binding fragment of any of embodiments 102-104, wherein the subject has received prior treatment with an inhibitor of PD-1 or PD-L1. 106. The antibody or antigen-binding fragment of embodiment 105, wherein the subject has undergone prior treatment with a PD-1 inhibitor. 107. The antibody or antigen-binding fragment of embodiment 101, wherein the melanoma is subcutaneous melanoma. 108. The antibody or antigen-binding fragment of embodiment 107, wherein the subcutaneous melanoma is ocular melanoma or mucosal melanoma. 109. The antibody or antigen-binding fragment of embodiment 101, wherein the melanoma is a non-cutaneous melanoma. 110. The antibody or antigen-binding fragment of embodiment 100, wherein the cancer is mesothelioma. 111. The antibody or antigen-binding fragment of embodiment 110, wherein the mesothelioma is selected from the group consisting of pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, and testicular mesothelioma. 112. The antibody or antigen-binding fragment of embodiment 111, wherein the mesothelioma is pleural mesothelioma. 113. The antibody or antigen-binding fragment of embodiment 112, wherein the subject has been pretreated with a platinum-based therapy. 114. The antibody or antigen-binding fragment of embodiment 113, wherein the platinum-based therapy is cisplatin. 115. The antibody or antigen-binding fragment of any of embodiments 112-114, wherein the subject has been previously treated with pemetrexed. 116. The antibody or antigen-binding fragment of embodiment 100, wherein the lung cancer is non-small cell lung cancer. 117. The antibody or antigen-binding fragment of embodiment 116, wherein the non-small cell lung cancer has a mutated form of the epidermal growth factor receptor (EGFR). 118. The antibody or antigen-binding fragment of embodiment 116, wherein the non-small cell lung cancer has wild-type EGFR. 119. The antibody or antigen-binding fragment of embodiment 118, wherein the subject has been pretreated with a platinum-based therapy. 120. The antibody or antigen-binding fragment of embodiment 118 or 119, wherein the subject has undergone prior treatment with an inhibitor of PD-1 or PD-L1. 121. The antibody or antigen-binding fragment of embodiment 120, wherein the subject has undergone prior treatment with an inhibitor of PD-1. 122. The antibody or antigen-binding fragment of embodiment 100, wherein the breast cancer is selected from the group consisting of HER2-positive, HER2-negative, estrogen receptor (ER)-positive, ER-negative, progesterone receptor (PR)-positive, PR-negative, and triple-negative breast cancer. 123. The antibody or antigen-binding fragment of embodiment 122, wherein the breast cancer is HER2-negative breast cancer. 124. The antibody or antigen-binding fragment of embodiment 123, wherein the subject has received one or more prior lines of treatment for HER2-negative breast cancer. 125. The antibody or antigen-binding fragment of embodiment 124, wherein one or more prior lines of therapy comprise treatment with a taxane. 126. The antibody or antigen-binding fragment of embodiment 124 or 125, wherein the subject is hormone receptor positive. 127. The antibody or antigen-binding fragment of embodiment 126, wherein the subject has undergone prior treatment with an inhibitor of CDK4 / 6. 128. The antibody or antigen-binding fragment of embodiment 126 or 127, wherein the subject has been pre-treated with hormone-directed therapy. 129. The antibody or antigen-binding fragment of embodiment 128, wherein the colorectal cancer is selected from the group consisting of colorectal adenocarcinoma, gastrointestinal stromal tumor, primary colorectal lymphoma, gastrointestinal carcinoid tumor, and leiomyosarcoma. 130. The antibody or antigen-binding fragment of embodiment 129, wherein the subject has received two or more prior lines of treatment for colorectal cancer. 131. The antibody or antigen-binding fragment of embodiment 100, wherein the pancreatic cancer is an exocrine cancer or a neuroendocrine cancer. 132. The antibody or antigen-binding fragment of embodiment 131, wherein the exocrine cancer is selected from the group consisting of pancreatic adenocarcinoma, acinar cell carcinoma, cystadenocarcinoma, pancreatoblastoma, adenosquamous carcinoma, signet ring cell carcinoma, hepatoid carcinoma, colloid carcinoma, undifferentiated carcinoma, and pancreatic mucinous cystic neoplasm. 133. The antibody or antigen-binding fragment of embodiment 132, wherein the pancreatic adenocarcinoma is pancreatic ductal adenocarcinoma. 134. The antibody or antigen-binding fragment of embodiment 132 or 133, wherein the subject has received one or more prior lines of treatment for pancreatic cancer. 135. The antibody or antigen-binding fragment of any of embodiments 90-134, wherein the antibody or antigen-binding fragment or antibody drug conjugate is in a pharmaceutical composition comprising the antibody or antigen-binding fragment or antibody drug conjugate and a pharmaceutically acceptable carrier. 136. The antibody or antigen-binding fragment of any of embodiments 90-135, wherein the subject is a human.

[0218] 137. Use of the antibody or antigen-binding fragment according to any of embodiments 1 to 17, or the antibody-drug conjugate according to any of embodiments 18 to 25, for the manufacture of a medicament for treating cancer in a subject. 138. The use of embodiment 137, wherein the subject has previously been treated with one or more therapeutic agents and has not responded to the treatment, and the one or more therapeutic agents are not the antibody, antigen-binding fragment, or antibody-drug conjugate. 139. The use of embodiment 137, wherein the subject has previously been treated with one or more therapeutic agents and has relapsed after treatment, and the one or more therapeutic agents are not the antibody, antigen-binding fragment, or antibody-drug conjugate. 140. The use of embodiment 137, wherein the subject has previously been treated with one or more therapeutic agents and experienced disease progression during treatment, and the one or more therapeutic agents are not said antibody, antigen-binding fragment, or antibody-drug conjugate. 141. The use according to any of embodiments 137 to 140, wherein the cancer is an advanced stage cancer. 142. The use according to embodiment 141, wherein the advanced cancer is stage 3 or stage 4 cancer. 143. The use according to embodiment 141 or 142, wherein the advanced cancer is metastatic cancer. 144. The use according to any of embodiments 137 to 143, wherein the cancer is a recurrent cancer. 145. The use according to any of embodiments 137 to 144, wherein the cancer is unresectable. 146. The use according to any of embodiments 137 to 145, wherein the subject has been pretreated with standard therapy for cancer and has failed the pretreatment. 147. The use according to any of embodiments 137 to 146, wherein the cancer is selected from the group consisting of melanoma, pancreatic cancer, mesothelioma, colorectal cancer, lung cancer, thyroid cancer, breast cancer, cholangiocarcinoma, esophageal cancer, and head and neck cancer. 148. The use according to embodiment 147, wherein the cancer is melanoma. 149. The use according to embodiment 148, wherein the melanoma is cutaneous melanoma. 150. The use according to embodiment 149, wherein the cutaneous melanoma is selected from the group consisting of superficial spreading melanoma, nodular melanoma, acral lentiginous melanoma, lentigo maligna melanoma, and desmoplastic melanoma. 151. The use according to embodiment 150, wherein the acral lentiginous melanoma is subungual melanoma. 152. The use according to any of embodiments 149-151, wherein the subject has undergone prior treatment with an inhibitor of PD-1 or PD-L1. 153. The use according to embodiment 152, wherein the subject has undergone prior treatment with a PD-1 inhibitor. 154. The use according to embodiment 148, wherein the melanoma is subcutaneous melanoma. 155. The use according to embodiment 154, wherein the subcutaneous melanoma is ocular melanoma or mucosal melanoma. 156. The use according to embodiment 148, wherein the melanoma is non-cutaneous melanoma. 157. The use according to embodiment 147, wherein the cancer is mesothelioma. 158. The use according to embodiment 157, wherein the mesothelioma is selected from the group consisting of pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, and testicular mesothelioma. 159. The use according to embodiment 158, wherein the mesothelioma is pleural mesothelioma. 160. The use of embodiment 159, wherein the subject has been pretreated with a platinum-based therapy. 161. The use according to embodiment 160, wherein the platinum-based therapy is cisplatin. 162. The use according to any of embodiments 158-161, wherein the subject has been pretreated with pemetrexed. 163. The use according to embodiment 147, wherein the lung cancer is non-small cell lung cancer. 164. The use according to embodiment 163, wherein the non-small cell lung cancer has a mutated form of the epidermal growth factor receptor (EGFR). 165. The use according to embodiment 163, wherein the non-small cell lung cancer has wild-type EGFR. 166. The use of embodiment 165, wherein the subject has been pretreated with a platinum-based therapy. 167. The use according to embodiment 165 or 166, wherein the subject has undergone prior treatment with an inhibitor of PD-1 or PD-L1. 168. The use according to embodiment 167, wherein the subject has undergone prior treatment with an inhibitor of PD-1. 169. The use according to embodiment 147, wherein the breast cancer is selected from the group consisting of HER2-positive, HER2-negative, estrogen receptor (ER)-positive, ER-negative, progesterone receptor (PR)-positive, PR-negative, and triple-negative breast cancer. 170. The use according to embodiment 169, wherein the breast cancer is HER2-negative breast cancer. 171. The use of embodiment 170, wherein the subject has received one or more prior lines of treatment for HER2-negative breast cancer. 172. The use according to embodiment 171, wherein one or more prior lines of treatment include treatment with a taxane. 173. The use according to embodiment 171 or 172, wherein the subject is hormone receptor positive. 174. The use according to embodiment 173, wherein the subject has undergone prior treatment with an inhibitor of CDK4 / 6. 175. The use according to embodiment 173 or 174, wherein the subject has been previously treated with hormone-directed therapy. 176. The use according to embodiment 147, wherein the colorectal cancer is selected from the group consisting of colorectal adenocarcinoma, gastrointestinal stromal tumor, primary colorectal lymphoma, gastrointestinal carcinoid tumor, and leiomyosarcoma. 177. The use of embodiment 176, wherein the subject has received two or more prior lines of treatment for colorectal cancer. 178. The use according to embodiment 147, wherein the pancreatic cancer is an exocrine or neuroendocrine cancer. 179. The use according to embodiment 178, wherein the exocrine cancer is selected from the group consisting of pancreatic adenocarcinoma, acinar cell carcinoma, cystadenocarcinoma, pancreatoblastoma, adenosquamous carcinoma, signet ring cell carcinoma, hepatoid carcinoma, colloid carcinoma, undifferentiated carcinoma, and pancreatic mucinous cystic neoplasm. 180. The use according to embodiment 179, wherein the pancreatic adenocarcinoma is pancreatic ductal adenocarcinoma. 181. The use of embodiment 179 or 180, wherein the subject has undergone one or more prior lines of treatment for pancreatic cancer. 182. The use according to any of embodiments 137 to 181, wherein the antibody or antigen-binding fragment or antibody-drug conjugate is in a pharmaceutical composition comprising the antibody or antigen-binding fragment or antibody-drug conjugate and a pharmaceutically acceptable carrier. 183. The use according to any of embodiments 137 to 182, wherein the subject is a human.

[0219] The present invention will be more fully understood by reference to the following examples, which, however, should not be construed as limiting the scope of the present invention. It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the scope of the claims. [Example]

[0220] [Example 1] CD288 expression in cancer cell lines Quantification of CD228 copy numbers on the cell surface of various cancer cell lines was determined using the DAKO QiFiKit flow cytometry indirect assay as described by the manufacturer (DAKO A / S, Glostrup, Denmark) using mouse CD228 mAb as the primary antibody and evaluated using an Attune NxT flow cytometer. The resulting numbers of CD228 molecules expressed per cell are shown in Table 1.

[0221] [Table 1] TIFF0007754715000035.tif87161

[0222] [Example 2] Immunohistochemical analysis of CD228 expression Tumor tissue arrays were obtained from commercial sources. Tumor formalin-fixed and paraffin-embedded (FFPE) tissues were purchased from US Biomax Inc. All samples were processed with a Bond-Max™ automated stainer (Leica).

[0223] FFPE slides sectioned on glass slides were deparaffinized and rehydrated using Bond™ Dewax solution (Leica, catalog no. AR9222) at 72°C. Antigen retrieval was performed using the EDTA-based Bond™ Epitope Retrieval Solution 2 (Leica, catalog no. AR9640) at 95–100°C for 20 min before incubation with a primary anti-CD228 antibody (Sigma, catalog no. HPA004880). Isotype-matched rabbit IgG1 was used as a negative control for background staining. Automated IHC staining was performed using either the Refine DAB kit or an alkaline phosphatase-based detection kit: the Bond™ Polymer AP Red Detection Kit (Leica, catalog no. DS9305). Slides were incubated with a rabbit monoclonal primary antibody against rabbit CD228 mAb at 1 μg / ml for 45 min, followed by a 30-min protein block (DAKO, catalog no. X0909). After chromogenic development, sections were counterstained with hematoxylin and coverslipped. Slides were evaluated and scored by a pathologist and imaged using a Zeiss Axiovert 200M microscope (Carl Zeiss, Inc., Thornwood, NY).

[0224] FIG. 1 shows the high levels of CD228 expression in melanoma cancer patient samples, providing a strong rationale for treating these tumors with CD228 ADCs.

[0225] Figure 2 shows high levels of CD228 expression in mesothelioma cancer patient samples, providing a strong rationale for treating these tumors with CD228 ADCs.

[0226] Figure 3 shows the high levels of CD228 expression in colorectal cancer patient samples, providing a strong rationale for treating these tumors with CD228 ADCs.

[0227] Figure 4 shows high levels of CD228 expression in triple-negative (HR-, PgR-, Her2-) breast cancer patient samples (top panel) and Her2-HR+ breast cancer patient samples (bottom panel), providing a strong rationale for treating these tumors with CD228 ADCs.

[0228] Figure 5 shows high levels of CD228 expression in pancreatic cancer patient samples, providing a strong rationale for treating these tumors with CD228 ADCs.

[0229] Figure 6 shows high levels of CD228 expression in squamous cell non-small cell lung cancer patient samples (top panel) and adenocarcinoma non-small cell lung cancer patient samples (bottom panel), providing a strong rationale for treating these tumors with CD228 ADCs.

[0230] Summary immunohistochemistry experiments were compared with CD228 RNA levels reported by The Cancer Genome Atlas for various tumor types. We determined the threshold for CD228 RNA positivity by applying the IHC prevalence of melanoma to TCGA. As can be seen in Figure 7, most tumor types have a close correlation between RNA expression and immunohistochemistry, except for HER2- / HR+ breast cancer. TNBC = triple-negative breast cancer. NSCLC = non-small cell lung cancer. Adeno = adenocarcinoma. Squamous = squamous cell carcinoma. TCGA = Cancer Genome Atlas.

[0231] [Example 3] Anti-CD228 antibody-drug conjugate Various anti-CD228 antibodies were conjugated to the drug MMAE via the linker MDpr-PEG(12)-gluc, resulting in an average drug loading of approximately 8 per antibody. The conjugation method is described in U.S. Patent Application Publication No. 2018 / 0092984. Tumor cells were incubated with CD228 antibody-drug conjugates (ADCs) for 96 to 144 hours at 37°C. A human IgG ADC was used as a negative control. Cell viability was measured using Cell Titer Glo according to the manufacturer's instructions. Fluorescence signals were measured using a Fusion HT fluorescent plate reader (Perkin Elmer, Waltham, MA). Data were normalized to untreated cells, and x50 values ​​were calculated using GraphPad software. Results were expressed as IC, the concentration of compound required to produce a 50% reduction in viability compared to vehicle-treated cells (control = 100%). 50 are reported in Table 2. The chimeric L49 (cL49) and murine (mL49) ADCs outperformed all other anti-CD228 ADCs, especially in cell lines with low CD228 expression.

[0232] [Table 2]

[0233] [Example 4] Humanization of mouse L49 antibody The murine antibody mL49 (Siemers et al., 1997, Bioconjug. Chem. 8:510-9) was used as the starting or donor antibody for humanization. Suitable human acceptor sequences were the genomic sequences provided by hIGHV4-59 and hIGHJ4 for the heavy chain and by hIGKV2-30 and hIGKJ2 for the light chain. The human acceptor sequences show 70 percent (heavy chain) and 84 percent (light chain) identity with the donor sequences in the variable region frameworks, when the CDRs are defined according to the Kabat numbering scheme.

[0234] Alignment of the donor sequences identified 26 positions in the heavy chain and 13 positions in the light chain where the human acceptor framework sequence differed from the donor framework sequence, and which, when CDRs are defined according to the Kabat numbering scheme, may directly contact the antigen, affect the conformation of the CDR, or affect the packing between the heavy and light chains, thereby affecting antibody binding. Three humanized heavy chains (HA, HB, and HC) and three humanized light chains (LA, LB, and LC) were generated by incorporating back mutations at different permutations of specific positions. See Figures 8-11 and Tables 3-6.

[0235] [Table 3]

[0236] [Table 4]

[0237] [Table 5]

[0238] [Table 6]

[0239] Next, we expressed humanized antibodies representing all permutations of these chains (nine possibilities) for the humanized heavy and light chains. After one week of incubation at the temperature and pH conditions shown in Table 7, we then compared the antibodies using peptide map analysis of the labile chemical modifications found in the L2 peptide for hL49 HALB (potential Asn(N)-asparagine deamidation) and hL49 HALC (potential Asp(D)-aspartic acid isomerization). hL49 HALC(N28D) eliminates the deamidation observed in hL49 HALB and also limits isomerization. Overall L2 peptide modifications are reduced from 13% to 2%.

[0240] [Table 7]

[0241] The binding curves of each antibody obtained were determined by competitive binding assay. Briefly, 1 × 10 antibodies stably expressing human CD228 were cultured per well of a 96-well v-bottom plate on ice. 5 RPMI-7951 cells were aliquoted. The cells were incubated for 1 hour with 5 nM AlexaFluor-647 (AF)-labeled parental mouse CD228 mAb and increasing concentrations (0.06 nM to 1000 nM) of unlabeled humanized CD228 mAb, along with various combinations of the humanized light chain LA-LB and the humanized heavy chain HA-HC. The cells were pelleted and washed three times with PBS / BSA. The cells were pelleted and resuspended in 125 μL of PBS / BSA. Fluorescence was analyzed by flow cytometry using the percent saturated fluorescent signal to determine the percent bound labeled mouse CD228 mAb. Binding curves for the recombinant human anti-CD228 antibodies are shown in Figures 12A-12F.

[0242] Next, the K for each antibody obtained D The binding affinity of human CD228 was determined by saturation binding assay. Briefly, 1 × 10 cells stably expressing human CD228 were cultured per well of a 96-well v-bottom plate. 5RPMI-7951 cells were aliquoted. Each CD228 antibody was added at concentrations ranging from 0.05 pM to 340 nM and incubated on ice for 60 minutes. Cells were pelleted and washed three times with PBS / BSA, followed by the addition of 10 μg / ml PE-labeled goat anti-human IgG secondary antibody and further incubation on ice for 60 minutes. Cells were pelleted, washed three times with PBS / BSA, and resuspended in 125 μL of PBS / BSA. Fluorescence was analyzed by flow cytometry using the percent of saturated fluorescent signal to determine percent binding, followed by the apparent K D The binding curves of the recombinant human anti-CD228 antibodies are shown in Figure 13, and the K values ​​of cL49ec (chimeric L49 with a S239C mutation in the light chain constant region), hL49 HALA G1, hL49 HALB G1, and hL49 HALC G1 were calculated. D are shown in Table 8. An antibody designated "HALC," "hL49," or "hL49-HALC," containing a heavy chain "HA" and a light chain "LC," was selected for use in all other experiments.

[0243] [Table 8]

[0244] [Example 5] hL49-HALC antibody drug conjugates with various drug linkers A. Antibody-Drug Conjugation hL49-HALC was conjugated to eight loads of either MDpr-PEG(12)-gluc-MMAE, auristatin T, tubulysin M, or lipophilic MMAF, two loads of either MC-VC-MMAE or MDpr-gluc-MMAE, or two loads of PBD. The conjugation method is described in U.S. Patent Application Publication No. 2018 / 0092984. All commercially available anhydrous solvents were used without further purification. PEG reagents were obtained from Quanta BioDesign (Powell, Ohio). Analytical thin-layer chromatography was performed on silica gel 60 F254 aluminum sheets (EMD Chemicals, Gibbstuwn, NJ). Radial chromatography was performed on a Chromatotron instrument (Harris Research, Palo Alto, Calif.). Column chromatography was performed on a Biotage Isolera One Flash Purification System (Charlotte, NC). Analytical HPLC was performed on a Varian ProStar 210 solvent delivery system equipped with a Varian ProStar 330 PDA detector. Samples were eluted on a C12 Phenomenex Synergi 2.0 × 150 mm, 4 μm, 80 Å reverse-phase column. The acidic mobile phase consisted of acetonitrile and water, both containing 0.05% trifluoroacetic acid or 0.1% formic acid (as indicated for each compound). Compounds were eluted with a linear gradient of acidic acetonitrile from 5% at 1 min to 95% at 11 min after injection, followed by an isocratic gradient of 95% acetonitrile up to 15 min (flow rate = 1.0 mL / min). LC-MS was performed on two different systems. LC-MS System 1 consisted of a ZMD Micromass mass spectrometer connected to an HP Agilent 1100 HPLC instrument equipped with a C12 Phenomenex Synergi 2.0 × 150 mm, 4 μm, 80 Å reverse-phase column. The acidic eluent consisted of a linear gradient of 5 to 95% acetonitrile over 10 min in 0.1% aqueous formic acid, followed by 5 min of isocratic 95% acetonitrile (flow rate = 0.4 mL / min).LC-MS System 2 consisted of a Waters Xevo G2 Tof mass spectrometer connected to a Waters 2695 Separation Module equipped with a Waters 2996 Photodiode Array Detector; the column, mobile phase, gradient, and flow rate were the same as those in LC-MS System 1. UPLC-MS was performed on a Waters SQ mass detector connected to an Acquity Ultra Performance LC equipped with an Acquity UPLC BEH C18 2.1 × 50 mm, 1.7 μm reversed-phase column. The acidic mobile phase (0.1% formic acid) consisted of a gradient of 3% acetonitrile / 97% water to 100% acetonitrile (flow rate = 0.5 mL / min). Preparative HPLC was performed on a Varian ProStar 210 solvent delivery system equipped with a Varian ProStar 330 PDA detector. The product was purified on a C12 Phenomenex Synergi 10.0 × 250 mm, 4 μm, 80 Å reverse-phase column eluting with 0.1% formic acid / water (solvent A) and 0.1% formic acid / acetonitrile (solvent B). The purification method consisted of the following gradient from solvent A to solvent B: 90:10 from 0 to 5 min; 90:10 to 10:90 from 5 to 80 min; followed by an isocratic 10:90 for 5 min. The flow rate was 4.6 mL / min with monitoring at 254 nm. Preparative HPLC for the compounds of Schemes 3 and 4 was performed using 0.1% trifluoroacetic acid in both mobile phases instead of 0.1% formic acid.

[0245] [ka]

[0246] (2S,3S,4S,5R,6S)-6-(2-(3-aminopropanamido)-4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-di Methyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (2): To a flask containing the known (Compound 8a of U.S. Patent Application Publication No. 2008 / 0241128 A1) glucuronide-MMAE intermediate 2 (40 mg, 26.8 μmol), 0.9 mL of methanol and 0.9 mL of tetrahydrofuran were added. The solution was then cooled in an ice bath, and lithium hydroxide monohydrate (6.8 mg, 161 μmol) was added dropwise as a solution in 0.9 mL of water. The reaction was then stirred on ice for 1.5 hours, at which point LC / MS indicated complete conversion to the product. Glacial acetic acid (9.2 μL, 161 μmol) was then added, and the reaction was concentrated to dryness. Preparative HPLC afforded the fully deprotected glucuronide-MMAE linker intermediate 3 (26 mg, 87%) as an oily residue. Analytical HPLC (0.1% formic acid): R 9.3 min. LC-MS System 1:t R 11.10 min, m / z(ES + ) Measured value 1130.48 (M+H) + , m / z(ES - ) Measurement value 1128.63 (MH) - .

[0247] [ka]

[0248] (S)-44-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39-azapentatetracontan-45-oic acid (4). To a flask containing N-Fmoc-lysine 3 (59 mg, 161 μmol), 2.9 mL of anhydrous dichloromethane was added, followed by methoxy-PEG-OSu (100 mg, 146 μmol). DIPEA (127 μL, 730 μmol) was then added, and the reaction was stirred under nitrogen at room temperature, followed by TLC and LC / MS. After 2 h, LC / MS showed conversion to product. The reaction solution was diluted in dichloromethane and purified by silica gel chromatography. The stationary phase was eluted with dichloromethane containing increasing amounts of methanol (0 to 20%) to give the desired product 4 (153 mg, 112%). UPLC-MS: R 1.77 min, m / z (ES + ) Measured value 939.58 (M+H) + .

[0249] (S)-2,5-Dioxopyrrolidin-1-yl 44-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39-azapentatetracontan-45-oate (5): A flask was charged with N-Fmoc-lysine(PEG)-OH (153 mg, 163 μmol) and 1.6 mL of anhydrous tetrahydrofuran. N-Hydroxoisuccinimide (28 mg, 245 μmol) was added, followed by diisopropylcarbodiimide (38 μL, 245 μmol). The reaction was sealed under nitrogen and stirred overnight. The crude reaction was diluted in dichloromethane and purified on silica gel, eluting with dichloromethane containing increasing amounts of methanol (0 to 10%) to give the desired activated ester 5 (155 mg). The material was carried forward without further characterization. UPLC-MS: R 1.92 min, m / z (ES + ) Measured value 1036.48 (M+H) + .

[0250] (2S,3S,4S,5R,6S)-6-(2-((S)-44-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-38,45-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39,46-diazanonatetracontanamide)-4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)- (1-Methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (6): The deprotected glucuronide-MMAE linker intermediate 2 (92 mg, 81 μmol) was dissolved in anhydrous dimethylformamide (1.6 mL) and added to a flask containing N-Fmoc-lysine(PEG)-OSu 5 (101 mg, 97 μmol). Next, diisopropylethylamine (70 μL, 405 μmol) was added, and the reaction was stirred at room temperature under nitrogen. After 4.5 h, LC-MS showed conversion to product. The product was purified by preparative HPLC to give Fmoc-Lys(PEG)-glucuronide-MMAE intermediate 6 (111 mg, 62% over two steps) as an oily residue. UPLC-MS: R 2.01 min, m / z (ES + ) Measured value 2050.92 (M+H) + .

[0251] (2S,3S,4S,5R,6S)-6-(2-((S)-44-amino-38,45-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39,46-diazanonatetracontanamide)-4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxo (isopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (7): Fmoc-Lys(PEG12)-glucuronide-MMAE intermediate 6 (111 mg, 54 μmol) was dissolved in 2.2 mL of anhydrous dimethylformamide, followed by the addition of 0.5 mL of piperidine. The reaction was stirred under nitrogen for 3 hours and then concentrated to dryness. The product was purified by preparative HPLC to give H-Lys(PEG12)-glucuronide-MMAE intermediate 7 (85 mg, 86%) as an oily residue. UPLC-MS: R 1.50 min, m / z (ES + ) Measured value 1829.31 (M+H) + .

[0252] (S)-2,5-Dioxopyrrolidin-1-yl 3-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (9): (S)-Nα-maleimido-Nβ-Boc-diaminopropanoic acid 8 (Nature Biotechnology, 2014, 32, 1059-1062) (400 mg, 1.4 mmol) was dissolved in 7 mL of anhydrous dimethylformamide. N-Hydroxysuccinimide (178 mg, 1.5 mmol) was added, followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (298 mg, 1.5 mmol). The reaction was stirred under nitrogen at room temperature for 3 hours. Aqueous workup was performed by diluting with 120 mL of water, and then the aqueous layer was extracted three times with 60 mL of ethyl acetate. The combined organic layers were then washed with brine, dried over sodium sulfate, and concentrated to dryness. The product was purified by flash column chromatography eluting with a mixture of hexane:ethyl acetate (50:50 to 0:100) to give (S)-Nα-maleimido-Nβ-Boc-diaminopropanoic acid NHS ester [MDpr(Boc)-OSu]9 (297 mg, 55%). LC-MS system 1:t R 12.23 min, m / z (ES + ) Measured value 282.0599 (M+H-Boc group) + LC-MS system 2:t R 11.30 min, m / z(ES + ) Measured value 2580.2515 (M+H) + .

[0253] [ka]

[0254] (2R / S,3S,4S,5R,6S)-6-(2-((S)-44-((S)-3-((tert-butoxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamide)-38,45-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39,46-diazanonatetracontanamide)-4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl (amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (10): MDpr(Boc)-OSu9 (20 mg, 53 μmol) was dissolved in 2.2 mL of anhydrous dimethylformamide and added to a flask containing H-Lys(PEG)-glucuronide-MMAE linker intermediate 7 (86 mg, 44 μmol). Next, diisopropylethylamine (15 μL, 88 μmol) was added, and the reaction was stirred under nitrogen at room temperature for 2.5 h. The reaction was quenched with 15 μL of glacial acetic acid and purified by preparative HPLC to give MDpr(Boc)-Lys(PEG)-glucuronide-MMAE intermediate 10 (37 mg, 40%) as a mixture of diastereomers. The diastereomers were separated by chiral chromatography. UPLC-MS: R 1.84 min, m / z (ES + ) Measured value 2095.44 (M+H) + .

[0255] (2R / S,3S,4S,5R,6S)-6-(2-((S)-44-((R)-3-amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamide)-38,45-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39,46-diazanonatetracontanamide)-4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenyl (Propan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (11). A flask containing MDpr(Boc)-Lys(PEG12)-glucuronide-MMAE intermediate 10 (34 mg, 16 μmol) was cooled to 0 °C in an ice bath under nitrogen. A 10% trifluoroacetic acid in dichloromethane solution (0.8 mL) was added dropwise. The reaction was then stirred at 0 °C for 2 h, at which point LC-MS indicated complete Boc deprotection. The reaction was then concentrated to a crude residue and purified by preparative HPLC to give MDpr-Lys(PEG)-glucuronide-MMAE linker 11 (22 mg, 68%). UPLC-MS: R 1.50 min, m / z (ES+) measured 1995.18 (M+H) + .

[0256] Compound 11 was conjugated via its interchain thiol to an anti-CD228 antibody using methods known in the art (see, e.g., U.S. Patent No. 7,659,241) with an average drug loading of 8 drugs per antibody.

[0257] B. Cytotoxicity of hL49-HALC ADC in vitro Tumor cells were incubated with each antibody-drug conjugate (ADC) for 96 to 144 hours at 37°C. A nonbinding ADC (referred to as h00 or IgG) was used as a negative control. Cell viability was measured using Cell Titer Glo according to the manufacturer's instructions. Fluorescence signals were measured using a Fusion HT fluorescent plate reader (Perkin Elmer, Waltham, MA). Data were normalized to untreated cells, and x50 values ​​were calculated using GraphPad software. Results were expressed as IC, the concentration of compound required to produce a 50% reduction in viability compared to vehicle-treated cells (control = 100%). 50 The IC50 values ​​are reported in Table 9. The hL49 ADC demonstrated single-digit ng / ml IC50 values ​​across a range of cell lines with CD228 expression ranging from 16,000 to 450,000. 50 Achieve value.

[0258] [Table 9]

[0259] In a similar experiment, the percentage of viable cells was determined after treatment with various concentrations of hL49 conjugated to different drug linkers with different amounts of MMAE. The resulting percentage of viable cells for A2058 cells treated with various antibody-drug conjugate (ADC) concentrations of hL49-MC-val-cit-PAB-MMAE (4), hL49-MP-gluc-MMAE (4), and hL49-MP-gluc-MMAE (8) is shown in Figure 14A. Eight-loaded MP-gluc-MMAE outperforms four-loaded MP-gluc-MMAE and the MC-val-cit-PAB-MMAE drug linker in vitro. Four-loaded MP-gluc-MMAE outperforms the MC-val-cit-PAB-MMAE drug linker in vitro, despite containing the same amount of the same drug (MMAE).

[0260] The resulting percent viable cells for A375 cells treated with various antibody-drug conjugate (ADC) concentrations of hL49-MC-val-cit-PAB-MMAE (4), hL49-MP-gluc-MMAE (4), and hL49-MP-gluc-MMAE (8) are shown in Figure 14B. Eight-loaded MP-gluc-MMAE outperforms four-loaded MP-gluc-MMAE and the MC-val-cit-PAB-MMAE drug linker in vitro. Four-loaded MP-gluc-MMAE outperforms the MC-val-cit-PAB-MMAE drug linker in vitro, despite containing the same amount of the same drug (MMAE).

[0261] The resulting percent viable cells for Colo-853 cells treated with various antibody-drug conjugate (ADC) concentrations of hL49-MC-val-cit-PAB-MMAE (4), hL49-MP-gluc-MMAE (4), and hL49-MP-gluc-MMAE (8) are shown in Figure 14C. 8-loaded MP-gluc-MMAE outperforms 4-loaded MP-gluc-MMAE and MC-val-cit-PAB-MMAE drug linkers in vitro.

[0262] C. In vivo activity of anti-CD228 ADCs with different drug linkers Nude (nu / nu) mice (7–8 mice / group) were treated with 5 × l0 6 Cultured A2058 tumor cells were implanted in 25% Matrigel. Dosing of 1 mg / kg, 3 mg / kg, or 6 mg / kg of the test ADC was performed until tumors reached 100 mm 3 The tumor volume was monitored using a caliper and was started when the tumor volume reached approximately 800-1000 mm (4 intraperitoneal injections, once every 4 days). 3 Animals were euthanized when tumor volume reached 0.05. Mean tumor volume plots continued for each group until one or more animals were euthanized. All animal procedures were performed in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility under protocols approved by the Institutional Animal Care and Use Committee.

[0263] The resulting tumor volumes over time for untreated mice and mice treated with 3 mg / kg of hL49, hL49-Auristatin T(8), hL49-Lipophilic MMAF(8), hL49-Tublysin M(8), and hL49-MDpr-PEG(12)-gluc-MMAE(8) are shown in Figure 15. Despite superior potency in vitro, Auristatin T and the lipophilic MMAF ADC were less active in vivo than hL49-MDpr-PEG(12)-gluc-MMAE(8).

[0264] The resulting tumor volumes over time for untreated mice and mice treated with 6 mg / kg IgG-MDpr-gluc-MMAE (2), 6 mg / kg hL49ec-MDpr-gluc-MMAE (2), 3 mg / kg hL49ec-MDpr-gluc-MMAE (2), 1 mg / kg hL49ec-MDpr-gluc-MMAE (2), 3 mg / kg IgG-MDpr-gluc-MMAE (4), 3 mg / kg hL49-MDpr-gluc-MMAE (4), and 3 mg / kg hL49-MDpr-gluc-MMAE (8) are shown in Figure 16. MMAE loaded with 8 PEGs was superior to MMAE loaded with 2 or 4 PEGs in vivo.

[0265] The resulting tumor volumes over time for untreated mice and mice treated with 1 mg / kg hL49-MC-val-cit-PAB-MMAE (4), 3 mg / kg hL49-MC-val-cit-PAB-MMAE (4), 1 mg / kg hL49-MDpr-gluc-MMAE (8), and 3 mg / kg hL49-MDpr-gluc-MMAE (8) are shown in Figure 17. 3 mg / kg hL49-MDpr-gluc-MMAE (8) outperforms 1 mg / kg hL49-MDpr-gluc-MMAE (8) or any concentration of hL49-MC-val-cit-PAB-MMAE (4) in vivo.

[0266] The resulting tumor volumes over time for untreated mice and mice treated with 1 mg / kg hL49-MC-val-cit-PAB-MMAE (4), 3 mg / kg hL49-MC-val-cit-PAB-MMAE (4), 1 mg / kg hL49-MDpr-gluc-MMAE (8), and 3 mg / kg hL49-MDpr-gluc-MMAE (8) are shown in Figure 18. 3 mg / kg hL49-MDpr-gluc-MMAE (8) outperforms 1 mg / kg hL49-MDpr-gluc-MMAE (8) or any concentration of hL49-MC-val-cit-PAB-MMAE (4) in vivo.

[0267] [Example 6] In vivo comparison of ADCs based on tubulysin M and MDpr-PEG(12)-gluc-MMAE hL49 conjugated to tubulysin M or MDpr-PEG(12)-gluc-MMAE demonstrated superior activity against A2058 cells compared with other ADCs, and these ADCs were therefore selected for further evaluation at different doses and with different tumor cell types.

[0268] Nude (nu / nu) mice (6–8 mice / group) were treated with 2.5 × 10 5 Culture A2058, 1 x 10 6 SK-MEL-5, 1 x 10 5 IGR-37, 1 x 10 6 Colo-853 or 1 x 10 6 5 × 10 HPAF-II tumor cells were implanted in 25% Matrigel into NOD / SCID / gc KO (NSG) mice. 5 Each PDX was implanted with 10 ... 3When tumor volume reached approximately 1000 mm, dosing with 0.3 mg / kg, 0.5 mg / kg, 1 mg / kg, or 3 mg / kg of the test ADC was initiated (single intraperitoneal injection). Tumor volume was monitored using calipers, and when tumor volume reached approximately 1000 mm, 3 Animals were euthanized when tumor volume reached 100%. For PDX studies, the study was terminated 28 days after the last dose, regardless of tumor size. Mean tumor volume plots continued for each group until one or more animals were euthanized. All animal procedures were performed in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility under protocols approved by the Institutional Animal Care and Use Committee.

[0269] The A2058 tumor volumes obtained over time for untreated mice and mice treated with 3 mg / kg IgG-MDpr-PEG(12)-gluc-MMAE(8), 3 mg / kg IgG-Tubulysin M(8), 1 mg / kg or 3 mg / kg hL49-Tubulysin M(8), or 1 mg / kg or 3 mg / kg hL49-MDpr-PEG(12)-gluc-MMAE(8) are shown in Figure 19. hL49-Tubulysin M and hL49-MDpr-PEG(12)-gluc-MMAE have similar complete responses (CRs) at 3 mg / kg, while hL49-MDpr-PEG(12)-gluc-MMAE is superior at 1 mg / kg.

[0270] The SK-MEL-5 tumor volumes obtained over time for untreated mice and mice treated with 3 mg / kg IgG-MDpr-PEG(12)-gluc-MMAE(8), 3 mg / kg IgG-Tulysin M(8), 0.3 mg / kg, 1 mg / kg, or 3 mg / kg hL49-Tulysin M(8), or 0.3 mg / kg, 1 mg / kg, or 3 mg / kg hL49-MDpr-PEG(12)-gluc-MMAE(8) are shown in Figure 20. hL49-MDpr-PEG(12)-gluc-MMAE is superior to hL49-Tulysin M for SK-MEL-5 tumors.

[0271] The IGR-37 tumor volumes obtained over time for untreated mice and mice treated with 1 mg / kg or 3 mg / kg of hL49-Tubulysin M(8), or 1 mg / kg or 3 mg / kg of hL49-MDpr-PEG(12)-gluc-MMAE(8) are shown in Figure 21. hL49-MDpr-PEG(12)-gluc-MMAE is superior to hL49-Tubulysin M for IGR-37 tumors.

[0272] The Colo-853 tumor volumes obtained over time for untreated mice and mice treated with 0.3, 1, or 3 mg / kg of hL49-MDpr-PEG(12)-gluc-MMAE(8), or 3 mg / kg of IgG-MDpr-PEG(12)-gluc-MMAE(8) are shown in Figure 22. Colo-853 tumors are responsive to treatment with hL49-MDpr-PEG(12)-gluc-MMAE.

[0273] The LU0697 squamous NSCLC PDX model tumor volumes over time for untreated mice and mice treated with 1 mg / kg or 3 mg / kg hL49-MDpr-PEG(12)-gluc-MMAE(8), or 3 mg / kg IgG-MDpr-PEG(12)-gluc-MMAE(8) are shown in Figure 23. LU0697 squamous NSCL PDX model tumors are responsive to treatment with hL49-MDpr-PEG(12)-gluc-MMAE.

[0274] The LU0697 adenocarcinoma NSCLC PDX model tumor volumes over time for untreated mice and mice treated with 1 mg / kg or 3 mg / kg hL49-MDpr-PEG(12)-gluc-MMAE(8), or 3 mg / kg IgG-MDpr-PEG(12)-gluc-MMAE(8) are shown in Figure 24. LU0697 adenocarcinoma NSCL PDX model tumors are responsive to treatment with hL49-MDpr-PEG(12)-gluc-MMAE.

[0275] MDA-MB-231 TNBC tumor volumes obtained over time for untreated mice and mice treated with 0.5 mg / kg or 1 mg / kg of hL49-MDpr-PEG(12)-gluc-MMAE(8), or 0.5 mg / kg or 1 mg / kg of IgG-MDpr-PEG(12)-gluc-MMAE(8) are shown in Figure 25. MDA-MB-231 TNBC tumors are responsive to treatment with hL49-MDpr-PEG(12)-gluc-MMAE.

[0276] HPAF-II tumor volumes obtained over time for untreated mice and mice treated with 3 mg / kg IgG-MDpr-PEG(12)-gluc-MMAE(8), or 0.3 mg / kg, 1 mg / kg, or 3 mg / kg hL49-MDpr-PEG(12)-gluc-MMAE(8) are shown in Figure 26. HPAF-II tumors are responsive to treatment with hL49-MDpr-PEG(12)-gluc-MMAE.

[0277] [Example 7] Triple-negative breast cancer mouse clinical trial The percent change in tumor volume in response to treatment with hL49-MDpr-PEG(12)-gluc-MMAE(8) was evaluated in 22 different PDX models of triple-negative breast cancer. NCr or nude mice were implanted with an empirically determined amount of tumor cells for each model. A dose of 3 mg / kg of hL49-MDpr-PEG(12)-gluc-MMAE(8) resulted in tumors growing to approximately 150-300 mm. 3Treatment was initiated when the tumor volume reached 100 mg / kg / day. Tumor volume was monitored using calipers. The percent change in tumor volume for each mouse was calculated using hL49-MDpr-PEG(12)-gluc-MMAE(8) either at the time of optimal response or 7 days after dosing and is shown in Figure 27. Treatment with hL49-MDpr-PEG(12)-gluc-MMAE(8) achieved a 60% response rate, with 31% of animals achieving a partial response and 29% achieving a complete response. All animal procedures were performed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care and under protocols approved by the Institutional Animal Care and Use Committee.

[0278] [Example 7-1] Patient-derived xenograft models of various CD228-expressing cancers Further experiments were performed to evaluate the ability of hL49-MDpr-PEG(12)-gluc-MMAE(8) to inhibit tumor growth in various CD228-expressing cancers, as described in Example 7. Patient-derived xenograft models were generated by isolating tumors from 60 human patients (22 patients with triple-negative breast cancer (TNBC), 3 patients with mesothelioma, and 35 patients with non-small cell lung cancer (NSCLC)) and implanting the tumors into immunodeficient mice as described in Example 7. After implantation, mice were treated with a single dose of hL49-MDpr-PEG(12)-gluc-MMAE(8). Blood was collected from mice 48 hours after treatment with hL49-MDpr-PEG(12)-gluc-MMAE(8) and used for pharmacokinetic evaluation. Tumor growth inhibition (TGI percent (%)) and percent change in tumor volume from baseline to best response (percent change Tvol (%)) were assessed in Figures 36A and 36B, respectively. As can be seen, administration of a single dose of hL49-MDpr-PEG(12)-gluc-MMAE(8) had antitumor activity in various tumor models.

[0279] [Example 8] In vitro assessment of antibody effector function Antibody-dependent cellular cytotoxicity (ADCC) activity51 Briefly, tumor cells were inoculated with 100 μCi of NaCl. 51 The cells were labeled with CrO4, washed, and pre-incubated with the test ADC before addition of effector (natural killer, NK) cells. Immunomagnetic beads (EasySep, StemCell Technologies, Vancouver, BC, Canada) were used to isolate NK (CD16)-positive cells from nonadherent peripheral blood mononuclear cells (PBMCs) obtained from a normal FcγRIIIA 158V / V donor (Lifeblood, Memphis, TN). + CD56 + ) cells were prepared. Viable NK cells were added to target cells at an effector-to-target cell ratio of 10:1. Human IgG1κ (Ancell, Bayport, MN) was used as a negative control in this assay. After 4 hours of incubation, the supernatant was collected and dried overnight on a Luma plate. Gamma radiation emitted from lysed cells was then detected using a TopCount Microplate Scintillation and Luminescence Counter (Perkin Elmer, Waltham, Massachusetts). The % specific lysis (ADCC activity) for two patients is shown in Figures 28A-28B. hL49-MDpr-PEG(12)-gluc-MMAE (hL49-5088) reduced ADCC activity compared to hL49 mAb.

[0280] [Example 9] Pharmacokinetic evaluation in mice and rats Nude mice were intravenously administered hL49-MDpr-PEG(12)-gluc-MMAE or cL235-MDpr-PEG(12)-gluc-MMAE, and Sprague-Dawley rats were intravenously administered hL49-MDpr-PEG(12)-gluc-MMAE. Plasma concentrations of the ADC were measured over time using a Tab ELISA with an anti-human antibody as the capture antibody. The results are shown in Figure 29A (mice) and Figure 29B (rats). The resulting PK parameters are shown in Table 10 (mice) and Table 11 (rats).

[0281] [Table 10]

[0282] [Table 11]

[0283] [Example 10] Additional anti-CD228 antibodies Additional anti-CD228 antibodies were conjugated to MDpr-PEG(12)-gluc-MMAE. These additional anti-CD228 antibodies (designated cL235 (see Rolland Y, Pigment Cell Melanoma Res 2009, 22:86-98) and Ab1-9) have binding affinities similar to hL49, unlike the commercially available antibodies tested in Table 2 (Santa Cruz Cat. No. 271633, R&D Cat. No. 893416, and Biolegend Cat. No. 363101).

[0284] A. In Vitro Cytotoxicity Tumor cells were incubated with CD228 antibody-drug conjugates (ADCs) for 96–144 h at 37°C. Nonbinding (h00–5088(8)) ADC was used as a negative control. Cell viability was measured using Cell Titer Glo according to the manufacturer's instructions. Fluorescence signals were measured on a Fusion HT fluorescent plate reader (Perkin Elmer, Waltham, MA). Data were normalized to untreated cells, and x50 values ​​were calculated using GraphPad software. Results were expressed as IC, the concentration of compound required to produce a 50% reduction in viability compared to vehicle-treated cells (control = 100%). 50 The percentage of viable cells remaining at the highest dose is reported in Table 12.

[0285] [Table 12]

[0286] [Table 13]

[0287] B. In vivo activity of additional anti-CD228 ADCs Nude (nu / nu) mice (6 mice / group) were injected with 1 × 10 6 Culture A375, 1 x 10 5 37 IGRs or 2.5 x 10 5 A2058 tumor cells were implanted in 25% Matrigel. 3 When tumor volume reached approximately 800 mm, administration of 1 mg / kg (A2058) or 3 mg / kg of the test ADC was initiated (single intraperitoneal dose injection). Tumor volume was monitored using calipers, and when tumor volume reached approximately 800 mm, 3 Animals were euthanized when tumor volume reached 0.05. Mean tumor volume plots continued for each group until one or more animals were euthanized. All animal procedures were performed in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility under protocols approved by the Institutional Animal Care and Use Committee.

[0288] The resulting A2058 tumor volumes over time for untreated mice and mice treated with various antibodies are shown in Figure 30A. Figure 30B shows the percentage of animals with tumors that increased less than four-fold over time for each treatment condition. The number of complete responses (CRs) and median time to tumor quadrupling for each ADC and each cell line are shown in Table 14.

[0289] [Table 14]

[0290] [Example 11] Linker cleavage and CD228 turnover A fluorescence assay was used to examine the rate of conjugate cleavage over time. The fluorescent moiety AF647 was conjugated to the anti-CD228 antibody hL49 either directly to the eight native cysteines or via a glucuronide linker. Additionally, a quenching reagent, Tide Quencher 5WS succinimidyl ester (TQ5WS), was added via lysine residues, approximately four per antibody. When both the quencher and AF647 are bound to the antibody, fluorescence is quenched. Either cleavage of AF647 from the antibody or degradation of the antibody results in the release of the AF647 molecule from the quencher and a subsequent increase in fluorescence. In both A375 cells (Figure 31A) and Colo-853 cells (Figure 31B), conjugation of AF647 to the antibody via a glucuronide linker results in a more rapid increase in fluorescence activity than direct conjugation of AF647 to the antibody.

[0291] A375 cells were treated with hL49 antibody conjugated to vcQF01, consisting of TQ5WS linked to a Cy5 fluorophore via a Val-Cit-PAB linker, at a ratio of approximately two molecules per antibody. Similar to the reagents described in the previous section, Cy5 remains quenched when intact on the antibody and becomes fluorescent only when cleaved from the TQ5WS quencher. Next, 2 μg / ml of hL49-vcQF01 was added and allowed to bind to the cells. For pulse treatments, the labeled hL49 antibody was washed after 30 minutes to remove unbound labeled hL49 antibody. For continuous treatments with labeled hL49 antibody, unbound labeled hL49 antibody was not washed from the cells. As shown in Figure 32, pulse treatment resulted in a rapid plateau in the Cy5 signal, while continuous exposure to labeled hL49 resulted in a steady increase in the Cy5 fluorophore signal. This demonstrates that additional CD228 can be added to the cell surface over a 24-hour period and then bound by the labeled hL49 antibody, internalized within the cell, and cleaved to release Cy5. In further experiments, the effect of protein synthesis on CD228 binding by fluorescently labeled hL49 antibody was examined by comparing the fluorescence intensity per cell over time in the presence and absence of cycloheximide. Protein synthesis was inhibited using cycloheximide (CHX). An increase in fluorescence signal over time occurred in both Colo-853 cells (Figure 33A) and A375 cells (Figure 33B) in the presence of cycloheximide, but was reduced compared to cells not treated with cycloheximide. This suggests that CD228 is recycled back to the cell surface even in the absence of protein synthesis. Together, these experiments demonstrate that CD228 is recycled and replenished to the cell surface, contributing to antibody-drug conjugate activity.

[0292] [Example 12] pH-dependent binding of ADCs The ability of various anti-CD228 ADCs to bind to CD228 was assessed at pH values ​​ranging from 4 to 7.5 using a standard ELISA protocol. Briefly, 100 ng of human CD228 (R&D Systems Custom02, Lot DCWR021505A) or BSA (Sigma, Catalog No. A7030-100G) was diluted in PBS and added to each well overnight at 4°C. The plate was then washed three times with PBS-T (EMD Millipore, Catalog No. 5246531EA). After washing, the plate was blocked with 3% (w / v) BSA in PBS-T for 1 hour at room temperature. Next, excess blocking buffer was removed, and the primary antibody was added at a 3-fold dilution in dilution buffer (0.15 M citrate-phosphate buffer, pH 4.0–7.5), starting at an antibody concentration of 60 nM. After 1 hour of incubation at room temperature, the plates were washed three times and then incubated with secondary antibody (goat anti-human IgG Fc-specific HRP conjugate, Jackson ImmunoResearch code number 109-035-098) in PBS-T containing 1% BSA. After 30 minutes of incubation at room temperature, the plates were washed three times. 100 μl of TMB substrate (Life Technologies, catalog number 002023) was then added to each well. After 10 minutes of incubation at room temperature, 100 μl of H2SO4 was added to each well to stop the reaction, and the plates were covered with clear plate seals and read on the Envision at 450 nM. The pH-dependent binding of hL49, cL235, CD228Ab1, CD228Ab2, and CD228Ab3, CD228Ab4 is shown in Figures 34A-34F. The resulting EC2 of each ADC was measured. 50 are shown in Table 15. hL49 is the only ADC that exhibits differential binding across the pH gradient.

[0293] [Table 15]

[0294] [Example 13] ADC internalization and catabolism Various additional anti-CD228 antibodies were evaluated for their ability to internalize and catabolize the fluorescent moiety AF647. A375 cells were treated with anti-CD228 antibodies conjugated to QF01, consisting of the quenching agent Tide Quencher 5WS succinimidyl ester (TQ5WS) linked to a Cy5 fluorophore via a glucuronide linker (gluc), at a ratio of approximately two molecules per antibody. Cy5 remains quenched when intact on the antibody and becomes fluorescent only when cleaved from the TQ5WS quencher. The labeled anti-CD228 antibodies were washed after 30 minutes to remove unbound labeled anti-CD228 antibodies. These anti-CD228 antibodies have binding affinities similar to that of hL49. Tumor cells were incubated with the anti-CD228 antibodies, and an imaging assay was performed to determine the fluorescence intensity per cell over time (Figure 35A). Similar experiments were performed using hL49 or other anti-CD228 antibodies conjugated to MDpr-PEG(12)-gluc-MMAE(8). After 24 hours, intracellular drug concentrations were measured for each ADC (Figure 35B). These experiments demonstrate that despite similar binding affinities, some antibodies, such as hL49, internalize faster and deliver drug to a greater extent than other antibodies. This suggests that hL49-MDpr-PEG(12)-gluc-MMAE(8) can deliver drug to tumor cells more effectively than other ADCs. The present invention encompasses, for example, the following embodiments: [Embodiment 1] An isolated anti-CD228 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 and the light chain variable region comprises: (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6 An antibody or antigen-binding fragment thereof comprising: [Embodiment 2] The antibody or antigen-binding fragment of embodiment 1, wherein the antibody is humanized. [Embodiment 3] A humanized anti-CD228 antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 7, with the proviso that position H27 is occupied by D, position H30 is occupied by T, position H47 is occupied by Y, position H71 is occupied by R, and position H78 is occupied by Y; and a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 8, with the proviso that position L2 is occupied by F, position L36 is occupied by Y, and position L46 is occupied by L. [Embodiment 4] The antibody or antigen-binding fragment of embodiment 3, further provided that position L28 is occupied by D. [Embodiment 5] A humanized anti-CD228 antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region comprising the three Kabat CDRs of SEQ ID NO: 7, in which position H27 is occupied by D, position H30 is occupied by T, position H47 is occupied by Y, position H71 is occupied by R, and position H78 is occupied by Y; and a light chain variable region comprising the three Kabat CDRs of SEQ ID NO: 8, in which position L2 is occupied by F, position L36 is occupied by Y, and position L46 is occupied by L. [Embodiment 6] An antibody or antigen-binding fragment described in any one of embodiments 1 to 5, wherein the heavy chain variable region comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 8. [Embodiment 7] An antibody or antigen-binding fragment described in any of embodiments 1 to 5, wherein the heavy chain variable region comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 8. [Embodiment 8] An antibody or antigen-binding fragment described in any of embodiments 1 to 5, wherein the heavy chain variable region comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 8. [Embodiment 9] An antibody or antigen-binding fragment described in any one of embodiments 1 to 5, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8. [Embodiment 10] An antibody or antigen-binding fragment described in any one of embodiments 1 to 9, which is an antigen-binding fragment. [Embodiment 11] The antibody or antigen-binding fragment described in embodiment 10, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fab'-SH, Fv, diabody, linear antibody, and single-chain antibody fragment. [Embodiment 12] The antibody or antigen-binding fragment according to any one of embodiments 1 to 9, which is a full-length antibody. [Embodiment 13] An antibody or antigen-binding fragment described in embodiment 12, in which the heavy chain variable region is fused to a heavy chain constant region and the light chain variable region is fused to a light chain constant region. [Embodiment 14] An antibody or antigen-binding fragment described in embodiment 13, wherein the heavy chain constant region is of the IgG1 isotype. [Embodiment 15] An antibody or antigen-binding fragment described in embodiment 13 or 14, wherein the heavy chain constant region has an amino acid sequence comprising SEQ ID NO: 17 and the light chain constant region has an amino acid sequence comprising SEQ ID NO: 18. [Embodiment 16] An antibody or antigen-binding fragment described in embodiment 13 or 14, wherein the heavy chain constant region is a mutated form of a native human constant region that has reduced binding to Fc gamma receptors compared to the native human constant region. [Embodiment 17] An antibody or antigen-binding fragment described in embodiment 13 or 14, wherein the heavy chain constant region has an amino acid sequence comprising SEQ ID NO: 19 (S239C) and the light chain constant region has an amino acid sequence comprising SEQ ID NO: 18. [Embodiment 18] An antibody-drug conjugate comprising the antibody or antigen-binding fragment of any one of embodiments 1 to 17, conjugated to a cytotoxic drug or a cytostatic drug. [Embodiment 19] An antibody-drug conjugate described in embodiment 18, wherein the antibody or antigen-binding fragment is conjugated to a cytotoxic or cytostatic drug via a linker. [Embodiment 20] The antibody-drug conjugate of embodiment 19, wherein the linker is an MDpr-PEG(12)-gluc linker. [Embodiment 21] The antibody-drug conjugate of any one of embodiments 18 to 20, wherein the cytotoxic or cytostatic drug is monomethyl auristatin. [Embodiment 22] The antibody-drug conjugate of embodiment 21, wherein the monomethyl auristatin is monomethyl auristatin E (MMAE).

[0082] [Embodiment 23] A linker is attached to monomethyl auristatin E to form a compound having the structure:

change

[0082] [Embodiment 38] A linker is attached to monomethyl auristatin E to form a compound having the structure:

change

Claims

1. An anti-CD228 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

8.

2. The antigen-binding fragment of claim 1, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fab'-SH, Fv, diabody, linear antibody, and single-chain antibody fragment.

3. The antibody of claim 1, which is a full-length antibody.

4. The antibody of claim 3, wherein the heavy chain variable region is fused to a heavy chain constant region and the light chain variable region is fused to a light chain constant region.

5. The antibody of claim 4, wherein the heavy chain constant region is of the IgG1 isotype.

6. The antibody of claim 4 or 5, wherein the heavy chain constant region has an amino acid sequence comprising SEQ ID NO: 17 and the light chain constant region has an amino acid sequence comprising SEQ ID NO:

18.

7. 6. The antibody of claim 4 or 5, wherein the heavy chain constant region is a mutated form of a native human constant region that has reduced binding to Fc gamma receptors compared to the native human constant region.

8. The antibody of claim 4 or 5, wherein the heavy chain constant region has an amino acid sequence comprising SEQ ID NO: 19 (S239C) and the light chain constant region has an amino acid sequence comprising SEQ ID NO:

18.

9. An antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 8 conjugated to a cytotoxic or cytostatic drug.

10. 10. The antibody drug conjugate of claim 9, wherein the antibody or antigen-binding fragment thereof is conjugated to a cytotoxic or cytostatic drug via a linker.

11. 11. The antibody drug conjugate of claim 9 or 10, wherein the cytotoxic or cytostatic drug is monomethyl auristatin.

12. 12. The antibody-drug conjugate of claim 11, wherein the monomethyl auristatin is monomethyl auristatin E (MMAE).

13. The linker is attached to monomethyl auristatin E to form the structure: 【Chemical 1】 wherein Ab is the antibody of claim 1, n is 12, and R PR is hydrogen and R 21 is CH 3 where p is a number between 1 and 16.

13. The antibody drug conjugate of claim 12, wherein the antibody drug conjugate comprises:

14. 14. The antibody drug conjugate of claim 13, wherein the average value of p in the population of antibody drug conjugates is 8.

15. A nucleic acid encoding the heavy chain variable region and the light chain variable region according to any one of claims 1 to 8.

16. A vector comprising the nucleic acid of claim 15.

17. A host cell comprising the nucleic acid of claim 16.

18. 20. A method for producing an anti-CD228 antibody or antigen-binding fragment thereof, comprising culturing the host cell of claim 17 under conditions suitable for producing the anti-CD228 antibody or antigen-binding fragment thereof.

19. 20. A method for producing an anti-CD228 antibody drug conjugate, comprising culturing the host cell of claim 17 under conditions suitable for producing an anti-CD228 antibody, isolating the anti-CD228 antibody produced from the host cell, and conjugating the anti-CD228 antibody to a cytotoxic or cytostatic drug.

20. 20. The method of claim 19, wherein the anti-CD228 antibody is conjugated to a cytotoxic or cytostatic drug via a linker.

21. 21. The method of claim 19 or 20, wherein the cytotoxic or cytostatic agent is monomethyl auristatin.

22. The linker is attached to monomethyl auristatin E to form the structure: 【Chemistry 2】 wherein Ab is the antibody of claim 1, n is 12, and R PR is hydrogen and R 21 is CH 3 where p is a number between 1 and 16.

22. The method of claim 21 , wherein the antibody-drug conjugate comprises:

23. 23. The method of claim 22, wherein the average value of p in the population of antibody drug conjugates is 8.

24. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 8, or the antibody-drug conjugate of any one of claims 9 to 14, for treating cancer in a subject.

25. 25. The composition of claim 24, wherein the subject has previously been treated with one or more therapeutic agents and has not responded to the treatment, and the one or more therapeutic agents are not the antibody, antigen-binding fragment, or antibody-drug conjugate.

26. 25. The composition of claim 24, wherein the subject has previously been treated with one or more therapeutic agents and has relapsed after treatment, and the one or more therapeutic agents are not the antibody, antigen-binding fragment, or antibody-drug conjugate.

27. 25. The composition of claim 24, wherein the subject has previously been treated with one or more therapeutic agents and experienced disease progression during treatment, and the one or more therapeutic agents are not the antibody, antigen-binding fragment, or antibody-drug conjugate.

28. The composition of any one of claims 24 to 27, wherein the cancer is an advanced stage cancer.

29. 29. The composition of claim 28, wherein the advanced cancer is (i) stage 3 or stage 4 cancer, and / or (ii) metastatic cancer.

30. The composition of any one of claims 24 to 29, wherein the cancer is (i) a recurrent cancer and / or (ii) an unresectable cancer.

31. The composition of any one of claims 24 to 30, wherein the subject has been pretreated with a standard therapy for cancer and has failed the pretreatment.

32. If the cancer is: (i) melanoma, cutaneous melanoma, non-cutaneous melanoma, ocular melanoma, or mucosal melanoma; (ii) pancreatic cancer, exocrine cancer, or neuroendocrine cancer; (iii) mesothelioma, pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, or testicular mesothelioma, (iv) colorectal cancer, colorectal adenocarcinoma, gastrointestinal stromal tumor, primary colorectal lymphoma, gastrointestinal carcinoid tumor, or leiomyosarcoma; (v) Lung cancer or non-small cell lung cancer, (vi) Breast cancer, HER2-positive, HER2-negative, estrogen receptor (ER)-positive, ER-negative, progesterone receptor (PR)-positive, PR-negative, or triple-negative breast cancer. (vii) thyroid cancer, (viii) bile duct cancer, (ix) esophageal cancer, and (x) Head and neck cancer The composition of any one of claims 24 to 31, selected from the group consisting of:

33. 33. The composition of claim 32, wherein the cutaneous melanoma is selected from the group consisting of superficial spreading melanoma, nodular melanoma, acral lentiginous melanoma, lentigo maligna melanoma, and desmoplastic melanoma.

34. 34. The composition of claim 33, wherein the acral lentiginous melanoma is subungual melanoma.

35. 35. The composition of claim 33 or 34, wherein the subject has undergone prior treatment with a PD-1 or PD-L1 inhibitor.

36. 33. The composition of claim 32, wherein the mesothelioma is pleural mesothelioma.

37. 37. The composition of claim 36, wherein the subject has been pretreated with a platinum-based therapy.

38. 38. The composition of claim 36 or 37, wherein the subject has been pretreated with pemetrexed.

39. 33. The composition of claim 32, wherein the non-small cell lung cancer has a mutated form of the epidermal growth factor receptor (EGFR).

40. 33. The composition of claim 32, wherein the non-small cell lung cancer has wild-type EGFR.

41. 41. The composition of claim 40, wherein the subject has been previously treated with a platinum-based therapy and / or with an inhibitor of PD-1 or PD-L1.

42. The composition of claim 32, wherein the breast cancer is HER2-negative breast cancer.

43. 43. The composition of claim 42, wherein the subject has undergone one or more prior lines of treatment for HER2-negative breast cancer.

44. The composition of claim 43, wherein the subject is hormone receptor positive, the subject has been previously treated with an inhibitor of CDK4 / 6, and / or the subject has been previously treated with hormone-directed therapy.

45. 33. The composition of claim 32, wherein the subject has undergone two or more prior lines of treatment for colorectal cancer.

46. 33. The composition of claim 32, wherein the exocrine cancer is selected from the group consisting of pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, acinar cell carcinoma, cystadenocarcinoma, pancreatoblastoma, adenosquamous carcinoma, signet ring cell carcinoma, hepatoid carcinoma, colloid carcinoma, undifferentiated carcinoma, and pancreatic mucinous cystic neoplasm.

47. 47. The composition of claim 46, wherein the subject has undergone one or more prior lines of treatment for pancreatic cancer.

48. The composition of any one of claims 24 to 47, wherein the antibody or antigen-binding fragment thereof, or antibody-drug conjugate is in a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, or antibody-drug conjugate, and a pharmaceutically acceptable carrier.

49. The composition of any one of claims 24 to 48, wherein the subject is a human.

50. 15. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 8, or the antibody-drug conjugate of any one of claims 9 to 14, in the manufacture of a medicament for treating a subject with cancer.

51. (a) an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or an antibody-drug conjugate according to any one of claims 9 to 14, and (b) A kit comprising instructions for using the antibody or antigen-binding fragment thereof or antibody-drug conjugate in a method for treating cancer in a subject.

52. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 8, or the antibody-drug conjugate of any one of claims 9 to 14, and one or more agents selected from the group consisting of physiologically acceptable carriers, diluents, excipients and adjuvants.

53. 53. Use of the pharmaceutical composition of claim 52 in the manufacture of a medicament for treating cancer in a subject.

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