Anti-cd36 antibodies and their use in treating cancer

Abstract

The claimed invention relates to the treatment of cancer by targeting CD36, a fatty acid receptor. The claimed invention also relates to the treatment of cancer metastasis by targeting CD36. The invention relates to the use of anti-CD36 antibodies as blockers or inhibitors of CD36 activity.

Description

Technical Field

[0001] This disclosure relates to the treatment of cancer, particularly the treatment of cancer metastases, and the control of said diseases. More specifically, this disclosure relates to the use of anti-CD36 antibodies for the treatment of cancer. This disclosure also relates to the use of anti-CD36 antibodies for the treatment of primary cancer, cancer metastases, or both. The treatment involves the use of full-length antibodies and fragments thereof. Background Technology

[0002] CD36 (HGNC: 1663, EntrezGene: 948, Ensembl: ENSG00000135218, OMIM: 173510, UniProtKB: P16671) is a receptor protein with several different known functions, as indicated by its various alternative names: it is known as determinant 36, platelet-reactive protein receptor, type I collagen receptor, leukocyte differentiation antigen CD36, platelet glycoprotein 4, or fatty acid translocase, etc. The Entrez Gene and UniProt / SwissProt summaries of the CD36 gene (http: / / www.genecards.org / cgi-bin / carddisp.pl?gene=CD36) describe this protein as the fourth major glycoprotein on the platelet surface, acting as a receptor for platelet-reactive proteins in platelets and various cell lines. Since platelet-reactive proteins are widely distributed proteins involved in various adhesion processes, this protein can act as a cell adhesion molecule. This protein binds to collagen and thromboretin, thereby mediating the anti-angiogenic effect of thromboretin. It also binds to anionic phospholipids and oxidized LDL. This protein directly mediates cell adhesion in erythrocytes parasitized by *Plasmodium falciparum* and binds long-chain fatty acids. This protein is a co-receptor for the TLR4-TLR6 heterodimer, which promotes inflammation in monocytes / macrophages. Upon binding to ligands (e.g., oxLDL or amyloid-β42), CD36 rapidly induces the formation of heterodimers of TLR4 and TLR6. The TLR4-TLR6 heterodimer is internalized and triggers an inflammatory response, leading to NF-κB-dependent production of CXCL1, CXCL2, and CCL9 cytokines (via the MYD88 signaling pathway), CCL5 cytokine production (via the TICAM1 signaling pathway), and IL-1β secretion. CD36 is also at the top of the signal transduction cascade. It takes up lipids from the extracellular environment and triggers their β-oxidation to obtain energy in the form of ATP (Coburn et al., 2000; Ibrahimi et al., 1999; Pepino et al., 2014).

[0003] CD36 has been previously associated with cancer, but its connection to therapeutic significance and mechanism of action remains unclear. WO03 / 032813 discloses an experiment showing that CD36 is one of the genes upregulated in renal cell carcinoma. Although no experiments against other types of cancer are shown, the application proposes that CD36 is a useful target for the diagnosis and / or treatment and even prevention of certain cancers, and is also considered a predictor of prognosis in tumor treatment. SCC is considered one of the possible cancer types that can be treated with CD36 antibodies or antagonists such as antisense RNA, but no evidence is provided of changes in CD36 expression in SCC, or in particular, the efficacy of CD36 antibodies or other antagonists in preventing or treating primary tumors or metastases. Based on the experiment shown in WO03 / 032813, spontaneous animal tumors are proposed for testing the efficacy of antibodies that specifically bind to proteins overexpressed in renal cell carcinoma, and, given that it is a highly invasive malignant tumor, feline oral SCC is proposed as a suitable model. However, this proposal was made without providing examples of the practical utility of the method. Furthermore, there is no evidence that any gene overexpressed in renal cell carcinoma is also overexpressed in feline oral SCC, particularly no data showing changes (increases or decreases) in CD36 expression levels in feline oral SCC, or any data suggesting that CD36 may be involved in the initiation, progression, or spread of metastasis in this type of cancer. Additionally, while comments have noted a low metastasis rate in feline oral SCC, this may be due to the short survival time of cats with this tumor.

[0004] Regarding metastasis, previous studies have shown that inhibition of CD36 (either via antibodies that neutralize its activity or via shRNA) has a significant impact on the initiation and progression of metastasis, thereby reducing metastatic penetrance and growth in all cell lines and patient-derived tumors tested. See US Publication No. 2019-0106503, which is incorporated herein by reference in its entirety. Summary of the Invention

[0005] This application discloses anti-CD36 antibodies and their use in treating cancer. In some embodiments, anti-CD36 antibodies are used to treat cancer metastases. In some embodiments, anti-CD36 antibodies are used to treat both primary tumors and cancer metastases. In some embodiments, the anti-CD36 antibody is an isolated antibody comprising one or more complementary determinant regions (CDRs) from SEQ ID NO:5 and SEQ ID NO:7 (i.e., from the ONA-0-v1 antibody). In some embodiments, the anti-CD36 antibody is a chimeric antibody comprising one or more CDR sequences from SEQ ID NO:5 and SEQ ID NO:7. In some embodiments, the anti-CD36 antibody is a humanized antibody comprising one or more CDR sequences from SEQ ID NO:5 and SEQ ID NO:7. In some embodiments, the anti-CD36 antibody comprises a VH having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of VH (SEQ ID NO: 11) in the ONA-O-v1 antibody. In some embodiments, the anti-CD36 antibody comprises a VL having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of VL (SEQ ID NO: 13) in the ONA-O-v1 antibody. In some embodiments, the anti-CD36 antibody is ONA-O-v1, which comprises a heavy chain as listed in SEQ ID NO: 5 and a light chain as listed in SEQ ID NO: 7. In some embodiments, the anti-CD36 antibody is a chimeric ONA-O-v1 IgG1 LALA antibody comprising the heavy chain listed in SEQ ID NO:21 and the light chain listed in SEQ ID NO:23 (i.e., the heavy and light chains derived from the 1G04 antibody). In some embodiments, the anti-CD36 antibody is a chimeric ONA-O-v1 IgG1 antibody comprising the heavy chain listed in SEQ ID NO:64 and the light chain listed in SEQ ID NO:23 (i.e., the heavy and light chains derived from the 1G06 antibody).

[0006] In some embodiments, the anti-CD36 antibody comprises a heavy chain and a light chain, wherein the heavy chain CDR1 region comprises SEQ ID NO:27, the heavy chain CDR2 region comprises SEQ ID NO:28, the heavy chain CDR3 region comprises SEQ ID NO:29, the light chain CDR1 region comprises SEQ ID NO:30, the light chain CDR2 region comprises SEQ ID NO:31, and the light chain CDR3 region comprises SEQ ID NO:32. In some embodiments, the anti-CD36 antibody comprises a heavy chain and a light chain, wherein the heavy chain CDR1 region comprises SEQ ID NO:37, the heavy chain CDR2 region comprises SEQ ID NO:38, the heavy chain CDR3 region comprises SEQ ID NO:29, the light chain CDR1 region comprises SEQ ID NO:30, the light chain CDR2 region comprises SEQ ID NO:31, and the light chain CDR3 region comprises SEQ ID NO:32. In some embodiments, the anti-CD36 antibody comprises a heavy chain and a light chain, wherein the heavy chain CDR1 region comprises SEQ ID NO:39, the heavy chain CDR2 region comprises SEQ ID NO:40, the heavy chain CDR3 region comprises SEQ ID NO:41, the light chain CDR1 region comprises SEQ ID NO:42, the light chain CDR2 region comprises SEQ ID NO:43, and the light chain CDR3 region comprises SEQ ID NO:32.

[0007] In some embodiments, the anti-CD36 antibody is a humanized antibody, wherein the heavy chain CDR region comprises: (a) SEQ ID NO: 37, 38, and 29; (b) SEQ ID NO: 44, 46, and 29; or (c) SEQ ID NO: 45, 47, and 29. In some embodiments of these embodiments, the light chain CDR region comprises SEQ ID NO: 30, 31, and 32. In some embodiments of these embodiments, the light chain CDR region comprises SEQ ID NO: 48, 31, and 32. In some embodiments of these embodiments, the light chain CDR region comprises SEQ ID NO: 48, 49, and 32. In some embodiments of these embodiments, the light chain CDR region comprises SEQ ID NO: 30, 50, and 32. In some embodiments of these embodiments, the heavy chain variable region comprises SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54; and the light chain variable region comprises SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58.

[0008] In some embodiments, the anti-CD36 antibody is a humanized antibody comprising: (a) a heavy chain variable region comprising SEQ ID NO:51 and a light chain variable region comprising SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 or SEQ ID NO:58; (b) a heavy chain variable region comprising SEQ ID NO:52 and a light chain variable region comprising SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 or SEQ ID NO:58; (c) a heavy chain variable region comprising SEQ ID NO:53 and a light chain variable region comprising SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 or SEQ ID NO:58; or (d) a heavy chain variable region comprising SEQ ID NO:54 and a light chain variable region comprising SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 or SEQ ID NO:58.

[0009] In some embodiments, the anti-CD36 antibody is an isolated antibody, a chimeric antibody, or a humanized antibody that binds to the same human CD36 epitope as an antibody comprising the light chain of SEQ ID NO:7 and the heavy chain of SEQ ID NO:5. In some embodiments, the anti-CD36 antibody is an isolated antibody, a chimeric antibody, or a humanized antibody that competes with an antibody comprising the heavy chain of SEQ ID NO:5 and the light chain of SEQ ID NO:7 for binding to human CD36.

[0010] In some embodiments, the antibody is substantially free of antibodies that do not specifically bind to CD36. In some embodiments, the antibody is substantially free of light chains containing the light chain CDR1 region, light chain CDR2 region, and light chain CDR3 region present in SEQ ID NO:9.

[0011] In some embodiments, the anti-CD36 antibody binds to human CD36. In some embodiments, the anti-CD36 antibody binds to human CD36 with an affinity greater than 10 nM.

[0012] In some embodiments, the anti-CD36 antibody further comprises a heavy chain constant region. In some embodiments, the antibody comprises an IgA or IgG heavy chain constant region. In some embodiments, the heavy chain constant region is selected from the group consisting of human immunoglobulin IgA1, IgA2, IgG1, IgG2, IgG3, or IgG4 heavy chain constant regions. In some embodiments, the heavy chain constant region comprises a constant region containing one or more mutations at amino acid positions E233, L234, L235, G236, N297, P331, and P329. In some embodiments, the heavy chain constant region comprises an IgG constant region containing an LALA mutation, which consists of a change from leucine to alanine at amino acid positions 234 and 235.

[0013] In some embodiments, the heavy chain constant region includes an IgG constant region containing mutations at amino acid positions L234, L235, and / or G236. In some embodiments, the heavy chain constant region includes an IgG constant region containing a set of mutations selected from the group consisting of: L234A, L235S, and G236R; L234G, L235S, and G236R; L234Q, L235S, and G236R; L234S, L235G, and G236R; L234S, L235T, and G236R; L234S, L235V, and G236R; L234T, L235Q, and G236R; L 234T, L235S and G236R; L234T, L235T and G236R; L234A and L235A; L234A, L235A and P329G; G236R and L328R; L234A and G237A; L234A, L235A and G237A; L234A and L235E; L235V, F243L, R292P, Y300L, P396L; D265A and P329A; L234A, L235A and K 322A; L234F, L235E and P331S; L234F, L235Q and K322Q; L234A, L235A, G237A, P238S, H268A, A330S and P331S; E233P, L234V, L235A, G236Δ, A327G, A330S and P331S; L235A and G236R; L235S and G236R; G236R; L234Q and L235S; L235G and G 236R; L234Q, L235S, and Δ236R; L234Q and L235S; L234Q, L235S, and G236R; L234Q, L235S, and G236R; L234Q, L235S, and G236R; L234Q, L235S, and G236R; L234Q, L235S, G236R, M252Y, S254T, and T256E; and L234Q, L235S, G236R, T250Q, and M428L. In some embodiments, the heavy chain constant region includes an IgG constant region containing L234G, L235S, and G236R mutations. In some embodiments, the heavy chain constant region includes an IgG constant region containing L234S, L235T, and G236R mutations. In some embodiments, the heavy chain constant region includes an IgG constant region containing L234S, L235V, and G236R mutations. In some embodiments, the heavy chain constant region includes an IgG constant region containing L234T, L235Q, and G236R mutations. In some embodiments, the heavy chain constant region includes an IgG constant region containing L234T, L235T, and G236R mutations.In some embodiments, the heavy chain constant region includes an IgG constant region containing L234A and L235A mutations. In some embodiments, the heavy chain constant region includes an IgG constant region containing L234A, L235A, and P329G mutations.

[0014] In some embodiments, the anti-CD36 antibody further comprises a light chain constant region. In some embodiments, the light chain constant region is selected from the group consisting of the light chain constant regions of human immunoglobulin kappa (κ) and lambda (λ). In some embodiments, the antibody comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is the human IgG1 heavy chain constant region, and wherein the light chain constant region is the human κ light chain constant region.

[0015] In some embodiments, the antibody is an antigen-binding fragment. In some embodiments, the antigen-binding fragment includes Fab, Fab', F(ab')2, single-chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, intracellular antibody, IgGΔCH2, microantibody, F(ab')3, tetraantibody, triantibody, biantibody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

[0016] Some embodiments are pharmaceutical compositions comprising the anti-CD36 antibody described herein and a pharmaceutically acceptable excipient. In some embodiments, at least 95% of the antibody in the pharmaceutical composition is afucosylated. In some embodiments, the pharmaceutical composition further comprises one or more other therapeutic agents. In some embodiments, the pharmaceutical composition further comprises a PD-1 inhibitor. Suitable PD-1 inhibitors include the anti-PD-1 antibody pembrolizumab, pidilizumab, or nivolumab. In some embodiments, the pharmaceutical composition further comprises a PD-L1 inhibitor, such as the anti-PD-L1 antibody atezolizumab, durvalumab, avelumab, or BMS-936559. In some embodiments, the pharmaceutical composition further comprises a CTLA-4 inhibitor, such as the anti-CTLA-4 antibody ipilimumab. In some embodiments, the pharmaceutical composition further comprises a chemotherapeutic agent, such as cisplatin.

[0017] Some embodiments are methods of administering the anti-CD36 antibody and a pharmaceutical composition containing the anti-CD36 antibody described herein. Some embodiments relate to methods of treating a patient with cancer, comprising administering to a subject in need a therapeutically effective amount of the antibody disclosed herein or a therapeutically effective amount of the pharmaceutical composition disclosed herein. In some embodiments, the cancer is oral squamous cell carcinoma, head and neck cancer, esophageal cancer, gastric cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, colon cancer, kidney cancer, prostate cancer, sarcoma, melanoma, leukemia, or lymphoma. Some embodiments are methods of treating a patient with one or more metastatic tumors, comprising administering to a subject in need a therapeutically effective amount of the antibody disclosed herein or a therapeutically effective amount of the pharmaceutical composition disclosed herein. In some embodiments, the metastatic tumor develops from oral squamous cell carcinoma, head and neck cancer, esophageal cancer, gastric cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, colon cancer, kidney cancer, prostate cancer, sarcoma, melanoma, leukemia, or lymphoma. In some embodiments, the metastatic tumor is located in cervical lymph nodes, liver, lungs, spleen, kidneys, or peritoneal wall. In some embodiments, as measured by IVIS imaging or H&E staining, the treatment reduces the size of metastatic tumors. In some embodiments, the treatment reduces the size of metastatic tumors in cervical lymph nodes, liver, lungs, spleen, kidneys, or peritoneal wall. In some embodiments, as measured by IVIS imaging or H&E staining, the treatment prevents or inhibits the formation or development of metastatic tumors. In some embodiments, the treatment prevents or inhibits the formation or development of metastatic tumors in cervical lymph nodes, liver, lungs, spleen, kidneys, or peritoneal wall. In some embodiments, the treatment reduces the number of metastatic tumors. In some embodiments, the patient is a human patient. In some embodiments, the treatment is effective in treating both primary and metastatic tumors.

[0018] In some embodiments, the method includes administering an anti-CD36 antibody, which is a full-length antibody, a single-chain antibody, an scFv, a Fab fragment, or an F(ab')2 fragment. In some embodiments, the method includes administering an anti-CD36 antibody as a full-length antibody. In some embodiments, the method includes administering an anti-CD36 antibody comprising the heavy chain of SEQ ID NO:21 and the light chain of SEQ ID NO:23. In some embodiments, the method includes administering an anti-CD36 antibody comprising the heavy chain of SEQ ID NO:64 and the light chain of SEQ ID NO:23.

[0019] In some embodiments, the method includes administering a second treatment in addition to the anti-CD36 antibody. In some embodiments, the second treatment administered is immunotherapy. In some embodiments, the administered immunotherapy is a PD-1 inhibitor, such as the anti-PD-1 antibody pembrolizumab, pildizumab, or nivolumab. In some embodiments, the administered immunotherapy is a PD-L1 inhibitor, such as the anti-PD-L1 antibody atezolizumab, durvalumab, avelumab, or BMS-936559. In some embodiments, the administered immunotherapy is a CTLA-4 inhibitor, such as the anti-CTLA-4 antibody ipilimumab. In some embodiments, the second treatment is a chemotherapy agent. In some embodiments, the administered chemotherapy agent is cisplatin.

[0020] In some embodiments, metastases in the subject are reduced or suppressed. In some embodiments, metastases in the subject that have spread to the cervical lymph nodes, liver, lungs, spleen, kidneys, or peritoneal wall are reduced or suppressed. In some embodiments where the method involves administering a second treatment in addition to an anti-CD36 antibody, the two treatments are administered sequentially. In some embodiments where the method involves administering a second treatment in addition to an anti-CD36 antibody, the two treatments are administered simultaneously.

[0021] Some embodiments encode isolated polynucleotides of the antibodies disclosed herein. In some embodiments, the isolated polynucleotide encodes the heavy chain in SEQ ID NO:5 and the light chain in SEQ ID NO:7. In some embodiments, the isolated polynucleotide comprises SEQ ID NO:6. In some embodiments, the isolated polynucleotide comprises SEQ ID NO:8. In some embodiments, the isolated polynucleotide encodes the heavy chain in SEQ ID NO:21 and the light chain in SEQ ID NO:23. In some embodiments, the isolated polynucleotide encodes the heavy chain in SEQ ID NO:64 and the light chain in SEQ ID NO:23. In some embodiments, the isolated polynucleotide comprises SEQ ID NO:22. In some embodiments, the isolated polynucleotide comprises SEQ ID NO:24.

[0022] Some embodiments are vectors containing the isolated polynucleotides disclosed herein. Other embodiments are cells containing the isolated polynucleotides or vectors disclosed herein. In some embodiments, the cells are selected from the group consisting of: *Escherichia coli*, *Pseudomonas*, *Bacillus*, *Streptomyces*, yeast, CHO, YB / 20, NSO, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, BW, LM, COS1, COS7, BSC1, BSC40, BMT10 cells, plant cells, insect cells, and human cells in tissue culture. In some embodiments, the cells lack the functional α-1,6-fucosyltransferase gene (FUT8).

[0023] Some embodiments are methods for preparing the antibodies disclosed herein. In some embodiments, the method of preparing antibodies includes expressing antibodies using cells containing isolated polynucleotides or vectors disclosed herein. In some embodiments, the method of preparing antibodies includes culturing cells containing isolated polynucleotides or vectors disclosed herein under conditions suitable for antibody expression and isolating the antibodies expressed therein. Attached Figure Description

[0024] Figure 1A This is a schematic diagram illustrating an experimental overview of the efficacy study of a commercial anti-CD36 antibody in a mouse model of oral cancer metastases using Detroit-562 cells, with and without cisplatin. Figure 1B The study details the research groups tested in the study, particularly the treatments and dosages given to each group.

[0025] Figure 2A-2C Results were provided relating to the effects of anti-CD36 antibody and / or cisplatin on primary tumors in a Detroit-562 mouse model of oral cancer metastases. Figure 2A The quantitative in vitro imaging of the primary tumor during treatment with anti-CD36 antibody and / or cisplatin is shown. Figure 2B A representative image of a primary tumor stained with H&E from the tongue of an orthotopically injected mouse is shown. Figure 2C The surface area of ​​the primary tumor at the end of the treatment regimen is shown. These figures illustrate that the tested anti-CD36 Ab, in combination with cisplatin, has at least additive antitumor activity in inhibiting the growth of primary tumors in oral cancer.

[0026] Figure 3Representative H&E staining images of lung metastases at the end of treatment with anti-CD36 antibody and / or cisplatin in the Detroit-562 mouse model of oral cancer metastases. The figure illustrates that mice treated with cisplatin (top right), anti-CD36 antibody (bottom left), or cisplatin and anti-CD36 antibody (bottom right) had fewer and smaller metastases than control mice (top left).

[0027] Figure 4A and 4B The figures quantify the number and size of lung metastases in the Detroit-562 mouse model of oral cancer metastases. These figures illustrate that mice treated with anti-CD36 antibody alone had smaller and fewer metastases than control mice. Mice treated with cisplatin alone had a similar number of metastases to control mice, although cisplatin did reduce the size of the metastases. Treatment with both anti-CD36 antibody and cisplatin resulted in a similar number of metastases as treatment with anti-CD36 antibody alone. However, treatment with both anti-CD36 antibody and cisplatin resulted in a greater reduction in the size of metastatic tumors compared to treatment with either anti-CD36 antibody or cisplatin alone.

[0028] Figure 5 This is a schematic diagram illustrating the structures of the ONA-0-v1 antibody, the ONA-0-v2 antibody, the 1G04 antibody (i.e., a chimeric IgG1 version of the ONA-0-v1 antibody with altered LALAFc), and the chimeric ONA-0-v2 IgG LALA antibody. In this diagram, the green portion represents the mouse IgA constant region sequence present in both ONA-0-v1 and ONA-0-v2. The gray portion represents the human IgG1 sequence used in the chimeric antibody; the red dots within the gray area represent the leucine-to-alanine mutation (i.e., the "LALA" change) at amino acid positions 234 and 245 within the IgG1 sequence. The yellow portion represents the variable region of ONA-0-v1. The blue portion represents the light chain variable region in ONA-0-v2 that differs from the light chain variable region of ONA-0-v1.

[0029] Figure 6 Protein gels containing reduced or non-reduced ONA-0 antibodies are described, with 2.5 μg of antibody added to each lane. Individual gels of ONA-0-v1, ONA-0-v2, 1G04, and chimeric ONA-0-v2 IgG LALA antibodies are shown.

[0030] Figure 7Data from an ELISA assay were presented, which tested the ability of 1G04 and the chimeric ONA-0-v2 IgG1 LALA antibody to bind to human and mouse CD36 proteins coated in microplates. These data indicate that 1G04 specifically binds to human and mouse CD36, while the chimeric ONA-0-v2 IgG1 LALA antibody does not specifically bind to human and mouse CD36.

[0031] Figure 8 Data from an ELISA assay were presented, which tested the ability of the ONA-0-v1 antibody and a commercial anti-CD36 antibody to bind to human and mouse CD36 proteins coated in microplates. These data indicate that both antibodies bind specifically to human and mouse CD36 in a similar manner.

[0032] Figure 9A and 9B FACS analysis data depicted the binding ability of ONA-0-v1, 1G04, and chimeric ONA-0-v2 IgG1 LALA antibodies to cells overexpressing human CD36, relative to commercially available anti-CD36 antibodies. These data showed that ONA-0-v1, 1G04, and the commercial anti-CD36 antibody specifically bound to human CD36, while the chimeric ONA-0-v2 IgG1 LALA antibody did not specifically bind to human CD36.

[0033] Figure 10 FACS analysis data depicting the binding ability of ONA-0-v1, 1G04, and chimeric ONA-0-v2 IgG1 LALA antibodies relative to commercially available anti-CD36 antibodies to cells overexpressing human CD36 were presented. These data indicate that conversion to chimeric antibody form does not alter the binding of ONO-0 antibodies when tested at a concentration of 100 nM.

[0034] Figure 11A This is a schematic diagram illustrating an experimental overview of the study on the efficacy of the ONA-0-v1 anti-CD36 antibody in a mouse model of oral cancer metastases using FaDu cells, with and without cisplatin. Figure 11B The study details the research groups tested in the study, particularly the treatments and dosages given to each group.

[0035] Figure 12A and 12B This image shows IVIS imaging of the primary tumor from a mouse model of oral cancer metastases using FaDu cells, in a study of the efficacy of the ONA-0-v1 anti-CD36 antibody. Figure 12A ) and H&E staining ( Figure 12BThe results showed that while cisplatin inhibited tumor growth in both tests, treatment with the administered dose of ONA-0-v1 did not have a statistically significant effect on the primary tumor compared to treatment with the isotype control antibody in this model.

[0036] Figure 13A and 13B The results of IVIS imaging of metastases from a mouse model of oral cancer metastases using FaDu cells in a study on the efficacy of the ONA-0-v1 anti-CD36 antibody are shown. These results indicate that treatment with ONA-0-v1 can inhibit the growth of metastases.

[0037] Figure 14 and Figure 15 The results of IVIS imaging of lymph node metastases from a mouse model of oral cancer metastases using FaDu cells are shown. Treatment with the ONA-0-v1 antibody inhibited metastatic tumor growth by more than 50% compared to the IgA isotype control, and the addition of ONA-0-v1 to cisplatin enhanced cisplatin's ability to inhibit metastatic tumor growth.

[0038] Figure 16 The results of IVIS imaging of lymph node metastases from a mouse model of oral cancer metastases using FaDu cells in a study on the efficacy of the ONA-0-v1 anti-CD36 antibody are shown. Treatment with cisplatin or ONA-0-v1 reduced metastases entering the lymph nodes, and the inhibition of exotropy by ONA-0-v1 was synergistic with the inhibition of exotropy by cisplatin.

[0039] Figure 17A and Figure 17B Measurements of body weight and platelet count were included during treatment with ONA-0-v1 and / or cisplatin. These data indicate that, unlike cisplatin, ONA-0-v1 treatment alone had no effect on body weight or platelet count in mice compared to isotype control mice.

[0040] Figure 18A This is a schematic diagram illustrating an experimental overview of the study on the efficacy of the ONA-0-v1 anti-CD36 antibody in an ovarian cancer mouse model using OVCAR-3 cells. Figure 18B Images are of primary tumors removed from test mice in this model. The top row shows tumors from mice injected with the solvent, and the bottom row shows tumors from mice injected with ONA-0-v1. Figure 18C The quantification of the weight of these primary tumors was presented, and it was shown that treatment with ONA-0-v1 resulted in a relative reduction in the weight of the primary tumors (** indicates unpaired t-test, p = 0.033). Figure 18D and Figure 18EHistological analysis results of the percentage of necrosis and the percentage of fibrosis / collagen in primary OVCAR-3 tumors are shown (* indicates unpaired t-test, p = 0.0287). Figure 18D and 18E Treatment with ONA-0-v1 showed that increased necrosis and fibrosis occurred in the tumors analyzed.

[0041] Figure 19A and 19B A representative image of a metastatic tumor formed in a mouse model of ovarian cancer using OVCAR-3 cells is shown. Figure 19A An exemplary metastatic tumor in the peritoneal wall is shown. Figure 19B An exemplary liver metastasis is shown. Each image includes a ruler marked in centimeters and a white arrow pointing to the metastasis.

[0042] Figure 20A , 20B 20C depicts the quantification of the number and size of metastases in the OVCAR-3 mouse model of ovarian cancer in control-treated mice and mice treated with ONA-0-v1. Figure 20A The total number of macroscopic metastases observed in any organ in control (“solvent”) mice (total of all solvent mice; n=9) and mice treated with ONA-0-v1 (total of all treated mice; n=8) is shown, and treatment with ONA-0-v1 reduced the number of metastases by more than 50%. Figure 20B and 20C Macroscopic quantification of the size of metastatic tumors in the peritoneal wall and liver is shown, respectively. Overall, Figure 20A , 20B 20C showed that ONA-0-v1 reduced the size and number of metastatic tumors in an ovarian cancer OVCAR-3 mouse model.

[0043] Figure 21A This is a schematic diagram illustrating an experimental overview of the efficacy study of the ONA-0-v1 anti-CD36 antibody in a mouse model of colon cancer using HCT-116 cells. In vivo quantification of luciferase luminescence from HCT-116 cells was performed during treatment (e.g., Figure 21B As shown; * indicates Mann-Whitney test, p = 0.0288), and in vitro quantification was performed after the experiment (e.g. Figure 21C (As shown). These data demonstrate that treatment with ONA-0-V1 in this colon cancer model reduced the size of the primary tumor.

[0044] Figure 22A , 22BFigures 22C and 22D show the effect of ONA-0-v1 treatment on the penetrance of metastatic tumors that have spread to various organs in a mouse model of colon cancer HCT-116 (as measured by ex vivo luminescence analysis of the organs). Figure 22A and 22B The results showed that treatment with ONA-0-v1 reduced the percentage of liver and lungs with metastases, while more organs were measured to be metastatic (*** indicates p < 0.0001; ** indicates p = 0.0032, two-tailed Fisher exact test).

[0045] Figure 23A , 23B 23C and 23D show the effect of ONA-0-v1 treatment on the number of HCT-116 cells in specific organs (i.e., metastases) in a mouse model of colon cancer, measured by in vitro luciferase-based luminescence analysis. These data demonstrate the effect of ONA-0-v1 treatment on the liver ( Figure 23A ),lung( Figure 23B ),spleen( Figure 23C ) and kidneys ( Figure 23D The light emission in the ) is reduced.

[0046] Figure 24 The effect of ONA-0-v1 treatment on body weight in a mouse model of HCT-116 colon cancer is shown. Mice treated with ONA-0-v1 were better able to maintain their body weight over time.

[0047] Figure 25A , 25B 25C, 25D, 25E, 25F, and 25G show the results of testing the effects of ONA-0-v1 and 1G04 anti-CD36 antibodies in an ovarian cancer OVCAR-3 mouse model relative to control mice. Figure 25A This is a schematic diagram illustrating an overview of the experiments conducted in this study. Figure 25B The changes in body weight of treated mice over time were depicted. Figure 25C-25G The results showed that both ONA-0-v1 and 1G04 reduced the number and size of metastatic tumors in treated mice.

[0048] Figure 26A , 26B 26C, 26D, 26E, 26F, and 26G illustrate the effect of 1G04 treatment on the number of HCT-116 cells in specific organs (i.e. metastases) in a mouse model of colon cancer, as measured by in vitro luciferase-luminescent analysis. Figure 26A This is a schematic diagram illustrating an overview of the experiments conducted in this study. Figure 26B The changes in body weight of treated mice over time were depicted. Figure 26C 1G04 showed that it reduced the total cancer cell burden in treated mice. Figure 26D-26G It was shown that treatment with ONA-0-v1 caused liver damage ( Figure 26D ),lung( Figure 26E ),spleen( Figure 26F ) and kidneys ( Figure 26G The luminescence of the light source is reduced.

[0049] Figure 27A and 27B Data from a luminescent fatty acid uptake assay using an isotype control antibody and 1G04 were presented. The kinetics of fatty acid uptake over time are illustrated. Figure 27A ) and the inhibition of fatty acid uptake at a given time ( Figure 27B ).

[0050] Figure 28A and 28B Data from an ELISA assay were depicted, which tested 1G04 and 1G06 anti-CD36 antibodies against mouse CD36 coated in microplates. Figure 28A ) and human CD36 ( Figure 28B The ability of these antibodies to bind to proteins. These data indicate that both antibodies bind specifically to CD36 in humans and mice in a similar manner.

[0051] Figure 29 The binding of the 1G04 and 1G06 anti-CD36 antibodies to cells overexpressing human CD36 is shown, as measured by FACS analysis. These data indicate that the two antibodies bind specifically to human CD36 in a similar manner.

[0052] Figure 30A , 30B Figures 30C, 30D, and 30E show the results of testing the 1G04 anti-CD36 antibody in a metastatic lung cancer A549 model relative to solvent-treated mice. Figure 30A This is a schematic diagram illustrating an overview of the experiments conducted in this study. Figure 30B The study details the research groups tested in the study, particularly the treatments and dosages given to each group. Figure 30C 1G04 was shown to reduce the total cancer cell load in treated mice, as measured by luminescence. Figure 30D and 30E The results showed a decrease in lung weight and luminescence in the isolated lung after treatment with 1G04.

[0053] Figure 31A , 31B Figures 31C, 31D, and 31E demonstrate the efficacy of 1G04 treatment in an MC38 syngeneic colon cancer model. Figure 31A This is a schematic diagram illustrating an overview of the experiments conducted in this study. Figure 31B The study details the research groups tested in the study, particularly the treatments and dosages given to each group. Figure 31C 1G04 was shown to reduce the total cancer cell load in treated mice, as measured by luminescence. Figure 31D The study showed a reduction in liver luminescence after 1G04 treatment, indicating a decrease in the level of metastatic tumors in the liver. Similarly, Figure 31E The results showed a decrease in lung luminescence after 1G04 treatment, indicating a reduction in the level of lung metastases.

[0054] Figure 32A , 32B The results of 32C demonstrate the efficacy of treating mice with 1G04 antiCD36 antibody carrying 4T1 breast cancer tumors. Figure 32A This is a schematic diagram illustrating an overview of the experiments conducted in this study. Figure 32B The study details the research groups tested in the study, particularly the treatments and dosages given to each group. Figure 32C The results showed that lung luminescence was reduced after 1G04 treatment compared to solvent therapy, indicating a decrease in the level of lung metastases. Detailed Implementation

[0055] This disclosure relates to anti-CD36 antibodies, nucleotides encoding anti-CD36 antibodies, pharmaceutical compositions comprising anti-CD36 antibodies, and methods of treating (e.g., reducing and / or inhibiting) cancer, particularly cancer metastases, using anti-CD36 antibodies. The disclosed anti-CD36 antibodies include both IgA and IgG antibodies, both of which are effective in the disclosed methods of treating cancer. The disclosed anti-CD36 antibodies are effective in treating primary tumors, metastatic cancers, or both primary and metastatic cancers.

[0056] Definitions of general terms and expressions

[0057] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, each term shall have the meaning described below unless otherwise expressly specified herein. Other definitions are set forth throughout the application.

[0058] The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target (e.g., protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof). As used herein, the term "antibody" encompasses polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, recombinant antibodies, bispecific antibodies, fusion proteins containing full-length antibodies or fragments thereof, fragments of such antibodies, and any other modified immunoglobulin molecule, provided it exhibits the desired biological activity. Antibodies can belong to any of the five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), which are based on their respective heavy chain constant domains called α, δ, ε, γ, and μ. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional conformations. Antibodies can be naked or conjugated with other molecules such as toxins, radioisotopes, etc.

[0059] The term "antibody fragment" refers to a portion of a complete antibody. "Antigen-binding fragment," "antigen-binding domain," or "antigen-binding region" refers to a portion of a complete antibody that binds to an antigen. An antigen-binding fragment may contain the antigen-determining region (e.g., complementarity-determining region (CDR)) of the complete antibody. Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single-chain antibodies. Antigen-binding fragments of antibodies can be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans, or can be artificially generated.

[0060] The terms "anti-CD36 antibody," "CD36 antibody," and "CD36-binding antibody" refer to antibodies that bind to CD36 with sufficient affinity, making them suitable for use as diagnostic and / or therapeutic agents targeting CD36. For example, as measured by a radioimmunoassay (RIA), the binding degree of an anti-CD36 antibody to unrelated non-CD36 proteins can be less than approximately 10% of the antibody's binding to CD36.

[0061] The terms "anti-PD-1 antibody," "PD-1 antibody," and "PD-1-binding antibody" refer to antibodies that bind to PD-1 with sufficient affinity, making them suitable for use as diagnostic and / or therapeutic agents targeting PD-1. For example, as measured by radioimmunoassay (RIA), the binding degree of an anti-PD-1 antibody to unrelated non-PD-1 proteins can be less than approximately 10% of the antibody's binding to PD-1.

[0062] "Separated antibodies" refers to a population of antibodies containing antibodies from a single species. For example, a specific isolated anti-CD36 antibody consists of a population of antibodies with a single heavy chain amino acid sequence and a single light chain amino acid sequence, which binds to a single CD36 epitope. However, isolated antibodies that specifically bind to CD36 may be cross-reactive with other antigens, such as CD36 molecules from different species. Furthermore, an antibody population can still be "isolated antibodies" even when contaminated with small amounts of other antibody species. Specifically, isolated antibodies may contain less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or contain no other antibody species.

[0063] "Monoclonal antibody" refers to a group of homologous antibodies or antigen-binding fragments that participate in the highly specific recognition and binding of a single antigenic determinant or epitope. This contrasts with polyclonal antibodies, which typically consist of different antibodies targeting different antigenic determinants. The term "monoclonal antibody" encompasses both full-length and complete monoclonal antibodies, as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins containing antibody moieties, and any other modified immunoglobulin molecules containing antigen recognition sites. Furthermore, "monoclonal antibody" refers to such antibodies and their antigen-binding fragments prepared through various methods, including but not limited to hybridoma, phage selection, recombinant expression, and transgenic animals.

[0064] As used herein, the terms “variable region” or “variable domain” are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, usually a portion of the light or heavy chain, typically about 110 to 120 amino acids or about 110 to 125 amino acids at the amino terminus of the mature heavy chain and about 90 to 115 amino acids in the mature light chain. Variable regions vary considerably in sequence between antibodies and are responsible for the binding and specificity of a particular antibody to its specific antigen. Sequence variability is concentrated in those regions called complementarity-determining regions (CDRs), while highly conserved regions within a variable domain are called scaffold regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of both the light and heavy chains are primarily responsible for antibody-antigen interactions and specificity. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region comprises a rodent or mouse CDR and a human scaffold region (FR). In certain embodiments, the variable region is a primate (e.g., a non-human primate) variable region. In some implementations, the variable region includes a rodent or mouse CDR and a primate (e.g., a non-human primate) skeletal region (FR).

[0065] The terms “VL” and “VL domain” are used interchangeably to refer to the variable region of the light chain of an antibody.

[0066] The terms "VH" and "VH domain" are used interchangeably to refer to the variable region of the heavy chain of an antibody.

[0067] The term "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues in the variable regions of the heavy and light chains of antibodies or their antigen-binding fragments. In some respects, CDRs can be determined according to the Kabat numbering system (see, for example, Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within the antibody heavy chain molecule are typically located at amino acid positions 31 to 35 (which may optionally include one or two additional amino acids after position 35 (referred to as 35A and 35B in the Kabat numbering scheme)) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, the CDRs within the antibody light chain molecule are typically located at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In specific embodiments, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.

[0068] Chothia refers to the position of a structural loop (Chothia and Lesk, J.Mol.Biol.196:901-917 (1987)). When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B exists, the loop ends at 32; if only 35A exists, the loop ends at 33; if both 35A and 35B exist, the loop ends at 34).

[0069] The AbM hypervariable region represents a trade-off between the Kabat CDR and the Chothia structural loop, and is used using OxfordMolecular's AbM antibody modeling software. In specific embodiments, the CDR of the antibodies described herein has been determined according to either the Chothia numbering scheme or the AbM numbering scheme.

[0070] Table 1 – CDR Numbers

[0071]

[0072] In some respects, the CDR region can be determined according to the IMGT numbering system (see, for example, Guidicelli et al., Nucl. Acids Res. 34: D781-D784 (2006); Lefranc et al., Dev. Comp. Immunol. 27: 55-77 (2003)). This numbering scheme unifies the numbering of antibody λ and κ light chains, heavy chains, and T cell receptor chains.

[0073] As used herein, the terms “constant region” and “constant domain” are interchangeable and have their usual meanings in the art. A constant region is an antibody portion, such as the carboxyl-terminal portion of the light and / or heavy chain, that does not directly participate in antibody-antigen binding but can exhibit various effector functions, such as interaction with Fc receptors. Compared to the variable domains of immunoglobulins, the constant regions of immunoglobulin molecules typically have a more conserved amino acid sequence. In some respects, antibody or antigen-binding fragments contain a constant region or a portion sufficient for antibody-dependent cell-mediated cytotoxicity (ADCC).

[0074] As used herein, when referring to antibodies, the term "heavy chain" is based on the amino acid sequence of a constant domain and can refer to any different type, such as α, δ, ε, γ, and μ, which generate IgA, IgD, IgE, IgG, and IgM antibodies, including IgG subclasses (e.g., IgG1, IgG2, IgG3, and IgG4) and IgA subclasses (e.g., IgA1 and IgA2). Heavy chain amino acid sequences are well known in the art. In this specific embodiment, the heavy chain is the human heavy chain.

[0075] As used herein, when referring to antibodies, the term "light chain" is based on the amino acid sequence of a constant domain and can refer to any different type, such as κ or λ. Light chain amino acid sequences are well known in the art. In this specific embodiment, the light chain is a human light chain.

[0076] The term "chimeric antibody" refers to a full-length antibody or its antigen-binding fragment, in which the amino acid sequence is derived from two or more species. Typically, the variable regions of the light and heavy chains correspond to the variable regions of antibodies derived from one mammal (e.g., mouse, rat, rabbit, etc.) that have the desired specificity, affinity, and ability, while the constant regions are homologous to sequences derived from another mammal (usually human) to avoid triggering an immune response in that species.

[0077] "Humanized antibody" refers to a chimeric antibody or antigen-binding fragment thereof comprising amino acid residues derived from a non-human CDR and amino acid residues derived from human backbone regions and constant regions. In some embodiments, the humanized antibody will comprise substantially all of at least one and typically two variable domains, wherein all or substantially all of the CDRs correspond to the CDRs of the non-human antibody, and all or substantially all of the FRs correspond to the FRs of the human antibody. The humanized antibody may optionally comprise at least a portion of the antibody constant region derived from a human antibody. The "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization. Typically, humanized antibodies are human immunoglobulins in which residues from the CDR are replaced by residues from a non-human species (e.g., mouse, rat, rabbit, hamster) with the desired specificity, affinity, and ability (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). Therefore, humanized antibodies are also called "CDR transplantation" antibodies. Examples of methods for generating humanized antibodies are described in U.S. Patent 5,225,539; Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994) and Roguska et al., Protein Eng. 9(10):895-904 (1996).

[0078] "Human antibody" refers to a full-length antibody or fragment thereof having variable regions in which both the FR and CDR are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, that constant region is also derived from a human germline immunoglobulin sequence. Human antibodies of this disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human backbone sequence. The terms "human antibody" and "fully human antibody" are used synonymously.

[0079] A “non-fucosylated” antibody or its antigen-binding fragment, or a “fucosylated” antibody or its antigen-binding fragment, refers to an IgG1 or IgG3 isotype antibody or its antigen-binding fragment lacking any fucose residues in the constant-region glycosylation at least 50% of the antibody population. Glycosylation of human IgG1 or IgG3 occurs at Asn297 as a core fucosylated biantennary complex oligosaccharide glycosylation, terminating with up to two Gal residues. In some embodiments, non-fucosylated antibodies lack fucose at Asn297. Depending on the amount of terminal Gal residues, these structures are designated as G0, G1 (1,6, or 1,3), or G2 glycan residues, see, for example, Raju, TS, BioProcess Int. 1:44-53 (2003). CHO-type glycosylation of antibody Fc is described, for example, Routier, F. FL, Glycoconjugate J. 14:201-207 (1997).

[0080] Methods for measuring fucose include any methods known in the art. For the purposes of this document, fucose can be detected by the method described in Example 1 of WO2015 / 017600 (which is incorporated herein by reference in its entirety). Briefly, glycan analysis can be performed by releasing glycans from antibodies (e.g., via enzymatic release), labeling the glycans with anthranilic acid (2-AA), and then purifying the labeled glycans. Normal-phase HPLC with fluorescence detection is used to separate the glycans and measure the relative amount of each glycan in the antibody. Glycans can be definitively identified as lacking or containing fucose by mass spectrometry. In some embodiments, fucose is undetectable in compositions comprising multiple non-fucosylated antibodies. In some embodiments, the non-fucosylated antibodies have enhanced ADCC activity, which can be measured by the assay provided in Example 12 herein. In some embodiments, the non-fucosylated antibodies have enhanced affinity for FcγRIIIA. In some embodiments, the non-fucosylated antibody has enhanced affinity for FcγRIIIA (V158). Affinity for FcγRIIIA or its alleles can be measured using the assay provided in Example 10 herein.

[0081] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, "binding affinity" as used herein refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be expressed using the dissociation constant (K0). DAffinity can be measured and / or represented in a variety of ways known in the art, including but not limited to the equilibrium dissociation constant (K). D ) and equilibrium association constant (K A K D By k off / k on The quotient is calculated, and K A By k on / k off The quotient is calculated from k. on This refers to, for example, the association rate constant between an antibody and an antigen, and k off This refers to, for example, the dissociation of antibodies from antigens. on and k off It can be determined using techniques known to those skilled in the art, for example... Or KinExA.

[0082] As used herein, "epitope" is a term in the art and refers to a localized region of an antigen that an antibody can specifically bind to. An epitope can be, for example, a continuous amino acid of a polypeptide (linear or continuous epitope), or an epitope can be, for example, derived together from two or more discontinuous regions of a polypeptide (conformally nonlinear, discontinuous, or non-linear epitopes). In some embodiments, antibody-bound epitopes can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any method known in the art (e.g., Giegé R et al., (1994) Acta Crystallogr D Biol Crystallogr 50 (Pt4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303). Antibody crystals bound to antigens can be studied using well-known X-ray diffraction techniques and optimized using computer software such as X-PLOR (Yale University, 1992, published by Molecular Simulations, Inc.; see, for example, MethEnzymol (1985), vols. 114 and 115, edited by Wyckoff HW et al.; US2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed. Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323). Mutagenesis mapping studies can be performed using any method known to those skilled in the art. See, for example, descriptions of mutagenesis techniques, including alanine scanning mutagenesis, in Champe M et al., (1995) J BiolChem 270:1388-1394 and Cunningham BC & Wells JA (1989) Science 244:1081-1085.

[0083] A CD36 antibody that "binds to the same epitope" as a reference CD36 antibody is an antibody that binds to the same CD36 amino acid residues as the reference CD36 antibody. The ability of a CD36 antibody to bind to the same epitope as a reference CD36 antibody can be determined by hydrogen / deuterium exchange assay (see Coales et al. Rapid Commun. Mass Spectrom. 2009; 23:639–647), FACS analysis combined with alanine scanning, cross-linked mass spectrometry (XL-MS), peptide scanning, or mutagenesis.

[0084] As used herein, the terms “immunospecific binding,” “immunospecific recognition,” “specific binding,” and “specific recognition” are similar terms in the context of antibodies. These terms indicate that an antibody binds to an epitope via its antigen-binding domain, and that this binding requires some complementarity between the antigen-binding domain and the epitope. Thus, an antibody that “specifically binds” to human CD36 (SEQ ID NO: 1) may also bind to CD36 from other species (e.g., non-human primates, mouse, and / or rat CD36) and / or CD36 proteins produced by other human alleles, but to less than about 10% of the binding to unrelated non-CD36 proteins, as measured, for example, by radioimmunoassay (RIA).

[0085] In specific implementations, this article provides antibodies that bind to CD36 in humans, cynomolgus monkeys, mice, and rats.

[0086] If an antibody preferentially binds to an epitope or overlapping epitope to such an extent that it partially blocks the binding of a reference antibody to the epitope, then the antibody is said to "competitively inhibit" the binding of the reference antibody to the given epitope. Competitive inhibition can be determined by any method known in the art, such as a competitive ELISA assay or a competitive FACS. It can be said that the antibody competitively inhibits at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% of the binding of the reference antibody to the given epitope.

[0087] As used herein, "substantially free" means that the substance is almost completely or completely absent. For example, a pharmaceutical composition that is substantially free of a particular antibody type means that the antibody type is almost completely or completely absent in the pharmaceutical composition under discussion. In this context, "substantially free" can mean that the antibody in the pharmaceutical composition is less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or that there is no antibody of the antibody type under discussion. Furthermore, "substantially free" contaminants can mean that the composition has been purified to contain very little other cellular material and / or chemicals (e.g., less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or no other cellular material and / or chemicals).

[0088] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers having amino acids of any length. Polymers may be linear or branched, may contain modified amino acids, and may have non-amino acids inserted. The term also covers amino acid polymers that are naturally occurring or modified by interventions such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation or modification, such as conjugation with a labeled component. This definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids) and other modifications known in the art. It should be understood that because the polypeptides of the present invention are antibody-based, in some embodiments, the polypeptide may be present as a single chain or an associated chain.

[0089] "Identity percentage" refers to the degree of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences). The identity percentage can be determined by aligning two sequences, introducing vacancies to maximize the identity between them. Alignments can be generated using procedures known in the art. For the purposes of this document, nucleotide sequence alignment can be performed using the blastn procedure with default parameters, and amino acid sequence alignment can be performed using the blastp procedure with default parameters (see National Center for Biotechnology Information (NCBI) on the World Wide Web, ncbi.nlm.nih.gov).

[0090] As used herein, the term "host cell" can be any type of cell, such as primary cells, cells in culture medium, or cells derived from a cell line. In specific embodiments, the term "host cell" refers to a cell transfected with a nucleic acid molecule and the offspring or potential offspring of such cells. For example, the offspring of such cells may differ from the parent cells transfected with nucleic acid molecules due to mutations or environmental influences that may occur in subsequent generations or the integration of nucleic acid molecules into the host cell genome.

[0091] The terms "pharmaceutical composition" and "pharmaceutical formulation" refer to preparations in a form in which the biological activity of the active ingredient is therapeutically effective, and which do not contain any additional components that would have unacceptable toxicity to the subject to which the composition or formulation is administered. The composition or formulation may be sterile.

[0092] As used herein, the term "administer" (administering, administration, etc.) refers to a method that enables the delivery of a drug (e.g., an anti-CD36 antibody) to a desired biological site of action. Administration techniques that can be used with the pharmaceuticals and methods described herein are found, for example, in Goodman and Gilman, *The Pharmacological Basis of Therapeutics*, current edition, Pergamon; and Remington's, Pharmaceutical Sciences, current edition, Mack Publishing, Easton, Pa. Administration means the physical introduction of a composition containing a therapeutic agent into a target using any of the various methods and delivery systems known to those skilled in the art. Preferred routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other non-parenteral routes, such as by injection or infusion. As used herein, the phrase "parenteral administration" refers to administration other than enteral and local administration, typically by injection, and including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intralesional, intracystic, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as intracorporeal electroporation. In some embodiments, the formulation is administered via a non-parenteral route, preferably orally. Other non-parenteral routes include local, epidermal, or mucosal administration, such as intranasal, vaginal, rectal, sublingual, or local administration. Administration may also be performed, for example, once, multiple times, and / or over one or more durations.

[0093] Administering in combination with one or more other therapeutic agents includes administering them simultaneously (in parallel) or consecutively in any order.

[0094] Combination therapy can provide a “synergistic effect,” meaning that the combined effect of using active agents together is greater than the sum of the effects of using active agents alone. A synergistic effect can be achieved when the active agents are: (1) co-formulated and administered or delivered simultaneously in a combined unit-dose formulation; (2) delivered sequentially, alternately, or in parallel as individual formulations; or (3) through other regimens. A synergistic effect can also be achieved when delivered in alternating regimens, where the active agents are administered or delivered sequentially, for example, by different injections using different syringes. The effect of a “synergistic combination” is greater than the sum of the effects of the individual active agents in the combination.

[0095] Combination therapy can provide an "additive" effect, meaning that the effect achieved when active agents are used together is equal to the sum of the effects of using the active agents alone.

[0096] As used herein, the terms "object" and "patient" are used interchangeably. An object can be an animal. In some embodiments, the object is a mammal, such as a non-human animal (e.g., a cow, pig, horse, cat, dog, rat, mouse, monkey, or other primate). In some embodiments, the object is a cynomolgus monkey. In some embodiments, the object is a human.

[0097] The term "therapeuticly effective amount" refers to the amount of a drug, such as an anti-CD36 antibody, that effectively achieves the desired therapeutic or preventative outcome. In some cases, the desired outcome is the disease or condition of the treated subject. In the case of cancer, a therapeutically effective amount of a drug can: reduce the number of cancer cells; reduce the number of cancer cells; reduce tumor size or burden; inhibit (i.e., to some extent slow down, and in some embodiments, stop) cancer cell infiltration into peripheral organs; inhibit (i.e., to some extent slow down, and in some embodiments, stop) tumor metastasis; inhibit tumor growth to some extent; alleviate one or more cancer-related symptoms to some extent; and / or result in a favorable response, such as progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), or in some cases, an increase in stable disease (SD), a decrease in progressive disease (PD), a shortening of time to progression (TTP), or any combination thereof. Insofar as the drug can prevent the growth and / or kill existing cancer cells, it can be cytotoxic and / or cellular.

[0098] Terms such as “treating” or “alleviating” or “to alleviate” refer to therapeutic measures that cure, slow down, alleviate, or reduce the symptoms of a diagnosed pathological condition or symptom and / or stop the progression of a diagnosed pathological condition or symptom. Therefore, those who require treatment include those who have been diagnosed with or are suspected of having the condition. In some implementations, the method according to the invention is considered to have successfully “treated” the cancer of the target if the patient exhibits one or more of the following: a reduction or complete absence of cancer cells; a reduction in tumor size; suppression or absence of cancer cell infiltration into peripheral organs, including, for example, suppression or absence of cancer spread to soft tissues and bone; suppression or absence of tumor metastasis; suppression or absence of tumor growth; relief of one or more symptoms associated with the specific cancer; a reduction in morbidity and mortality; an improvement in quality of life; a reduction in tumorigenicity, frequency, or capacity; a reduction in the number or frequency of cancer stem cells in the tumor; differentiation of tumorigenic cells into a non-tumorigenic state; an increase in progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), stable disease (SD), a reduction in progressive disease (PD), a shortening of time to progression (TTP), or any combination thereof. In the context of metastatic cancer, treatment also refers to preventing the development of new metastatic tumors, reducing the size of metastatic tumors, or eliminating existing metastatic tumors.

[0099] "Cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. "Cancer" or "cancer tissue" can include tumors. Uncontrolled cell division and growth lead to the formation of malignant tumors that invade adjacent tissues and can also metastasize to distant sites of the body via the lymphatic system or bloodstream. After metastasis, a distant tumor can be said to "derive" from the pre-metastatic tumor. Such distant tumors are also called "metastatic tumors" or "metastatic tumors."

[0100] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in connection with this disclosure. For example, the Concise Dictionary of Biomedicine and Molecular Biology (Juo, Pei-Show, 2nd edition, 2002, CRC Press); The Dictionary of Cell and Molecular Biology (3rd edition, 1999, Academic Press); and the Oxford Dictionary of Biochemistry and Molecular Biology (Revised Edition, 2000, Oxford University Press) provide general dictionaries for those skilled in the art of the use of many of the terms used in this disclosure.

[0101] Units, prefixes, and symbols are represented in their forms accepted by the International System of Units (SI). Numerical ranges include those defining the range. The headings provided herein are not intended to limit any aspect of this disclosure, which can be obtained by referring to the entire specification. Therefore, the terms defined immediately thereafter are more fully defined by reference to the entire specification.

[0102] Unless otherwise expressly provided, the singular forms “a,” “an,” and “the” used in this disclosure and claims include the plural forms.

[0103] It should be understood that wherever the language “comprising” is used herein to describe an embodiment, similar embodiments described as “consisting of” and / or “substantially consisting of” are also provided. In this disclosure, “comprises”, “containing”, and “having” can have the meanings given to them under U.S. patent law, and can mean “includes”, etc.; “substantially consisting of” or “substantially consisting of” also have the meanings given to them under U.S. patent law, and the term is open-ended, allowing for more content than is cited, provided that the essential or novel features of the cited content are not altered by the presence of more content than listed, excluding prior art embodiments.

[0104] Unless otherwise specified or apparent from the context, as used herein, the term “or” is understood to be inclusive. The term “and / or” as used in phrases such as “A and / or B” is intended to include “A and B”, “A or B”, “A”, and “B”. Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0105] The terms “about” or “substantially include” refer to a value or combination within an acceptable range of error for a particular value or combination as determined by a person skilled in the art, which will depend in part on how the value or combination is measured or determined, i.e., the limitations imposed on the measurement system. For example, “about” or “substantially include” may mean within one or more standard deviations according to practice in the art. Alternatively, “about” or “substantially include” may mean a range up to 20%. Furthermore, particularly with respect to biological systems or processes, these terms may mean up to an order of magnitude or up to five times the value. When a particular value or combination is provided in the application and claims, unless otherwise stated, the meaning of “about” or “substantially include” should be assumed to be within an acceptable range of error for that particular value or combination.

[0106] Any composition or method provided herein may be combined with one or more of any other compositions and methods provided herein.

[0107] Anti-CD36 antibody

[0108] In one specific aspect, this document provides full-length antibodies (e.g., monoclonal antibodies, such as chimeric antibodies, humanized antibodies, or human antibodies) that specifically bind to CD36 (e.g., human CD36) and their antigen-binding fragments. The amino acid sequences of human, cynomolgus monkey, rhesus monkey, mouse, and rat CD36 are known in the art and are also provided herein as shown in SEQ ID NO:1-4, as follows.

[0109] Human CD36 (SEQ ID NO:1; UNIPROT P16671):

[0110] MGCDRNCGLIAGAVIGAVLAVFGGILMPVGDLLIQKTIKKQVVLEEGTIAFKNWVKTGTEVYRQFWIFDVQNPQEVMMNSSNIQVKQRGPYTYRVRFLAKENVTQDAEDNTVSFLQPNGAIFEPSLSVGTEADNFTVLNLAVAAASHIYQNQFVQMILNSLINKSKSSMFQVRTLRELLWGYRDPFLSLVPYPVTTTVGLFYPYNNTADGVYKVFNGKDNISKVAIIDTYKGKRNLSYWESHCDMINGTDAASFPPFVEKSQVLQFFSSDICRSIYAVFESDVNLKGIPVYRFVLPSKAFASPVENPDNYCFCTEKIISKNCTSYGVLDISKCKEGRPVYISLPHFLYASPDVSEPIDGLNPNEEEHRTYLDIEPITGFTLQFAKRLQVNLLVKPSEKIQVLKNLKRNYIVPILWLNETGTIGDEKANMFRSQVTGKINLLGLIEMILLSVGVVMFVAFMISYCACRSKTIK

[0111] Cynomolgus monkey / Rhesus monkey CD36 (SEQ ID NO: 2; UNIPROT Q4R6B4 and Q6J512, respectively):

[0112] MGCDRNCGLITGAVIGAVLAVFGGILMPVGDMLIQKTIKKEVVLEEGTIAFKNWVKTGTEIYRQFWIFDVQNPQEVMMNSSNIQVKQRGPYTYRVRFLAKENITQDPKDNTVSFLQPNGAIFEPSLSVGTEADNFTVLNLAVAAASHIYPNPFVQVVLNSLINKSKSSMFQVRTLRELLWGYTDPFLSLVPYPVSTRVGMFYPYNNTADGVYKVFNGKDSISKVAIIDTYKGKRNLSYWESYCDMINGTDAASFPPFVEKSQVLQFFSSDICRSIYAVFESDVNLKGIPVYRFVLPSKAFASPVQNPDNHCFCTEKIISKNCTSYGVLDISKCKEGKPVYISLPHFLYASPDVSETIDGLNPNEEEHRTYLDIEPITGFTLQFAKRLQVNLLVKPSNKIQVLKRLKRNYIVPILWLNETGTIGDEKAKMFRSQVTGKINLLGLIEMILLSVGVVMFVAFMISYCACRSKTIK

[0113] Mouse CD36 (SEQ ID NO: 3; UNIPROT Q08857):

[0114] MGCDRNCGLIAGAVIGAVLAVFGGILMPVGDMLIEKTIKREVVLEEGTTAFKNWVKTGTTVYRQFWIFDVQNPDDVAKNSSKIKVKQRGPYTYRVRYLAKENITQDPEDHTVSFVQPNGAIFEPSLSVGTEDDNFTVLNLAVAAAPHIYQNSFVQVVLNSLIKKSKSSMFQTRSLKELLWGYKDPFLSLVPYPISTTVGVFYPYNDTVDGVYKVFNGKDNISKVAIIESYKGKRNLSYWPSYCDMINGTDAASFPPFVEKSRTLRFFSSDICRSIYAVFGSEIDLKGIPVYRFVLPANAFASPLQNPDNHCFCTEKVISNNCTSYGVLDIGKCKEGKPVYISLPHFLHASPDVSEPIEGLHPNEDEHRTYLDVEPITGFTLQFAKRLQVNILVKPARKIEALKNLKRPYIVPILWLNETGTIGDEKAEMFKTQVTGKIKLLGMVEMALLGIGVVMFVAFMISYCACKSKNGK

[0115] Rat CD36 (SEQ ID NO: 4; UNIPROT Q07969):

[0116] MGCDRNCGLITGAVIGAVLAVFGGILMPVGDLLIEKTIKREVVLEEGTIAFKNWVKTGTTVYRQFWIFDVQNPEEVAKNSSKIKVKQRGPYTYRVRYLAKENITQDPKDSTVSFVQPN GAIFEPSLSVGTENDNFTVLNLAVAAAPHIYTNSFVQGVLNSLIKKSKSSMFQTRSLKELLWGYKDPFLSLVPYPISTTVGVFYPYNNTVDGVYKVFNGKDNISKVAIIDTYKGKRNL SYWESYCDMINGTDAASFPPFVEKSQTLRFFSDICRSIYAVFESEVNLKGIPVYRFVLPANAFASPLQNPDNHCFCTEKVISNNCTSYGVLDIGKCKEGKPVYISLPHFLHASPDVS EPIEGLNPNEDEHRTYLDVEPITGFTLQFAKRLQVNILVKPARKIEALKNLKRPYIVPILWLNETGTIGDEKAEMFRNQVTGKIKLLGLVEMVLLGVGVVMFVAFMISYCACRSKNGK

[0117] In some embodiments, the antibody described herein binds to human CD36. In some embodiments, the antibody binds to human and cynomolgus monkey CD36. In some embodiments, the antibody binds to human and mouse CD36. In some embodiments, the antibody binds to human, mouse, and rat CD36. In some embodiments, the antibody binds to human, cynomolgus monkey, mouse, and rat CD36.

[0118] The anti-CD36 antibody of the present invention comprises a full-length antibody, a single-chain antibody, and an scFv, Fab, or F(ab')2 fragment. In some embodiments, the anti-CD36 inhibitor is a full-length antibody. In some embodiments, the CD36 inhibitor is a humanized antibody. In some embodiments, the CD36 inhibitor is a human antibody. In some embodiments, the anti-CD36 antibody is ONA-O-v1 or ONA-O-v2. The amino acid sequence of ONA-O-v1 is provided as SEQ ID NO:5 (heavy chain) and SEQ ID NO:7 (light chain). The amino acid sequence of ONA-O-v2 is provided as SEQ ID NO:5 (heavy chain) and SEQ ID NO:9 (light chain). The ONA-O-v1 and ONA-O-v2 antibodies share the same constant region and the same heavy chain variable region, but differ in that they contain different light chain variable regions. Figure 5 Schematic diagrams of ONA-0-v1 and ONA-0-v2 are provided.

[0119] Embodiments of the present invention also include antibody fragments derived from ONA-O-v1 or ONA-O-v2, including but not limited to Fab, Fab', F(ab')2, single-chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, intracellular antibody, IgGΔCH2, microantibody, F(ab')3, tetraantibody, triantibody, biantibody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc. The antibody fragments can be generated using any technique known to those skilled in the art. In some embodiments, the antibody fragment further includes a portion that extends the in vivo half-life of the antibody. This portion is also referred to as a "half-life extension portion." Any portion known to those skilled in the art for extending the in vivo half-life of an antibody fragment can be used. For example, the half-life extension portion may include an Fc region, a polymer, albumin or albumin-binding protein, or a compound. The polymer may include natural or synthetic, optionally substituted linear or branched polyalkylene, polyolefin, polyoxyethylene, polysaccharide, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, methoxy polyethylene glycol, lactose, amylose, dextran, glycogen, or derivatives thereof. Substituents may include one or more hydroxyl, methyl, or methoxy groups. In some embodiments, Fab, Fab', F(ab')2, or scFv may be modified by adding one or more C-terminal amino acids to attach a half-life extension moiety. In some embodiments, the half-life extension moiety is polyethylene glycol or human serum albumin. In some embodiments, Fab, Fab', F(ab')2, or scFv is fused to the Fc region.

[0120] In some embodiments, the antibody binds to CD36 and comprises one or more CDRs of ONA-O-v1 (as determined by the Chothia, Kabat, or IMGT antibody numbering scheme). In some embodiments, the antibody is a humanized antibody comprising one or more of the six CDRs in SEQ ID NO:27-32. In some embodiments, the antibody is a humanized antibody comprising one or more of the six CDRs in SEQ ID NO:37, 38, and 29-32. In some embodiments, the antibody is a humanized antibody comprising one or more of the six CDRs in SEQ ID NO:39-43 and 32. In some embodiments, the heavy chain sequence comprises CDR regions GYTFTDY (heavy chain CDR1; SEQ ID NO:27), YPGSGN (heavy chain CDR2; SEQ ID NO:28), and GIGGGFGMDY (heavy chain CDR3; SEQ ID NO:29). In some embodiments, the heavy chain sequence comprises the CDR regions DYYIN (heavy chain CDR1; SEQ ID NO:37), RIYPGSGNTYYNEKFKG (heavy chain CDR2; SEQ ID NO:38), and GIGGGFGMDY (heavy chain CDR3; SEQ ID NO:29). In some embodiments, the heavy chain sequence comprises the CDR regions GYTFTDYY (heavy chain CDR1; SEQ ID NO:39), IYPGSGNT (heavy chain CDR2; SEQ ID NO:40), and ARGIGGGFGMDY (heavy chain CDR3; SEQ ID NO:41). In some embodiments, the light chain variable region comprises the CDR regions KASQSVSDDVA (light chain CDR1; SEQ ID NO:30), YASNRYT (light chain CDR2; SEQ ID NO:31), and QQDYSSPLT (light chain CDR3; SEQ ID NO:32). In some implementations, the light chain variable region includes CDR regions QSVSDD (light chain CDR1; SEQ ID NO:42), YAS (light chain CDR2; SEQ ID NO:43), and QQDYSSPLT (light chain CDR3; SEQ ID NO:32).

[0121] In some embodiments, the antibody binds to CD36 and comprises a variant of one or more CDRs of ONA-O-v1 (as determined by the Chothia, Kabat, or IMGT antibody numbering scheme). In some embodiments, the antibody contains DYYMH (SEQ ID NO:44) or DYYMN (SEQ ID NO:45) as a variant of the ONA-O-v1 heavy chain CDR1 region. In some embodiments, the antibody contains RIYPGSGNTYYNEKFQG (SEQ ID NO:46) or RIYPGSGNTYYNEKFTG (SEQ ID NO:47) as a variant of the ONA-O-v1 heavy chain CDR2 region. In some embodiments, the antibody contains QASQSVSDDVA (SEQ ID NO:48) as a variant of the ONA-O-v1 light chain CDR1 region. In some embodiments, the antibody contains YASNLYT (SEQ ID NO:49) or YASNRYS (SEQ ID NO:50) as a variant of the ONA-O-v1 light chain CDR2 region.

[0122] In some embodiments, the antibody is a humanized antibody comprising one or more CDRs of ONA-0-v1 or variants of ONA-0-v1 CDRs (as determined by the Chothia, Kabat, or IMGT antibody numbering scheme). Exemplary embodiments of antibodies comprising one or more CDRs of ONA-0-v1 or variants of ONA-0-v1 CDRs (as determined according to the Kabat numbering scheme) are provided in Table 2 below.

[0123] Table 2 – CDRs in ONA-0-1 and humanized variants of ONA-0-v1

[0124]

[0125]

[0126] In some embodiments, the antibody is a humanized antibody comprising a humanized variant of the ONA-0-v1 antibody. In some embodiments, the humanized variant of the ONA-0-v1 antibody comprises a humanized heavy chain variable region comprising SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54. In some embodiments, the humanized variant of the ONA-0-v1 antibody comprises a humanized light chain variable region comprising SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58. In some embodiments, the humanized variant of the ONA-0-v1 antibody comprises a humanized heavy chain variable region containing SEQ ID NO:51 and a humanized light chain variable region containing SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58. In some embodiments, the humanized variant of the ONA-0-vl antibody includes a humanized heavy chain variable region containing SEQ ID NO:52 and a humanized light chain variable region containing SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58. In some embodiments, the humanized variant of the ONA-0-vl antibody includes a humanized heavy chain variable region containing SEQ ID NO:53 and a humanized light chain variable region containing SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58. In some embodiments, the humanized variant of the ONA-0-vl antibody includes a humanized heavy chain variable region containing SEQ ID NO:54 and a humanized light chain variable region containing SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58.

[0127] In some embodiments, the antibody binds to CD36 and comprises one or more of the six CDRs of ONA-O-v2 (as determined by the Chothia, Kabat, or IMGT antibody numbering scheme). In some embodiments, the antibody is a humanized antibody comprising one or more of the six CDRs listed in SEQ ID NO:27-29 and 33-35. In some embodiments, the heavy chain sequence comprises the CDR regions GYTFTDY (heavy chain CDR1; SEQ ID NO:27), YPGSGN (heavy chain CDR2; SEQ ID NO:28), and GIGGGFGMDY (heavy chain CDR3; SEQ ID NO:29). In some embodiments, the light chain variable region comprises the CDR regions KASENVVTYVS (light chain CDR1; SEQ ID NO:33), GASNRYT (light chain CDR2; SEQ ID NO:34), and GQGYSYPYT (light chain CDR3; SEQ ID NO:35). In some implementations, the antibody is a humanized antibody containing one or more of the six CDRs of ONA-O-v2.

[0128] In some embodiments, the antibody described herein binds to human CD36 and comprises the ONA-0-v1 VH sequence provided as SEQ ID NO:11. In some embodiments, the antibody binds to human CD36 and comprises the ONA-0-v1 VL sequence provided as SEQ ID NO:13. In some embodiments, the antibody binds to human CD36 and comprises the VL sequence provided as SEQ ID NO:20. In some embodiments, the antibody is a chimeric antibody comprising the VH sequence provided as SEQ ID NO:11 and the VL sequence provided as SEQ ID NO:13. In some embodiments, the antibody is a chimeric antibody comprising the heavy chain sequence provided as SEQ ID NO:21 and the light chain provided as SEQ ID NO:23, such as the 1G04 antibody. In some embodiments, the antibody is a chimeric antibody comprising the heavy chain sequence provided as SEQ ID NO:64 and the light chain provided as SEQ ID NO:23, such as the 1G06 antibody.

[0129] In some embodiments, the anti-CD36 antibody is a bispecific antibody. The term "bispecific" means that the antibody in question can specifically bind to at least two different epitopes or antigens. Typically, a bispecific antibody contains two antigen-binding sites, each specific to a different epitope or antigen. Thus, in some embodiments, the bispecific anti-CD36 antibody also binds to a second epitope or antigen. In some embodiments, the bispecific antibody specifically binds to CD36 and specifically binds to a T cell receptor antigen. In some embodiments, the bispecific antibody specifically binds to CD36 and specifically binds to CD3. In some embodiments, the bispecific anti-CD36 antibody comprises one or more CDRs from the ONA-O-v1 antibody. Embodiments of the invention include methods for recruiting T cells to tumors using such bispecific antibodies. In some embodiments of these methods, the recruited T cells lyse tumor cells while bypassing antigen presentation via the major histocompatibility complex. Exemplary methods for preparing and using bispecific antibodies can be found in WO2016 / 141287A1, which is incorporated herein by reference in its entirety.

[0130] Table 3 below provides the amino acid sequences associated with the ONA-0-v1 antibody, the ONA-0-v2 antibody, and other embodiments.

[0131] Table 3 – Amino Acid Sequences

[0132]

[0133]

[0134]

[0135]

[0136] In some embodiments, the antibody described herein binds to CD36, comprises six CDRs of ONA-0-v1 (i.e., SEQ ID NOs: 27-32), and comprises VH and VL, wherein the VH comprises at least 80% identical sequence to the VH sequence of ONA-0-v1 (SEQ ID NO: 11), and the VL comprises at least 80% identical sequence to the VL sequence of ONA-0-v1 (SEQ ID NO: 13). In some embodiments of these embodiments, the antibody comprises a VH containing at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the VH sequence of ONA-0-v1 (SEQ ID NO: 11). In some of these embodiments, the antibody comprises a VL containing at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the VL sequence (SEQ ID NO: 13) of ONA-0-v1.

[0137] In some embodiments, the antibody described herein binds to CD36, comprises six CDRs of ONA-0-v1 (i.e., SEQ ID NOs: 27-32), and comprises a heavy chain and a light chain, the heavy chain comprising at least 80% identical sequence to the heavy chain sequence of ONA-0-v1 (SEQ ID NO: 5), and the light chain comprising at least 80% identical sequence to the light chain sequence of ONA-0-v1 (SEQ ID NO: 7). In some embodiments of these embodiments, the antibody comprises a heavy chain containing at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the heavy chain sequence of ONA-0-v1 (SEQ ID NO: 5). In some of these embodiments, the antibody comprises a light chain containing at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the heavy chain sequence (SEQ ID NO:7) of ONA-0-v1.

[0138] In another respect, this article provides antibodies that bind to the same CD36 epitope (e.g., human CD36 epitope) as the antibodies described herein (e.g., ONA-0-v1).

[0139] Competitive binding assays can be used to determine whether two antibodies bind to overlapping epitopes. Competitive binding can be determined in an assay that inhibits the specific binding of the test immunoglobulin to a reference antibody and a common antigen such as CD36. There are many types of competitive binding assays, such as: competitive FACS; solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competitive assay (see Stahli C et al., (1983) Methods Enzymol 9:242-253); solid-phase direct biotin-avidin EIA (see Kirkland TN et al., (1986) J Immunol 137:3614-9); solid-phase direct labeling assay, solid-phase direct labeling sandwich assay (see Harlow E & Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor); solid-phase direct labeling RIA using I-125 labeling (see Morel GA et al., (1988) Mol Immunol 25(1):7-15); solid-phase direct biotin-avidin EIA (Cheung RC et al.). (al., (1990) Virology 176:546-52); and directly labeled RIA (Moldenhauer G et al., (1990) Scand J Immunol 32:77-82). Typically, such assays involve the use of a purified antigen (e.g., CD36, such as human CD36) that binds to a solid surface or a cell carrying either an unlabeled test immunoglobulin or a labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of labeling bound to the solid surface or cell in the presence of the test immunoglobulin. Typically, the test immunoglobulin is present in excess. Typically, when a competitive antibody is present in excess, it inhibits the specific binding of the reference antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or more. Competitive binding assays can be configured using labeled antigens or labeled antibodies in a wide variety of different forms. In a common version of this assay, the antigen is immobilized on a 96-well plate. The ability of the unlabeled antibody to block the binding of the labeled antibody to the antigen is then measured using either radiolabeling or enzyme labeling.For further details, see, for example, Wagener C et al., (1983) J Immunol 130:2308-2315; Wagener C et al., (1984) J Immunol Methods 68:269-274; Kuroki M et al., (1990) Cancer Res 50:4872-4879; Kuroki M et al., (1992) Immunol Invest 21:523-538; Kuroki M et al., (1992) Hybridoma 11:391-407 and, as mentioned above, Antibodies: Laboratory Manual, edited by Ed Harlow E & Lane D, pp. 386-389.

[0140] In one implementation, surface plasmon resonance is used. Competitive assays are performed, for example by a “tandem method,” such as that described in Abdiche YN et al., (2009) Analytical Biochem 386:172-180, whereby the CD36 antigen is immobilized on the surface of a chip (e.g., a CM5 sensor chip), and then an anti-CD36 antibody is run on that chip. To determine whether an antibody competes with the anti-CD36 antibody described herein, the anti-CD36 antibody is first run on the chip surface to saturation, and then a potential competing antibody is added. The binding of the competing antibody can then be determined and quantified relative to a non-competitive control.

[0141] In one implementation, Fortebio Octet competitive binding is used to determine whether a CD36 antibody competitively inhibits the binding of another CD36 antibody to CD36.

[0142] In another aspect, this document provides competitive inhibition (e.g., in a dose-dependent manner) of the binding of the antibody described herein (e.g., ONA-0-v1) to CD36 (e.g., human CD36), as determined using an assay known to those skilled in the art or described herein (e.g., competitive ELISA assay, or suspension array, or surface plasmon resonance assay).

[0143] Preferably, anti-CD36 antibodies regulate CD36 activity, antagonizing or blocking CD36. Antibodies that block or inhibit CD36 activity can be full-length antibodies. Antibody analogs or fragments, such as single-chain antibodies, single-chain variable domain fragments (scFv), F(ab')2 fragments (obtainable by pepsin digestion of antibody molecules), or Fab fragments (obtainable by reducing the disulfide bonds of the F(ab')2 fragment), can also be used. When the subject is human, humanized antibodies can be used.

[0144] Because CD36 has several known functions, antibodies can be selected to inhibit all known functions of CD36, including its interactions with platelet-reactive proteins, collagen, and fatty acids, or to inhibit only specific functions of CD36 (e.g., blocking only the uptake of fatty acids and oxidized LDL). Therefore, in some embodiments, anti-CD36 antibodies block CD36-mediated uptake of fatty acids and / or oxidized LDL. In some embodiments, anti-CD36 antibodies block CD36-mediated uptake of fatty acids and / or oxLDL while having little or no effect on CD36 binding to TSP-1. And in some embodiments, anti-CD36 antibodies block CD36-mediated uptake of fatty acids and / or oxLDL while having little or no effect on CD36's role as a ligand for TSP-1. In some embodiments, anti-CD36 antibodies block at least about 10%, at least about 15%, at least about 20%, at least about 25%, or at least about 30% of CD36-mediated uptake of fatty acids and / or oxidized LDL relative to an untreated control. In some implementations, the anti-CD36 antibody blocks at least about 17% of CD36-mediated fatty acid and / or oxidized LDL uptake.

[0145] When the target of treatment is a human, any known anti-CD36 antibody or antibody that can be prepared for human administration can be used. Antibodies already generated in a non-human immune system (e.g., in mice) (such as those used in the assays of this application) may require humanization to enable their administration to humans to avoid adverse reactions. Humanized antibodies are antibodies originally generated in a non-human species, typically monoclonal antibodies, and their protein sequences have been modified to increase their similarity to naturally occurring human antibody variants, thus preserving the minimal sequence derived from non-human immunoglobulins. Even after humanization, the amino acid sequence of humanized antibodies is partially different from that of naturally occurring human antibodies. Several methods for antibody humanization are known to those skilled in the art (e.g., Almagro and Fransson (2008) reviewed them), including: humanization by generating mouse-human (mouse Fab spliced ​​with human Fc) chimeras, which can be further humanized by selectively altering the amino acid sequence of the Fab portion; insertion of one or more CDR fragments of a "donor" (non-human antibody) by replacing the corresponding fragment of the human antibody, which can be done using recombinant DNA technology to create constructs that can be expressed in mammalian cell cultures, or even by creating antibody gene libraries (usually derived from human RNA isolated from peripheral blood and displayed by microorganisms or viruses (e.g., in bacteriophages), or even cell-free extracts (e.g., in ribosomes), for example using recombinant DNA technology to select appropriate intermediates (usually antibody fragments, such as Fab or scFv) and obtain complete antibodies without using non-human mammals. Some patent documents focus on humanization methods, such as US6054297 assigned to Genentech; US5225539 and US4816397 are also useful references, which are incorporated herein by reference in their entirety.

[0146] Methods for obtaining monoclonal antibodies are well known to those skilled in the art. Generally, antibodies against the CD36 receptor can be produced by administering the full CD36 protein, or fragments thereof, or epitopes, to a host animal different from the mammal seeking therapeutic efficacy, according to known methods (e.g., those mentioned in the classic laboratory manual, “Antibodies: A Laboratory Manual, Second Edition,” edited by E.A. Greenfield, 2014). In particular, monoclonal antibodies can be prepared and isolated using any technique that provides the production of antibody molecules through a continuous cell line in culture, such as hybridoma techniques originally described by Kohler and Milstein (1975), human B-cell hybridoma techniques (Cote et al., 1983), or EBV hybridoma techniques (Cole et al., 1985). Other methods for preparing clonal cell lines and the monoclonal antibodies expressed therefrom, and their antigen-binding fragments, are well known in the art (see, for example, Chapter 11 of *Short Protocols in Molecular Biology*, 5th edition, (2002), Ausubel FM et al., as described above). Alternatively, as described above, Fab and / or scFv expression libraries can be constructed to allow for rapid identification of fragments with the desired specificity for the CD36 receptor.Examples of phage display methods that can be used to manufacture the antibodies or fragments described herein include Brinkman U et al., (1995) J Immunol Methods 182:41-50; Ames RS et al., (1995) J Immunol Methods 184:177-186; Kettleborough CA et al., (1994) Eur J Immunol 24:952-958; Persic L et al., (1997) Gene 187:9-18; Burton DR & Barbas CF (1994) Advan Immunol 57:191-280; PCT Publication No. PCT / GB91 / 001134; International Publication Nos. WO90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 1 The methods disclosed in WO 95 / 15982, WO 95 / 20401 and WO 97 / 13844; and U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108.

[0147] For the design of antibodies with specific specificity, it is advantageous to allocate resources to the annotated NCBI reference sequence (NC_000007.14, Homo sapiens annotation version: 107, i.e., the current version as of September 29, 2015) or UniProtKB P16671 to select specific domains or regions of the antibody to be targeted or mutated as immunogens before antibody generation, if necessary.

[0148] To achieve a therapeutic effect, anti-CD36 antibodies, acting as inhibitors of CD36 activity, are preferably administered at a therapeutically effective dose. The effective dose can be precisely determined based on individual patient factors, including patient size, age, cancer stage, and the nature of the inhibitor (e.g., expression construct, antisense oligonucleotide, antibody, or fragment thereof). Therefore, those skilled in the art can readily determine the dose from this disclosure and their knowledge. Multiple doses can also be administered to the subject during a specific treatment period, e.g., daily, weekly, monthly, every two months, every three months, or every six months. Therapeuticly effective plasma levels can also be achieved through daily administration of multiple doses. In some dosing regimens, the initial dose received by the subject at a first point in time is higher than one or more subsequent doses or a maintenance dose is received. For repeated administration over several days or longer, depending on the condition, treatment typically continues until the desired effect is achieved. Progression of such treatment can be easily monitored using routine techniques and assays.

[0149] Depending on the type and severity of the disease, an anti-CD36 antibody dose of approximately 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) can be a candidate initial dose for administration to a patient. This dose can be administered, for example, by single or multiple administrations alone, or by continuous infusion. Daily doses may range from approximately 1 μg / kg to 100 mg / kg or more. An exemplary dose of anti-CD36 antibody is in the range of approximately 0.005 mg / kg to approximately 10 mg / kg. In other examples, the dosage may also include administration of about 1 μg / kg body weight, about 5 μg / kg body weight, about 10 μg / kg body weight, about 50 μg / kg body weight, about 100 μg / kg body weight, about 200 μg / kg body weight, about 350 μg / kg body weight, about 500 μg / kg body weight, about 1 mg / kg body weight, about 5 mg / kg body weight, about 10 mg / kg body weight, about 50 mg / kg body weight, about 100 mg / kg body weight, about 200 mg / kg body weight, about 350 mg / kg body weight, about 500 mg / kg body weight, to about 1000 mg / kg body weight or more, and any range derived therefrom. Based on the figures above, in examples of ranges derived from the figures listed herein, administration of about 5 mg / kg body weight to about 100 mg / kg body weight, about 5 μg / kg body weight to about 500 mg / kg body weight, etc., is per administration. Therefore, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg or 10 mg / kg (or any combination thereof) can be administered to the patient.

[0150] For systemic administration, the effective therapeutic dose can initially be estimated through in vitro assays (e.g., cell culture assays). The dose can then be formulated in animal models to achieve circulating concentration ranges (including IC50 values ​​as determined in cell culture).50 This information can be used to more accurately determine the effective dose for humans. The initial dose can also be estimated from in vivo data (e.g., animal models) using techniques well-known in the art. Those skilled in the art can readily optimize human administration based on animal data. The dosage and interval can be adjusted independently to provide sufficient plasma levels of the anti-CD36 antibody to maintain therapeutic efficacy. Plasma levels can be measured, for example, by HPLC.

[0151] Anti-CD36 antibodies can be fused to or conjugated (e.g., covalently or non-covalently) with detectable labels or substances. Examples of detectable labels or substances include: enzyme labels, such as glucose oxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (121In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, as well as biotin. Antibodies with such labels can be used to detect CD36 protein (e.g., human CD36).

[0152] It has been reported that antibodies with reduced fucose content have increased affinity for Fc receptors (such as FcγRIIIA). Therefore, in some embodiments, the antibodies described herein have reduced fucose content or lack fucose (i.e., "unfucosylated"). Such antibodies can be produced using techniques known to those skilled in the art. For example, such antibodies can be expressed in cells lacking or devoid of fucosylation capacity. In one specific example, a cell line with two alleles of α1,6-fucosyltransferase knocked out can be used to produce antibodies with reduced fucose content. The Lonza system is an example of such a system that can be used to produce antibodies with reduced fucose content. Alternatively, antibodies with reduced or no fucose content can be produced, for example, by: (i) culturing cells under conditions that prevent or reduce fucosylation; (ii) removing fucose post-translationally (e.g., with fucosidase); (iii) adding the desired carbohydrate post-translationally, for example, after recombinant expression of a non-glycosylated glycoprotein; or (iv) purifying the glycoprotein to select for antibodies that are not fucosylated. See, for example, methods for producing antibodies with no or reduced fucose content in Longmore GD & Schachter H (1982) Carbohydr Res 100:365-92 and Imai-Nishiya H et al., (2007) BMC Biotechnol. 7:84.

[0153] In some embodiments, the CD36 antibody exhibits enhanced in vitro ADCC activity compared to a fucosylated CD36 antibody having the same amino acid sequence. In some embodiments, the specific cleavage induced by the non-fucosylated CD36 antibody is at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 65, at least 70, or at least 75 percentage points higher than that induced by the fucosylated CD36 antibody.

[0154] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein, thereby producing an Fc region variant. The Fc region variant may contain a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) with amino acid modifications (e.g., substitutions) at one or more amino acid positions. In some embodiments, the Fc domain contains one or more amino acid substitutions that reduce binding to Fc receptors, particularly Fcγ receptors. In some embodiments, the Fc domain belongs to the human IgG1 subclass having amino acid mutations L234A, L235A, and / or P329G (according to the Kabat EU index number). In some embodiments, the Fc domain belongs to the human IgG1 subclass having amino acid mutations L234G, L235S, and G236R. In some embodiments, the Fc domain belongs to the human IgG1 subclass having amino acid mutations L234S, L235T, and G236R. In some embodiments, the Fc domain belongs to the human IgG1 subclass having amino acid mutations L234S, L235V, and G236R. In some embodiments, the Fc domain is a human IgG1 subclass with amino acid mutations L234T, L235Q, and G236R. In some embodiments, the Fc domain is a human IgG1 subclass with amino acid mutations L234T, L235T, and G236R. In some embodiments, the Fc domain is a human IgG1 subclass with amino acid mutations L234A and L235A.

[0155] The Fc domain endows the antibodies of the present invention with favorable pharmacokinetic properties, including a long serum half-life that facilitates good accumulation in target tissues and a favorable tissue-to-blood distribution ratio. However, the Fc domain may also cause the antibodies of the present invention to undesirably target cells expressing Fc receptors rather than preferred antigen-carrying cells. Therefore, in specific embodiments, the Fc domain of the antibodies of the present invention exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to the native IgG Fc domain, particularly the IgG1 FC domain or the IgG4 Fc domain. More specifically, the Fc domain is the IgG1 FC domain.

[0156] In certain aspects, the Fc domain is engineered to have reduced binding affinity to the Fc receptor and / or reduced effector function compared to the unengineered Fc domain. In one such embodiment, the Fc domain exhibits less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% binding affinity to the Fc receptor compared to the native IgG1 Fc domain, and / or less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% effector function compared to the native IgG1 Fc domain. In one embodiment, the Fc domain substantially does not bind to the Fc receptor and / or induce effector function. In a specific embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more particularly human FcγRIIIa, FcγRI, or FcγRIIa, most particularly human FcγRIIIa. In one embodiment, the Fc receptor is an inhibitory Fc receptor. In a specific embodiment, the Fc receptor is an inhibitory human Fcγ receptor, more particularly human FcγRIIIB. In one embodiment, the effector function is one or more of CDC, ADCC, ADCP, and cytokine secretion. In a specific embodiment, the effector function is ADCC. In one embodiment, the Fc domain exhibits a substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to the native IgG1 Fc domain. A substantially similar binding affinity to FcRn is achieved when the Fc domain exhibits greater than about 70%, particularly greater than about 80%, more particularly greater than about 90% of the binding affinity of the native IgG1 Fc domain to FcRn. In some embodiments, the binding affinity to complement components, particularly to C1q, is also reduced. In one aspect, the binding affinity to the neonatal Fc receptor (FcRn) is not reduced.

[0157] In some embodiments, the Fc domain of the antibody of the present invention is engineered to have reduced effector functions compared to the unengineered Fc domain. Reduced effector functions may include, but are not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent phagocytosis (ADCP), reduced cytokine secretion, reduced antigen uptake by antigen-presenting cells mediated by immune complexes, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced dendritic cell maturation, or reduced T cell sensitization.

[0158] Antibodies with reduced effector function include those with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant with residues 265 and 297 replaced by alanine (US Patent No. 7,332,581). Certain antibody variants with increased or decreased binding to FcR have been described (e.g., US Patent No. 6,737,056; WO2004 / 056312, and Shields, R. Letal., J. Biol. Chem. 276(2001)6591-6604).

[0159] In one aspect of the invention, the Fc domain contains an amino acid substitution at one or more of positions E233, L234, L235, G236, N297, P331, and P329. In some aspects, the Fc domain contains amino acid substitutions L234A and L235A (“LALA”). In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In one aspect, the Fc domain contains an amino acid substitution at position P329. In a more specific aspect, the amino acid substitution is P329A or P329G, particularly P329G. In one embodiment, the Fc domain contains an amino acid substitution at position P329 and other amino acid substitutions selected from the group consisting of E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a more specific embodiment, the Fc domain comprises amino acid mutations L234A, L235A, and P329G (“P329GLALA”). As described in PCT patent application number WO 2012 / 130831 A1, the amino acid-substituted “P329G LALA” combination almost completely eliminates Fcγ receptor binding of the human IgG1 Fc domain. The document also describes methods for preparing such mutant Fc domains and methods for determining their properties (e.g., Fc receptor binding or effector function). Such antibodies are IgG1 with mutant L234A and L235A or with mutant L234A, L235A, and P329G (according to EU index number Kabat et al., Kabat et al., Sequences of Proteins of Immunological Interest (5th edition, Public Health Service, National Institutes of Health, Bethesda, Maryland, 1991)).

[0160] In some aspects of the invention, the heavy chain constant region comprises an IgG constant region containing mutations at amino acid positions L234, L235, and / or G236. Mutant groups that may be particularly advantageous for use with anti-CD36 antibodies include embodiments in which the heavy chain constant region comprises an IgG constant region containing a group of mutations selected from the group consisting of: L234A, L235S, and G236R; L234G, L235S, and G236R; L234Q, L235S, and G236R; L234S, L235G, and G236R; L234S, L235T, and G236R; L234S, L235V, and G236R; L234T, L235Q and G236R; L234T, L235S and G236R; L234T, L235T and G236R; L234A and L235A; L234A, L235A and P329G; G236R and L328R; L234A and G237A; L234A, L235A and G237A; L234A and L235E; L235V, F243L, R292P, Y300L, P396L; D265A and P329A; L234A L235A and K322A; L234F, L235E and P331S; L234F, L235Q and K322Q; L234A, L235A, G237A, P238S, H268A, A330S and P331S; E233P, L234V, L235A, G236Δ, A327G, A330S and P331S; L235A and G236R; L235S and G236R; G236R; L234Q and L235S; L23 5G and G236R; L234Q, L235S and Δ236R; L234Q and L235S; L234Q, L235S and G236R; L234Q, L235S and G236R; L234Q, L235S and G236R; L234Q, L235S and G236R; L234Q, L235S and G236R; L234Q, L235S, G236R, M252Y, S254T and T256E; and L234Q, L235S, G236R, T250Q and M428L. In some embodiments, the heavy chain constant region includes an IgG constant region containing L234G, L235S and G236R mutations. In some embodiments, the heavy chain constant region includes an IgG constant region containing L234S, L235T and G236R mutations. In some embodiments, the heavy chain constant region includes an IgG constant region containing L234S, L235V, and G236R mutations. In some embodiments, the heavy chain constant region includes an IgG constant region containing L234T, L235Q, and G236R mutations. In some embodiments, the heavy chain constant region includes an IgG constant region containing L234T, L235T, and G236R mutations.In some embodiments, the heavy chain constant region includes an IgG constant region containing L234A and L235A mutations. In some embodiments, the heavy chain constant region includes an IgG constant region containing L234A, L235A, and P329G mutations.

[0161] In one aspect, the antibody of the present invention comprises (all positions according to the EU index of Kabat): (i) a homodimeric Fc region of a human IgG1 subclass, optionally having mutants P329G, L234A, and L235A; or (ii) a homodimeric Fc region of a human IgG4 subclass, optionally having mutants P329G, S228P, and L235E; or (iii) a homodimeric Fc region of a human IgG1 subclass, optionally having mutants P329G, L234A, and L235E. L235A, I253A, H310A, and H435A or optionally having mutations P329G, L234A, L235A, H310A, H433A, and Y436A; or (iv) heterodimeric Fc regions, wherein one Fc region polypeptide contains the mutation T366W, and the other Fc region polypeptide contains the mutations T366S, L368A, and Y407V, or one Fc region polypeptide contains the mutations T366W and Y349C, and the other Fc region polypeptide contains the mutations T366S, L368A, and Y407V. The Fc region polypeptide contains mutants T366S, L368A, Y407V, and S354C, or one Fc region polypeptide contains mutants T366W and S354C, while the other Fc region polypeptide contains mutants T366S, L368A, Y407V, and Y349C; or (v) a heterodimeric Fc region of a human IgG1 subclass, wherein both Fc region polypeptides contain mutants P329G, L234A, and L235A, and one Fc region polypeptide contains mutant T366S, L368A, Y407V, and Y349C. 6W, while another Fc region polypeptide contains mutants T366S, L368A, and Y407V, or one Fc region polypeptide contains mutants T366W and Y349C, and another Fc region polypeptide contains mutants T366S, L368A, Y407V, and S354C, or one Fc region polypeptide contains mutants T366W and S354C, and another Fc region polypeptide contains mutants T366S, L368A, Y407V, and Y349C.

[0162] In one aspect, the Fc domain is the IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain containing an amino acid substitution at position S228 (Kabat number), specifically the amino acid substitution S228P. In a more specific embodiment, the Fc domain is an IgG4 Fc domain containing the amino acid substitutions L235E and S228P and P329G. This amino acid substitution reduces Fab arm exchange of IgG4 antibodies in vivo (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). Therefore, in one aspect, an antibody is provided comprising (all positions according to Kabat's EU index) heterodimeric Fc regions of human IgG4 subclasses, wherein both Fc region polypeptides contain mutants P329G, S228P, and L235E, and one Fc region polypeptide contains mutant T366W while the other Fc region polypeptide contains mutants T366S, L368A, and Y407V; or one Fc region polypeptide contains mutants T366W and Y349C while the other Fc region polypeptide contains mutants T366S, L368A, Y407V, and S354C; or one Fc region polypeptide contains mutants T366W and S354C while the other Fc region polypeptide contains mutants T366S, L368A, Y407V, and Y349C.

[0163] Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn) (responsible for transferring maternal IgG to the fetus) are described in US 2005 / 0014934 (Guyer, R. Let et al., J. Immunol. 117 (1976) 587-593 and Kim, J. Ke et al., J. Immunol. 24 (1994) 2429-2434). These antibodies contain an Fc region having one or more substitutions that improve the binding of the Fc region to the FcRn. Such Fc variants include those with substitutions at one or more residues of the Fc region: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (e.g., substitution of Fc region residue 434 (US Patent No. 7,371,826)). See also Duncan, A.R. and Winter, G., Nature 322 (1988) 738-740; US Patent Nos. 5,648,260; 5,624,821 and WO 94 / 29351, which contain other examples involving Fc region variants.

[0164] Binding to the Fc receptor can be readily determined, for example, by ELISA or by surface plasmon resonance (SPR) using a standard instrument (e.g., a BIAcore instrument (GE Healthcare)), and the Fc receptor can be obtained, for example, through recombinant expression. Suitable such binding assays are described herein. Alternatively, the binding affinity of the Fc domain or cell-activating antibodies containing the Fc domain to the Fc receptor can be assessed using cell lines known to express a specific Fc receptor, such as human NK cells expressing the FcγIIIa receptor. The effector function of the Fc domain or the antibodies of the present invention containing the Fc domain can be measured by methods known in the art. Suitable assays for measuring ADCC are described herein. Other examples of in vitro assays for assessing ADCC activity of target molecules are described in U.S. Patent Nos. 5,500,362; Hellstrom et al., Proc Natl Acad Sci USA 83,7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82,1499-1502 (1985); U.S. Patent Nos. 5,821,337; Bruggemann et al., J Exp Med 166,1351-1361 (1987). Alternatively, non-radioactive assays (see, for example, ACTI for flow cytometry) can be used. TM Non-radioactive cytotoxicity assays (Cell Technology, Inc., Mountain View, California); and CytoTox Non-radioactive cytotoxicity assays (Promega, Madison, Wisconsin). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the target molecule can be assessed in vivo, for example in animal models (such as those disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998)).

[0165] Nucleotides encoding anti-CD36 antibodies

[0166] In some respects, this document provides a polynucleotide comprising: a nucleotide sequence encoding an antibody or its domains (e.g., variable light chain region and / or variable heavy chain region) that specifically binds to the CD36 (e.g., human CD36) antigen as described herein; and a vector, for example, a vector comprising such polynucleotide for recombinant expression in host cells (e.g., Escherichia coli and mammalian cells).

[0167] In certain respects, this document provides for polynucleotides comprising a nucleotide sequence encoding an antibody that specifically binds to a CD36 polypeptide (e.g., human CD36) and comprising an amino acid sequence as described herein, as well as antibodies encoding antibodies that competitively bind to such antibodies (e.g., in a dose-dependent manner) to the CD36 polypeptide, or antibodies encoding antibodies that bind to the same epitope as such antibodies.

[0168] In some respects, this document provides a polynucleotide comprising a nucleotide sequence encoding the light or heavy chain of the antibody described herein. The polynucleotide may comprise a nucleotide sequence encoding the heavy chain of the VH or CDR of the antibody described herein. The polynucleotide may comprise a nucleotide sequence encoding the light chain of the VL or CDR of the antibody described herein.

[0169] In certain embodiments, this document provides a polynucleotide comprising a nucleotide sequence encoding an anti-CD36 antibody, the anti-CD36 antibody comprising three VH chain CDRs, for example, VH CDR1, VHCDR2, and VH CDR3 of any antibody described herein. In certain embodiments, this document provides a polynucleotide comprising three VL chain CDRs, for example, VL CDR1, VL CDR2, and VL CDR3 of any antibody described herein. In certain embodiments, this document provides a polynucleotide comprising a nucleotide sequence encoding an anti-CD36 antibody, the anti-CD36 antibody comprising: three VH chain CDRs, for example, VH CDR1, VH CDR2, and VH CDR3 of any antibody described herein; and three VL chain CDRs, for example, VL CDR1, VL CDR2, and VL CDR3 of any antibody described herein.

[0170] In certain embodiments, this document provides a polynucleotide comprising a nucleotide sequence encoding an anti-CD36 antibody or a fragment thereof, wherein the anti-CD36 antibody or fragment thereof comprises a VH domain, for example, containing FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, which comprises the amino acid sequence described herein. In specific embodiments, this document provides a polynucleotide comprising a nucleotide sequence encoding an anti-CD36 antibody or a fragment thereof, wherein the anti-CD36 antibody or fragment thereof comprises a VL domain, for example, containing FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, which comprises the amino acid sequence described herein.

[0171] This document also provides polynucleotides encoding the anti-CD36 antibody or its domains described herein, which are optimized, for example, through codon / RNA optimization, substitution with a heterologous signaling sequence, and elimination of mRNA instability elements. Methods for generating optimized nucleic acids encoding the anti-CD36 antibody or its domains (e.g., heavy chain, light chain, VH domain, or VL domain) for recombinant expression by introducing codon changes (e.g., codon changes encoding the same amino acid due to the degeneracy of the genetic code) and / or eliminating repressive regions in the mRNA can be performed using the optimization methods described in U.S. Patent Nos. 5,965,726; 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, respectively.

[0172] In some embodiments, this document provides polynucleotides encoding any antibody or antibody fragment described in this application. Exemplary nucleotide sequences and other embodiments encoding the ONA-0-v1 antibody and the ONA-0-v2 antibody are provided in Table 4 below.

[0173] Table 4 – Nucleotide Sequences

[0174]

[0175]

[0176]

[0177]

[0178] Polynucleotides encoding the antibodies or their domains described herein can be generated from nucleic acids of a suitable source (e.g., hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells that produce the target antibody. Such PCR amplification methods can be used to obtain nucleic acids containing sequences encoding the light chain and / or heavy chain of an antibody. Such PCR amplification methods can be used to obtain nucleic acids containing sequences encoding variable light chain regions and / or variable heavy chain regions of an antibody. The amplified nucleic acids can be cloned into vectors for expression in host cells and further cloned, for example, to generate chimeric antibodies and humanized antibodies.

[0179] The polynucleotides described herein can be in, for example, RNA or DNA form. DNA includes cDNA, genomic DNA, and synthetic DNA, and can be double-stranded or single-stranded. If single-stranded, the DNA can be a coding strand or a non-coding (antisense) strand. In some embodiments, the polynucleotide is cDNA or DNA lacking one or more introns. In some embodiments, the polynucleotide is a non-naturally occurring polynucleotide. In some embodiments, the polynucleotide is recombinant-derived. In some embodiments, the polynucleotide is isolated. In some embodiments, the polynucleotide is substantially pure. In some embodiments, the polynucleotide is purified from a natural component.

[0180] Antibody production

[0181] Antibodies that specifically bind to CD36 (e.g., human CD36) can be generated by any method known in the art for synthesizing full-length antibodies or antigen-binding fragments thereof, such as by chemical synthesis or by recombinant expression techniques. Unless otherwise stated, the methods described herein employ conventional techniques from molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields of expertise. These techniques are described, for example, in the references cited herein and are fully explained therein. See, for example, Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates) annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: A Laboratory Approach, Cold Spring Harbor Laboratory Press.

[0182] In one aspect, this document provides a method for preparing antibodies that specifically bind to CD36 (e.g., human CD36), the method comprising culturing the cells or host cells described herein. In another aspect, this document provides a method for preparing antibodies that specifically bind to CD36 (e.g., human CD36), the method comprising expressing (e.g., recombinantly expressing) the antibody using the cells or host cells described herein (e.g., cells or host cells containing a polynucleotide encoding the antibody described herein). In a specific embodiment, the cells are isolated cells. In a specific embodiment, exogenous polynucleotides have been introduced into the cells. In a specific embodiment, the method further includes the step of purifying the antibody obtained from the cells or host cells.

[0183] Pharmaceutical Composition

[0184] This document provides compositions comprising, in physiologically acceptable carriers, excipients, or stabilizers, an anti-CD36 antibody of the desired purity described herein (Remington Pharmaceutical Sciences, Mack Publishing, Easton, PA). The acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the amounts and concentrations used.

[0185] In various embodiments, the composition comprising the anti-CD36 antibody is provided as a formulation having a pharmaceutically acceptable carrier (see, for example, Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000).

[0186] The pharmaceutical compositions described herein can be used to block CD36 activity. The pharmaceutical compositions described herein can be used to treat conditions such as cancer. Examples of cancers that can be treated according to the methods described herein include, but are not limited to, solid tumors and their metastases. In some embodiments, the pharmaceutical compositions described herein can be used to treat oral squamous cell carcinoma, head and neck cancer, esophageal cancer, gastric cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, colon cancer, kidney cancer, prostate cancer, sarcoma, melanoma, leukemia, or lymphoma. In some embodiments, the pharmaceutical compositions described herein can be used to treat metastases that develop from oral squamous cell carcinoma, head and neck cancer, esophageal cancer, gastric cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, colon cancer, kidney cancer, prostate cancer, sarcoma, melanoma, leukemia, or lymphoma. In some embodiments, the pharmaceutical compositions described herein can be used to treat primary tumors and metastases that develop from oral squamous cell carcinoma, head and neck cancer, esophageal cancer, gastric cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, colon cancer, kidney cancer, prostate cancer, sarcoma, melanoma, leukemia, or lymphoma.

[0187] In one embodiment, the pharmaceutical composition described herein is used as a medicine. In one embodiment, the pharmaceutical composition described herein is used as a diagnostic agent, for example, to detect the presence of CD36 in a sample obtained from a patient (e.g., a human patient).

[0188] Compositions intended for in vivo administration can be sterile. This can be easily achieved through filtration, for example, using a sterile filter membrane.

[0189] In some embodiments, the pharmaceutical composition comprises isolated antibodies. In some embodiments, the pharmaceutical composition is substantially free of other antibodies. In some embodiments, the pharmaceutical composition is substantially free of ONA-O-v2 antibody.

[0190] In some embodiments, a pharmaceutical composition is provided comprising an anti-CD36 antibody as described herein and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition is provided comprising a non-fucosylated anti-CD36 antibody as described herein and a pharmaceutically acceptable carrier.

[0191] In specific embodiments, such pharmaceutical compositions comprise non-fucosylated anti-CD36 antibodies, for example, wherein at least 80% of the antibody in the composition is non-fucosylated. Antibodies with a reduced fucosylation content in the Fc region of the glycan moiety can exhibit higher ADCC activity due to increased affinity for Fc receptors (e.g., FcγRIIIA) compared to fully fucosylated antibodies (Niwa R et al., Clinical Cancer Research 11(6):2327-36(2005)). In some embodiments, CD36 antibodies exhibit enhanced in vitro ADCC activity compared to fucosylated CD36 antibodies having the same amino acid sequence. In specific embodiments, such pharmaceutical compositions comprise non-fucosylated anti-CD36 antibodies, for example, wherein at least 50% of the antibody in the composition is non-fucosylated. In specific embodiments, such pharmaceutical compositions comprise non-fucosylated anti-CD36 antibodies, for example, wherein at least 60% of the antibody in the composition is non-fucosylated. In specific embodiments, such pharmaceutical compositions comprise non-fucosylated anti-CD36 antibodies, for example, wherein at least 70% of the antibodies in the composition are non-fucosylated. In specific embodiments, such pharmaceutical compositions comprise non-fucosylated anti-CD36 antibodies, for example, wherein at least 80% of the antibodies in the composition are non-fucosylated. In specific embodiments, such pharmaceutical compositions comprise non-fucosylated anti-CD36 antibodies, for example, wherein at least 85% of the antibodies in the composition are non-fucosylated. In specific embodiments, such pharmaceutical compositions comprise non-fucosylated anti-CD36 antibodies, for example, wherein at least 90% of the antibodies in the composition are non-fucosylated. In specific embodiments, such pharmaceutical compositions comprise non-fucosylated anti-CD36 antibodies, for example, wherein at least 95% of the antibodies in the composition are non-fucosylated. In specific embodiments, such pharmaceutical compositions comprise non-fucosylated anti-CD36 antibodies, for example, wherein at least 96% of the antibodies in the composition are non-fucosylated. In specific embodiments, such pharmaceutical compositions comprise non-fucosylated anti-CD36 antibodies, for example, wherein at least 97% of the antibodies in the composition are non-fucosylated. In specific embodiments, such pharmaceutical compositions comprise non-fucosylated anti-CD36 antibodies, for example, wherein at least 98% of the antibodies in the composition are non-fucosylated. In specific embodiments, such pharmaceutical compositions comprise non-fucosylated anti-CD36 antibodies, for example, wherein at least 99% of the antibodies in the composition are non-fucosylated. In specific embodiments, such pharmaceutical compositions comprise non-fucosylated anti-CD36 antibodies, wherein fucose is undetectable in the composition.

[0192] The method disclosed herein

[0193] In some embodiments, the present invention provides a method for treating mammalian cancer using a combination of anti-CD36 antibodies and a second therapy. In some embodiments, the cancer is selected from the group consisting of: oral squamous cell carcinoma, head and neck cancer, esophageal cancer, gastric cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, colon cancer, kidney cancer, prostate cancer, sarcoma, melanoma, leukemia, and lymphoma. In one embodiment, the cancer is oral squamous cell carcinoma. In some embodiments, the cancer is ovarian cancer. In other embodiments, the cancer is melanoma. In further embodiments, the cancer is any cancer disclosed herein. In some embodiments, the cancer is metastatic cancer. In some embodiments, the cancer is both a primary tumor and metastatic cancer. In some embodiments, the mammal is a human.

[0194] In some embodiments, the anti-CD36 antibody is a full-length antibody, a single-chain antibody, or an scFv, Fab, or F(ab')2 fragment. In one embodiment, the CD36 inhibitor is an antibody. In some embodiments, the CD36 inhibitor is a humanized antibody. In some embodiments, the CD36 inhibitor is the antibody disclosed herein. In some embodiments, the CD36 inhibitor is a commercially available anti-CD36 antibody, such as antibody JC63.1. In one embodiment, the CD36 inhibitor is shRNA or iRNA, siRNA, or antisense RNA or DNA.

[0195] In some embodiments, the second treatment is immunotherapy. In one embodiment, the immunotherapy is a PD-1 inhibitor. In another embodiment, the PD-1 inhibitor is an anti-PD-1 antibody. In one embodiment, the anti-PD-1 antibody is pembrolizumab (KEYTRUDA; MK-3475), pildizumab (CT-011), or nivolumab (OPDIVO; BMS-936558). In another embodiment, the immunotherapy is a PD-L1 inhibitor. In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody. In another embodiment, the anti-PD-L1 antibody is atezolizumab (Tecentriq or RG7446), durvalumab (Imfinzi or MEDI4736), or avelumab (Bavencio or BMS-936559). In one embodiment, the immunotherapy is a CTLA-4 inhibitor. In another embodiment, the CTLA-4 inhibitor is an anti-CTLA-4 antibody. In one embodiment, the anti-CTLA-4 antibody is ipilimumab or its antigen-binding fragment.

[0196] In one implementation, the second treatment is a chemotherapy agent. In one implementation, the chemotherapy agent is cisplatin. In some implementations, the chemotherapy agent comprises one of the anticancer drugs or combinations of anticancer drugs listed in Table 5.

[0197] Table 5 – Chemotherapy Agents

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204] In some embodiments, the present invention provides a method for treating mammalian cancers using a combination of a CD36 inhibitor and an anti-PD-1 antibody. In some embodiments, the cancer is selected from the group consisting of: oral squamous cell carcinoma, head and neck cancer, esophageal cancer, gastric cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, colon cancer, kidney cancer, prostate cancer, sarcoma, melanoma, leukemia, and lymphoma. In some embodiments, the cancer is oral squamous cell carcinoma. In some embodiments, the cancer is ovarian cancer. In other embodiments, the cancer is melanoma. In further embodiments, the cancer is any other cancer disclosed herein. In one embodiment, the cancer is metastatic cancer. In some embodiments, the cancer is both primary and metastatic. In embodiments, the CD36 inhibitor is an antibody, a single-chain antibody, or an scFv, Fab, or F(ab')2 fragment. In one embodiment, the CD36 inhibitor is an antibody. In embodiments, the CD36 inhibitor is a humanized antibody. In some embodiments, the CD36 inhibitor is an antibody disclosed herein. In some embodiments, the CD36 inhibitor is a commercial anti-CD36 antibody, such as antibody JC63.1. In one embodiment, the CD36 inhibitor is shRNA or iRNA, siRNA, or antisense RNA or DNA. In one embodiment, the anti-PD-1 antibody is pembrolizumab (KEYTRUDA; MK-3475), pildizumab (CT-011), or nivolumab (OPDIVO; BMS-936558).

[0205] Examples of cancers and / or malignancies that can be treated using the methods of this invention include liver cancer, hepatocellular carcinoma (HCC), bone cancer, pancreatic cancer, skin cancer, oral cancer, head and neck cancer, breast cancer, lung cancer, small cell lung cancer, NSCLC, malignant melanoma of the skin or eye, Merkel cell carcinoma (MCC), squamous cell carcinoma of the skin (cSCC), kidney cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, anal region cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, squamous cell carcinoma of the head and neck (SCCHN), and non-Hodgkin's lymphoma. Lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, lymphocytic lymphoma, urothelial carcinoma, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system (CNS) vegetations, primary CNS lymphoma, tumor angiogenesis, spinal cord axon tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma. Sarcoma), epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers (including asbestos-induced cancers), hematologic malignancies (including, for example, multiple myeloma, B-cell lymphoma, Hodgkin's lymphoma / primary mediastinal B-cell lymphoma, non-Hodgkin's lymphoma, acute myeloid lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-cell lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma, and precursor T-cell lymphoma), and any combination of said cancers. The invention is also applicable to the treatment of metastatic cancer. In some embodiments, the cancer is oral squamous cell carcinoma. In some embodiments, the cancer is ovarian cancer. In other implementations, the cancer is melanoma.

[0206] In implementation, the antibody can be administered systemically, such as intraperitoneally, and can be in a suitable suspension form, such as an aqueous suspension in water or another suitable liquid (e.g., a saline solution).

[0207] For antibody administration, the dosage ranges from approximately 0.0001 mg / kg to 100 mg / kg based on host body weight, more typically from 0.01 mg / kg to 5 mg / kg. For example, the dosage can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, or 10 mg / kg body weight, or within the range of 1-10 mg / kg. Exemplary treatment regimens require administration once weekly, once every two weeks, once every three weeks, once every four weeks, once monthly, once every three months, or once every three to six months. In some embodiments, the antibody is administered at a steady or fixed dose. In embodiments, the antibody is administered at any dosage described for antibodies in the art.

[0208] Anti-PD-1 and anti-PD-L1 antibodies

[0209] As used herein, the terms “programmed cell death protein 1”, “programmed cell death protein 1”, “protein PD-1”, “PD-1”, “PD1”, “PDCD1”, “hPD-1”, and “hPD-1” are used interchangeably and include variants, isotypes, species homologs of human PD-1, and analogs that share at least one common epitope with PD-1. The complete PD-1 sequence can be found in GenBank accession number U64863.

[0210] Programmed cell death 1 (PD-1) is a cell surface signaling receptor that plays a crucial role in the regulation of T cell activation and tolerance (Keir ME, et al., Annu. Rev. Immunol. 2008; 26:677-704). PD-1 is a type I transmembrane protein that, along with BTLA, CTLA-4, ICOS, and CD28, constitutes the CD28 family of T cell co-stimulatory receptors. PD-1 is primarily expressed on activated T cells, B cells, and myeloid cells (Dong H., et al., Nat. Med. 1999; 5:1365-1369; Agata et al. as mentioned above; Okazaki et al. (2002) Curr. Opin. Immunol. 14:391779-82; Bennett et al. (2003) J Immunol 170:711-8). PD-1 is also expressed on natural killer (NK) cells (Terme M., et al., Cancer Res. 2011; 71:5393-5399). The binding of PD-1 to its ligands PD-L1 and PD-L2 leads to phosphorylation of tyrosine residues in the tyrosine repressor domain of proximal intracellular immune receptors, subsequently recruiting the phosphatase SHP-2, ultimately resulting in downregulation of T cell activation. An important role of PD-1 is to limit the activity of T cells in peripheral tissues during inflammatory responses to infection, thereby limiting the progression of autoimmunity (Pardoll DM, Nat. Rev. Cancer 2012; 12:252-264). Evidence for this negative regulatory effect comes from the discovery that PD-1-deficient mice develop lupus-like autoimmune diseases (including arthritis and nephritis) and cardiomyopathy (Nishimura H., et al., Immunity, 1999; 11:141-151; and Nishimura H., et al., Science, 2001; 291:319-322). In the tumor setting, the result is the development of immune resistance within the tumor microenvironment. PD-1 is highly expressed on tumor-infiltrating lymphocytes, and PD-1 ligands are upregulated on the cell surface of many different tumors (Dong H., et al., Nat. Med. 2002; 8:793-800). Multiple mouse cancer models have demonstrated that ligand binding to PD-1 leads to immune evasion. Furthermore, the blocking of this interaction leads to antitumor activity (Topalian SL, et al. NEJM 2012; 366(26):2443-2454; Hamid O., et al., NEJM 2013; 369:134-144).Furthermore, inhibition of the PD-1 / PD-L1 interaction has been shown to mediate potent antitumor activity in preclinical models (US Patent Nos. 8,008,449 and 7,943,743).

[0211] The initial members of the PD-1 family, CD28 and ICOS, were discovered through their functional role in enhancing T cell proliferation upon the addition of monoclonal antibodies (Hutloff et al. Nature (1999); 397:263-266; Hansen et al. Immunogenics (1980); 10:247-260). PD-1 was identified by screening for differential expression in apoptotic cells (Ishida et al. EMBO J (1992); 11:3887-95). Other members of this family, CTLA-4 and BTLA, were discovered by screening for differential expression in cytotoxic T lymphocytes and TH1 cells, respectively. CD28, ICOS, and CTLA-4 all possess unpaired cysteine ​​residues, allowing for homodimerization. In contrast, PD-1 is considered to exist as a monomer, lacking the unpaired cysteine ​​residue signature found in other CD28 family members.

[0212] The PD-1 gene is a 55 kDa type I transmembrane protein and is part of the Ig gene superfamily (Agata et al. (1996) Int Immunol 8:765-72). PD-1 contains a proximal tyrosine-based immunoreceptor repressive motif (ITIM) and a distal tyrosine-based switch motif (ITSM) (Thomas, ML (1995) J Exp Med 181:1953-6; Vivier, E and Daeron, M (1997) Immunol Today 18:286-91). Although structurally similar to CTLA-4, PD-1 lacks the MYPPPY motif (SEQ ID NO:36), which is crucial for the binding of B7-1 and B7-2. Two ligands for PD-1, PD-L1 and PD-L2, have been identified. PD-L1 and PD-L2 have been shown to downregulate T cell activation upon binding to PD-1 (Freeman et al. (2000) J Exp Med 192:1027-34; Latchman et al. (2001) Nat Immunol 2:261-8; Carter et al. (2002) Eur J Immunol 32:634-43). Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1 but not to other CD28 family members. PD-L1 is abundant in various human cancers (Dong et al. (2002) Nat. Med. 8:787-9). The interaction between PD-1 and PD-L1 leads to a reduction in tumor-infiltrating lymphocytes, a decrease in T-cell receptor-mediated proliferation, and immune evasion by cancer cells (Dong et al. (2003) J. Mol. Med. 81:281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi et al. (2004) Clin. Cancer Res. 10:5094-100). Immunosuppression can be reversed by inhibiting the local interaction between PD-1 and PD-L1, and the effect is additive when the interaction between PD-1 and PD-L2 is also blocked (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99:12293-7; Brown et al. (2003) J. Immunol. 170:1257-66).

[0213] Consistent with PD-1 being a repressive member of the CD28 family, animals lacking PD-1 develop various autoimmune phenotypes, including autoimmune cardiomyopathy and lupus-like syndrome with arthritis and nephritis (Nishimura et al. (1999) Immunity 11:141-51; Nishimura et al. (2001) Science 291:319-22). Furthermore, PD-1 has been found to play a role in autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease (GVHD), type 1 diabetes, and rheumatoid arthritis (Salama et al. (2003) J Exp Med 198:71-78; Prokunina and Alarcon-Riquelme (2004) Hum Mol Genet 13:R143; Nielsen et al. (2004) Lupus 13:510). In mouse B-cell tumor lines, the ITSM of PD-1 has been shown to be crucial for blocking BCR-mediated Ca.sup.2+ flux and tyrosine phosphorylation of downstream effector molecules (Okazaki et al. (2001) PNAS 98:13866-71).

[0214] Programmed death protein ligand-1 (PD-L1) is one of two cell surface glycoprotein ligands of PD-1 (the other being PD-L2). PD-L1 downregulates T cell activation and cytokine secretion upon binding to PD-1. As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isotypes, and species homologs of hPD-L1, as well as analogs that share at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found in GenBank accession number Q9NZQ7.

[0215] Some embodiments of the present invention include combinations of anti-PD-1 antibodies or anti-PD-L1 antibodies with anti-CD36 antibodies. PD-1 is a key immune checkpoint receptor expressed by activated T cells and B cells and mediates immunosuppression. PD-1 is a member of the CD28 family of receptors, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. Two cell surface glycoprotein ligands for PD-1, namely programmed death protein ligand 1 (PD-L1) and programmed death protein ligand 2 (PD-L2), have been identified. They are expressed on antigen-presenting cells and in many cancers and have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1. Inhibition of the PD-1 / PD-L1 interaction mediates potent antitumor activity in preclinical models.

[0216] Human monoclonal antibodies (HuMAb) that bind specifically to PD-1 with high affinity have been disclosed in U.S. Patent Nos. 8,008,449 and 8,779,105. Other anti-PD-1 mAbs have been described, for example, in U.S. Patent Nos. 6,808,710, 7,488,802, 8,168,757, and 8,354,509 and PCT Publications WO2012 / 145493 and WO2016 / 168716. Each anti-PD-1 HuMAb disclosed in U.S. Patent No. 8,008,449 has been shown to exhibit one or more of the following characteristics: (a) as determined by surface plasmon resonance using the Biacore biosensor system, at a ratio of 1 x 10 -7 M or smaller K D (a) binds to human PD-1; (b) substantially does not bind to human CD28, CTLA-4, or ICOS; (c) increases T cell proliferation in mixed lymphocyte reaction (MLR) assays; (d) increases interferon-γ production in MLR assays; (e) increases IL-2 secretion in MLR assays; (f) binds to human PD-1 and cynomolgus monkey PD-1; (g) inhibits the binding of PD-L1 and / or PD-L2 to PD-1; (h) stimulates antigen-specific memory responses; (i) stimulates Ab responses; and (j) inhibits tumor cell growth in vivo. Anti-PD-1 antibodies used in this invention comprise mAbs that specifically bind to human PD-1 and exhibit at least one, preferably at least five, of the aforementioned characteristics.

[0217] The anti-human PD-1 antibody (or the VH and / or VL domains derived from such anti-human PD-1 antibody) suitable for use in this invention can be produced using methods well known in the art. Alternatively, anti-PD-1 antibodies recognized in the art can be used. For example, monoclonal antibodies 5C4 (referred to herein as nivolumab or BMS-936558), 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, as described in WO2006 / 121168 (whose teachings are incorporated herein by reference in their entirety), 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4 can be used. Other known PD-1 antibodies include Lambrulizumab (MK-3475) described in WO2008 / 156712 and AMP-514 described in WO2012 / 145493. Other known anti-PD-1 antibodies and other PD-1 inhibitors include those described in WO2009 / 014708, WO03 / 099196, WO2009 / 114335, and WO2011 / 161699. Another known anti-PD-1 antibody is pidilimumab (CT-011). Antibodies that compete with any of these antibodies or inhibitors for binding to PD-1 may also be used.

[0218] In one implementation, the anti-PD-1 antibody is nivolumab. Nivolumab (also known as...) BMS-936558 (formerly known as 5C4, BMS-936558, MDX-1106, or ONO-4538) is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitor antibody that selectively blocks interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking the downregulation of anti-tumor T cell function (US Patent No. 8,008,449; Wang et al., 2014 Cancer Immunol Res. 2(9):846-56). In another embodiment, the anti-PD-1 antibody or a fragment thereof cross-competes with nivolumab. In other embodiments, the anti-PD-1 antibody or a fragment thereof binds to the same epitopes as nivolumab. In some embodiments, the anti-PD-1 antibody has the same CDR as nivolumab.

[0219] In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized monoclonal IgG4 (S228P) antibody targeting the human cell surface receptor PD-1 (programmed death protein-1 or programmed cell death protein-1). Pembrolizumab is described, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587.

[0220] In another embodiment, the anti-PD-1 antibody cross-competes with pembrolizumab. In some embodiments, the anti-PD-1 antibody and pembrolizumab bind to the same epitopes. In some embodiments, the anti-PD-1 antibody has the same CDR as pembrolizumab. In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (also known as...) Lambrolizumab (MK-3475) is a humanized monoclonal IgG4 antibody targeting the human cell surface receptor PD-1 (programmed death protein-1, or programmed cell death protein-1). Pembrolizumab is described, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587; see http: / / www.cancer.gov / drugdictionary?cdrid=695789 (last accessed: May 25, 2017). Pembrolizumab has been approved by the FDA for the treatment of relapsed or refractory melanoma.

[0221] In other embodiments, the anti-PD-1 antibody cross-competes with MEDI0608. In other embodiments, the anti-PD-1 antibody binds to the same epitopes as MEDI0608. In some embodiments, the anti-PD-1 antibody has the same CDR as MEDI0608. In other embodiments, the anti-PD-1 antibody is MEDI0608 (formerly known as AMP-514), which is a monoclonal antibody. MEDI0608 is described, for example, in U.S. Patent No. 8,609,089 or http: / / www.cancer.gov / drugdictionary?cdrid=756047 (last accessed May 25, 2017).

[0222] In other embodiments, the anti-PD-1 antibody cross-competes with BGB-A317. In some embodiments, the anti-PD-1 antibody and BGB-A317 bind to the same epitopes. In some embodiments, the anti-PD-1 antibody and BGB-A317 have the same CDR. In some embodiments, the anti-PD-1 antibody is BGB-A317, which is a humanized monoclonal antibody. BGB-A317 is described in U.S. Publication No. 2015 / 0079109.

[0223] Anti-PD-1 antibodies that can be used in the disclosed compositions also include isolated antibodies that specifically bind to human PD-1 and cross-competitively bind to human PD-1 with nivolumab (see, for example, U.S. Patents 8,008,449 and 8,779,105; International Publication No. WO2013 / 173223). The ability of antibodies to cross-competitively bind to antigens indicates that these antibodies bind to the same epitope region on the antigen and spatially prevent other cross-competing antibodies from binding to that specific epitope region. Because they bind to the same epitope region on PD-1, these cross-competing antibodies are expected to have very similar functional properties to nivolumab. Cross-competing antibodies can be readily identified based on their ability to cross-competitively bind to nivolumab in standard PD-1 binding assays such as Biacore assays, ELISA assays, or flow cytometry (see, for example, International Publication No. WO2013 / 173223).

[0224] In some embodiments, the antibody that cross-competes with nivolumab to bind to human PD-1 or to the same epitope region of human PD-1 is a mAb. For administration to human subjects, these cross-competing antibodies can be chimeric antibodies, humanized antibodies, or human antibodies. Such chimeric, humanized, or human mAbs can be prepared and isolated using methods well known in the art.

[0225] Anti-PD-1 antibodies that can be used with the compositions of the present invention disclosed herein also include the antigen-binding portion of the antibody described above. It has been sufficiently demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments comprising two Fab fragments linked by disulfide bonds in a hinge region; (iii) Fd fragments consisting of VH and CH1 domains; and (iv) Fv fragments consisting of VL and VH domains of a single arm of the antibody.

[0226] The anti-PD-1 antibody suitable for the disclosed compositions is an antibody that binds to PD-1 with high specificity and affinity, blocks the binding of PD-L1 and / or PD-L2, and inhibits the immunosuppressive effect of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, the anti-PD-1 “antibody” comprises an antigen-binding portion or fragment that binds to the PD-1 receptor and exhibits functional properties similar to those of a full-fledged antibody in inhibiting ligand binding and upregulating the immune system. In some embodiments, the anti-PD-1 antibody cross-competitively binds to human PD-1 with nivolumab. In other embodiments, the anti-PD-1 antibody is a chimeric, humanized, or human monoclonal antibody or a portion thereof. In some embodiments, the antibody is a humanized antibody. In other embodiments, the antibody is a human antibody. Antibodies of IgG1, IgG2, IgG3, or IgG4 isotypes may be used.

[0227] In some embodiments, the anti-PD-1 antibody comprises a heavy chain constant region of a human IgG1 or IgG4 isotype. In some other embodiments, the sequence of the IgG4 heavy chain constant region of the anti-PD-1 antibody contains an S228P mutation, which replaces a serine residue in the hinge region with a proline residue typically found at the corresponding position on IgG1 isotype antibodies. This mutation present in nivolumab prevents the exchange of the Fab arm with endogenous IgG4 antibodies while preserving low affinity for the Fc receptor associated with activation of wild-type IgG4 antibodies (Wang et al., 2014). In other embodiments, the antibody comprises a light chain constant region, which is a human κ or λ constant region. In other embodiments, the anti-PD-1 antibody is a mAb or its antigen-binding portion. In some embodiments of any of the treatment methods described herein, administration of an anti-PD-1 antibody, which is nivolumab, is included. In other embodiments, the anti-PD-1 antibody is pembrolizumab. In other embodiments, the anti-PD-1 antibody is selected from human antibodies 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4 described in U.S. Patent No. 8,008,449. In other embodiments, the anti-PD-1 antibody is MEDI0608 (formerly AMP-514), AMP-224, or pildizumab (CT-011). Other known PD-1 antibodies include, for example, lambolizumab (MK-3475) described in WO2008 / 156712 and AMP-514 described in WO2012 / 145493. Other known anti-PD-1 antibodies and other PD-1 inhibitors include, for example, those described in WO2009 / 014708, WO03 / 099196, WO2009 / 114335, and WO2011 / 161699. In one embodiment, the anti-PD-1 antibody is REGN2810. In one embodiment, the anti-PD-1 antibody is PDR001. Another known anti-PD-1 antibody is pildizumab (CT-011). Each of the above references is incorporated herein by reference. Antibodies that compete with any of these antibodies or inhibitors for binding to PD-1 may also be used.

[0228] Other anti-PD-1 monoclonal antibodies have been described, for example, in U.S. Patent Nos. 6,808,710, 7,488,802, 8,168,757 and 8,354,509, U.S. Publication No. 2016 / 0272708 and PCT Publications Nos. WO 2012 / 145493, WO 2008 / 156712, WO 2015 / 112900, WO 2012 / 145493, WO 2015 / 112800, WO 2014 / 206107, WO 2015 / 35606, WO 2015 / 085847, WO 2014 / 179664, WO 2017 / 020291, WO 2017 / 020858, WO 2016 / 197367, WO 2017 / 024515、WO 2017 / 025051、WO 2017 / 123557、WO 2016 / 106159、WO 2014 / 194302、WO 2017 / 040790、WO 2017 / 133540、WO 2017 / 132827、WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 106061, WO 2017 / 19846, WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825 and WO 2017 / 133540, each of which is incorporated herein by reference.

[0229] In some implementations, the anti-PD-1 antibody is selected from the group consisting of: nivolumab (also known as...) 5C4, BMS-936558, MDX-1106 and ONO-4538), pembrolizumab (Merck, also known as...) Lambrolizumab and MK-3475; see WO2008 / 156712), PDR001 (Novartis; see WO2015 / 112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO2012 / 145493), Cimipril (Regeneron; also known as REGN-2810; see WO2015 / 112800), JS001 (TAIZHOU JUNSHI PHARMA; see Si-Yang Liu et al.) al., J. Hematol. Oncol. 10:136 (2017)), BGB-A317 (Beigene; see WO2015 / 35606 and US2015 / 0079109), INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; see WO2015 / 085847; Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO2014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; see Si-Yang Liu et al. (al., J. Hematol. Oncol. 10:136(2017)), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics; see WO2014 / 194302), AGEN2034 (Agenus; see WO2017 / 040790), MGA012 (Macrogenics; see WO2017 / 19846), and IBI308 (Innovent; see WO2017 / 024465, WO2017 / 025016, WO2017 / 132825, and WO2017 / 133540). Each of the above references is incorporated herein by reference.

[0230] In this embodiment, the anti-PD-1 antibody is a bispecific antibody. In this embodiment, the second treatment is a PD-1 inhibitor. In this embodiment, the PD-1 inhibitor is a small molecule.

[0231] Because anti-PD-1 antibodies and anti-PD-L1 antibodies target the same signaling pathway and have shown similar levels of efficacy in a variety of cancers in clinical trials, anti-PD-L1 antibodies can replace anti-PD-1 antibodies in any treatment or composition disclosed herein.

[0232] The anti-human PD-L1 antibody (or the VH and / or VL domains derived from such anti-human PD-L1 antibody) suitable for use in this invention can be produced using methods well known in the art. Alternatively, anti-PD-L1 antibodies recognized in the art can be used. For example, the human anti-PD-L1 antibody disclosed in U.S. Patent No. 7,943,743 (the contents of which are incorporated herein by reference) can be used. Such anti-PD-L1 antibodies include 3G10, 12A4 (also known as BMS-936559), 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4. Other art-recognized anti-PD-L1 antibodies that may be used include those described, for example, in U.S. Patent Nos. 7,635,757 and 8,217,149, U.S. Publication No. 2009 / 0317368, and PCT Publications Nos. WO2011 / 066389 and WO2012 / 145493, each of which is incorporated herein by reference. Other examples of anti-PD-L1 antibodies include atezolizumab (TECENTRIQ; RG7446) or durvalumab (IMFINZI; MEDI4736). Antibodies that compete with any of these art-recognized antibodies or inhibitors for binding to PD-L1 may also be used.

[0233] Examples of anti-PD-L1 antibodies that can be used in the methods of this disclosure include the antibody disclosed in U.S. Patent No. 9,580,507, which is incorporated herein by reference. The anti-PD-L1 human monoclonal antibody disclosed in U.S. Patent No. 9,580,507 has been shown to exhibit one or more of the following characteristics: (a) as determined by surface plasmon resonance using a Biacore biosensor system, at a ratio of 1 x 102 -7 M or smaller K D (a) binds to human PD-L1; (b) increases T cell proliferation in a mixed lymphocyte reaction (MLR) assay; (c) increases interferon-γ production in an MLR assay; (d) increases IL-2 secretion in an MLR assay; (e) stimulates an antibody response; and (f) reverses the effects of T regulatory cells on T cell effector cells and / or dendritic cells. Anti-PD-L1 antibodies that can be used in this invention comprise monoclonal antibodies that specifically bind to human PD-L1 and exhibit at least one, or in some embodiments at least five, of the aforementioned characteristics.

[0234] In some embodiments, the anti-PD-L1 antibody is BMS-936559 (formerly 12A4 or MDX-1105) (see, for example, U.S. Patent No. 7,943,743; WO2013 / 173223). In other embodiments, the anti-PD-L1 antibody is MPDL3280A (also known as RG7446 and atezolizumab) (see, for example, Herbst et al. 2013 J Clin Oncol 31(suppl):3000; U.S. Patent No. 8,217,149), MEDI4736 (Khleif, 2013, in: Proceedings from the European Cancer Congress; 27 September to 1 October 2013; Amsterdam, Netherlands, Abstract 802) or MSB0010718C (also known as averuzumab; see US2014 / 0341917). In some embodiments, the antibody that cross-competes with the aforementioned reference PD-L1 antibody to bind to human PD-L1 or binds to the same epitope region of human PD-L1 with the aforementioned reference PD-L1 antibody is a mAb. For administration to human subjects, these cross-competing antibodies may be chimeric antibodies, or they may be humanized antibodies or human antibodies. Such chimeric, humanized, or human mAbs can be prepared and isolated using methods well known in the art. In some embodiments, the anti-PD-L1 antibody is selected from the group consisting of: BMS-936559 (also known as 12A4, MDX-1105; see, for example, U.S. Patent Nos. 7,943,743 and WO2013 / 173223), atezolizumab (Roche; also known as... MPDL3280A, RG7446; see US8,217,149; also see Herbstet et al. (2013) J Clin Oncol 31(suppl):3000), Dvalumab (AstraZeneca; also known as IMFINZI) TM MEDI-4736; see, for example, WO2011 / 066389), avirumab (Pfizer; also known as...) MSB-0010718C (see, for example, WO2013 / 079174), STI-1014 (Sorrento; see, for example, WO2013 / 181634), CX-072 (Cytomx; see, for example, WO2016 / 149201), KN035 (3D Med / Alphamab; see Zhang et al., Cell Discov. 7:3 (March 2017)), LY3300054 (Eli Lilly and Company; see, for example, WO2017 / 034916), and CK-301 (Checkpoint Therapeutics; see Gorelik et al., AACR:Abstract 4606 (Apr 2016)). The above references are incorporated herein by reference.

[0235] In some implementations, the PD-L1 antibody is atezolizumab. Atezolizumab is a fully humanized IgG1 monoclonal anti-PD-L1 antibody.

[0236] In some implementations, the PD-L1 antibody is divoruzumab (IMFINZI). TM Dvorumab is a human IgG1κ monoclonal anti-PD-L1 antibody.

[0237] In some implementations, the PD-L1 antibody is avermectin. Avirumab is a human IgG1λ monoclonal anti-PD-L1 antibody.

[0238] In other embodiments, the anti-PD-L1 monoclonal antibody is selected from the group consisting of 28-8, 28-1, 28-12, 29-8, 5H1 and any combination thereof.

[0239] Anti-PD-L1 antibodies that can be used in the disclosed methods also include isolated antibodies that specifically bind to human PD-L1 and cross-competitively bind to human PD-L1 with any of the anti-PD-L1 antibodies disclosed herein (e.g., atezolizumab, durvalumab, and / or avelumab). In some embodiments, the anti-PD-L1 antibody binds to the same epitope as any of the anti-PD-L1 antibodies described herein (e.g., atezolizumab, durvalumab, and / or avelumab). The ability of the antibodies to cross-competitively bind to the antigen indicates that these antibodies bind to the same epitope region on the antigen and spatially prevent other cross-competing antibodies from binding to that particular epitope region. Because the cross-competing antibodies bind to the same epitope region on PD-L1, these cross-competing antibodies are expected to have very similar functional properties to the reference antibody (e.g., atezolizumab and / or avelumab). Cross-competitive antibodies can be easily identified based on their ability to cross-competite with atezolizumab and / or avelumumab in standard PD-L1 binding assays such as Biacore assays, ELISA assays, or flow cytometry (see, for example, WO2013 / 173223).

[0240] In some embodiments, the antibody that cross-competes with atezolizumab, durvalumab, and / or avelumab for binding to human PD-L1, or that binds to the same epitope region on the antibody as atezolizumab, durvalumab, and / or avelumab for binding to human PD-L1, is a monoclonal antibody. For administration to human subjects, these cross-competing antibodies are chimeric antibodies, engineered antibodies, or humanized or human antibodies. Such chimeric, engineered, humanized, or human monoclonal antibodies can be prepared and isolated using methods well known in the art.

[0241] The anti-PD-L1 antibody that can be used in the disclosed method of the present invention further includes the antigen-binding portion of the antibody described above. It has been sufficiently demonstrated that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody.

[0242] Anti-PD-L1 antibodies suitable for the disclosed methods or compositions are antibodies that bind to PD-L1 with high specificity and affinity, block PD-1 binding, and inhibit the immunosuppressive effects of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, an anti-PD-L1 “antibody” comprises an antigen-binding portion or fragment that binds to PD-L1 and exhibits functional properties similar to those of a full-fledged antibody in inhibiting receptor binding and upregulating the immune system. In some embodiments, the anti-PD-L1 antibody cross-competes with atezolizumab, durvalumab, and / or avelumab for binding to human PD-L1.

[0243] Anti-CTLA-4 antibody

[0244] In some embodiments, the implementation includes the use of an anti-CTLA-4 antibody. In one embodiment, the anti-CTLA-4 antibody binds to and inhibits CTLA-4. In some embodiments, the anti-CTLA-4 antibody is ipilimumab (YERVOY), tramemumab (CP-675,206), AAGEN-1884, or ATOR-1015.

[0245] Other implementation methods

[0246] An isolated antibody that binds to CD36 includes a light chain CDR1 region, a light chain CDR2 region, a light chain CDR3 region, a heavy chain CDR1 region, a heavy chain CDR2 region, and a heavy chain CDR3 region; wherein the heavy chain CDR3 region is the heavy chain CDR3 region present as determined according to the Kabat numbering scheme in SEQ ID NO:5.

[0247] 1. A chimeric antibody that binds to CD36, comprising a light chain CDR1 region, a light chain CDR2 region, a light chain CDR3 region, a heavy chain CDR1 region, a heavy chain CDR2 region, and a heavy chain CDR3 region; wherein the heavy chain CDR3 region is the heavy chain CDR3 region present in SEQ ID NO:5 as determined according to the Kabat numbering scheme.

[0248] 2. A humanized antibody that binds to CD36, comprising a light chain CDR1 region, a light chain CDR2 region, a light chain CDR3 region, a heavy chain CDR1 region, a heavy chain CDR2 region, and a heavy chain CDR3 region, wherein the heavy chain CDR3 region is the heavy chain CDR3 region present in SEQ ID NO:5 as determined according to the Kabat numbering scheme.

[0249] 3. A separated antibody that binds to CD36, comprising the light chain CD1 region, light chain CD2 region, light chain CD3 region, heavy chain CD1 region, heavy chain CD2 region, and heavy chain CD3 region.

[0250] The light chain CDR1 region, the light chain CDR2 region, and the light chain CDR3 region are the light chain CDR1 region, light chain CDR2 region, and light chain CDR3 region as defined in SEQ ID NO:7 according to the Kabat numbering scheme; and

[0251] The heavy chain CDR1 region, the heavy chain CDR2 region, and the heavy chain CDR3 region are the heavy chain CDR1 region, heavy chain CDR2 region, and heavy chain CDR3 region as defined in SEQ ID NO:5 according to the Kabat numbering scheme.

[0252] 4. A chimeric antibody that binds to CD36, comprising a light chain CDR1 region, a light chain CDR2 region, a light chain CDR3 region, a heavy chain CDR1 region, a heavy chain CDR2 region, and a heavy chain CDR3 region.

[0253] The light chain CDR1 region, the light chain CDR2 region, and the light chain CDR3 region are the light chain CDR1 region, light chain CDR2 region, and light chain CDR3 region as defined in SEQ ID NO:7 according to the Kabat numbering scheme; and

[0254] The heavy chain CDR1 region, the heavy chain CDR2 region, and the heavy chain CDR3 region are the heavy chain CDR1 region, heavy chain CDR2 region, and heavy chain CDR3 region as defined in SEQ ID NO:5 according to the Kabat numbering scheme.

[0255] 5. A humanized antibody that binds to CD36, comprising light chain CD1, light chain CD2, light chain CD3 regions, heavy chain CD1, heavy chain CD2, and heavy chain CD3 regions.

[0256] The light chain CDR1 region, the light chain CDR2 region, and the light chain CDR3 region are the light chain CDR1 region, light chain CDR2 region, and light chain CDR3 region as defined in SEQ ID NO:7 according to the Kabat numbering scheme; and

[0257] The heavy chain CDR1 region, the heavy chain CDR2 region, and the heavy chain CDR3 region are the heavy chain CDR1 region, heavy chain CDR2 region, and heavy chain CDR3 region as defined in SEQ ID NO:5 according to the Kabat numbering scheme.

[0258] 6. The antibody according to any one of embodiments 1 to 3, wherein the heavy chain CDR1 region contains SEQ ID NO:27, the heavy chain CDR2 region contains SEQ ID NO:28, the heavy chain CDR3 region contains SEQ ID NO:29, the light chain CDR1 region contains SEQ ID NO:30, the light chain CDR2 region contains SEQ ID NO:31, and the light chain CDR3 region contains SEQ ID NO:32.

[0259] 7. The antibody according to any one of embodiments 1 to 3, wherein the heavy chain CDR1 region contains SEQ ID NO:37, the heavy chain CDR2 region contains SEQ ID NO:38, the heavy chain CDR3 region contains SEQ ID NO:29, the light chain CDR1 region contains SEQ ID NO:30, the light chain CDR2 region contains SEQ ID NO:31, and the light chain CDR3 region contains SEQ ID NO:32.

[0260] 8. The antibody according to any one of embodiments 1 to 3, wherein the heavy chain CDR1 region contains SEQ ID NO:39, the heavy chain CDR2 region contains SEQ ID NO:40, the heavy chain CDR3 region contains SEQ ID NO:41, the light chain CDR1 region contains SEQ ID NO:42, the light chain CDR2 region contains SEQ ID NO:43, and the light chain CDR3 region contains SEQ ID NO:32.

[0261] 9. The humanized antibody according to Embodiment 3, wherein the heavy chain CDR region comprises:

[0262] (a) SEQ ID NO: 37, 38 and 29;

[0263] (b) SEQ ID NO: 44, 46 and 29; or

[0264] (c)SEQ ID NO:45, 47 and 29.

[0265] 10. The humanized antibody according to embodiment 3 or 10, wherein the light chain CDR region comprises SEQ ID NO: 30, 31 and 32.

[0266] 11. The humanized antibody according to embodiment 3 or 10, wherein the light chain CDR region comprises SEQ ID NO: 48, 31 and 32.

[0267] 12. The humanized antibody according to embodiment 3 or 10, wherein the light chain CDR region comprises SEQ ID NO: 48, 49 and 32.

[0268] 13. The humanized antibody according to embodiment 3 or 10, wherein the light chain CDR region comprises SEQ ID NO: 30, 50 and 32.

[0269] 14. The humanized antibody according to any one of embodiments 3 and 10 to 14, wherein the heavy chain variable region comprises SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54; and wherein the light chain variable region comprises SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 or SEQ ID NO:58.

[0270] 15. The humanized antibody according to embodiment 15, wherein the humanized antibody comprises:

[0271] (a) The heavy chain variable region containing SEQ ID NO:51 and the light chain variable region containing SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 or SEQ ID NO:58;

[0272] (b) The heavy chain variable region containing SEQ ID NO:52 and the light chain variable region containing SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 or SEQ ID NO:58;

[0273] (c) A heavy chain variable region comprising SEQ ID NO:53 and a light chain variable region comprising SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 or SEQ ID NO:58; or

[0274] (d) The heavy chain variable region containing SEQ ID NO:54 and the light chain variable region containing SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 or SEQ ID NO:58.

[0275] 16. An isolated antibody that binds to the same human CD36 epitope as an antibody comprising the light chain in SEQ ID NO:7 and the heavy chain in SEQ ID NO:5.

[0276] 17. An isolated antibody that competes with an antibody comprising the light chain in SEQ ID NO:7 and the heavy chain in SEQ ID NO:5 for binding to human CD36.

[0277] 18. The antibody according to any one of embodiments 1 to 18, wherein the antibody is substantially free of antibodies that do not specifically bind to CD36.

[0278] 19. An antibody according to any one of embodiments 1 to 19, wherein the antibody substantially does not contain light chains comprising light chain CDR1, light chain CDR2 and light chain CDR3 regions as defined in SEQ ID NO:9 according to the Kabat numbering scheme.

[0279] 20. The antibody according to any one of embodiments 1 to 20, wherein the antibody binds to human CD36.

[0280] 21. An antibody according to any of embodiments 1 to 21, wherein, if measured using SPR data fitted in a 1-to-1 model, the antibody has a Kc of less than 10 nM. D It combines with human CD36.

[0281] 22. An antibody according to any one of embodiments 1, 2, 4, 5, 7 to 9 or 19 to 22, wherein the antibody comprises VH having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO:11.

[0282] 23. The antibody according to embodiment 23, wherein the antibody comprises VH containing the amino acid sequence of SEQ ID NO:11.

[0283] 24. An antibody according to any one of embodiments 1, 2, 4, 5, 7 to 9 or 19 to 22, wherein the antibody comprises a VL having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO:13.

[0284] 25. The antibody according to embodiment 25, wherein the antibody comprises a VL containing the amino acid sequence of SEQ ID NO:13.

[0285] 26. The antibody according to any one of embodiments 1 to 26, wherein the antibody further comprises a heavy chain constant region.

[0286] 27. The antibody according to embodiment 27, wherein the heavy chain constant region is selected from the group consisting of: human immunoglobulin IgA1, IgA2, IgG1, IgG2, IgG3 or IgG4 heavy chain constant regions.

[0287] 28. The antibody according to embodiment 28, wherein the antibody comprises the constant region of the IgG1 heavy chain.

[0288] 29. The antibody according to embodiment 29, wherein the heavy chain constant region comprises an IgG constant region, and the IgG constant region contains amino acid substitutions for L234A and L235A (“LALA”).

[0289] 30. The antibody according to embodiment 29, wherein the heavy chain constant region comprises an IgG constant region containing amino acid substitutions selected from the group consisting of: L234G, L235S, and G236R; L234S, L235T, and G236R; L234S, L235V, and G236R; L234T, L235Q, and G236R; L234T, L235T, and G236R; L234A and L235A; and L234A, L235A, and P329G.

[0290] 31. The antibody according to embodiment 28, wherein the antibody comprises the IgG4 heavy chain constant region.

[0291] 32. The antibody according to embodiment 31, wherein the heavy chain constant region comprises an IgG constant region, and the IgG constant region contains an amino acid substitution S228P.

[0292] 33. The antibody according to any one of embodiments 1 to 33, wherein the antibody further comprises a light chain constant region.

[0293] 34. The antibody according to embodiment 34, wherein the light chain constant region is selected from the group consisting of the light chain constant regions of human immunoglobulin κ and λ.

[0294] 35. The antibody according to any one of embodiments 1 to 35, wherein the antibody further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is a human IgG1 heavy chain constant region, and wherein the light chain constant region is a human κ light chain constant region.

[0295] 36. The antibody according to any one of embodiments 1 to 2, 4 to 5 or 17 to 36, wherein the antibody comprises the light chain in SEQ ID NO:23 and the heavy chain in SEQ ID NO:21.

[0296] 37. The antibody according to any one of embodiments 1 to 2, 4 to 5 or 17 to 36, wherein the antibody comprises the light chain in SEQ ID NO:23 and the heavy chain in SEQ ID NO:64.

[0297] 38. The antibody according to any one of embodiments 1 to 36, wherein the antibody is an antigen-binding fragment.

[0298] 39. The antigen-binding fragment according to embodiment 39, wherein the antigen-binding fragment comprises Fab, Fab', F(ab')2, single-chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, intracellular antibody, IgGΔCH2, microantibody, F(ab')3, tetraantibody, triantibody, biantibody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2 or scFv-Fc.

[0299] 40. A pharmaceutical composition comprising the antibody described in any one of embodiments 1 to 40 and a pharmaceutically acceptable excipient.

[0300] 41. The pharmaceutical composition according to embodiment 41, wherein at least 95% of the antibody in the composition is non-fucosylated.

[0301] 42. The pharmaceutical composition according to embodiment 41 or 42 further comprises a PD-1 inhibitor.

[0302] 43. The pharmaceutical composition according to embodiment 43, wherein the PD-1 inhibitor is an anti-PD-1 antibody.

[0303] 44. The pharmaceutical composition according to embodiment 44, wherein the anti-PD-1 antibody is pembrolizumab, pildizumab, or nivolumab.

[0304] 45. The pharmaceutical composition according to any one of embodiments 41 to 45 further comprises a PD-L1 inhibitor.

[0305] 46. ​​The pharmaceutical composition according to embodiment 46, wherein the PD-L1 inhibitor is an anti-PD-L1 antibody.

[0306] 47. The pharmaceutical composition according to embodiment 47, wherein the anti-PD-L1 antibody is atezolizumab, durvalumab, avelumab, or BMS-936559.

[0307] 48. The pharmaceutical composition according to any one of embodiments 41 to 48 further comprises a CTLA-4 inhibitor.

[0308] 49. The pharmaceutical composition according to embodiment 49, wherein the CTLA-4 inhibitor is an anti-CTLA-4 antibody.

[0309] 50. The pharmaceutical composition according to embodiment 50, wherein the anti-CTLA-4 antibody is ipilimumab.

[0310] 51. The pharmaceutical composition according to any one of embodiments 41 to 51, wherein the composition further comprises a chemotherapeutic agent.

[0311] 52. The pharmaceutical composition according to embodiment 52, wherein the chemotherapeutic agent is cisplatin.

[0312] 53. The pharmaceutical composition according to any one of embodiments 41 to 53, wherein the antibody is substantially free of light chains comprising light chain CDR1, light chain CDR2 and light chain CDR3 regions as defined in SEQ ID NO:9 according to the Kabat numbering scheme.

[0313] 54. A method of treating a patient with cancer, comprising administering to a subject in need a therapeutically effective amount of an antibody as described in any one of embodiments 1 to 40, or a therapeutically effective amount of a pharmaceutical composition as described in any one of embodiments 41 to 54.

[0314] 55. The method according to embodiment 55, wherein the cancer is oral squamous cell carcinoma, head and neck cancer, esophageal cancer, gastric cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, colon cancer, kidney cancer, prostate cancer, sarcoma, melanoma, leukemia, or lymphoma.

[0315] 56. A method of treating one or more metastatic tumors in a patient, comprising administering to a subject in need a therapeutically effective amount of an antibody as described in any one of embodiments 1 to 40, or a therapeutically effective amount of a pharmaceutical composition as described in any one of embodiments 41 to 54.

[0316] 57. The method according to embodiment 57, wherein the metastatic tumor develops from oral squamous cell carcinoma, head and neck cancer, esophageal cancer, gastric cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, colon cancer, kidney cancer, prostate cancer, sarcoma, melanoma, leukemia, or lymphoma.

[0317] 58. The method according to embodiment 58, wherein, as measured by IVIS imaging or H&E staining, the treatment reduces the size of metastatic tumors.

[0318] 59. The method according to any one of embodiments 57 to 59, wherein, as measured by IVIS imaging or H&E staining, the treatment inhibits the formation or development of metastatic tumors.

[0319] 60. The method according to any one of embodiments 55 to 60, wherein the anti-CD36 antibody blocks CD36-mediated uptake of fatty acids and / or oxLDL, while having little or no effect on the binding of CD36 to TSP-1.

[0320] 61. The method according to any one of embodiments 55 to 61, wherein the patient is a human patient.

[0321] 62. The method according to any one of embodiments 55 to 62, wherein the anti-CD36 antibody is a full-length antibody, a single-chain antibody, scFv, a Fab fragment, or an F(ab')2 fragment.

[0322] 63. The method according to any one of embodiments 55 to 63, wherein the anti-CD36 antibody is a full-length antibody.

[0323] 64. The method according to embodiment 64, wherein the anti-CD36 antibody comprises the light chain in SEQ ID NO:23 and the heavy chain in SEQ ID NO:21.

[0324] 65. The method according to embodiment 64, wherein the anti-CD36 antibody comprises the light chain in SEQ ID NO:23 and the heavy chain in SEQ ID NO:64.

[0325] 66. The method according to any one of embodiments 55 to 66, wherein the method further includes administering a second treatment.

[0326] 67. The method according to embodiment 67, wherein the second treatment is immunotherapy.

[0327] 68. The method according to embodiment 68, wherein the immunotherapy is a PD-1 inhibitor.

[0328] 69. The method according to embodiment 69, wherein the PD-1 inhibitor is an anti-PD-1 antibody.

[0329] 70. The method according to embodiment 70, wherein the anti-PD-1 antibody is pembrolizumab, pildizumab, or nivolumab.

[0330] 71. The method according to embodiment 68, wherein the immunotherapy is a PD-L1 inhibitor.

[0331] 72. The method according to embodiment 72, wherein the PD-L1 inhibitor is an anti-PD-L1 antibody.

[0332] 73. The method according to embodiment 73, wherein the anti-PD-L1 antibody is atezolizumab, durvalumab, avelumab, or BMS-936559.

[0333] 74. The method according to embodiment 68, wherein the immunotherapy is a CTLA-4 inhibitor.

[0334] 75. The method according to embodiment 75, wherein the CTLA-4 inhibitor is an anti-CTLA-4 antibody.

[0335] 76. The method according to embodiment 76, wherein the anti-CTLA-4 antibody is ipilimumab.

[0336] 77. The method according to embodiment 67, wherein the second treatment is a chemotherapeutic agent.

[0337] 78. The method according to embodiment 78, wherein the chemotherapeutic agent is cisplatin.

[0338] 79. The method according to any one of embodiments 55 to 79, wherein metastatic tumors are reduced or suppressed in the subject.

[0339] 80. The method according to any one of embodiments 67 to 80, wherein two treatments are applied sequentially.

[0340] 81. The method according to any one of embodiments 67 to 80, wherein two treatments are applied simultaneously.

[0341] 82. A method for treating a subject suffering from cancer expressing CD36, comprising administering a therapeutically effective amount of the anti-CD36 antibody according to any one of embodiments 1 to 40 to the subject.

[0342] 83. An antibody for use according to embodiment 83, wherein the cancer is oral squamous cell carcinoma, head and neck cancer, esophageal cancer, gastric cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, colon cancer, kidney cancer, prostate cancer, sarcoma, melanoma, leukemia, or lymphoma.

[0343] 84. An antibody for use according to embodiment 83 or 84, wherein the cancer is metastatic cancer.

[0344] 85. An antibody for use according to any one of embodiments 83 to 85, wherein, as measured by IVIS imaging or H&E staining, the treatment reduces the size of metastatic tumors.

[0345] 86. An antibody for use according to any one of embodiments 83 to 86, wherein, as measured by IVIS imaging or H&E staining, the treatment inhibits the formation or development of metastatic tumors.

[0346] 87. An antibody for use according to any one of embodiments 83 to 87, wherein the anti-CD36 antibody blocks CD36-mediated uptake of fatty acids and / or oxLDL, while having little or no effect on the binding of CD36 to TSP-1.

[0347] 88. An antibody for any one of the uses described in embodiments 83 to 88, wherein the use is combined with a second treatment.

[0348] 89. An antibody for use according to embodiment 89, wherein the second treatment is immunotherapy.

[0349] 90. An antibody for use according to embodiment 90, wherein the immunotherapy is an anti-PD-1 antibody, an anti-PL-L1 antibody, or an anti-CTLA-4 antibody.

[0350] 91. An antibody for use according to embodiment 89, wherein the second treatment is a chemotherapeutic agent.

[0351] 92. An antibody for use according to embodiment 92, wherein the chemotherapeutic agent is cisplatin.

[0352] 93. Use of the antibody according to any one of embodiments 1 to 40 in the preparation of a medicament for treating a person suffering from a cancer expressing CD36.

[0353] 94. Use of the antibody according to embodiment 94, wherein the cancer is oral squamous cell carcinoma, head and neck cancer, esophageal cancer, gastric cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, colon cancer, kidney cancer, prostate cancer, sarcoma, melanoma, leukemia, or lymphoma.

[0354] 95. Use of the antibody according to embodiment 94 or 95, wherein the cancer is metastatic cancer.

[0355] 96. Use of the antibody according to any one of embodiments 94 to 96, wherein, as measured by IVIS imaging or H&E staining, the treatment reduces the size of metastatic tumors.

[0356] 97. Use of the antibody according to any one of embodiments 94 to 97, wherein, as measured by IVIS imaging or H&E staining, the treatment inhibits the formation or development of metastatic tumors.

[0357] 98. Use of the antibody according to any one of embodiments 94 to 98, wherein the anti-CD36 antibody blocks CD36-mediated uptake of fatty acids and / or oxLDL while having little or no effect on the binding of CD36 to TSP-1.

[0358] 99. Use of the antibody according to any one of embodiments 94 to 99, wherein the use is combined with a second treatment.

[0359] 100. Use of the antibody according to embodiment 100, wherein the second treatment is immunotherapy.

[0360] 101. Use of the antibody according to embodiment 101, wherein the immunotherapy is an anti-PD-1 antibody, an anti-PL-L1 antibody, or an anti-CTLA-4 antibody.

[0361] 102. Use of the antibody according to embodiment 100, wherein the second treatment is a chemotherapeutic agent.

[0362] 103. Use of the antibody according to embodiment 103, wherein the chemotherapeutic agent is cisplatin.

[0363] 104. An isolated polynucleotide encoding the antibody described in any one of embodiments 1 to 40.

[0364] 105. The isolated polynucleotide according to embodiment 105, which encodes the light chain in SEQ ID NO:7 and the heavy chain in SEQ ID NO:5.

[0365] 106. The isolated polynucleotide according to embodiment 105 or 106, comprising SEQ ID NO:8.

[0366] 107. The isolated polynucleotide according to any one of embodiments 105 to 107, comprising SEQ ID NO:6.

[0367] 108. The isolated polynucleotide according to embodiment 105 or 106, comprising SEQ ID NO:24.

[0368] 109. The isolated polynucleotide according to any one of embodiments 105 to 107, comprising SEQ ID NO:22.

[0369] 110. A carrier comprising the isolated polynucleotide described in any one of embodiments 105 to 110.

[0370] 111. A cell comprising the isolated polynucleotide of any one of embodiments 105 to 110 or the vector of embodiment 111.

[0371] 112. The cells according to embodiment 112 are selected from the group consisting of: Escherichia coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NSO, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, BW, LM, COS1, COS7, BSC1, BSC40, BMT10 cells, plant cells, insect cells, and human cells in tissue culture.

[0372] 113. The cell according to embodiment 112 or 113, wherein the cell lacks the functional α-1,6-fucosyltransferase gene (FUT8).

[0373] 114. A method for preparing an antibody capable of specifically binding to CD36, comprising expressing the antibody in any of the cells described in embodiments 112 to 114.

[0374] 115. A method for preparing an antibody capable of specifically binding to CD36, comprising culturing the cells described in any one of embodiments 112 to 115 and isolating the antibody expressed therein.

[0375] 116. Use of the antibody according to any one of embodiments 1 to 40 for the preparation of a pharmaceutical composition.

[0376] 117. Use of the antibody and pharmaceutically acceptable excipient or carrier according to any one of embodiments 1 to 40 for the preparation of a pharmaceutical composition.

[0377] 118. The method according to any one of embodiments 57 to 82, wherein the metastatic tumor is present in one or more of the liver, lungs, spleen, kidneys, cervical lymph nodes or peritoneal wall.

[0378] 119. An antibody for use according to any one of embodiments 83 to 93, wherein the metastatic cancer includes metastatic tumors in one or more of the liver, lungs, spleen, kidneys, cervical lymph nodes, or peritoneal wall.

[0379] 120. Use of the antibody according to any one of embodiments 94 to 104, wherein the metastatic cancer includes metastatic tumors in one or more of the liver, lungs, spleen, kidneys, cervical lymph nodes, or peritoneal wall.

[0380] 121. A method of treating a patient’s primary and metastatic tumors, comprising administering to a subject in need a therapeutically effective amount of an antibody as described in any one of embodiments 1 to 40, or a therapeutically effective amount of a pharmaceutical composition as described in any one of embodiments 41 to 54.

[0381] 122. The method according to embodiment 122, wherein the cancer is oral squamous cell carcinoma, head and neck cancer, esophageal cancer, gastric cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, colon cancer, kidney cancer, prostate cancer, sarcoma, melanoma, leukemia, or lymphoma.

[0382] 123. The method according to embodiment 122 or 123, wherein the metastatic tumor develops from oral squamous cell carcinoma, head and neck cancer, esophageal cancer, gastric cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, colon cancer, kidney cancer, prostate cancer, sarcoma, melanoma, leukemia, or lymphoma.

[0383] 124. The method according to any one of embodiments 122 to 124, wherein the treatment reduces the size of metastatic tumors, as measured by IVIS imaging or H&E staining.

[0384] 125. The method according to any one of embodiments 122 to 125, wherein the treatment reduces the size of the primary tumor.

[0385] 126. The method according to any one of embodiments 122 to 126, wherein, as measured by IVIS imaging or H&E staining, the treatment inhibits the formation or development of metastatic tumors.

[0386] 127. The method according to any one of embodiments 122 to 127, wherein the anti-CD36 antibody blocks CD36-mediated uptake of fatty acids and / or oxLDL, while having little or no effect on the binding of CD36 to TSP-1.

[0387] 128. The method according to any one of embodiments 122 to 128, wherein the patient is a human patient.

[0388] 129. The method according to any one of embodiments 122 to 129, wherein the anti-CD36 antibody is a full-length antibody, a single-chain antibody, scFv, a Fab fragment, or an F(ab')2 fragment.

[0389] 130. The method according to any one of embodiments 122 to 130, wherein the anti-CD36 antibody is a full-length antibody.

[0390] 131. The method according to embodiment 131, wherein the anti-CD36 antibody comprises the light chain in SEQ ID NO:23 and the heavy chain in SEQ ID NO:21.

[0391] 132. The method according to embodiment 131, wherein the anti-CD36 antibody comprises the light chain in SEQ ID NO:23 and the heavy chain in SEQ ID NO:64.

[0392] 133. The method according to any one of embodiments 122 to 133, wherein the method further includes administering a second treatment.

[0393] 134. The method according to embodiment 134, wherein the second treatment is immunotherapy.

[0394] 135. The method according to embodiment 135, wherein the immunotherapy is a PD-1 inhibitor.

[0395] 136. The method according to embodiment 136, wherein the PD-1 inhibitor is an anti-PD-1 antibody.

[0396] 137. The method according to embodiment 137, wherein the anti-PD-1 antibody is pembrolizumab, pildizumab, or nivolumab.

[0397] 138. The method according to embodiment 135, wherein the immunotherapy is a PD-L1 inhibitor.

[0398] 139. The method according to embodiment 139, wherein the PD-L1 inhibitor is an anti-PD-L1 antibody.

[0399] 140. The method according to embodiment 140, wherein the anti-PD-L1 antibody is atezolizumab, durvalumab, avelumab, or BMS-936559.

[0400] 141. The method according to embodiment 135, wherein the immunotherapy is a CTLA-4 inhibitor.

[0401] 142. The method according to embodiment 142, wherein the CTLA-4 inhibitor is an anti-CTLA-4 antibody.

[0402] 143. The method according to embodiment 143, wherein the anti-CTLA-4 antibody is ipilimumab.

[0403] 144. The method according to embodiment 134, wherein the second treatment is a chemotherapeutic agent.

[0404] 145. The method according to embodiment 145, wherein the chemotherapeutic agent is cisplatin.

[0405] 146. The method according to any one of embodiments 122 to 146, wherein metastatic tumors are reduced or suppressed in the subject.

[0406] 147. The method according to any one of embodiments 134 to 147, wherein two treatments are applied sequentially.

[0407] 148. The method according to any one of embodiments 134 to 147, wherein two treatments are applied simultaneously.

[0408] Example

[0409] Example 1: Animal Research

[0410] Unless otherwise stated, the animal studies disclosed in the following examples were conducted using the following materials and methods.

[0411] NOD scid gamma(NSG)(NOD.Cg-Prkdc scid II2rg tm1Wjl / SzJ) mice were purchased from Charles River and internally hybridized. All mice were housed under a 12-hour light / 12-hour dark cycle and SPF conditions, and all procedures were evaluated and approved by the CEEA (Ethics Committee for Animal Experimentation) of the Government of Catalonia. Intralingual injection of SCCs was performed as previously described (Oskarsson et al., 2014; Nieman et al., 2011). Briefly, mice were anesthetized by intraperitoneal injection of a mixture of 50 mg / kg ketamine and 0.5 mg / kg medemidin, and SCC cells resuspended in 30 μl PBS were injected into the tongue of each mouse using a BD ultrafine 6 mm needle. The luciferase bioluminescence signal of the mice was monitored immediately after injection (T0) and weekly thereafter using a Xenogen IVISImaging System-100 (Caliper Life Sciences). Briefly, 50 μl of 5 mg / ml PBS was injected into the animals via retro-orbital injection. -1 Diluted D-fluorescein (Promega). Continuous administration of isoflurane gas was provided to ensure anesthesia of the animals during imaging. Data were quantized using Living Image software version 4.4 (Caliper Life Sciences). Quantization was calculated using unsaturated pixels. Minimum and maximum chromaticity values ​​are shown in the image.

[0412] To treat mice in vivo with neutralizing anti-CD36 antibodies, mice were intraperitoneally injected with 100 μl of physiological serum containing: 5 μg, 10 μg, or 20 μg of neutralizing monoclonal anti-CD36 antibody JC63.1 (CAYMAN, CAY-10009893-500); 5 μg, 10 μg, or 20 μg of neutralizing monoclonal anti-CD36 ONA-0-v1 (IgA or IgG isotype); or 5 μg, 10 μg, or 20 μg of the corresponding control IgA (mouse IgA, kappa[S107], Abcam, ab37322) or IgG antibody. These doses correspond to 0.25 mg / kg, 0.5 mg / kg, and 1 mg / kg, respectively. All antibodies were azide-free and free of added preservative compounds.

[0413] For each experiment, once the experimental group reached the humane endpoint according to the approved CEEA protocol (4-6 weeks after in situ injection, once the mice began to lose weight due to the growth of oral lesions), the mice were sacrificed and then subjected to cell analysis.

[0414] Total blood samples were collected from mice from the inferior vena cava and then processed in the experimental toxicology and ecotoxicology unit (PCB) according to standard procedures.

[0415] Animal tissues were collected and fixed overnight at room temperature (RT) with 4% paraformaldehyde (PFA), then embedded in OCT and frozen at -80°C or dehydrated and embedded in paraffin. Toxicological studies were performed at a histopathology facility according to standard procedures.

[0416] Histological analysis. For analysis, 8 μm frozen or dewaxed antigen-retrieval sections (in boiling 0.01 M, pH 6.0 citric acid for 10 min) were permeated in 0.25% Triton X-100 / PBS for 25 min and blocked in 0.25% gelatin / PBS for 90 min. Hematoxylin and eosin (H&E) staining was performed according to standard protocols. Images were acquired using a Nikon E600+Olympus DP72, Leica SPE, and Leica TCS SP5 confocal microscope. Representative images were selected for each case.

[0417] For all experiments, the appropriate sample size was determined based on the results of the pilot study. No statistical methods were used to determine the sample size. All animals that met the appropriate experimental conditions during the experiment were included in the analysis. Based on the results of the pilot study, male and female homologous groups and their control littermates aged 8 to 12 weeks were used for the experimental studies. On day 7 post-injection, animals were randomly assigned to groups based on the luminescence intensity of the primary tumor or cervical lymph node metastases. Data are typically presented as mean ± sem. Statistical significance was analyzed using Prism 6 software (GraphPad) with two-tailed t-tests, Mann-Whitney U tests, Fisher's exact test, or hypergeometric tests. A p-value less than or equal to 0.05 was considered significant.

[0418] Example 2: Treatment of cancer with or without cisplatin using anti-CD36 antibodies

[0419] The effects of anti-CD36 antibodies with or without cisplatin were studied in NSG mice (immunodeficient). Figure 1A An experimental overview of these studies is provided. These studies included only male mice, although a similar trend was observed using female mice (this data was not reported). All mice were inoculated with commercially available Detroit 562 (ATCC) cancer cells (transduced with a retroviral vector expressing luciferase and green fluorescent protein (Luc-GFP)). Detroit 562 cells were derived from metastatic sites of pharyngeal cancer (i.e., derived from oral cancer). Prior to inoculation, Detroit 562 cells were cultured in a humidified incubator at 37°C and 5% CO2, supplemented with 5 μg / ml of [unspecified substance]. -1 Grow in EMEM (LONZA) containing penicillin / streptomycin and 10% FBS (GIBCO).

[0420] For each mouse, 50,000 Detroit 562 cells were injected in situ. Previous tests have shown that in untreated NSG mice, 100% of mice injected with Detroit 562 cells developed large primary tumors, and 81% of the injected mice developed lymph node metastases within one week of injection.

[0421] Nine days after inoculation with cancer cells, the mice were treated. The mice were divided into four different treatment groups. Figure 1B As shown, the treatment group is:

[0422] Group 1: IgA allotype control (day 1 to day 23 n=9; day 29 n=6);

[0423] Group 2: IgA isotype control plus cisplatin (n=5);

[0424] Group 3: Commercial anti-CD36 antibody (JC63.1) (days 1 to 23, n=6; day 29, n=4);

[0425] Group 4: Commercial anti-CD36 antibody (JC63.1) plus cisplatin (n=5).

[0426] Antibody treatment was administered daily via intraperitoneal (ip) at a dose of 1 mg / kg. Cisplatin was administered twice weekly at a dose of 2 mg / kg (groups 2 and 4). Mice not receiving cisplatin (groups 1 and 3) were instead given an equal volume of PBS. Mice were observed weekly using an in vivo imaging system (IVIS) during treatment. Additionally, mouse weight was measured twice weekly to update the appropriate dosage. Mice were sacrificed when their weight decreased to a level consistent with ethically approved guidelines or at the end of the treatment period. After sacrifice, organs and tissues were collected for immunohistochemical analysis.

[0427] from Figure 2A-2C It can be seen that anti-CD36 Ab therapy and cisplatin have at least additive antitumor activity in inhibiting the growth of primary oral cancer tumors. Figure 2A The results showed that, as measured by the relative intensity of luciferase-induced luminescence in treated mice compared to control mice, mice treated with anti-CD36 antibody and cisplatin were better able to suppress tumor growth than mice treated with control antibody (IgA) and cisplatin. Figure 2B This image shows a representative image of a primary tumor that developed in the tongue after in situ injection of Detroit 562 cells. Figure 2C The study showed that the tumor surface area of ​​primary tumors was reduced in mice treated with anti-CD36 antibody and cisplatin, or control antibody (IgA) and cisplatin.

[0428] Figure 3 Representative images of lung metastases present in mice inoculated with and treated with Detroit 562 cancer cells as described above are shown. These images illustrate that mice treated with cisplatin (top right), a commercial anti-CD36 antibody (JC63.1; bottom left), or cisplatin and JC63.1 (bottom right) had fewer and smaller metastases than control mice (top left). Furthermore, the number of lung metastases ( Figure 4A ) and size ( Figure 4BQuantitative analysis showed that mice treated with JC63.1 alone had smaller and fewer metastases than control mice. Mice treated with cisplatin alone had a similar number of metastases to control mice, although cisplatin did reduce the size of the metastases. Treatment with JC63.1 and cisplatin resulted in a similar number of metastases in mice as treatment with JC63.1 alone. However, treatment with JC63.1 and cisplatin resulted in a greater reduction in the size of metastatic tumors compared to treatment with JC63.1 or cisplatin alone.

[0429] Example 3: Construction of chimeric antibodies

[0430] Novel chimeric antibodies were generated based on ONA-0-v1 and ONA-0-v2 antibodies using standard molecular biology techniques. In short, the variable domains of the ONA-0-v1 and ONA-0-v2 antibodies were codon-optimized for expression in human cells, and NheI and AvaI restriction sites were designed at the 5' and 3' ends. The variable domains were synthesized and then cloned into expression vectors containing the corresponding constant domain sequences of the human IgG1-LALA heavy chain, mouse IgA heavy chain, or human κ light chain. After sequence validation, sufficient plasmids were prepared for transfection using the Plasmid Plus purification kit (Qiagen).

[0431] The chimeric ONA-0-v1 IgG1 LALA antibody comprises the heavy chain in SEQ ID NO:21 and the light chain in SEQ ID NO:23, and is named 1G04. A similar chimeric ONA-0-v2 IgG1 LALA antibody comprises the heavy chain in SEQ ID NO:21 and the light chain in SEQ ID NO:25. Exemplary polynucleotides encoding 1G04 are provided as SEQ ID NO:22 (encoding the heavy chain) and SEQ ID NO:24 (encoding the light chain). Exemplary polynucleotides encoding the chimeric ONA-0-v2 IgG1 LALA antibody are SEQ ID NO:22 (encoding the heavy chain) and SEQ ID NO:26 (encoding the light chain).

[0432] Figure 5 This diagram illustrates the sequence and structure of ONA-0-v1, ONA-0-v2, 1G04, and the chimeric ONA-0-v2 IgG1 LALA antibody. The diagram shows that the light chain variable region (shown in blue) is the only differing region between ONA-0-v1 and ONA-0-v2. Furthermore, the diagram further illustrates that the chimeric version of the ONA-0 antibody includes a human IgG1 Fc tail with a LALA mutation (shown in red) replacing the mouse IgA Fc tail.

[0433] Example 4: Characterization of anti-CD36 antibody

[0434] HEK 293 (human embryonic kidney 293) mammalian cells were passaged to the optimal stage for transient transfection. Cells were transiently transfected with heavy and light chain expression vectors and cultured for another 6 days.

[0435] The culture medium was harvested by centrifugation at 4000 rpm and filtered through a 0.22 μm filter. For IgG antibodies, the first step of purification was performed by protein A affinity chromatography, followed by elution with citrate buffer at pH 3.0. For mouse IgA antibodies, the first step of purification was ConA agarose affinity chromatography, followed by elution with 0.1 M Tris, 0.1 M NaCl, and 0.5 M glucopyranoside buffer (pH 7.6). The purified antibody was then buffer-exchanged into phosphate-buffered saline (PBS) using a PD10 desalting column (GE Healthcare). Antibody concentration was determined by UV spectroscopy, and the antibody was concentrated as needed. Antibody purity was determined by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) using an X-cell SureLock system with 4–12% Bis-Tris NuPAGE gel and NuPAGE MES buffer (Thermo). If necessary, the sample was reduced using NuPAGE sample reducing agent. The stained protein gels showing purified ONA-0-v1, ONA-0-v2, IgG04, and chimeric ONA-0-v2 (IgG1-LALA) are as follows: Figure 6 As shown.

[0436] For ELISA assays, 96-well ELISA plates were coated overnight with recombinant human CD36 (Sino Biological, reference 10752-H08H) or mouse CD36 (Sino Biological, reference 50422-M08H) protein at a concentration of 0.25 mg / ml. After washing twice with PBS, the plates were blocked at 37°C for 1 hour with PBS solution of 4% skim milk. The blocking solution was discarded, and the plates were washed five times with 200 μl / well of PBS-Tween 20 (0.5%). Primary antibodies were either omitted (as in control blocking solution) or mixed into the blocking solution at several dilutions from 0.01 nM to 0.5 μM. The primary antibody solution was added to the wells and incubated overnight at 4°C. After washing the plate five times with 200 μL / well of PBS-Tween 20 (0.5%), the plate was incubated with goat anti-mouse HRP conjugated antibody (Abcam, refer to ab97235) diluted 1:2000 in blocking solution at room temperature for 1 hour. The plate was then washed five times with 200 μL / well of PBS-Tween 20 (0.5%), incubated with 100 μL / well of TMB, and detected at 630 nm using a spectrophotometer after 10, 30, or 60 minutes.

[0437] like Figure 7 The ELISA assays showed that ONA-O-v1 specifically binds to human CD36 (top image; magenta circle) and mouse CD36 (bottom image; magenta circle). Based on these ELISA data, the K+ binding potential of ONA-O-v1 to human CD36 was estimated. D Approximately 0.04 nM, for the K+ of mouse CD36 D Approximately 0.1 nM. Conversely, Figure 7 It was also shown that ONA-0-v2 did not interact with human CD36 (top; gray square) or mouse CD36 (bottom; gray square) in the same ELISA assay.

[0438] like Figure 8 The ELISA assays showed that the ONA-0-v1 antibody and the commercially available anti-CD36 antibody (JC63.1) exhibited similar affinity and binding properties for human CD36 (top) and mouse CD36 (bottom).

[0439] like Figure 28A and Figure 28B The ELISA assays showed that the 1G04 and 1G06 antibodies exhibited similar affinity and binding properties to human CD36 and mouse CD36.

[0440] For FACS analysis, cells were treated with trypsin, collected in 15 mL tubes, and diluted in wash buffer (PBS solution of 2% FBS). Cells were then centrifuged at 1500 rpm for 5 minutes at 4°C, the supernatant was discarded, and the cells were resuspended in fresh wash buffer. Anti-CD36 antibody was either omitted (as in control wash buffer treatment) or serially diluted in wash buffer (up to 100 nM) and added to the cells. Cells were incubated on ice for 1 hour. Cells were then centrifuged at 1500 rpm for 5 minutes at 4°C, the supernatant was discarded, and the cells were resuspended in fresh wash buffer. Finally, cells were incubated with goat anti-mouse IgA (BV421 rat anti-mouse IgA, Becton Dickinson, reference 743293) diluted 1:100 in wash buffer, washed, and analyzed by FACS.

[0441] like Figure 9A and 9B FACS assays showed that both ONA-O-v1 and 1G04 specifically bound to cells overexpressing human CD36. Figure 9AA commercial anti-CD36 antibody (JC63.1) similarly bound to cells overexpressing human CD36. However, similar to what was observed in ELISA assays, no interaction was observed between the chimeric ONA-O-v2 IgG1 LALA antibody and cells overexpressing human CD36 in FACS assays. Figure 9B ).

[0442] like Figure 10 The FACS assay showed that when the antibody was used at a concentration of 100 nM, ONA-0-v1 and 1G04 bound equivalently to cells overexpressing human CD36.

[0443] like Figure 29 FACS assays showed that the 1G04 and 1G06 antibodies bound equivalently to cells overexpressing human CD36.

[0444] The affinity of ONA-0-v1, 1G04, and a commercial anti-CD36 antibody (JC63.1) for human CD36 was also measured using a Biacore T200 via surface plasmon resonance (SPR). 1G04 was analyzed using a protein A capture surface with a capture range of 100–150 RU and an antigen titration range of 3.3–333 nM. Mouse antibody ONA-0-V1 and a commercial anti-CD36 antibody were analyzed using an anti-mouse IgA capture surface with a capture range of 160–180 RU and an antigen titration range of 3.3–333 nM. For each individual analysis cycle, titrations of five antigen concentrations were injected onto the captured antibody, and then the dissociation of the complex was measured. A dual-reference method was employed, where data from reference surfaces (fc1 and fc3, respectively) from uncaptured antibodies were subtracted from the antibody-bound capture surfaces (fc2 and fc4). Each antigen titration cycle included a buffer blank injection, which was then subtracted from the analyte injection cycle to correct for minor variations in antibody capture surface density. All analyses were performed at 25°C, and the sample rack was maintained at 6°C during experimental runs. Each experiment was run at least three times, and the reported mean binding constant was generated from at least two independent assays. All analyses were performed in 40 μL / min PBS-T run buffer.

[0445] In SPR analysis, commercial anti-CD36 antibodies and ONA-0-V1 showed similar K... D The values ​​are as follows, with 1G04 showing the tightest bond. The SPR results are shown in Tables 6, 7, and 8 below.

[0446] Table 6 – Mean kinetic data of antibody-CD36 interaction measured by SPR (fitted with a 1-to-1 model)

[0447] Commercial anti-CD36 4.18E+05 1.96E-03 4.71E-09 ONA-0-v1(IgA) 6.32E+05 3.54E-03 5.85E-09 1G04(IgG1) 1.00E+05 1.59E-04 1.74E-09

[0448] Table 7 – Average kinetic data of antibody-CD36 interaction measured by SPR (fitted with a 2-state model)

[0449] Commercial anti-CD36 8.69E+04 1.80E-03 5.72E-04 3.32E-04 7.61E-09 ONA-0-v1(IgA) 1.08E+05 2.30E-03 5.58E-04 3.93E-04 8.72E-09 1G04(IgG1) 1.57E+05 3.24E-03 1.31E-03 1.64E-04 2.78E-09

[0450] Table 8 – Mean kinetic data of antibody-CD36 interaction measured by SPR (fitted with a bivalent model)

[0451]

[0452] To measure the ability of anti-CD36 antibodies to alter CD36-driven fatty acid uptake, commercially available SCC-25 cells (ATCC) were modified by stable transduction with a CD36-expressing retroviral vector and a luciferase-expressing lentiviral vector. These cells, derived from tongue squamous cell carcinoma, were grown in serum-free keratinocyte medium (KSFM) supplemented with 5 μg / ml penicillin / streptomycin, 0.025 mg / ml bovine pituitary extract, and 0.2 μg / ml hEGF. Fatty acid uptake was assessed using a commercially available bioluminescently labeled long-chain fatty acid analog (SwissLumix) as a substrate. For assays, cells were placed in 96-well plates and stimulated with 100 μM palmitate for 48 hours starting the next day. 10 μg / ml of an allotype control antibody or 1G04 was added before palmitate stimulation, and the results were updated over the following days. To quantify uptake kinetics, substrate was added to the plate after washing with PBS 1X, and readings were taken over time using a Synergy H1M microplate reader. The analysis showed that treatment with 1G04 blocked fatty acid uptake over time, such as… Figure 27A As shown in the figure. A comparison of fatty acid uptake 256 seconds after substrate addition showed that 1G04 inhibited fatty acid uptake by approximately 17% compared to control cells. Figure 27B *** = p = 0.0010).

[0453] Example 5: Treatment of cancer with or without cisplatin using ONA-0-v1 anti-CD36 antibody

[0454] The combined effects of ONA-0-v1 anti-CD36 antibody with or without cisplatin were studied in NSG mice (immunodeficient). Figure 11AAn experimental overview of these studies is provided. These studies included male and female mice. All mice were inoculated with commercially available FaDu (ATCC) cancer cells (transduced using a retroviral vector expressing luciferase and green fluorescent protein (Luc-GFP)). FaDu cells were derived from squamous cell carcinoma (i.e., oral cancer). Prior to inoculation, FaDu cells were cultured in a humidified incubator at 37°C and 5% CO2, supplemented with 5 μg / ml of [unspecified substance]. -1 Grow in EMEM (LONZA) containing penicillin / streptomycin and 10% FBS (GIBCO).

[0455] For each mouse, 100,000 FaDu cells were injected in situ. Previous tests have shown that in untreated NSG mice, 100% of mice injected with FaDu cells developed large primary tumors, and 91% of the injected mice developed lymph node metastases within one week of injection.

[0456] Nine days after inoculation with cancer cells, the mice were treated. The mice were divided into four different treatment groups. Figure 11B As shown, the treatment group is:

[0457] Group 1: IgA isotype control (n=7);

[0458] Group 2: IgA isotype control plus cisplatin (n=8);

[0459] Group 3: Anti-CD36 antibody ONA-0-v1 (n=8);

[0460] Group 4: Anti-CD36 antibody ONA-0-v1 plus cisplatin (n=8).

[0461] Antibody treatment was administered daily via intraperitoneal (ip) at a dose of 1 mg / kg. Cisplatin was administered twice weekly at a dose of 2 mg / kg (groups 2 and 4). Mice not receiving cisplatin (groups 1 and 3) were instead administered an equal volume of PBS. Mice were observed weekly using an in vivo imaging system (IVIS) during treatment. Additionally, mouse body weight was measured twice weekly to update the appropriate dosage. At the end of treatment, mice were sacrificed, and organs and tissues were collected for immunohistochemical analysis.

[0462] like Figure 12A and 12BAs shown, as measured by IVIS imaging and H&E staining of the primary tumor, treatment with the anti-CD36 antibody ONA-0-v1 in combination with cisplatin had similar efficacy to cisplatin alone. In this model, treatment with ONA-0-v1 alone at a dose of 1 mg / kg had no statistically significant effect on the primary tumor compared to treatment with an allotype control antibody. In contrast, as measured by relative intensity in IVIS imaging, Figure 13A and 13B The study showed that treatment with ONA-0-v1 alone could inhibit the growth of lymph node metastases. Furthermore, as measured by relative intensity in IVIS imaging, treatment with ONA-0-v1 in combination with cisplatin resulted in almost complete inhibition of lymph node metastasis growth.

[0463] Treatment with the ONA-0-v1 antibody inhibited the growth of lymph node metastases. Figure 14 A representative IVIS image of NSG mice vaccinated on day 7 after in situ injection of FaDu cells, just before the start of treatment, is shown. Lymph node metastases in these mice are indicated by circled areas, and the intensity of the luciferase signal is represented by a heatmap. Figure 14 The quantification of lymph node metastases present in all mouse groups on day 7 was also shown. Initial intensity was identical in all groups. Further IVIS imaging was performed at the treatment endpoint, as shown in the figure. Figure 15 As shown in the left figure, as measured by the ratio of IVIS imaging intensity between the treatment endpoint and the starting point, treatment with the ONA-0-v1 antibody inhibited metastatic tumor growth by more than 50% compared to the IgA isotype control. Furthermore, similarly... Figure 15 As shown in the right figure, adding ONA-0-v1 to cisplatin enhances cisplatin's ability to inhibit the growth of metastatic tumors. The combination of ONA-0-v1 and cisplatin resulted in almost complete inhibition of tumor growth in lymph node metastases.

[0464] like Figure 16 As shown, treatment with the ONA-0-v1 antibody also inhibited the exotropy of metastases into lymph nodes. Lymph node metastases appeared in all control mice. Treatment with either cisplatin or ONA-0-v1 prevented lymph node metastases in one of the eight test mice in each treatment group. Furthermore, the inhibition of exotropy by ONA-0-v1 was synergistic with that by cisplatin, as the combination of cisplatin and ONA-0-v1 prevented any metastases in five of the eight test mice.

[0465] During treatment, NSG mice tolerated ONA-0-v1 antibody therapy well. Figure 17A and Figure 17BAs shown, compared to the isotype control mice, ONA-0-v1 treatment alone had no effect on mouse body weight or platelet count. ONA-0-v1 treatment also did not significantly enhance cisplatin-mediated weight loss or cisplatin-mediated platelet count reduction.

[0466] Example 6: Antitumor efficacy of anti-CD36 antibody combined with PD1 inhibition in C57Bl6 / J mice carrying melanoma tumors derived from YUMM1.7 cells.

[0467] 250,000 YUMM1.7 cells were suspended in PBS and subcutaneously injected into the flank of 8-12 week old C57Bl6 / J mice. When the tumor reached 50-100 mm... 3 When the average volume was reached, the mice were randomly grouped and treatment began.

[0468] The experimental groups are shown in Table 9 below.

[0469] Table 9 – Treatment groups treated with anti-CD36 antibodies and anti-PD-1 antibodies

[0470]

[0471] All antibodies were administered intraperitoneally (IP) three times a week at a concentration of 10 mg / kg. Mouse body weight and tumor volume were monitored three times a week, and behavior and survival were monitored daily. When the tumor reached 1.500 mm... 3 At maximum volume, mice were euthanized and tissues were collected. Primary tumors were weighed and measured again with calipers. Lungs and livers were embedded in paraffin for H&E staining and blinded analysis of metastatic lesions. Expected results will show additive or synergistic effects of anti-CD36 antibodies (e.g., 1G04) and anti-PD-1 antibodies in treating cancer in the YUMM1.7 melanoma mouse model.

[0472] Example 7: Treatment of ovarian cancer with ONA-0-v1 anti-CD36 antibody

[0473] The effects of ONA-0-v1 anti-CD36 antibody on ovarian cancer were studied in NSG mice (immunodeficient). Figure 18A The document provides an overview of the experiments conducted in these studies. These studies included only female mice. All mice were inoculated with commercially available OVCAR-3 (ATCC) cancer cells. OVCAR-3 cells are derived from human progressive ovarian adenocarcinoma (i.e., derived from ovarian cancer). Prior to inoculation, OVCAR-3 cells were cultured in a humidified incubator at 37°C and 5% CO2, supplemented with 5 μg / ml of [unspecified substance]. -1 Growth in RPMI-1640 containing penicillin / streptomycin, 0.01 mg / ml bovine insulin and 20% FBS (GIBCO).

[0474] For each mouse, an OVCAR-3 xenograft was implanted in situ. For example... Figure 18B As shown, NSG mice implanted with OVCAR-3 cells developed large primary tumors. Mice implanted with OVCAR-3 also developed metastatic tumors in the peritoneal wall and liver. Exemplary metastatic tumors from inoculated mice are shown in [the image / description]. Figure 19A and 19B middle.

[0475] Treatment of the implanted mice began 23 days after OVCAR-3 tumor graft implantation. The inoculated mice were randomly assigned to one of two treatment groups: a solvent injection control (n=9) or ONA-0-v1 treatment (n=9). Antibody treatment was administered daily via intraperitoneal (ip) at a dose of 3 mg / kg, while control mice received an equal volume of solvent via the same protocol. Mice were sacrificed at the end of the treatment period. After sacrifice, organs and tissues were collected for immunohistochemical analysis.

[0476] like Figure 18B and 18C As shown, treatment with ONA-0-v1 resulted in smaller tumors in the OVCAR-3 mouse model of ovarian cancer. Figure 18C Quantification of this effect showed that OVCAR-3 treatment reduced tumor weight from an average of 1.844g to an average of 1.058g, a reduction of 43%. These data indicate that ONA-0-v1 inhibits tumor growth and / or promotes tumor cell destruction during treatment.

[0477] Histological analysis of primary tumors in mice treated with solvent and mice treated with ONA-0-v1 was also performed. First, the tumors were analyzed by visual examination and pathological quantification to determine the percentage of necrosis. The results of this analysis are as follows: Figure 18D As shown, ONA-0-v1 increased from approximately 24.4% to approximately 40.71% (* = p = 0.0287). This increase indicates that the treated tumors exhibited higher cell death. Primary tumors in treated and ONA-0-v1-treated mice were also analyzed by Sirius red staining to determine the percentage of collagen-forming and fibrotic regions. The results of this analysis show... Figure 18E The study showed that ONA-0-v1 increased the SR-positive area from 16.9% to 22.5% (*=p=0.0457). This increase indicates that ONA-0-v1 treatment increased fibrosis and necrosis, suggesting that the treated tumors were not only smaller but also composed of fewer tumor cells.

[0478] Figure 20A , 20BFigures 20C show the results of quantifying metastatic tumors in mice treated with ONA-0-v1. Figure 20A The results showed that the total number of metastatic tumors in mice treated with ONA-0-v1 was reduced by more than 50% compared to mice treated with solvent. The total number of metastatic tumors was determined by visual examination of the organs. Figure 20B and Figure 20C The results of a macroscopic analysis of the size of metastases in the peritoneal wall and liver are shown separately. The size of metastases was measured visually. In the solvent-treated group, 48% of the animals had large metastases (>5 mm) in the peritoneal wall, 41% had small metastases (1 mm–2 mm), and 11% had no metastases. In animals treated with ONA-0-v1, no large metastases were detected, 38% had small metastases, and 63% had no metastases. In the liver, the percentage of mice without metastases increased from 22% in the solvent-treated group to 50% in the treated group. In animals with liver metastases, the percentage of large metastases decreased from 16% to 6%, and the percentage of small metastases decreased from 62% to 44%. ONA-0-v1 treatment altered the size of peritoneal wall metastases, resulting in the complete disappearance of large metastases, and more mice showing no peritoneal metastases at all. Figure 20B Similarly, treatment with ONA-0-v1 altered the size of liver metastases, resulting in fewer large metastases being detected, and more mice showing no liver metastases at all. Figure 20C Overall, Figure 20A , 20B 20C showed that ONA-0-v1 is effective in reducing the formation and growth of metastases from ovarian cancer.

[0479] Example 8: Treatment of colon cancer with ONA-0-v1 anti-CD36 antibody

[0480] The effects of ONA-0-v1 anti-CD36 antibody on colon cancer were studied in BALB / c nude mice (immunodeficient). Figure 21A An experimental overview of these studies is provided. These studies included only female mice. All mice were inoculated with commercially available HCT-116 (ATCC) cancer cells transduced with a retroviral vector expressing luciferase. HCT-116 cells were derived from human colorectal cancer (i.e., colon cancer). Prior to inoculation, HCT-116 cells were cultured in a humidified incubator at 37°C and 5% CO2, supplemented with 5 μg / ml of [unspecified substance]. -1 It was grown in McCoy's 5A medium containing penicillin / streptomycin and 10% FBS (GIBCO).

[0481] For each mouse, 2 × 10⁶ mice were injected orally. 6HCT-116 cells were administered. Each mouse was imaged post-inoculation and one week later, and liver metastases were confirmed by in vitro chemiluminescence immunoassay before treatment initiation. Treatment began 14 days after HCT-116 cell inoculation. Inoculated mice were assigned to one of two treatment groups: a solvent injection control (n=10) or ONA-0-v1 treatment (n=10). Antibody treatment was administered daily via intraperitoneal (ip) at a dose of 3 mg / kg, while control mice received an equal volume of solvent via the same protocol. All mice were imaged by IVIS on days 7, 14, and 21 after treatment initiation. Mice were sacrificed at the end of treatment (day 25). After sacrifice, organs and tissues were collected for necropsy, in vitro IVIS, and histopathological examination.

[0482] Figure 21B and Figure 21C IVIS imaging of a primary tumor formed at the HCT-116 injection site is shown. Figure 21B The changes in total tumor bioluminescence in vivo over time were shown, and treatment with ONA-0-v1 by day 21 was shown to reduce tumor growth compared to the solvent-treated control (* = p = 0.0288). Figure 21C The bioluminescence of tumors on day 25, measured by ex vivo imaging, is shown after mouse euthanasia. Again, bioluminescence was observed in tumors compared to the solvent-treated control (mean radiation 2.15 x 10⁻⁶). 10 ONA-0-v1 treatment reduced tumor growth (average radiation 1.51*10). 10 ).

[0483] Figure 22A , 22B Figures 22C and 22D show the results of quantifying metastatic tumors in mice treated with ONA-0-v1 and solvent. The penetrance of metastatic tumors in the liver, lung, spleen, and kidney was quantified by in vitro luminescence, where organs that did not show luminescence were characterized as metastatic-free. Figure 22A The results showed that treatment with ONA-0-v1 reduced the percentage of mice with tumors in their livers from 90% to 60% (* = p < 0.0001). Similarly, Figure 22B The results showed that treatment with ONA-0-v1 reduced the percentage of mice with tumors in their lungs from 80% to 60% (*=p=0.0032).

[0484] The luminescence of metastatic tumors in the liver, lungs, spleen, and kidneys was also quantified. Figure 23A , 23B The results of this quantification are shown in 23C and 23D. The organs to be examined were removed from the mice and examined using IVIS. Figure 23B and 23DThe results showed that treatment with ONA-0-v1 induced a reduction in isolated lung size (1.23*10) in treated mice. 7 Up to 1.24*10 6 ()( Figure 23B ) and kidney (4.26*10 6 Up to 1.08*10 6 ()( Figure 23D The luminescence in these organs was almost completely eliminated, reflecting the complete or near-complete elimination of metastases in these organs. Similarly, Figure 23A and 23C The results showed that treatment with ONA-0-v1 resulted in a reduction of 1.41*10⁻⁶ livers in isolated mice. 8 Up to 9.02*10 7 ) and spleen (3.77*10 8 Up to 1.79*10 8 The reduced luminescence of ONA-0-v1 reflects a decrease in the size and / or number of lung metastases. These data suggest that ONA-0-v1 is an effective inhibitor of the spread and growth of metastases in colorectal cancer.

[0485] The weight of mice inoculated with HCT-116 was also tracked throughout the experiment. Figure 24 The results showed that, starting from day 18, the average body weight of mice treated with ONA-0-v1 was higher than that of control mice. For example, on day 18, the body weight of control mice was 84.3% of their starting body weight, while that of mice treated with ONA-0-v1 was 91.1% of their starting body weight. This reflects that ONA-0-v1 mice are healthier and better able to fight colon cancer tumors.

[0486] Example 9: Treatment of ovarian cancer with ONA-0-v1 and 1G04 anti-CD36 antibodies

[0487] The effects of ONA-0-v1 and 1G04 anti-CD36 antibodies on ovarian cancer were studied in NSG mice (immunodeficient). Figure 25A An experimental overview of these studies is provided. These studies included only female mice. All mice were inoculated with commercially available OVCAR-3 (ATCC) cancer cells. OVCAR-3 cells are derived from human progressive ovarian adenocarcinoma (i.e., derived from ovarian cancer). For each mouse, an OVCAR-3 xenograft was implanted in situ. Prior to inoculation, OVCAR-3 cells were cultured in a humidified incubator at 37°C and 5% CO2, supplemented with 5 μg / ml of [unspecified substance]. -1 Growth in RPMI-1640 containing penicillin / streptomycin, 0.01 mg / ml bovine insulin and 20% FBS (GIBCO).

[0488] Treatment of the implanted mice began 7 days after OVCAR-3 tumor graft implantation. The inoculated mice were randomly assigned to one of three treatment groups: solvent injection control (n=9), ONA-0-v1 treatment (n=9), or 1G04 treatment (n=9). ONA-0-v1 antibody was administered daily via intraperitoneal (ip) injection at a dose of 3 mg / kg. TIW (three-times-weekly) 1G04 antibody was administered via ip injection at a dose of 10 mg / kg. Control mice received an equal volume of solvent daily. Figure 25B As can be seen, the body weight of mice remained the same throughout the treatment period in all three treatment groups. Mice were sacrificed at the end of the treatment period. After sacrifice, organs and tissues were collected for necropsy and histopathological analysis.

[0489] Figures 25C to 25G The results of quantifying metastatic tumors in treated mice are shown. Figure 25C The total number of metastases under each treatment condition is shown. The total number of metastases was determined by visual examination of the organs. This analysis showed that the number of metastases in mice treated with ONA-0-v1 was reduced by approximately 45% compared to mice treated with solvent (52 metastases in the solvent group vs. 29 metastases in the treatment group). The total number of metastases in mice treated with 1G04 was also reduced by approximately 35% compared to mice treated with solvent (52 metastases in the solvent group vs. 34 metastases in the treatment group).

[0490] Figure 25D and Figure 25EThe results of a macroscopic analysis of metastatic tumor size in the peritoneal wall and liver are shown separately. Metastatic tumor size was measured visually. Treatment with ONA-0-v1 or 1G04 reduced the observed metastatic tumor size, resulting in fewer large (>5 mm) and medium (1–2 mm) metastatic tumors. In the peritoneal wall of solvent-treated animals, 26% of mice had metastatic tumors >5 mm, 39% had 2–5 mm, and 13% had 1–2 mm. In the peritoneal wall of ONA-0-v1-treated animals, 19% of mice had metastatic tumors >5 mm, 19% had 2–5 mm, and 19% had 1–2 mm. In the peritoneal wall of 1G04-treated animals, 7% of mice had metastatic tumors >5 mm, 11% had 2–5 mm, and 49% had 1–2 mm. Furthermore, the livers of treated mice exhibited a similar pattern. In the livers of animals treated with solvent, 5% of mice had metastases of 2-5 mm, 25% had metastases of 1-2 mm, and 25% had metastases <1 mm. In the livers of animals treated with ONA-0-v1, 6% of mice had metastases of 2-5 mm, 17% had metastases of 1-2 mm, and no mice had metastases <1 mm. Mice treated with 1G04 had no metastases of 2-5 mm, 11% had metastases of 1-2 mm, and no mice had metastases <1 mm. Furthermore, treatment with ONA-0-v1 or 1G04 increased the percentage of animals without metastases in the peritoneal wall and liver. 22% of the solvent-treated mice, 44% of the ONA-0-v1-treated mice, and 33% of the 1G04-treated mice had no metastases in the peritoneal wall. 44% of mice treated with solvent, 78% of mice treated with ONA-0-v1, and 89% of mice treated with 1G04 had no liver metastases.

[0491] Figure 25F Microscopic analysis of the penetrance of lung metastases is shown. Similar to the peritoneal wall and liver, treatment with ONA-0-v1 or 1G04 increased the percentage of animals without lung metastases (from 33% in the solvent group to 44% in the ONA-0-v1 group and 66% in the 1G04 group, respectively). Furthermore, as... Figure 25G The data quantified that treatment with ONA-0-v1 or 1G04 reduced the number of lung metastases per mouse (mean number of metastases was 3.6 in the solvent treatment group, 1.6 in the ONA-0-v1 group, and 1.2 in the 1G04 group).

[0492] In general, Figures 25C to 25GThe results showed that both ONA-0-v1 (mouse IgA antibody) and 1G04 (chimeric IgG1 antibody) effectively reduced the formation and growth of metastases from ovarian cancer.

[0493] Example 10: Treatment of colon cancer with 1G04 anti-CD36 antibody

[0494] The effects of 1G04 antiCD36 antibody on colon cancer were studied in BALB / c nude mice (immunodeficient). Figure 26A An experimental overview of these studies is provided. These studies included only female mice. All mice were inoculated with commercially available HCT-116 (ATCC) cancer cells transduced with a retroviral vector expressing luciferase. HCT-116 cells were derived from human colorectal cancer (i.e., colon cancer). Prior to inoculation, HCT-116 cells were cultured in a humidified incubator at 37°C and 5% CO2, supplemented with 5 μg / ml of [unspecified substance]. -1 It was grown in McCoy's 5A medium containing penicillin / streptomycin and 10% FBS (GIBCO).

[0495] For each mouse, 2 × 10⁶ mice were injected orally. 6 HCT-116 cells were administered. Each mouse was imaged post-inoculation and one week later, and liver metastases were confirmed by in vitro chemiluminescence immunoassay before treatment initiation. Treatment began 12 days post-inoculation with HCT-116 cells. Inoculated mice were assigned to one of two treatment groups: a solvent injection control (n=10) or 1G04 treatment (n=10). Antibody treatment was administered three times weekly via intraperitoneal (ip) at a dose of 10 mg / kg, while control mice received an equal volume of solvent via the same protocol. All mice were imaged by IVIS one day before treatment initiation, and on days 7, 14, and 21 after treatment initiation. Mice were sacrificed at the end of treatment (day 25). After sacrifice, organs and tissues were collected for necropsy, in vitro IVIS, and histopathological examination.

[0496] like Figure 26B As shown, mice treated with 1G04 were able to maintain their weight better during the treatment process. Figure 26C The results of whole-animal bioluminescence imaging over time are shown, representing readings of the growth of luciferase-containing tumor cells in mice. Bioluminescence imaging revealed that 1G04 reduced whole-animal luminescence, thus slowing the in vivo growth of HCT-116 tumor cells after injection.

[0497] Figure 26D , 26E 26F and 26G show the results of quantifying metastatic tumors in mice treated with 1G04 and solvent. After the organs to be examined were removed from the mice, the liver was analyzed using in vitro luminescence via IVIS. Figure 26D ),lung( Figure 26E ),spleen( Figure 26F ) and kidneys ( Figure 26G The luminescence of metastatic tumors in the lungs was quantified. In each organ, 1G04 treatment reduced luminescence, reflecting a reduction in the size and / or number of metastatic tumors. The mean luminescence values ​​observed in the liver, lungs, spleen, and kidneys of solvent-treated mice were 1.69 × 10⁻⁶. 8 5.38*10 6 2.66*10 8 and 4.11*10 7 The average luminescence values ​​observed in the liver, lungs, spleen, and kidneys of mice treated with 1G04 were 1.07*10⁻⁶. 8 1.68*10 6 1.83*10 7 and 1.46*10 7 These data indicate that 1G04 is an effective inhibitor of the spread and growth of metastatic tumors in colorectal cancer.

[0498] In general, Figures 26D to 26G The results showed that 1G04 is effective in reducing the formation and growth of metastases from colorectal cancer.

[0499] Example 11: Treatment of lung cancer with 1G04 anti-CD36 antibody

[0500] The effects of 1G04 anti-CD36 antibody on lung cancer were studied in NSG mice (immunodeficient). Figure 30A An experimental overview of these studies is provided. These studies included only female mice. All mice were inoculated with commercially available A549-luc2 (ATCC) cancer cells (a modified version of A549 cells stably transduced using a lentiviral vector expressing luciferase). A549 cells are derived from lung cancer (i.e., lung cancer-derived cells) and were therefore used as part of a mouse model of lung cancer. Prior to inoculation, A549 cells were cultured in a humidified incubator at 37°C and 5% CO2, supplemented with 5 μg / ml of [unspecified substance]. -1 It was grown in F-12K medium containing penicillin / streptomycin and 10% FBS (GIBCO).

[0501] For each mouse, 1 × 10⁻⁶ mice were injected via tail vein injection. 6 A549 cells were administered. Each mouse was imaged post-inoculation and one week later, and lung metastases were confirmed by chemiluminescence immunoassay before treatment initiation. Treatment began 8 days post-inoculation of A549 cells. Figure 30BAs detailed, the inoculated mice were divided into two treatment groups: a solvent injection control group (n=11) or a 1G04 treatment group (n=11). Antibody treatment was administered three times weekly via intraperitoneal (ip) at a dose of 10 mg / kg, while control mice received an equal volume of solvent via the same protocol. All mice were imaged via IVIS one day before treatment began and weekly thereafter. Mice were sacrificed at the end of the treatment period (day 61). After sacrifice, organs and tissues were collected for necropsy and in vitro IVIS.

[0502] Figure 30C The results of whole-animal bioluminescence imaging over time are shown, with decreased fluorescence observed in mice treated with 1G04. This indicates that 1G04 treatment reduced the growth of injected A549 tumor cells in vivo (** = p = 0.0002). At the endpoint, the lungs of mice treated with the 1G04 antibody were smaller than those of mice treated with the control solvent. Figure 30D This indicates that less tumor growth occurred. The average lung weight observed in solvent-treated mice was 0.90 g, compared to 0.72 g in mice treated with 1G04 (a 20% reduction). Figure 30E As shown, at the endpoint, the lungs of animals treated with 1G04 also exhibited less luminescence (2.11*10). 8 Up to 1.39*10 8 These results indicate that 1G04 inhibits the growth of metastatic tumors in lung cancer.

[0503] Example 12: Treatment of colon cancer with 1G04 anti-CD36 antibody

[0504] The effects of 1G04 antiCD36 antibody on lung cancer were studied in C57BL / 6 mice (immunely active). Figure 31A An experimental overview of these studies is provided. These studies included only female mice. All mice were inoculated with commercially available MC-38 cancer cells transduced with a luciferase-expressing vector. MC-38 cells are derived from mouse colon adenocarcinoma (i.e., colon cancer). Prior to inoculation, MC-38 cells were cultured in a humidified incubator at 37°C and 5% CO2, supplemented with 5 μg / ml of [unspecified substance]. -1 It was grown in DMEM medium containing penicillin / streptomycin and 10% FBS (GIBCO).

[0505] For each mouse, 1 × 10⁻⁶ cells were injected intrasplenically. 6 MC-38 cells were used. Each mouse was imaged 4 days later, and liver metastases were confirmed by in vitro chemiluminescence on day 5 post-inoculation, before the start of treatment. Figure 31BAs detailed, the inoculated mice were divided into two treatment groups: a solvent injection control group (n=13) or a 1G04 treatment group (n=10). Antibody treatment was administered three times weekly via intraperitoneal (ip) at a dose of 10 mg / kg, while control mice received an equal volume of solvent via the same protocol. All mice were imaged by IVIS one day before treatment began and twice weekly thereafter. Mice were sacrificed at the end of the treatment period (day 60). After sacrifice, organs and tissues were collected for necropsy and in vitro IVIS.

[0506] Whole-animal bioluminescence imaging during the study showed that 1G04 treatment reduced luminescence, indicating a decrease in tumor growth (* = p = 0.003). Figure 31C In vitro luminescence analysis showed that mice treated with 1G04 had liver tissue with a concentration of 1.41 x 10⁻⁶ cells / mL. 9 Up to 6.67*10 4 ) and lungs (7.23*10 6 Up to 6.78*10 4 All of them exhibited low luminescence (respectively) Figure 31D and 31E In summary, 1G04 showed efficacy in reducing the size of metastatic tumors in colorectal cancer.

[0507] Example 13: Treatment of breast cancer with 1G04 anti-CD36 antibody

[0508] The effects of 1G04 anti-CD36 antibody on breast cancer were studied in BALB / c mice (immunely active). Figure 32A An experimental overview of these studies is provided. These studies included only female mice. All mice were inoculated with commercially available 4T1 cancer cells (ATCC) transduced with a luciferase-expressing vector. The 4T1 cells were derived from mouse mammary tissue (i.e., from breast cancer). Prior to inoculation, the 4T1 cells were cultured in a humidified incubator at 37°C and 5% CO2, supplemented with 5 μg / ml of [unspecified substance]. -1 It was grown in RPMI medium containing penicillin / streptomycin, 2 mM L-glutamine and 10% FBS (GIBCO).

[0509] For each mouse, 4 × 10 4 4T1 cells were orally inoculated into the mammary fat pad. Treatment began 5 days after 4T1 cell inoculation. Mice were assigned to one of two treatment groups: a solvent injection control (n=10) or 1G04 treatment (n=10). Antibody treatment was administered three times weekly via intraperitoneal (ip) at a dose of 10 mg / kg, while control mice received an equal volume of solvent ( Figure 32BMice were euthanized at the end of the treatment period (day 22). After euthanasia, organs and tissues were collected for autopsy and in vitro IVIS.

[0510] Compared with solvent-treated mice, mice treated with 1G04 showed a reduction in luminescence in their lungs (2.49*10). 5 Up to 5.96*10 4 , Figure 32C This indicates that anti-CD36 treatment reduced the size of metastatic tumors and / or reduced metastasis to distant organs. sequence list <110> ONA Therapeutics Co., Ltd. S. Aznar Benita V. Van Hollen M. de Frias Sanchez B. Morancho Armison S. Guardiola Bagan <120> Anti-CD36 antibodies and their use in cancer treatment <130> 4427.005PC03 <150> US 63 / 117,529 <151> 2020-11-24 <150> US 62 / 986,174 <151> 2020-03-06 <150> EP 20382166.5 <151> 2020-03-06 <160> 59 <170> PatentIn version 3.5 <210> 1 <211> 472 <212> PRT <213> Artificial Sequence <220> <223> Human CD36 <400> 1 Met Gly Cys Asp Arg Asn Cys Gly Leu Ile Ala Gly Ala Val Ile Gly 1 5 10 15 Ala Val Leu Ala Val Phe Gly Gly Ile Leu Met Pro Val Gly Asp Leu 20 25 30 Leu Ile Gln Lys Thr Ile Lys Lys Gln Val Val Leu Glu Glu Gly Thr 35 40 45 Ile Ala Phe Lys Asn Trp Val Lys Thr Gly Thr Glu Val Tyr Arg Gln 50 55 60 Phe Trp Ile Phe Asp Val Gln Asn Pro Gln Glu Val Met Met Asn Ser 65 70 75 80 Ser Asn Ile Gln Val Lys Gln Arg Gly Pro Tyr Thr Tyr Arg Val Arg 85 90 95 Phe Leu Ala Lys Glu Asn Val Thr Gln Asp Ala Glu Asp Asn Thr Val 100 105 110 Ser Phe Leu Gln Pro Asn Gly Ala Ile Phe Glu Pro Ser Leu Ser Val 115 120 125 Gly Thr Glu Ala Asp Asn Phe Thr Val Leu Asn Leu Ala Val Ala Ala 130 135 140 Ala Ser His Ile Tyr Gln Asn Gln Phe Val Gln Met Ile Leu Asn Ser 145 150 155 160 Leu Ile Asn Lys Ser Lys Ser Ser Met Phe Gln Val Arg Thr Leu Arg 165 170 175 Glu Leu Leu Trp Gly Tyr Arg Asp Pro Phe Leu Ser Leu Val Pro Tyr 180 185 190 Pro Val Thr Thr Thr Val Gly Leu Phe Tyr Pro Tyr Asn Asn Thr Ala 195 200 205 Asp Gly Val Tyr Lys Val Phe Asn Gly Lys Asp Asn Ile Ser Lys Val 210 215 220 Ala Ile Ile Asp Thr Tyr Lys Gly Lys Arg Asn Leu Ser Tyr Trp Glu 225 230 235 240 Ser His Cys Asp Met Ile Asn Gly Thr Asp Ala Ala Ser Phe Pro Pro 245 250 255 Phe Val Glu Lys Ser Gln Val Leu Gln Phe Phe Ser Ser Asp Ile Cys 260 265 270 Arg Ser Ile Tyr Ala Val Phe Glu Ser Asp Val Asn Leu Lys Gly Ile 275 280 285 Pro Val Tyr Arg Phe Val Leu Pro Ser Lys Ala Phe Ala Ser Pro Val 290 295 300 Glu Asn Pro Asp Asn Tyr Cys Phe Cys Thr Glu Lys Ile Ile Ser Lys 305 310 315 320 Asn Cys Thr Ser Tyr Gly Val Leu Asp Ile Ser Lys Cys Lys Glu Gly 325 330 335 Arg Pro Val Tyr Ile Ser Leu Pro His Phe Leu Tyr Ala Ser Pro Asp 340 345 350 Val Ser Glu Pro Ile Asp Gly Leu Asn Pro Asn Glu Glu Glu His Arg 355 360 365 Thr Tyr Leu Asp Ile Glu Pro Ile Thr Gly Phe Thr Leu Gln Phe Ala 370 375 380 Lys Arg Leu Gln Val Asn Leu Leu Val Lys Pro Ser Glu Lys Ile Gln 385 390 395 400 Val Leu Lys Asn Leu Lys Arg Asn Tyr Ile Val Pro Ile Leu Trp Leu 405 410 415 Asn Glu Thr Gly Thr Ile Gly Asp Glu Lys Ala Asn Met Phe Arg Ser 420 425 430 Gln Val Thr Gly Lys Ile Asn Leu Leu Gly Leu Ile Glu Met Ile Leu 435 440 445 Leu Ser Val Gly Val Val Met Phe Val Ala Phe Met Ile Ser Tyr Cys 450 455 460 Ala Cys Arg Ser Lys Thr Ile Lys 465 470 <210> 2 <211> 472 <212> PRT <213> Artificial Sequence <220> <223> Cynomolgus monkey / Rhesus monkey CD36 <400> 2 Met Gly Cys Asp Arg Asn Cys Gly Leu Ile Thr Gly Ala Val Ile Gly 1 5 10 15 Ala Val Leu Ala Val Phe Gly Gly Ile Leu Met Pro Val Gly Asp Met 20 25 30 Leu Ile Gln Lys Thr Ile Lys Lys Glu Val Val Leu Glu Glu Gly Thr 35 40 45 Ile Ala Phe Lys Asn Trp Val Lys Thr Gly Thr Glu Ile Tyr Arg Gln 50 55 60 Phe Trp Ile Phe Asp Val Gln Asn Pro Gln Glu Val Met Met Asn Ser 65 70 75 80 Ser Asn Ile Gln Val Lys Gln Arg Gly Pro Tyr Thr Tyr Arg Val Arg 85 90 95 Phe Leu Ala Lys Glu Asn Ile Thr Gln Asp Pro Lys Asp Asn Thr Val 100 105 110 Ser Phe Leu Gln Pro Asn Gly Ala Ile Phe Glu Pro Ser Leu Ser Val 115 120 125 Gly Thr Glu Ala Asp Asn Phe Thr Val Leu Asn Leu Ala Val Ala Ala 130 135 140 Ala Ser His Ile Tyr Pro Asn Pro Phe Val Gln Val Val Leu Asn Ser 145 150 155 160 Leu Ile Asn Lys Ser Lys Ser Ser Met Phe Gln Val Arg Thr Leu Arg 165 170 175 Glu Leu Leu Trp Gly Tyr Thr Asp Pro Phe Leu Ser Leu Val Pro Tyr 180 185 190 Pro Val Ser Thr Arg Val Gly Met Phe Tyr Pro Tyr Asn Asn Thr Ala 195 200 205 Asp Gly Val Tyr Lys Val Phe Asn Gly Lys Asp Ser Ile Ser Lys Val 210 215 220 Ala Ile Ile Asp Thr Tyr Lys Gly Lys Arg Asn Leu Ser Tyr Trp Glu 225 230 235 240 Ser Tyr Cys Asp Met Ile Asn Gly Thr Asp Ala Ala Ser Phe Pro Pro 245 250 255 Phe Val Glu Lys Ser Gln Val Leu Gln Phe Phe Ser Ser Asp Ile Cys 260 265 270 Arg Ser Ile Tyr Ala Val Phe Glu Ser Asp Val Asn Leu Lys Gly Ile 275 280 285 Pro Val Tyr Arg Phe Val Leu Pro Ser Lys Ala Phe Ala Ser Pro Val 290 295 300 Gln Asn Pro Asp Asn His Cys Phe Cys Thr Glu Lys Ile Ile Ser Lys 305 310 315 320 Asn Cys Thr Ser Tyr Gly Val Leu Asp Ile Ser Lys Cys Lys Glu Gly 325 330 335 Lys Pro Val Tyr Ile Ser Leu Pro His Phe Leu Tyr Ala Ser Pro Asp 340 345 350 Val Ser Glu Thr Ile Asp Gly Leu Asn Pro Asn Glu Glu Glu His Arg 355 360 365 Thr Tyr Leu Asp Ile Glu Pro Ile Thr Gly Phe Thr Leu Gln Phe Ala 370 375 380 Lys Arg Leu Gln Val Asn Leu Leu Val Lys Pro Ser Asn Lys Ile Gln 385 390 395 400 Val Leu Lys Arg Leu Lys Arg Asn Tyr Ile Val Pro Ile Leu Trp Leu 405 410 415 Asn Glu Thr Gly Thr Ile Gly Asp Glu Lys Ala Lys Met Phe Arg Ser 420 425 430 Gln Val Thr Gly Lys Ile Asn Leu Leu Gly Leu Ile Glu Met Ile Leu 435 440 445 Leu Ser Val Gly Val Val Met Phe Val Ala Phe Met Ile Ser Tyr Cys 450 455 460 Ala Cys Arg Ser Lys Thr Ile Lys 465 470 <210> 3 <211> 472 <212> PRT <213> Artificial Sequence <220> <223> Mouse CD36 <400> 3 Met Gly Cys Asp Arg Asn Cys Gly Leu Ile Ala Gly Ala Val Ile Gly 1 5 10 15 Ala Val Leu Ala Val Phe Gly Gly Ile Leu Met Pro Val Gly Asp Met 20 25 30 Leu Ile Glu Lys Thr Ile Lys Arg Glu Val Val Leu Glu Glu Gly Thr 35 40 45 Thr Ala Phe Lys Asn Trp Val Lys Thr Gly Thr Thr Val Tyr Arg Gln 50 55 60 Phe Trp Ile Phe Asp Val Gln Asn Pro Asp Asp Val Ala Lys Asn Ser 65 70 75 80 Ser Lys Ile Lys Val Lys Gln Arg Gly Pro Tyr Thr Tyr Arg Val Arg 85 90 95 Tyr Leu Ala Lys Glu Asn Ile Thr Gln Asp Pro Glu Asp His Thr Val 100 105 110 Ser Phe Val Gln Pro Asn Gly Ala Ile Phe Glu Pro Ser Leu Ser Val 115 120 125 Gly Thr Glu Asp Asp Asn Phe Thr Val Leu Asn Leu Ala Val Ala Ala 130 135 140 Ala Pro His Ile Tyr Gln Asn Ser Phe Val Gln Val Val Leu Asn Ser 145 150 155 160 Leu Ile Lys Lys Ser Lys Ser Ser Met Phe Gln Thr Arg Ser Leu Lys 165 170 175 Glu Leu Leu Trp Gly Tyr Lys Asp Pro Phe Leu Ser Leu Val Pro Tyr 180 185 190 Pro Ile Ser Thr Thr Val Gly Val Phe Tyr Pro Tyr Asn Asp Thr Val 195 200 205 Asp Gly Val Tyr Lys Val Phe Asn Gly Lys Asp Asn Ile Ser Lys Val 210 215 220 Ala Ile Ile Glu Ser Tyr Lys Gly Lys Arg Asn Leu Ser Tyr Trp Pro 225 230 235 240 Ser Tyr Cys Asp Met Ile Asn Gly Thr Asp Ala Ala Ser Phe Pro Pro 245 250 255 Phe Val Glu Lys Ser Arg Thr Leu Arg Phe Phe Ser Ser Asp Ile Cys 260 265 270 Arg Ser Ile Tyr Ala Val Phe Gly Ser Glu Ile Asp Leu Lys Gly Ile 275 280 285 Pro Val Tyr Arg Phe Val Leu Pro Ala Asn Ala Phe Ala Ser Pro Leu 290 295 300 Gln Asn Pro Asp Asn His Cys Phe Cys Thr Glu Lys Val Ile Ser Asn 305 310 315 320 Asn Cys Thr Ser Tyr Gly Val Leu Asp Ile Gly Lys Cys Lys Glu Gly 325 330 335 Lys Pro Val Tyr Ile Ser Leu Pro His Phe Leu His Ala Ser Pro Asp 340 345 350 Val Ser Glu Pro Ile Glu Gly Leu His Pro Asn Glu Asp Glu His Arg 355 360 365 Thr Tyr Leu Asp Val Glu Pro Ile Thr Gly Phe Thr Leu Gln Phe Ala 370 375 380 Lys Arg Leu Gln Val Asn Ile Leu Val Lys Pro Ala Arg Lys Ile Glu 385 390 395 400 Ala Leu Lys Asn Leu Lys Arg Pro Tyr Ile Val Pro Ile Leu Trp Leu 405 410 415 Asn Glu Thr Gly Thr Ile Gly Asp Glu Lys Ala Glu Met Phe Lys Thr 420 425 430 Gln Val Thr Gly Lys Ile Lys Leu Leu Gly Met Val Glu Met Ala Leu 435 440 445 Leu Gly Ile Gly Val Val Met Phe Val Ala Phe Met Ile Ser Tyr Cys 450 455 460 Ala Cys Lys Ser Lys Asn Gly Lys 465 470 <210> 4 <211> 472 <212> PRT <213> Artificial Sequence <220> <223> Rat CD36 <400> 4 Met Gly Cys Asp Arg Asn Cys Gly Leu Ile Thr Gly Ala Val Ile Gly 1 5 10 15 Ala Val Leu Ala Val Phe Gly Gly Ile Leu Met Pro Val Gly Asp Leu 20 25 30 Leu Ile Glu Lys Thr Ile Lys Arg Glu Val Val Leu Glu Glu Gly Thr 35 40 45 Ile Ala Phe Lys Asn Trp Val Lys Thr Gly Thr Thr Val Tyr Arg Gln 50 55 60 Phe Trp Ile Phe Asp Val Gln Asn Pro Glu Glu Val Ala Lys Asn Ser 65 70 75 80 Ser Lys Ile Lys Val Lys Gln Arg Gly Pro Tyr Thr Tyr Arg Val Arg 85 90 95 Tyr Leu Ala Lys Glu Asn Ile Thr Gln Asp Pro Lys Asp Ser Thr Val 100 105 110 Ser Phe Val Gln Pro Asn Gly Ala Ile Phe Glu Pro Ser Leu Ser Val 115 120 125 Gly Thr Glu Asn Asp Asn Phe Thr Val Leu Asn Leu Ala Val Ala Ala 130 135 140 Ala Pro His Ile Tyr Thr Asn Ser Phe Val Gln Gly Val Leu Asn Ser 145 150 155 160 Leu Ile Lys Lys Ser Lys Ser Ser Met Phe Gln Thr Arg Ser Leu Lys 165 170 175 Glu Leu Leu Trp Gly Tyr Lys Asp Pro Phe Leu Ser Leu Val Pro Tyr 180 185 190 Pro Ile Ser Thr Thr Val Gly Val Phe Tyr Pro Tyr Asn Asn Thr Val 195 200 205 Asp Gly Val Tyr Lys Val Phe Asn Gly Lys Asp Asn Ile Ser Lys Val 210 215 220 Ala Ile Ile Asp Thr Tyr Lys Gly Lys Arg Asn Leu Ser Tyr Trp Glu 225 230 235 240 Ser Tyr Cys Asp Met Ile Asn Gly Thr Asp Ala Ala Ser Phe Pro Pro 245 250 255 Phe Val Glu Lys Ser Gln Thr Leu Arg Phe Phe Ser Ser Asp Ile Cys 260 265 270 Arg Ser Ile Tyr Ala Val Phe Glu Ser Glu Val Asn Leu Lys Gly Ile 275 280 285 Pro Val Tyr Arg Phe Val Leu Pro Ala Asn Ala Phe Ala Ser Pro Leu 290 295 300 Gln Asn Pro Asp Asn His Cys Phe Cys Thr Glu Lys Val Ile Ser Asn 305 310 315 320 Asn Cys Thr Ser Tyr Gly Val Leu Asp Ile Gly Lys Cys Lys Glu Gly 325 330 335 Lys Pro Val Tyr Ile Ser Leu Pro His Phe Leu His Ala Ser Pro Asp 340 345 350 Val Ser Glu Pro Ile Glu Gly Leu Asn Pro Asn Glu Asp Glu His Arg 355 360 365 Thr Tyr Leu Asp Val Glu Pro Ile Thr Gly Phe Thr Leu Gln Phe Ala 370 375 380 Lys Arg Leu Gln Val Asn Ile Leu Val Lys Pro Ala Arg Lys Ile Glu 385 390 395 400 Ala Leu Lys Asn Leu Lys Arg Pro Tyr Ile Val Pro Ile Leu Trp Leu 405 410 415 Asn Glu Thr Gly Thr Ile Gly Asp Glu Lys Ala Glu Met Phe Arg Asn 420 425 430 Gln Val Thr Gly Lys Ile Lys Leu Leu Gly Leu Val Glu Met Val Leu 435 440 445 Leu Gly Val Gly Val Val Met Phe Val Ala Phe Met Ile Ser Tyr Cys 450 455 460 Ala Cys Arg Ser Lys Asn Gly Lys 465 470 <210> 5 <211> 462 <212> PRT <213> Artificial Sequence <220> ONA-0-v1 heavy chain <400> 5 Gln Val Gln Leu Lys Gln Ser Gly Ala Asp Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Ala Arg Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Glu Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Ile Gly Gly Gly Phe Gly Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser Glu Ser Ala Arg Asn Pro Thr Ile Tyr 115 120 125 Pro Leu Thr Leu Pro Pro Val Leu Cys Ser Asp Pro Val Ile Ile Gly 130 135 140 Cys Leu Ile His Asp Tyr Phe Pro Phe Gly Thr Met Asn Val Thr Trp 145 150 155 160 Gly Lys Ser Gly Lys Asp Ile Thr Thr Val Asn Phe Pro Pro Ala Leu 165 170 175 Ala Ser Gly Gly Arg Tyr Thr Met Ser Ser Gln Leu Thr Leu Pro Ala 180 185 190 Val Glu Cys Pro Glu Gly Glu Ser Val Lys Cys Ser Val Gln His Asp 195 200 205 Ser Asn Pro Val Gln Glu Leu Asp Val Asn Cys Ser Pro Thr Pro Pro 210 215 220 Pro Pro Ile Thr Ile Pro Ser Cys Gln Pro Ser Leu Ser Leu Gln Arg 225 230 235 240 Pro Ala Leu Glu Asp Leu Leu Leu Gly Ser Asp Ala Ser Ile Thr Cys 245 250 255 Thr Leu Asn Gly Leu Arg Asn Pro Glu Gly Ala Ala Phe Thr Trp Glu 260 265 270 Pro Ser Thr Gly Lys Asp Ala Val Gln Lys Lys Ala Ala Gln Asn Ser 275 280 285 Cys Gly Cys Tyr Ser Val Ser Ser Val Leu Pro Gly Cys Ala Glu Arg 290 295 300 Trp Asn Ser Gly Ala Ser Phe Lys Cys Thr Val Thr His Pro Glu Ser 305 310 315 320 Gly Thr Leu Thr Gly Thr Ile Ala Lys Val Thr Val Asn Thr Phe Pro 325 330 335 Pro Gln Val His Leu Leu Pro Pro Pro Ser Glu Glu Leu Ala Leu Asn 340 345 350 Glu Leu Leu Ser Leu Thr Cys Leu Val Arg Ala Phe Asn Pro Lys Glu 355 360 365 Val Leu Val Arg Trp Leu His Gly Asn Glu Glu Leu Ser Pro Glu Ser 370 375 380 Tyr Leu Val Phe Glu Pro Leu Lys Glu Pro Gly Glu Gly Ala Thr Thr 385 390 395 400 Tyr Leu Val Thr Ser Val Leu Arg Val Ser Ala Glu Thr Trp Lys Gln 405 410 415 Gly Asp Gln Tyr Ser Cys Met Val Gly His Glu Ala Leu Pro Met Asn 420 425 430 Phe Thr Gln Lys Thr Ile Asp Arg Leu Ser Gly Lys Pro Thr Asn Val 435 440 445 Ser Val Ser Val Ile Met Ser Glu Gly Asp Gly Ile Cys Tyr 450 455 460 <210> 6 <211> 1386 <212> DNA <213> Artificial Sequence <220> <223> ONA-0 heavy chain <400> 6 caagtgcagc tgaagcagtc cggagctgat ctggtgagac ccggagccag cgtgaagctg 60 agctgcaagg ccagcggcta caccttcacc gactactaca tcaactgggt gaagcagagg 120 cccggccaag gactggagtg gatcgctaga atctaccccg gctccggcaa tacatactac 180 aacgagaagt tcaaaggcaa ggccacactg accgccgaga agagcagcag caccgcctac 240 atgcagctga gctctctgac ctccgaggac agcgccgtgt acttttgcgc cagaggcatc ggaggcggat tcggcatgga ttactggggc caaggcacct ccgtgaccgt ctcgagcgaa 360 tcggccagaa accccactat ctaccctctg accctgcctc ctgtcctgtg ttccgacccc 420 gtgatcatcg gatgcctgat ccacgactac ttccctttcg gcaccatgaa cgtgacctgg 480 gggaagtcgg gaaaggacat tactaccgtg aacttcccac cggccctggc gtcggggggt 540 cgctacacca tgtccagcca gcttactctg cccgctgtgg agtgccccga aggagagtca gtgaagtgct ccgtgcaaca tgactccaac ccggtccagg aattggacgt caattgctcc ccgactccgc ctccgcctat cacgatccca agctgccagc cctccctgag cctccagcgg 720 ccagccctgg aggatcttct gctgggctcc gacgcctcca ttacatgcac tctgaacggc 780 ctgagaacc cggaaggggc ggcctttact tggggagccct ccaccgggga ggatgcggtc 840 cagaagaagg cagcccaaaa ttcctgcgga tgctactcag tgtctagcgt gctgcctggt 960. tgtgccgaac ggtggaactc cggagcgtca ttcaagtgta ccgtgaccca ccctgagtcc ggaactctga ccggcaccat cgccaaggtc accgtgaaca cctttccgcc acaagtgcac 1020 ctcctgccgc cgccgtcgga ggaactcgct ctgaacgagt tgctctcgct gacttgtctc 1080 gtgcgcgcct tcaaccctaa ggaggtgctc gtgcgctggc tgcatggcaa cgaagaactg 1140 tcccccgaat cgtacctggt gttcgaaccg ctgaaagagc ccggagaggg tgcaaccacc 1200 taccttgtga cgagcgtgct ccgggtgtcc gccgaaacct ggaagcaggg cgaccagtac 1260 agctgcatgg tcggccacga ggccctcccc atgaacttca ctcagaaaac cattgatagg 1320 ttgtccggaa agcccaccaa cgtgtcagtg tccgtgatta tgagcgaagg agatggaatc 1380 tgctat 1386 <210> 7 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v1 light chain <400> 7 Ser Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly 1 5 10 15 Asp Arg Ile Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asp Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 8 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> ONA-0-v1 light chain <400> 8 tccatcgtga tgacccagac ccccaagttt ctgctggtgt ccgccggaga cagaatcacc 60 atcacatgca aggccagcca gagcgtgagc gatgacgtgg cttggtacca gcagaagccc 120 ggccagagcc ctaagctgct gatctactac gccagcaata gatacaccgg agtgcccgat 180 agattcaccg gcagcggcta cggcaccgac ttcaccttca caatctccac cgtgcaagcc 240 gaggatctgg ccgtgtactt ctgtcagcaa gactactcca gccctctgac cttcggagcc 300 ggcaccaagc tcgagatcaa gcgcgcagat gctgctccta ccgtgagcat cttcccgccg 360 tccagcgaac aactcactag cggaggcgcg tcagtggtct gcttccttaa caatttctac 420 cctaaggaca tcaacgtcaa gtggaagatt gacggatcgg aacgccagaa cggagtgctg 480 aactcatgga ctgatcagga ttccaaagac tcgacttact ccatgtccag caccctgacc 540 ctgaccaaag acgagtacga aaggcacaac tcgtacacgt gcgaagccac ccacaagact 600 tccacctcgc ccatcgtgaa gtccttcaat cgcaatgagt gc 642 <210> 9 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v2 light chain <400> 9 Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Met Ser Val Gly 1 5 10 15 Glu Arg Val Thr Leu Thr Cys Lys Ala Ser Glu Asn Val Val Thr Tyr 20 25 30 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 35 40 45 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala 50 55 60 Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Gly Tyr Ser Tyr Pro Tyr 65 70 75 80 85 90 95 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 10 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> ONA-0-v2 light chain <400> 10 aacatcgtga tgacccaaag ccccaagagc atgagcatgt ccgtgggcga gagagtgaca 60 ctgacatgca aggccagcga gaacgtggtg acctacgtga gctggtacca gcagaagccc 120 gaacagagcc ctaagctgct gatctacgga gcctccaata gatataccgg cgtgcccgac 180 agattcaccg gcagcggcag cgccaccgat ttcacactga ccatcagcag cgtgcaagcc 240 gaggatctgg ctgactacca ctgcggccaa ggctacagct acccctacac cttcggcggc 300 ggcaccaagc tcgagatcaa gcgcgcagat gctgctccta ccgtgagcat cttcccgccg 360 tccagcgaac aactcactag cggaggcgcg tcagtggtct gcttccttaa caatttctac 420 cctaaggaca tcaacgtcaa gtggaagatt gacggatcgg aacgccagaa cggagtgctg 480 aactcatgga ctgatcagga ttccaaagac tcgacttact ccatgtccag caccctgacc 540 ctgaccaaag acgagtacga aaggcacaac tcgtacacgt gcgaagccac ccacaagact 600 tccacctcgc ccatcgtgaa gtccttcaat cgcaatgagt gc 642 <210> 11 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v1 VH <400> 11 Gln Val Gln Leu Lys Gln Ser Gly Ala Asp Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Ala Arg Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Glu Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Ile Gly Gly Gly Phe Gly Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115 <210> 12 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> ONA-0-v1 VH <400> 12 caagtgcagc tgaagcagtc cggagctgat ctggtgagac ccggagccag cgtgaagctg 60 agctgcaagg ccagcggcta caccttcacc gactactaca tcaactgggt gaagcagagg 120 cccggccaag gactggagtg gatcgctaga atctaccccg gctccggcaa tacatactac 180 aacgagaagt tcaaaggcaa ggccacactg accgccgaga agagcagcag caccgcctac 240 atgcagctga gctctctgac ctccgaggac agcgccgtgt acttttgcgc cagaggcatc 300 ggaggcggat tcggcatgga ttactggggc caaggcacct ccgtgaccgt ctcgagc 357 <210> 13 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v1 VL <400> 13 Ser Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly 1 5 10 15 Asp Arg Ile Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asp Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 14 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> ONA-0-v1 VL <400> 14 tccatcgtga tgacccagac ccccaagttt ctgctggtgt ccgccggaga cagaatcacc 60 atcacatgca aggccagcca gagcgtgagc gatgacgtgg cttggtacca gcagaagccc 120 ggccagagcc ctaagctgct gatctactac gccagcaata gatacaccgg agtgcccgat 180 agattcaccg gcagcggcta cggcaccgac ttcaccttca caatctccac cgtgcaagcc 240 gaggatctgg ccgtgtactt ctgtcagcaa gactactcca gccctctgac cttcggagcc 300 ggcaccaagc tcgagatcaa g 321 <210> 15 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v2 VL <400> 15 Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Met Ser Val Gly 1 5 10 15 Glu Arg Val Thr Leu Thr Cys Lys Ala Ser Glu Asn Val Val Thr Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Gly Tyr Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 16 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> ONA-0-v2 VL <400> 16 aacatcgtga tgacccaaag ccccaagagc atgagcatgt ccgtgggcga gagagtgaca 60 ctgacatgca aggccagcga gaacgtggtg acctacgtga gctggtacca gcagaagccc 120 gaacagagcc ctaagctgct gatctacgga gcctccaata gatataccgg cgtgcccgac 180 agattcaccg gcagcggcag cgccaccgat ttcacactga ccatcagcag cgtgcaagcc 240 gaggatctgg ctgactacca ctgcggccaa ggctacagct acccctacac cttcggcggc 300 ggcaccaagc tcgagatcaa g 321 <210> 17 <211> 463 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v1B heavy chain <400> 17 Gln Val Gln Leu Lys Gln Ser Gly Ala Asp Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Ala Arg Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Glu Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Ile Gly Gly Gly Phe Gly Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser Glu Ser Ala Arg Asn Pro Thr Ile Tyr 115 120 125 Pro Leu Thr Leu Pro Arg Ala Leu Ser Ser Asp Pro Val Ile Ile Gly 130 135 140 Cys Leu Ile His Asp Tyr Phe Pro Ser Gly Thr Met Asn Val Thr Trp 145 150 155 160 Gly Lys Ser Gly Lys Asp Ile Thr Thr Val Asn Phe Pro Pro Ala Leu 165 170 175 Ala Ser Gly Gly Gly Tyr Thr Met Ser Ser Gln Leu Thr Leu Pro Ala 180 185 190 Val Glu Cys Pro Glu Gly Glu Ser Val Lys Cys Ser Val Gln His Asp 195 200 205 Ser Asn Ala Val Gln Glu Leu Asp Val Lys Cys Ser Gly Pro Pro Pro 210 215 220 Pro Cys Pro Pro Cys Pro Pro Ser Cys His Pro Ser Leu Ser Leu Gln 225 230 235 240 Arg Pro Ala Leu Glu Asp Leu Leu Leu Gly Ser Asp Ala Ser Leu Thr 245 250 255 Cys Thr Leu Asn Gly Leu Arg Asn Pro Glu Gly Ala Val Phe Thr Trp 260 265 270 Glu Pro Ser Thr Gly Lys Asp Ala Val Gln Lys Lys Ala Val Gln Asn 275 280 285 Ser Cys Gly Cys Tyr Ser Val Ser Ser Val Leu Pro Gly Cys Ala Glu 290 295 300 Arg Trp Asn Ser Gly Ala Ser Phe Lys Cys Thr Val Thr His Pro Glu 305 310 315 320 Ser Asp Thr Leu Thr Gly Thr Ile Ala Lys Ile Thr Val Asn Thr Phe 325 330 335 Pro Pro Gln Val His Leu Leu Pro Pro Pro Ser Glu Glu Leu Ala Leu 340 345 350 Asn Glu Leu Val Ser Leu Thr Cys Leu Val Arg Ala Phe Asn Pro Lys 355 360 365 Glu Val Leu Val Arg Trp Leu His Gly Asn Glu Glu Leu Ser Pro Glu 370 375 380 Ser Tyr Leu Val Phe Glu Pro Leu Lys Glu Pro Gly Glu Gly Ala Thr 385 390 395 400 Thr Tyr Leu Val Thr Ser Val Leu Arg Val Ser Ala Glu Leu Trp Lys 405 410 415 Gln Gly Asp Gln Tyr Ser Cys Met Val Gly His Glu Ala Leu Pro Met 420 425 430 Asn Phe Thr Gln Lys Thr Ile Asp Arg Leu Ser Gly Lys Pro Thr Asn 435 440 445 Val Ser Val Ser Val Ile Met Ser Glu Gly Asp Gly Ile Cys Tyr 450 455 460 <210> 18 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v1B light chain <400> 18 Ser Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly 1 5 10 15 Asp Arg Ile Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asp Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 19 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v2B light chain <400> 19 Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Met Ser Val Gly 1 5 10 15 Glu Arg Val Thr Leu Thr Cys Lys Ala Ser Glu Asn Val Val Thr Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Gly Tyr Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 20 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v1B VL <400> 20 Ser Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly 1 5 10 15 Asp Arg Ile Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asp Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 21 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> 1G04 heavy chain <400> 21 Gln Val Gln Leu Lys Gln Ser Gly Ala Asp Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Ala Arg Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Glu Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Ile Gly Gly Gly Phe Gly Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 22 <211> 1347 <212> DNA <213> Artificial Sequence <220> <223> 1G04 heavy chain <400> 22 caagtgcagc tgaagcagtc cggagctgat ctggtgagac ccggagccag cgtgaagctg 60 agctgcaagg ccagcggcta caccttcacc gactactaca tcaactgggt gaagcagagg 120 cccggccaag gactggagtg gatcgctaga atctaccccg gctccggcaa tacatactac 240. aacgagaagt tcaaaggcaa ggccacactg accgccgaga agagcagcag caccgcctac atgcagctga gctctctgac ctccgaggac agcgccgtgt acttttgcgc cagaggcatc ggaggcggat tcggcatgga ttactggggc caaggcacct ccgtgaccgt ctcgagcgcc 360 agcaccaaag gtccatccgt gtttccgctc gccccgtcct caaagtcgac ctccggaggc 420 actgccgccc tgggctgcct tgtcaaggac tatttccccg aacctgtcac ggtgtcctgg 480 aacagcggcg ctctgacttc cggagtgcac accttccccg ccgtcctgca atccagcggc 540 ctgtactcac tgtcatccgt tgtgactgtc ccgtcgtcca gcctgggaac ccaaacctac atttgcaacg tgaatcacaa accatcga accaaggtcg father cgagccgaag tcatgcgaca agactcacac ctgtccgcct tgcccggcgc cagaagcggc cggcggccct 720 tcggtgtttt tgtttccgcc gaagccgaag gacactctga tgatctcacg cactccagag 780 gtgacttgcg tggtggtcga tgtttcgcac gaggacccgg aagtgaatt caactggtat 840 gtcgacgggg tggaagtgca taatgccaag acgaagccga gggaggaaca gtacaactcc 900 acctacagag tggtttcagt ccttaccgtc ctccatcaag attggctgaa cggaaaggag 960 tacaaatgta aggtgtcgaa caaagcgttg ccggccccta tcgaaaagac tatcagcaag 1020 gccaaaggac agccgcggga gccgcaagtg tacaccctcc cgccttcgcg ggacgagctg 1080 accaagaatc aggtgtccct tacttgcctg gtgaagggat tctacccctc ggatatcgca 1140 gtcgaatggg aatcgaatgg acagccagaa aacaactaca agaccactcc cccggtgctc 1200 gactccgacg gttccttctt cctgtactcg aagctgaccg tggacaaatc acgctggcag 1260 cagggaaacg tgtttagctg cagcgtgatg catgaggcgc tgcataatca ctacacccag 1320 aagtcactct cgctcagccc agggaag 1347 <210> 23 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> 1G04 light chain; 1G06 light chain <400> 23 Ser Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly 1 5 10 15 Asp Arg Ile Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asp Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 24 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> 1G04 light chain <400> 24 tccatcgtga tgacccagac ccccaagttt ctgctggtgt ccgccggaga cagaatcacc 60 atcacatgca aggccagcca gagcgtgagc gatgacgtgg cttggtacca gcagaagccc 120 ggccagagcc ctaagctgct gatctactac gccagcaata gatacaccgg agtgcccgat 180 agattcaccg gcagcggcta cggcaccgac ttcaccttca caatctccac cgtgcaagcc 240 gaggatctgg ccgtgtactt ctgtcagcaa gactactcca gccctctgac cttcggagcc 300 ggcaccaagc tcgagatcaa gagaactgtg gccgcgccgt cagtgtttat cttccctcca 360 tcggatgaac agcttaagtc cggcacggcg tctgtggtct gcctgctcaa taacttttac 420 cctagggaag ctaaagtcca atggaaagtg gataacgccc tgcagtcagg aaacagccag 480 gaatcggtta ccgaacagga cagcaaggac agcacttact ccttgtcgtc gactcttact 540 ctgagcaagg ccgattacga gaagcacaag gtctacgcct gcgaggtcac ccatcaggga 600 ctctcgtccc cggtgaccaa atccttcaat agaggcgaat gc 642 <210> 25 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v2 ch IgG1 LALA light chain <400> 25 Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Met Ser Val Gly 1 5 10 15 Glu Arg Val Thr Leu Thr Cys Lys Ala Ser Glu Asn Val Val Thr Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Gly Tyr Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 26 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> ONA-0-v2 ch IgG1 LALA light chain <400> 26 aacatcgtga tgacccaaag ccccaagagc atgagcatgt ccgtgggcga gagagtgaca 60 ctgacatgca aggccagcga gaacgtggtg acctacgtga gctggtacca gcagaagccc 120 gaacagagcc ctaagctgct gatctacgga gcctccaata gatataccgg cgtgcccgac 180 agattcaccg gcagcggcag cgccaccgat ttcacactga ccatcagcag cgtgcaagcc 240 gaggatctgg ctgactacca ctgcggccaa ggctacagct acccctacac cttcggcggc 300 ggcaccaagc tcgagatcaa gagaactgtg gccgcgccgt cagtgtttat cttccctcca 360 tcggatgaac agcttaagtc cggcacggcg tctgtggtct gcctgctcaa taacttttac 420 cctagggaag ctaaagtcca atggaaagtg gataacgccc tgcagtcagg aaacagccag 480 gaatcggtta ccgaacagga cagcaaggac agcacttact ccttgtcgtc gactcttact 540 ctgagcaagg ccgattacga gaagcacaag gtctacgcct gcgaggtcac ccatcaggga 600 ctctcgtccc cggtgaccaa atccttcaat agaggcgaat gc 642 <210> 27 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v1 VH CDR1 Chothia <400> 27 Gly Tyr Thr Phe Thr Asp Tyr 1 5 <210> 28 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v1 VH CDR2 Chothia <400> 28 Tyr Pro Gly Ser Gly Asn 1 5 <210> 29 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v1 VH CDR3 Chothia; ONA-0-v1 VH CDR3 Kabat; CDR-H3 ONA-0-v1, ONA-0-v1 Humanization v1-v16 <400> 29 Gly Ile Gly Gly Gly Phe Gly Met Asp Tyr 1 5 10 <210> 30 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v1 VL CDR1 Chothia; ONA-0-v1 VL CDR1 Kabat; CDR-L1 ONA-0-v1, ONA-0-v1 Humanized v3 / v4 / v7 / v8 / v11 / v12 / v15 / v16 <400> 30 Lys Ala Ser Gln Ser Val Ser Asp Asp Val Ala 1 5 10 <210> 31 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v1 VL CDR2 Chothia; ONA-0-v1 VL CDR2 Kabat; CDR-L2 ONA-0-v1, ONA-0-v1 Humanized v1 / v3 / v5 / v7 / v9 / v11 / v13 / v15 <400> 31 Tyr Ala Ser Asn Arg Tyr Thr 1 5 <210> 32 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v1 VL CDR3 Chothia; ONA-0-v1 VL CDR3 Kabat; ONA-0-v1 VL CDR3 IMGT; CDR-L3 ONA-0-v1, ONA-0-v1 Humanization v1-v16 <400> 32 Gln Gln Asp Tyr Ser Ser Pro Leu Thr 1 5 <210> 33 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v2 VL CDR1 Chothia <400> 33 Lys Ala Ser Glu Asn Val Val Thr Tyr Val Ser 1 5 10 <210> 34 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v2 VL CDR2 Chothia <400> 34 Gly Ala Ser Asn Arg Tyr Thr 1 5 <210> 35 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v2 VL CDR3 Chothia <400> 35 Gly Gln Gly Tyr Ser Tyr Pro Tyr Thr 1 5 <210> 36 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 36 Met Tyr Pro Pro Pro Tyr 1 5 <210> 37 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v1 VH CDR1 Kabat; CDR-H1 ONA-0-v1, ONA-0-v1 Humanization v1 / v2 / v3 / v4 / v9 / v10 / v11 / v12 <400> 37 Asp Tyr Tyr Ile Asn 1 5 <210> 38 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v1 VH CDR2 Kabat; CDR-H2 ONA-0-v1, ONA-0-v1 Humanization v1 / v2 / v3 / v4 / v9 / v10 / v11 / v12 <400> 38 Arg Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 39 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v1 VH CDR1 IMGT <400> 39 Gly Tyr Thr Phe Thr Asp Tyr Tyr 1 5 <210> 40 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v1 VH CDR2 IMGT <400> 40 Ile Tyr Pro Gly Ser Gly Asn Thr 1 5 <210> 41 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v1 VH CDR3 IMGT <400> 41 Ala Arg Gly Ile Gly Gly Gly Phe Gly Met Asp Tyr 1 5 10 <210> 42 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v1 VL CDR1 IMGT <400> 42 Gln Ser Val Ser Asp Asp 1 5 <210> 43 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> ONA-0-v1 VL CDR2 IMGT <400> 43 Tyr Ala Ser 1 <210> 44 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 humanized variant Kabat; CDR-H1 ONA-0-v1 humanized v5 / v6 / v7 / v8 <400> 44 Asp Tyr Tyr Met His 1 5 <210> 45 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 humanized variant Kabat; CDR-H1 ONA-0-v1 humanized v13 / v14 / v15 / v16 <400> 45 Asp Tyr Tyr Met Asn 1 5 <210> 46 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 humanized variant Kabat; CDR-H2 ONA-0-v1 humanized v5 / v6 / v7 / v8 <400> 46 Arg Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe Gln 1 5 10 15 Gly <210> 47 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 humanized variant Kabat; CDR-H2 ONA-0-v1 humanized v13 / v14 / v15 / v16 <400> 47 Arg Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe Thr 1 5 10 15 Gly <210> 48 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 humanized variant Kabat; CDR-L1 ONA-0-v1 humanized v1 / v2 / v5 / v6 / v9 / v10 / v13 / v14 <400> 48 Gln Ala Ser Gln Ser Val Ser Asp Asp Val Ala 1 5 10 <210> 49 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VL CDR2 humanized variant Kabat; CDR-L2 ONA-0-v1 humanized v2 / v6 / v10 / v14 <400> 49 Tyr Ala Ser Asn Leu Tyr Thr 1 5 <210> 50 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VL CDR2 humanized variant Kabat; CDR-L2 ONA-0-v1 humanized v4 / v8 / v12 / v16 <400> 50 Tyr Ala Ser Asn Arg Tyr Ser 1 5 <210> 51 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Humanized ONA-0-v1 VH Variant 1 <400> 51 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Ala Arg Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Glu Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Ile Gly Gly Gly Phe Gly Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 52 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Humanized ONA-0-v1 VH variant 2 <400> 52 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Ala Arg Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ile Gly Gly Gly Phe Gly Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 53 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Humanized ONA-0-v1 VH variant 3 <400> 53 Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Ala Arg Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Gly Phe 50 55 60 Lys Gly Arg Phe Val Leu Ser Ala Glu Lys Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Ile Gly Gly Gly Phe Gly Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 54 <211> 119 <212> PRT <213> Artificial Sequence <220> Humanized ONA-0-v1 VH variant 4 <400> 54 Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Ala Arg Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Gly Phe 50 55 60 Thr Gly Arg Phe Val Phe Ser Ala Asp Lys Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ile Gly Gly Gly Phe Gly Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 55 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Humanized ONA-0-v1 VL variant 1 <400> 55 Ser Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Val Ser Asp Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 56 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Humanized ONA-0-v1 VL variant 2 <400> 56 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Val Ser Asp Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Leu Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 57 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Humanized ONA-0-v1 VL variant 3 <400> 57 Ser Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Ser Asp Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 58 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Humanized ONA-0-v1 VL variant 4 <400> 58 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Ser Asp Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 59 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> 1G06 heavy chain <400> 59 Gln Val Gln Leu Lys Gln Ser Gly Ala Asp Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Ala Arg Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Glu Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Ile Gly Gly Gly Phe Gly Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ser Thr Arg Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys

Claims

1. An isolated antibody that binds to CD36, comprising a light chain CDR1 region, a light chain CDR2 region, a light chain CDR3 region, a heavy chain CDR1 region, a heavy chain CDR2 region, and a heavy chain CDR3 region; wherein the heavy chain CDR1 region is composed of SEQ ID NO: 39, the heavy chain CDR2 region is composed of SEQ ID NO: 40, the heavy chain CDR3 region is composed of SEQ ID NO: 41, the light chain CDR1 region is composed of SEQ ID NO: 42, the light chain CDR2 region is composed of SEQ ID NO: 43, and the light chain CDR3 region is composed of SEQ ID NO:

32.

2. An isolated antibody that binds to CD36, comprising a light chain CDR1 region, a light chain CDR2 region, a light chain CDR3 region, a heavy chain CDR1 region, a heavy chain CDR2 region, and a heavy chain CDR3 region; wherein the heavy chain CDR1 region is composed of SEQ ID NO: 37, the heavy chain CDR2 region is composed of SEQ ID NO: 38, the heavy chain CDR3 region is composed of SEQ ID NO: 29, the light chain CDR1 region is composed of SEQ ID NO: 30, the light chain CDR2 region is composed of SEQ ID NO: 31, and the light chain CDR3 region is composed of SEQ ID NO:

32.

3. The antibody according to claim 1 or 2, wherein, The antibody binds to human CD36.

4. The antibody according to claim 1 or 2, wherein, If measured using SPR data fitted with a 1-to-1 model, the antibody has a Kc of less than 10 nM. D It combines with human CD36.

5. The antibody according to claim 1 or 2, wherein, The antibody comprises VH consisting of the amino acid sequence of SEQ ID NO:

11.

6. The antibody according to claim 1 or 2, wherein, The antibody comprises a VL consisting of the amino acid sequence of SEQ ID NO:

13.

7. The antibody according to claim 1 or 2, wherein the antibody further comprises a heavy chain constant region.

8. The antibody according to claim 7, wherein, The heavy chain constant region is selected from the group consisting of: human immunoglobulin IgA1, IgA2, IgG1, IgG2, IgG3 or IgG4 heavy chain constant regions.

9. The antibody according to claim 8, wherein the antibody comprises the IgG1 heavy chain constant region.

10. The antibody according to claim 9, wherein, The heavy chain constant region includes an IgG constant region containing amino acid substitutions selected from the group consisting of: L234G, L235S, and G236R; L234S, L235T, and G236R; L234S, L235V, and G236R; L234T, L235Q, and G236R; L234T, L235T, and G236R; L234A and L235A; and L234A, L235A, and P329G.

11. The antibody according to claim 8, wherein the antibody comprises the IgG4 heavy chain constant region.

12. The antibody according to claim 11, wherein, The heavy chain constant region includes an IgG constant region, which contains an amino acid substitution S228P.

13. The antibody according to claim 1 or 2, wherein, The antibody also contains a light chain constant region.

14. The antibody according to claim 13, wherein, The light chain constant region is selected from the group consisting of the light chain constant regions of human immunoglobulin κ and λ.

15. The antibody according to claim 1 or 2, wherein, The antibody further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is the human IgG1 heavy chain constant region, and wherein the light chain constant region is the human κ light chain constant region.

16. The antibody according to claim 1 or 2, wherein, The antibody comprises the light chain in SEQ ID NO: 23 and the heavy chain in SEQ ID NO:

21.

17. The antibody according to claim 1 or 2, wherein, The antibody comprises the light chain in SEQ ID NO: 23 and the heavy chain in SEQ ID NO:

64.

18. The antibody according to claim 1 or 2, wherein the antibody is an antigen-binding fragment.

19. The antibody according to claim 18, wherein, The antigen-binding fragments include Fab, Fab', F(ab')2, single-chain Fv (scFv), disulfide-linked Fv, IgGΔCH2, F(ab')3, tetraantibody, triantibody, biantibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

20. The antibody according to claim 1 or 2, wherein the antibody is a bispecific antibody.

21. A pharmaceutical composition comprising the antibody of any one of claims 1 to 20 and a pharmaceutically acceptable excipient.

22. The pharmaceutical composition according to claim 21, wherein, At least 95% of the antibody in the composition is non-fucosylated.

23. The pharmaceutical composition according to claim 21 or 22, further comprising a PD-1 inhibitor.

24. The pharmaceutical composition according to claim 23, wherein, The PD-1 inhibitor is an anti-PD-1 antibody.

25. The pharmaceutical composition according to claim 24, wherein, The anti-PD-1 antibody is pembrolizumab, pildizumab, or nivolumab.

26. The pharmaceutical composition according to claim 21 or 22, further comprising a PD-L1 inhibitor.

27. The pharmaceutical composition according to claim 26, wherein, The PD-L1 inhibitor is an anti-PD-L1 antibody.

28. The pharmaceutical composition according to claim 27, wherein, The anti-PD-L1 antibody is atezolizumab, durvalumab, avelumab, or BMS-936559.

29. The pharmaceutical composition according to claim 21 or 22, further comprising a CTLA-4 inhibitor.

30. The pharmaceutical composition according to claim 29, wherein, The CTLA-4 inhibitor is an anti-CTLA-4 antibody.

31. The pharmaceutical composition according to claim 30, wherein, The anti-CTLA-4 antibody is ipilimumab.

32. The pharmaceutical composition according to claim 21 or 22, wherein, The composition also contains a chemotherapeutic agent.

33. The pharmaceutical composition according to claim 32, wherein, The chemotherapy agent is cisplatin.

34. Use of the antibody according to any one of claims 1 to 20 in the preparation of a medicament for treating cancers expressing CD36, wherein, The cancer in question is oral squamous cell carcinoma, ovarian cancer, lung cancer, breast cancer, or colon cancer.

35. The use according to claim 34, wherein, The cancer in question is metastatic cancer.

36. The use according to claim 35, wherein, The metastatic cancer includes metastatic tumors in one or more of the liver, lungs, spleen, kidneys, cervical lymph nodes, or peritoneal wall; and / or wherein the metastatic tumors are oral squamous cell carcinoma, ovarian cancer, lung cancer, breast cancer, or colon cancer.

37. The use according to claim 34, wherein, As measured by IVIS imaging or H&E staining, the treatment reduced the size of metastatic tumors.

38. The use according to claim 34, wherein, As measured by IVIS imaging or H&E staining, the treatment inhibits the formation or development of metastatic tumors.

39. The use according to claim 34, wherein, The antibody blocks CD36-mediated uptake of fatty acids and / or oxLDL.

40. The use according to claim 34, wherein, The intended use is combined with the second treatment.

41. The use according to claim 40, wherein, The second treatment is: a) Immunotherapy; or b) Chemotherapy agents.

42. The use according to claim 41, wherein, The immunotherapy is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody; and / or the chemotherapeutic agent is cisplatin.

43. An isolated polynucleotide encoding an antibody according to any one of claims 1 to 20.

44. The isolated polynucleotide according to claim 43, wherein, The polynucleotide is selected from the group consisting of: a) Polynucleotides encoding the light chain in SEQ ID NO: 7 and the heavy chain in SEQ ID NO: 5; b) Polynucleotides containing SEQ ID NO: 8 and SEQ ID NO: 6; c) Polynucleotides containing SEQ ID NO: 24 and SEQ ID NO:

22.

45. A carrier comprising the isolated polynucleotide of claim 43.

46. ​​A cell comprising the isolated polynucleotide of claim 43 or the vector of claim 45.

47. The cell according to claim 46, selected from the group consisting of: *Escherichia coli*, *Pseudomonas*, *Bacillus*, *Streptomyces*, yeast, CHO, YB / 20, NSO, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, BW, LM, COS1, COS7, BSC1, BSC40, BMT10 cells, plant cells, insect cells, and human cells in tissue culture; and / or wherein, The cells lack the functional α-1,6-fucosyltransferase gene (FUT8).

48. A method for preparing an antibody capable of specifically binding to CD36, comprising expressing the antibody in the cells of claim 46, or alternatively, comprising culturing the cells of claim 46 and isolating the antibody expressed therein.

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