Anti-TMEM30a (CDC50a) antibodies as a novel therapeutic strategy

Description

PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 ANTI-TMEM30A (CDC50A) ANTIBODIES AS A NOVEL THERAPEUTIC STRATEGY CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 553,450, filed on February 14, 2024, the disclosure of which is incorporated by reference in its entirety for all purposes. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under Grant Nos. R01 CA139083 and R01 CA239462 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND

[0003] Transmembrane Protein 30A (TMEM30A), which is also referred to as CDC50A, has been identified as a key regulator of the ATP-dependent flippase activity that regulates cell surface expression of phosphatidylserine (PS) in human cells (Segawa, Katsumori et al. “Caspase-mediated cleavage of phospholipid flippase for apoptotic phosphatidylserine exposure.” Science (New York, N.Y.) vol. 344,6188 (2014): 1164-8. doi:10.1126 / science.1252809). PS is normally restricted to the inner cytoplasmic leaflet of the plasma membrane. In human cells (but not in platelets), TMEM30A is a component of the P4ATPase flippase complex that maintains PS distribution to the internal cytoplasmic leaflet, thus preventing externalization of the PS “Eat Me” pro-phagocytic signal that is recognized by macrophages (Ennishi, Daisuke et al. “TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma.” Nature medicine vol. 26,4 (2020): 577-588. doi:10.1038 / s41591-020-0757-z; Takatsu, Hiroyuki et al. “Phospholipid flippase activities and substrate specificities of human type IV P-type ATPases localized to the plasma membrane.” The Journal of biological chemistry vol. 289,48 (2014): 33543-56. doi:10.1074 / jbc.M114.593012; Kato, Utako et al. “Role for phospholipid flippase complex of ATP8A1 and CDC50A proteins in cell KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 migration.” The Journal of biological chemistry vol. 288,7 (2013): 4922-34. doi:10.1074 / jbc.M112.402701). Depletion of TMEM30A led to increased surface expression of PS and phagocytosis of TMEM30A-deficient cells by macrophages (Segawa, Katsumori et al. “Flippases and scramblases in the plasma membrane.” Cell cycle (Georgetown, Tex.) vol. 13,19 (2014): 2990-1. doi:10.4161 / 15384101.2014.962865; Segawa, Katsumori, and Shigekazu Nagata. “An Apoptotic 'Eat Me' Signal: Phosphatidylserine Exposure.” Trends in cell biology vol. 25,11 (2015): 639-650. doi:10.1016 / j.tcb.2015.08.003). This system is conserved in Cryptococcus neoformans in which TMEM30A mediates fungal resistance to macrophage killing (Huang, Wei et al. “Lipid Flippase Subunit Cdc50 Mediates Drug Resistance and Virulence in Cryptococcus neoformans.” mBio vol. 7,3 e00478-16. 10 May. 2016, doi:10.1128 / mBio.00478-16).

[0004] In addition, there is evidence that TMEM30A has a role in regulating cell migration, including tumor cell migration (Wang, Jiao et al. “A biosystems approach to identify the molecular signaling mechanisms of TMEM30A during tumor migration.” PloS one vol. 12,6 e0179900.22 Jun.2017, doi:10.1371 / journal.pone.0179900), in the organization of endothelial cells to generate new blood vessels (Zhang, Shanshan et al. “TMEM30A deficiency in endothelial cells impairs cell proliferation and angiogenesis.” Journal of cell science vol.132,7 jcs225052.3 Apr.2019, doi:10.1242 / jcs.225052), tumor angiogenesis, and in the regulation of the inflammatory response in macrophages, whereby reduction in TMEM30A expression enhances the macrophage mediated inflammatory response (van der Mark, Vincent A et al. “Phospholipid flippases attenuate LPS-induced TLR4 signaling by mediating endocytic retrieval of Toll-like receptor 4.” Cellular and molecular life sciences : CMLS vol.74,4 (2017): 715-730. doi:10.1007 / s00018-016-2360-5).

[0005] TMEM30A is implicated as a mediator of therapeutic resistance in a variety of cancers, including acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), multiple myeloma (MM), lymphoma, lung cancer, colon cancer, and hepatocellular carcinoma (HCC). Expression of TMEM30A has been demonstrated to contribute to oxaliplatin resistance in colorectal cancer. Gene silencing of TMEM30A expression improves oxaliplatin sensitivity of HCT 116 and SW480 colorectal cancer cells (Harradine et al., “Functional Genomics Reveals Diverse Cellular Processes that Modulate Tumor Cell Response to Oxaliplatin Molecular Cancer Research 20119(2):173-182). TMEM30A knockout cell lines and TMEM30A mutated primary cells were associated with increased chemotherapy accumulation in diffuse large B- KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 cell lymphoma, suggesting that pharmacologic disruption of TMEM30A may also promote chemotherapeutic sensitivity in lymphoma and potentially other tumor types (Ennishi, E. et al., “TMEM30A loss of function mutations drive lymphomagenesis and confer therapeutic exploitable vulnerability in B-cell lymphoma. Nature Medicine 2020. 26, 577-588). Recent evidence demonstrates that mutations of TMEM30A in tumor cells correlates with the resistance of lymphoma cells to CD19 CAR-T Cell Therapy (Sworder et al., “Determinants of Resistance to Engineered T Cell Therapies Targeting CD19 in Large B-cell Lymphoma Cancer Cell 2023 January 941(1):210-225). Elevated expression of TMEM30A as well as mutations thereof can be used as a biomarker of adverse prognosis in cancer. SUMMARY

[0006] This section highlights certain features of the invention, but is not intended to be a comprehensive summary of all aspects of the invention.

[0007] The present disclosure provides anti-TMEM30A antibody compositions and methods for the treatment of cancer. In other embodiments, the antibodies are used for the treatment of inflammatory disorders, including autoimmune disease, and fungal infections.

[0008] In one aspect, provided herein is an antibody that binds to the extracellular domain of human TMEM30A on cancer cells. In some embodiments, the antibody comprises (i) a heavy chain variable region (VH) comprising an HCDR1 comprising a sequence DYAMH (SEQ ID NO: 1), an HCDR2 comprising a sequence VISTYSGNTNYNQKFKG (SEQ ID NO: 2), and an HCDR3 comprising a sequence YYRYDGETMDY (SEQ ID NO: 3); and (ii) a light chain variable region (VL) comprising an LCDR1 comprising a sequence SASSSVSYMH (SEQ ID NO: 4), an LCDR2 comprising a sequence RTSNLAS (SEQ ID NO: 5), and an LCDR3 comprising a sequence QQRSSYPLT (SEQ ID NO: 6). In some embodiments, antibody binding to the extracellular domain of human TMEM30A promotes macrophage-mediated phagocytosis of tumor cells.

[0009] In some embodiments, the antibody comprises (i) a heavy chain variable region (VH) comprising an HCDR1 comprising a sequence DYAMH (SEQ ID NO: 1), an HCDR2 comprising a sequence VISTYSGNTNYNQKFKG (SEQ ID NO: 2), and an HCDR3 comprising a sequence YYRYDGETMDY (SEQ ID NO: 3); and (ii) a light chain variable region (VL) comprising an LCDR1 comprising a sequence SASSSVSYMH (SEQ ID NO: 4), KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 an LCDR2 comprising a sequence RTSNLAS (SEQ ID NO: 5), and an LCDR3 comprising a sequence QQRSSYPLT (SEQ ID NO: 6).

[0010] In some embodiments, the antibody comprises (i) a heavy chain variable region (VH) comprising an HCDR1 comprising a sequence NYGVH (SEQ ID NO: 9), an HCDR2 comprising a sequence VIWAGGSTHYNSTLMS (SEQ ID NO: 10), and an HCDR3 comprising a sequence VSPPGYFDV (SEQ ID NO: 11); and (ii) a light chain variable region (VL) comprising an LCDR1 comprising a sequence TASSSVSSSYLH (SEQ ID NO: 12), an LCDR2 comprising a sequence STSNLAS (SEQ ID NO: 13), and an LCDR3 comprising a sequence HQYHRSPLT (SEQ ID NO: 14).

[0011] In some embodiments, the antibody comprises (i) a heavy chain variable region (VH) comprising an HCDR1 comprising a sequence RYWMS (SEQ ID NO: 17), an HCDR2 comprising a sequence EINPDSSTINYTPSLKD (SEQ ID NO: 18), and an HCDR3 comprising a sequence RLVYYAMDY (SEQ ID NO: 19); and (ii) a light chain variable region (VL) comprising an LCDR1 comprising a sequence SASQGINNYLN (SEQ ID NO: 20), an LCDR2 comprising a sequence YTSSLHS (SEQ ID NO: 21), and an LCDR3 comprising a sequence QQYSKLPYT (SEQ ID NO: 22).

[0012] In some embodiments, the antibody VHcomprises an amino acid sequence having at least 80% identity or at least, 85%, 90%, or 95% identity) to SEQ ID NO: 7. In some embodiments, the antibody VLcomprises an amino sequence having at least 80% identity or at least 85%, 90%, or 95% identity ) to SEQ ID NO: 8. In some embodiments, the antibody VH comprises SEQ ID NO: 7 and the antibody VL comprises SEQ ID NO: 8.

[0013] In some embodiments, the antibody VHcomprises an amino acid sequence having at least 80% identity or at least 85%, 90%, or 95% identity to SEQ ID NO: 15. In some embodiments, the antibody VL comprises an amino sequence having at least 80% identity, or at least 85%, 90%, or 95% identity, to SEQ ID NO: 16. In some embodiments, the antibody VH comprises SEQ ID NO: 15 and the antibody VL comprises SEQ ID NO: 16.

[0014] In some embodiments, the antibody the VHcomprises an amino acid sequence having at least 80% identity, or at least 85%, 90%, or 95% identity, to SEQ ID NO: 23. In some embodiments, the antibody VL comprises an amino sequence having at least 80% identity, or at least 85%, 90%, or 95% identity, to SEQ ID NO: 24. In some embodiments, the antibody VH comprises SEQ ID NO: 23 and the antibody VL comprises SEQ ID NO: 24. KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2

[0015] In some embodiments, the antibody is linked to a cytotoxic agent or a detectable label.

[0016] In another aspect, provided herein is a nucleic acid encoding an antibody of the present disclosure. In some embodiments, the nucleic acid sequence is operably linked to a heterologous promoter.

[0017] In another aspect, provided herein is an expression vector comprising a nucleic acid of the present disclosure.

[0018] In another aspect, provided herein is a cell expressing an antibody of the present disclosure, comprising a nucleic acid or an expression vector of the present disclosure.

[0019] In another aspect, provided herein is a method of producing an antibody, the method comprising culturing a cell comprising a nucleic acid or an expression vector of the present disclosure, under conditions in which the nucleic acid encoding the VH and the nucleic acid encoding the VL are expressed.

[0020] In another aspect, provided herein is a method of treating a disease in a subject comprising administering an antibody of the present disclosure to a subject. In some embodiments, the disease is a cancer. In some embodiments, the cancer is selected from the group consisting of hepatocellular carcinoma, non-Hodgkin lymphoma, central nervous system (CNS) lymphoma, multiple myeloma (MM), acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), non-small lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma, and lung small cell carcinoma (small cell lung cancer; SCLC), and ovarian cancer. In some embodiments, the cancer is resistant to lenalidomide.

[0021] In some embodiments, an antibody of the present disclosure is administered for the treatment of an inflammatory disorder or an autoimmune disorder. In some embodiments, the antibody is administered for the treatment of an infection, e.g., a fungal infection.

[0022] In another aspect, provided herein is a method for killing cancer cells in a subject comprising contacting the cancer cells in the subject in vivo with an antibody of the present disclosure. In some embodiments, the method further comprises contacting the cancer cells with a therapeutic molecule in vivo selected from the group consisting of a protein kinase inhibitor, a chemotherapeutic drug, and an immunotherapeutic drug. In some embodiments, the protein kinase inhibitor is an inhibitor of mTOR. In some embodiments, the inhibitor of mTOR is AZD2014. In some embodiments, the protein kinase inhibitor is a serine / threonine kinase KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 inhibitor, a tyrosine kinase inhibitor, or a protein kinase C inhibitor. In some embodiments, the protein kinase inhibitor is a broad spectrum protein kinase inhibitor such as staurosporine.

[0023] In another aspect, provided herein is a method for reducing inflammation in a subject having an inflammatory condition comprising contacting cells in the subject in vivo with an antibody of the present disclosure, wherein binding of the antibody to the cells reduces inflammation in the subject.

[0024] In another aspect, provided herein is a method for increasing phagocytosis in a subject comprising administering the antibody of the present disclosure to the subject, wherein binding of the antibody to cells increases phagocytosis. In some embodiments, the cells are selected from the group consisting of tumor cells, cancer cells, macrophages, endothelial cells, and monocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figures 1A-C show high-resolution array-comparative genomic hybridization (CGH) comparative analysis of the paired genomes of relapsed central nervous system (CNS) lymphoma cells. In Figure 1A, cells were isolated from a patient (CV-R1, pre-lenalidomide) with relapsed secondary CNS lymphoma. Figure 1A, top panel, shows analysis for cells before treatment with lenalidomide (“Pre-Len,” in the setting of relapse after radiation, intravenous administration and intraventricular administration of methotrexate and / or rituximab, and intravenous administration of cyclophosphamide, adriamycin, vincristine and prednisone. Figure 1A, bottom panel, shows analysis for cells at subsequent relapse (CV-R2, post lenalidomide, “Post-Len”) after treatment with lenalidomide monotherapy in vivo, demonstrating focal high level DNA copy gain at 6q13 (arrow), a locus encoding TMEM30A. Figure 1B shows focal high level copy number gain at 6q13 and differentially upregulated genes encoded by this locus, as demonstrated by differential RNA-Seq analysis of transcriptomes isolated from CV-R1 compared to CV-R2. Transcripts differentially upregulated at this locus in association with resistance included TMEM30A, Cox7A2, Col12A1, and FILIP1. Figure 1C shows upregulation of TMEM30A protein by lenalidomide resistant relapsed CNS lymphoma tumor cells according to immunohistochemical analysis. There was no upregulation of Cox7A2 protein expression by the resistant tumor cells. KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2

[0026] Figure 2A shows upregulation of TMEM30A in paired specimens of treatment- refractory CNS lymphoma in autopsy specimens compared to diagnostic biopsy specimen according to immunohistochemical analysis.

[0027] Figure 2B shows marked upregulation of TMEM30A gene expression in lymphoma from autopsy specimen compared to brain biopsy according to in situ hybridization using fluorescence probes. DAPI was used to localize tumor cell nuclei.

[0028] Figures 3A-C show that TMEM30A is expressed in a variety of refractory cancers. Figure 3A shows RNA expression across different cancer cells (see x-axis) according to the Cancer Genome Atlas Program (TCGA). Figure 3B shows immunohistochemical detection of TMEM30A expression by hepatocellular carcinoma cells. (Representative of each of 63 consecutive cases of hepatocellular carcinoma analyzed.) Figure 3C shows expression of TMEM30A in non-small cell lung cancer cells and in normal lung cells.

[0029] Figures 4A-C show TMEM30A knockdown via two silencing short hairpin RNAs (shRNAs) – TMEM30A.KD.1 and TMEM30A.KD.2. Figures 4A-B show that both shRNAs cause marked delay in in vitro tumor growth and cell survival of HepG2 cells. Figure 4C shows the impact of the shRNAs on TMEM30A expression (52 kDa) by Western blot.

[0030] Figures 5A-B show Western blots of monoclonal antibodies (mAbs) probed with human TMEM30A extracellular domain. In Figure 5A, TMEM30A extracellular domain peptide antigen was used. In Figure 5B, human acute myeloid leukemia (AML) cells expressing TMEM30A extracellular domain were used. A band at the apparent molecular weight of 51 kDa corresponding to TMEM30A was observed.

[0031] Figure 6 shows immunoreactivity of an exemplary anti-TMEM30A monoclonal antibody (mAb), mAb2, against human HepG2 cells in culture (left panel), and species cross- reactivity using an immunohistochemical assay with murine tissues (mouse brain), demonstrating TMEM30A expression by mouse astrocytes and endothelia (right panel).

[0032] Figure 7 shows that an exemplary anti-human TMEM30A mAb promoted annexin V expression by HepG2 cells in culture by fluorescence microscopy.

[0033] Figure 8A shows that an exemplary anti-human TMEM30A mAb promotes the synergistic cell death of human HepG2 cells in culture when treated in combination with the mTOR inhibitor, AZD2014. KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2

[0034] Figure 8B shows the effect of an exemplary anti-TMEM30A on Hep3B cells. Hep3B cells exhibited marked cytotoxic response when treated with AZD2014 and anti-TMEM30A mAb.

[0035] Figures 9A-B show that an exemplary anti-human TMEM0A mAb, but not control IgG1, promoted phagocytosis of human AML MV4-11 macrophages. Figure 9A shows fluorescence microscopy images. Figure 9B shows quantification of phagocytosis events per field of image.

[0036] Figure 10 shows that an exemplary anti-human TMEM0A mAb (right panels), but not control IgG1 (left panels), promoted lamellipodia formation in human Raji lymphoma cells in the presence of M1 macrophages (top panels) and in the presence of MCF-10 cells (not stained, bottom panels).

[0037] Figure 11A shows that an exemplary anti-TMEM0A mAb (right panels), but not control IgG1 (left panels), promotes lamellipodia formation in human Hep3B hepatocellular carcinoma cells (HCC), in the presence of M1 macrophages (upper right). 25-50 micron diameter fragments of Hep3B cells were internalized by phagosomes (indicated by arrows). The top and bottom portions of the right panel share the same field of view. The top and bottom portions of the left panel share the same field of view.

[0038] Figure 11B shows that an exemplary anti-human TMEM30A mAb promotes the enlargement of M1 macrophages after 18 hours of phagocytosis of Hep3B hepatocellular carcinoma cells.

[0039] Figure 12A shows that the exemplary anti-human TMEM30A mAb (anti-T) attenuated Hep3B tumor progression in RAG- / - mice. The exemplary anti-human TMEM30A mAb was co-administered with interferon gamma.

[0040] Figure 12B shows a representative example of smaller liver tumors from mice treated with anti-TMEM30A mAb.

[0041] Figures 12C-D show that an exemplary anti-human TMEM30A mAb administered twice weekly, without administration of interferon gamma, as a single agent, markedly human attenuates hepatocellular carcinoma (HCC; Hep3B) tumor progression in RAG- / - mice. Figure 12C shows exemplary bioluminescence in mice. Figure 12D shows quantification of bioluminescence. KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2

[0042] Figure 13 shows high expression of TMEM30A by ovarian cancer (16 / 16 consecutive cases) compared to normal ovary (4 consecutive cases) as demonstrated by immunohistochemistry using an exemplary anti-TMEM30A monoclonal antibody (3D7E) of the present disclosure.

[0043] Figure 14A shows that an exemplary anti-TMEM30A monoclonal antibody of the present disclosure (mAb 3D7E6) recognizes murine TMEM30A in murine A20 lymphoma cells as demonstrated by western immunoblotting.

[0044] Figure 14B shows that treatment of A20 lymphoma cells in vitro for four hours with anti-TMEM30A (mAb 3D7E6) (10 micrograms / ml) results in significantly increased annexin V expression as demonstrated by immunofluorescence microscopy (Blue, DAPI; red, annexin V).

[0045] Figure 14C shows quantification of Annexin V induction comparing control, anti- TMEM30A monotherapy (mAb 3D7E6), staurosporine monotherapy, and combination anti- TMEM30A plus staurosporine (four hour in vitro drug treatment)

[0046] Figures 15A-15B show that an exemplary anti-human TMEM30A mAb of the present disclosure (mAb 3D7E6) attenuates tumor progression of human non-small cell lung cancer (NSCLC) in RAG- / - mice. Figure 15A shows exemplary bioluminescence curve and tumor growth curves in mice. Figure 15B shows that mice bearing NSCLC tumors experienced early weight loss when treated with control IgG1 antibody compared to mice treated with exemplary anti-human TMEM30A mAb.

[0047] Figure 16 shows that the anti-human TMEM30A mAb (mAb 3D7E6) reduces the efficiency of Raji lymphoma cell chemotaxis in response to the chemokine SDF-1 in a transwell migration assay.

[0048] Figures 17A-17B show that treatment of metastatic non-small cell lung cancer model (NCI H460) with anti-TMEM30A mAb is associated with reduced tumor angiogenesis. Figure 17A: Control IgG1. Scale bar is 25 microns. Figure 17B: Anti- TMEM30A mAb. Scale bar is 25 microns. KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 DETAILED DESCRIPTION I. Terminology

[0049] As used herein, the term “TMEM30A” refers to a polypeptide that is encoded by a TMEM30A gene. The human TMEM30A gene is located on chromosome 6q13-14.3 and functions as an accessory component of various P4-ATPase flippase complexes. TMEM30A is a transmembrane protein that can interact with many flippases, including, for example, ATP8A1, ATP8A2, ATP8B1, ATP8B2, ATP8B4, ATP10A, ATP10B, ATP10D, ATP11A, ATP11B and ATP11C. TMEM30A is also known as C6orf67, CDC50A, cell cycle control protein 50A (CC50A), PT-ATPase flippase complex beta subunit TMEM30A, and transmembrane protein 30A. Human TMEM30A protein sequence encoded by the TMEM30A gene are available under Uniprot accession number Q9NV96 (or Q9NV96-1). There are three isoforms of human TMEM30A – Q9NV96 (or Q9NV96-1), Q9NV96-2, and Q9NV96-3; the Q9NV96 (or Q9NV96-1) sequence is the canonical sequence. A TMEM30A gene nucleotide sequence is available under GenBank accession number AL080250.11. A TMEM30A cDNA nucleotide sequence is available under GenBank accession number AK001718.1. As a component of the flippase complex, TMEM30A catalyzes the hydrolysis of ATP coupled to the transport of aminophospholipids from the outer to the inner leaflet of various membranes and ensures the maintenance of asymmetric distribution of phospholipids. TMEM30A may be involved in regulation of neurite outgrowth. When TMEM30A is reconstituted to liposomes, it predominantly transports phosphatidylserine (PS); it transports phosphatidylethanolamine (PE) to a lesser extent. TMEM30A from other species are known. For example, the TMEM30A protein sequences from Bos taurus, Rattus norvegicus, Mus musculus, Pongo abelii, Macaca mulatta, Felix catus, and Pan paniscus are available under Uniprot accession numbers Q17QL5, Q6AY41, Q8VEK0, Q5R6C0, I0FTB2, M3XDP7, and A0A2R9A0S8 respectively.

[0050] As used herein, the term “antibody” as used here means an isolated or recombinant binding agent that comprises the necessary variable region sequences or antigen-binding region to specifically bind an antigenic epitope. Therefore, an “antibody” as used herein is any form of antibody of any class or subclass or fragment thereof that exhibits the desired biological activity, e.g., binding a specific target antigen. Thus, it is used in the broadest sense and includes, but is not limited to, a monoclonal antibody (including full-length monoclonal antibodies), human antibodies, chimeric antibodies, single domain antibodies, such as VHH, NANOBODIES®, diabodies, camelid-derived antibodies, monovalent antibodies, bivalent KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments including, but not limited to scFv, Fab, and the like so long as they exhibit the desired biological activity.

[0051] The “isotype” or “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4. The antibodies described herein can be of any of these classes or subclasses.

[0052] Antibodies can exist as intact immunoglobulins or as any of a number of well- characterized fragments that include specific antigen-binding activity. Such fragments can be produced by digestion with various peptidases. Pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)’2,a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)’2may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)’2dimer into an Fab’ monomer. The Fab’ monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments are often synthesized de novo using recombinant DNA methodology.

[0053] Antibodies or antigen-binding molecules of the disclosure further includes one or more immunoglobulin chains that are chemically conjugated to, or expressed as, fusion proteins with other proteins. The term “antibody” additionally encompasses bispecific and multispecific antibodies as well as any other monovalent, bivalent, or multivalent antibody format. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites.

[0054] A “monoclonal antibody” refers to a clonal preparation of antibodies with a single binding specificity and affinity for a given epitope on an antigen.

[0055] A “chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region, CDR, or portion thereof) is linked to a constant region of a different or altered class, effector function and / or species, or an entirely different molecule which confers new properties to the chimeric antibody (e.g., an enzyme, toxin, hormone, growth factor, drug, etc.); or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 having a different or altered antigen specificity (e.g., CDR and framework regions from different species).

[0056] A “humanized” antibody is an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts. In one embodiment, some, most or all of the amino acids outside the CDR domains are replaced with amino acids corresponding to the human immunoglobulin germline, while amino acids within one or more CDR regions are unchanged. In some embodiments, one or more CDR residues may be altered, e.g., to provide a sequence closer to germline or to replace a residue that may impede expression or production of the antibody.

[0057] The amino acid sequences of the CDRs and framework regions can be determined using various well known definitions in the art, e.g., Kabat, Chothia, international ImMunoGeneTics database (IMGT), and AbM (see, e.g., Johnson et al., supra; Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992, structural repertoire of the human VH segments J. Mol. Biol.227, 799-817; Al-Lazikani et al., J.Mol.Biol 1997, 273(4)). Definitions of antigen combining sites are also described in the following: Ruiz et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219–221 (2000); and Lefranc,M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan 1;29(1):207-9 (2001); MacCallum et al, Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); and Martin et al, Proc. Natl Acad. Sci. USA, 86, 9268–9272 (1989); Martin, et al, Methods Enzymol., 203, 121–153, (1991); Pedersen et al, Immunomethods, 1, 126, (1992); and Rees et al, In Sternberg M.J.E. (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141–172 1996). Reference to CDRs as determined by Kabat numbering are based, for example, on Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, MD (1991)). Chothia CDRs are determined as defined by Chothia (see, e.g.,Chothia and Lesk J. Mol. Biol.196:901-917 (1987)).

[0058] The term “specifically bind” refers to a molecule (e.g., antibody or antibody fragment) that binds to a target with at least 2-fold greater affinity than non-target compounds, KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 e.g., at least 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, or 100-fold greater affinity. For example, an antibody that specifically binds to TMEM30A typically binds to TMEM30A with at least 2-fold greater affinity than a non-TMEM30A target. The term “specific binding,” “specifically binds to,” or “is specific for” a particular target, as used herein, can be exhibited, for example, by a molecule (e.g., an antibody) having an equilibrium dissociation constant, KD, for the target (e.g., a ligand) of, e.g., 10-2M or smaller, e.g., 10-3M, 10-4M, 10-5M, 10-6M, 10-7M, 10-8M, 10-9M, 10-10M, 10-11M, or 10-12M. For example, an antibody that specifically binds to TMEM30A has a KD of less than 100 nM or less than 10 nM. In some embodiments, an antibody binds to TMEM30A with a KD that is at least 100-fold greater than its affinity for a non-TMEM30A target.

[0059] “Epitope" or “antigenic determinant” refers to a site on an antigen to which an antibody binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol.66, Glenn E. Morris, Ed (1996).

[0060] The term “valency” as used herein refers to the number of different binding sites of an antibody for an antigen. A monovalent antibody comprises one binding site for an antigen. A multivalent antibody comprises multiple binding sites.

[0061] The terms “polynucleotide” and “nucleic acid” are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides. The terms include RNA, DNA, and synthetic forms and mixed polymers of the above. In particular embodiments, a nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form or analog of either type of nucleotide, and combinations thereof. A reference to a nucleic acid sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. Reference to a “polynucleotide” or “nucleic acid” that encodes a polypeptide sequence also includes codon-optimized nucleic KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 acids and nucleic acids that comprise alternative codons that encode the same polypeptide sequence. Nucleic acids may be referred to by their commonly accepted single-letter codes.

[0062] The term “vector” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. A “vector” also refers to a recombinant construct in which a nucleic acid sequence of interest is inserted into the vector. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0063] “Polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0064] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, -carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0065] “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 identical or associated, e.g., naturally contiguous, sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to another of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes silent variations of the nucleic acid. One of skill will recognize that in certain contexts each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, silent variations of a nucleic acid which encodes a polypeptide is implicit in a described sequence with respect to the expression product, but not with respect to actual probe sequences.

[0066] A “conservative” substitution as used herein refers to a substitution of an amino acid such that charge, hydrophobicity, and / or size of the side group chain is maintained. Illustrative sets of amino acids that may be substituted for one another include (i) positively-charged amino acids Lys, Arg and His; (ii) negatively charged amino acids Glu and Asp; (iii) aromatic amino acids Phe, Tyr and Trp; (iv) nitrogen ring amino acids His and Trp; (v) large aliphatic nonpolar amino acids Val, Leu and Ile; (vi) slightly polar amino acids Met and Cys; (vii) small-side chain amino acids Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gln and Pro; (viii) aliphatic amino acids Val, Leu, Ile, Met and Cys; and (ix) small hydroxyl amino acids Ser and Thr. Reference to the charge of an amino acid in this paragraph refers to the charge at physiological pH.

[0067] The phrase “percent identical,” “percent identity,” or equivalents used in the context of two nucleic acids or polypeptides, refers to a sequence that has at least a specified level of identity, e.g., at least 50% sequence identity with a reference sequence (e.g., any SEQ ID NO included herein). Alternatively, percent identity can be any integer from 50% to 100%. Some embodiments include at least: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, compared to a reference sequence using the programs described herein, e.g., BLAST using standard parameters, as described below.

[0068] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0069] A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well- known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection.

[0070] Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol.215: 403-410 and Altschul et al. (1977) Nucleic Acids Res.25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0071] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10-5, and most preferably less than about 10-20.

[0072] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0073] As used herein the phrase “heterologous” refers to what is not found in nature. The term “heterologous sequence” refers to a sequence not normally found in a given cell in nature. As such, a heterologous nucleotide or protein sequence may be: (a) foreign to its host cell (i.e., is exogenous to the cell); (b) naturally found in the host cell (i.e., endogenous) but present at an unnatural quantity in the cell (i.e., greater or lesser quantity than naturally found in the host cell); or (c) be naturally found in the host cell but positioned outside of its natural locus.

[0074] The term “recombinant” or “engineered” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non- recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

[0075] The term “isolated,” when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state. It can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames that flank the gene and encode a protein other than the gene of interest. The term “purified” denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.

[0076] As used herein, the term “pharmaceutically acceptable carrier” refers to an excipient or diluent in a pharmaceutical composition. The pharmaceutically acceptable carrier must be compatible with the other ingredients of the formulation and not deleterious to the recipient. In the present disclosure, the pharmaceutically acceptable carrier must provide adequate pharmaceutical stability to the active ingredient. The nature of the carrier differs with the mode of administration. For example, for intravenous administration, an aqueous solution carrier is generally used; for oral administration, a solid carrier is preferred.

[0077] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, e.g., a mammal, such as a primate. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0078] The terms “treat” and “treatment” refer to both therapeutic treatment and prophylactic or preventive measures, wherein the object is to prevent or slow down an undesired physiological change or disorder. For purpose of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. In other embodiments the terms “treat”, “treatment” and “treating” refer to the inhibition of the progression of a proliferative disorder, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. In other embodiments the terms “treat”, “treatment” and “treating” refer to the reduction or stabilization of tumor size or cancerous cell count.

[0079] The term “effective amount” as used herein, refers to the amount of an anti- TMEM30A antibody or variant thereof as described in the present disclosure that is sufficient to effect treatment, prognosis, or diagnosis of a disease associated with TMEM30A-expressing cells (e.g., a TMEM30A-positive cancer cell), as described herein, when administered to a subject. For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of therapeutic effect at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2- fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control. A therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. Dosage regiments may be adjusted to provide the optimum therapeutic response. An effective amount is also one in which any toxic or detrimental effects (i.e., side effects) of the antibody are minimized and / or outweighed by the beneficial effects. In determining the effective amount of antibodies to be administered, a physician may evaluate circulating plasma levels of immune cells or antibodies in the body and any antibody-related toxicity.

[0080] The terms “dose” and “dosage” are used interchangeably herein. A dose refers to the amount of active ingredient given to an individual at each administration. As used herein, the dose can refer to the concentration of the antibody or associated components, e.g., the amount of therapeutic agent or dosage of radiolabel. The dose will vary depending on a number of factors, including frequency of administration; size and tolerance of the individual; severity of the condition; risk of side effects; the route of administration; and the imaging modality of the detectable moiety (if present). One of skill in the art will recognize that the dose can be KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 modified depending on the above factors or based on therapeutic progress. The term “dosage form” refers to the particular format of the pharmaceutical, and depends on the route of administration. For example, a dosage form can be in a liquid, e.g., a saline solution for injection.

[0081] The term "co-administer" refers to the simultaneous presence of two active agents in the blood of an individual. Active agents that are co-administered can be concurrently or sequentially delivered.

[0082] As used in herein, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “an antibody” optionally includes a combination of two or more such antibodies, and the like.

[0083] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. For example, for KD and IC50 values ± 20%, ± 10%, or ± 5%, are within the intended meaning of the recited value. II. Introduction

[0084] The present disclosure relates to antibodies and variants thereof that bind to TMEM30A. The antibodies modulate TMEM30A activity in certain proliferative diseases, e.g., a cancer, an inflammatory disorder, and an autoimmune disorder.

[0085] The anti-TMEM30A antibodies of the present disclosure include variants, e.g., truncations and mutations, and antibody fragments, e.g., Fab, F(ab’)2, Fv, and scFv, and any variation thereof that can bind to the extracellular domain of human TMEM30A and regulate flippase activity. In some embodiments the antibody binds to mouse TMEM30A or TMEM30A from another species.

[0086] In some embodiments, the anti-TMEM30A antibody modulates TMEM30A activity in various cancers that express TMEM30A, to block pro-survival signaling in the cancer cells. In some embodiments, the anti-TMEM30A antibody potentiates immunotherapeutic interventions, pro-apoptotic interventions, and chemotherapeutic effects that promote tumor cell death. In some embodiments the anti-TMEM30A antibody blocks tumor migration, chemotaxis, and metastasis. In some embodiments the anti-TMEM30A antibody blocks tumor angiogenesis. The anti-TMEM30A antibody treats inflammatory disorders and / or autoimmune 20 KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 disorders via the attenuation of the inflammatory effects of macrophages that express TMEM30A.

[0087] In some embodiments, an anti-TMEM30A antibody of the present disclosure disrupts or affects one or more functions of TMEM30A. In some embodiments, the anti-TMEM30A antibody affects phospholipid flippase function. In some embodiments, the anti-TMEM30A antibody induces annexin V expression (a marker of cell death). In some embodiments, the anti-TMEM30A antibody synergizes with an mTOR inhibitor in vitro to induce cell death in cancer cells. In some embodiments, the anti-TMEM30A antibody induces the formation of filopodia and / or lamellipodia of cancer cells. In some embodiments, the anti-TMEM30A antibody facilitates phagocytosis of cancer cells, including tumor cells. In some embodiments, the phagocytosis is mediated by macrophages, e.g., M1-polarized macrophages. In some embodiments, the anti-TMEM30A antibody reduces the rate of tumor formation. In some embodiments, the anti-TMEM30A antibody kills tumor cells. In some embodiments, the anti- TMEM30A antibody has apparent low toxicity when administered to a subject.

[0088] In some embodiments, the anti-TMEM30A antibody (e.g., a monoclonal antibody, mAb) recognizes murine TMEM30A protein at 51 kDa on western blot in murine A20 lymphome cells. In some embodiments, the anti-TMEM30A monoclonal antibody induces annexin V expression in murine A20 lymphoma cells in vitro. In some embodiments, when the anti-TMEM30A monoclonal antibody is administered in combination with the protein kinase inhibitor staurosporine, annexin V expression is synergistically induced.

[0089] In some embodiments, an anti-TMEM30A antibody described herein functions as a blocking antibody by blocking the TMEM30A from interacting with or binding to one or more of its binding partners. Thus, in some embodiments, where TMEM30A is a component of a flippase complex, the anti-TMEM30A antibody blocks TMEM30A from interacting with or binding to the other subunits of the flippase complex. In some embodiments, where TMEM30A is the extracellular component of a flippase complex, the anti-TMEM30A antibody blocks TMEM30A, i.e., flippase, from binding to a ligand, e.g., lipids. In some embodiments, the anti- TMEM30A is an inhibitor of flippase. III. Anti-TMEM30A Antibodies

[0090] Descriptions of anti-TMEM30A antibodies below are grouped by the clone names provided in Tables 1-3 for convenience only. KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 Clone 3D7E6

[0091] In some embodiments, the anti-TMEM30A antibody binding domain comprises at least one, at least two, or at least three heavy chain complementarity determining regions (CDRs) of a variable domain sequence of SEQ ID NO: 7. In some embodiments, the anti- TMEM30A antibody binding domain comprises at least one, at least two, or at least three light chain CDRs of a variable domain sequence of SEQ ID NO: 8. In some embodiments, the CDRs are as defined by Kabat. In some embodiments, the CDRs are as defined by Chothia. In some embodiments, CDRs are as defined by AbM. In some embodiments, CDRs are as defined by IMGT. In some embodiments, the antibody comprises an HCDR1, HCDR2, and HCDR3 of the VH region of SEQ ID NO: 7 and an LCDR1, LCDR2, and LCDR3 of the VL region of SED ID NO: 8 as defined by Chothia. In some embodiments, the antibody comprises an HCDR1, HCDR2, and HCDR3 of the VH region of SEQ ID NO: 7 and an LCDR1, LCDR2, and LCDR3 of the VL region of SED ID NO: 8 as defined by AbM. In some embodiments, the antibody comprises an HCDR1, HCDR2, and HCDR3 of the VH region of SEQ ID NO: 7 and an LCDR1, LCDR2, and LCDR3 of the VL region of SED ID NO: 8 as defined by IMGT.

[0092] In some embodiments, an anti-TMEM30A antibody binding domain of the present disclosure comprises a heavy chain CDR1 (HCDR1) sequence comprising SEQ ID NO: 1. In some embodiments, the anti-TMEM30A antibody binding domain comprises an HCDR2 sequence comprising SEQ ID NO: 2. In some embodiments, the anti-TMEM30A antibody binding domain comprises an HCDR3 sequence comprising SEQ ID NO: 3. In some embodiments, the anti-TMEM30A antibody binding domain comprises at least two heavy chain CDR sequences selected from SEQ ID NO: 1, 2, and 3. In typical embodiments, the anti- TMEM30A antibody binding domain comprises an HCDR1 sequence comprising SEQ ID NO: 1, an HCDR2 sequence comprising SEQ ID NO: 2, and an HCDR3 sequence comprising SEQ ID NO: 3.

[0093] In some embodiments, an anti-TMEM30A antibody binding domain of the present disclosure comprises a light chain CDR1 (LCDR1) sequence comprising SEQ ID NO: 4. In some embodiments, the anti-TMEM30A antibody binding domain comprises an LCDR2 sequence comprising SEQ ID NO: 5. In some embodiments, the anti-TMEM30A antibody binding domain comprises an LCDR3 sequence comprising SEQ ID NO: 6. In some embodiments, the anti-TMEM30A antibody binding domain comprises at least two light chain KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 CDR sequences selected from SEQ ID NO: 4, 5, and 6. In typical embodiments, the anti- TMEM30A antibody binding domain comprises an LCDR1 sequence comprising SEQ ID NO: 4, an LCDR2 sequence comprising SEQ ID NO: 5, and an LCDR3 sequence comprising SEQ ID NO: 6.

[0094] In some embodiments, the anti-TMEM30A antibody binding domain comprises a HCDR1, HCDR2, and HCDR3, in which one of the HCDRs comprises a substitution relative to the corresponding HCDR as set forth in SEQ ID NO: 1, 2, or 3. In some embodiments, the anti-TMEM30A antibody binding domain comprises an HCDR1, HCDR2, and HCDR3, in which two of the HCDRs each comprise a substitution relative to the corresponding HCDR as set forth in SEQ ID NO: 1, 2, or 3.

[0095] In some embodiments, the anti-TMEM30A antibody binding domain comprises a LCDR1, LCDR2, and LCDR3, in which one of the LCDRs comprises a substitution relative to the corresponding LCDR as set forth in SEQ ID NO: 4, 5, or 6. In some embodiments, the anti- TMEM30A antibody binding domain comprises an LCDR1, LCDR2, and LCDR3, in which two of the LCDRs each comprise a substitution relative to the corresponding LCDR as set forth in SEQ ID NO: 4, 5, or 6.

[0096] In some embodiments, the antibody comprises a VHregion comprising an HCDR1 sequence comprising SEQ ID NO: 1, an HCDR2 sequence comprising SEQ ID NO: 2, and an HCDR3 sequence comprising SEQ ID NO: 3; and a VLregion comprising an LCDR1 sequence comprising SEQ ID NO: 4, an LCDR2 sequence comprising SEQ ID NO: 5, and an LCDR3 comprising sequence SEQ ID NO: 6.

[0097] In some embodiments, the anti-TMEM30A antibody binding domain comprises a heavy chain variable region having at least 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to the VH amino acid sequence of a variable region sequence of SEQ ID NO: 7. In some embodiments, the anti- TMEM30A antibody variable domain comprises substitutions, insertions, or deletions in the framework of a variable region as shown in SEQ ID NO: 7.

[0098] In some embodiments, the anti-TMEM30A antibody VHregion comprises a heavy chain framework 1 (FR-H1) sequence defined by Kabat as set forth in SEQ ID NO: 7. In some embodiments, the anti-TMEM30A antibody has 1, 2, 3, 4, or 5 amino acid substitutions relative to the FR1 sequence as defined by Kabat of the VH region of SEQ ID NO: 7. In some KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 embodiments, the VHFR1 region comprises at least 80%, at least 85%, at least 90%, or at least 95% identity to the FR1 region as defined by Kabat of SEQ ID NO: 7. In some embodiments, the anti-TMEM30A antibody VHregion comprises a heavy chain FR2 sequence defined by Kabat as set forth in SEQ ID NO: 7. In some embodiments, the anti-TMEM30A antibody has 1, 2, 3, 4, or 5 amino acid substitutions relative to the FR2 sequence as defined by Kabat of the VH region of SEQ ID NO:7. In some embodiments, the VH FR2 region comprises least 80%, at least 85%, at least 90%, or at least 95% identity to the FR2 region as defined by Kabat of SEQ ID NO: 7. In some embodiments, the anti-TMEM30A antibody VH region comprises a heavy chain FR3 sequence defined by Kabat as set forth in SEQ ID NO: 7. In some embodiments, the anti-TMEM30A antibody has 1, 2, 3, 4, or 5 amino acid substitutions relative to the FR3 sequence as defined by Kabat of the VH region of SEQ ID NO: 7. In some embodiments, the VH FR3 region comprises least 80%, at least 85%, at least 90%, or at least 95% identity to the FR3 region as defined by Kabat of SEQ ID NO: 7. In some embodiments, the anti-TMEM30A antibody VHregion comprises a heavy chain FR4 sequence defined by Kabat as set forth in SEQ ID NO: 7. In some embodiments, the anti-TMEM30A antibody has 1, 2, or 3 amino acid substitutions relative to the FR4 sequence as defined by Kabat of the VHregion of SEQ ID NO: 7. In some embodiments, the VHFR4 region comprises least 80%, at least 85%, at least 90%, or at least 95% identity to the FR4 region as defined by Kabat of SEQ ID NO: 7.

[0099] In some embodiments, the anti-TMEM30A antibody binding domain comprises a light chain variable region having at least 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to the VLamino acid sequence of a variable region sequence of SEQ ID NO: 8. In some embodiments, the anti- TMEM30A antibody variable domain comprises substitutions, insertions, or deletions in the framework of a variable region as shown in SEQ ID NO: 8.

[0100] In some embodiments, the anti-TMEM30A antibody VLregion comprises a light chain framework 1 (FR-L1) sequence defined by Kabat as set forth in SEQ ID NO: 8. In some embodiments, the anti-TMEM30A antibody has 1, 2, 3, 4, or 5 amino acid substitutions relative to the FR1 sequence as defined by Kabat of the VLregion of SEQ ID NO:8. In some embodiments, the VL FR1 region comprises least 80%, at least 85%, at least 90%, or at least 95% identity to the FR1 region as defined by Kabat of SEQ ID NO: 8. In some embodiments, the anti-TMEM30A antibody VH region comprises a light chain FR2 sequence defined by KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 Kabat as set forth in SEQ ID NO:8. In some embodiments, the anti-TMEM30A antibody has 1, 2, 3, 4, or 5 amino acid substitutions relative to the FR2 sequence as defined by Kabat of the VLregion of SEQ ID NO: 8. In some embodiments, the VLFR2 region comprises least 80%, at least 85%, at least 90%, or at least 95% identity to the FR2 region as defined by Kabat of SEQ ID NO: 8. In some embodiments, the anti-TMEM30A antibody VLregion comprises a light chain FR3 sequence defined by Kabat as set forth in SEQ ID NO: 8. In some embodiments, the anti-TMEM30A antibody has 1, 2, 3, 4, or 5 amino acid substitutions relative to the FR3 sequence as defined by Kabat of the VL region of SEQ ID NO: 8. In some embodiments, the VL FR3 region comprises least 80%, at least 85%, at least 90%, or at least 95% identity to the FR3 region as defined by Kabat of SEQ ID NO: 8. In some embodiments, the anti-TMEM30A antibody VL region comprises a light chain FR4 sequence defined by Kabat as set forth in SEQ ID NO: 8. In some embodiments, the anti-TMEM30A antibody has 1, 2, or 3 amino acid substitutions relative to the FR4 sequence as defined by Kabat of the VL region of SEQ ID NO: 8. In some embodiments, the VLFR4 region comprises least 80%, at least 85%, at least 90%, or at least 95% identity to the FR4 region as defined by Kabat of SEQ ID NO:8.

[0101] In some embodiments, the anti-TMEM30A antibody binding domain comprises a VH comprising SEQ ID NO: 7 and a VL comprising SEQ ID NO: 8. Clone 7G8G1

[0102] In some embodiments, the anti-TMEM30A antibody binding domain comprises at least one, at least two, or at least three heavy chain CDRs of a variable domain sequence of SEQ ID NO: 15. In some embodiments, the anti-TMEM30A antibody binding domain comprises at least one, at least two, or at least three light chain CDRs of a variable domain sequence of SEQ ID NO: 16. In some embodiments, the CDRs are as defined by Kabat. In some embodiments, the CDRs are as defined by Chothia. In some embodiments, CDRs are as defined by AbM. In some embodiments, CDRs are as defined by IMGT. In some embodiments, the antibody comprises an HCDR1, HCDR2, and HCDR3 of the VHregion of SEQ ID NO: 15 and an LCDR1, LCDR2, and LCDR3 of the VLregion of SED ID NO: 16 as defined by Chothia. In some embodiments, the antibody comprises an HCDR1, HCDR2, and HCDR3 of the VHregion of SEQ ID NO: 15 and an LCDR1, LCDR2, and LCDR3 of the VLregion of SED ID NO: 16 as defined by AbM. In some embodiments, the antibody comprises an HCDR1, KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 HCDR2, and HCDR3 of the VHregion of SEQ ID NO: 15 and an LCDR1, LCDR2, and LCDR3 of the VLregion of SED ID NO: 16 as defined by IMGT.

[0103] In some embodiments, an anti-TMEM30A antibody binding domain of the present disclosure comprises a heavy chain CDR1 (HCDR1) sequence comprising SEQ ID NO: 9. In some embodiments, the anti-TMEM30A antibody binding domain comprises an HCDR2 sequence comprising SEQ ID NO: 10. In some embodiments, the anti-TMEM30A antibody binding domain comprises an HCDR3 sequence comprising SEQ ID NO: 11. In some embodiments, the anti-TMEM30A antibody binding domain comprises at least two heavy chain CDR sequences chosen from SEQ ID NOS: 9, 10, and 11. In typical embodiments, the anti-TMEM30A antibody binding domain comprises an HCDR1 sequence comprising SEQ ID NO: 9, an HCDR2 sequence comprising SEQ ID NO: 10, and an HCDR3 sequence comprising SEQ ID NO: 11.

[0104] In some embodiments, an anti-TMEM30A antibody binding domain comprises a light chain CDR1 (LCDR1) sequence comprising SEQ ID NO: 12. In some embodiments, the anti-TMEM30A antibody binding domain comprises an LCDR2 sequence comprising SEQ ID NO: 13. In some embodiments, the anti-TMEM30A antibody binding domain comprises an LCDR3 sequence comprising SEQ ID NO: 14. In some embodiments, the anti-TMEM30A antibody binding domain comprises at least two at least two light chain CDR sequences chosen from SEQ ID NOS: 12, 13, and 14. In typical embodiments, the anti-TMEM30A antibody binding domain comprises an LCDR1 sequence comprising SEQ ID NO: 12, an LCDR2 sequence comprising SEQ ID NO: 13 and an LCDR3 sequence comprising SEQ ID NO: 14.

[0105] In some embodiments, the anti-TMEM30A antibody binding domain comprises a HCDR1, HCDR2, and HCDR3, in which one of the HCDRs comprises a substitution relative to the corresponding HCDR as set forth in SEQ ID NO: 9, 10, or 11. In some embodiments, the anti-TMEM30A antibody binding domain comprises an HCDR1, HCDR2, and HCDR3, in which two of the HCDRs each comprise a substitution relative to the corresponding HCDR as set forth in SEQ ID NO: 9, 10, or 11.

[0106] In some embodiments, the anti-TMEM30A antibody binding domain comprises a LCDR1, LCDR2, and LCDR3, in which one of the LCDRs comprises a substitution relative to the corresponding LCDR as set forth in SEQ ID NO: 12, 13, or 14. In some embodiments, the anti-TMEM30A antibody binding domain comprises an LCDR1, LCDR2, and LCDR3, in KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 which two of the LCDRs each comprise a substitution relative to the corresponding LCDR as set forth in SEQ ID NO: 12, 13, or 14.

[0107] In some embodiments, the antibody comprises a VH region comprising an HCDR1 sequence comprising SEQ ID NO: 9, an HCDR2 sequence comprising SEQ ID NO: 10, and an HCDR3 sequence comprising SEQ ID NO: 11; and a VLregion comprising an LCDR1 sequence comprising SEQ ID NO: 12, an LCDR2 sequence comprising SEQ ID NO: 13, and an LCDR3 sequence comprising SEQ ID NO: 14.

[0108] In some embodiments, the anti-TMEM30A antibody binding domain comprises a heavy chain variable region having at least 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to the VHamino acid sequence of a variable region sequence of SEQ ID NO: 15. In some embodiments, the anti- TMEM30A antibody variable domain comprises substitutions, insertions, or deletions in the framework of a variable region as shown in SEQ ID NO: 15.

[0109] In some embodiments, the anti-TMEM30A antibody VHregion comprises a heavy chain framework 1 (FR-H1) sequence defined by Kabat as set forth in SEQ ID NO: 15. In some embodiments, the anti-TMEM30A antibody has 1, 2, 3, 4, or 5 amino acid substitutions relative to the FR1 sequence as defined by Kabat of the VHregion of SEQ ID NO: 15. In some embodiments, the VHFR1 region comprises at least 80%, at least 85%, at least 90%, or at least 95% identity to the FR1 region as defined by Kabat of SEQ ID NO: 15. In some embodiments, the anti-TMEM30A antibody VH region comprises a heavy chain FR2 sequence defined by Kabat as set forth in SEQ ID NO: 15. In some embodiments, the anti-TMEM30A antibody has 1, 2, 3, 4, or 5 amino acid substitutions relative to the FR2 sequence as defined by Kabat of the VH region of SEQ ID NO: 15. In some embodiments, the VH FR2 region comprises least 80%, at least 85%, at least 90%, or at least 95% identity to the FR2 region as defined by Kabat of SEQ ID NO: 15. In some embodiments, the anti-TMEM30A antibody VH region comprises a heavy chain FR3 sequence defined by Kabat as set forth in SEQ ID NO: 15. In some embodiments, the anti-TMEM30A antibody has 1, 2, 3, 4, or 5 amino acid substitutions relative to the FR3 sequence as defined by Kabat of the VHregion of SEQ ID NO: 15. In some embodiments, the VHFR3 region comprises least 80%, at least 85%, at least 90%, or at least 95% identity to the FR3 region as defined by Kabat of SEQ ID NO: 15. In some embodiments, the anti-TMEM30A antibody VHregion comprises a heavy chain FR4 sequence defined by 27 KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 Kabat as set forth in SEQ ID NO: 15. In some embodiments, the anti-TMEM30A antibody has 1, 2, or 3 amino acid substitutions relative to the FR4 sequence as defined by Kabat of the VHregion of SEQ ID NO: 15. In some embodiments, the VHFR4 region comprises least 80%, at least 85%, at least 90%, or at least 95% identity to the FR4 region as defined by Kabat of SEQ ID NO: 15.

[0110] In some embodiments, the anti-TMEM30A antibody binding domain comprises a light chain variable region having at least 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to the VLamino acid sequence of a variable region sequence of SEQ ID NO: 16. In some embodiments, the anti- TMEM30A antibody variable domain comprises substitutions, insertions, or deletions in the framework of a variable region as shown in SEQ ID NO: 16.

[0111] In some embodiments, the anti-TMEM30A antibody VLregion comprises a light chain framework 1 (FR-L1) sequence defined by Kabat as set forth in SEQ ID NO: 16. In some embodiments, the anti-TMEM30A antibody has 1, 2, 3, 4, or 5 amino acid substitutions relative to the FR1 sequence as defined by Kabat of the VLregion of SEQ ID NO: 16. In some embodiments, the VL FR1 region comprises least 80%, at least 85%, at least 90%, or at least 95% identity to the FR1 region as defined by Kabat of SEQ ID NO: 16. In some embodiments, the anti-TMEM30A antibody VH region comprises a light chain FR2 sequence defined by Kabat as set forth in SEQ ID NO: 16. In some embodiments, the anti-TMEM30A antibody has 1, 2, 3, 4, or 5 amino acid substitutions relative to the FR2 sequence as defined by Kabat of the VL region of SEQ ID NO: 16. In some embodiments, the VL FR2 region comprises least 80%, at least 85%, at least 90%, or at least 95% identity to the FR2 region as defined by Kabat of SEQ ID NO: 16. In some embodiments, the anti-TMEM30A antibody VLregion comprises a light chain FR3 sequence defined by Kabat as set forth in SEQ ID NO: 16. In some embodiments, the anti-TMEM30A antibody has 1, 2, 3, 4, or 5 amino acid substitutions relative to the FR3 sequence as defined by Kabat of the VLregion of SEQ ID NO: 16. In some embodiments, the VLFR3 region comprises least 80%, at least 85%, at least 90%, or at least 95% identity to the FR3 region as defined by Kabat of SEQ ID NO: 16. In some embodiments, the anti-TMEM30A antibody VLregion comprises a light chain FR4 sequence defined by Kabat as set forth in SEQ ID NO: 16. In some embodiments, the anti-TMEM30A antibody has 1, 2, or 3 amino acid substitutions relative to the FR4 sequence as defined by Kabat of the VL region of SEQ ID NO: 16. In some embodiments, the VL FR4 region comprises least 80%, at 28 KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 least 85%, at least 90%, or at least 95% identity to the FR4 region as defined by Kabat of SEQ ID NO: 16.

[0112] In some embodiments, the anti-TMEM30A antibody binding domain comprises a VH comprising SEQ ID NO: 15, and a VLcomprising SEQ ID NO: 16. Clone 6A7H6

[0113] In some embodiments, the anti-TMEM30A antibody binding domain comprises at least one, at least two, or at least three heavy chain CDRs of a variable domain sequence of SEQ ID NO: 23. In some embodiments, the anti-TMEM30A antibody binding domain comprises at least one, at least two, or at least three light chain CDRs of a variable domain sequence of SEQ ID NO: 24. In some embodiments, the CDRs are as defined by Kabat. In some embodiments, the CDRs are as defined by Chothia. In some embodiments, CDRs are as defined by AbM. In some embodiments, CDRs are as defined by IMGT. In some embodiments, the antibody comprises an HCDR1, HCDR2, and HCDR3 of the VHregion of SEQ ID NO: 23 and an LCDR1, LCDR2, and LCDR3 of the VLregion of SED ID NO: 24 as defined by Chothia. In some embodiments, the antibody comprises an HCDR1, HCDR2, and HCDR3 of the VHregion of SEQ ID NO: 23 and an LCDR1, LCDR2, and LCDR3 of the VLregion of SED ID NO: 24 as defined by AbM. In some embodiments, the antibody comprises an HCDR1, HCDR2, and HCDR3 of the VHregion of SEQ ID NO: 23 and an LCDR1, LCDR2, and LCDR3 of the VLregion of SED ID NO: 24 as defined by IMGT.

[0114] In some embodiments, an anti-TMEM30A antibody binding domain of the present disclosure comprises a heavy chain CDR1 (HCDR1) sequence comprising SEQ ID NO: 17. In some embodiments, the anti-TMEM30A antibody binding domain comprises an HCDR2 sequence comprising SEQ ID NO: 18. In some embodiments, the anti-TMEM30A antibody binding domain comprises an HCDR3 sequence comprising SEQ ID NO: 19. In some embodiments, the anti-TMEM30A antibody binding domain comprises at least two heavy chain CDR sequences chosen from SEQ ID NOS: 19, 18, and 19. In typical embodiments, the anti-TMEM30A antibody binding domain comprises an HCDR1 sequence comprising SEQ ID NO: 17, an HCDR2 sequence comprising SEQ ID NO: 18, and an HCDR3 sequence comprising SEQ ID NO: 19.

[0115] In some embodiments, an anti-TMEM30A antibody binding domain comprises a light chain CDR1 (LCDR1) sequence comprising SEQ ID NO: 20. In some embodiments, the KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 anti-TMEM30A antibody binding domain comprises an LCDR2 sequence comprising SEQ ID NO: 21. In some embodiments, the anti-TMEM30A antibody binding domain comprises an LCDR3 sequence comprising SEQ ID NO: 22. In some embodiments, the anti-TMEM30A antibody binding domain comprises at least two light chain CDR sequences chosen from SEQ ID NOS: 20, 21, and 22. In typical embodiments, the anti-TMEM30A antibody binding domain comprises an LCDR1 sequence comprising SEQ ID NO: 20, an LCDR2 sequence comprising SEQ ID NO: 21, and an LCDR3 sequence comprising SEQ ID NO: 22.

[0116] In some embodiments, the anti-TMEM30A antibody binding domain comprises a HCDR1, HCDR2, and HCDR3, in which one of the HCDRs comprises a substitution relative to the corresponding HCDR as set forth in SEQ ID NO: 17, 18, or 19. In some embodiments, the anti-TMEM30A antibody binding domain comprises an HCDR1, HCDR2, and HCDR3, in which two of the HCDRs each comprise a substitution relative to the corresponding HCDR as set forth in SEQ ID NO: 17, 18, or 19.

[0117] In some embodiments, the anti-TMEM30A antibody binding domain comprises a LCDR1, LCDR2, and LCDR3, in which one of the LCDRs comprises a substitution relative to the corresponding LCDR as set forth in SEQ ID NO: 20, 21, or 22. In some embodiments, the anti-TMEM30A antibody binding domain comprises an LCDR1, LCDR2, and LCDR3, in which two of the LCDRs each comprise a substitution relative to the corresponding LCDR as set forth in SEQ ID NO: 20, 21, or 22.

[0118] In some embodiments, the antibody comprises a VH region comprising an HCDR1 sequence SEQ ID NO: 17, an HCDR2 sequence SEQ ID NO: 18, and an HCDR3 sequence SEQ ID NO: 19; and a VL region comprising an LCDR1 sequence SEQ ID NO: 20, an LCDR2 sequence SEQ ID NO: 21, and an LCDR3 sequence SEQ ID NO: 22.

[0119] In some embodiments, the anti-TMEM30A antibody binding domain comprises a heavy chain variable region having at least 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to the VH amino acid sequence of a variable region sequence of SEQ ID NO: 23. In some embodiments, the anti- TMEM30A antibody variable domain comprises substitutions, insertions, or deletions in the framework of a variable region as shown in SEQ ID NO: 23.

[0120] In some embodiments, the anti-TMEM30A antibody VH region comprises a heavy chain framework 1 (FR-H1) sequence defined by Kabat as set forth in SEQ ID NO: 23. In some KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 embodiments, the anti-TMEM30A antibody has 1, 2, 3, 4, or 5 amino acid substitutions relative to the FR1 sequence as defined by Kabat of the VHregion of SEQ ID NO: 23. In some embodiments, the VHFR1 region comprises at least 80%, at least 85%, at least 90%, or at least 95% identity to the FR1 region as defined by Kabat of SEQ ID NO: 23. In some embodiments, the anti-TMEM30A antibody VHregion comprises a heavy chain FR2 sequence defined by Kabat as set forth in SEQ ID NO: 23. In some embodiments, the anti-TMEM30A antibody has 1, 2, 3, 4, or 5 amino acid substitutions relative to the FR2 sequence as defined by Kabat of the VH region of SEQ ID NO: 23. In some embodiments, the VH FR2 region comprises least 80%, at least 85%, at least 90%, or at least 95% identity to the FR2 region as defined by Kabat of SEQ ID NO: 23. In some embodiments, the anti-TMEM30A antibody VH region comprises a heavy chain FR3 sequence defined by Kabat as set forth in SEQ ID NO: 23. In some embodiments, the anti-TMEM30A antibody has 1, 2, 3, 4, or 5 amino acid substitutions relative to the FR3 sequence as defined by Kabat of the VH region of SEQ ID NO: 23. In some embodiments, the VHFR3 region comprises least 80%, at least 85%, at least 90%, or at least 95% identity to the FR3 region as defined by Kabat of SEQ ID NO: 23. In some embodiments, the anti-TMEM30A antibody VHregion comprises a heavy chain FR4 sequence defined by Kabat as set forth in SEQ ID NO: 23. In some embodiments, the anti-TMEM30A antibody has 1, 2, or 3 amino acid substitutions relative to the FR4 sequence as defined by Kabat of the VHregion of SEQ ID NO: 23. In some embodiments, the VHFR4 region comprises least 80%, at least 85%, at least 90%, or at least 95% identity to the FR4 region as defined by Kabat of SEQ ID NO: 23.

[0121] In some embodiments, the anti-TMEM30A antibody binding domain comprises a light chain variable region having at least 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to the VLamino acid sequence of a variable region sequence of SEQ ID NO: 24. In some embodiments, the anti- TMEM30A antibody variable domain comprises substitutions, insertions, or deletions in the framework of a variable region as shown in SEQ ID NO: 24.

[0122] In some embodiments, the anti-TMEM30A antibody VLregion comprises a light chain framework 1 (FR-L1) sequence defined by Kabat as set forth in SEQ ID NO: 24. In some embodiments, the anti-TMEM30A antibody has 1, 2, 3, 4, or 5 amino acid substitutions relative to the FR1 sequence as defined by Kabat of the VL region of SEQ ID NO: 24. In some embodiments, the VL FR1 region comprises least 80%, at least 85%, at least 90%, or at least KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 95% identity to the FR1 region as defined by Kabat of SEQ ID NO: 24. In some embodiments, the anti-TMEM30A antibody VHregion comprises a light chain FR2 sequence defined by Kabat as set forth in SEQ ID NO: 24. In some embodiments, the anti-TMEM30A antibody has 1, 2, 3, 4, or 5 amino acid substitutions relative to the FR2 sequence as defined by Kabat of the VLregion of SEQ ID NO: 24. In some embodiments, the VLFR2 region comprises least 80%, at least 85%, at least 90%, or at least 95% identity to the FR2 region as defined by Kabat of SEQ ID NO: 24. In some embodiments, the anti-TMEM30A antibody VL region comprises a light chain FR3 sequence defined by Kabat as set forth in SEQ ID NO: 24. In some embodiments, the anti-TMEM30A antibody has 1, 2, 3, 4, or 5 amino acid substitutions relative to the FR3 sequence as defined by Kabat of the VL region of SEQ ID NO: 24. In some embodiments, the VL FR3 region comprises least 80%, at least 85%, at least 90%, or at least 95% identity to the FR3 region as defined by Kabat of SEQ ID NO: 24. In some embodiments, the anti-TMEM30A antibody VL region comprises a light chain FR4 sequence defined by Kabat as set forth in SEQ ID NO: 24. In some embodiments, the anti-TMEM30A antibody has 1, 2, or 3 amino acid substitutions relative to the FR4 sequence as defined by Kabat of the VLregion of SEQ ID NO: 24. In some embodiments, the VLFR4 region comprises least 80%, at least 85%, at least 90%, or at least 95% identity to the FR4 region as defined by Kabat of SEQ ID NO: 24.

[0123] In some embodiments, the anti-TMEM30A antibody binding domain comprises a VHcomprising SEQ ID NO: 23, and a VLcomprising SEQ ID NO: 24.

[0124] In some embodiments, the anti-TMEM30A antibody of the present disclosure, e.g., as described in the preceding paragraphs in this section, binds to a region of the extracellular domain of human TMEM30A comprising SEQ ID NO: 25 with a KDof less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 500 pM, less than about 200 pM, less than about 100 pM, less than about 75 pM, or less than about 50 pM, e.g., as measured by bio-layer interferometry. In typical embodiments, KDis measured by surface plasmon resonance at 37ºC under standard conditions. a. Bispecific Antibodies

[0125] In some embodiments, the antibody is a bispecific antibody, i.e., an antibody that can bind to epitopes of two different target antigens, or to two different epitopes for the same target KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 antigen. In some embodiments, the bispecific antibody binds to TMEM30A and a second antigen. In some embodiments, the second antigen is CD3, CD19, CD20, Her2 / neu, epidermal growth factor receptor (EGFR), PD-L1, CD206, vascular endothelial growth factor receptor (VEGFR), integrin V 3, mesothelin, GD2, or ALPPL2. In some embodiments, the bispecific antibody binds to the second antigen via an antibody fragment, a natural ligand of the second antigen, or a variant thereof. In some embodiments, the antibody is a bispecific T cell engager (BiTE). b. Humanized Antibodies

[0126] In some embodiments, the antibody is a humanized antibody, i.e., an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts. See, e.g., Morrison et al., PNAS USA, 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988); Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun., 31(3):169-217 (1994). Techniques for humanizing antibodies are well known in the art and are described in e.g., U.S. Patent Nos. 4,816,567; 5,530,101; 5,859,205; 5,585,089; 5,693,761; 5,693,762; 5,777,085; 6,180,370; 6,210,671; and 6,329,511; WO 87 / 02671; EP Patent Application 0173494; Jones et al. (1986) Nature 321:522; and Verhoyen et al. (1988) Science 239:1534. Humanized antibodies are further described in, e.g., Winter and Milstein (1991) Nature 349:293. For example, polynucleotides comprising a first sequence coding for humanized immunoglobulin framework regions and a second sequence set coding for the desired immunoglobulin complementarity determining regions can be produced synthetically or by combining appropriate cDNA and genomic DNA segments. Human constant region DNA sequences can be isolated in accordance with well-known procedures from a variety of human cells.

[0127] In some cases, transfer of a CDR to a human framework leads to a loss of specificity for the humanized antibody. In these cases, back mutations can be introduced into the framework regions of the human portion of the antibody. Methods of making back mutations are described in, e.g., Co et al., PNAS USA 88;2269-2273 (1991) and WO 90 / 07861. KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 c. Antibody Formats

[0128] As discussed above, an anti-TMEM30A antibody of the present disclosure is incorporated into a bivalent antibody or a multivalent antibody that binds to the same, or a different, antigen. In some embodiments, an anti-TMEM30A antibody is incorporated into a bispecific antibody or multispecific antibody that binds to the antigen at different epitopes, or that binds to different antigens. In some embodiments, such an antibody can comprise an Fc region. In some embodiments, an anti-TMEM30A antibody is present as an antigen binding domain of a larger molecule, e.g., present as an antigen binding domain of a chimeric antigen receptor, such as a chimeric T-cell receptor-like polypeptide, or synthetic Notch receptor.

[0129] Other antigen-binding fragments or antibody proteins include bivalent scFv (diabody), bispecific scFv antibodies where the antibody molecule recognizes two different epitopes, single binding domains (dAbs), and minibodies.

[0130] In some embodiments, an antibody of the present disclosure comprises at least one constant region domain. In general, the Fc region of an antibody refers to a polypeptide comprising the CH3, CH2, and at least a portion of the hinge region of a constant domain of an antibody. In some embodiments, an Fc region can include a CH4 domain that can be found in certain antibody classes. In some embodiments, an Fc region can comprise the entire hinge region of a constant domain of an antibody. In some embodiments, an antibody comprises an Fc region and a CH1 region. In some embodiments, the antibody comprises an Fc region, a CH1 region and a Ckappa / lambda region.

[0131] In some embodiments, an anti-TMEM30A antibody of the present disclosure lacks a CH1, CH2, CH3, and / or CH4 antibody region. In some embodiments, an anti-TMEM30A antibody lacks an Fc region and / or lacks Fc-mediated effector functions, e.g., antibody- dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement fixation, and / or FcRn-mediated recycling. In some instances, an anti- TMEM30A antibody without an Fc region has a shorter half-life and / or has a lower propensity to aggregate compared to an anti-TMEM30A antibody with an Fc region. Chimeric receptors constructs comprising an anti-TMEM30A antibody

[0132] In a further aspect, the disclosure provides genetically modified immune cells, such as T cells, or in some instances natural killer (NK) cells, that express a chimeric receptor comprising an anti-TMEM30A antibody of the present disclosure. KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2

[0133] Chimeric antigen receptors (CARs) are recombinant receptor constructs comprising an extracellular antigen-binding domain joined to a transmembrane domain, and further linked to an intracellular signaling domain (e.g., an intracellular T cell signaling domain of a T cell receptor) that transduces a signal to elicit a function. In certain embodiments, immune cells (e.g., T cells or NK cells are genetically modified to express CARs that comprise a antigen binding domain of a TMEM30A antibody of the present disclosure and have the functionality of effector cells (e.g., cytotoxic and / or memory functions of T cells or NK cells).

[0134] In a standard CAR, the components include an extracellular targeting domain, a transmembrane domain and intracellular signaling / activation domain, which are typically linearly constructed as a single fusion protein. In the present invention, the extracellular region comprises an anti-TMEM30A antibody as described herein. The intracellular region may contain a signaling domain of a TCR complex, and / or one or more costimulatory signaling domains, such as those from CD28, 4-1BB (CD137) and OX-40 (CD134). For example, a "first-generation CAR" generally has a CD3-zeta signaling domain. Additional costimulatory intracellular domains may also be introduced (e.g., second and third generation CARS) and further domains including homing and suicide domains may additionally be included in CAR constructs. CAR components are further described below. Extracellular domain

[0135] A chimeric antigen receptor of the present disclosure comprises an extracellular antigen-binding domain that comprises an anti-TMEM30A antibody antigen binding domain having CDRs of an antibody as described herein. A CAR construct may also comprise a sequence that encodes a signal peptide to target the extracellular domain to the cell surface. Hinge domain

[0136] In some embodiments, the CAR may include one or more hinge domains that link the antigen binding domain comprising an antigen binding domain of a TMEM30A antibody of the present invention and the transmembrane domain for positioning the antigen binding domain. Such a hinge domain may be derived either from a natural, synthetic, semi- synthetic, or recombinant source. The hinge domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region, e.g., a naturally occurring human immunglobulin hinge region, or an altered immunoglobulin hinge region. Illustrative hinge KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 domains suitable for use in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8 alpha, CD4, CD28, PD1, CD152, and CD7, which may be wild-type hinge regions from these molecules or may be altered. Transmembrane domain

[0137] Any transmembrane suitable for use in a CAR construct may be employed. Such transmembrane domains, include, but are not limited to, all or part of the transmembrane domain of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD 11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGAl, VLAl, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB 1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100, (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME, (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKG2D, or NKG2C.

[0138] A transmembrane domain incorporated into a CAR construct may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. Intracellullar signaling domain

[0139] A CAR construct of the present disclosure includes one or more intracellular signaling domains, also referred to herein as co-stimulatory domains, or cytoplasmic domains that activate or otherwise modulate an immune cell, (e.g., a T lymphocyte, NK cell, iNKT cell, macrophage, gamma delta T cell). The intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been introduced. In one embodiment, a co-stimulatory domain is used that increases CAR immune T cell cytokine production. In another embodiment, a co- stimulatory domain is used that facilitates immune cell (e.g., T cell) replication. In still another embodiment, a co-stimulatory domain is used that prevents CAR immune cell (e.g., T KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 cell) exhaustion. In another embodiment, a co-stimulatory domain is used that increases immune cell (e.g., T cell) antitumor activity. In still a further embodiment, a co-stimulatory domain is used that enhances survival of CAR immune cells (e.g., T cells) (e.g., post-infusion into patients).

[0140] Examples of intracellular signaling domains for use in a CAR include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.

[0141] A primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs.

[0142] Examples of IT AM containing primary intracellular signaling domains include those of CD3 zeta, common FcR gamma, Fc gamma Rlla, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In one embodiment, a CAR comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3- zeta.

[0143] An intracellular signaling domain of a CAR can comprise a primary intracellular signaling domain only, or may comprise additional desired intracellular signaling domain(s) useful in the context of a CAR of the invention. For example, the intracellular signaling domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling domain. The costimulatory signaling domain refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that binds to CD83, and the like. For example, CD27 costimulation has been demonstrated to enhance expansion, effector function, and survival of human CART cells in vitro and augments human T cell persistence and antitumor activity in vivo (Song et al. Blood.2012; 119(3):696-706). Further examples KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB 1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAMl, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM, (SLAMFl, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, and CD 19a.

[0144] In some embodiments, a CAR may be designed to exhibit conditional expression, e.g., designed as an inducible CAR, or may otherwise comprise a mechanism for reversibly expressing the CAR, or controlling CAR activity to largely restrict it to a desired environment. In some embodiments, the CAR may comprise a safety switch gene. In some embodiments, the CAR may be an on-switch CAR or an off-switch CAR (see, e.g., Jan et al, Sci Transl Med. Jan 6:13(575):eabb6295, 2021).

[0145] In some embodiments, the CAR-expressing cell uses a split CAR. The split CAR approach is described in more detail in publications WO2014 / 055442 and WO2014 / 055657. Briefly, a split CAR system comprises a cell expressing a first CAR having a first antigen binding domain and a costimulatory domain (e.g., 41BB), and the cell also expresses a second CAR having a second antigen binding domain and an intracellular signaling domain (e.g., CD3 zeta). When the cell encounters the first antigen, the costimulatory domain is activated, and the cell proliferates. When the cell encounters the second antigen, the intracellular signaling domain is activated and cell-killing activity begins. Thus, the CAR- expressing cell is only fully activated in the presence of both antigens.

[0146] In some embodiments, a host cell, e.g., a T cell, can be engineered such that a synthetic Notch receptor comprising an extracellular domain that targets one antigen induces the expression of a CAR that targets a second antigen. Such systems are described, e.g., in U.S.. Patent Application Publication No.20190134093; see also, synNotch polypeptides as described in US20160264665, each incorporated herein by reference. In some embodiments, a synNotch comprises a binding domain of an anti-TMEM30A antibody of the present KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 disclosure. In some embodiments, the binding domains is incorporated into a CAR, the expression of which is activated by a synNotch expressed by the host cell.

[0147] In some embodiments, a cell expressing a CAR comprising a TMEM30A binding expresses a second CAR, e.g., a second CAR that includes a different antigen binding domain, e.g., that binds to the same target or a different target.

[0148] In some embodiments, a cell expressing a TMEM30A binding domain of an antibody of the present disclosure comprises an alternative chimeric antigen receptor, such as an HLA-Independent TCR-based Chimeric Antigen Receptor (also known as “HIT-CAR”, e.g., those disclosed in International Patent Application No. PCT / US19 / 017525), T cell receptor fusion constructs (TRuCs) (e.g., those disclosed in Baeuerle et al., “Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response,” Nature Comm.10: 2087 (2019), synthetic T cell receptor and antigen receptors (STARs) (e.g., those disclosed in Liu et al. Science Translational Medicine 13(586):eabb5191, 2021), antibody-T- cell receptor (AbTCR) (e.g., those disclosed in Xu et al. Cell Discovery (2018) 4:62), and T cell antigen coupler (TAC) (e.g., those disclosed in Helsen et al. Nature Communications (2018);9:3049). d. Antibody Conjugates

[0149] An anti-TMEM30A antibody of the present disclosure can also be conjugated or linked, either directly or indirectly, to therapeutic and / or imaging / detectable moieties. For example, in some embodiments, an anti-TMEM30A antibody is conjugated to an agent including, but not limited to, a detectable label or marker, a cytotoxic agent, an imaging agent, a therapeutic agent, or an oligonucleotide. Methods for conjugating or linking an antibody to a desired molecule moiety are well known in the art. The moiety may be linked to the antibody covalently or by non-covalent linkages.

[0150] In some embodiments, an anti-TMEM30A antibody is conjugated to a cytotoxic moiety or other moiety that inhibits cell proliferation. In some embodiments, the anti- TMEM30A antibody is conjugated to a cytotoxic agent including, but not limited to, e.g., deruxtecan, ricin A chain, doxorubicin, daunorubicin, a maytansinoid, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, methotrexact, actinomycin, a diphtheria toxin, extotoxin A from Pseudomonas, Pseudomonas exotoxin40, abrin, abrin A chain, modeccin A chain, alpha sarcin, gelonin, KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 mitogellin, restrictocin, cobran venom factor, a ribonuclease, engineered Shiga toxin, phenomycin, enomycin, curicin, crotin, calicheamicin, Saponaria officinalis inhibitor, glucocorticoid, auristatin, auromycin, yttrium, bismuth, combrestatin, duocarmycins, dolastatin, cc1065, or a cisplatin. In some embodiments, the anti-TMEM30A antibody may be linked to an agent such as an enzyme inhibitor, a proliferation inhibitor, a lytic agent, a DNA or RNA synthesis inhibitors, a membrane permeability modifier, a DNA metabolite, a dichloroethylsulfide derivative, a protein production inhibitor, a ribosome inhibitor, or an inducer of apoptosis.

[0151] In some embodiments, an anti-TMEM30A antibody is linked to a radionuclide, an iron-related compound, a dye, a fluorescent agent, or an imaging agent. In some embodiments, an anti-TMEM30A antibody may be linked to agents, such as, but not limited to, metals; metal chelators; lanthanides; lanthanide chelators; radiometals; radiometal chelators; positron- emitting nuclei; microbubbles (for ultrasound); liposomes; molecules microencapsulated in liposomes or nanosphere; monocrystalline iron oxide nanocompounds; magnetic resonance imaging contrast agents; light absorbing, reflecting and / or scattering agents; colloidal particles; fluorophores, such as near-infrared fluorophores. IV. Polynucleotides and Cells for Antibody Production

[0152] Recombinant polynucleotides encoding any of the anti-TMEM30A antibodies disclosed herein are provided. In some embodiments, the polynucleotide comprises a sequence that encodes for an anti-TMEM30A antibody comprising any combination of CDRs disclosed herein. In some embodiments, the polynucleotide comprises a sequence that encodes for an anti-TMEM30A antibody comprising any combination of VH and VL disclosed herein.

[0153] DNA constructs or expression vectors encoding an anti-TMEM30A antibody of the present disclosure and comprising a promoter that drives expression of the antibody may be introduced into cells such that the cells express the anti-TMEM30A antibody. In many embodiments, the nucleic acid sequence encoding an anti-TMEM30A antibody is operably linked to a heterologous promoter. Preferred promoters for controlling transcription from vectors in host cells are known to one of ordinary skill in the art. The polynucleotides can be prepared according to standard methods known to those of skill in the art. Also, using the sequence information provided herein, the anti-TMEM30A antibody can be chemically synthesized using well known methods of peptide synthesis. KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2

[0154] Molecular cloning techniques to achieve these ends are known in the art. A wide variety of cloning and in vitro amplification methods are suitable for the construction of recombinant nucleic acids. Examples of these techniques and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al. (1989) Molecular Cloning – A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook); and Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel). Methods of producing recombinant immunoglobulins are also known in the art. See, Cabilly, U.S. Patent No. 4,816,567; and Queen et al. (1989) Proc. Natl Acad. Sci. USA 86: 10029-10033.

[0155] Many methods for introducing nucleic acids and viral vectors (e.g., viral particles) into a target cell are known to one of ordinary skill in the art. Non-limiting examples of suitable methods include electroporation (e.g., nucleofection), viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, microparticle- or nanoparticle-mediated nucleic acid delivery, and the like. In some embodiments, a viral vector may be used, such as an adenovirus, adeno-associated virus (AAV), lentivirus vector, a vaccinia virus vector, or any of a number of different vectors.

[0156] Production of an anti-TMEM30A antibody comprises culturing a host cell that comprises the nucleic acid encoding the antibody with culture conditions that are suitable for expressing the antibody in the host cell. Optionally, the antibody may be isolated and / or purified from the host cell or the host cell culture. An anti-TMEM30A antibody can be produced by a variety of recombinant DNA techniques, including by expression in transfected cells (e.g., E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO, and HeLa cells lines, and immortalized eukaryotic cells, such as myeloma or hybridoma cells). Suitable source cells for the DNA sequences and host cells for immunoglobulin expression and secretion can be obtained from a number of sources, such as the American Type Culture Collection (Catalogue of Cell Lines and Hybridomas, Fifth edition (1985) Rockville, Md). KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2

[0157] The various anti-TMEM30A antibodies or antigen-binding fragments described herein can be produced by enzymatic or chemical modification of the intact antibodies, or synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv), or identified using yeast or phage display libraries (see, e.g., McCafferty et al., Nature 348:552- 554, 1990; Boder, et al (2000) Proc. Natl. Acad. Sci. U. S. A. 97:10701). For example, minibodies can be generated using methods described in the art, e.g., Vaughan and Sollazzo, Comb Chem High Throughput Screen. 4:417-302001. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol.79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992). Single chain antibodies can be identified using phage display libraries, yeast display, or ribosome display libraries, gene shuffled libraries. Such libraries can be constructed from synthetic, semi-synthetic or native and immunocompetent sources. V. Methods of Use a. Therapeutic Methods

[0158] Provided herein are methods for the treatment of certain disease with an anti- TMEM30A antibody of the present disclosure. In some embodiments, the anti-TMEM30A antibody modulates TMEM30A activity, e.g., phospholipid flippase function, in cells implicated in diseases. In some embodiments, the disease is a cancer, an inflammatory disorder, or an autoimmune disorder.

[0159] In some embodiments, the anti-TMEM30A antibody is administered to a patient such that the antibody comes into contact with a cancer cell or a tumor cell. Without being bound by theory, in some embodiments, treatment with an anti-TMEM30A antibody reduces angiogenesis and / or cell proliferation, In some embodiments, treatment with an anti- TMEM30A antibody results in slower tumor growth. In some embodiments, treatment with an anti-TMEM30A antibody results in cancer cell death. In some embodiments, treatment with an anti-TMEM30A antibody results in phagocytosis of cancer cells. In some embodiments, treatment with an anti-TMEM30A antibody promotes antibody-dependent cellular phagocytosis of tumor cells by M1-polarized macrophages. In some embodiments, treatment with an anti-TMEM30A antibody kills tumor cells. In some embodiments, treatment with an anti-TMEM30A antibody prevents tumor migration, tumor-associated angiogenesis or metastasis in a patient with cancer. KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2

[0160] In some embodiments, treatment with an anti-TMEM30A antibody in combination with a protein kinase inhibitor is used to additively or synergistically kill tumor cells in a patient. Protein kinase used in combination with an anti-TMEM30A antibody include, for example, without limitations, inhibitors of serine / threonine protein kinases, protein kinase C inhibitors, tyrosine kinase inhibitors, and mTOR inhibitors (e.g., AZD2014). In some embodiments, the protein kinase inhibitor is a broad spectrum protein kinase inhibitor such as staurosporine, or the multi-targeted staurosporine-derived protein kinase inhibitor midostaurin. In some embodiments, the protein kinase inhibitor is an alternative inhibitor that exhibits activity against protein kinase C and other serine / threonine kinases and tyrosine kinases. These inhibitors include hydroxystaurosporine (UCN-010), lestaurtinib, enzastaurin, ruboxistaurin, sotrastaurin, balanol, and chelerythrine.

[0161] Additional protein kinase inhibitors can also be used. In some embodiments, the protein kinase inhibitor is a multi-kinase inhibitor that has activity against epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGF) and other tyrosine kinases. Such an inhibitor includes, but is not limited to, gefitinib, erlotinib, afatinib, osimertinib, lapatinib, neratinib, sorafenib, sunitinib, tivozanib, pazopinib, axitinib, cabozantinib, vandatenib, regorafenib, or lenvatini. In some embodiments, the protein kinase inhibitor is a Bruton Tyrosine Kinase (BTK) inhibitor, for example, ibrutinib, acalibrutinib, zanubrutinib, tirabrutinib, orelabrutinib, pirtobrutinib, spebrutinib, tolebrutinib, evobrutinib, elsubrutinib, nemtabrutinib, fenebrutinib, vecabrutinib, rilzabrutinib, remibrutinib, rocbrutinib, docirbrutinib (AS-1763), TT-01488, or a BTK degrader such as NX-2127, NX- 5948, and BGB16673; DFCI-002-05 (HCK and BTK degrader), or DFCI-002-06 (HCK and BTK degrader). In some embodiments, the protein kinase inhibitor is an SYK inhibitor, such as fosamatinib, endospletinib, cerdulatinib, TAK-659, or lanrapleni. In other embodiments, the inhibitor is a Flt-3 (+ / - IRAK4) inhibitor such as gilteritinib (Flt-3), quizartinib, crenolanib, emavusertib (CA-4948) (Flt-3 and IRAK4 and CLK), lomonitinib (ZE46-0134) (Flt-3 and IRAK4), tuspetinib, or fostamatib. Additional inhibitors that can be administered with a TMEM30A antibody of the present disclosure include inhibitors of MET and / or ALK and / or ROS: crizotinib, ceritinib, alectinib brigatinib, lorlatinib, unecritinib (TQ-B3101), or iruplinalkib (WX-0593); inhibitors of JAK, such as ruxolitinib, tofacitinib, oclacitinib, baricitinib, peficitinib, upadacitinib, fedratinib, delgocitinib, filgotinib, abrocitinib, pacritinib, deucravacitinib, ritlecitinib, momelotinib, golidocitinib, deuruxolitinib, OB756, CHZ868, KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 rovadicitinib, or flonoltinib maleate; inhibitors of RAF and / or MEK, such as vemurafenib, dabrafenib, trametinib, cobimetinib, binimetinib, selumetinib, belvarafenib, PLX4032, PLX4720, ulixertinib, taselisib, or pimasertib; inhibitors of PI3 kinase such as idelalisib, duvelisib, alpelisib, umbralisib, leniolisib, buparlisib, dactolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, fimepinostat, gedatolisib, linperlisib nemiralisib, pictilisib, pilaralisib, samotolisib, seletalisib, serabelisib, sonolisib, tenalisib, voxtalisib, AZD8186, GSK2636771, SF1126, Acalisib, Omipalisib, AZD8835, CAL263, GSK1059615, mEN1611, PWT33597, TG100-115, or ZSTK474; inhibitors of cyclin dependent kinases, such as ribociclib, abemaciclib, or flavopiridol; inhibitors of mTOR kinases, such as AZD2014 (vistusertib), AZD8055, sapanisertib, rapamycin, temsirolimus, sirolimus, everolimus, deforolimus, dactolisib, voxtalisib, BGT226, SF1126, PKI-587, or NVPBE235; inhibitors of PIM kinases such as nuvisertib TP-3654 (PIM1), SEL24, AZD1208, or PIM447; inhibitors of FAK kinases, such as APG2449, IN10018, defactinib, GSK2256098, or conteltinib; CDK9 inhibitors such as PRT2527, QHRD107, or alvocidib; AKT inhibitors such as AKT044, capivasertib, or ipatasertib; an aurora kinase inhibitor alisertib (MLN8237); a casein kinase I inhibitor / degrader BMS-986397 or INNO-220; an RIP kinase inhibitor GSK2982772, or an IRAK1 / 4 degrader: Jh-XIII-05-1 or KT-474.

[0162] Cancer”, “tumor,” “transformed” and like terms include precancerous, neoplastic, transformed, and cancerous cells, and can refer to a solid tumor, or a non-solid cancer. Cancer includes both benign and malignant neoplasms (abnormal growth). The term “cancer” can thus refer to carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, solid and lymphoid cancers, etc. Examples of different types of cancer include, but are not limited to, lung cancer (e.g., non-small cell lung cancer or NSCLC), ovarian cancer, prostate cancer, colorectal cancer, urothelial cancer, liver cancer (i.e., hepatocellular carcinoma), renal cancer (i.e., renal cell carcinoma), bladder cancer, breast cancer, thyroid cancer, pleural cancer, pancreatic cancer, uterine cancer, endometrial cancer, cervical cancer, testicular / testis cancer, anal cancer, pancreatic cancer, bile duct cancer, gastrointestinal carcinoid tumors, esophageal cancer, gall bladder cancer, appendix cancer, small intestine cancer, stomach (gastric) cancer, cancer of the central nervous system, skin cancer, choriocarcinoma; head and neck cancer, blood cancer, osteogenic sarcoma, fibrosarcoma, neuroblastoma, glioma, melanoma, B-cell lymphoma, non-Hodgkin's lymphoma, Burkitt’s lymphoma, Small Cell lymphoma, Large Cell lymphoma, central nervous system (CNS) lymphoma, monocytic leukemia, KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 myelogenous leukemia, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myeloid leukemia (CML), and multiple myeloma. In some embodiments, the antibody compositions and methods described herein can be used for treating cancer. In some embodiments, the cancer is a refractory cancer, i.e., a cancer that did not respond or was resistant to previous or current treatment. Any of the exemplary cancers disclosed herein may be a refractory cancer.

[0163] In some embodiments, the cancer is a liver cancer; e.g., hepatocellular carcinoma (HCC). In some embodiments, the cancer is a lymphoma, e.g., non-Hodgkin lymphoma or central nervous system (CNS) lymphoma. In some embodiments, the cancer is multiple myeloma (MM). In some embodiments, the cancer is a leukemia, e.g., acute lymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML). In some embodiments, the cancer is a lung cancer, e.g., non-small lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma, and lung small cell carcinoma (or small cell lung cancer; SCLC).

[0164] In some embodiments, the anti-TMEM30A antibody is used to treat inflammation related to inflammatory disorders or autoimmune disorders. In some embodiments, the anti- TMEM30A antibody is administered to a patient such that the antibody comes into contact with immune cells that expresses TMEM30A. In some embodiments, the anti-TMEM30A antibody reduces the inflammation caused by immune cells that express TMEM30A. In some embodiments, the anti-TMEM30A attenuates Toll-like receptor 4 (TLR4) signaling. In some embodiments, the anti-TMEM30A antibody kills or reduces the number of immune cells that express TMEM30A. In some embodiments, the immune cells are macrophages and monocytes.

[0165] In general, inflammatory disorders (or inflammatory diseases) are conditions where a patient experiences chronic inflammation that does not resolve and the body does not return to healthy homeostasis. Chronic inflammation can persist for months or years and contribute to further tissue injury and disease. Examples of inflammatory disorder include type 1 diabetes, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, multi-organ autoimmune syndromes, chronic obstructive pulmonary disease (COPD), asthma, fibrotic disorders, such as airway fibrosis, idiopathic pulmonary fibrosis, non-specific interstitial pneumonia, post- infectious lung fibrosis, diffuse alveolar damage, collagen-vascular disease associated lung fibrosis, drug-induced lung fibrosis, silicosis, asbestos-related lung fibrosis, respiratory bronchiolitis, follicular bronchiolitis, respiratory bronchiolitis, interstitial lung disease, KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 desquamative interstitial fibrosis, cryptogenic organizing pneumonia, chronic hypersensitivity pneumonia, and graft-versus host disease of a lung transplant.

[0166] In some embodiments, the inflammatory disorder is an autoimmune disorder (or autoimmune disease). Examples of autoimmune disorders include as organ-tissue autoimmune diseases (e.g., Raynaud's syndrome), inflammatory bowel disease, scleroderma, myasthenia gravis, transplant rejection, endotoxin shock, sepsis, psoriasis, eczema, dermatitis, multiple sclerosis, autoimmune thyroiditis, uveitis, systemic lupus erythematosis, Addison's disease, autoimmune polyglandular disease (also known as autoimmune polyglandular syndrome), peripheral neuropathy, and Grave's disease.

[0167] In some embodiments, the anti-TMEM30A antibody is used to treat fungal infections. Fungal infections (or mycosis) are diseases caused by a fungus and can be found on skin, nails, mouth, throat, lungs, urinary tract, and other parts of the human body. The lipid flippase subunit TMEM30A mediates drug resistance and virulence in certain fungi, e.g., Cryptococcus neoformans. Huang, W, Liano G et al. mBio. 2016 May 10; 7(3): e00478-16. In some embodiments, the anti-TMEM30A antibody is administered to a patient such that the antibody comes into contact with immune cells that express TMEM30A. In some embodiments, the anti- TMEM30A antibody potentiates (e.g., enhances or activates) the inflammation caused by immune cells that express TMEM30A. In some embodiments, the immune cells are macrophages and monocytes. In some embodiments, the anti-TMEM30A antibody antagonizes yeast virulence and / or potentiates anti-fungal therapy.

[0168] Fungal infections can occur in places where the body traps moisture or have a lot of friction. Fungal infections can be more prevalent in subjects with poor circulation, diabetes, or a weakened immune system. Examples of fungal infections include cryptococcal meningitis (fungal meningitis), ringworm (dermatophytosis), onychomycosis, candidiasis (including oral thrush, diaper rash, vaginal yeast infections (vulvovaginitis), esophageal candidiasis, and candida intertrigo), tinea versicolor or pityriasis versicolor, sporotrichosis (rose gardener’s disease), chromoblastomycosis, eumycetoma, histoplasmosis, coccidioidomycosis (Valley fever), blastomycosis, aspergillosis, candida urinary tract infection, invasive candidiasis (including candidemia and endophthalmitis), pneumocystis pneumonia (JPJ), mucormycosis (including rhinocerebral mucormycosis, pulmonary mucormycosis, gastrointestinal mucormycosis, cutaneous mucormycosis, and disseminated KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 mucormycosis), and cryptococcosis. In some cases, the fungal infection is caused by dermatophytes, mucormycetes, Cryptococcus neoformans, Cryptococcus gattii, Candida sp., Candida albicans, Malassezia, Sporothix, Coccidioides, Blastomyces, Aspergillus, Pneumocystis jirovecii, or environmental fungi. b. Diagnostic Methods

[0169] Provided herein are diagnostic methods using anti-TMEM30A antibodies of the present disclosure. In some embodiments, an anti-TMEM30A antibody may be used to determine the presence or the amount of TMEM30A in a subject, or cells or tissue derived from a subject. In some embodiments, the anti-TMEM30A antibody may be used to determine prognosis of a disease or a disorder in a subject. In some embodiments, binding of the anti- TMEM30A antibody to the cell or tissue indicates the subject has an adverse prognosis. In some embodiments, detection of TMEM30A levels that are higher than expected for a healthy subject indicates that the subject has an adverse prognosis.

[0170] In some embodiments, binding of the anti-TMEM30A antibody to the cell or tissue indicates that the cell or tissue may be therapeutically sensitivity to the anti-TMEM30A antibody. In some embodiments, detection of TMEM30A levels in the cell or tissue that are higher than expected for a healthy counterpart indicates that the cell or tissue may be therapeutically sensitivity to the anti-TMEM30A antibody. In some embodiments, where the anti-TMEM30A antibody binds to the cell or tissue, or where TMEM30A levels in the cell or tissue are higher than expected for a healthy counterpart, the anti-TMEM30A antibody is used to treat a disease in the subject. In some embodiments, the disease is a cancer, an inflammatory disorder, or an autoimmune disorder.

[0171] In many methods, a cell or tissue is first obtained from the subject. Exemplary cells or tissues relating to a cancer prognosis that may be used include tumor cells, cancer cells, macrophages, and endothelial cells. In some embodiments, these cells are isolated from the cancer microenvironment. Exemplary cells or tissues relating to an inflammatory disorder or autoimmune disorder prognosis that may be used include immune cells such as macrophages and monocytes. KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 c. Administration of Anti-TMEM Antibodies to Patients

[0172] In general, a treatment method comprises administering the anti-TMEM30A antibody as a pharmaceutical composition to a patient in a therapeutically effective amount using a dosing regimen suitable for treatment of the disease or the disorder.

[0173] The anti-TMEM30A antibody may be administered with one or more additional therapeutic agents, e.g., radiation therapy, chemotherapeutic agents, and / or immunotherapeutic agents. As used herein, administered “in combination” means that two (or more) different treatments are delivered to the subject for the treatment of the cancer, e.g., AML, e.g., the two or more treatments are administered after the subject has been diagnosed with the cancer. In some embodiments, there may be overlap in the time frames in which the two therapeutic agents are administered. In other embodiments, one treatment protocol ends before the second begins. In some embodiment, treatment may be more effective because of combined administration.

[0174] In some embodiments, the anti-TMEM30A antibody is administered in conjunction with an mTOR inhibitor. In some embodiments, the mTOR inhibitor is AZD2014, rapamycin, sirolimus, temsirolimus, everolimus, ridaforolimus, umirolimus, zotarolimus, or an analog thereof.

[0175] In some embodiments, the anti-TMEM30A antibody is administered in conjunction with an agent that targets an antigen on a cancer cell, e.g., an immune checkpoint antigen. In one aspect, the agent is a biologic therapeutic or a small molecule. In another aspect, the agent is a monoclonal antibody, a humanized antibody, a human antibody, a fusion protein or a combination thereof. In certain embodiments, the agents inhibit, e.g., by blocking ligand binding to receptor, a checkpoint antigen that may be PD1, PDL1, CTLA-4, ICOS, PDL2, IDO1, IDO2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, GITR, HAVCR2, LAG3, KIR, LAIR1, LIGHT, MARCO, OX-40, SLAM, , 2B4, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137 (4-1BB), CD160, CD39, VISTA, TIGIT, a SIGLEC, CGEN-15049, 2B4, CHK 1, CHK2, A2aR, B-7 family ligands or a combination thereof. In some embodiments, the agent targets PD-1, e.g., an antibody that blocks PD-L1 binding to PD-1 or otherwise inhibits PD-1. In some embodiments, agent targets CTLA-4. In some embodiments, the targets LAG3. In some embodiments, the agents targets TIM3. In some embodiments, the agents target ICOS. KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2

[0176] In some embodiments, the anti-TMEM30A antibody is administered with a chemotherapeutic agent. In some embodiments, co-administration of the anti-TMEM30A antibody with a chemotherapeutic agent blocks cancer growth by increasing chemotherapeutic sensitivity and / or chemotherapeutic agent uptake. Examples of cancer chemotherapeutic agents include alkylating agents such as temozolomide, thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil; folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2',2"-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside; cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 and carboplatin and oxaliplatin; vinblastine; docetaxel, platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid derivatives such as bexarotene, alitretinoin; denileukin diftitox; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0177] In some embodiments, the anti-TMEM30A antibody is administered to a cancer patient wherein the cancer demonstrates resistance to current or previous treatment, e.g., with an immunomodulatory imide drug (IMiD), such as lenalidomide. A cancer is deemed resistant to treatment when the patient does not appear to experience a therapeutic effect from the treatment. In some embodiments, when the cancer is resistant to treatment, the patient does not demonstrate amelioration of the disease or symptoms of the disease. In some embodiments, when the cancer is resistant to treatment, the treatment does not decrease the growth rate of cancer cells. In some embodiments, resistance to treatment is indicated by worsening symptoms or cancer recurrence (relapse) in the patient.

[0178] In some embodiments, the IMiD-resistant cancer is a liver cancer; e.g., hepatocellular carcinoma (HCC). In some embodiments, the IMiD-resistant cancer is a lymphoma, e.g., non- Hodgkin lymphoma or central nervous system (CNS) lymphoma. In some embodiments, the IMiD-resistant cancer is multiple myeloma (MM). In some embodiments, the IMiD-resistant cancer is a leukemia, e.g., acute lymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML).

[0179] In some embodiments, the anti-TMEM30A antibody is administered in conjunction with an anti-fungal agent. In some embodiments, coadministration of the anti-TMEM30A antibody with an anti-fungal agent increases sensitivity to the anti-fungal agent. In some embodiments, the anti-fungal agent is caspofungin, clotrimazole (Canesten®), econazole, miconazole, terbinafine (Lamisil), fluconazole (Diflucan), ketoconazole (Daktarin®), nystatin (Nystan™), or amphotericin.

[0180] In some embodiments, the anti-TMEM30A antibody is administered to potentiate (e.g., enhance or activate) the immune response, e.g., in T cells or NK cells. In some embodiments, the anti-TMEM30A antibody is administered in conjunction with a therapeutic agent or a therapeutic treatment method, e.g., an immunomodulatory imide drug (IMiD), such KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 as lenalidomide; an immune checkpoint inhibitor; a chimeric antigen receptor (CAR) T cell therapy; or CAR NK cell therapy. In some embodiments, administration of the anti-TMEM30A antibody enhances disruption of phospholipid flippase activity, thereby modifying structure of the cell membrane. In some embodiments, administration of the anti-TMEM30A antibody facilitates entry of perforin and granzyme B entry into disease cells or target cells. In some embodiments, administration of the anti-TMEM30A antibody facilitates release of cytotoxic granules by T cells or NK cells. In some embodiments, administration of the anti-TMEM30A antibody potentiates (e.g., enhance or activate) the inflammatory response in macrophages.

[0181] In some embodiments, the anti-TMEM30A antibody is a blocking antibody. In some embodiments, the anti-TMEM30A antibody blocks TMEM30A, i.e., flippase, from binding to a ligand, e.g., lipids. In some embodiments, the anti-TMEM30A antibody is an inhibitor of TMEM30A, i.e., flippase. VI. Pharmaceutical Compositions

[0182] An anti-TMEM30A antibody is provided in a solution suitable for administration to the subject, such as a sterile isotonic aqueous solution for injection. The anti-TMEM30A antibody is dissolved or suspended at a suitable concentration in an acceptable carrier or excipient. In some embodiments, the carrier is aqueous, e.g., water, saline, phosphate buffered saline, and the like. The compositions may contain auxiliary pharmaceutical substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, and the like. The compositions can be formulated for use in a variety of drug delivery systems. Suitable formulations for use in the present invention are found, e.g., in Remington: The Science and Practice of Pharmacy, 21st Edition, Philadelphia, PA. Lippincott Williams & Wilkins, 2005.

[0183] Pharmaceutical compositions are administered to a patient in an amount sufficient to cure or at least partially arrest the disease or symptoms of the disease, and its complications. An amount adequate to accomplish this is defined as a “therapeutically effective dose.” A therapeutically effective dose is determined by monitoring a patient’s response to therapy. Typical benchmarks indicative of a therapeutically effective dose include the amelioration of symptoms of the disease in the patient. Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health, including other factors such as age, weight, gender, administration route, etc. Single or multiple administrations of the KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 antibody may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the methods provide a sufficient quantity of anti-TMEM30A antibody to effectively treat the patient.

[0184] An anti-TMEM30A antibody can be administered by any suitable means, including, for example, parenteral, intrapulmonary, and intranasal administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, the antibody may be administered by insufflation. In an illustrative embodiment, the antibody may be stored at 10 mg / ml in sterile isotonic aqueous saline solution for injection at 4°C and is diluted in either 100 ml or 200 ml 0.9% sodium chloride for injection prior to administration to the patient. In some embodiments, the antibody is administered by intravenous infusion over the course of 1 hour at a dose of between 0.01 and 25 mg / kg. In other embodiments, the antibody is administered by intravenous infusion over a period of between 15 minutes and 2 hours. In still other embodiments, the administration procedure is via sub-cutaneous bolus injection.

[0185] The dose of anti-TMEM30A antibody is chosen in order to provide effective therapy for the patient and is in the range of less than 0.01 mg / kg body weight to about 25 mg / kg body weight or in the range 1 mg – 2 g per patient. Preferably the dose is in the range 0.1 – 10 mg / kg or approximately 50 mg – 1000 mg / patient. The dose may be repeated at an appropriate frequency which may be in the range once per day to once every three months, or every six months, depending on the pharmacokinetics of the antibody (e.g., half-life of the antibody in the circulation) and the pharmacodynamic response (e.g., the duration of the therapeutic effect of the antibody). In some embodiments, the in vivo half-life of between about 7 and about 25 days and antibody dosing is repeated between once per week and once every 3 months or once every 6 months. In other embodiments, the antibody is administered approximately once per month. EXAMPLES

[0186] The following technical section represents certain aspects of the present disclosure. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Overview

[0187] The Examples that follow relate to TMEM30A expression in cancer cells. The Examples demonstrate the efficacy of exemplary anti-TMEM30A antibodies in binding to TMEM30A and in promoting cell death in cancer cells. Example 1 – TMEM30A is Upregulated in Lymphoma

[0188] This Example relates to the upregulation of the cell surface protein TMEM30A (CDC50A) in lymphoma. Whole exome sequencing of tumor-specific genomes of autopsy specimens identified novel mutations in TMEM30A that were not detected in paired diagnostic specimens of the lymphoma. In one case, high level focal amplification of the locus of chromosome 6q encoding TMEM30A in a lymphoma specimen was isolated from the patient at final relapse. This focal chromosomal amplification was associated with lenalidomide resistance because this focal chromosomal amplification was not detected in the paired specimen isolated from the patient before exposure to lenalidomide (Figure 1A). Transcriptional profiling using RNA-seq and immunohistochemistry demonstrated selective upregulation of the expression of TMEM30A by lymphoma cells in specimens isolated at final relapse and upon lenalidomide resistance (Figure 1B-C). Significant upregulation of TMEM30A protein expression was found by immunohistochemistry in relapsed CNS lymphoma specimens compared to diagnostic specimens of primary CNS lymphoma. Selective high tumor expression of TMEM30A transcripts was also confirmed in autopsy specimens using in situ hybridization. (Figures 2A-B). Example 2 – TMEM30A is Expressed in Cancer

[0189] This Example discusses TMEM30A’s impact on resistance to treatment in several cancers. According to the Cancer Genome Atlas Program (TCGA), TMEM30A was expressed by in glioma, thyroid cancer, lung cancer, liver cancer, pancreatic cancer, head and neck cancer, stomach cancer, colorectal cancer, urothelial cancer, renal cancer, prostate cancer, testis cancer, breast cancer, cervical cancer, ovarian cancer, endometrial cancer, and melanoma (Figure 3A). A TCGA dataset (N=197 patients) demonstrated that high TMEM30A expression correlated with shorter survival in AML (p<0.03), particularly in AML with mixed-lineage leukemia (MLL) genetic abnormalities that have a dismal prognosis. In addition, in B-cell ALL, KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 TMEM30A expression correlated with cases with poor prognosis, the Ph1+ (BCR / ABL1) genotype, as well as persistent minimal residual disease. High expression of TMEM30A also correlated with poor survival in MM patients treated with bortezomib (p<0.05). Further, according to immunohistochemical analysis of TMEM30A expression in tissues, high expression of TMEM30A expression was found in HCC (Figure 3B) compared to normal liver cells (not shown).

[0190] Given the high expression of TMEM30A in lung cancers based on TCGA data (Figure 3A), TMEM30A expression was determined in diagnostic specimens of non-small cell lung cancers. Using an immunohistochemical assay for TMEM30A, 13 out of 14 cases of lung cancer with high tumor cell expression, including two cases of squamous cell carcinoma, had marked TMEM30A immunoreactivity in the membrane and the cytoplasm, with strong expression in the invasive, leading edge of tumors. Representative figures are shown in Figure 3C. Using an anti-TMEM30A monoclonal antibody of the present disclosure in an immunohistochemistry assay, 74% of lung cancer cases analyzed (40 / 54 cases) were shown to express high TMEM30A in the lung tumor cells compared to each of 5 specimens of normal lung. There was reproducibly scant to absent TMEM30A expression by epithelial cells in normal lung. In addition, using the same anti-TMEM30A monoclonal antibody in an immunohistochemistry assay, 100% of ovarian cancer cases analyzed (16 / 16 cases) expressed high TMEM30A in the ovarian tumor cells compared to each of 4 specimens of normal ovary (Figure 13). The results suggest that TMEM30A is a biomarker of adverse prognosis expressed in tumor cells, macrophages, and endothelia in the cancer microenvironment. TMEM30A may be a candidate “Don’t Eat Me” signal expressed in cancer cells. TMEM30A may also regulate the threshold of cancer cells to undergo programmed cell death or apoptosis or other types of cell death in response to chemotherapy and / or radiation. Example 3 – TMEM30A Knockdown Attenuated Proliferation of Liver Cancer Cells

[0191] This Example relates to the role of TMEM30A in the HepG3b HCC liver cancer cell line. HepG3b HCC cells were treated for 12 days with TMEM30A.KD.1 or TMEM30A.KD.2, two shRNA’s that target TMEM30A. N=3 for each of control, TMEM30A.KD.1, and TMEM30A.KD.2. The shRNAs decreased TMEM30A expression (Figure 4C) and significantly attenuated proliferation of liver cancer cells (Figures 4A-B). p-values in Figure 4B were determined from repeated measure of two-way ANOVA test. KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 Example 4 – Anti-TMEM30A mAbs Bind to TMEM30A

[0192] This Example discusses the testing of ten IgG1 mAbs for TMEM30A binding. The anti-TMEM30A mAbs were raised against a peptide containing amino acids 120-325 of the human TMEM30A protein sequence from the extracellular domain of TMEM30A. This domain is highly conserved between humans and mice with 86% sequence identity. Amino acid residues 120-325 of human TMEM30A are as follows: MYYGLSNFYQNHRRYVKSRDDSQLNGDSSALLNPSKECEPYRRNEDKPIAPC GAIANSMFNDTLELFLIGNDSYPIPIALKKKGIAWWTDKNVKFRNPPGGDNLE ERFKGTTKPVNWLKPVYMLDSDPDNNGFINEDFIVWMRTAALPTFRKLYRLI ERKSDLHPTLPAGRYSLNVTYNYPVHYFDGRKRMILSTISWMGGKNPFL (SEQ ID NO: 25).

[0193] As shown by Western blots, the anti-TMEM30A antibodies 3D7B6, 3D7E6, 6A7A6, 6A7D6, 6A7H6, 6G10G1, 2H6F7, 4B5C7, 7G8E6, and 7G8G1 bound to the TMEM30A extracellular domain peptide antigen (Lanes 1-10 in Figure 5A). Further, the anti-TMEM30A antibody mAb23D7E6 bound to human AML cells that expressed TMEM30A extracellular domain (Figure 5B). Each mAb demonstrated immunoreactivity with a TMEM30A-positive HCC cell line, HepG2. As shown in Figure 6, the anti-TMEM30A mAb 3D7E6 bound to HepG2 cells (left panel) and mouse brain tissue (right panel) that express TMEM30A. Example 5 - Anti-TMEM30A mAb 3D7E6 Induced Annexin V Expression

[0194] Anti-TMEM30A mAbs were tested for potential biological activity in cancer. HepG2 cells were incubated with the anti-TMEM30A mAb 3D7E6 at 10 g / mL for 18 hours. As shown in Figure 7 (bottom right), mAb 3D7E6 can induce annexin V expression in HepG2 cells. Annexin V is commonly used as a marker for apoptotic cells as it binds to PS residues that are exposed on the surface of apoptotic cells. Thus, the results demonstrate that anti- TMEM30A mAbs can induce apoptosis of cancer cells. Example 6 – Anti-TMEM30A mAb Promoted Cell Death in Liver Cancer Cells In Vitro

[0195] Because TMEM30A signaling may involve mTOR, a synergistic relationship between mTOR inhibition and TMEM30A perturbation was tested. In this Example, a pharmacological inhibitor of mTORC1 and 2, AZD2014, was used to treat cancer cells with anti-TMEM30A antibodies. KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2

[0196] The anti-TMEM30A mAb 3D7E6 was administered at 10 g / mL with a 50 nM of AZD2014 to HepG2 cells. After 72 hours, cell death was observed (Figure 8A).

[0197] Hep3B cells were also incubated for four days with 10 g / mL of 3D7E6 and 50 nM of AZD2014 (AZD in Figure 8B). There were triplicates for each of the four conditions shown in Figure 8B: Control antibody (Ab), anti-TMEM30A 3D7E6, AZD and Control Ab, and AZD and anti-TMEM30A 3D7E6. Viable Hep3B cells were quantified. Hep3B cells exhibited marked cytotoxic response when treated with 3D7E6 and AZD2014. The two-sided T-test p- value is < 1.2E-4 when the combination treatment was compared to the others. Example 7 – Anti-TMEM30A mAb Promoted Phagocytosis in AML Cells In Vitro

[0198] Anti-TMEM30A mAbs were tested for ability to induce phagocytosis. As shown in Figures 9A-B, four hours of incubation of the lead candidate anti-TMEM30A mAb, 3D7E6, at 10 g / mL promoted phagocytosis of AML MV4-11 by macrophages but not in MCF-10 non- malignant breast cancer cells. Similar results were observed in murine A20 B cell lymphoma cells that were also treated with mAb 3D7E6.

[0199] Next, the mAb 3D7E6 was tested for induction of phagocytosis of lymphoma cells. Co-cultures of human Raji lymphoma cells with macrophages or MCF-10 cells were treated with 10 g / mL of mAb 3D7E6. After 3.5 hours, the mAb 3D7E6 (Figure 10A, right panels), but not control IgG1 (Figure 10A, left panels), promoted lamellipodia formation in human Raji lymphoma cells, in the presence of both M1 macrophages (Figure 10A, top right panel) and MCF-10 cells (Figure 10A, bottom right panel). Notably, lamellipodia formation in Raji cells induced by mAb 3D7E6 was consistently directed towards co-cultured macrophages, suggesting a chemotactic response. In contrast, although Raji cells co-cultured MCF-10 cells also demonstrated lamellipodia formation, the lamellipodia were not directed toward the MCF- 10 cells. This observation suggests that anti-TMEM30A mAbs can promote a macrophage- specific chemotactic response, which may play a role in tumor phagocytosis by macrophages.

[0200] Next, mAb 3D7E6 was tested for induction of phagocytosis of Hep3B HCC cells by M1 macrophages. M1-polarized macrophages were purified from peripheral blood mononuclear cells isolated from healthy volunteers. Co-cultures of Hep3B cells and macrophages were treated for four hours with 10 g / mL mAb 3D7E6. mAb 3D7E6 induced macrophage-mediated phagocytosis of Hep3B cells (Figure 11A, upper right; Iba1 immunoreactivity).25-50 mM fragments of Hep3B cells were internalized by phagosomes, as KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 indicated by arrows in Figure 11A. The bottom right arrow in Figure 11A indicates lamellipodium of a Hep3B cell interacting with a macrophage. Cytoplasmic phagocytic cuffs in macrophages around Hep3B fragments were reproducibly evident in images that excluded green fluorescence of Hep3B cells. Phagocytic cuffs were detected in ~20% of M1 macrophages exposed to mAb 3D7E6 (Figure 11A, right panels) but not in M1 macrophages exposed to control IgG (Figure 11A, left panels).

[0201] Further, the macrophages treated with mAb 3D7E6 for 18 hours also exhibited a significant shift in size distribution compared to macrophages exposed to control mAb. As shown in Figure 11B, the median macrophage size (area, determined by ImageJ) was two-fold larger in the presence of mAb 3D7E6 (P<0.01), consistent with cell size expansion associated with sustained induced-phagocytosis.

[0202] Taken together, the results demonstrate that anti-TMEM30A mAbs can induce phagocytosis of cancer cells by macrophages. Example 8 – Testing of Anti-TMEM30A mAb for Efficacy in a Mouse Model for Liver Cancer

[0203] The impact of the anti-TMEM30A mAb 3D7E6 on tumor growth was tested. A 100 g / dose of mAb 3D7E6 was administered to tumor-bearing, RAG- / -, BALB / c mice and nontumor-bearing, RAG- / -, BALB / c mice. The mice had tumors that were intraperitoneal hepatocellular carcinoma Hep3B cells; the mice are an orthotopic model of hepatocellular carcinoma. The 100 g / dose of mAb 3D7E6 was administered with or without 50 g / kg interferon gamma. mAb 3D7E6 and / or interferon gamma were administered twice weekly for 30 days. Tumor growth, as quantified by bioluminescence, was significantly delayed in mice treated with mAb 3D7E6 with interferon gamma (Figure 12A) and without interferon gamma (Figures 12C-D). Tumor growth rates were confirmed by gross histopathologic analysis of tumors from treated mice. As shown in Figure 12B, the livers of mice treated with mAb 3D7E6 had smaller tumors than the livers of untreated mice (control liver). p-value was 0.006 as determined from repeated measure of two-way ANOVA test. Co-administration of murine interferon gamma did not enhance the efficacy of ant-TMEM30A monoclonal antibodies. The results demonstrate that anti-TMEM30A mAbs can reduce tumor growth in liver cancer. KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 Example 9 – Testing of Anti-TMEM30A mAb for Safety in Mice

[0204] mAb 3D7E6 was tested for toxicity in BALB-c mice. mAb 3D7E6 was administered at 100 g / dose twice weekly for two weeks. Analysis of peripheral blood from treated mice demonstrated no decline in blood counts, including leukocyte subsets, red blood cells or platelets. The results demonstrate that anti-TMEM30A mAbs do not appear to have adverse effects in treated mice as determined by peripheral blood analysis. Example 10 – Anti-human TMEM30A mAb Binds to Murine TMEM30A and Induces Cell Death

[0205] This example demonstrates that anti-human TMEM30A mAb (mAb 3D7E6) also recognizes murine TMEM30A protein at 51 kDa on a western blot in murine A20 cells. See Figure 14A. Detection of the 51-kDa band in murine A20 cells is demonstrated both in wild type (wt) murine A20 cells as well as in murine A20 cells lentivirally transduced to overexpress murine TMEM30A gene. Band of similar molecular weight is also recognized by immunoblotting using a commercially available polyclonal anti-TMEM30A polyclonal antibody.

[0206] The anti-TMEM30A antibody was tested for its ability to induce cell death in murine A20 cells with and without co-administration with staurosporine. The cells were incubated with the anti-TMEM30A antibody for four hours. As shown in Figures 14B-14C, in vitro treatment with the exemplary anti-TMEM30A monoclonal antibody (mAb 3D7E6) induces annexin V expression in murine A20 lymphoma cells in vitro. Annexin V is a marker of programmed cell death. Further, when the cells were incubated for four hours with the anti-TMEM30A antibody in combination with the protein kinase inhibitor staurosporine, the level of annexin V expression was increased. (Each condition was performed in triplicate.) Staurosporine inhibits protein Kinase C, as well as other serine / threonine kinases and tyrosine kinases. The results suggest that the anti-TMEM30A mAb and staurosporine work synergistically to induce cell death. Example 11 – Anti-human TMEM30A Antibody Delays Progression of Lung Cancer In a Mouse Model

[0207] This example demonstrates the anti-human TMEM30A mAb (mAb 3D7E6) delays progression of human non-small cell lung cancer (NSCLC) in the NCI RAG- / - mouse model for metastatic NSCLC. The mAb was administered twice weekly as a single agent. The antibody attenuated tumor progression of cancer in the mice (Figure 15A). As shown in KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 Figure 15B, the mice treated with the mAb had prolonged survival by >60% compared to mice treated with a control IgG1 antibody. The mice bearing NSCLC tumors (NCI-H460 tumors) experienced early weight loss when treated with control IgG1 antibody compared to mice treated with the anti-TMEM30A mAb. In contrast, the mice treated with mAb demonstrated delayed tumor progression. The results demonstrate that the anti-human TMEM30A antibody delays progression of lung cancer in a mouse model. Example 11 – Anti-human TMEM30A mAb Reduces Raji Cell Chemotaxis in Response to Chemokine SDF-1

[0208] This Example demonstrates that the anti-human TMEM30A mAb (mAb 3D7E6) reduces chemotaxis in Raji lymphoma cells in the presence of SDF-1 (10 ng / mL). A 7-hour migration transwell assay was performed in triplicate conditions.

[0209] As shown in Figure 16, Raji cells treated with the anti-human TMEM30A mAb (mAb 3D7E6) and SDF-1 had reduced chemotaxis as compared to Raji cells treated with SDF-1 and control IgG1. The results demonstrate that the anti-human TMEM30A mAb (mAb 3D7E6) significantly reduced the efficiency of Raji lymphoma cell chemotaxis (by about 50%) in response to the chemokine SDF-1. The results indicate that an anti-human TMEM30A mAb may be used to antagonize tumor cell metastasis in animal models and in human patients in the clinic. Example 12 – Anti-human TMEM30A mAb Reduces Tumor Angiogenesis In Vivo

[0210] This example demonstrates that treatment of tumor cells with anti-human TMEM30A mAb (mAb 3D7E6) reduces tumor-associated angiogenesis. A metastatic non- small cell lung cancer (NSCLC) model using NCI-H460 tumor cells in RAG- / - mice was used. The anti-human TMEM30A mAb (mAb 3D7E6) was administered twice weekly in the mice. The von Willebrand factor (vWF) immunoreactivity assay was used.

[0211] Figure 17A shows a non-small cell lung cancer metastatic lesion from a mouse treated twice weekly with control IgG1 mAb. The mouse was sacrificed on day 26 after tumor implantation. Dense tumor-associated vascular density is demonstrated by strong immunoreactivity for vWF, a marker of vasculature and tumor vessels.

[0212] Figure 17B shows a non-small cell lung cancer metastatic lesion from a mouse treated twice weekly with anti-TMEM30A mAb. The mouse was sacrificed on day 44 after tumor implantation. Markedly reduced tumor-associated vascular density is demonstrated by KILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 weak immunoreactivity for vWF, consistent with an effect of anti-TMEM30A mAb in reducing tumor-associated angiogenesis. The results demonstrate that the anti-TMEM30A mAb reduces tumor angiogenesis in vivo.

[0213] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. VII. TMEM30A ANTIBODY SEQUENCESKILPATRICK TOWNSEND 780457241PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2KILPATRICK TOWNSEND 780457241

Claims

PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 WHAT IS CLAIMED IS:

1. An antibody that binds to the extracellular domain of human TMEM30A on cancer cells, wherein: (a) upon binding to the extracellular domain of human TMEM30A, the antibody promotes macrophage-mediated phagocytosis of tumor cells; and (b) the antibody comprises: (i) a heavy chain variable region (VH) comprising: an HCDR1 comprising a sequence DYAMH (SEQ ID NO: 1), an HCDR2 comprising a sequence VISTYSGNTNYNQKFKG (SEQ ID NO: 2), and an HCDR3 comprising a sequence YYRYDGETMDY (SEQ ID NO: 3); (ii) a light chain variable region (VL) comprising: an LCDR1 comprising a sequence SASSSVSYMH (SEQ ID NO: 4), an LCDR2 comprising a sequence RTSNLAS (SEQ ID NO: 5), and an LCDR3 comprising a sequence QQRSSYPLT (SEQ ID NO: 6); or (i) a heavy chain variable region (VH) comprising: an HCDR1 comprising a sequence NYGVH (SEQ ID NO: 9), an HCDR2 comprising a sequence VIWAGGSTHYNSTLMS (SEQ ID NO: 10), and an HCDR3 comprising a sequence VSPPGYFDV (SEQ ID NO: 11); (ii) a light chain variable region (VL) comprising: an LCDR1 comprising a sequence TASSSVSSSYLH (SEQ ID NO: 12), an LCDR2 comprising a sequence STSNLAS (SEQ ID NO: 13), and an LCDR3 comprising a sequence HQYHRSPLT (SEQ ID NO: 14); or (i) a heavy chain variable region (VH) comprising: an HCDR1 comprising a sequence RYWMS (SEQ ID NO: 17), an HCDR2 comprising a sequence EINPDSSTINYTPSLKD (SEQ ID NO: 18), and an HCDR3 comprising a sequence RLVYYAMDY (SEQ ID NO: 19);PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 (ii) a light chain variable region (VL) comprising: an LCDR1 comprising a sequence SASQGINNYLN (SEQ ID NO: 20), an LCDR2 comprising a sequence YTSSLHS (SEQ ID NO: 21), and an LCDR3 comprising a sequence QQYSKLPYT (SEQ ID NO: 22).

2. The antibody of claim 1, wherein the VH comprises an amino acid sequence having at least 80% identity or at least, 85%, 90%, or 95% identity) to SEQ ID NO:

7.

3. The antibody of claim 1 or 2, wherein the VL comprises an amino sequence having at least 80% identity or at least 85%, 90%, or 95% identity ) to SEQ ID NO:

8.

4. The antibody of claim 2 or 3, wherein the VHcomprises SEQ ID NO: 7 and the VLcomprises SEQ ID NO:

8.

5. The antibody of claim 1, wherein the VHcomprises an amino acid sequence having at least 80% identity or at least 85%, 90%, or 95% identity to SEQ ID NO:

15.

6. The antibody of claim 1 or 5, wherein the VLcomprises an amino sequence having at least 80% identity, or at least 85%, 90%, or 95% identity, to SEQ ID NO:

16.

7. The antibody of claim 5 or 6, wherein the VH comprises SEQ ID NO: 15 and the VL comprises SEQ ID NO:

16.

8. The antibody of claim 1, wherein the VH comprises an amino acid sequence having at least 80% identity, or at least 85%, 90%, or 95% identity, to SEQ ID NO:

23.

9. The antibody of claim 1 or 8, wherein the VL comprises an amino sequence having at least 80% identity, or at least 85%, 90%, or 95% identity, to SEQ ID NO:PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 10. The antibody of claim 8 or 9, wherein the VHcomprises SEQ ID NO: 23 and the VLcomprises SEQ ID NO:

24.

11. The antibody of any one of claims 1 to 10, wherein the antibody is linked to a cytotoxic agent or a detectable label.

12. A nucleic acid encoding the antibody of any one of claims 1 to 11.

13. The nucleic acid of claim 12, wherein the nucleic acid sequence is operably linked to a heterologous promoter.

14. An expression vector comprising a nucleic acid of claim 12 or 13.

15. A cell expressing the antibody of any one of claims 1 to 11, or comprising the nucleic acid of claim 12 or 13 or the expression vector of claim 14.

16. A method of producing an antibody, the method comprising culturing a cell comprising the nucleic acid claim 12 or 13, or the expression vector of claim 14, under conditions in which the nucleic acid encoding the VHand the nucleic acid encoding the VLare expressed.

17. A method of treating a disease in a subject comprising administering the antibody of any one of claims 1 to 11 to the subject.

18. The method of claim 17, wherein the disease is a cancer.

19. The method of claim 18, wherein the cancer is selected from the group consisting of hepatocellular carcinoma, non-Hodgkin lymphoma, central nervous system (CNS) lymphoma, multiple myeloma (MM), acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), non-small lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma, lung small cell carcinoma (small cell lung cancer; SCLC), and ovarian cancer.

20. The method of claim 18 or 19, wherein the cancer is resistant to lenalidomide.PATENT Attorney Docket No.: 081906-1486452-260510PC Client Reference No.: SF-2020-136-2 21. The method of claim 17, wherein the disease is an inflammatory disorder or an autoimmune disorder.

22. The method of claim 17, wherein the disease is a fungal infection.

23. A method of killing cancer cells in a subject comprising contacting cancer cells in the subject in vivo with an antibody of any one of claims 1 to 11.

24. The method of claim 23, further comprising contacting the cancer cells with a therapeutic molecule in vivo selected from the group consisting of a chemotherapeutic drug, an immunotherapeutic drug, and a protein kinase inhibitor.

25. The method of claim 24, wherein the protein kinase inhibitor is an inhibitor of mTOR.

26. The method of the immediately preceding claim, wherein the inhibitor of mTOR is AZD2014.

27. The method of claim 24, wherein protein kinase inhibitor is a serine / threonine kinase inhibitor, a tyrosine kinase inhibitor, protein kinase C inhibitor, or a multi-target protein kinase inhibitor.

28. The method of claim 24, wherein the protein kinase inhibitor is staurosporine or midostaurin.

29. A method for reducing inflammation in a subject having an inflammatory condition comprising contacting cells in the subject in vivo with an antibody of any one of claims 1 to 11, wherein binding of the antibody to the cells reduces inflammation in the subject.

30. A method for increasing phagocytosis in a subject comprising administering the antibody of any one of claims 1 to 11 to the subject, wherein binding of the antibody to cells increases phagocytosis.

31. The method of any one of claims 23-30, wherein the cells are selected from the group consisting of tumor cells, cancer cells, macrophages, endothelial cells, and monocytes.

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