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

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the following U.S. Provisional Application Nos.: 62 / 801,590 filed on February 5, 2019; 62 / 824,923 filed on March 27, 2019; 62 / 879,660 filed on July 29, 2019; 62 / 882,016 filed on August 2, 2019; and 62 / 934,424 filed on November 12, 2019; the contents of which are incorporated herein by reference in their entirety.

[0003] ASCII Text File Sequence Listing Submission

[0004] The contents of the following submitted ASCII text file are incorporated herein by reference in their entirety: Computer Readable Form (CRF) of the Sequence Listing (File Name: 761682001340SEQLIST.TXT, Record Date: January 21, 2020, Size: 28KB). Technical Field

[0005] The present invention relates to novel anti-CD228 antibodies and antibody-drug conjugates and methods of using such anti-CD228 antibodies and antibody-drug conjugates to treat cancer. Background Art

[0006] CD228 (also known as melanin transferrin, MELTF, p97 and MF12) is a glycosylphosphatidylinositol-anchored glycoprotein and was originally identified as a 97 kDa cell surface marker for malignant melanoma cells. CD228 is overexpressed on most clinical melanoma isolates and is also observed in many human cancers. CD228 has been shown to be expressed in a variety of cancers. CD228 belongs to the transferrin family of iron-binding proteins.

[0007] Melanoma, also known as malignant melanoma, is a type of cancer that develops from melanocytes, which are cells that contain pigment. Melanoma is the most dangerous type of skin cancer. In 2015, 59,800 people died of the 3.1 million people with active disease and melanoma. Surgery can be effective for early-stage melanoma, but may not be a treatment option for disease that has metastasized to distant organs. Melanoma often spreads to lymph nodes in the region and then to other locations. Attempts to improve survival by surgically removing lymph nodes are accompanied by many complications with no overall survival benefit. Immunotherapy, chemotherapy, and radiation therapy have all been used but are generally incurable, especially in advanced melanoma. When there are distant metastases, the cancer is generally considered incurable. The five-year survival rate for stage IV disease is 15%-20%. Therefore, there is a need for improved treatments for melanoma.

[0008] All references cited herein, including patent applications, patent publications, and scientific literature, are hereby incorporated by reference in their entirety to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference. Summary of the invention

[0009] Provided herein are isolated anti-CD228 antibodies or antigen-binding fragments thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises:

[0010] (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1;

[0011] (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and

[0012] (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and

[0013] Wherein the light chain variable region comprises:

[0014] (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4;

[0015] (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and

[0016] (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody is humanized.

[0017] Also provided herein are humanized anti-CD228 antibodies or antigen-binding fragments 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.

[0018] Also provided herein are humanized anti-CD228 antibodies or antigen-binding fragments thereof, comprising a heavy chain variable region comprising the three Kabat CDRs of SEQ ID NO:7, wherein 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, wherein position L2 is occupied by F, position L36 is occupied by Y and position L46 is occupied by L.

[0019] In some any of the embodiments herein, 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. In some any of the embodiments herein, the heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 8. In some any of the embodiments herein, the antibody or antigen-binding fragment is an antigen-binding fragment. In some any of the embodiments herein, the antibody or antigen-binding fragment is a full-length antibody.

[0020] Also provided herein is an antibody-drug conjugate comprising an antibody or antigen-binding fragment provided herein conjugated to a cytotoxic agent or cytostatic agent. In some embodiments, the linker is a MDpr-PEG(12)-gluc linker. In some embodiments of any of the present invention, the cytotoxic agent or cytostatic agent is monomethyl auristatin. In some embodiments, the monomethyl auristatin is monomethyl auristatin E (MMAE). In some embodiments of any of the present invention, the linker is attached to monomethyl auristatin E, thereby forming an antibody-drug conjugate having the following structure:

[0021]

[0022] Where Ab is antibody hL49, n is 12, R PR is hydrogen, R 21 is CH3, and p represents a number from 1 to 16. In some of any of the embodiments herein, the antibody-drug conjugate is hL49-MDpr-PEG(12)-gluc-MMAE.

[0023] Also provided herein is a nucleic acid encoding the heavy chain variable region and / or light chain variable region of the antibody described herein. Also provided herein is a vector comprising the nucleic acid provided herein. Also provided herein is a host cell comprising the nucleic acid provided herein.

[0024] Also provided herein is a method of producing an anti-CD228 antibody or antigen-binding fragment provided herein, the method comprising culturing a host cell provided herein under conditions suitable for producing the anti-CD228 antibody or antigen-binding fragment thereof.

[0025] Also provided herein is a method for producing an anti-CD228 antibody-drug conjugate provided herein, the method comprising culturing a host cell provided herein 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 agent or a cytostatic agent.

[0026] Also provided herein is a method for treating cancer in a subject, the method 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 melanoma, pancreatic cancer, mesothelioma, colorectal cancer, lung cancer, thyroid cancer, breast cancer, bile duct cancer, esophageal cancer and head and neck cancer. In some embodiments, the subject is human.

[0027] Also provided herein are kits comprising: (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.

[0028] Also provided herein is a pharmaceutical composition comprising the antibody or antigen-binding fragment provided herein or the antibody-drug conjugate provided herein and one or more agents selected from physiologically acceptable carriers, diluents, excipients and adjuvants. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The patent or application file contains at least one drawing printed in color. Copies of this patent or patent application publication with one or more color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0030] Figure 1 shows CD228 protein expression analysis of melanoma cancer patient samples by IHC.

[0031] FIG. 2 shows CD228 protein expression analysis of mesothelioma cancer patient samples by IHC.

[0032] FIG3 shows CD228 protein expression analysis of colorectal cancer patient samples by IHC.

[0033] Figure 4 shows the CD228 protein expression analysis of breast cancer patient samples by IHC. The upper panel shows the CD228 protein expression analysis of triple-negative breast cancer patient samples by IHC. The lower panel shows the CD228 protein expression analysis of Her2-HR+ breast cancer patient samples by IHC.

[0034] FIG. 5 shows CD228 protein expression analysis of pancreatic cancer patient samples by IHC.

[0035] Figure 6 shows the CD228 protein expression analysis of non-small cell lung cancer patient samples by IHC. The upper panel shows the CD228 protein expression analysis of squamous NSCLC cancer patient samples by IHC. The lower panel shows the CD228 protein expression analysis of adenocarcinoma NSCLC cancer patient samples by IHC.

[0036] Figure 7 shows a comparison of the percentage of patient samples positive for CD228 expression as determined by IHC and by RNA levels reported by The Cancer Genome Atlas for various tumor types.

[0037] Figure 8 shows an alignment of the heavy chain variable region amino acid sequence of the parental murine anti-CD228 monoclonal antibody, designated Mu L49 vH (SEQ ID NO: 21), with the human receptor sequence, designated Hu IGHV4-59 / HJ4 (SEQ ID NO: 23), and humanized forms of the L49 antibody, designated hvHA (SEQ ID NO: 7), hvHB (SEQ ID NO: 24), and hvHC (SEQ ID NO: 25). CDR positions were assigned using both the Kabat and IMGT numbering schemes.

[0038] Figure 9 shows an alignment of the heavy chain variable region amino acid sequences of humanized versions of the L49 antibody, designated hvHA (SEQ ID NO: 7), hvHB (SEQ ID NO: 24), and hvHC (SEQ ID NO: 25). The CDR positions were designated using both the Kabat and IMGT numbering schemes.

[0039] Figure 10 shows an alignment of the light chain variable region amino acid sequence of the parental murine anti-CD228 monoclonal antibody, designated Mu L49 vL (SEQ ID NO: 31), with the human receptor sequence, designated Hu IGKV2-30 / KJ2 (SEQ ID NO: 32), and humanized forms of the L49 antibody, designated hvLA (SEQ ID NO: 33), hvLB (SEQ ID NO: 34), and hvLC (SEQ ID NO: 35). CDR positions were assigned using both the Kabat and IMGT numbering schemes.

[0040] Figure 11 shows an alignment of the light chain variable region amino acid sequences of humanized versions of the L49 antibody, designated hvLA (SEQ ID NO: 33), hvLB (SEQ ID NO: 34), and hvLC (SEQ ID NO: 35). The CDR positions were designated using both the Kabat and IMGT numbering schemes.

[0041] Figures 12A-12F show the results of competition binding studies of recombinant humanized anti-CD228 antibodies, the parental murine antibody (referred to as mL49), and a chimeric antibody (cL49ec).

[0042] Figure 13 shows the results of saturation binding studies for recombinant humanized anti-CD228 antibodies.

[0043] Figures 14A-14C show the percentage of live 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).

[0044] Figure 15 shows the A2058 tumor volume over time for untreated mice and mice treated with 3 mg / kg hL49-HALC, hL49-HALC-Auristatin T(8), hL49-HALC-lipophilic MMAF(8), hL49-HALC-tubulin M(8), and hL49-HALC-MDpr-PEG(12)-gluc-MMAE(8).

[0045] Figure 16 shows the 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).

[0046] Figure 17 shows the 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).

[0047] Figure 18 shows the 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).

[0048] Figure 19 shows the 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-Tubulin M(8), 1 mg / kg or 3 mg / kg hL49-Tubulin M(8), or 1 mg / kg or 3 mg / kg hL49-MDpr-PEG(12)-gluc-MMAE(8).

[0049] Figure 20 shows the 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-Tubulin M(8), 0.3 mg / kg, 1 mg / kg or 3 mg / kg hL49-Tubulin M(8), or 0.3 mg / kg, 1 mg / kg or 3 mg / kg hL49-MDpr-PEG(12)-gluc-MMAE(8).

[0050] FIG. 21 shows the IGR-37 tumor volume over time for untreated mice and mice treated with 1 mg / kg or 3 mg / kg hL49-Tubulin M(8) or 1 mg / kg or 3 mg / kg hL49-MDpr-PEG(12)-gluc-MMAE(8).

[0051] FIG. 22 shows Colo-853 tumor volume over time for untreated mice and mice treated with 0.3, 1, or 3 mg / kg hL49-MDpr-PEG(12)-gluc-MMAE(8) or 3 mg / kg IgG-MDpr-PEG(12)-gluc-MMAE(8).

[0052] FIG. 23 shows the 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).

[0053] FIG. 24 shows the 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).

[0054] FIG. 25 shows the 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).

[0055] Figure 26 shows the HPAF-II tumor volume over time for untreated mice and mice treated with 3 mg / kg IgGhL49-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).

[0056] FIG. 27 shows the percent change in tumor volume in response to treatment with hL49-MDpr-PEG(12)-gluc-MMAE(8) in 22 different mouse triple-negative breast cancer PDX models.

[0057] 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.

[0058] Figure 29A shows the ADC plasma concentrations over time in nude mice. Figure 29B shows the ADC plasma concentrations over time in rats.

[0059] Figure 30A shows the A2058 tumor volume over time for untreated mice and mice treated with various CD228 antibodies. Figure 30B shows the percentage of animals with tumors that increased <4-fold over time for each treatment condition.

[0060] Figures 31A-31B show the rate of conjugate cleavage over time in A375 and Colo-853 cells.

[0061] Figure 32 shows the recruitment of CD228 on the cell surface over time by comparing the rate of conjugate cleavage over time in cells treated with fluorescently labeled hL49 antibody with either pulses or continuous treatment with labeled antibody.

[0062] FIG. 33A-B show the mean fluorescence intensity per cell over time in cells incubated with fluorescently labeled hL49 antibody in the presence or absence of cycloheximide to inhibit protein synthesis.

[0063] Figures 34A-34F show the binding of various anti-CD228 antibodies to CD228 at pH values ​​ranging from 4 to 7.4.

[0064] Figures 35A-35B show the ability of antibodies with similar binding affinities to internalize and catabolize drugs.

[0065] Figures 36A-36B show that a single dose of hL49-MDpr-PEG(12)-gluc-MMAE(8) has anti-tumor activity in patient-derived tumor (PDX) models. DETAILED DESCRIPTION

[0066] I. Definitions

[0067] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning described below. Additional definitions are set forth throughout the application.

[0068] The term "and / or" as used herein is considered to be a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to cover each of the following aspects: 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).

[0069] It is to be understood that the aspects and embodiments of the present invention described herein include "comprising the various aspects and embodiments," "consisting of the various aspects and embodiments," and / or "consisting essentially of the various aspects and embodiments."

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present disclosure belongs. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press, provide a general dictionary of many terms used in the present disclosure for the skilled person.

[0071] Units, prefixes and symbols are all expressed in a form acceptable to the International System of Units (SI). Numerical ranges include numbers that limit the ranges. The headings provided herein are not limitations on the various aspects of the disclosure, which can be obtained as a whole by reference to the specification. Therefore, the terms defined immediately below can be more fully defined by reference to the specification as a whole.

[0072] The terms "CD228," "p97," "melanotransferrin," "MELTF," and "MF12" are used interchangeably herein and, unless otherwise indicated, include any variants, isoforms, and homologs of human CD228 that are normally expressed by cells or expressed on cells transfected with the CD228 gene.

[0073] The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, a pair of light (L) low molecular weight chains and a pair of heavy (H) chains, all four of which are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd edition Raven Press, NY (1989)). In short, each heavy chain typically consists of a heavy chain variable region (abbreviated herein as V H or VH) and the heavy chain constant region (C H The heavy chain constant region is typically composed of three domains, namely, C H 1. C H 2 and C H The heavy chains are usually linked to each other via disulfide bonds in the so-called "hinge region". Each light chain typically consists of a light chain variable region (abbreviated herein as V L or VL) and the light chain constant region (C L The light chain constant region consists of a domain C L The constant region of the heavy chain is a region of the IgG1 heavy chain, which is a member of the heavy chain constant region. ...

[0074] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding the antibody to an antigen. The variable regions of the heavy and light chains of a native antibody (V H and V L) can be further subdivided into regions of hypervariability (or hypervariable regions, which may be hypervariable when in the form of sequence and / or structure-defined loops), also called complementarity determining regions (CDRs), which are interspersed with more conserved regions, called framework regions (FRs). The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR", as known in the art, and refer to non-contiguous sequences of amino acids within an antibody variable region that confer antigen specificity and / or binding affinity. Typically, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3), and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). "Framework region" and "FR" are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. Typically, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region, and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region. H and V L In a polypeptide, the three CDRs and four FRs are typically arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mot. Biol., 195, 901-917 (1987)).

[0075] In the context of the present invention, the term "antibody" (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of any of them that has the ability to specifically bind to an antigen under typical physiological conditions, wherein the half-life is a significant period of time, such as at least about 30 min, at least about 45 min, at least about one hour (h), at least about two hours, at least about four hours, at least about eight hours, at least about 12 hours (h), about 24 hours or longer, about 48 hours or longer, about three, four, five, six, seven days or longer, etc., or any other relevant functionally defined period of time (such as a time sufficient to induce, promote, enhance, and / or regulate a physiological response associated with an antibody that binds to an antigen and / or a time sufficient to recruit effector activity of the antibody). The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The constant region of the antibody (Ab) can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system (such as C1q, the first component in the classical pathway of complement activation). The antibody may also be a bispecific antibody, a diabody, a multispecific antibody or a similar molecule.

[0076] The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules recombinantly produced with a single primary amino acid sequence. A monoclonal antibody composition exhibits a single binding specificity and affinity for a specific epitope. Therefore, the term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity with a variable region and a constant region derived from a human germline immunoglobulin sequence. Human monoclonal antibodies can be produced by hybridomas, including B cells obtained from transgenic or transchromosomal non-human animals (such as transgenic mice) with a genome comprising a human heavy chain transgene and a light chain transgene, fused to immortalized cells.

[0077] "Isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic 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, the isolated antibody that specifically binds to CD228 can have cross-reactivity with other antigens (such as CD228 molecules from different species). In addition, the isolated antibody can be substantially free of other cellular substances and / or chemicals. In one embodiment, the isolated antibody includes an antibody conjugate attached to another agent (e.g., a small molecule drug). In some embodiments, the isolated anti-CD228 antibody includes a conjugate of an anti-CD228 antibody and a small molecule drug (e.g., MMAE or MMAF).

[0078] "HuMAb" refers to an antibody having a variable region in which both FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been transplanted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used synonymously.

[0079] The term "humanized antibody" as used herein refers to a genetically engineered non-human antibody containing a human antibody constant domain and modified to contain a non-human variable domain with a high level of sequence homology to a human variable domain. This can be achieved by transplanting the six non-human antibody complementary determining regions (CDRs) that form the antigen binding site together to the homologous human receptor framework region (FR) (see WO 92 / 22653 and EP 0629240). In order to completely reconstruct the binding affinity and specificity of the parent antibody, it may be necessary to replace the framework residues from the parent antibody (i.e., non-human antibody) with human framework regions (reverse mutations). Structural homology modeling may help identify the amino acid residues that are important for the binding properties of antibodies in the framework region. Therefore, a humanized antibody may include a non-human CDR sequence, optionally containing one or more amino acid reverse mutations of a non-human amino acid sequence, and a completely human constant region. Optionally, other amino acid modifications (which are not necessarily reverse mutations) may be applied to obtain a humanized antibody with preferred features (such as affinity and biochemical properties).

[0080] The term "chimeric antibody" as used herein refers to an antibody in which the variable region is derived from a non-human species (e.g., from a rodent) and the constant region is derived from a different species (e.g., human). Chimeric antibodies can be produced by antibody engineering. "Antibody engineering" is a general term for antibody modification of different species, and it is a process well known to those skilled in the art. In particular, chimeric antibodies can be produced by using standard DNA techniques as described in Sambrook et al., 1989, Molecular Cloning: Alaboratory Manual, New York: Cold Spring Harbor Laboratory Press, Chapter 15. Therefore, the chimeric antibody can be a genetic or enzyme engineered recombinant antibody. Producing chimeric antibodies is within the knowledge of technicians, and therefore, the production of chimeric antibodies according to the present invention can be carried out by other methods except as described herein. Chimeric monoclonal antibodies for therapeutic applications have been developed to reduce the immunogenicity of antibodies. They can typically contain non-human (e.g., mouse) variable regions specific for the target antigen and human constant antibody heavy and light chain domains. The term "variable region" or "variable domain" used in the context of a chimeric antibody refers to the region comprising the CDRs and framework regions of the heavy and light chains of an immunoglobulin.

[0081] "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.

[0082] 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 an 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 antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with one antigen-binding site; and a residual "Fc" fragment, a name that reflects the ability to crystallize readily. Pepsin treatment produces a F(ab')2 fragment, which has two antigen-binding sites and is still able to cross-link antigen.

[0083] "Percentage (%) of 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 the amino acid residues in a reference polypeptide sequence after aligning the sequences and introducing gaps (if necessary) in order to achieve maximum percentage of sequence identity and not considering any conservative substitutions as part of sequence identity. Alignment for determining percentage of amino acid sequence identity can be achieved in a variety of ways well known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment in the total length of the compared sequences. For example, the % sequence identity of a given amino acid sequence A to / with / relative to a given amino acid sequence B (or this can be expressed in phrases as a given amino acid sequence A having or comprising a specific % sequence identity to / with / relative to a given amino acid sequence B) is calculated as follows:

[0084] Multiply the fraction X / Y by 100

[0085] where X is the number of amino acid residues scored as identical matches by the sequence in the program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % sequence identity of A to B will not be equal to the % sequence identity of B to A.

[0086] As used herein, the terms "binding", "bind" or "specific binding" in the context of binding of an antibody to a predetermined antigen typically refers to binding with an affinity corresponding to about 10 Å when measured, for example, by biolayer interferometry (BLI) in an Octet HTX instrument using the antibody as ligand and the antigen as analyte. -6 M or smaller (e.g. 10-7 M or smaller, such as about 10 -8 M or smaller, such as about 10 -9 M or smaller, about 10 -10 M or less or about 10 -11 M or even smaller) D , and wherein the antibody binds to the predetermined antigen with an affinity that corresponds to a K greater than its K for binding to a nonspecific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen. D At least ten times (such as at least 100 times, for example at least 1,000 times, such as at least 10,000 times, for example at least 100,000 times) lower K D The combined K D The lower degree depends on the antibody's K D , so when the antibody's K D When it is very low, the K D Lower than the K for binding to nonspecific antigen D The degree of specificity may be at least 10,000-fold (ie, the antibody is highly specific).

[0087] As used herein, the term "K D "(M)" refers to the dissociation equilibrium constant for a particular antibody-antigen interaction. As used herein, affinity and K D is inversely related, i.e., higher affinity means lower K D , and lower affinity is intended to refer to higher K D .

[0088] The term "ADC" refers to an antibody-drug conjugate, which in the context of the present invention refers to an anti-CD228 antibody coupled to a drug moiety (eg, MMAE or MMAF) as described in this application.

[0089] The abbreviations "vc" and "val-cit" refer to the dipeptide valine-citrulline.

[0090] The abbreviation "PAB" refers to suicide spacer:

[0091]

[0092] The abbreviation "MC" refers to the maleimidocaproyl extender:

[0093]

[0094] The abbreviation "MP" refers to the maleimidopropionyl extender:

[0095]

[0096] "Cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. "Cancer" or "cancerous tissue" can include tumors. Unregulated cell division and growth lead to the formation of malignant tumors, which invade neighboring tissues and may also metastasize to distant parts of the body via the lymphatic system or bloodstream. After metastasis, the distant tumor can be said to have "derived from" the pre-metastatic tumor.

[0097] The term "antibody-dependent cellular toxicity" or ADCC is a mechanism of inducing cell death that relies on the interaction of antibody-coated target cells with immune cells (also referred to as effector cells) that have lytic activity. Such effector cells include natural killer cells, monocytes / macrophages, and neutrophils. Effector cells are attached to one or more Fc effector domains of Ig, which bind to target cells via their antigen binding sites. The death of antibody-coated target cells occurs as a result of effector cell activity.

[0098] The term "antibody-dependent cellular phagocytosis" or ADCP refers to the process by which antibody-coated cells are internalized, in whole or in part, by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) bound to one or more Fc effector domains of the Ig.

[0099] The term "complement dependent cytotoxicity" or CDC refers to a mechanism of inducing cell death in which one or more Fc effector domains of a target-binding antibody activate a series of enzymatic reactions that ultimately lead to the formation of pores in the target cell membrane. Typically, antigen-antibody complexes (such as those on antibody-coated target cells) bind and activate complement component Clq, which in turn activates the complement cascade, leading to target cell death. Activation of complement may also result in the deposition of complement components on the target cell surface, which promote ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0100] "Cytostatic effect" refers to the inhibition of cell proliferation. "Cytostatic agent" refers to an agent that has a cytostatic effect on cells, thereby inhibiting the growth and / or expansion of a specific cell subpopulation. Cytostatic agents can be conjugated to antibodies or administered in combination with antibodies.

[0101] "Treatment" or "therapy" of a subject refers to any type of intervention or treatment performed on a subject, or the administration of an active agent to a subject, with the purpose of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of symptoms, complications, conditions or biochemical markers associated with a disease. In some embodiments, the disease is cancer.

[0102] "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 (such as mice, rats, and guinea pigs). In some embodiments, the subject is a human. The terms "subject" and "patient" and "individual" are used interchangeably herein.

[0103] An "effective amount" or "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the drug, when used alone or in combination with another therapeutic agent, that protects a subject from the onset of disease or promotes regression of disease as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or the prevention of impairment or disability due to disease distress. The ability of a therapeutic agent to promote regression of disease can be evaluated using a variety of methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by measuring agent activity in in vitro assays.

[0104] For example, for treating tumors, a therapeutically effective amount of an anticancer agent in a treated subject (e.g., one or more treated subjects) inhibits cell growth 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% relative to an untreated subject (e.g., one or more untreated subjects). In some embodiments, a therapeutically effective amount of an anticancer agent in a treated subject (e.g., one or more treated subjects) inhibits cell growth or tumor growth by 100% relative to an untreated subject (e.g., one or more untreated subjects).

[0105] In other embodiments of the present disclosure, tumor regression may be observed and persist 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.

[0106] A therapeutically effective amount of a drug includes a "prophylactically effective amount," which is any amount of a drug (e.g., an anti-CD228 antibody-drug conjugate) that inhibits the development or recurrence of a cancer when administered alone or in combination with an anticancer agent to a subject at risk of developing cancer (e.g., a subject with a precancerous condition) or suffering from a recurrence of cancer. In some embodiments, the prophylactically effective amount completely prevents the development or recurrence of cancer. "Inhibiting" the development or recurrence of cancer means reducing the likelihood of cancer development or recurrence, or completely preventing the development or recurrence of cancer.

[0107] As used herein, "subtherapeutic dose" refers to a dose of a therapeutic compound (e.g., anti-CD228 antibody-drug conjugate) that is lower than the usual or typical dose of the therapeutic compound when administered alone to treat a hyperproliferative disease (e.g., cancer).

[0108] An "immune-related response pattern" refers to a clinical response pattern frequently observed in cancer patients treated with immunotherapeutics that produce antitumor effects either by inducing a cancer-specific immune response or by modifying innate immune processes. This response pattern is characterized by a beneficial treatment effect following an initial increase in tumor burden or the appearance of new lesions, which in the evaluation of traditional chemotherapeutics would be classified as disease progression and would be synonymous with drug failure. Therefore, appropriate evaluation of immunotherapeutics may require long-term monitoring of the effects of these agents on the target disease.

[0109] For example, an "anticancer agent" promotes cancer regression in a subject. In some embodiments, a therapeutically effective amount of a drug promotes cancer regression to the extent of eliminating cancer. "Promoting cancer regression" means that an effective amount of a drug administered alone or in combination with an anti-tumor agent results in a reduction in tumor growth or size, tumor necrosis, a reduction in the severity of at least one disease symptom, an increase in the frequency and duration of disease-free periods, or the prevention of damage or disability caused by disease distress. In addition, the terms "effective" and "effectiveness" with respect to treatment include both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cell, organ and / or organism level caused by the administration of the drug.

[0110] A "sustained response" refers to a persistent effect on reducing tumor growth after treatment has stopped. For example, the tumor size may remain the same or smaller than it was at the beginning of the administration period. In some embodiments, the duration of a sustained response is at least the same as the duration of treatment, or at least 1.5, 2.0, 2.5, or 3 times longer than the duration of treatment.

[0111] As used herein, "complete response" or "CR" means the disappearance of all target lesions; "partial response" or "PR" means a decrease of at least 30% in the sum of the longest diameters (SLD) of the target lesions, relative to the baseline SLD; and "stable disease" or "SD" means that the target lesions have neither shrunk sufficiently to qualify as a PR nor increased sufficiently to qualify as a PD, relative to the smallest SLD since the start of treatment.

[0112] As used herein, "progression-free survival" or "PFS" refers to the length of time during and after treatment that the disease being treated (e.g., cancer) does not get worse. Progression-free survival can include the amount of time a patient experiences a complete response or a partial response, as well as the amount of time a patient experiences stable disease.

[0113] As used herein, "overall response rate" or "ORR" refers to the sum of the complete response (CR) rate and the partial response (PR) rate.

[0114] As used herein, "overall survival" or "OS" refers to the percentage of individuals in a group who are likely to survive after a specific duration.

[0115] The phrase "pharmaceutically acceptable" indicates that the substance or composition must be compatible chemically and / or toxicologically with the other ingredients making up the formulation and / or the mammal to be treated therewith.

[0116] The phrase "pharmaceutically acceptable salt" used herein refers to a pharmaceutically acceptable organic or inorganic salt of the compounds of the present invention. Exemplary salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, succinates, maleates, gentisates, fumarates, gluconates, glucuronates, sugarates, formates, benzoates, glutamates, methanesulfonates ("mesylate"), ethanesulfonates, benzenesulfonates, p-toluenesulfonates, pamoic acid (i.e., 4,4'-methylene-bis-(2-hydroxy-3-naphthoic acid)) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. Pharmaceutically acceptable salts may involve comprising another molecule, such as acetate ions, succinate ions, or other counterions. The counterion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt can have more than one charged atom in its structure. If multiple charged atoms are part of a pharmaceutically acceptable salt, there can be multiple counterions. Thus, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterions.

[0117] "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 of the anti-CD228 antibody-drug conjugate include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion (e.g., intravenous infusion). As used herein, the phrase "parenteral administration" means a mode of administration other than enteral and topical administration, typically by injection, and includes but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. Therapeutic agents may be administered via non-parenteral routes or orally. Other non-parenteral routes include topical, epidermal or mucosal administration routes, such as intranasal, vaginal, rectal, sublingual or topical. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0118] The terms "baseline" or "baseline value" used interchangeably herein may refer to the measurement or characterization of symptoms before or at the beginning of the administration of therapy (e.g., an anti-CD228 antibody-drug conjugate as described herein). The baseline value can be compared to a reference value to determine the reduction or improvement of the symptoms of a CD228-related disease (e.g., cancer) considered herein. The terms "reference" or "reference value" used interchangeably herein may refer to the measurement or characterization of symptoms after the administration of the therapy (e.g., an anti-CD228 antibody-drug conjugate as described). The reference value may be measured one or more times during a dosage regimen or treatment cycle or at the completion of a dosage regimen or treatment cycle. A "reference value" may be an absolute value; a relative value; a value with an upper and / or lower limit; a series of values; an average value; a median value; a mean; or a value compared to a baseline value.

[0119] Similarly, a "baseline value" can be an absolute value; a relative value; a value with an upper and / or lower limit; a range of values; an average value; a median value; a mean value; or a value compared to a reference value. Reference values ​​and / or baseline values ​​can be obtained from one individual, two different individuals, or a group of individuals (e.g., a group of two, three, four, five or more individuals).

[0120] As used herein, the term "monotherapy" means that the anti-CD228 antibody-drug conjugate is the only anticancer agent administered to the subject during the treatment cycle. However, other therapeutic agents may be administered to the subject. For example, anti-inflammatory agents or other agents may be administered to a subject with cancer during the monotherapy period to treat symptoms associated with the cancer (but not the underlying cancer itself), including, for example, inflammation, pain, weight loss, and general discomfort.

[0121] As used herein, an "adverse event" (AE) is any unfavorable and usually unintended or undesirable sign (including abnormal laboratory findings), symptom or disease associated with the use of a drug therapy. A drug therapy may have one or more associated AEs, and each AE may have the same or different levels of severity. Reference to a method that can "modify adverse events" means a treatment regimen that reduces the incidence and / or severity of one or more AEs associated with the use of a different treatment regimen.

[0122] As used herein, a “serious adverse event” or “SAE” is an adverse event that meets one of the following criteria:

[0123] Fatal or life-threatening. As used in the definition of a serious adverse event, “life-threatening” means an event in which the patient is at risk of death at the time of the event; it does not mean an event that, if more serious, could hypothetically result in death.

[0124] · Causes lasting or significant disability / incapacity

[0125] Constitutes a congenital anomaly / birth defect

[0126] Medically significant, defined as an event that harms the patient or may require medical or surgical intervention to prevent one of the above outcomes. Medical and scientific judgment must be exercised in deciding whether an AE is “medically significant.”

[0127] · Requires hospitalization of the patient or prolongation of an existing hospitalization, excluding the following: 1) routine treatment or monitoring of underlying medical conditions not related to worsening of the condition; 2) elective or pre-planned treatment of pre-existing conditions that are unrelated to the indication for the study and have not worsened since signing the informed consent; and 3) social reasons and temporary care in the absence of any worsening of the patient's overall condition.

[0128] The use of alternatives (e.g., "or") should be understood to mean 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 stated or listed components.

[0129] The term "about" or "consisting essentially of..." refers to a value or composition that is within an acceptable error range for a particular value or composition determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" or "consisting essentially of..." can mean within 1 or more than 1 standard deviation. Alternatively, "about" or "consisting essentially of..." can mean a range of up to 20%. In addition, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. When a particular value or composition is provided in the application and claims, unless otherwise stated, it should be assumed that the meaning of "about" or "consisting essentially of..." is within an acceptable error range for that particular value or composition.

[0130] References herein to "about" a value or parameter include (and describe) embodiments directed to that value or parameter per se. For example, descriptions referring to "about X" include and describe "X".

[0131] As described herein, any concentration range, percentage range, ratio range or integer range should be understood to include any integer values ​​within the range, and where appropriate, fractions thereof (such as tenths and hundredths of integers), unless otherwise specified.

[0132] Various aspects of the disclosure are described in further detail in the following subsections.

[0133] II. Overview

[0134] The present invention provides antibodies that specifically bind to CD228. The present invention is based in part on the following discovery: antibody-drug conjugates targeting CD228 (including PEGylated-MMAE antibody-drug conjugates) are particularly effective in killing cells expressing CD228+. 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 carcinoma, breast cancer and cervical cancer.

[0135] III. Target molecules

[0136] Unless otherwise indicated, CD228 refers to human CD228. The exemplary human protein sequence was assigned UniProt ID NO. P08582.

[0137] IV. Antibodies of the Invention

[0138] The present invention provides antibodies, such as humanized antibodies, derived from mouse antibody L49. L49 is a murine immunoglobulin G1 (IgG1) monoclonal antibody against CD228, which was derived from BALB / c mice immunized with lung cancer and melanoma cell lines (Siemers et al., 1997, Bioconjug. Chem. 8:510-9).

[0139] The binding affinity (i.e., dissociation constant K) of the humanized version of the mouse L49 antibody D ) is preferably within five times or two times the binding affinity of the mouse antibody L49 for human CD228. The humanized L49 antibody specifically binds to human CD228 as the mouse antibody from which it is derived. These antibodies bind to CD228 in their native form and in a recombinantly expressed form, for example, from Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells. The affinity of the preferred humanized L49 antibody for human CD228 is equal to or greater than (i.e., greater than the error limit of the measurement) the affinity of L49 for human CD228 (e.g., 1.1-5 times, 1.1-3 times, 1.5-3 times, 1.7 to 2.3 times, or 1.7-2.1 times, or about twice the affinity of L49). The preferred humanized L49 antibody binds to the same epitope and / or competes with mouse L49 for binding to human CD228.

[0140] In animal models or clinical trials, preferred antibodies of the invention inhibit cancer (e.g., cell growth, metastasis, and / or lethality to an organism) as shown on cancer cells propagated in culture. Animal models can be formed by implanting human tumor cell lines expressing CD228 into appropriate immunodeficient rodent strains (e.g., athymic nude mice or SCID mice). These tumor cell lines can be established as solid tumors in immunodeficient rodent hosts by subcutaneous injection or as disseminated tumors by intravenous injection.

[0141] Once established in a host, these tumor models can be used to evaluate the therapeutic efficacy of anti-CD228 antibodies or conjugated forms thereof as described in the Examples.

[0142] Typically, the anti-CD228 antibody and / or anti-CD228 antibody-drug conjugate of the present disclosure binds to CD228, such as human CD228, and exerts cytostatic and cytotoxic effects on malignant cells (such as cancer cells). The anti-CD228 antibody of the present disclosure is preferably monoclonal, and can be a multispecific antibody, a human antibody, a humanized antibody or a chimeric antibody, a single-chain antibody, a Fab fragment, a F(ab') fragment, a fragment produced by a Fab expression library, and a CD228 binding fragment of any of the above. In some embodiments, the anti-CD228 antibody of the present disclosure specifically binds to CD228. The immunoglobulin molecule of the present disclosure can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecules.

[0143] 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, and includes, but is not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), and V-containing L or V H Fragments of domains. Antigen-binding fragments comprising single-chain antibodies may comprise one or more variable regions alone or in combination with all or part of the following: hinge region, CH1, CH2, CH3 and CL domains. The present disclosure also includes antigen-binding fragments comprising any combination of one or more variable regions and hinge region, CH1, CH2, CH3 and CL domains. In some embodiments, the anti-CD228 antibody or antigen-binding fragment thereof is an antibody of a human, mouse (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken.

[0144] The anti-CD228 antibodies of the present disclosure can be monospecific, bispecific, trispecific or have greater multispecificity. Multispecific antibodies can be specific for different epitopes of CD228, or can be specific for both CD228 and heterologous proteins. See, for example, PCT Publication WO 93 / 17715; WO 92 / 08802; WO 91 / 00360; WO 92 / 05793; Tutt et al., 1991, J. Immunol. 147: 60 69; U.S. Pat. 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.

[0145] The anti-CD228 antibodies of the present disclosure may be described or specified in terms of the specific CDRs they contain. The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those 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 Igsuperfamily V-like domains," Dev Comp Immunol, 2003 Jan; 27(1): 55-77 ("IMGT" numbering scheme); Honegger A and Plückthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun 8; 309(3): 657-70 ("Aho" numbering scheme); 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, a "CDR" or "complementarity determining region" or a separately designated CDR (e.g., CDR-H1, CDR-H2, CDR-H3) of a given antibody or region thereof (e.g., a variable region thereof) is understood to encompass one (or specific) CDR as defined by any of the above schemes. For example, in stating that a specific CDR (e.g., CDR-H3) contains a given V. H or V L In the case of an amino acid sequence of a corresponding CDR in a variable region amino acid sequence, it is understood that such CDR has the sequence of a corresponding CDR (e.g., CDR-H3) within the variable region as defined by any of the above schemes. A scheme for identifying a specific CDR or CDRs (e.g., CDRs defined by the Kabat, Chothia, AbM, or IMGT methods) can be specified.

[0146] The CDR sequences of the anti-CD228 antibodies and anti-CD228 antibody-drug conjugates described herein are according to the Kabat numbering scheme described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD.

[0147] 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) a 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) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and / or wherein the light chain variable region comprises (i) a 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) a CDR-L3 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.

[0148] The anti-CD228 antibodies described herein may include any suitable framework variable domain sequence, provided that the antibody retains the ability to bind CD228 (e.g., human CD228). As used herein, the heavy chain framework region is referred to as "HC-FR1-FR4", and the light chain framework region is referred to as "LC-FR1-FR4". In some embodiments, the anti-CD228 antibodies include heavy chain variable domain framework sequences of SEQ ID NO: 9, 10, 11, and 12 (HC-FR1, HC-FR2, HC-FR3, and HC-FR4, respectively). In some embodiments, the anti-CD228 antibodies include light chain variable domain framework sequences of SEQ ID NO: 13, 14, 15, and 16 (LC-FR1, LC-FR2, LC-FR3, and LC-FR4, respectively).

[0149] In some embodiments of the anti-CD228 antibodies described herein, the heavy chain variable domain comprises

[0150] QVQLQESGPGLVKPSETLSLTCTVSGDSITSGYWNWIRQPPGKGLEYIGYISDSGITYYNPSLKSRVTISRDTSKNQYSLKLSSVTAADTAVYYCARRTLATYYAMDYWGQGTLVTVSS (SEQ ID NO: 7), and the light chain variable domain comprises the amino acid sequence of DFVMTQSPLSLPVTLGQPASISCRASQSLVHSDGNTYLHWYQQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPPTFGQGTKLEIK (SEQ ID NO: 8).

[0151] In some embodiments of the anti-CD228 antibodies described herein, the heavy chain CDR sequences comprise the following:

[0152] a) CDR-H1 (SGYWN (SEQ ID NO: 1));

[0153] b) CDR-H2 (YISDSGITYYNPSLKS (SEQ ID NO: 2)); and

[0154] c) CDR-H3 (RTLATYYAMDY (SEQ ID NO: 3)).

[0155] In some embodiments of the anti-CD228 antibodies described herein, the heavy chain FR sequence comprises the following:

[0156] a) HC-FR1(QVQLQESGPGLVKPSETLSLTCTVSGDSIT(SEQ ID NO:9));

[0157] b) HC-FR2(WIRQPPGKGLEYIG(SEQ ID NO:10));

[0158] c) HC-FR3 (RVTISRDTSKNQYSLKLSSVTAADTAVYYCAR (SEQ ID NO: 11)); and

[0159] d) HC-FR4 (WGQGTLVTVSS (SEQ ID NO: 12)).

[0160] In some embodiments of the anti-CD228 antibodies described herein, the light chain CDR sequences comprise the following:

[0161] a) CDR-L1(RASQSLVHSDGNTYLH(SEQ ID NO:4));

[0162] b) CDR-L2 (RVSNRFS (SEQ ID NO: 5)); and

[0163] c) CDR-L3 (SQSTHVPPT (SEQ ID NO: 6)).

[0164] In some embodiments of the anti-CD228 antibodies described herein, the light chain FR sequence comprises the following:

[0165] a) LC-FR1 (DFVMTQSPLSLPVTLGQPASISC (SEQ ID NO: 13));

[0166] b) LC-FR2(WYQQRPGQSPRLLIY(SEQ ID NO:14));

[0167] c) LC-FR3 (GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC (SEQ ID NO: 15)); and

[0168] d) LC-FR4 (FGQGTKLEIK (SEQ ID NO: 16)).

[0169] 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:

[0170] (a) a heavy chain variable domain comprising:

[0171] (1) HC-FR1 comprising the amino acid sequence of SEQ ID NO: 9;

[0172] (2) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1;

[0173] (3) HC-FR2 comprising the amino acid sequence of SEQ ID NO: 10;

[0174] (4) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2;

[0175] (5) HC-FR3 comprising the amino acid sequence of SEQ ID NO: 11;

[0176] (6) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and

[0177] (7) HC-FR4 comprising the amino acid sequence of SEQ ID NO: 12,

[0178] and / or

[0179] (b) a light chain variable domain comprising:

[0180] (1) LC-FR1 comprising the amino acid sequence of SEQ ID NO: 13;

[0181] (2) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4;

[0182] (3) LC-FR2 comprising the amino acid sequence of SEQ ID NO: 14;

[0183] (4) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5;

[0184] (5) LC-FR3 comprising the amino acid sequence of SEQ ID NO: 15;

[0185] (6) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6; and

[0186] (7) LC-FR4 comprising the amino acid sequence of SEQ ID NO:16.

[0187] In one aspect, provided herein are anti-CD228 antibodies and / or anti-CD228 antibody-drug conjugates comprising a heavy chain variable domain comprising an amino acid sequence of SEQ ID NO: 7 or a light chain variable domain comprising an 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. In one aspect, provided herein are anti-CD228 antibodies comprising a heavy chain variable domain comprising an amino acid sequence of SEQ ID NO: 7 and a light chain variable domain comprising an 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.

[0188] 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 is cyclized to form pyroglutamic acid. In certain embodiments, the 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 relative 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, substitutions, insertions or deletions (e.g., 1, 2, 3, 4 or 5 amino acids) occur in regions outside of CDR (i.e., in FR). In some embodiments, the anti-CD228 antibody comprises a heavy chain variable domain sequence of SEQ ID NO: 7, which includes post-translational modifications of the sequence. In some embodiments, the N-terminal glutamine of the heavy chain variable domain is cyclized to form pyroglutamic acid. In a specific embodiment, 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.

[0189] 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 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: 8 contains substitutions (e.g., conservative substitutions), insertions or deletions relative 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, substitution, insertion or deletion (e.g., 1, 2, 3, 4 or 5 amino acids) occurs in a region outside the CDR (i.e., in the FR). In some embodiments, the anti-CD228 antibody comprises a light chain variable domain sequence of SEQ ID NO: 8, including post-translational modifications of the sequence. In a specific embodiment, the light chain variable domain comprises one, two or three CDRs selected from the following: (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 4, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 6.

[0190] In some embodiments, the anti-CD228 antibody and / or the anti-CD228 antibody-drug conjugate comprises a heavy chain variable domain as in any embodiment provided above and a light chain variable domain as in any embodiment provided above. In one embodiment, the antibody comprises a heavy chain variable domain sequence of SEQ ID NO:7 and a light chain variable domain sequence of SEQ ID NO:8, including post-translational modifications of those sequences. In some embodiments, the N-terminal glutamine of the heavy chain variable domain is cyclized to form pyroglutamic acid.

[0191] 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.

[0192] In some embodiments, the anti-CD228 antibody and / or the 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 is cyclized to form pyroglutamic acid.

[0193] In some embodiments, the anti-CD228 antibody or anti-CD228 antibody of the anti-CD228 antibody-drug conjugate is a monoclonal antibody.

[0194] The anti-CD228 antibodies of the invention can also be described or specified based on their binding affinity to CD228 (e.g., human CD228). Preferred binding affinities include dissociation constants or K D Less than 5x10 -2 M, 10 -2 M, 5x10 -3 M, 10 -3 M, 5x10 - 4 M, 10 -4 M, 5x10 -5 M, 10 -5 M, 5x10 -6 M, 10 -6 M, 5x10 -7 M, 10 -7 M, 5x10 -8 M, 10 -8 M, 5x10 -9 M, 10 -9 M, 5x10 - 10 M, 10 -10 M, 5x10 -11 M, 10 -11M, 5x10 -12 M, 10 -12 M, 5x10 -13 M, 10 -13 M, 5x10 -14 M, 10 -14 M, 5x10 -15 M, or 10 -15 Those of M.

[0195] In some embodiments, the binding of the anti-CD228 antibodies of the present invention is pH dependent, such that the antibodies exhibit different binding across a pH gradient. In some embodiments, the anti-CD228 antibodies exhibit maximum binding between a pH of about 5.5 and a pH of about 6.3. In some embodiments, the anti-CD228 antibodies exhibit maximum binding at a pH of about 5.6. In some embodiments, the anti-CD228 antibodies exhibit maximum binding at a pH of about 6.3. In some embodiments, the anti-CD228 antibodies exhibit minimum binding at a pH of about 5.1 or less.

[0196] There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, with heavy chains called α, δ, ε, γ and μ, respectively. The γ and α categories are further divided into subclasses, for example, people express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. IgG1 antibodies can exist as multiple polymorphic variants called allotypes (reviewed in Jefferis and Lefranc 2009. mAbs Vol. 1 No. 4 1-7), any of which is suitable for use in some embodiments herein. Common allotype variants in the human population are those named by letters a, f, n, z or a combination thereof. In any embodiment herein, the antibody may include a heavy chain Fc region, and the heavy chain Fc region includes a human IgG Fc region. In other embodiments, the human IgG Fc region includes human IgG1.

[0197] In some embodiments, the anti-CD228 antibody and / or the anti-CD228 antibody-drug conjugate comprises a heavy chain variable domain as in any of the embodiments provided above and a light chain variable domain as in any of the embodiments provided above. In one embodiment, the antibody comprises ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(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 those sequences.In another embodiment, the antibody comprises ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:19) and a light chain constant region containing the amino acid sequence of TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:18), including post-translational modifications of those sequences. SEQ ID NO:19 contains a serine to cysteine ​​substitution at amino acid position 239 of the human IgG1 isotype. The presence of additional cysteine ​​residues allows interchain disulfide bond formation. This interchain disulfide bond formation may cause steric hindrance, thereby reducing the affinity of the Fc region-FcγR binding interaction. Cysteine ​​residues introduced in or near the Fc region of the IgG constant region can also serve as a site for conjugation with a therapeutic agent (i.e., a conjugated cytotoxic drug using a thiol-specific reagent such as a maleimide derivative of the drug). The presence of the therapeutic agent causes steric hindrance, 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 FcγRI receptors (see, e.g., US 6,624,821, US 5,624,821).

[0198] In some embodiments, the anti-CD228 antibody or the anti-CD228 antibody of the antibody-drug conjugate is the humanized antibody hL49 HALC. The hL49 HALC comprises a heavy chain variable region sequence of SEQ ID NO:7 and a 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 or the anti-CD228 antibody of the antibody-drug conjugate is the humanized antibody hL49. hL49 comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:7, a light chain variable region comprising an amino acid sequence of SEQ ID NO:8, a heavy chain constant region comprising an amino acid sequence of SEQ ID NO:17, and a light chain constant region comprising an amino acid sequence of SEQ ID NO:18.

[0199] Antibodies also include modified derivatives, i.e., modified by covalent attachment of any type of molecule to the antibody such that the covalent attachment does not prevent the antibody from binding to CD228 or exerting a cytostatic or cytotoxic effect on HD cells. For example, but not limiting, antibody derivatives include antibodies that have been modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, etc. Any of a variety of chemical modifications may be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, the derivatives may contain one or more non-classical amino acids.

[0200] Humanized Antibodies

[0201] Humanized antibodies are genetically engineered antibodies in which the CDRs from a non-human "donor" antibody are grafted into a human "acceptor" antibody sequence (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 sequence can be, for example, a mature human antibody sequence, a complex of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. The preferred acceptor sequence for the heavy chain is the germline V H Exon V H l-2 (also referred to in the literature as HV1-2) (Shin et al., 1991, EMBO J. 10:3641-3645) and for the hinge region (J H ) is 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 as KV2-30 in the literature) and for the hinge region, the preferred acceptor sequence is exon JK-4 (Hieter et al., 1982, J. Biol. Chem. 257: 1516-1522). Therefore, a humanized antibody is an antibody having some or all CDRs from a donor antibody completely or substantially and variable region framework sequences and constant regions (if present) from human antibody sequences completely or substantially. Similarly, a humanized heavy chain has at least one, two, and usually all three CDRs from a donor antibody heavy chain and heavy chain variable region framework sequences and heavy chain constant regions (if present) that are substantially from human heavy chain variable region framework and constant region sequences. Similarly, humanized light chains have at least one, two, and usually all three CDRs from the donor antibody light chain completely or substantially, and light chain variable region framework sequences and light chain constant regions (if present) substantially from human light chain variable region framework and constant region sequences. In addition to nanobodies and dAbs, humanized antibodies comprise humanized heavy chains and humanized light chains. When at least 60%, 85%, 90%, 95% or 100% of the corresponding residues (as defined by Kabat) between each CDR are identical, the CDRs in the humanized antibodies are substantially from the corresponding CDRs in non-human antibodies. When at least 85%, 90%, 95% or 100% of the corresponding residues defined by Kabat are identical, the variable region framework sequences of the antibody chains or the constant regions of the antibody chains are substantially from human variable region framework sequences or human constant regions, respectively.

[0202] Although humanized antibodies typically incorporate all six CDRs (preferably as defined by Kabat) from a mouse antibody, they can also be made with less than all CDRs (e.g., at least 3, 4, or 5 CDRs) from a mouse 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).

[0203] Based on the possible influence on CDR conformation and / or binding to antigen, certain amino acids from human variable region framework residues can be selected for substitution. The study of such possible influence is carried out by modeling, examining the characteristics of amino acids at specific positions or empirical observation of substitution or mutagenesis of specific amino acids.

[0204] For example, when the amino acid between a murine variable region framework residue and a selected human variable region framework residue differs, the human framework amino acid can be substituted with the equivalent framework amino acid from the mouse antibody, provided that the amino acid is reasonably expected to:

[0205] (1) Direct non-covalent binding to antigens,

[0206] (2) adjacent to the CDR region,

[0207] (3) otherwise interacts with the CDR region (e.g., within about 6 Å of the CDR region); or

[0208] (4) Mediate the interaction between heavy and light chains.

[0209] One aspect of the present invention provides a humanized form of mouse antibody L49. One such humanized variant of mouse antibody L49 is named HALC. HALC comprises a mature heavy chain variable region containing the amino acid sequence of SEQ ID NO:7 and a mature light chain variable region containing 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. The humanized antibodies of the present invention include variants of HALC humanized antibodies, wherein the humanized heavy chain mature variable region shows at least 90%, 95% or 99% identity to SEQ ID NO:7, and the humanized light chain mature variable region shows at least 90%, 95% or 99% sequence identity to SEQ ID NO:8. Preferably, in such antibodies, some or all of the back mutations in HALC are retained. In other words, at least 1, 2, 3, 4 or preferably all 5 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 region of such humanized antibodies is identical or substantially identical to the CDR region of the mouse donor antibody. In a preferred embodiment, the light chain CDR1 position L28 is occupied by D. The CDR region can be defined by any conventional definition (e.g., Chothia), but is preferably defined by Kabat. In one embodiment, the humanized antibody comprises a heavy chain containing 3 CDRs of SEQ ID NO:7 and a variable region framework having at least 95% identity with the variable region framework of SEQ ID NO:7. In another embodiment, the humanized antibody comprises a light chain containing 3 CDRs of SEQ ID NO:8 and a variable region framework having at least 95% identity with the variable region framework of SEQ ID NO:8. In another embodiment, the humanized antibody comprises a heavy chain comprising the 3 CDRs of SEQ ID NO: 7 and a variable region framework having at least 95% identity with the variable region framework of SEQ ID NO: 7, and a light chain comprising the 3 CDRs of SEQ ID NO: 8 and a variable region framework having at least 95% identity with the variable region framework of SEQ ID NO: 8. In one embodiment, the humanized antibody comprises a heavy chain comprising the 3 CDRs of SEQ ID NO: 7 and a variable region framework having at least 98% identity with the variable region framework of SEQ ID NO: 7. In another embodiment, the humanized antibody comprises a light chain comprising the 3 CDRs of SEQ ID NO: 8 and a variable region framework having at least 98% identity with the variable region framework of SEQ ID NO: 8.In another embodiment, the humanized antibody comprises a heavy chain comprising 3 CDRs of SEQ ID NO: 7 and a variable region framework having at least 98% identity with the variable region framework of SEQ ID NO: 7, and a light chain comprising 3 CDRs of SEQ ID NO: 8 and a variable region framework having at least 98% identity with the variable region framework of SEQ ID NO: 8. In one embodiment, the humanized antibody comprises a heavy chain comprising 3 CDRs of SEQ ID NO: 7 and a variable region framework having at least 99% identity with the variable region framework of SEQ ID NO: 7. In another embodiment, the humanized antibody comprises a light chain comprising 3 CDRs of SEQ ID NO: 8 and a variable region framework having at least 99% identity with the variable region framework of SEQ ID NO: 8. In another embodiment, the humanized antibody comprises a heavy chain and a light chain, wherein the heavy chain contains the three CDRs of SEQ ID NO:7 and a variable region framework that is at least 99% identical to the variable region framework of SEQ ID NO:7, and the light chain contains the three CDRs of SEQ ID NO:8 and a variable region framework that is at least 99% identical to the variable region framework of SEQ ID NO:8.

[0210] Compared with mouse antibody L49, humanized antibody HALC includes asparagine to aspartic acid substitution at amino acid position L28, which is in light chain CDR1. This substitution eliminates the deamidation observed in humanized L49 variant HALB and has limited isomerization. In some embodiments, in any antibody described herein, the light chain variable region 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.

[0211] To the extent that the humanized antibody exhibits any variation compared to the exemplary HALC humanized antibody, one possibility for such additional variation is additional back mutations in the variable region framework. However, such additional back mutations are not preferred because they generally do not improve affinity and the introduction of more mouse residues may increase the risk of immunogenicity.

[0212] Another possible variation is to replace certain residues in the CDR of mouse antibody with corresponding residues from people CDR sequences, typically from the CDR of the human acceptor sequence for designing exemplary humanized antibodies. In some antibodies, only a part of CDR (i.e., a subset of CDR residues required for binding, referred to as SDR) is required to retain the binding in humanized antibodies. CDR residues that do not contact antigen and are not in SDR (e.g., residues H60-H65 in CDR H2 are generally not required) can be based on previous studies (Chothia, J.Mol.Biol.196:901,1987) from the region of Kabat CDR located outside the Chothia hypervariable loop by molecular modeling and / or relying on experience or as described in Gonzales et al., Mol.Immunol.41:863 (2004) to identify. In such humanized antibodies, in the absence of one or more donor CDR residues or the position of omitting the entire donor CDR, the amino acid occupying the position can be the amino acid occupying the corresponding position (by Kabat numbering) in the acceptor antibody sequence. The number of such replacements of the acceptor pair donor amino acid included in the CDR reflects the balance of competitive considerations. Such replacements are potentially advantageous in reducing the number of mouse amino acids in the humanized antibody and thus reducing potential immunogenicity. However, replacements may also cause changes in affinity, and preferably avoid significant reductions in affinity. The position of replacement and the amino acid to be replaced in the CDR can also be selected empirically.

[0213] Although not preferred, other amino acid substitutions can be made, for example, in framework residues that do not contact the CDRs, or even in some potential CDR contact residues within the CDRs. Typically, the substitutions made in the variant humanized sequences are conservative with respect to the substituted HALC amino acids. Preferably, the substitutions relative to HALC (whether conservative or not) have no significant effect on the binding affinity or potency of the humanized mAb, i.e., its ability to bind to human CD228 and inhibit cancer cell growth.

[0214] The variants typically differ from the heavy and light chain mature variable region sequences of HALC by a small number (eg, typically no more than 1, 2, 3, 5 or 10) of substitutions, deletions or insertions in either the light or heavy chain mature variable region or both.

[0215] Selection of constant regions

[0216] The heavy chain and light chain variable region of humanized antibody can be connected with at least a portion of human constant region.The selection part of constant region depends on whether antibody dependent cell-mediated cytotoxicity, antibody dependent cell phagocytosis and / or complement dependent cellular toxicity are needed.For example, human isotype IgG1 and IgG3 have strong complement dependent cellular toxicity, and human isotype IgG2 has weak complement dependent cellular toxicity and human IgG4 lacks complement dependent cellular toxicity.Human IgG1 and IgG3 also induce stronger cell-mediated effector function than human IgG2 and IgG4.The light chain constant region can be λ or κ.Antibody can be expressed as a tetramer containing two light chains and two heavy chains, as separated heavy chain, light chain, such as Fab, Fab', F(ab')2 and Fv, or as single-chain antibody, wherein heavy chain and light chain variable domain are connected by spacer.

[0217] Human constant regions show allotypic and heterotypic variation between different individuals, that is, the constant regions may differ at one or more polymorphic positions in different individuals. Heterotypes differ from allotypes in that serum recognizing heterotypes binds to non-polymorphic regions of one or more other isotypes.

[0218] One or more amino acids at the amino or carboxyl termini of the light and / or heavy chains (such as the C-terminal lysine of the heavy chain) may be deleted or derivatized as part or all of the molecule. Substitutions may be made in the constant region 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 the half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004).

[0219] Exemplary substitutions include amino acid substitutions that introduce natural amino acids to cysteine ​​residues at amino acid positions 234, 235, 237, 239, 267, 298, 299, 326, 330 or 332, preferably S239C mutations in human IgG1 isotypes (US20100158909). The presence of additional cysteine ​​residues allows interchain disulfide bond formation. This interchain disulfide bond formation may cause steric hindrance, thereby reducing the affinity of Fc region-Fc γ R binding interactions. One or more cysteine ​​residues introduced in the Fc region of IgG constant region or near the Fc region can also serve as a site conjugated with a therapeutic agent (i.e., using a thiol-specific reagent (such as a maleimide derivative of a drug) coupled cytotoxic drug). The presence of a therapeutic agent causes steric hindrance, thereby further reducing the affinity of Fc region-Fc γ R binding interactions. Other substitutions at any of positions 234, 235, 236 and / or 237 reduce affinity for the Fcy receptor, particularly the FcyRI receptor (see, e.g., US 6,624,821, US 5,624,821).

[0220] The in vivo half-life of an antibody may also affect its effector function. The half-life of an antibody can be increased or decreased to alter its therapeutic activity. FcRn is a receptor that is structurally similar to MHC class I antigens and non-covalently associates 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 (pH of intracellular vesicles) but not at pH 7.4 (pH of blood); this interaction enables IgG to recycle 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 with these substitutions have longer serum half-lives. Therefore, compared to unmodified IgG1, these modified IgG1 molecules may be able to perform their effector functions over a longer period of time, and thus exert their therapeutic efficacy. Other exemplary substitutions for increasing binding to FcRn include Gin at position 250 and / or Leu at position 428. EU numbering is used for all positions in the constant region.

[0221] Oligosaccharides covalently attached to the conserved Asn297 are involved in the ability of the Fc region of IgG to bind FcyR (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 a bisecting N-acetylglucosamine modification (Umana et al., 1999, Nat. Biotechnol. 17:176-180; Davies et al., 2001, Biotech. Bioeng. 74:288-94) to this glycoform 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 improve the binding between IgG Fc and FcyR, thereby enhancing Ig-mediated ADCC activity. In some embodiments, the anti-CD228 antibody or the anti-CD228 antibody of the antibody-drug conjugate described herein has a glycan attached to the conserved Asn297 residue of the constant region, wherein the numbering of the 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 Edition Public Health Service, National Institutes of Health, Bethesda, MD. (1991). In some embodiments, the glycan is bi-antennary. In some embodiments, the glycan is core-fucosylated. In some embodiments, the glycan has zero terminal galactose residues. In some embodiments, the glycan is bi-antennary and core-fucosylated. In some embodiments, the glycan is bi-antennary and has zero terminal galactose residues. In some embodiments, the glycan is core-fucosylated and has zero terminal galactose residues. In some embodiments, the glycan is bi-antennary, core-fucosylated and has zero galactose residues. In some embodiments, the glycan is bi-antennary, core-fucosylated and has zero galactose residues.In some embodiments, in an anti-CD228 antibody or a population of anti-CD228 antibodies of an antibody-drug conjugate described herein, the conserved Asn297 residue of the constant region (wherein the 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 a biantennary core-fucosylated glycan with a zero-terminal galactose residue.

[0222] Systematic substitution of solvent-exposed amino acids in the Fc region of human IgG1 has generated IgG variants with altered FcγR binding affinity (Shields et al., 2001, J. Biol. Chem. 276: 6591-604). A subset of these variants, including substitutions of Thr256 / Ser298, Ser298 / Glu333, Ser298 / Lys334, or Ser298 / Glu333 Lys334 to Ala, showed increased binding affinity to FcγRs and ADCC activity when compared to the parental IgG1 (Shields et al., 2001, J. Biol. Chem. 276: 6591-604; Okazaki et al., 2004, J. Mol. Biol. 336: 1239-49).

[0223] The complement fixation activity (both Clq 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 on the human IgG2 backbone can convert antibody isotypes that bind poorly to Clq and are severely deficient in complement activation activity into antibody isotypes that can both bind Clq and mediate CDC (Idusogie et al., 2001, J. Immunol. 166: 2571-75). Several other methods have also been used to improve the complement fixation activity of antibodies. For example, transplanting an 18 amino acid carboxyl terminal tail fragment of IgM to the carboxyl terminus of IgG greatly enhances its CDC activity. Even for IgG4, which does not normally have detectable CDC activity, this enhancement is observed (Smith et al., 1995, J. Immunol. 154: 2226-36). In addition, substitution of Ser444 near the carboxyl terminus of the IgG1 heavy chain with Cys induced tail-to-tail dimerization of IgG1, with CDC activity increased 200-fold compared to monomeric IgG1 (Shopes et al., 1992, J. Immunol. 148: 2918-22). In addition, bispecific diabody constructs specific for C1q also conferred CDC activity (Kontermann et al., 1997, Nat. Biotech. 15: 629-31).

[0224] Complement activity can be reduced by mutating at least one of the amino acid residues 318, 320, and 322 of the heavy chain to a residue with a different side chain, such as Ala. Other alkyl-substituted nonionic residues, such as Gly, He, Leu, or Val, or aromatic nonpolar residues, such as Phe, Tyr, Trp, and Pro, replacing any of the three residues also reduce or eliminate Clq binding. Ser, Thr, Cys, and Met can be used at residues 320 and 322 but not at 318 to reduce or eliminate Clq binding activity.

[0225] Replacing residue 318 (Glu) with a polar residue can alter but not eliminate Clq binding activity. Replacing residue 297 (Asn) with Ala results in removal of lytic activity, but only slightly reduces (approximately 3 times weaker) affinity for Clq. This change destroys the glycosylation site and the presence of carbohydrates required for complement activation. Any other substitution at this site also destroys the glycosylation site. The following mutations and any combination thereof also reduce Clq binding: D270A, K322A, P329A and P31 IS (see WO06 / 036291).

[0226] References to human constant regions include constant regions with any natural allotype or any substitution of residues occupying polymorphic positions in natural allotypes. In addition, there may be up to 1, 2, 5 or 10 mutations relative to the natural human constant region, such as those indicated above, to reduce Fcγ receptor binding or increase binding to FcRN.

[0227] In some embodiments, the anti-CD228 and / or anti-CD228 antibody-drug conjugate antibodies described herein comprise a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the anti-CD228 and / or anti-CD228 antibody-drug conjugate antibodies described herein comprise a light chain constant region comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-CD228 and / or anti-CD228 antibody-drug conjugate antibodies 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 and / or anti-CD228 antibody-drug conjugate antibodies described herein comprise a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-CD228 and / or anti-CD228 antibody-drug conjugate antibodies 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.

[0228] V. Expression of recombinant antibodies

[0229] Humanized antibodies are typically produced by recombinant expression. Recombinant polynucleotide constructs typically include expression control sequences operably connected to the coding sequence of the antibody chain, including naturally associated or heterologous promoter regions. Preferably, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of nucleotide sequences and collection and purification of cross-reactive antibodies.

[0230] Mammalian cells are preferred hosts for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). Many suitable host cell lines capable of secreting complete heterologous proteins have been developed in the art, and the suitable host cell lines include CHO cell lines (e.g., DG44), various COS cell lines, HeLa cells, HEK293 cells, L cells, and myeloma (including Sp2 / 0 and NS0) that do not produce antibodies. Preferably, the cells are non-human. The expression vectors used for these cells may include expression control sequences, such as replication origins, promoters, enhancers (Queen et al., Immunol. Rev. 89: 49 (1986)); and necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription termination sequences. Preferred expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papilloma virus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0231] Once expressed, the antibodies may be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, and the like (see generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0232] VI. Nucleic Acids

[0233] The present invention also provides nucleic acids encoding any one of the above-mentioned humanized heavy chains and light chains. Typically, the nucleic acid also encodes a signal peptide fused to a mature heavy chain and light chain. The coding sequence on the nucleic acid can be operably connected to a regulatory sequence to ensure the expression of the coding sequence, such as a promoter, an enhancer, a ribosome binding site, a transcription termination signal, etc. The nucleic acid encoding the heavy chain and light chain can exist in a separated form, or can be cloned into one or more vectors. The nucleic acid can be synthesized by, for example, solid-state synthesis or PCR of overlapping oligonucleotides. The nucleic acid encoding the heavy chain and light chain can, for example, be connected as a continuous nucleic acid in an expression vector, or can be separated, for example, each cloned into its own expression vector.

[0234] In some aspects, nucleic acids encoding anti-CD228 antibodies or antigen-binding fragments thereof as described herein are also provided herein. Vectors comprising nucleic acids encoding anti-CD228 antibodies or antigen-binding fragments thereof as described herein are also provided herein. Host cells expressing nucleic acids encoding anti-CD228 antibodies or antigen-binding fragments thereof as described herein are also provided herein. Host cells comprising vectors are also provided herein, wherein the vectors contain nucleic acids encoding anti-CD228 antibodies or antigen-binding fragments thereof as described herein.

[0235] 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 antibody is 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. Patent No. 5,981,216, WO 87 / 04462, EP 323997, U.S. Patent No. 5,591,639, U.S. Patent No. 5,658,759, EP 338841, U.S. Patent No. 5,879,936 and U.S. Patent No. 5,891,693.

[0236] The monoclonal anti-CD228 antibodies described herein can be produced, for example, by the hybridoma method first described by Kohler et al., Nature, 256, 495 (1975), or can be produced by recombinant DNA methods. Monoclonal antibodies can also be isolated from phage antibody libraries using, for example, Clackson et al., Nature, 352, 624-628 (1991) and Marks et al., J Mol, Biol., 222 (3): 581-597 (1991) described in the technique. Monoclonal antibodies can be obtained from any suitable source. Thus, for example, monoclonal antibodies can be obtained from hybridomas prepared from mouse spleen B cells, which are obtained from mice immunized with target antigens (e.g., in the form of cells expressing antigens on the surface, or nucleic acids encoding target antigens). Monoclonal antibodies can also be obtained from hybridomas of antibody-expressing cells derived from immunized humans or non-human mammals (such as rats, dogs, primates, etc.).

[0237] VII. Antibody-drug conjugates

[0238] Anti-CD228 antibodies can be conjugated with cytotoxic or cytostatic moieties (including pharmaceutically compatible salts thereof) to form antibody drug conjugates (ADCs). Particularly suitable moieties for conjugation with antibodies are cytotoxic agents (e.g., chemotherapeutic agents), prodrug converting enzymes, radioisotopes or compounds or toxins (these moieties are collectively referred to as therapeutic agents). For example, anti-CD288 antibodies can be conjugated with cytotoxic agents such as chemotherapeutic agents or toxins (e.g., cytostatic or cytocidal agents, such as abrin, ricin A, Pseudomonas exotoxin or diphtheria toxin).

[0239] Anti-CD228 antibodies can be conjugated with prodrug converting enzymes. Prodrug converting enzymes can be recombinantly fused to the antibody or chemically conjugated using known methods. Exemplary prodrug converting enzymes are carboxypeptidase G2, β-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, β-lactamase, β-glucosidase, nitroreductase and carboxypeptidase A.

[0240] The techniques for conjugating therapeutic agents to proteins, particularly to antibodies, are well known. (See, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al., ed., Alan R. Liss, Inc., 1985); Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (Robinson et al., ed., Marcel Dekker, Inc., 2nd ed. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al., ed., 1985); “Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy,” in Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al., ed., Academic Press, 1985); and Thorpe et al., 1982, Immunol. Rev. 62: 119-58. See also, e.g., PCT Publication WO 89 / 12624.)

[0241] The therapeutic agent can be conjugated in a manner that reduces its activity unless it is cleaved from the antibody (e.g., by hydrolysis, by antibody degradation, or by a cleavage agent). Such a therapeutic agent is attached to the antibody with a cleavable linker that is sensitive to cleavage in the intracellular environment of a cancer cell expressing CD228, but substantially insensitive to the extracellular environment, such that when the conjugate is internalized by a cancer cell expressing CD228, it is cleaved from the antibody (e.g., in an endosome or in a lysosomal environment or in a caveolae environment, for example, due to pH sensitivity or protease sensitivity).

[0242] Typically, ADC includes a joint region between a therapeutic agent and an anti-CD228 antibody. As described above, typically, the joint is cleavable under intracellular conditions, so that the cleavage of the joint releases the therapeutic agent from the antibody in the intracellular environment (e.g., in a lysosome or endosome or caveolae). The joint can be, for example, a peptidyl joint cut by an intracellular peptidase or protease (including a lysosome or endosomal protease). Typically, the peptidyl joint is at least two amino acids long or at least three amino acids long. The cutting agent can include cathepsins B and D and plasmin (see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123). The most typical is a peptidyl joint that can be cut by an enzyme present in cells expressing CD228. For example, a peptidyl joint (e.g., a joint comprising a Phe-Leu or Gly-Phe-Leu-Gly peptide (SEQ ID NO: 30)) that can be cut by a thiol-dependent protease cathepsin-B that is highly expressed in cancer tissue can be used. Other such linkers are described, for example, in U.S. Pat. No. 6,214,345. In a specific embodiment, the peptidyl linker that can be cleaved by an intracellular protease includes a Val-Cit linker or a Phe-Lys dipeptide (see, for example, U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin using a Val-Cit linker). An advantage of using intracellular proteolysis to release the therapeutic agent is that the agent is generally attenuated upon conjugation, and the serum stability of the conjugate is generally high.

[0243] The cleavable linker can be pH sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, pH-sensitive linkers are hydrolyzable under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiothiocarbamides, cis-aconitamides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes can be used. (See, e.g., 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 those in blood), but are unstable when they are below pH 5.5 or 5.0 (pH similar to lysosomes). In certain embodiments, the hydrolyzable linker is a thioether linker (eg, a thioether attached to the therapeutic agent via an acylhydrazone bond) (see, eg, US Pat. No. 5,622,929).

[0244] Other linkers (e.g., disulfide linkers) are cleavable under reducing conditions. Disulfide linkers include those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio) propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio) butyrate), and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio) toluene), SPDB, and SMPT. {See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW Vogel, ed., Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)}

[0245] The linker can also be a malonate 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 can also be a malonate 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).

[0246] The linker may also be a non-cleavable linker such as a maleimido-alkylene- or maleimido-aryl linker that is directly attached to the therapeutic agent (eg, drug). The active drug-linker is released by degradation of the antibody.

[0247] Typically, the linker is 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%, even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the linker in a sample of ADC is cleaved when the ADC is present in an extracellular environment (e.g., in plasma).

[0248] Whether a linker is substantially insensitive to the extracellular environment can be determined, for example, by incubating (a) ADC ("ADC sample") and (b) an equimolar amount of an unconjugated antibody or therapeutic agent ("control sample") 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 the amount present in the control sample as measured, for example, by high performance liquid chromatography.

[0249] The linker can also promote cellular internalization. When conjugated to a therapeutic agent (i.e., in the context of a linker-therapeutic agent portion of an ADC or ADC derivative as described herein), the linker can promote cellular internalization. Alternatively, when conjugated to both a therapeutic agent and an anti-CD228 antibody, the linker can promote cellular internalization (i.e., in the context of an ADC as described herein).

[0250] The anti-CD228 antibody can be conjugated to a linker via a heteroatom of the antibody. These heteroatoms can be present in the antibody in its natural state, or can be introduced into the antibody. In some aspects, the anti-CD228 antibody will be conjugated to a linker via a nitrogen atom of a lysine residue. In other aspects, the anti-CD228 antibody will be conjugated to a linker via a sulphur atom of a cysteine ​​residue. The cysteine ​​residue can be a naturally occurring or engineered residue in the antibody. Methods for conjugating a linker and a drug-linker to an antibody via lysine and cysteine ​​residues are known in the art.

[0251] Exemplary antibody-drug conjugates include antibody-drug conjugates based on auristatin (i.e., the drug component is an auristatin drug). Auristatin binds to tubulin, has been shown to interfere with microtubule dynamics as well as nuclear and cell division, and has anti-cancer activity. Typically, an antibody-drug conjugate based on auristatin comprises 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 released by degradation of the antibody). Auristatins include auristatin T, MMAF, and MMAE. The synthesis and structure 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.

[0252] Other exemplary antibody-drug conjugates include maytansinoid antibody-drug conjugates (i.e., the drug component is a maytansinoid drug) and benzodiazepine antibody-drug conjugates (i.e., the drug component is a benzodiazepine (e.g., pyrrolo[l,4]benzodiazepine dimer (PBD dimer), indolebenzodiazepine dimer, and oxazolidinebenzodiazepine dimer)).

[0253] In some embodiments, the PBD dimer used in the present invention is represented by Formula I. The preferred stereochemistry of the PBD dimer is as shown in Formula Ia:

[0254]

[0255] or a pharmaceutical salt, solvate or a solvate of the salt; wherein the subscript n is 1 or 3.

[0256] Solvates of formula (I) and (Ia) are typically formed by adding water or an alcohol solvent to the imine functional group of one or both PBD monomers to form one or more carbinolamines and / or carbinolamine ethers. For example, an imine (N=C), carbinolamine (NH—CH(OH)) or carbinolamine ether (NH—CH(OMe)) may be present at the N10-C11 position, as shown in the following formulas I' and Ia':

[0257]

[0258] in:

[0259] (a)R 10 Yes H, R 11 is OH or ORA, where R A It is saturated C 1-4 Alkyl (preferably methyl); or

[0260] (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

[0261] (c)R 10 One of them is H, R 11 Is OH or OR A , where R A It is saturated C 1-4 alkyl (preferably methyl); and another R 10 and R 11 A nitrogen-carbon double bond is formed between the nitrogen atom and the carbon atom to which they are bound.

[0262] The PBD dimer of formula I or Ia (or its pharmaceutical salt, solvate or solvate of the salt) is typically connected to the antibody via a linker unit LU. The linker unit is used to release the PBD dimer of formula I or Ia (or its pharmaceutical salt, solvate or solvate of the salt) at the target site (for example, inside a cancer cell). The PBD drug-linker compound used for the present invention is represented by the following formula II (preferred stereochemistry, as shown in IIa), wherein LU is a linker unit. The linker unit can be, for example, a cleavable peptide linker unit (for example, a linker comprising a valine-alanine peptide) or a cleavable disulfide linker unit:

[0263]

[0264] or a pharmaceutical salt, solvate or a solvate of the salt; wherein the subscript n is 1 or 3.

[0265] Preferred PBD drug-linker compounds for use in the present invention are represented by the following Formula III:

[0266] or a pharmaceutical salt, solvate or a solvate of said salt; wherein subscript n is 1 or 3 and subscript m is an integer from 2 to 5.

[0267] The PBD drug-linker is conjugated to an anti-CD228 antibody to produce an antibody-drug conjugate targeting CD228. For example, the antibody can be conjugated to a drug-linker of Formula II or Formula III. Exemplary antibody-drug conjugates targeting C2248 are shown in Formulas IV, IVa, and IVb below:

[0268]

[0269]

[0270] or a pharmaceutical salt, solvate or a solvate of said salt; wherein subscript n is 1 or 3; subscript m is an integer from 2 to 5; and subscript p is from 1 to 4.

[0271] 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 provides an antibody drug conjugate with reduced plasma clearance and increased anti-tumor activity in a xenograft model compared to a non-PEGylated control. Thus, a particularly advantageous drug-linker for attachment to an antibody of the invention is as follows:

[0272]

[0273] or a pharmaceutically acceptable salt thereof.

[0274] The preferred stereochemistry of this drug-linker is shown in Formula Va below:

[0275]

[0276] or a pharmaceutically acceptable salt thereof, wherein for Formula V and Va, Z represents an organic moiety having a reactive site that is capable of reacting with a functional group on the antibody to form a covalent attachment thereto, n ranges from 8 to 36, and most preferably ranges from 8 to 14 (most preferably 12), R 21 It is a capping unit of a polyethylene glycol moiety, preferably -CH3 or -CH2CH2CO2H.

[0277] The preferred Z moiety is a maleimide-containing moiety. A particularly preferred Z moiety is shown in the following drug-linker:

[0278]

[0279] or a pharmaceutically acceptable salt thereof.

[0280] The preferred stereochemistry of such drug-linkers is shown below:

[0281]

[0282] or a pharmaceutically acceptable salt thereof, wherein for Formula VI, VIa, VII and VIIa, n ranges from 8 to 36, and most preferably ranges from 8 to 14 (most preferably 12), R PR is hydrogen or a protecting group (eg, an acid-labile protecting group such as BOC), R 21 It is a capping unit of a polyethylene glycol moiety, preferably -CH3 or -CH2CH2CO2H.

[0283] As mentioned above, R PRCan be hydrogen or protecting group. As used herein, protecting group refers to the group that selectively temporarily or permanently blocks the reaction site in the multifunctional compound. If the protecting group can prevent or avoid the premature loss of undesirable side reactions or protecting groups under the reaction conditions required for the desired chemical transformation in other places of the molecule and during the purification of the newly formed molecule when necessary, and can be removed under the conditions of not adversely affecting the structure of the newly formed molecule or the stereochemical integrity, then the protecting group is a suitable protecting group. 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 primary or secondary amino groups into their corresponding carbamates, and include tert-butyl, allyl and benzyl carbamates.

[0284] As mentioned above, R 21 is a capping unit of a polyethylene glycol moiety. As will be appreciated by those skilled in the art, the polyethylene glycol unit can be capped with a variety of organic moieties (typically those that are relatively non-reactive). Alkyl and substituted alkyl groups are preferred.

[0285] Typically, there are 1 to 16 drug-linkers attached to each antibody.

[0286] Referring to antibody-drug conjugates targeting CD228, the subscript p represents drug load, and depending on the context, may represent the number of molecules of drug-linker molecules attached to individual antibody molecules, and therefore is an integer value; or may represent the average drug load, and therefore may be an integer or non-integer value, but typically is a non-integer value. The average drug load represents the average number of drug-linker molecules per antibody in the population. Usually, but not always, when we refer to antibodies, such as monoclonal antibodies, we refer to a population of antibody molecules. In a composition comprising a population of antibody-drug conjugate molecules, the average drug load is an important quality attribute because it determines the amount of drug that can be delivered to the target cell. The percentage of unconjugated antibody molecules in the composition is included in the average drug load value.

[0287] In preferred aspects of the invention, when referring to a composition comprising a population of antibody-drug conjugate compounds, the average drug load is from 1 to about 16, preferably from about 2 to about 14, more preferably from about 2 to about 10. For PBD antibody drug conjugates, such as those exemplified herein, a particularly preferred average drug load is about 2. In some aspects, the actual drug load of individual antibody molecules in the population of antibody-drug conjugate compounds is from 1 to 4, 1 to 3, or 1 to 2, with the majority of the drug load being 2. In preferred aspects, an average drug load of 2 is achieved via site-specific conjugation technology (e.g., engineered cysteine ​​introduced into an antibody including position 239 according to the EU index numbering system).

[0288] For MMAE PEGylated ADCs, such as those exemplified herein, a particularly preferred average drug load is about 8. In an exemplary embodiment, the drug-linker is conjugated to a cysteine ​​residue of a reduced interchain disulfide. In some aspects, the actual drug load of a single antibody molecule in a population of antibody-drug conjugate compounds is from 1 to 10 (or from 6 to 10 or from 6 to 8), with the majority of the drug load being 8. For example, if, in addition to the interchain disulfide, the drug-linker is also conjugated to an introduced cysteine ​​residue (such as a cysteine ​​residue introduced at position 239 according to the EU index), a higher drug load can be achieved.

[0289] An exemplary ADC includes the following formula:

[0290]

[0291]

[0292] or a pharmaceutically acceptable salt thereof, wherein n ranges from 8 to 36 and most preferably ranges from 8 to 14 (most preferably 12), R PR is hydrogen or a protecting group (eg, an acid labile protecting group such as BOC), R 21 is a capping unit of a polyethylene glycol moiety, preferably -CH3 or -CH2CH2CO2H, Ab represents an anti-CD228 antibody, and 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 represents an average drug loading from about 4 or about 6 to about 14, preferably about 8 when referring to a population of antibody molecules.

[0293] As described above, the PEG (polyethylene glycol) portion of the drug linker can range from 8 to 36, however, PEG of 12 ethylene oxide units has 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. Therefore, 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.

[0294] Polydisperse PEG, monodisperse PEG and discrete PEG can be used to prepare the pegylated antibody drug conjugate of the present invention. Polydisperse PEG is a heterogeneous mixture of size and molecular weight, and monodisperse PEG is typically purified from a heterogeneous mixture, thereby providing a single chain length and molecular weight. The preferred PEG unit is a discrete PEG, i.e., a compound synthesized in a stepwise manner rather than via a polymerization process. Discrete PEG provides a single molecule with a defined and specified chain length. As the subscript "p", when referring to a population of antibody-drug conjugates, the value of the subscript "n" can be an average, and can be an integer or a non-integer.

[0295] In a preferred embodiment, the covalent attachment of the antibody to the drug-linker is accomplished by the interaction of the sulfhydryl functional group of the antibody with the maleimide functional group of the drug linker to form a sulfur-substituted succinimide. The sulfhydryl functional group can be present on the ligand unit in the natural state of the ligand, for example, in a naturally occurring residue (interchain disulfide residue), or can be introduced into the ligand via chemical modification or by bioengineering or a combination of the two. It should be understood that the antibody-substituted succinimide can exist in one or more hydrolyzed forms. For example, in a preferred embodiment, the ADC is composed of a succinimide moiety, which, when bonded to the antibody, is represented by the following structure:

[0296] Or it is composed of its corresponding acid amide portion, which is represented by the following structure when bonded to an antibody:

[0297]

[0298] The wavy line indicates the connection to the rest of the drug-linker.

[0299] Useful classes of cytotoxic agents conjugated to anti-CD228 antibodies include, for example, anti-tubulin agents, DNA minor groove binders, DNA replication inhibitors, chemosensitizers, and the like. Other exemplary classes of cytotoxic agents include anthracyclines, auristatins, camptothecins, duocarmycins, etoposide, maytansinoids, and vinca alkaloids. Some exemplary cytotoxic agents include auristatins (e.g., auristatin T, auristatin E, AFP, monomethyl auristatin F (MMAF), lipophilic monomethyl auristatin F, monomethyl auristatin E (MMAE)), DNA minor groove binders (e.g., enediynes and lexitropsin), duocarmycins, taxanes (e.g., paclitaxel and docetaxel), vinca alkaloids, nicotinamide phosphoribosyltransferase inhibitors (NAMPTi), tubulysin M, doxorubicin, morpholino-doxorubicin, and cyclomorpholino-doxorubicin.

[0300] The cytotoxic agent can be a chemotherapeutic agent, such as, for example, doxorubicin, paclitaxel, melphalan, vinca alkaloids, methotrexate, mitomycin C, or etoposide. The agent can also be a CC-1065 analog, calicheamicin, maytansine, an analog of Aplysia 10, rhizobactin, or sea anemone toxin.

[0301] The cytotoxic agent may also 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 p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatins include auristatin T, AFP, MMAF and MMAE. The synthesis and structure of various auristatins are described, for example, in US 2005-0238649 and US 2006-0074008.

[0302] The cytotoxic agent can be a DNA minor groove binder. (See, e.g., U.S. Pat. No. 6,130,237.) For example, the minor groove binder can be a CBI compound or an enediyne (e.g., calicheamicin).

[0303] The cytotoxic or cytostatic agent may be an anti-tubulin agent. Examples of anti-tubulin agents include taxanes (e.g., (paclitaxel)), (docetaxel), T67 (Tularik), vinca alkaloids (e.g., vincristine, vinblastine, vindesine and vinorelbine) and auristatins (e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEVB). Exemplary auristatins are shown below in Formulas III-XIII. Other suitable anti-tubulin agents include, for example, baccatin derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine and colcemid, estramustine, cryptophysin, simadotin, maytansinoids, combretin, discodermoide and eleuthrobin.

[0304] The cytotoxic agent can be a maytansinoid, i.e. another group of anti-tubulin agents (e.g., DM1, DM2, DM3, DM4). For example, the maytansinoid can be a drug linker containing maytansine or maytansine (e.g., DM-1 or DM-4) (ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res.).

[0305] In some embodiments, the anti-CD228 antibody of the invention is conjugated to monomethyl auristatin E via a MDpr-PEG(12)-gluc linker to form an antibody-drug conjugate having the following structure:

[0306]

[0307] or a pharmaceutically acceptable salt thereof, wherein n ranges from 8 to 36 and most preferably ranges from 8 to 14 (most preferably 12), R PR is hydrogen or a protecting group (eg, an acid labile protecting group such as BOC), R 21 is a capping unit of a polyethylene glycol moiety, preferably -CH3 or -CH2CH2CO2H, Ab represents an anti-CD228 antibody, and 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 represents an average drug load from about 4 or about 6 to about 14, preferably about 8 when referring to a population of antibody molecules. 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 having an average drug load of about 8 drug-linkers per antibody.

[0308] VIII. Therapeutic Applications

[0309] The antibodies of the present invention can be used to treat cancer in a subject alone or as its anti-CD228 antibody-drug conjugate. Some such cancers show detectable CD228 levels measured at protein (e.g., by immunoassay using one of the exemplified antibodies) or mRNA levels. Some such cancers show elevated CD228 levels relative to non-cancerous tissues of the same type (preferably from the same patient). Exemplary CD228 levels on treatable cancer cells are 5000-500,000 CD228 molecules per cell, but higher or lower levels can be treated. Optionally, the level of CD228 in cancer is measured before treatment. In some embodiments, the subject has previously been treated with one or more therapeutic agents and has no response to the treatment, wherein the one or more therapeutic agents are not the antibody, antigen binding fragment, or antibody-drug conjugate. In some embodiments, the subject has previously been treated with one or more therapeutic agents and relapsed after the treatment, wherein the one or more therapeutic agents are not the antibody, antigen binding fragment, or antibody-drug conjugate. In some embodiments, the subject has previously been treated with one or more therapeutic agents and has experienced disease progression during treatment, wherein the one or more therapeutic agents are not the antibody, antigen binding fragment, or antibody-drug conjugate. In some embodiments, the cancer is an advanced cancer. In some embodiments, the advanced cancer is a stage 3 or stage 4 cancer. In some embodiments, the advanced cancer is a metastatic cancer. In some embodiments, the cancer is a recurrent cancer. In some embodiments, the subject has received a previous treatment with a standard of care therapy for the cancer, and the previous treatment failed. In some embodiments, the subject is a human.

[0310] Examples of cancers associated with CD228 expression and treatable include melanoma and other cancers, including pancreatic cancer, lung cancer (such as non-small cell lung cancer), thyroid cancer, esophageal cancer, head and neck cancer, breast cancer (such as triple-negative breast cancer), colorectal cancer, mesothelioma and bile duct cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used in a method for treating melanoma in a subject. In some embodiments, the melanoma is a skin melanoma. In some embodiments, the skin melanoma is selected from superficial spreading melanoma, nodular melanoma, acral lentiginous nevus melanoma, malignant lentigo melanoma and desmoplastic melanoma. In some embodiments, the skin melanoma is a superficial spreading melanoma. In some embodiments, the skin melanoma is a nodular melanoma. In some embodiments, the skin melanoma is an acral lentiginous nevus melanoma. In some embodiments, the acral lentiginous nevus melanoma is a subungual melanoma. In some embodiments, the skin melanoma is lentigo maligna melanoma. In some embodiments, the skin melanoma is desmoplastic melanoma. In some embodiments, the subject has received prior therapy for skin melanoma with an inhibitor of PD-1 or PD-L1. In some embodiments, the subject has received prior therapy with an inhibitor of PD-1. In some embodiments, the inhibitor of PD-1 is selected from: nivolumab ( BMS-936558 or MDX-1106), pembrolizumab ( In some embodiments, the subject has received prior therapy with an inhibitor of PD-L1. In some embodiments, the inhibitor of PD-L1 is selected from atezolizumab ( M PDL3280A), Avelumab Durvalumab and BMS-936559. In some embodiments, the melanoma is a subcutaneous melanoma. In some embodiments, the subcutaneous melanoma is an ocular melanoma or a mucosal melanoma. In some embodiments, the melanoma is a non-cutaneous melanoma. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used in a method of 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 exocrine pancreatic cancer is selected from pancreatic adenocarcinoma, acinar cell carcinoma, cystadenocarcinoma, pancreatoblastoma, adenosquamous carcinoma, signet ring carcinoma, hepatoid carcinoma, colloid carcinoma, undifferentiated carcinoma, and pancreatic mucinous cystic tumor. In some embodiments, the subject has received one or more prior lines of treatment for the exocrine pancreatic cancer. In some embodiments, the subject has received one prior line of treatment for the exocrine pancreatic cancer. In some embodiments, the subject has received more than one prior line of treatment 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 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 tumor. In some embodiments, the pancreatic cancer is neuroendocrine carcinoma. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used in a method for treating lung cancer in a subject. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used in a method for treating non-small cell lung cancer in a subject. In some embodiments, the non-small cell lung cancer has a mutant 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 received a previous therapy for the non-small cell lung cancer using a platinum-based therapy. In some embodiments, the platinum-based therapy is selected from carboplatin, cisplatin, oxaliplatin, nedaplatin, tetranitrate triplatin, pheniplatin, 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 tetranitrate triplatin. In some embodiments, the platinum-based therapy is pheniplatin.In some embodiments, the platinum-based therapy is picoplatin. In some embodiments, the platinum-based therapy is satraplatin. In some embodiments, the subject has received prior therapy for the non-small cell lung cancer with an inhibitor of PD-1 or PD-L1. In some embodiments, the subject has received prior therapy with an inhibitor of PD-1. In some embodiments, the PD-1 inhibitor is selected from: nivolumab (. BMS-936558 or M DX-1106), pembrolizumab ( In some embodiments, the subject has received prior therapy with an inhibitor of PD-L1. In some embodiments, the inhibitor of PD-L1 is selected from atezolizumab ( MPDL3280A), Avelumab Durvalumab and BMS-936559. In some embodiments, the subject has received a prior therapy for the non-small cell lung cancer using a platinum-based therapy and an inhibitor of PD-1 or PD-L1. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used in a method for treating thyroid cancer in a subject. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used in a method for treating esophageal cancer in a subject. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used in a method for treating head and neck cancer in a subject. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used in a method for treating breast cancer in a subject. In some embodiments, the breast cancer is selected from 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 HER2 positive breast cancer. In some embodiments, the breast cancer is HER2 negative breast cancer. In some embodiments, the subject has received one or more prior treatment lines for the HER2 negative breast cancer. In some embodiments, the one or more prior treatment lines include treatment with taxanes. In some embodiments, the taxane is selected from 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 received prior therapy with an inhibitor of CDK4 / 6. In some embodiments, the subject with HER2-negative, hormone receptor-positive breast cancer has received prior therapy 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 present invention is used in a method for treating triple-negative breast cancer in a subject. Triple-negative breast cancer is a term for cancers that lack detectable estrogen and progesterone receptors and lack overexpression of HER2 / neu. In some embodiments, the antibody or antibody-drug conjugate of the invention is used in a method of treating colorectal cancer in a subject. In some embodiments, the colorectal cancer is selected from 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 a gastrointestinal carcinoid tumor. In some embodiments, the colorectal cancer is a leiomyosarcoma. In some embodiments, the subject has received two or more previous lines of treatment for the colorectal cancer. In some embodiments, the subject has received two previous lines of treatment for the colorectal cancer. In some embodiments, the subject has received more than two lines of treatment for the colorectal cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used in a method for treating mesothelioma in a subject. In some embodiments, the mesothelioma is selected from pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, and testicular mesothelioma. In some embodiments, the mesothelioma is pleural mesothelioma. In some embodiments, the subject has received a previous therapy for the pleural mesothelioma using a platinum-based therapy. In some embodiments, the platinum-based therapy is selected from carboplatin, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenaplatin, picoplatin, and satraplatin.

[0311] 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 phenanthridine platinum. In some embodiments, the platinum-based therapy is picoplatin. In some embodiments, the platinum-based therapy is satraplatin. In some embodiments, the subject has received prior therapy 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 antibody or antibody-drug conjugate of the present invention is used in a method for treating bile duct cancer. The treatment can be applied to patients with primary or metastatic tumors of these kinds. The treatment can also be applied to patients who are refractory to conventional treatment or who have relapsed after responding to such treatment. In some embodiments, the subject is human.

[0312] The antibody of the present invention (such as humanized antibody) is administered alone or as its conjugate with an effective regimen, which means that the onset can be delayed, the severity can be reduced, the dosage, route of administration and frequency of administration of at least one sign or symptom of the cancer can be suppressed, further deteriorated and / or improved. If the patient already has cancer, the regimen can be referred to as a therapeutically effective regimen. If the patient has an increased risk of developing cancer relative to the general population, but has not yet experienced symptoms, the regimen can be referred to as a preventive effective regimen. In some cases, treatment or preventive efficacy can be observed in a single patient relative to historical controls or past experience in the same patient. In other cases, treatment or preventive efficacy in preclinical or clinical trials can be demonstrated relative to a control population of untreated patients.

[0313] An exemplary dosage of the monoclonal antibody is 0.1 mg / kg to 50 mg / kg of patient body weight, 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. An exemplary dosage of a monoclonal antibody or its antibody drug conjugate is 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 of subject body weight, or 0.1-20 mg / kg body weight or 0.5-5 mg / kg body weight (e.g., 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg or 10 mg / kg) or as a fixed dose of 10-1500 mg or 200-1500 mg. In some methods, a dosage of at least 1.5 mg / kg, at least 2 mg / kg or at least 3 mg / kg is administered to the patient once every three weeks or longer. The dosage depends on factors such as the frequency of administration, the patient's condition and response to previous treatment (if any), whether the treatment is preventive or therapeutic, and whether the disorder is acute or chronic.

[0314] Administration can be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular. Administration can also be directly localized to the tumor. Administration to the systemic circulation is preferred by intravenous or subcutaneous administration. Intravenous administration can be performed, for example, by infusion for a period of time (e.g., 30-90 min) or by a single bolus injection.

[0315] The frequency of administration depends on factors such as the half-life of the antibody or conjugate in the circulation, the patient's condition, and the route of administration. The frequency can be once a day, once a week, once a month, once a quarter, or at irregular intervals in response to changes in the patient's condition or the progression of the cancer being treated. In a continuous treatment process, an exemplary frequency of intravenous administration is between twice a week and once a quarter, but it can also be administered at a higher or lower frequency. In a continuous treatment process, other exemplary frequencies of intravenous administration are between once a week or three times every four weeks, but it can also be administered at a higher or lower frequency. For subcutaneous administration, an exemplary frequency of administration is once a day to once a month, but it can also be administered at a higher or lower frequency.

[0316] The number of dosages administered depends on the nature of the cancer (e.g., whether acute symptoms or chronic symptoms are present) and the reaction of the disorder to treatment. For acute disorders or acute exacerbations of chronic disorders, between 1 and 10 doses are generally sufficient. Sometimes, a single bolus dose (optionally in a separated form) is sufficient for acute disorders or acute exacerbations of chronic disorders. For the recurrence or acute exacerbations of acute disorders, repeated treatment is possible. For chronic disorders, the antibody can be administered at regular intervals, for example, once a week, once every two weeks, once a month, once a quarter, once every six months, for at least 1, 5 or 10 years or the life span of the patient.

[0317] Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under GMP conditions. The pharmaceutical composition can be provided in unit dosage form (i.e., a dose for a single administration). The pharmaceutical composition can be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the selected route of administration. For injection, the antibody can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or saline or acetate buffer (to reduce discomfort at the injection site). The solution may contain a formulation agent, such as a suspending agent, a stabilizer, and / or a dispersant. Alternatively, prior to use, the antibody can be in lyophilized form for use with a suitable vehicle (e.g., sterile pyrogen-free water). The concentration of the antibody in the liquid formulation can be, for example, 1-100 mg / ml, such as 10 mg / ml.

[0318] Treatment with the antibodies of the invention can be combined with chemotherapy, radiation, stem cell therapy, surgery, other treatments that are effective for the disorder being treated. Useful classes of other agents 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, anti-tubulin agents (e.g., auristatins), DNA minor groove binders, 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, etoposide, fluorinated pyrimidines, ionophores, lexistatins, nitrosoureas, cisplatin, preformed compounds, purine antimetabolites, puromycin, radiosensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, and the like.

[0319] Treatment with an anti-CD228 antibody or antibody-drug conjugate, optionally in combination with any of the other agents or regimens described above, alone or as an antibody drug conjugate, may increase the median progression-free survival or overall survival time of patients with tumors (e.g., melanoma, pancreatic cancer, non-small lung cancer, thyroid cancer, head and neck cancer, triple-negative breast cancer, colorectal cancer, mesothelioma, bile duct cancer), especially in the case of relapsed or refractory tumors, by at least 30% or 40%, but 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 + partial) in patients with tumors by at least 30% or 40%, but 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.

[0320] Typically, in a clinical trial (e.g., Phase II, Phase II / III, or Phase III trial), the aforementioned increase in median progression-free survival and / or response rate of patients treated with standard therapy plus an anti-CD228 antibody alone or as a conjugate relative to a control group of patients receiving standard therapy alone (or plus placebo) is statistically significant, e.g., at the p=0.05 or 0.01 or even 0.001 level. Complete and partial response rates are determined by objective criteria commonly used in cancer clinical trials (e.g., as listed or accepted by the National Cancer Institute and / or the Food and Drug Administration).

[0321] IX. Products and Kits

[0322] On the other hand, a product or kit is provided, the product or kit comprising an anti-CD228 antibody or anti-CD228 antibody-drug conjugate as described herein. The product or kit may also include instructions for use of anti-CD228 antibodies or anti-CD228 antibody-drug conjugates as described herein in the method of the present invention. Therefore, in certain embodiments, the product or kit comprises instructions for use of anti-CD228 antibodies or anti-CD228 antibody-drug conjugates as described herein in the method for treating cancer (e.g., melanoma and other cancers, 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 bile duct cancer) in a subject, the method comprising administering an effective amount of anti-CD228 antibodies or anti-CD228 antibody-drug conjugates as described herein to the subject. 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 bile duct cancer. In some embodiments, the subject is a human.

[0323] The article or kit may also include a container. Suitable containers include, for example, bottles, vials (e.g., dual-chamber vials), syringes (such as single-chamber or dual-chamber syringes), and test tubes. In some embodiments, the container is a vial. The container may be made of various materials (e.g., glass or plastic). The container holds the preparation.

[0324] The product or kit may also include a label or package insert on or associated with the container, which may indicate instructions for reconstructing and / or using the preparation. The label or package insert may further indicate that the preparation may be used for or intended for subcutaneous, intravenous (e.g., intravenous infusion) or other modes of administration to treat cancer (e.g., melanoma and other cancers, 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 bile duct cancer) in the subject. The container containing the preparation may be a single-use vial or a multiple-use vial, which allows repeated administration of the reconstructed preparation. The product or kit may also include a second container containing a suitable diluent. The product or kit may also include other materials desired from a commercial, therapeutic and user perspective, including other buffers, diluents, filters, needles, syringes and package inserts printed with instructions for use.

[0325] The article or kit herein also optionally comprises a container containing a second drug, wherein the anti-CD228 antibody or anti-CD228 antibody-drug conjugate is a first drug, and the article or kit further comprises instructions on a label or package insert for treating a subject with an effective amount of the second drug. In some embodiments, the second drug is used to eliminate or reduce the severity of one or more adverse events.

[0326] In some embodiments, the anti-CD228 antibody or anti-CD228 antibody-drug conjugate is present in the container as a lyophilized powder. In some embodiments, the lyophilized powder is in an airtight container (such as a vial, an ampoule or a pouch), and the container indicates the amount of the active agent. In the case of administering the drug by injection, an ampoule of sterile water for injection or saline can, for example, be optionally provided as a part of the test kit so that the ingredients can be mixed before administration. If necessary, such test kits may also include one or more various conventional drug components, such as containers with one or more pharmaceutically acceptable carriers, other containers, etc., which are clear to those skilled in the art. The test kit may also include a printed instruction as an insert or as a label, which indicates the amount of the component to be administered, the guidance of the administration guide and / or the mixing component.

[0327] X. Other Applications

[0328] Anti-CD228 antibodies described herein, such as humanized anti-CD228 antibodies, can be used to detect CD228 in the context of clinical diagnosis or treatment or in research. The expression of CD228 on cancer provides an indication that the cancer can be treated with the antibodies of the present invention. The antibodies can also be sold as reagents for laboratory studies in detecting cells carrying CD228 and their responses to various stimuli. In such uses, monoclonal antibodies can be labeled with fluorescent molecules, spin-labeled molecules, enzymes or radioisotopes, and can be provided in the form of a kit together with all reagents required for the determination of CD228. The antibodies described herein can be used to detect CD228 protein expression and determine whether cancer can be treated with CD228 ADC. For example, hL49 (HALC) can be used to detect CD228 expression on 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, for example, to purify CD228 by affinity chromatography.

[0329] All patent applications, websites, other publications, accession numbers, etc. cited above or below are incorporated by reference in their entirety for all purposes, to the extent that each individual item is specifically and individually indicated to be incorporated by reference. If different versions of a sequence are associated with accession numbers at different times, it means that the version is associated with the accession number at the effective filing date of the present application. The effective filing date means the earlier of the actual filing date or the filing date of the priority application with respect to the accession number (if applicable). Similarly, if different versions of a publication, website, etc. are published at different times, unless otherwise stated, it refers to the version published on the effective filing date closest to the application. Unless otherwise specifically stated, any feature, step, element, embodiment or aspect of the present invention may be used in combination with any other. Although the present invention has been described in some detail by way of illustration and example for the purpose of clarity and understanding, it is clear that certain changes and modifications may be implemented within the scope of the appended claims.

[0330] XI. Variable Domain Sequences

[0331] For each variable region sequence below, the CDRs according to the Kabat numbering scheme are underlined and the CDRs according to the IMGT numbering scheme are in bold and italics.

[0332] Mouse L49 vH

[0333]

[0334] Mu IGHV3-8 vH

[0335]

[0336] Hu IGHV4-59 / HJ4

[0337]

[0338] HkDJ

[0339]

[0340] HkDJ

[0341]

[0342] HkDJ

[0343]

[0344] L49

[0345]

[0346] Mu IGKV1-110 vL

[0347]

[0348] Hu IGKV2-30 / KJ2

[0349]

[0350] oeLh

[0351]

[0352] oeLh

[0353]

[0354] LC

[0355]

[0356] XII. Exemplary Embodiments

[0357] Embodiments provided herein include:

[0358] 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 comprises:

[0359] (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1;

[0360] (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and

[0361] (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and

[0362] Wherein the light chain variable region comprises:

[0363] (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4;

[0364] (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and

[0365] (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO:6.

[0366] 2. The antibody or antigen-binding fragment of embodiment 1, wherein the antibody is humanized.

[0367] 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, provided 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, provided that position L2 is occupied by F, position L36 is occupied by Y and position L46 is occupied by L.

[0368] 4. The antibody or antigen-binding fragment of embodiment 3, wherein the condition is further that position L28 is occupied by D.

[0369] 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, wherein 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, wherein position L2 is occupied by F, position L36 is occupied by Y and position L46 is occupied by L.

[0370] 6. An antibody or antigen-binding fragment according to 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.

[0371] 7. An antibody or antigen-binding fragment according to any one 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.

[0372] 8. An antibody or antigen-binding fragment according to any one 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.

[0373] 9. The antibody or antigen-binding fragment of 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.

[0374] 10. The antibody or antigen-binding fragment of any one of embodiments 1 to 9, wherein the antibody or antigen-binding fragment is an antigen-binding fragment.

[0375] 11. The antibody or antigen-binding fragment according to embodiment 10, wherein the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fab'-SH, Fv, diabodies, linear antibodies and single-chain antibody fragments.

[0376] 12. The antibody or antigen-binding fragment of any one of embodiments 1 to 9, wherein the antibody or antigen-binding fragment is a full-length antibody.

[0377] 13. An antibody or antigen-binding fragment according to embodiment 12, wherein the heavy chain variable region is fused to the heavy chain constant region, and the light chain variable region is fused to the light chain constant region.

[0378] 14. The antibody or antigen-binding fragment of embodiment 13, wherein the heavy chain constant region is of the IgG1 isotype.

[0379] 15. The antibody or antigen-binding fragment of embodiment 13 or embodiment 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.

[0380] 16. An antibody or antigen-binding fragment according to embodiment 13 or embodiment 14, wherein the heavy chain constant region is a mutated form of a native human constant region that has reduced binding to Fcγ receptors relative to the native human constant region.

[0381] 17. An antibody or antigen-binding fragment according to embodiment 13 or embodiment 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.

[0382] 18. An antibody-drug conjugate comprising the antibody or antigen-binding fragment according to any one of embodiments 1 to 17 conjugated to a cytotoxic or cytostatic agent.

[0383] 19. The antibody-drug conjugate of embodiment 18, wherein the antibody or antigen-binding fragment is conjugated to the cytotoxic agent or cytostatic agent via a linker.

[0384] 20. The antibody-drug conjugate of embodiment 19, wherein the linker is a MDpr-PEG(12)-gluc linker.

[0385] 21. The antibody-drug conjugate of any one of embodiments 18 to 20, wherein the cytotoxic or cytostatic agent is monomethyl auristatin.

[0386] 22. The antibody-drug conjugate of embodiment 21, wherein the monomethyl auristatin is monomethyl auristatin E (MMAE).

[0387] 23. The antibody-drug conjugate of embodiment 22, wherein the linker is attached to monomethyl auristatin E, thereby forming an antibody-drug conjugate having the following structure:

[0388]

[0389] Where Ab is antibody hL49, n is 12, R PR is hydrogen, R 21 is CH3, and p represents a number from 1 to 16.

[0390] 24. An antibody-drug conjugate according to embodiment 23, wherein the average value of p in the population of antibody-drug conjugates is about 8.

[0391] 25. The antibody-drug conjugate of any one of embodiments 18 to 24, wherein the antibody-drug conjugate is hL49-MDpr-PEG(12)-gluc-MMAE.

[0392] 26. A nucleic acid encoding a heavy chain variable region and / or a light chain variable region as defined in any one of embodiments 1 to 17.

[0393] 27. A vector comprising the nucleic acid according to embodiment 26.

[0394] 28. The vector of embodiment 27, wherein the vector is an expression vector.

[0395] 29. A host cell comprising the nucleic acid according to embodiment 26.

[0396] 30. The host cell of embodiment 29, wherein the host cell is a Chinese Hamster Ovary (CHO) cell.

[0397] 31. A method of producing an anti-CD228 antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell according to embodiment 29 or embodiment 30 under conditions suitable for producing the anti-CD228 antibody or the antigen-binding fragment thereof.

[0398] 32. The method according to embodiment 31 further comprises isolating the anti-CD228 antibody or antigen-binding fragment thereof produced by the host cell.

[0399] 33. A method for producing an anti-CD228 antibody-drug conjugate, the method comprising culturing the host cell according to embodiment 29 or embodiment 30 under conditions suitable for producing anti-CD228 antibodies; isolating the anti-CD228 antibodies produced from the host cells; and conjugating the anti-CD228 antibodies to a cytotoxic agent or a cytostatic agent.

[0400] 34. A method according to embodiment 33, wherein the anti-CD228 antibody is conjugated to the cytotoxic agent or cytostatic agent via a linker.

[0401] 35. A method according to embodiment 34, wherein the linker is a MDpr-PEG(12)-gluc linker.

[0402] 36. The method of any one of embodiments 33 to 35, wherein the cytotoxic or cytostatic agent is monomethyl auristatin.

[0403] 37. The method of embodiment 36, wherein the monomethyl auristatin is monomethyl auristatin E (MMAE).

[0404] 38. The method of embodiment 37, wherein the linker is attached to monomethyl auristatin E, thereby forming an antibody-drug conjugate having the following structure:

[0405]

[0406] Where Ab is antibody hL49, n is 12, R PR is hydrogen, R 21 is CH3, and p represents a number from 1 to 16.

[0407] 39. A method according to embodiment 38, wherein the average value of p in the population of antibody-drug conjugates is about 8.

[0408] 40. The method of any one of embodiments 33 to 39, wherein the antibody-drug conjugate is hL49-MDpr-PEG(12)-gluc-MMAE.

[0409] 41. A method of treating cancer in a subject, the method comprising administering to the subject the antibody or antigen-binding fragment of any one of Embodiments 1 to 17 or the antibody-drug conjugate of any one of Embodiments 18 to 25.

[0410] 42. A method according to embodiment 41, wherein the subject has previously been treated with one or more therapeutic agents and has not responded to the treatment, wherein the one or more therapeutic agents are not the antibody, antigen-binding fragment, or antibody-drug conjugate.

[0411] 43. A method according to embodiment 41, wherein the subject has been previously treated with one or more therapeutic agents and relapses after said treatment, wherein the one or more therapeutic agents are not the antibody, antigen-binding fragment, or antibody-drug conjugate.

[0412] 44. A method according to embodiment 41, wherein the subject has previously been treated with one or more therapeutic agents and experienced disease progression during treatment, wherein the one or more therapeutic agents are not the antibody, antigen-binding fragment, or antibody-drug conjugate.

[0413] 45. The method of any one of embodiments 41 to 44, wherein the cancer is an advanced cancer.

[0414] 46. ​​The method of embodiment 45, wherein the advanced cancer is stage 3 or stage 4 cancer.

[0415] 47. The method of embodiment 45 or 46, wherein the advanced cancer is metastatic cancer.

[0416] 48. The method of any one of embodiments 41 to 47, wherein the cancer is a recurrent cancer.

[0417] 49. The method of any one of embodiments 41 to 48, wherein the cancer is unresectable.

[0418] 50. A method according to any one of embodiments 41 to 49, wherein the subject has received prior treatment with standard of care therapy for the cancer, but the prior treatment has failed.

[0419] 51. A method according to any one of embodiments 41 to 50, wherein the cancer is selected from melanoma, pancreatic cancer, mesothelioma, colorectal cancer, lung cancer, thyroid cancer, breast cancer, bile duct cancer, esophageal cancer and head and neck cancer.

[0420] 52. The method of embodiment 51, wherein the cancer is melanoma.

[0421] 53. The method of embodiment 52, wherein the melanoma is cutaneous melanoma.

[0422] 54. The method of embodiment 53, wherein the cutaneous melanoma is selected from superficial spreading melanoma, nodular melanoma, acral lentigo melanoma, lentigo maligna melanoma, and desmoplastic melanoma.

[0423] 55. The method of embodiment 54, wherein the acral lentiginous melanoma is a subungual melanoma.

[0424] 56. The method of any one of embodiments 53 to 55, wherein the subject has received prior therapy with an inhibitor of PD-1 or PD-L1.

[0425] 57. A method according to embodiment 56, wherein the subject has received prior therapy with an inhibitor of PD-1.

[0426] 58. The method of embodiment 52, wherein the melanoma is subcutaneous melanoma.

[0427] 59. The method of embodiment 58, wherein the subcutaneous melanoma is ocular melanoma or mucosal melanoma.

[0428] 60. The method of embodiment 52, wherein the melanoma is non-cutaneous melanoma.

[0429] 61. The method of embodiment 51, wherein the cancer is mesothelioma.

[0430] 62. The method of embodiment 61, wherein the mesothelioma is selected from pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, and testicular mesothelioma.

[0431] 63. The method of embodiment 62, wherein the mesothelioma is pleural mesothelioma.

[0432] 64. The method of embodiment 63, wherein the subject has received prior therapy with a platinum-based therapy.

[0433] 65. The method of embodiment 64, wherein the platinum-based therapy is cisplatin.

[0434] 66. The method of any one of embodiments 63 to 65, wherein the subject has received prior therapy with pemetrexed.

[0435] 67. The method of embodiment 51, wherein the lung cancer is non-small cell lung cancer.

[0436] 68. A method according to embodiment 67, wherein the non-small cell lung cancer has a mutated form of epidermal growth factor receptor (EGFR).

[0437] 69. A method according to embodiment 67, wherein the non-small cell lung cancer has wild-type EGFR.

[0438] 70. The method of embodiment 69, wherein the subject has received prior therapy with a platinum-based therapy.

[0439] 71. The method of embodiment 69 or 70, wherein the subject has received prior therapy with an inhibitor of PD-1 or PD-L1.

[0440] 72. The method of embodiment 71, wherein the subject has received prior therapy with an inhibitor of PD-1.

[0441] 73. A method according to embodiment 51, wherein the breast cancer is selected from HER2-positive, HER2-negative, estrogen receptor (ER)-positive, ER-negative, progesterone receptor (PR)-positive, PR-negative and triple-negative breast cancer.

[0442] 74. The method of embodiment 73, wherein the breast cancer is HER2-negative breast cancer.

[0443] 75. The method of embodiment 74, wherein the subject has received one or more prior lines of treatment for the HER2-negative breast cancer.

[0444] 76. The method of embodiment 75, wherein the one or more prior lines of treatment comprised treatment with a taxane.

[0445] 77. The method of embodiment 75 or 76, wherein the subject is hormone receptor positive.

[0446] 78. The method of embodiment 77, wherein the subject has received prior therapy with an inhibitor of CDK4 / 6.

[0447] 79. The method of embodiment 77 or 78, wherein the subject has received prior therapy with hormone-directed therapy.

[0448] 80. A method according to embodiment 51, wherein the colorectal cancer is selected from colorectal adenocarcinoma, gastrointestinal stromal tumor, primary colorectal lymphoma, gastrointestinal carcinoid tumor and leiomyosarcoma.

[0449] 81. The method of embodiment 80, wherein the subject has received two or more prior lines of treatment for the colorectal cancer.

[0450] 82. The method of embodiment 51, wherein the pancreatic cancer is an exocrine cancer or a neuroendocrine cancer.

[0451] 83. A method according to embodiment 82, wherein the exocrine cancer is selected from pancreatic adenocarcinoma, acinar cell carcinoma, cystadenocarcinoma, pancreatoblastoma, adenosquamous carcinoma, signet ring carcinoma, hepatoid carcinoma, colloid carcinoma, undifferentiated carcinoma and pancreatic mucinous cystic neoplasm.

[0452] 84. A method according to embodiment 83, wherein the pancreatic adenocarcinoma is pancreatic ductal adenocarcinoma.

[0453] 85. The method of embodiment 83 or 84, wherein the subject has received one or more prior lines of treatment for the pancreatic cancer.

[0454] 86. A method according to any one of embodiments 41 to 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.

[0455] 87. The method of any one of embodiments 41 to 86, wherein the subject is human.

[0456] 88. A kit comprising:

[0457] (a) the antibody or antigen-binding fragment according to any one of Embodiments 1 to 17 or the antibody-drug conjugate according to any one of Embodiments 18 to 25; and

[0458] (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.

[0459] 89. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of embodiments 1 to 17 or the antibody-drug conjugate of any one of embodiments 18 to 25 and one or more agents selected from physiologically acceptable carriers, diluents, excipients and adjuvants.

[0460] 90. The antibody or antigen-binding fragment of any one of embodiments 1 to 17 or the antibody-drug conjugate of any one of embodiments 18 to 25 for use in treating cancer in a subject.

[0461] 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 said treatment, wherein said one or more therapeutic agents are not said antibody, antigen-binding fragment, or antibody-drug conjugate.

[0462] 92. The antibody or antigen-binding fragment of embodiment 90, wherein the subject has been previously treated with one or more therapeutic agents and relapsed following said treatment, wherein said one or more therapeutic agents are not said antibody, antigen-binding fragment, or antibody-drug conjugate.

[0463] 93. The antibody or antigen-binding fragment of embodiment 90, wherein 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 the antibody, antigen-binding fragment, or antibody-drug conjugate.

[0464] 94. The antibody or antigen-binding fragment of any one of embodiments 90 to 93, wherein the cancer is an advanced cancer.

[0465] 95. The antibody or antigen-binding fragment of embodiment 94, wherein the advanced cancer is stage 3 or stage 4 cancer.

[0466] 96. The antibody or antigen-binding fragment of embodiment 94 or 95, wherein the advanced cancer is a metastatic cancer.

[0467] 97. The antibody or antigen-binding fragment of any one of embodiments 90 to 96, wherein the cancer is a recurrent cancer.

[0468] 98. The antibody or antigen-binding fragment of any one of embodiments 90 to 97, wherein the cancer is unresectable.

[0469] 99. The antibody or antigen-binding fragment of any one of embodiments 90 to 98, wherein the subject has received prior treatment with standard of care therapy for the cancer, but the prior treatment has failed.

[0470] 100. The antibody or antigen-binding fragment of any one of embodiments 90 to 99, wherein the cancer is selected from melanoma, pancreatic cancer, mesothelioma, colorectal cancer, lung cancer, thyroid cancer, breast cancer, bile duct cancer, esophageal cancer, and head and neck cancer.

[0471] 101. The antibody or antigen-binding fragment of embodiment 100, wherein the cancer is melanoma.

[0472] 102. The antibody or antigen-binding fragment of embodiment 101, wherein the melanoma is cutaneous melanoma.

[0473] 103. The antibody or antigen-binding fragment of embodiment 102, wherein the cutaneous melanoma is selected from superficial spreading melanoma, nodular melanoma, acral lentigo melanoma, lentigo maligna melanoma, and desmoplastic melanoma.

[0474] 104. The antibody or antigen-binding fragment of embodiment 103, wherein the acral lentiginous melanoma is subungual melanoma.

[0475] 105. The antibody or antigen-binding fragment of any one of embodiments 102 to 104, wherein the subject has received prior therapy with an inhibitor of PD-1 or PD-L1.

[0476] 106. The antibody or antigen-binding fragment of embodiment 105, wherein the subject has received prior therapy with an inhibitor of PD-1.

[0477] 107. The antibody or antigen-binding fragment of embodiment 101, wherein the melanoma is subcutaneous melanoma.

[0478] 108. The antibody or antigen-binding fragment of embodiment 107, wherein the subcutaneous melanoma is ocular melanoma or mucosal melanoma.

[0479] 109. The antibody or antigen-binding fragment of embodiment 101, wherein the melanoma is non-cutaneous melanoma.

[0480] 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.

[0481] 112. The antibody or antigen-binding fragment of embodiment 111, wherein the mesothelioma is pleural mesothelioma.

[0482] 113. The antibody or antigen-binding fragment of Embodiment 112, wherein the subject has received prior therapy with a platinum-based therapy.

[0483] 114. The antibody or antigen-binding fragment of Embodiment 113, wherein the platinum-based therapy is cisplatin.

[0484] 115. The antibody or antigen-binding fragment of any one of embodiments 112 to 114, wherein the subject has received prior therapy with pemetrexed.

[0485] 116. The antibody or antigen-binding fragment of embodiment 100, wherein the lung cancer is non-small cell lung cancer.

[0486] 117. An antibody or antigen-binding fragment according to embodiment 116, wherein the non-small cell lung cancer has a mutated form of epidermal growth factor receptor (EGFR).

[0487] 118. An antibody or antigen-binding fragment according to embodiment 116, wherein the non-small cell lung cancer has wild-type EGFR.

[0488] 119. The antibody or antigen-binding fragment of Embodiment 118, wherein the subject has received prior therapy with a platinum-based therapy.

[0489] 120. The antibody or antigen-binding fragment of Embodiment 118 or 119, wherein the subject has received prior therapy with an inhibitor of PD-1 or PD-L1.

[0490] 121. The antibody or antigen-binding fragment of embodiment 120, wherein the subject has received prior therapy with an inhibitor of PD-1.

[0491] 122. An antibody or antigen-binding fragment according to embodiment 100, wherein the breast cancer is selected from HER2-positive, HER2-negative, estrogen receptor (ER)-positive, ER-negative, progesterone receptor (PR)-positive, PR-negative and triple-negative breast cancer.

[0492] 123. The antibody or antigen-binding fragment of embodiment 122, wherein the breast cancer is HER2-negative breast cancer.

[0493] 124. The antibody or antigen-binding fragment of embodiment 123, wherein the subject has received one or more prior lines of treatment for the HER2-negative breast cancer.

[0494] 125. The antibody or antigen-binding fragment of Embodiment 124, wherein the one or more prior lines of treatment comprised treatment with a taxane.

[0495] 126. The antibody or antigen-binding fragment of embodiment 124 or 125, wherein the subject is hormone receptor positive.

[0496] 127. The antibody or antigen-binding fragment of embodiment 126, wherein the subject has received prior therapy with an inhibitor of CDK4 / 6.

[0497] 128. The antibody or antigen-binding fragment of Embodiment 126 or 127, wherein the subject has received prior therapy with hormone-directed therapy.

[0498] 129. An antibody or antigen-binding fragment according to embodiment 128, wherein the colorectal cancer is selected from colorectal adenocarcinoma, gastrointestinal stromal tumor, primary colorectal lymphoma, gastrointestinal carcinoid tumor and leiomyosarcoma.

[0499] 130. The antibody or antigen-binding fragment of embodiment 129, wherein the subject has received two or more prior lines of treatment for the colorectal cancer.

[0500] 131. The antibody or antigen-binding fragment of embodiment 100, wherein the pancreatic cancer is an exocrine cancer or a neuroendocrine cancer.

[0501] 132. An antibody or antigen-binding fragment according to embodiment 131, wherein the exocrine cancer is selected from pancreatic adenocarcinoma, acinar cell carcinoma, cystadenocarcinoma, pancreatoblastoma, adenosquamous carcinoma, signet ring carcinoma, hepatoid carcinoma, colloid carcinoma, undifferentiated carcinoma and pancreatic mucinous cystic neoplasm.

[0502] 133. The antibody or antigen-binding fragment of embodiment 132, wherein the pancreatic adenocarcinoma is pancreatic ductal adenocarcinoma.

[0503] 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 the pancreatic cancer.

[0504] 135. The antibody or antigen-binding fragment of any one of embodiments 90 to 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.

[0505] 136. The antibody or antigen-binding fragment of any one of embodiments 90 to 135, wherein the subject is human.

[0506] 137. Use of the antibody or antigen-binding fragment of any one of embodiments 1 to 17 or the antibody-drug conjugate of any one of embodiments 18 to 25 for the manufacture of a medicament for treating cancer in a subject.

[0507] 138. The use according to embodiment 137, wherein the subject has previously been treated with one or more therapeutic agents and has not responded to the treatment, wherein the one or more therapeutic agents are not the antibody, antigen-binding fragment or antibody-drug conjugate.

[0508] 139. The use according to embodiment 137, wherein the subject has been previously treated with one or more therapeutic agents and relapsed after said treatment, wherein said one or more therapeutic agents are not said antibody, antigen-binding fragment or antibody-drug conjugate.

[0509] 140. The use according to embodiment 137, wherein the subject has previously been treated with one or more therapeutic agents and experienced disease progression during treatment, wherein the one or more therapeutic agents are not the antibody, antigen-binding fragment or antibody-drug conjugate.

[0510] 141. The use according to any one of embodiments 137 to 140, wherein the cancer is an advanced cancer.

[0511] 142. The use according to embodiment 141, wherein the advanced cancer is stage 3 or stage 4 cancer.

[0512] 143. The method of embodiment 141 or 142, wherein the advanced cancer is a metastatic cancer. 144. The use of any one of embodiments 137 to 143, wherein the cancer is a recurrent cancer.

[0513] 145. The use according to any one of embodiments 137 to 144, wherein the cancer is unresectable.

[0514] 146. The use of any one of embodiments 137 to 145, wherein the subject has received prior treatment with standard of care therapy for the cancer, but the prior treatment has failed.

[0515] 147. The use according to any one of embodiments 137 to 146, wherein the cancer is selected from melanoma, pancreatic cancer, mesothelioma, colorectal cancer, lung cancer, thyroid cancer, breast cancer, bile duct cancer, esophageal cancer, and head and neck cancer.

[0516] 148. The use according to embodiment 147, wherein the cancer is melanoma.

[0517] 149. The use according to embodiment 148, wherein the melanoma is cutaneous melanoma.

[0518] 150. The use according to embodiment 149, wherein the cutaneous melanoma is selected from superficial spreading melanoma, nodular melanoma, acral lentigo melanoma, lentigo maligna melanoma and desmoplastic melanoma.

[0519] 151. The use according to embodiment 150, wherein the acral lentiginous melanoma is subungual melanoma.

[0520] 152. The use according to any one of embodiments 149 to 151, wherein the subject has received prior therapy with an inhibitor of PD-1 or PD-L1.

[0521] 153. The use according to embodiment 152, wherein the subject has received prior therapy with an inhibitor of PD-1.

[0522] 154. The use according to embodiment 148, wherein the melanoma is subcutaneous melanoma.

[0523] 155. The use according to embodiment 154, wherein the subcutaneous melanoma is ocular melanoma or mucosal melanoma.

[0524] 156. The use according to embodiment 148, wherein the melanoma is non-cutaneous melanoma.

[0525] 157. The use according to embodiment 147, wherein the cancer is mesothelioma.

[0526] 158. The use according to embodiment 157, wherein the mesothelioma is selected from pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma and testicular mesothelioma.

[0527] 159. The use according to embodiment 158, wherein the mesothelioma is pleural mesothelioma.

[0528] 160. The use according to embodiment 159, wherein the subject has received prior therapy with a platinum-based therapy.

[0529] 161. The use according to embodiment 160, wherein the platinum-based therapy is cisplatin.

[0530] 162. The use of any one of embodiments 158 to 161, wherein the subject has received prior therapy with pemetrexed.

[0531] 163. The use according to embodiment 147, wherein the lung cancer is non-small cell lung cancer.

[0532] 164. The use according to embodiment 163, wherein the non-small cell lung cancer has a mutated form of epidermal growth factor receptor (EGFR).

[0533] 165. The use according to embodiment 163, wherein the non-small cell lung cancer has wild-type EGFR.

[0534] 166. The use according to embodiment 165, wherein the subject has received prior therapy with a platinum-based therapy.

[0535] 167. The use according to embodiment 165 or 166, wherein the subject has received prior therapy with an inhibitor of PD-1 or PD-L1.

[0536] 168. The use according to embodiment 167, wherein the subject has received prior therapy with an inhibitor of PD-1.

[0537] 169. The use according to embodiment 147, wherein the breast cancer is selected from HER2-positive, HER2-negative, estrogen receptor (ER)-positive, ER-negative, progesterone receptor (PR)-positive, PR-negative, and triple-negative breast cancer.

[0538] 170. The use according to embodiment 169, wherein the breast cancer is HER2-negative breast cancer.

[0539] 171. The use according to embodiment 170, wherein the subject has received one or more prior lines of treatment for the HER2-negative breast cancer.

[0540] 172. The use according to embodiment 171, wherein the one or more prior lines of treatment comprised treatment with a taxane.

[0541] 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 received prior therapy with an inhibitor of CDK4 / 6.

[0542] 175. The use according to embodiment 173 or 174, wherein the subject has received prior therapy with hormone-directed therapy.

[0543] 176. The use according to embodiment 147, wherein the colorectal cancer is selected from colorectal adenocarcinoma, gastrointestinal stromal tumors, primary colorectal lymphoma, gastrointestinal carcinoid tumors and leiomyosarcoma.

[0544] 177. The use according to embodiment 176, wherein the subject has received two or more prior lines of treatment for the colorectal cancer.

[0545] 178. The use according to embodiment 147, wherein the pancreatic cancer is an exocrine cancer or a neuroendocrine cancer.

[0546] 179. The use according to embodiment 178, wherein the exocrine cancer is selected from pancreatic adenocarcinoma, acinar cell carcinoma, cystadenocarcinoma, pancreatoblastoma, adenosquamous carcinoma, signet ring carcinoma, hepatoid carcinoma, colloid carcinoma, undifferentiated carcinoma and pancreatic mucinous cystic neoplasm.

[0547] 180. The use according to embodiment 179, wherein the pancreatic adenocarcinoma is pancreatic ductal adenocarcinoma.

[0548] 181. The use according to embodiment 179 or 180, wherein the subject has received one or more prior lines of treatment for the pancreatic cancer.

[0549] 182. The use according to any one of embodiments 137 or 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.

[0550] 183. The use of any one of embodiments 137 to 182, wherein the subject is a human.

[0551] The present invention will be more fully understood by reference to the following examples. However, these examples should not be interpreted as limiting the scope of the present invention. It should be understood that the embodiments and embodiments described herein are for illustrative purposes only, and the various modifications or changes made according to them will be known to those skilled in the art, and should be included in the spirit and scope of the application and within the scope of the appended claims.

[0552] Example

[0553] Example 1: CD288 expression in cancer cell lines

[0554] Quantification of CD228 copy number on the cell surface of various cancer cell lines was determined using mouse CD228 mAb as primary antibody and DAKO QiFiKit flow cytometry indirect assay as described by the manufacturer (DAKO A / S, Glostrup Municipality, Denmark) and evaluated with Attune NxT flow cytometer. The resulting number of CD228 molecules expressed per cell is shown in Table 1.

[0555] Table 1: CD228 molecules per cell for various cell lines

[0556]

[0557]

[0558] Example 2: Immunohistochemical analysis of CD228 expression

[0559] Tumor tissue arrays were obtained from commercial sources. Formalin-fixed and paraffin-embedded (FFPE) tumor tissues were purchased from Biomax Inc., USA. All samples were analyzed in Bond-Max TM The samples were processed using a fully automatic staining machine (Leica).

[0560] Using Bond TM FFPE slides sectioned on glass slides were dewaxed and rehydrated at 72°C using Dewaxing Solution (Leica, Cat. No. AR9222). TM Antigen retrieval was performed with Epitope Retrieval Solution 2 (Leica, catalog number AR9640) for 20 min, and then incubated with primary anti-CD228 antibody (Sigma; catalog number HPA004880). Isotype-matched rabbit IgG1 was used as a negative control for background staining. For automated IHC staining, we used the Refine DAB kit or the alkaline phosphatase-based detection kit: Bond TM Polymer AP Red Detection Kit (Leica, catalog number DS9305). Slides were incubated with 1 μg / ml of rabbit monoclonal primary antibody against rabbit CD228 mAb for 45 min, with protein blocking for the initial 30 min (DAKO catalog number X0909). After chromogen development, sections were counterstained with hematoxylin and coverslipped. Slides were evaluated and scored by a pathologist, and images were captured using a Zeiss Axiovert 200M microscope (Carl Zeiss, Inc., Thornwood, NY).

[0561] Figure 1 shows high levels of CD228 expression in melanoma cancer patient samples, providing strong evidence for the use of CD228 ADCs to treat these tumors.

[0562] Figure 2 shows high levels of CD228 expression in mesothelioma cancer patient samples, providing strong evidence for the use of CD228 ADCs to treat these tumors.

[0563] Figure 3 shows high levels of CD228 expression in colorectal cancer patient samples, providing a strong argument for the use of CD228 ADCs to treat these tumors.

[0564] Figure 4 shows high levels of CD228 expression in triple-negative (HR-, PgR-, Her2-) breast cancer patient samples (upper panels) and Her2-HR+ breast cancer patient samples (lower panels), providing strong arguments for the use of CD228 ADCs to treat these tumors.

[0565] Figure 5 shows high levels of CD228 expression in pancreatic cancer patient samples, providing strong evidence for the use of CD228 ADCs to treat these tumors.

[0566] Figure 6 shows high levels of CD228 expression in squamous non-small cell lung cancer patient samples (upper panels) and adenocarcinoma non-small cell lung cancer patient samples (lower panels), providing strong evidence for the use of CD228 ADCs to treat these tumors.

[0567] For various tumor types, the summary of immunohistochemistry experiments was compared with CD228 RNA levels as reported by The Cancer Genome Atlas. We determined the threshold for CD228 RNA positivity by applying the IHC prevalence of melanoma to TCGA. As can be seen from 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 = The Cancer Genome Atlas.

[0568] Example 3: Anti-CD228 Antibody Drug Conjugate

[0569] A variety of anti-CD228 antibodies were conjugated to the drug MMAE via the linker MDpr-PEG(12)-gluc, resulting in an average drug load of about 8 per antibody. The conjugation method is described in U.S. Publication No. 2018 / 0092984. Tumor cells were incubated with CD228 antibody drug conjugates (ADCs) at 37°C for 96-144 hours. 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 on a Fusion HT fluorescence plate reader (PerkinElmer, Waltham, MA). Data were normalized for untreated cells and x50 values ​​were calculated using Graph Pad software. Results are reported as IC in Table 2 50 , ie, the concentration of compound required to produce a 50% reduction in viability compared to vehicle-treated cells (control = 100%). Chimeric L49 (cL49) and mouse (mL49) ADCs outperformed all other anti-CD228 ADCs, particularly in cell lines with lower CD228 expression.

[0570] Table 2: IC of anti-CD228 antibody-drug conjugates against various cancer cells 50

[0571]

[0572] Example 4: Humanization of mouse L49 antibody

[0573] Mouse antibody mL49 (Siemers et al., 1997, Bioconjug. Chem. 8: 510-9) was used as a starting point or donor antibody for humanization. Suitable human acceptor sequences are genomic sequences provided by hIGHV4-59 and hIGHJ4 for heavy chains and by hIGKV2-30 and hIGKJ2 for light chains. When CDRs are defined according to the Kabat numbering scheme, human acceptor sequences show 70 (heavy chain) and 84 (light chain) percent identity with donor sequences in the variable region framework.

[0574] When defining CDR according to the Kabat numbering scheme, the comparison of donor sequences identified 26 positions in the heavy chain and 13 positions in the light chain, at which positions, the human acceptor framework sequence is different from the donor framework sequence, and may affect antibody binding due to direct contact with antigen, thereby affecting the conformation of CDR or affecting the stacking between the heavy chain and the light chain. Three humanized heavy chains (HA, HB and HC) and three humanized light chains (LA, LB and LC) were made, and back mutations were incorporated to the different displacements of specific positions. Referring to Fig. 8-Fig. 11 and Table 3-Table 6.

[0575] Table 3: Humanizing mutations in hL49 heavy chain variants

[0576]

[0577] Table 4: Specific murine framework mutations in hL49 heavy chain variants

[0578] Variants 27 30 40 47 48 67 71 78 82B 91 people% HkDJ D T Y R Y 88.8 HkDJ D T F Y M I R Y 85.7 HkDJ D T F Y M I R Y F N 83.7

[0579] Table 5: Humanizing mutations in hL49 kappa light chain variants

[0580] vK variant KV exon receptor sequence Mouse donor framework residues Human receptor CDR residues oeLh IGKV2-30 / KJ2 L2 none oeLh IGKV2-30 / KJ2 L2,L36,L46 none LC IGKV2-30 / KJ2 L2,L36,L46 L28

[0581] Table 6: Specific murine framework mutations in hL49 kappa light chain variants

[0582] Variants 2 36 46 people% oeLh F 91.0 oeLh F Y L 89.0 LC F Y L 90.0

[0583] Humanized antibodies representing each of the permutations (9 possibilities) of these chains for the humanized heavy and light chains were then expressed. The antibodies were then compared using peptide localization analysis of labile chemical modifications found in the L2 peptides of hL49 HALB (potential for Asn(N)-asparagine deamidation) and hL49 HALC (potential for Asp(D)-aspartate isomerization) after incubation for 1 week under the temperature and pH conditions as shown in Table 7. hL49 HALC (N28D) eliminates the deamidation observed in hL49 HALB and has limited isomerization. Overall L2 peptide modifications dropped from 13% to 2%.

[0584] Table 7: Peptide localization analysis of humanized anti-CD228 antibodies

[0585]

[0586] The binding curve of each resulting antibody was determined by competitive binding assay. Briefly, 1x10 5 RPMI-7951 cells stably expressing human CD228 were equally divided in each well of a 96-well v-bottom plate on ice. The cells were incubated for 1 hour with various combinations of parental mouse CD228 mAb labeled with 5nM AlexaFluor-647 (AF) and increasing concentrations (from 0.06nM to 1000nM) of unlabeled humanized CD228 mAb and humanized light chain LA-LB and humanized heavy chain HA-HC. The cells were precipitated and washed 3 times with PBS / BSA. The cells were precipitated and resuspended in 125 μL of PBS / BSA. Fluorescence was analyzed by flow cytometry, and the percentage of labeled mouse CD228 mAb binding was determined using the percentage of saturated fluorescence signal. The binding curves of recombinant human anti-CD228 antibodies are shown in Figures 12A-12F.

[0587] The K of each resulting antibody was then determined by saturation binding assay. D In short, 1x10 5 RPMI-7951 cells stably expressing human CD228 were equally divided in each well of a 96-well v-bottom plate. Each CD228 antibody was added at a concentration ranging from 0.05pM to 340nM and incubated on ice for 60 minutes. The cells were pelleted and washed 3 times with PBS / BSA, followed by the addition of 10ug / ml of PE-labeled anti-human IgG goat secondary antibody and incubated on ice for another 60 minutes. The cells were pelleted and washed 3 times with PBS / BSA and resuspended in 125μL of PBS / BSA. Fluorescence was analyzed by flow cytometry, and the percentage of saturated fluorescence signal was used to determine the percentage of binding, followed by calculation of apparent K DThe binding curves of the recombinant human anti-CD228 antibodies are shown in FIG13 , and the K values ​​for cL49ec (chimeric L49 with S239C mutation in the light chain constant region), hL49 HALAG1, hL49HALB G1, and hL49HALC G1 are shown in FIG14 . D Shown in Table 8. Antibodies called "HALC", "hL49" or "hL49-HALC" comprising a heavy chain "HA" and a light chain "LC" were selected for all other experiments.

[0588] Table 8: K of humanized anti-CD228 antibodies D

[0589] cL49 hL49 HALA G1 hL49 HALB G1 hL49 HALC G1 <![CDATA[K D (nM)]]> 5.8 5.3 7.8 4.0

[0590] Example 5: hL49-HALC Antibody Drug Conjugates with Various Drug Linkers

[0591] A. Antibody Drug Conjugation

[0592] hL49-HALC was conjugated with 8 loads of MDpr-PEG (12) -gluc-MMAE, Auristatin T, tubulysin M or lipophilic MMAF, 2 loads of MC-VC-MMAE or MDpr-gluc-MMAE or 2 loads of PBD. The conjugation method is described in U.S. Publication No. 2018 / 0092984. All commercially available anhydrous solvents can be used without further purification. PEG reagents were purchased from Quanta BioDesign (Powell, Ohio). Analytical thin layer chromatography was performed on silica gel 60F254 aluminum sheets (EMD Chemicals, Gibbstown, New Jersey). Radial chromatography was performed on a Chromatotron device (Harris Research, Palo Alto, California). Column chromatography was performed on a Biotage Isolera One rapid purification system (Charlotte, North Carolina). Analytical HPLC was performed on a Varian ProStar 210 solvent delivery system equipped with a Varian ProStar 330PDA detector. The samples were passed through a C12 Phenomenex Synergi 2.0×150mm, 4μm, Reverse phase column elution. 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 after injection to 95% at 11 min, followed by isocratic 95% acetonitrile to 15 min (flow rate = 1.0 mL / min). LC-MS was performed on two different systems. LC-MS system 1 was interfaced to a Phenomenex Synergi 2.0×150 mm, 4 μm, equipped with a C12 The HPLC-MS system 1 was composed of a ZMD Micromass mass spectrometer with an HP Agilent 1100 HPLC instrument equipped with a reverse phase column. The acidic eluent consisted of a linear gradient of acetonitrile from 5% to 95% in 0.1% formic acid in water for 10 min, followed by isocratic 95% acetonitrile for 5 min (flow rate = 0.4 mL / min). LC-MS system 2 consisted of a Waters Xevo G2 Tof mass spectrometer interfaced to a Waters 2695 Separations Module with a Waters 2996 photodiode array detector; the chromatographic column, mobile phase, gradient and flow rate were the same as for LC-MS system 1. UPLC-MS was performed on a Waters SQ mass detector interfaced to an Acquity Ultra Performance LC equipped with an Acquity UPLC BEH C18 2.1×50 mm, 1.7 μm reverse 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 eluted on a C12 Phenomenex Synergi 10.0×250 mm, 4 μm, using 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Reverse phase column purification. The purification method consisted of the following gradient of solvent A to solvent B: 90:10 from 0 to 5 min; 90:10 to 10:90 from 5 min to 80 min; followed by isocratic 10:90 for 5 min. The flow rate was 4.6 mL / min with monitoring at 254 nm. Preparative HPLC for compounds in Schemes 3 and 4 was performed using 0.1% trifluoroacetic acid instead of 0.1% formic acid in both mobile phases.

[0593] Solution 1

[0594]

[0595] (2S,3S,4S,5R,6S)-6-(2-(3-aminopropionamido)-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 (2): to a mixture containing a known (described in US 2008 / 0241128) Compound 8a) in A1) glucuronide-MMAE intermediate 2 (40 mg, 26.8 μmol) was added to a flask with 0.9 mL of methanol and 0.9 mL of tetrahydrofuran. The solution was then cooled in an ice bath, and lithium hydroxide monohydrate (6.8 mg, 161 μmol) was added dropwise in 0.9 mL of water as a solution. The reaction was then stirred on ice for 1.5 h, at which point LC / MS showed complete conversion to product. Glacial acetic acid (9.2 μL, 161 μmol) was then added, and the reaction was concentrated to dryness. Preparative HPLC provided fully deprotected glucuronide-MMAE linker intermediate 3 (26 mg, 87%) as an oily residue. Analytical HPLC (0.1% formic acid): t R 9.3min. LC-MS system 1: t R 11.10min, m / z (ES + ) Measured value is 1130.48 (M+H) + , m / z(ES - ) The measured value is 1128.63 (MH) - .

[0596] Solution 2.

[0597]

[0598] (S)-44-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-39-azapentatetradecan-45-oic acid (4): α-Fmoc-lysine 3 (59 mg, 161 μmol) was added to a flask with 2.9 mL of anhydrous dichloromethane, followed by methoxy-PEG12-OSu (100 mg, 146 μmol). DIPEA (127 μL, 730 μmol) was then added and the reaction was stirred at room temperature under nitrogen, 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 and increasing amounts of methanol (0% to 20%) to provide the desired product 4 (153 mg, 112%). UPLC-MS: t R 1.77min, m / z (ES + ) Measured value is 939.58 (M+H) + .

[0599] (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-dodeca-39-azapentatetradecanoate-45-oic acid ester (5): Place N α -Fmoc-Lysine (PEG12) -OH 4 (153 mg, 163 μmol) and 1.6 mL of anhydrous tetrahydrofuran. N-hydroxysuccinimide (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 eluted with dichloromethane and increasing amounts of methanol (0% to 10%) to provide the desired activated ester 5 (155 mg). The material was continued without further characterization. UPLC-MS: t R 1.92min, m / z (ES + ) Measured value is 1036.48 (M+H) + .

[0600] (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-dodeca-39,46-diazanonatetraconylamide)-4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)

[0136] Deprotected glucuronide-MMAE linker intermediate 2 (92 mg, 81 μmol) was dissolved in anhydrous dimethylformamide (1.6 mL) and added to a flask containing Na-Fmoc-lysine (PEG12)-OSu 5 (101 mg, 97 μmol). Diisopropylethylamine (70 μL, 405 μmol) was then 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 afford Fmoc-Lys(PEG12)-glucuronide-MMAE intermediate 6 (111 mg, 62% after two steps) as an oily residue. UPLC-MS: t R 2.01min, m / z (ES + ) The measured value is 2050.92 (M+H) + .

[0601] (2S,3S,4S,5R,6S)-6-(2-((S)-4-amino-38,45-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-39,46-diazanonatetraconamido)-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

[0136] 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 provide H-Lys(PEG12)-glucuronide-MMAE intermediate 7 (85 mg, 86%) as an oily residue. UPLC-MS: t R 1.50min, m / z (ES + ) Measured value is 1829.31 (M+H) + .

[0602] (S)-2,5-dioxopyrrolidin-1-yl 3-((tert-butyloxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (9): (S)-N α -Maleimido-N β -Boc- diaminopropionic acid 8 (Nature Biotechnology, 2014, 32, 1059-1062) (400mg, 1.4mmol) was dissolved in 7mL of anhydrous dimethylformamide. N-hydroxysuccinimide (178mg, 1.5mmol) was added, followed by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (298mg, 1.5mmol). The reaction was stirred at room temperature under nitrogen for 3 hours. Aqueous treatment was achieved by dilution into 120mL of water; the aqueous layer was then extracted three times with 60mL of ethyl acetate. The combined organic layer was then washed with brine, dried over sodium sulfate, and concentrated to dryness. The product was purified by flash column chromatography, eluting hexane: a mixture of ethyl acetate (50:50 to 0:100) to provide (S)-N α -Maleimido-N β-Boc-diaminopropionic acid NHS ester [MDpr(Boc)-OSu]9 (297 mg, 55%). LC-MS system 1: t R 12.23min, m / z (ES + ) The measured value is 282.0599 (M+H-Boc group) + LC-MS system 2: t R 11.30min, m / z (ES + ) The measured value is 2580.2515 (M+H) + .

[0603] Solution 3.

[0604]

[0605] (2R / S,3S,4S,5R,6S)-6-(2-((S)-44-((S)-3-((tert-butyloxycarbonyl)amino)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionamido)-38,45-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-39,46-diazanonatetraconamido)-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)-OSu 9 (20 mg, 53 μmol) was dissolved in 2.2 mL of anhydrous dimethylformamide and added to a flask containing H-Lys(PEG12)-glucuronide-MMAE linker intermediate 7 (86 mg, 44 μmol). Diisopropylethylamine (15 μL, 88 μmol) was then added and the reaction was stirred at room temperature under nitrogen for 2.5 h. The reaction was quenched with 15 μL of glacial acetic acid and purified by preparative HPLC to provide MDpr(Boc)-Lys(PEG12)-glucuronide-MMAE intermediate 10 (37 mg, 40%) as a mixture of diastereomers. The diastereomers can be separated by chiral chromatography. UPLC-MS: t R 1.84min, m / z (ES + ) The measured value is 2095.44 (M+H) + .

[0606] (2R / S,3S,4S,5R,6S)-6-(2-((S)-44-((R)-3-amino-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionamido)-38,45-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-39,46-diazanonatetraconamido)-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 (11): A flask containing MDpr(Boc)-Lys(PEG12)-glucuronide-MMAE intermediate 10 (34 mg, 16 umol) was cooled to 0°C in an ice bath under nitrogen. A solution of 10% trifluoroacetic acid in dichloromethane (0.8 mL) was added dropwise. The reaction was then stirred at 0°C for 2 h, at which time LC-MS showed complete Boc deprotection. The reaction was then concentrated to a crude residue and purified by preparative HPLC to provide MDpr-Lys(PEG12)-glucuronide-MMAE linker 11 (22 mg, 68%). UPLC-MS: t R 1.50min, m / z (ES + ) The measured value is 1995.18 (M+H) + .

[0607] Compound 11 was conjugated to anti-CD228 antibody via its interchain thiol using methods known in the art (see, eg, US Pat. No. 7,659,241) at an average drug loading of 8 drugs per antibody.

[0608] B. In vitro cytotoxicity of hL49-HALC ADC

[0609] Tumor cells were incubated with each antibody drug conjugate (ADC) at 37°C for 96-144 hours. Non-binding (referred to as h00 or 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 on a Fusion HT fluorescence plate reader (Perkin Elmer, Waltham, Massachusetts). Data were normalized for untreated cells and x50 values ​​were calculated using Graph Pad software. Results are reported as IC in Table 9 50 , the concentration of compound required to produce a 50% reduction in viability compared to vehicle-treated cells (control = 100%). The hL49 ADC achieved single-digit ng / ml IC across a panel of cell lines with CD228 expression ranging from 16,000 to 450,000 50 value.

[0610] Table 9: IC of anti-CD228 antibody-drug conjugates against various cancer cells 50

[0611]

[0612] In a similar experiment, the percentage of viable cells was determined after treatment with different concentrations of hL49 conjugated with different drug linkers with different amounts of MMAE. The percentage of viable cells obtained for A2058 cells treated with hL49-MC-val-cit-PAB-MMAE (4), hL49-MP-gluc-MMAE (4), and hL49-MP-gluc-MMAE (8) at different antibody-drug conjugate (ADC) concentrations is shown in Figure 14A. The 8-load MP-gluc-MMAE outperformed the 4-load MP-gluc-MMAE and MC-val-cit-PAB-MMAE drug linkers in vitro. Despite containing the same amount of the same drug (MMAE), the 4-load MP-gluc-MMAE outperformed the MC-val-cit-PAB-MMAE drug linker in vitro.

[0613] The percentage of viable cells for A375 cells treated with hL49-MC-val-cit-PAB-MMAE (4), hL49-MP-gluc-MMAE (4), hL49-MP-gluc-MMAE (8) at different antibody-drug conjugate (ADC) concentrations is shown in Figure 14B. The 8-load MP-gluc-MMAE outperformed the 4-load MP-gluc-MMAE and MC-val-cit-PAB-MMAE drug linkers in vitro. Despite containing the same amount of the same drug (MMAE), the 4-load MP-gluc-MMAE outperformed the MC-val-cit-PAB-MMAE drug linker in vitro.

[0614] The resulting percentage of viable cells for Colo-853 cells treated with hL49-MC-val-cit-PAB-MMAE (4), hL49-MP-gluc-MMAE (4), hL49-MP-gluc-MMAE (8) at different antibody-drug conjugate (ADC) concentrations are shown in Figure 14C. The 8-loaded MP-gluc-MMAE outperformed the 4-loaded MP-gluc-MMAE and MC-val-cit-PAB-MMAE drug linkers in vitro.

[0615] C. In vivo activity of anti-CD228 ADCs with various drug linkers

[0616] Place 5x10 6 Cultured A2058 tumor cells were implanted into nude (nu / nu) mice (7-8 animals / group). When the tumors reached 100 mm 3 At 4 pm, 1 mg / kg, 3 mg / kg or 6 mg / kg of the test ADC was started (q4d x 4 intraperitoneal injections). Tumor volume was monitored using a caliper and was assessed when the tumor volume reached approximately 800-1000 mm 3 Animals were euthanized when the tumor volume reached 1. The mean tumor volume for each group was plotted continuously 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 according to protocols approved by the Institutional Animal Care and Use Committee.

[0617] The resulting tumor volumes over time for untreated mice and mice treated with 3 mg / kg hL49, hL49-Auristatin T (8), hL49-lipophilic MMAF (8), hL49-Tubulin M (8), and hL49-MDpr-PEG (12)-gluc-MMAE (8) are shown in Figure 15. Despite superior potency in vitro, Auristatin T and lipophilic MMAF ADCs were less active in vivo than hL49-MDpr-PEG (12)-gluc-MMAE (8).

[0618] 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. In vivo, 8 loadings of MMAE with PEG were superior to 2 loadings or 4 loadings of MMAE with PEG.

[0619] 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. In vivo, 3 mg / kg hL49-MDpr-gluc-MMAE(8) was superior to 1 mg / kg hL49-MDpr-gluc-MMAE(8) or any concentration of hL49-MC-val-cit-PAB-MMAE(4).

[0620] 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. In vivo, 3 mg / kg hL49-MDpr-gluc-MMAE(8) was superior to 1 mg / kg hL49-MDpr-gluc-MMAE(8) or any concentration of hL49-MC-val-cit-PAB-MMAE(4).

[0621] Example 6: In vivo comparison of tubulysin M and MDpr-PEG(12)-gluc-MMAE

[0622] hL49 conjugated with tubulysin M or MDpr-PEG(12)-gluc-MMAE showed superiority over other ADCs against A2058 cells, so these ADCs were selected for further evaluation at different doses and using different tumor cell types.

[0623] Place 2.5x10 5 A2058, 1x10 6 SK-MEL-5, 1x10 5 IGR-37, 1x10 6 Colo-853 or 1x10 6 HPAF-II tumor cells were implanted into nude (nu / nu) mice (6-8 animals / group). 5x10 5 Cultured MDA-MB-231 tumor cells were implanted into NOD / SCID / gc KO (NSG) mice. For PDX models, the LU0697 squamous NSCLC model was grown in NOD / SCID mice and the LU5200 adenocarcinoma NSCLC model was grown in BALB / c nude mice (3 animals / group). When tumors reached approximately 100 mm 3 At 4 hr, dosing with 0.3 mg / kg, 0.5 mg / kg, 1 mg / kg or 3 mg / kg of the test ADC was started (single intraperitoneal injection). Tumor volume was monitored using a caliper and was repeated when the tumor volume reached approximately 1000 mm 3Animals were euthanized at 4 dpi. For PDX studies, they were sacrificed 28 days after the final dose, regardless of tumor size. The mean tumor volume for each group continued to be plotted until one or more animals were euthanized. All animal procedures were performed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care according to protocols approved by the Institutional Animal Care and Use Committee.

[0624] The resulting 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-Tubulin M(8), 1 mg / kg or 3 mg / kg hL49-Tubulin M(8), or 1 mg / kg or 3 mg / kg hL49-MDpr-PEG(12)-gluc-MMAE(8) is shown in Figure 19. hL49-Tubulin M and hL49-MDpr-PEG(12)-gluc-MMAE had similar complete response (CR) rates at 3 mg / kg, but hL49-MDpr-PEG(12)-gluc-MMAE was superior at 1 mg / kg.

[0625] The resulting SK-MEL-5 tumor volumes over time for untreated mice and mice treated with 3 mg / kg IgG-MDpr-PEG(12)-gluc-MMAE(8), 3 mg / kg IgG-Tubulin M(8), 0.3 mg / kg, 1 mg / kg or 3 mg / kg hL49-Tubulin 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 was superior to hL49-Tubulin M for SK-MEL-5 tumors.

[0626] The resulting IGR-37 tumor volumes over time for untreated mice and mice treated with 1 mg / kg or 3 mg / kg hL49-Tubulin M (8) or 1 mg / kg or 3 mg / kg hL49-MDpr-PEG (12) -gluc-MMAE (8) are shown in Figure 21. hL49-MDpr-PEG (12) -gluc-MMAE was superior to hL49-Tubulin M for IGR-37 tumors.

[0627] The resulting Colo-853 tumor volumes over time for untreated mice and mice treated with 0.3, 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 22. Colo-853 tumors responded to treatment with hL49-MDpr-PEG(12)-gluc-MMAE.

[0628] The resulting 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. The LU0697 squamous NSCL PDX model tumors responded to treatment with hL49-MDpr-PEG(12)-gluc-MMAE.

[0629] The resulting 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. The LU0697 adenocarcinoma NSCL PDX model tumors responded to treatment with hL49-MDpr-PEG(12)-gluc-MMAE.

[0630] The resulting MDA-MB-231 TNBC tumor volumes 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) are shown in Figure 25. MDA-MB-231 TNBC tumors responded to treatment with hL49-MDpr-PEG(12)-gluc-MMAE.

[0631] The resulting HPAF-II tumor volumes 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 responded to treatment with hL49-MDpr-PEG(12)-gluc-MMAE.

[0632] Example 7: Triple Negative Breast Cancer Mouse Clinical Trial

[0633] The percent change in tumor volume in response to treatment with hL49-MDpr-PEG(12)-gluc-MMAE(8) was evaluated in 22 different mouse triple-negative breast cancer PDX models. An empirically determined number of tumor cells for each model was implanted into NCr or nude mice. When tumors reached approximately 150-300 mm 3 At 14:00, 3 mg / kg of hL49-MDpr-PEG(12)-gluc-MMAE(8) was started. Tumor volume was monitored using a caliper. The percentage change in tumor volume for each mouse was calculated 7 days after the optimal response time or administration with hL49-MDpr-PEG(12)-gluc-MMAE(8) and is shown in Figure 27. Treatment with hL49-MDpr-PEG(12)-gluc-MMAE(8) achieved a 60% response rate, with 31% of the animals achieving a partial response and 29% of the animals achieving a complete response. All animal procedures were performed in a facility approved by the Association for Assessment and Accreditation of Laboratory Animal Care according to a protocol approved by the Institutional Animal Care and Use Committee.

[0634] Example 7-1: Patient-derived xenograft models of various CD228-expressing cancers

[0635] Additional experiments were performed as described in Example 7 to evaluate the ability of hL49-MDpr-PEG(12)-gluc-MMAE(8) to inhibit tumor growth in various CD228-expressing cancers. Patient-derived xenograft models were generated by isolating tumors from 60 human patients (triple negative breast cancer (TNBC) = 22 patients, mesothelioma = 3 patients, and non-small cell lung cancer (NSCLC) = 35 patients) 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 drawn from mice 48 hours after treatment with hL49-MDpr-PEG(12)-gluc-MMAE(8) and used for pharmacokinetic assessments. The percentage of tumor growth inhibition (TGI percentage (%)) and the percentage change in tumor volume from baseline to best response (Tvol percentage change (%)) were evaluated in Figures 36A and 36B, respectively. It can be seen that administration of a single dose of hL49-MDpr-PEG(12)-gluc-MMAE(8) has antitumor activity in various tumor models.

[0636] Example 8: In vitro evaluation of antibody effector function

[0637] Use Standards 51 Antibody-dependent cellular cytotoxicity (ADCC) activity was measured using a Cr-release assay. Briefly, tumor cells were treated with 100 μCi Na 51 CrO4 labeled, washed and pre-incubated with the test ADC. NK (CD16 + CD56 + ) cells. Live NK cells were added to target cells at a ratio of 10:1 effector cells to target cells. In this assay, human IgG1κ (Ancell, Bayport, Minnesota) was used as a negative control. After incubation for 4 hours, the supernatant was collected and dried overnight on a Luma plate. The γ radiation emitted by the lysed cells was then detected using a TopCount microplate scintillation and luminescence counter (Perkin Elmer, Waltham, Massachusetts). The specific lysis % (ADCC activity) of the two patients is shown in Figures 28A-28B. Compared with hL49 mAb, hL49-MDpr-PEG (12) -gluc-MMAE (hL49-5088) has reduced ADCC activity.

[0638] Example 9: Pharmacokinetic Evaluation in Mice and Rats

[0639] hL49-MDpr-PEG(12)-gluc-MMAE or cL235-MDpr-PEG(12)-gluc-MMAE were administered intravenously to nude mice, and hL49-MDpr-PEG(12)-gluc-MMAE was administered intravenously to Sprague-Dawley rats. The plasma concentration of the ADC over time was measured using a Tab ELISA with an anti-human antibody as the capture antibody. The results are shown in Figure 29 A (mice) and Figure 29 B (rats). The resulting PK parameters are shown in Table 10 (mice) and Table 11 (rats).

[0640] Table 10: Pharmacokinetic (PK) parameters of hL49-MDpr-PEG(12)-gluc-MMAE in mice

[0641] <![CDATA[AUC inf / dose]]> <![CDATA[C max / dose]]> <![CDATA[T max ]]> half life (day*kg*μg / mL / mg) (kg*μg / mL / mg) (sky) (sky) 41 8.8 0.25 4.7 50 8.1 1.0 4.4 63 13.3 0.25 5.1

[0642] Table 11: Pharmacokinetic (PK) parameters of hL49-MDpr-PEG(12)-gluc-MMAE in rats

[0643]

[0644]

[0645] Example 10: Additional anti-CD228 antibodies

[0646] Additional anti-CD228 antibodies were conjugated to MDpr-PEG(12)-gluc-MMAE. These additional anti-CD228 antibodies (referred to as cL235 (see Rolland Y., Pigment Cell Melanoma Res 2009, 22:86-98) and Ab1-9) had binding affinities similar to those of hL49, which were different from the commercial antibodies tested in Table 2 (Santa Cruz catalog number 271633, R&D catalog number 893416, and Biolegend catalog number 363101).

[0647] A. In vitro cytotoxicity

[0648] Tumor cells were incubated with CD228 antibody drug conjugates (ADCs) at 37°C for 96-144 hours. Non-binding (h00-5088(8)) ADCs were used as negative controls. Cell viability was measured using Cell Titer Glo according to the manufacturer's instructions. Fluorescence signals were measured on a Fusion HT fluorescence plate reader (Perkin Elmer, Waltham, MA). Data were normalized to untreated cells and x50 values ​​were calculated using Graph Pad software. Results are reported as IC in Table 12. 50 , ie, the concentration of compound required to produce a 50% reduction in viability compared to vehicle-treated cells (control = 100%). The percentage of viable cells remaining at the highest dose is shown in Table 13.

[0649] Table 12: IC of anti-CD228 antibody-drug conjugates against various cancer cells 50

[0650]

[0651] Table 13: Percentage of viable cells remaining at the highest dose of anti-CD228 antibody-drug conjugate for various cancer cells

[0652]

[0653]

[0654] B. In vivo activity of additional anti-CD228 ADCs

[0655] Place 1x10 6 A375 cultured, 1x10 5 IGR37 or 2.5x10 5 A2058 tumor cells were implanted into nude (nu / nu) mice (6 animals / group). When the tumor reached 100 mm 3 At 4 pm, dosing with 1 mg / kg (A2058) or 3 mg / kg of the test ADC was started (single intraperitoneal injection). Tumor volume was monitored using a caliper and was determined when the tumor volume reached approximately 800 mm 3Animals were euthanized at 4 hr. The mean tumor volume for each group was plotted continuously until one or more animals were euthanized. All animal procedures were performed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care according to protocols approved by the Institutional Animal Care and Use Committee.

[0656] 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 <4-fold over time for each treatment condition. The number of complete responses (CRs) and the median time for tumors to quadruple for each ADC and each cell line are shown in Table 14.

[0657] Table 14: In vivo responses to additional anti-CD228 ADCs

[0658]

[0659] Example 11: Linker cleavage and CD228 turnover

[0660] The time-dependent conjugate cleavage rate was studied using fluorescence determination. Fluorescence moiety AF647 was directly conjugated to 8 natural cysteines of anti-CD228 antibody hL49 or conjugated to anti-CD228 antibody hL49 via a glucuronide linker. In addition, quenching agent Tide Quencher 5WS succinimidyl ester (TQ5WS) was added via lysine residues so that each antibody had approximately 4. When both quencher and AF647 were attached to antibody, fluorescence was quenched. AF647 molecules were released from quencher from antibody cutting or antibody degradation, and subsequent fluorescence increase. In A375 cells (Figure 31A) and Colo-853 cells (Figure 31B), compared to the direct conjugation of AF647 to antibody, the conjugation of AF647 to antibody via a glucuronide linker caused a faster increase in fluorescence activity.

[0661] A375 cells were treated with hL49 antibodies conjugated to vcQF01, which consisted of TQ5WS linked to a Cy5 fluorophore via a Val-Cit-PAB linker, at a ratio of approximately 2 molecules per antibody. Similar to the reagents described in the previous section, Cy5 remains quenched when intact on the antibody and only fluoresces when cleaved from the TQ5WS quencher. 2 μg / ml of hL49-vcQF01 was then added and allowed to bind to the cells. For pulse treatment, the labeled hL49 antibody was washed after 30 minutes to remove unbound labeled hL49 antibody. For continuous treatment with labeled hL49 antibody, unbound labeled hL49 antibody was not washed out of the cells. As shown in Figure 32, pulse treatment resulted in a rapid plateau of the Cy5 signal, while continuous exposure to labeled hL49 resulted in a steady increase in the signal of the Cy5 fluorophore. This shows that additional CD228 is added to the cell surface within 24 hours, and can then be bound by the labeled hL49 antibody, internalized into the cell, and cut to release Cy5. In further experiments, by comparing the fluorescence intensity of each cell that changes over time in the presence and absence of cycloheximide, the impact of protein synthesis on the binding of CD228 by the fluorescently labeled hL49 antibody was studied. Cycloheximide (CHX) is used to inhibit protein synthesis. In the presence of cycloheximide in Colo-853 (Figure 33 A) and A375 (Figure 33 B cells), the increase of fluorescence signal over time occurred, but the fluorescence signal decreased compared with the cells not treated with cycloheximide. This shows that even in the absence of protein synthesis, CD228 is also recycled back to the cell surface. These experiments jointly prove that CD228 is recycled and supplemented on the cell surface, which contributes to the activity of antibody-drug conjugates.

[0662] Example 12: pH-dependent binding of ADC

[0663] The ability of various anti-CD228 ADCs to bind to CD228 at pH values ​​ranging from 4 to 7.5 was evaluated using a standard ELISA protocol. In brief, 100 ng of human CD228 (R&D Systems Custom02; batch number DCWR021505A) or BSA (Sigma; catalog number 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 number 5246531EA). After washing, the plate was blocked for 1 hour at room temperature with 3% (w / v) BSA in PBS-T. Excess blocking buffer was then removed, and the primary antibody was added at a 3-fold dilution starting from an antibody concentration of 60 nM in a dilution buffer (0.15 M citrate-phosphate buffer pH 4.0-7.5). After incubation at room temperature for 1 hour, the plate was washed 3 times and then incubated with a secondary antibody (goat anti-human IgG Fc specific HRP conjugated, Jackson ImmunoResearch code 109-035-098) in PBS-T with 1% BSA. After incubation at room temperature for 30 minutes, the plate was washed 3 times. Then 100 μl of TMB substrate (Life Technologies; catalog number 002023) was added to each well. After incubation at room temperature for 10 minutes, 100 μl of H2SO4 was added to each well to stop the reaction, covered with a transparent plate sealer on the plate and read at 450nM on Envision. The pH-dependent binding of hL49, cL235, CD228Ab1, CD228Ab2 and CD228Ab3, CD228Ab4 is shown in Figures 34A-34F. The resulting EC for each ADC 50 Shown in Table 15. hL49 was the only ADC that exhibited differential binding across a pH gradient.

[0664] Table 15: EC in nM for each ADC 50

[0665] oeLh cL235 CD228Ab1 CD228Ab2 CD228Ab3 CD228Ab4 pH 4 - 0.809 0.404 0.188 2.887 0.158 pH 4.55 - 0.727 0.295 0.158 0.194 0.150 pH 5.1 - 0.756 0.285 0.146 0.123 0.209 pH 5.6 21.820 0.740 0.254 0.196 0.162 0.223 pH 5.9 5.307 0.838 0.243 0.186 0.161 0.235 pH 6.3 2.963 0.749 0.285 0.202 0.166 0.217 pH 6.66 1.542 0.757 0.221 0.146 0.152 0.161 pH 7.1 1.099 0.724 0.238 0.140 0.196 0.170 pH 7.4 0.856 0.767 0.359 0.110 0.294 0.093 pH 7.5 1.178 0.694 0.294 0.169 0.415 0.148

[0666] Example 13: Internalization and catabolism of ADC

[0667] The internalization of various other anti-CD228 antibodies and the ability to degrade the fluorescent moiety AF647 were assessed. A375 cells were treated with anti-CD228 antibodies conjugated to QF01, which was composed of a quencher Tide Quencher 5WS succinimidyl ester (TQ5WS) connected to a Cy5 fluorophore via a glucuronide linker (gluc) at a ratio of about 2 molecules per antibody. Cy5 remained quenched when intact on the antibody and only fluoresced when cut from the TQ5WS quencher. After 30 minutes, the labeled anti-CD228 antibodies were washed to remove unbound labeled anti-CD228 antibodies. The binding affinity that these anti-CD228 antibodies had was similar to that of hL49. Tumor cells were incubated with anti-CD228 antibodies, and imaging was performed to determine the fluorescence intensity of each cell that changes 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, the intracellular drug concentration for each ADC was measured (Figure 35B). These experiments showed that despite similar binding affinity, some antibodies (such as hL49) internalized faster and delivered drug to a greater extent than other antibodies. This suggests that hL49-MDpr-PEG(12)-gluc-MMAE(8) can deliver drugs to tumor cells more effectively than other ADCs. Sequence Listing <110> sigen co., ltd. <120> Anti-CD228 antibodies and antibody-drug conjugates <130> 76168-20013.40 <140> Not yet assigned <141> Submit together <150> US 62 / 801,590 <151> 2019-02-05 <150> US 62 / 824,923 <151> 2019-03-27 <150> US 62 / 879,660 <151> 2019-07-29 <150> US 62 / 882,016 <151> 2019-08-02 <150> US 62 / 934,424 <151> 2019-11-12 <160> 35 <170> FastSEQ version 4.0 for Windows <210> 1 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 1 Ser Gly Tyr Trp Asn 1 5 <210> 2 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 2 Tyr Ile Ser Asp Ser Gly Ile Thr Tyr Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 3 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 3 Arg Thr Leu Ala Thr Tyr Tyr Ala Met Asp Tyr 1 5 10 <210> 4 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 4 Arg Ala Ser Gln Ser Leu Val His Ser Asp Gly Asn Thr Tyr Leu His 1 5 10 15 <210> 5 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 5 Arg Val Ser Asn Arg Phe Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 6 Ser Gln Ser Thr His Val Pro Pro Thr 1 5 <210> 7 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 7 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Tyr Ile 35 40 45 Gly Tyr Ile Ser Asp Ser Gly Ile Thr Tyr Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Arg Thr Leu Ala Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 8 <211> 112 <212> PRT <213> artificial sequence <220> <223> synthetic construction <400> 8 Asp Phe Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ala Ser Gln Ser Leu Val His Ser 20 25 30 Asp Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 9 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 9 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Thr 20 25 30 <210> 10 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 10 Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Tyr Ile Gly 1 5 10 <210> 11 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 11 Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu Lys 1 5 10 15 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 12 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 12 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 13 <211> twenty three <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 13 Asp Phe Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys 20 <210> 14 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 14 Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr 1 5 10 15 <210> 15 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 15 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys 20 25 30 <210> 16 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 16 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 17 <211> 329 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 17 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 <210> 18 <211> 106 <212> PRT <213> artificial sequence <220> <223> synthetic construction <400> 18 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65 70 75 80 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 19 <211> 329 <212> PRT <213> artificial sequence <220> <223> synthetic construction <400> 19 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Cys Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 <210> 20 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 20 Asp Phe Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ala Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 21 <211> 119 <212> PRT <213> Mus musculus <400> 21 Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Asp Ser Gly Ile Thr Tyr Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu 65 70 75 80 Gln Leu Asn Phe Val Thr Ala Glu Asp Thr Ala Thr Tyr Asn Cys Ala 85 90 95 Arg Arg Thr Leu Ala Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115 <210> 22 <211> 97 <212> PRT <213> Mus musculus <400> 22 Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu 65 70 75 80 Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg <210> 23 <211> 112 <212> PRT <213> Homo sapiens <400> 23 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> twenty four <211> 119 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> twenty four Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Lys Gly Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Asp Ser Gly Ile Thr Tyr Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Arg Thr Leu Ala Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 25 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Synthesize construction <400> 25 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Lys Gly Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Asp Ser Gly Ile Thr Tyr Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu 65 70 75 80 Lys Leu Ser Phe Val Thr Ala Ala Asp Thr Ala Val Tyr Asn Cys Ala 85 90 95 Arg Arg Thr Leu Ala Thr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 26 <211> 112 <212> PRT <213> Mus musculus <400> 26 Asp Phe Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ala Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 27 <211> 100 <212> PRT <213> Mus musculus <400> 27 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro 100 <210> 28 <211> 112 <212> PRT <213> Homo sapiens <400> 28 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95 Thr His Trp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 29 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 29 Asp Phe Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ala Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 30 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 30 Gly Phe Leu Gly 1 <210> 31 <211> 113 <212> PRT <213> Mus musculus <400> 31 Asp Phe Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ala Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 32 <211> 113 <212> PRT <213> Homo sapiens <400> 32 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95 Thr His Trp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 33 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 33 Asp Phe Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ala Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 34 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 34 Asp Phe Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ala Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Angry <210> 35 <211> 113 <212> PRT <213> artificial sequence <220> <223> synthetic structure <400> 35 Asp Phe Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ala Ser Gln Ser Leu Val His Ser 20 25 30 Asp Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg

Claims

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 comprises: (i) CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 1; (ii) CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 2; and (iii) CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 3; and Wherein the light chain variable region comprises: (i) CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 4; (ii) CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 5; and (iii) CDR-L3 consisting of the amino acid sequence of SEQ ID NO:

6.

2. The antibody or antigen-binding fragment of claim 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, provided that the heavy chain variable region comprises: CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 1; CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 2; A CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 3; and 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, wherein the amino acid positions are according to Kabat; and A light chain variable region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 8, provided that the light chain variable region comprises: A CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 4; CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 5; A CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 6; and Position L2 is occupied by F, position L36 is occupied by Y and position L46 is occupied by L, wherein the amino acid positions are according to Kabat.

4. The antibody or antigen-binding fragment of claim 3, wherein the condition is further that position L28 is occupied by D.

5. The antibody or antigen-binding fragment according to any one of claims 1 to 4, 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.

6. The antibody or antigen-binding fragment according to any one of claims 1 to 4, 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.

7. The antibody or antigen-binding fragment according to any one of claims 1 to 4, 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.

8. The antibody or antigen-binding fragment according to any one of claims 1 to 4, 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.

9. The antibody or antigen-binding fragment of any one of claims 1 to 8, wherein the antibody or antigen-binding fragment is an antigen-binding fragment.

10. The antibody or antigen-binding fragment according to claim 9, 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.

11. The antibody or antigen-binding fragment of any one of claims 1 to 8, wherein the antibody or antigen-binding fragment is a full-length antibody.

12. The antibody or antigen-binding fragment of claim 11, 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.

13. The antibody or antigen-binding fragment of claim 12, wherein the heavy chain constant region is of the IgG1 isotype.

14. The antibody or antigen-binding fragment of claim 12 or 13, 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.

15. The antibody or antigen-binding fragment of claim 12 or 13, wherein the heavy chain constant region is a mutated form of a native human constant region that has reduced binding to Fcγ receptors relative to the native human constant region.

16. The antibody or antigen-binding fragment of claim 12 or 13, 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.

17. An antibody-drug conjugate comprising the antibody or antigen-binding fragment according to any one of claims 1 to 16 conjugated to a cytotoxic agent or a cytostatic agent.

18. The antibody-drug conjugate of claim 17, wherein the antibody or antigen-binding fragment is conjugated to the cytotoxic or cytostatic agent via a linker.

19. The antibody-drug conjugate of claim 18, wherein the linker is a MDpr-PEG(12)-gluc linker.

20. The antibody-drug conjugate of any one of claims 17 to 19, wherein the cytotoxic or cytostatic agent is monomethyl auristatin.

21. The antibody-drug conjugate of claim 20, wherein the monomethyl auristatin is monomethyl auristatin E (MMAE).

22. The antibody-drug conjugate of claim 21, wherein the linker is attached to monomethyl auristatin E, thereby forming an antibody-drug conjugate having the following structure: ， Where Ab is antibody hL49, n is 12, R PR is hydrogen, R 21 is CH3, and p represents a number from 1 to 16.

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

24. The antibody-drug conjugate of any one of claims 17 to 23, wherein the antibody-drug conjugate is hL49-MDpr-PEG(12)-gluc-MMAE.

25. A nucleic acid encoding a heavy chain variable region and / or a light chain variable region as defined in any one of claims 1 to 16.

26. A vector comprising the nucleic acid according to claim 25.

27. The vector according to claim 26, wherein the vector is an expression vector.

28. A host cell comprising the nucleic acid of claim 25.

29. The host cell of claim 28, wherein the host cell is a Chinese Hamster Ovary (CHO) cell.

30. A method for producing an anti-CD228 antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell according to claim 28 or 29 under conditions suitable for producing the anti-CD228 antibody or the antigen-binding fragment thereof.

31. The method of claim 30, further comprising isolating the anti-CD228 antibody or antigen-binding fragment thereof produced by the host cell.

32. A method for producing an anti-CD228 antibody-drug conjugate, the method comprising culturing the host cell of claim 28 or 29 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 agent or a cytostatic agent.

33. The method of claim 32, wherein the anti-CD228 antibody is conjugated to the cytotoxic or cytostatic agent via a linker.

34. The method of claim 33, wherein the linker is a MDpr-PEG(12)-gluc linker.

35. The method of any one of claims 32 to 34, wherein the cytotoxic or cytostatic agent is monomethyl auristatin.

36. The method of claim 35, wherein the monomethyl auristatin is monomethyl auristatin E (MMAE).

37. The method of claim 36, wherein the linker is attached to monomethyl auristatin E, thereby forming an antibody-drug conjugate having the following structure: ， Where Ab is antibody hL49, n is 12, R PR is hydrogen, R 21 is CH3, and p represents a number from 1 to 16.

38. The method of claim 37, wherein the average value of p in the population of antibody-drug conjugates is about 8.

39. The method of any one of claims 32 to 38, wherein the antibody-drug conjugate is hL49-MDpr-PEG(12)-gluc-MMAE.

40. Use of an antibody-drug conjugate according to any one of claims 17 to 24 in the preparation of a medicament for treating a CD228-expressing cancer in a subject, wherein the cancer is selected from the group consisting of melanoma, mesothelioma, non-small cell lung cancer, and triple-negative breast cancer.

41. The use of claim 40, wherein the subject has been previously treated with one or more therapeutic agents and has not responded to said treatment, wherein said one or more therapeutic agents are not said antibody, antigen-binding fragment or antibody-drug conjugate.

42. The use of claim 40, wherein the subject has been previously treated with one or more therapeutic agents and relapses following said treatment, wherein said one or more therapeutic agents are not said antibody, antigen binding fragment, or antibody-drug conjugate.

43. The use of claim 40, wherein 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 the antibody, antigen-binding fragment, or antibody-drug conjugate.

44. The use according to any one of claims 40 to 43, wherein the cancer is an advanced cancer.

45. The use according to claim 44, wherein the advanced cancer is stage 3 or stage 4 cancer.

46. ​​Use according to claim 44 or 45, wherein the advanced cancer is a metastatic cancer.

47. The use according to any one of claims 40 to 46, wherein the cancer is a recurrent cancer.

48. The use according to any one of claims 40 to 47, wherein the cancer is unresectable.

49. The use of any one of claims 40 to 48, wherein the subject has received prior treatment with a standard of care therapy for the cancer, but the prior treatment has failed.

50. The use according to claim 40, wherein the melanoma is cutaneous melanoma.

51. The use according to claim 50, 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.

52. The use according to claim 51, wherein the acral lentiginous melanoma is a subungual melanoma.

53. The use according to any one of claims 50 to 52, wherein the subject has received prior therapy with an inhibitor of PD-1 or PD-L1.

54. The use according to claim 53, wherein the subject has received prior therapy with an inhibitor of PD-1.

55. The use according to claim 40, wherein the melanoma is subcutaneous melanoma.

56. The use according to claim 55, wherein the subcutaneous melanoma is an ocular melanoma or a mucosal melanoma.

57. The use according to claim 40, wherein the melanoma is a non-cutaneous melanoma.

58. The use according to claim 40, wherein the mesothelioma is selected from the group consisting of pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma and testicular mesothelioma.

59. Use according to claim 58, wherein the mesothelioma is pleural mesothelioma.

60. The use of claim 59, wherein the subject has received prior therapy with a platinum-based therapy.

61. The use according to claim 60, wherein the platinum-based therapy is cisplatin.

62. The use according to any one of claims 59 to 61, wherein the subject has received prior therapy with pemetrexed.

63. The use according to claim 40, wherein the non-small cell lung cancer has a mutated form of epidermal growth factor receptor (EGFR).

64. The use of claim 40, wherein the non-small cell lung cancer has wild-type EGFR.

65. The use of claim 64, wherein the subject has received prior therapy with a platinum-based therapy.

66. The use according to claim 64 or 65, wherein the subject has received prior therapy with an inhibitor of PD-1 or PD-L1.

67. The use according to claim 66, wherein the subject has received prior therapy with an inhibitor of PD-1.

68. The use of claim 40, wherein the subject has received one or more prior lines of treatment for HER2-negative breast cancer.

69. The use of claim 68, wherein the one or more prior lines of treatment comprised treatment with a taxane.

70. The use according to claim 68 or 69, wherein the subject is hormone receptor positive.

71. The use of claim 70, wherein the subject has received prior therapy with an inhibitor of CDK4 / 6.

72. The use of claim 70 or 71, wherein the subject has received prior therapy with hormone-directed therapy.

73. The use according to any one of claims 40 to 72, 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.

74. The use according to any one of claims 40 to 73, wherein the subject is a human.

75. A kit comprising: (a) the antibody-drug conjugate according to any one of claims 17 to 24; and (b) instructions for using the antibody-drug conjugate for the use according to any one of claims 40 to 74.

76. A pharmaceutical composition comprising the antibody or antigen-binding fragment according to any one of claims 1 to 16 or the antibody-drug conjugate according to any one of claims 17 to 24 and one or more agents selected from physiologically acceptable carriers, diluents, excipients and adjuvants.

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