Anti-CD36 antibodies and uses thereof

Abstract

The present disclosure provides antibodies that specifically bind to human CD36 protein (hCD36) and can reduce, inhibit, and / or completely block CD36-mediated immunomodulatory effects and / or ligand transport into cells. Examples of CD36-mediated effects include, among others, transport of oxidized lipids into CD8 T cells, which can cause cell death, and fatty acid transport in regulatory T cells, which increases regulatory T cell survival and immune suppression in the tumor microenvironment (TME). The present disclosure also provides methods of using the antibodies (and compositions thereof) to treat various diseases and conditions that respond to reducing, inhibiting, and / or blocking CD36-dependent ligand transport into cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 317,160, filed March 7, 2022, the entirety of which is incorporated herein by reference.

[0002] The present disclosure relates to antibodies that bind to CD36 and methods of using such antibodies.

[0003] Reference to sequence listing An official copy of the Sequence Listing is submitted contemporaneously herewith as an XML file in WIPO Standard ST.26 format with the file name "09793-005WO1.xml", creation date February 26, 2023, and size 75,330 bytes. The Sequence Listing submitted via the USPTO Patent Center is a part of this specification and is incorporated by reference in its entirety. [Background technology]

[0004] Macrophages and other myeloid cells play a prominent role in generating and maintaining the immunosuppressive environment of tumors, and a distinct microenvironment exists in which tumor-associated macrophages (TAMs) suppress immune responses and promote other processes, including angiogenesis, cancer cell motility, and metastasis (Lewis & Pollard, 2006; Pollard, 2008; Wu et al., 2020). For example, it was almost 20 years ago that production of vascular endothelial growth factor by TAMs was proposed to play a role in lymphatic metastasis of several human cancers (e.g., Pepper et al., 2003), and it was in 2006 that clodronate-encapsulated liposome-mediated TAM depletion was demonstrated to inhibit tumor growth in experimental models through a mechanism that likely involves the reduction of tumor angiogenesis (Zeisberger et al., 2006). Furthermore, macrophages are thought to not only promote the egress of tumour cells from primary tumours but also promote dissemination at distant metastatic sites (Joyce & Pollard, 2009; Psaila & Lyden, 2009).

[0005] Although our understanding of the various pro-tumorigenic (often referred to as M2) and anti-tumorigenic M1 macrophages and other myeloid cells is still growing (see, e.g., Wu et al., 2020), it has long been recognized that high numbers of TAMs are associated with poor patient survival (with a few notable exceptions) and are an independent prognostic factor in many cancers (Lewis & Pollard, 2006). Furthermore, myeloid cells called myeloid-derived immunosuppressive cells (MDSCs) are found in the peripheral blood and tumor tissues of cancer patients, and depleting or otherwise targeting MDSCs in mouse models results in improved immune responses and delayed tumor growth (Goedegebuure et al., 2011). MDSCs have been described as somewhat diverse, yet still divided into monocytic versus granulocytic subsets with distinct immunosuppressive mechanisms. Among other roles, tumor MDSCs have been implicated in the recruitment and maintenance of immunosuppressive regulatory T cells and have been suggested to promote angiogenesis and metastasis (Goedegebuure et al., 2011; Oh et al., 2013).A CD45-positive but CD14-negative subset of human peripheral blood cells, at least similar to currently known MDSCs, was described by Barrett et al. (2007) to express CD36 and the immunosuppressive cytokine interleukin-10.

[0006] CD36 is a transmembrane cell surface protein also known as platelet glycoprotein 4, fatty acid translocase (FAT) and scavenger receptor class B member 3 (SCARB3), among other names. CD36 binds and transports fatty acids into cells, but also binds several other ligands, such as apoptotic cells, low density lipoproteins, phospholipids and their oxidized forms (Pepino et al., 2014; Wang & Li, 2019). CD36 also has a distinct thrombospondin binding domain (Pepino et al., 2014; Wang & Li, 2019). Both of these two distinct functional events, thrombospondin binding versus fatty acid binding, can result in different downstream signaling events that may differ depending on the cell type and various other CD36 and / or ligand interaction partners (Pepino et al., 2014; Wang & Li, 2019). Fatty acid transport by CD36, if not required for cell differentiation state or cell survival, is involved in the metabolic "wiring" of various cells. For example, fatty acid transport by CD36 has been shown to be required for M2 macrophage differentiation through a mechanism related to the level or state of endoplasmic reticulum stress (Oh et al., 2012).

[0007] CD36 has also been shown to be crucial for the activation of M2 macrophages, as measured by markers of immunosuppressive capacity such as PDL-2 expression (Huang et al., 2014). CD36-mediated lipid transport has also been implicated in the acquisition of immunosuppressive effectors during MDSC differentiation (Al-Khami et al., 2017). CD36-deficient mice had reduced numbers of tumor-resident MDSCs in all (lung and colon) tumor models tested when compared to tumor-bearing wild-type mice, which was reflected in tumors of wild-type chimeric mice that were CD36-deficient in their bone marrow (Al-Khami et al., 2017). Most recently, it was shown that TAMs were significantly reduced in melanoma and myeloma models of CD36-deficient mice compared to those in wild-type mice, and that such TAMs in wild-type mice had elevated CD36 expression and lipid accumulation compared to normal macrophages (Su et al., 2020).

[0008] Given that CD36-mediated uptake of fatty acids or other ligands is important for the biology of M2 TAMs and MDSCs, blocking CD36 ligand trafficking may reduce tumor growth by reducing the immunosuppressive capacity, angiogenesis, and / or metastasis that would otherwise be promoted by M2 TAMs and MDSCs. Indeed, mouse tumor models have shown that tumor growth (lung and colon) is significantly reduced in CD36-deficient mice and that this is due to the loss of CD36 in bone marrow-derived cells (Al-Khami et al., 2017).

[0009] Besides myeloid cells, it has been reported more recently that CD36 is expressed on a subset of tumor-resident regulatory T cells, and that blocking CD36 with an antibody that inhibits CD36-mediated fatty acid transport results in the reduction of melanoma growth in experimental models (Wang et al., 2020). Furthermore, it has been reported that CD36 deficiency on regulatory T cells in mice alone is sufficient to cause the reduction of melanoma growth (Wang et al., 2020). More recently, it has been shown that CD36 expression by tumor-resident CD8 T cells impairs their antitumor capacity and leads to increased tumor CD8 T cell death through lipid peroxidation leading to ferroptosis (Ma et al., 2021). Previous reports have also described CD36 on tumor-infiltrating CD8 T cells and its deleterious effects on such CD8 T cells (Xu et al., 2020), which was later published in peer-review format (Xu et al., 2021).

[0010] There are also numerous reports on the role of ligand transport by CD36 expressed on tumor cells. For example, CD36 has been associated with tamoxifen resistance in breast cancer (Liang et al., 2018), chemotherapy resistance in melanoma (Alioa et al., 2019), and HER2-targeted therapy resistance in lung cancer (Feng et al., 2019). As early as 1993, CD36 was linked to adriamycin resistance in a screen for adriamycin-resistant sublines of the human myeloid leukemia cell line K562, but did not confer adriamycin resistance by itself, even though CD36 was overexpressed in the adriamycin-sensitive parental cell line (Sugimoto et al., 1993). CD36 is also involved in the self-renewal, survival, and / or increased proliferation of glioblastoma stem cells (Hale et al., 2014) and leukemia stem cells (Ye et al., 2016). Thus, CD36 appears to be involved in chemoresistance by leukemic stem cells in the adipose tissue niche (Ye et al., 2016). Glioblastoma formation using patient-derived glioblastoma stem cells in xenograft mouse models was significantly reduced when CD36 was "knocked down" in the stem cells by RNA interference, and proliferation of glioblastoma stem cells was increased in a CD36-dependent manner by exposure to oxidized low-density lipoprotein (Hale et al., 2014). CD36 was also shown to be a useful biomarker for brain malignancies and to be negatively correlated with patient prognosis (Hale et al., 2014).

[0011] More recently, CD36 was shown to be important for prostate cancer progression and tumor growth in mouse models, and reducing CD36 expression in prostate cancer cells by RNA interference, or alternatively blocking CD36-mediated lipid transport using antibodies, led to reduced cell migration ability and reduced tumor growth in mouse models (Watt et al., 2019).

[0012] CD36 has also been proposed as a prognostic metastasis biomarker in various cancers (reviewed by Enciu et al., 2018), and has been shown to promote metastasis of oral squamous cell carcinoma (OSCC), breast cancer, and melanoma cells in mouse models (PCT / EP2016 / 073208; Pascual et al., 2017). OSCC metastasis was seen after cell injection into an orthotopic site (tongue), and this metastasis was reduced by an antibody blocking fatty acid transport by CD36. Melanoma and breast cancer cells were injected into the blood of mice so that they could potentially colonize the metastatic niche, and this metastasis was reduced when cells in which CD36 had been "knocked down" by RNA interference were used (PCT / EP2016 / 073208; Pascual et al., 2017). As mentioned above, CD36 is also expected to be involved in intravasation of tumor cells from the primary tumor site into lymphatic and / or blood circulation, primarily through its role on myeloid cells of the immune system. Fatty acid transport by CD36 on hepatocellular carcinoma cells has also been shown to induce increased epithelial-mesenchymal transition, and chemical inhibition of CD36-mediated fatty acid transport reduced such phenotype as well as migration of hepatocellular carcinoma cells (Nath et al., 2015).

[0013] All the above describes the situation where the transport of fatty acids or other ligands into cells by CD36 is involved in the metabolic adaptation or differentiation of various cell types, but it should be emphasized that more direct immunosuppressive effects can also be brought about by CD36 signaling. Some of these have been mentioned above, but it should be emphasized that apoptotic cell binding via CD36 normally promotes homeostatic anti-inflammatory processes and that it has been proposed that apoptotic cell binding via CD36 is at least partially responsible for the IL-10 production by macrophages (among other immunosuppressive effects on macrophages) that follows apoptotic cell binding (Chung et al., 2007). CD36-mediated ligand transport can also bring about immunosuppressive metabolites: the increased production of arachidonic acid as a result of CD36 ligand-induced intracellular signaling provides substrates for the production of prostaglandins (e.g. Kuda et al., 2011), some of which are known to have a wide range of immunomodulatory effects (see, e.g. Wang & Dubois, 2006; Mizuno et al., 2019). Indeed, immunosuppressive prostaglandin E2 is produced in many cancers by tumor cells and by other immune system cells (Wang & Dubois, 2006; Mizuno et al., 2019), including myeloid cells and inducible regulatory T cells (Whiteside & Jackson, 2013). Furthermore, mouse macrophages have at least been shown to produce prostaglandin E2 in a CD36-dependent manner, but not in cancer models (Almeida et al., 2014).

[0014] Beyond the many roles of CD36 in cancer, CD36 ligand trafficking clearly plays an important pathological role in foam cell formation in atherosclerosis (e.g., Zhao et al., 2018), non-alcoholic fatty liver disease (e.g., Rada et al., 2020), and other conditions. Thus, antibodies that interfere with CD36 binding / trafficking of apoptotic cells or other ligands have several promising applications. [Prior art documents] [Patent documents]

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Summary of the Invention

Problems to be Solved by the Invention

[0017] The present disclosure provides anti-CD36 antibodies that specifically bind to human CD36 with high affinity. The antibodies can reduce, inhibit, and / or completely block immunomodulatory effects mediated by CD36, including CD36-mediated fatty acid transport (e.g., cellular uptake of oxidized low density lipoprotein, or "oxLDL"). [Means for solving the problem]

[0018] In at least one embodiment, the present disclosure provides a method for producing a cellular membrane comprising: (i) a first light chain complementarity determining region (CDR-L1), a second light chain complementarity determining region (CDR-L2), and a third light chain complementarity determining region (CDR-L3), and / or (ii) a first heavy chain complementarity determining region (CDR-H1), a second heavy chain complementarity determining region (CDR-H2), and a third heavy chain complementarity determining region (CDR-H3). The present invention provides an anti-CD36 antibody comprising: (a) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 3, 21, 24, or 27; (b) CDR-H2 comprises the amino acid sequence of SEQ ID NO: 4, 28, 31, 34, 37, 40, or 43; (c) CDR-H3 comprises the amino acid sequence of SEQ ID NO:5; (d) CDR-L1 comprises an amino acid sequence selected from SEQ ID NO:7; (e) CDR-L2 comprises an amino acid sequence selected from SEQ ID NO: 8, 12, or 15; (f) CDR-L3 comprises an amino acid sequence selected from SEQ ID NO: 9, 13, or 18.

[0019] In at least one embodiment of the anti-CD36 antibody of the disclosure: (a) CDR-H1 comprises the amino acid sequence of SEQ ID NO:3, CDR-H2 comprises the amino acid sequence of SEQ ID NO:4, CDR-H3 comprises the amino acid sequence of SEQ ID NO:5, CDR-L1 comprises the amino acid sequence of SEQ ID NO:7, CDR-L2 comprises the amino acid sequence of SEQ ID NO:8, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:9; (b) CDR-H1 comprises the amino acid sequence of SEQ ID NO:3, CDR-H2 comprises the amino acid sequence of SEQ ID NO:4, CDR-H3 comprises the amino acid sequence of SEQ ID NO:5, CDR-L1 comprises the amino acid sequence of SEQ ID NO:7, CDR-L2 comprises the amino acid sequence of SEQ ID NO:12, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:13; (c) CDR-H1 comprises the amino acid sequence of SEQ ID NO:3, CDR-H2 comprises the amino acid sequence of SEQ ID NO:4, CDR-H3 comprises the amino acid sequence of SEQ ID NO:5, CDR-L1 comprises the amino acid sequence of SEQ ID NO:7, CDR-L2 comprises the amino acid sequence of SEQ ID NO:15, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:13; (d) CDR-H1 comprises the amino acid sequence of SEQ ID NO:3, CDR-H2 comprises the amino acid sequence of SEQ ID NO:4, CDR-H3 comprises the amino acid sequence of SEQ ID NO:5, CDR-L1 comprises the amino acid sequence of SEQ ID NO:7, CDR-L2 comprises the amino acid sequence of SEQ ID NO:15, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:18; (e) CDR-H1 comprises the amino acid sequence of SEQ ID NO:21, CDR-H2 comprises the amino acid sequence of SEQ ID NO:4, CDR-H3 comprises the amino acid sequence of SEQ ID NO:5, CDR-L1 comprises the amino acid sequence of SEQ ID NO:7, CDR-L2 comprises the amino acid sequence of SEQ ID NO:15, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:18; (f) CDR-H1 comprises the amino acid sequence of SEQ ID NO:24, CDR-H2 comprises the amino acid sequence of SEQ ID NO:4, CDR-H3 comprises the amino acid sequence of SEQ ID NO:5, CDR-L1 comprises the amino acid sequence of SEQ ID NO:7, CDR-L2 comprises the amino acid sequence of SEQ ID NO:15, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:18; (g) CDR-H1 comprises the amino acid sequence of SEQ ID NO:27, CDR-H2 comprises the amino acid sequence of SEQ ID NO:28, CDR-H3 comprises the amino acid sequence of SEQ ID NO:5, CDR-L1 comprises the amino acid sequence of SEQ ID NO:7, CDR-L2 comprises the amino acid sequence of SEQ ID NO:15, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:18; (h) CDR-H1 comprises the amino acid sequence of SEQ ID NO:3, CDR-H2 comprises the amino acid sequence of SEQ ID NO:31, CDR-H3 comprises the amino acid sequence of SEQ ID NO:5, CDR-L1 comprises the amino acid sequence of SEQ ID NO:7, CDR-L2 comprises the amino acid sequence of SEQ ID NO:15, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:18; (i) CDR-H1 comprises the amino acid sequence of SEQ ID NO:3, CDR-H2 comprises the amino acid sequence of SEQ ID NO:34, CDR-H3 comprises the amino acid sequence of SEQ ID NO:5, CDR-L1 comprises the amino acid sequence of SEQ ID NO:7, CDR-L2 comprises the amino acid sequence of SEQ ID NO:15, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:18; (j) CDR-H1 comprises the amino acid sequence of SEQ ID NO:3, CDR-H2 comprises the amino acid sequence of SEQ ID NO:37, CDR-H3 comprises the amino acid sequence of SEQ ID NO:5, CDR-L1 comprises the amino acid sequence of SEQ ID NO:7, CDR-L2 comprises the amino acid sequence of SEQ ID NO:15, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:18; (k) CDR-H1 comprises the amino acid sequence of SEQ ID NO:3, CDR-H2 comprises the amino acid sequence of SEQ ID NO:40, CDR-H3 comprises the amino acid sequence of SEQ ID NO:5, CDR-L1 comprises the amino acid sequence of SEQ ID NO:7, CDR-L2 comprises the amino acid sequence of SEQ ID NO:15, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:18; or (l) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 24, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 43, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 5, CDR-L1 comprises the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 15, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 18.

[0020] In at least one embodiment, the present disclosure provides a method for producing a cellular membrane comprising: (i) the sequences of the first heavy chain complementarity determining region (CDR-H1), the second heavy chain complementarity determining region (CDR-H2), and the third heavy chain complementarity determining region (CDR-H3) are from a VH region having an amino acid sequence selected from SEQ ID NOs: 2, 20, 23, 26, 30, 33, 36, 39, and 42; CDR-H1, CDR-H2, and CDR-H3; and (ii) the sequences of the first light chain complementarity determining region (CDR-L1), the second light chain complementarity determining region (CDR-L2), and the third light chain complementarity determining region (CDR-L3) are from a VL region having an amino acid sequence selected from SEQ ID NOs: 6, 11, 14, and 17; CDR-L1, CDR-L2, and CDR-L3 and providing an anti-CD36 antibody comprising Here, CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 are according to the Kabat numbering.

[0021] In at least one embodiment of the anti-CD36 antibody: (a) the VH amino acid sequence is SEQ ID NO:2 and the VL amino acid sequence is SEQ ID NO:6; (b) the VH amino acid sequence is SEQ ID NO:2 and the VL amino acid sequence is SEQ ID NO:11: (c) the VH amino acid sequence is SEQ ID NO:2 and the VL amino acid sequence is SEQ ID NO:14; (d) the VH amino acid sequence is SEQ ID NO:2 and the VL amino acid sequence is SEQ ID NO:17; (e) the VH amino acid sequence is SEQ ID NO:20 and the VL amino acid sequence is SEQ ID NO:17; (f) the VH amino acid sequence is SEQ ID NO:23 and the VL amino acid sequence is SEQ ID NO:17; (g) the VH amino acid sequence is SEQ ID NO:26 and the VL amino acid sequence is SEQ ID NO:17; (h) the VH amino acid sequence is SEQ ID NO: 30 and the VL amino acid sequence is SEQ ID NO: 17; (i) the VH amino acid sequence is SEQ ID NO: 33 and the VL amino acid sequence is SEQ ID NO: 17; (j) the VH amino acid sequence is SEQ ID NO: 36 and the VL amino acid sequence is SEQ ID NO: 17; (k) the VH amino acid sequence is SEQ ID NO:39 and the VL amino acid sequence is SEQ ID NO:17; or (l) The VH amino acid sequence is SEQ ID NO: 42 and the VL amino acid sequence is SEQ ID NO: 17.

[0022] In at least one embodiment of the anti-CD36 antibody of the disclosure, the antibody comprises a heavy chain variable domain (V) having at least 90% identity to a sequence selected from SEQ ID NOs: 2, 20, 23, 26, 30, 33, 36, 39, or 42. H ) amino acid sequence; and / or a light chain variable domain (V) having at least 90% identity to a sequence selected from SEQ ID NOs: 6, 11, 14, or 17. L ) amino acid sequence.

[0023] In at least one embodiment of the anti-CD36 antibody of the disclosure: (a) the antibody comprises a VH amino acid sequence having at least 90% identity to SEQ ID NO:2 and a VL amino acid sequence having at least 90% identity to SEQ ID NO:6; (b) the antibody comprises a VH amino acid sequence having at least 90% identity to SEQ ID NO:2 and a VL amino acid sequence having at least 90% identity to SEQ ID NO:11; (c) the antibody comprises a VH amino acid sequence having at least 90% identity to SEQ ID NO:2 and a VL amino acid sequence having at least 90% identity to SEQ ID NO:14; (d) the antibody comprises a VH amino acid sequence having at least 90% identity to SEQ ID NO:2 and a VL amino acid sequence having at least 90% identity to SEQ ID NO:17; (e) the antibody comprises a VH amino acid sequence having at least 90% identity to SEQ ID NO: 20 and a VL amino acid sequence having at least 90% identity to SEQ ID NO: 17; (f) the antibody comprises a VH amino acid sequence having at least 90% identity to SEQ ID NO: 23 and a VL amino acid sequence having at least 90% identity to SEQ ID NO: 17; (g) the antibody comprises a VH amino acid sequence having at least 90% identity to SEQ ID NO:26 and a VL amino acid sequence having at least 90% identity to SEQ ID NO:17; (h) the antibody comprises a VH amino acid sequence having at least 90% identity to SEQ ID NO: 30 and a VL amino acid sequence having at least 90% identity to SEQ ID NO: 17; (i) the antibody comprises a VH amino acid sequence having at least 90% identity to SEQ ID NO: 33 and a VL amino acid sequence having at least 90% identity to SEQ ID NO: 17; (j) the antibody comprises a VH amino acid sequence having at least 90% identity to SEQ ID NO: 36 and a VL amino acid sequence having at least 90% identity to SEQ ID NO: 17; (k) the antibody comprises a VH amino acid sequence having at least 90% identity to SEQ ID NO: 39 and a VL amino acid sequence having at least 90% identity to SEQ ID NO: 17; or (l) the antibody comprises a VH amino acid sequence having at least 90% identity to SEQ ID NO: 42 and a VL amino acid sequence having at least 90% identity to SEQ ID NO: 17.

[0024] In at least one embodiment of the anti-CD36 antibody of the disclosure, The antibody comprises a heavy chain (HC) amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 44, 48, 50, 51, 52, 53, 54, 55, 56, or 57, and / or a light chain (LC) amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 45, 46, 47, or 49.

[0025] In at least one embodiment of the anti-CD36 antibody of the disclosure, The antibody: (a) an HC amino acid sequence having at least 90% identity to SEQ ID NO: 44, and an LC amino acid sequence having at least 90% identity to SEQ ID NO: 45; (b) an HC amino acid sequence having at least 90% identity to SEQ ID NO: 44, and an LC amino acid sequence having at least 90% identity to SEQ ID NO: 46; (c) an HC amino acid sequence having at least 90% identity to SEQ ID NO: 44, and an LC amino acid sequence having at least 90% identity to SEQ ID NO: 47; (d) an HC amino acid sequence having at least 90% identity to SEQ ID NO: 44, and an LC amino acid sequence having at least 90% identity to SEQ ID NO: 49; (e) an HC amino acid sequence having at least 90% identity to SEQ ID NO: 48, and an LC amino acid sequence having at least 90% identity to SEQ ID NO: 49; (f) an HC amino acid sequence having at least 90% identity to SEQ ID NO:50, and an LC amino acid sequence having at least 90% identity to SEQ ID NO:49; (g) an HC amino acid sequence having at least 90% identity to SEQ ID NO:51, and an LC amino acid sequence having at least 90% identity to SEQ ID NO:49; (h) an HC amino acid sequence having at least 90% identity to SEQ ID NO:52, and an LC amino acid sequence having at least 90% identity to SEQ ID NO:49; (i) an HC amino acid sequence having at least 90% identity to SEQ ID NO:53, and an LC amino acid sequence having at least 90% identity to SEQ ID NO:49; (j) an HC amino acid sequence having at least 90% identity to SEQ ID NO:54, and an LC amino acid sequence having at least 90% identity to SEQ ID NO:49; (k) an HC amino acid sequence having at least 90% identity to SEQ ID NO:55, and an LC amino acid sequence having at least 90% identity to SEQ ID NO:49; (l) an HC amino acid sequence having at least 90% identity to SEQ ID NO:56, and an LC amino acid sequence having at least 90% identity to SEQ ID NO:49; or (m) an HC amino acid sequence having at least 90% identity to SEQ ID NO:57, and an LC amino acid sequence having at least 90% identity to SEQ ID NO:49. Includes.

[0026] In at least one embodiment of the anti-CD36 antibody of the disclosure: (a) The antibody was diluted to 1 × 10 -8 M or less, 1×10 -9 M or less, 1×10 -10M or less; optionally, wherein the binding affinity is equal to or less than the equilibrium dissociation constant (K D ) measured by; (b) This antibody was diluted to 1 × 10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less; optionally, wherein the binding affinity is equal to or less than the equilibrium dissociation constant (K D ) measured by; (c) The antibody was diluted to 1 × 10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less; optionally, wherein the binding affinity is equal to or less than the equilibrium dissociation constant (K D ) measured by; (d) the antibody inhibits CD36-dependent oxidized LDL uptake by at least 65%, at least 75%, at least 85%, at least 95%, or at least 100% in F293 cells that overexpress surface human CD36; optionally, where at an oxLDL concentration of 1-10 μg / mL, the antibody has an IC of 5 nM or less, 1 nM or less, 0.5 nM or less, or 0.1 nM or less. 50 having; (e) the antibody inhibits CD36-dependent oxidized LDL uptake in U937 cells by at least 65%, at least 75%, at least 85%, at least 95%, or at least 100%; optionally, where at an oxLDL concentration of 1-10 μg / mL, the antibody has an IC of 5 nM or less, 1 nM or less, 0.5 nM or less, or 0.1 nM or less. 50 and / or (f) the antibody inhibits CD36-dependent oxidized LDL uptake in murine CD45+ TILs by at least 65%, at least 75%, at least 85%, at least 95%, or at least 100%; optionally, where at an oxLDL concentration of 1-10 μg / mL, the antibody has an IC of 5 nM or less, 1 nM or less, 0.5 nM or less, or 0.1 nM or less. 50 has.

[0027] The disclosure also provides embodiments of the anti-CD36 antibodies disclosed herein, including the following embodiments: (i) the antibody is a human, humanized, or chimeric antibody; (ii) the antibody is a fusion to a protein; optionally, a fusion to an immunostimulatory cytokine, such as IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, or IFN-α; (iii) the antibody is a full-length antibody of class IgG, optionally, wherein the class IgG antibody has an isotype selected from IgG1, IgG2, IgG3, and IgG4; (iv) the antibody is an Fc region variant, optionally an effector (v) the antibody is an antibody fragment, optionally selected from the group consisting of F(ab')2, Fab', Fab, Fv, single domain antibody (VHH), and scFv; (vi) the antibody comprises an immunoconjugate, optionally wherein the immunoconjugate comprises a therapeutic agent for treating a CD36-mediated disease or condition; or (vii) the antibody is a multispecific antibody, optionally a bispecific antibody.

[0028] In at least one embodiment, the disclosure provides an isolated polynucleotide, or a vector comprising a polynucleotide, wherein the polynucleotide sequence encodes an anti-CD36 antibody of the disclosure, or a polypeptide chain of an anti-CD36 antibody of the disclosure. In at least one embodiment, the isolated polynucleotide or vector comprises a sequence encoding a polypeptide of an anti-CD36 antibody of the disclosure. In at least one embodiment of the isolated polynucleotide or vector, the encoded polypeptide chain is: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, 21, 24, or 27; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 4, 28, 31, 34, 37, 40, or 43; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5; (b) a CDR-L1 comprising an amino acid sequence selected from SEQ ID NO: 7; a CDR-L2 comprising an amino acid sequence selected from SEQ ID NO: 8, 12, or 15; and a CDR-L3 comprising an amino acid sequence selected from SEQ ID NO: 9, 13, or 18; (c) a heavy chain variable domain (V) having at least 90% identity to a sequence selected from SEQ ID NO: 2, 20, 23, 26, 30, 33, 36, 39, or 42. H ) amino acid sequence; (d) a light chain variable domain (V) having at least 90% identity to a sequence selected from SEQ ID NO: 6, 11, 14, or 17. L ) amino acid sequence; (e) a heavy chain (HC) amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 44, 48, 50, 51, 52, 53, 54, 55, 56, or 57; and / or (f) a light chain (LC) amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 45, 46, 47, or 49. The amino acid sequence comprises:

[0029] In at least one embodiment, the disclosure also provides an isolated host cell comprising a polynucleotide or vector encoding an anti-CD36 antibody of the disclosure, or a polypeptide chain of the anti-CD36 antibody. In at least one embodiment, the disclosure also provides a method of producing an anti-CD36 antibody of the disclosure, comprising culturing a host cell comprising a polynucleotide or vector encoding the anti-CD36 antibody, such that the antibody is produced.

[0030] In at least one embodiment, the present disclosure provides a pharmaceutical composition comprising an anti-CD36 antibody of the present disclosure and a pharma- ceutically acceptable carrier; optionally, wherein the composition further comprises a chemotherapeutic agent, and / or an antibody comprising specificity for an immune checkpoint molecule.

[0031] In at least one embodiment, the present disclosure provides a method of treating a CD36-mediated disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-CD36 antibody of the present disclosure or administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present disclosure; optionally, wherein the disease is cancer; optionally, wherein the cancer is colon cancer, pancreatic cancer, ovarian cancer, HCC, renal cancer, breast cancer, lung cancer, gastric cancer, melanoma, head and neck cancer, and oral cancer.

[0032] In at least one embodiment, the present disclosure provides a method for treating cancer in a subject comprising administering to the subject a CD36 antagonist and a chemotherapeutic agent and / or an antibody comprising specificity for an immune checkpoint molecule; optionally, wherein the CD36 antagonist comprises an anti-CD36 antibody, shRNA, siRNA, miRNA, a small molecule inhibitor of CD36, or a combination thereof.

[0033] The novel features and advantages of the present disclosure will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the present disclosure are utilized, and the accompanying drawings (also referred to herein as "Figures" and "FIGs"), in which: [Brief description of the drawings]

[0034] [Figure 1A] FIG. 1A depicts ELISA results for an exemplary anti-CD36 antibody of the disclosure (12P109, A8A) in full length human IgG1 format binding to recombinant human CD36.ECD (FIG. 1A). ELISA was performed as described in Example 1. [Figure 1B] FIG. 1B depicts ELISA results of an exemplary anti-CD36 antibody of the disclosure (12P109, A8A) in full length human IgG1 format binding to recombinant mouse CD36.ECD (FIG. 1B). ELISA was performed as described in Example 1. [Figure 1C]FIG. 1C depicts ELISA results for an exemplary anti-CD36 antibody of the disclosure (12P109, A8A) in full length human IgG1 format binding to recombinant human CD36.ECD (FIG. 1C). ELISA was performed as described in Example 1. [Figure 1D] FIG. 1D depicts ELISA results of an exemplary anti-CD36 antibody of the disclosure (12P109, A8A) in full length human IgG1 format binding to recombinant mouse CD36.ECD (FIG. 1D). ELISA was performed as described in Example 1. [Figure 2A] ELISA results for exemplary anti-CD36 antibodies of the disclosure (12P109, and 117 variants) binding to recombinant human CD36.ECD. ELISA was performed as described in Example 1. [Figure 2B] ELISA results for exemplary anti-CD36 antibodies (117 variant) of the disclosure binding to recombinant human CD36.ECD. ELISA was performed as described in Example 1. [Figure 2C] ELISA results for exemplary anti-CD36 antibodies (117 variant) of the disclosure binding to recombinant human CD36.ECD. ELISA was performed as described in Example 1. [Figure 2D] ELISA results for exemplary anti-CD36 antibodies (117 variant) of the disclosure binding to recombinant human CD36.ECD. ELISA was performed as described in Example 1. [Figure 2E] ELISA results for exemplary anti-CD36 antibodies (117 variant) of the disclosure binding to recombinant human CD36.ECD. ELISA was performed as described in Example 1. [Figure 2F] ELISA results for exemplary anti-CD36 antibodies (117 variant) of the disclosure binding to recombinant human CD36.ECD. ELISA was performed as described in Example 1. [Figure 3A] FIG. 1 depicts plots showing results from SEC-UPLC analysis of anti-CD36 antibodies (12P109, A8A, and 117 variants), as described in Example 1. [Figure 3B] FIG. 1 depicts plots showing results from SEC-UPLC analysis of anti-CD36 antibodies (117 variant), as described in Example 1. [Figure 3C] FIG. 1 depicts plots showing results from SEC-UPLC analysis of anti-CD36 antibodies (12P109 and 117 variants), as described in Example 1. [Figure 3D] FIG. 1 depicts plots showing results from SEC-UPLC analysis of anti-CD36 antibodies (12P109 and 117 variants), as described in Example 1. [Figure 3E] FIG. 1 depicts plots showing results from SEC-UPLC analysis of anti-CD36 antibodies (12P109, A8A, and 117 variants), as described in Example 1. [Figure 4A] FIG. 1 depicts a plot showing the results from flow cytometric cell surface binding analysis of an exemplary anti-CD36 antibody (117 variant) of the disclosure to stable F293 cells overexpressing human CD36 ("hCD36"), as described in Example 2. [Figure 4B] FIG. 1 depicts a plot showing the results from flow cytometric cell surface binding analysis of an exemplary anti-CD36 antibody (117 variant) of the disclosure to stable F293 cells overexpressing human CD36 ("hCD36"), as described in Example 2. [Figure 4C] FIG. 1 depicts a plot showing the results from flow cytometric cell surface binding analysis of an exemplary anti-CD36 antibody (117 variant) of the disclosure to stable F293 cells overexpressing rhesus CD36, as described in Example 2. [Figure 4D] FIG. 1 depicts a plot showing the results from flow cytometric cell surface binding analysis of an exemplary anti-CD36 antibody (117 variant) of the disclosure to stable F293 cells overexpressing rhesus CD36, as described in Example 2. [Figure 4E]FIG. 1 depicts a plot showing the results from flow cytometric cell surface binding analysis of an exemplary anti-CD36 antibody (117 variant) of the disclosure to stable F293 cells overexpressing mouse CD36 ("mCD36"), as described in Example 2. [Figure 4F] FIG. 1 depicts a plot showing the results from flow cytometric cell surface binding analysis of an exemplary anti-CD36 antibody (117 variant) of the disclosure to stable F293 cells overexpressing mouse CD36 ("mCD36"), as described in Example 2. [Figure 5A] FIG. 1 depicts plots of extracted flow cytometry data showing the activity of anti-CD36 antibodies of the disclosure in blocking oxLDL binding by U937 cells, as described in Example 3. [Figure 5B] FIG. 1 depicts plots of extracted flow cytometry data showing the activity of anti-CD36 antibodies of the disclosure in blocking oxLDL uptake by U937 cells, as described in Example 3. [Figure 6A] FIG. 13 depicts plots extracted from flow cytometry data illustrating the oxLDL uptake blocking activity exhibited by anti-CD36 antibodies (12P109 and 117 variants) in F293 / hCD36 cells, as described in Example 3. [Figure 6B] FIG. 13 depicts plots extracted from flow cytometry data illustrating the oxLDL uptake blocking activity exhibited by anti-CD36 antibody (117 variant) in F293 / hCD36 cells, as described in Example 3. [Figure 6C] FIG. 13 depicts plots extracted from flow cytometry data illustrating the oxLDL uptake blocking activity exhibited by anti-CD36 antibodies (12P109 and 117 variants) in F293 / mCD36 cells, as described in Example 3. [Figure 7A]FIG. 13 depicts plots of data showing the ability of anti-CD36 antibodies of the disclosure (12P109, A8A) to inhibit oxLDL uptake in TILs from a syngeneic mouse model of colon cancer (CT26), as described in Example 4. [Figure 7B] FIG. 13 depicts plots of data showing the ability of anti-CD36 antibodies (117 variant) of the present disclosure to inhibit oxLDL uptake in TILs from a syngeneic mouse model of colon cancer (MC38), as described in Example 4. [Figure 7C] FIG. 13 depicts a plot of data showing the ability of anti-CD36 antibodies (117 variant) of the disclosure to inhibit oxLDL uptake in TILs from a syngeneic mouse model of liver cancer (BNL 1MEA.7R.1), as described in Example 4. [Figure 7D] FIG. 13 depicts a plot of data showing the ability of anti-CD36 antibodies (117 variant) of the present disclosure to inhibit oxLDL uptake in TILs from a syngeneic mouse model of lung cancer (LL2), as described in Example 4. [Figure 7E] FIG. 13 depicts a plot of data showing the ability of anti-CD36 antibodies (117 variant) of the disclosure to inhibit oxLDL uptake in TILs from a syngeneic mouse model of melanoma (B16F10), as described in Example 4. [Figure 8A] FIG. 13 depicts plots of data illustrating the ability of anti-CD36 antibodies of the disclosure (12P109, and 117 variants) to inhibit M2 macrophage polarization, as described in Example 5. [Figure 8B] FIG. 13 depicts plots of data illustrating the ability of anti-CD36 antibodies of the disclosure (12P109, and 117 variants) to inhibit M2 macrophage polarization, as described in Example 5. [Figure 8C] FIG. 13 depicts plots of data illustrating the ability of anti-CD36 antibodies (117 variant) of the disclosure to inhibit oxLDL-induced M2 macrophage activation, as described in Example 5. [Figure 8D]FIG. 13 depicts plots of data illustrating the ability of anti-CD36 antibodies (117 variant) of the disclosure to inhibit oxLDL-induced M2 macrophage activation, as described in Example 5. [Figure 8E] FIG. 13 depicts plots of data illustrating the ability of anti-CD36 antibodies (117 variant) of the disclosure to inhibit oxLDL-induced M2 macrophage activation, as described in Example 5. [Figure 8F] FIG. 13 depicts plots of data illustrating the ability of anti-CD36 antibodies (117 variant) of the disclosure to inhibit oxLDL-induced M2 macrophage activation, as described in Example 5. [Figure 9] FIG. 1 depicts the results of tumor growth curves measured by bioluminescence in MYCOE / p53KO HCC model mice treated with anti-CD36 antibody 117_30DA, as described in Example 6. [Figure 10A] Figure 10 depicts the results measured in β-catenin OE / MYCOE HCC model mice treated with anti-CD36 antibody 117_DA57E as described in Example 6. Figure 10A shows the results of tumor growth curves measured by bioluminescence. [Figure 10B] FIG. 10 depicts results measured in β-catenin OE / MYCOE HCC model mice treated with anti-CD36 antibody 117_DA57E as described in Example 6. FIG. 10B shows a plot of the endpoint liver mass results. [Figure 10C] Figure 10 depicts the results measured in β-catenin OE / MYCOE HCC model mice treated with anti-CD36 antibody 117_DA57E as described in Example 6. Figure 10C shows a plot of the results of the analysis of plasma ALT (alanine transaminase) activity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] The present disclosure provides antibodies, including humanized antibodies, that specifically bind to CD36 with high affinity, thereby inhibiting, reducing, and / or completely blocking the function of CD36 as a cell surface protein involved in immune regulation, in particular the function of CD36-mediated fatty acid transport and / or oxidized lipid transport (e.g., uptake of oxLDL) among various roles of CD36, such as those detailed in the Background section, including but not limited to the role of CD36 in regulating the function and maintenance or survival (or in some cases, death) of tumor cells, tumor-associated macrophages (TAMs), MDSCs, regulatory T cells, and CD8 T cells.

[0036] Thus, it is contemplated that any of the compositions or formulations comprising the anti-CD36 antibodies of the present disclosure may be used as a therapy for the treatment of diseases mediated by the function of CD36 in fatty acid transport, such as cancer. Additionally, it is contemplated that the anti-CD36 antibodies of the present disclosure may be used as a therapy in combination with other therapies, such as cell therapy, cytokines and other biologics or drugs that modify the tumor microenvironment and / or enhance immune responses, antibody drug conjugates, antibodies that modulate immune cells, and / or antibodies that target immune checkpoint molecules, including, but not limited to, PD1, PD-L1, LAG3, CTLA-4, A2AR, TIM-3, BTLA, CD276, CD328, VTCN1, IDO, KIR, NOX2, VISTA, OX40, CD27, CD28, CD40, CD122, CD137, GITR, ICOS.

[0037] Overview of terms and techniques For purposes of the description herein and the appended claims, the singular forms "a" and "an" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a protein" includes two or more proteins, and a reference to "a compound" refers to two or more compounds. It is further noted that the claims may be written to exclude any optional elements. Thus, this statement is intended to serve as a prerequisite for using exclusive language, such as "solely," "only," or for using a "negative" limitation in connection with the recitation of claim elements. The use of "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and are not intended to be limiting. It should be further understood that when the term "comprising" is used in describing various embodiments, those of ordinary skill in the art will understand that in some specific cases, the embodiments can alternatively be described using the language "consisting essentially of" or "consisting of."

[0038] Where a range of values ​​is provided, it is understood that, unless expressly stated otherwise, each intervening integer between the upper and lower limits of that range, and each tenth of each intervening integer, unless expressly stated otherwise, as well as any other stated or intervening value within the stated range, are encompassed within the invention. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either (i) or both (ii) of those included limits are also encompassed within the invention. For example, "1 to 50" includes "2 to 25," "5 to 20," "25 to 50," "1 to 10," etc.

[0039] Generally, the nomenclature used herein, and the techniques and procedures described herein, include those well understood and commonly used by those of skill in the art, such as conventional techniques and methodologies described in Sambrook et al., Molecular Cloning-A Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989 (hereinafter "Sambrook"); Current Protocols in Molecular Biology, edited by F. M. Ausubel et al., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (revised in 2011) (hereinafter "Ausubel"); Antibody Engineering, Vols. 1 and 2, edited by R. Kontermann and S. Dubel, Springer-Verlag, Berlin and Heidelberg (2010); Monoclonal Antibodies: Methods and Protocols, edited by V. Ossipow and N. Fischer, 2nd ed., Humana Press (2014); Therapeutic Antibodies: From Bench to Clinic, edited by Z. An, J. Wiley & Sons, Hoboken, NJ (2009); and Phage Display, edited by Tim Clackson and Henry B. Lowman, Oxford University Press, United Kingdom (2004).

[0040] All publications, patents, patent applications, and other documents referenced in this disclosure are herein incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and other document was individually indicated to be incorporated by reference for all purposes.

[0041] Unless otherwise defined, all technical and scientific terms used herein shall have the meaning commonly understood by those skilled in the art to which the present invention pertains. It is to be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. For purposes of interpreting this disclosure, the following explanation of terms shall apply, and where appropriate, terms used in the singular shall also include the plural and vice versa.

[0042] "CD36," as used herein, refers to the CD36 protein, and as used herein, includes human, cynomolgus monkey, and mouse CD36 proteins, and any isoforms of these proteins. The amino acid sequences of various exemplary CD36 proteins are known in the art and are provided in Table 1 below and in the attached sequence listing.

[0043] "CD36-mediated condition" or "CD36-mediated disease" as used herein includes any pathology associated with the specific binding of a ligand to the cell surface protein CD36. For example, the specific binding of lipids and / or fatty acids by CD36 acts to regulate or enhance the immunosuppressive capacity of TAMs and regulatory T cells in the tumor microenvironment. Thus, CD36-mediated disease can include, but is not limited to, any disease or condition that is mediated by and / or responds to CD36 antagonists or inhibitors, including, but not limited to, cancer.

[0044] "Antibody," as used herein, refers to a molecule comprising one or more polypeptide chains that specifically binds to or is immunologically reactive with a particular antigen. Exemplary antibodies of the present disclosure include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (e.g., fusion proteins), multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies (e.g., single-arm antibodies), multivalent antibodies, antigen-binding fragments (e.g., Fab', F(ab')2, Fab, Fv, rIgG, and scFv fragments), and synthetic antibodies (or antibody mimetics).

[0045] An "anti-CD36 antibody" or an "antibody that binds CD36" refers to an antibody that binds CD36 with sufficient affinity such that the antibody is useful as a therapeutic and / or diagnostic agent for targeting CD36. In some embodiments, the extent of binding of an anti-CD36 specific antibody to an unrelated, non-CD36 antigen is less than about 20%, less than about 15%, less than about 10%, or less than about 5% of the binding of the antibody to CD36, as measured, for example, by radioimmunoassay (RIA) or surface plasmon resonance (SPR). In some embodiments, the anti-CD36 antibodies of the present disclosure have a dissociation constant (K D ), <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <1 pM (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 Up to M, for example, 10 -9 M to 10 -13 The amino acid sequences of exemplary CD36 proteins of the disclosure are provided in Table 2 below and in the attached sequence listing.

[0046] "Full length antibody," "intact antibody," or "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody or having a heavy chain that contains an Fc region as defined herein.

[0047] "Antibody fusion" refers to an antibody that is covalently conjugated (or fused) to a polypeptide or protein, usually via a linker to the end of the light chain (LC) or heavy chain (HC) of the antibody. Exemplary antibody fusions contemplated by the present disclosure may include an anti-CD36 antibody fused via a linker to a protein that is a T cell activating or immune stimulating cytokine, such as IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, or IFN-α.

[0048] "Antibody fragment" refers to a portion of a full-length antibody that is capable of binding to the same antigen as the full-length antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; monovalent or single-armed antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0049] The "class" of an antibody refers to the type of constant domain or region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, several of which are further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0050] "Variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a natural antibody are (V H and V L ) have an overall similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR) (see, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91). H Domain or V LIn some cases, a single domain may be sufficient to confer antigen-binding specificity. Furthermore, an antibody that binds to a particular antigen can be identified by dividing the V domain by the V domain derived from the antibody that binds that antigen. H Domain or V L The domains were isolated using the complementary V L Domain or V H Libraries of domains can be screened individually (see, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0051] "Hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops ("hypervariable loops"). Generally, naturally occurring antibodies contain six HVRs; heavy chain variable domain V H (HVR-H1, HVR-H2, HVR-H3) and the light chain variable domain V L(HVR-L1, HVR-L2, HVR-L3). HVRs generally contain amino acid residues from hypervariable loops and / or from "complementarity determining regions" (CDRs). Several delineations of hypervariable regions are used herein and are encompassed herein. Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Instead, Chothia refers to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. The "contact" hypervariable regions are based on an analysis of the available complex crystal structures. The residue ranges of the hypervariable regions defined under these systems are listed in the table below.

[0052] [Table 1]

[0053] In addition to the above systems, HVRs and CDRs can be identified using the international ImMunoGeneTics information system, called IMGT / V-Quest and described in Brochet, X. et al., Nucl. Acids Res. 36, W503-508 (2008). PMID: 18503082; and available online at www.imgt.org / IMGT_vquest / input. IMGT / V-Quest uses the IMGT unique numbering to analyze alignments to the variable region nucleotide sequences of the closest germline V genes, thereby identifying HVRs and CDRs.

[0054] Hypervariable regions (HVRs), as used herein, may include extended or alternative hypervariable regions, such as: 27-32, 27-36, 24-34, or 24-38 (HVR-L1); 50-52, 54-56, 50-56, or 54-60 (HVR-L2); 89-97, or 93-101 (HVR-L3); H and 97-110, 97-112, 99-110, or 99-112 (H3). The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

[0055] "Complementarity determining region" or "CDR" as used herein refers to the region within the HVR of a variable domain that has the highest sequence variability and / or is involved in antigen recognition. Generally, naturally occurring antibodies have six CDRs; three in the heavy chain variable domain and one in the V H (CDR-H1, CDR-H2, CDR-H3), and three in the light chain variable domain, V L(CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs are present at each of the following variable domain amino acid residue positions: 24-34, 27-32, 27-36, 24-38 (CDR-L1); 50-56, 50-52, 54-56, or 54-60 (CDR-L2); 89-97, or 93-101 (CDR-L3); 31-35, or 26-33 (CDR-H1), 50-66, or 51-58 (CDR-H2); and 99-112, 99-110, 97-112, or 97-110 (CDR-H3).

[0056] "Framework" or "FR" refers to variable domain residues other than HVR residues. The FR of a variable domain generally consists of four domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally refer to the V H (or V L ) in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0057] Unless otherwise indicated, HVR, CDR, FR residues, and other residue positions in the variable domain are numbered herein according to Kabat et al., supra.

[0058] "Native antibody" refers to a naturally occurring immunoglobulin molecule. For example, a native IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain contains a variable region (V), also called a variable heavy domain or heavy chain variable domain. H ), followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain contains a variable region (V), also called a variable light domain or a light chain variable domain. L ), followed by a constant light chain (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0059] As used herein, a "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope, excluding possible variant antibodies (e.g., variant antibodies that contain naturally occurring mutations or arise during the production of the monoclonal antibody and are generally present in minor amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the term "monoclonal" refers to the character of the antibody obtained from a population of substantially homogeneous antibodies, and should not be interpreted as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, and such and other exemplary methods for producing monoclonal antibodies are described herein.

[0060] A "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular origin or species, while the remainder of the heavy and / or light chain is derived from a different origin or species.

[0061] A "humanized antibody" refers to a chimeric antibody that comprises amino acid sequences derived from non-human CDRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody can optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0062] "Human antibody" refers to an antibody that possesses an amino acid sequence that corresponds to that of an antibody produced by a human or a human cell, or from a repertoire of human antibodies or other non-human sources that utilize human antibody-encoding sequences. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0063] The "human consensus framework" is a set of human immunoglobulin V L Or V H In selecting framework sequences, the framework represents the most commonly occurring amino acid residues. L Sequence or V H The selection of sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup similar to those described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Bethesda MD (1991), NIH Publication 91-3242, Vols. 1-3. In one embodiment, the V L For V, the subgroup is subgroup kappa I as per Kabat et al., supra. H For , the subgroup is subgroup III as per Kabat et al., supra.

[0064] As used herein, an "acceptor human framework" refers to a light chain variable domain (VV) derived from a human immunoglobulin framework or a human consensus framework. L ) framework or heavy chain variable domain (V HAn acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence as thereto, or the acceptor human framework may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the acceptor human framework comprises the amino acid sequence of a V L The acceptor human framework is V L The sequence is identical to a human immunoglobulin framework sequence or a human consensus framework sequence.

[0065] "Fc region" refers to a dimeric complex comprising the C-terminal polypeptide sequence of an immunoglobulin heavy chain, where the C-terminal polypeptide sequence is a sequence obtainable by papain digestion of an intact antibody. The Fc region may comprise a native Fc sequence or a variant Fc sequence. Although the boundaries of the Fc sequence of an immunoglobulin heavy chain vary, the human IgG heavy chain Fc sequence is usually defined as extending from an amino acid residue at about position Cys226 or from about position Pro230 to the carboxyl terminus of the Fc sequence. However, the C-terminal lysine (Lys447) of the Fc sequence may or may not be present. The Fc sequence of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally a CH4 domain.

[0066] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR binds IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcR, as used herein, also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 1 17:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), as well as regulating immunoglobulin homeostasis. FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995).

[0067] A "multispecific antibody" is an antibody that has at least two different binding sites, each site having a different binding specificity. A multispecific antibody may be a full-length antibody or an antibody fragment, and the different binding sites can each bind to a different antigen, or the different binding sites can bind to two different epitopes of the same antigen.

[0068] "Fv fragment" refers to an antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which is covalent in nature, e.g., in scFv. The three CDRs of each variable domain interact to form the V H -V L It is in this configuration that defines an antigen-binding site on the surface of the dimer. Collectively, the six CDRs, or a subset thereof, confer antigen-binding specificity to the antibody. However, a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, although usually with a lower affinity than the entire binding site.

[0069] "Fab fragment" refers to an antibody fragment containing the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. "F(ab')2 fragment" comprises a pair of Fab fragments that are generally covalently linked near the carboxy termini of the Fab by hinge cysteines between the Fab. Other chemical couplings of antibody fragments are known in the art.

[0070] "Antigen-binding arm," as used herein, refers to the component of an antibody having the ability to specifically bind to a target molecule of interest. Typically, an antigen-binding arm is a complex of immunoglobulin polypeptide sequences, such as CDRs and / or immunoglobulin light and heavy chain variable domain sequences.

[0071] "Single-chain Fv" or "scFv" refers to the V H and V L Fv refers to an antibody fragment comprising V domains, where these domains are present in a single polypeptide chain. Generally, an Fv polypeptide comprises an Fv polypeptide that enables the scFv to form the desired antigen-binding structure. H Domain and V L It further comprises a polypeptide linker between the domains.

[0072] "Affinity" 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). "Binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally measured by the equilibrium dissociation constant (K D Affinity can be measured by common methods known in the art, including those described herein. Specific exemplary and illustrative embodiments for measuring binding affinity are described below.

[0073] "Specifically binds" or "specific binding" means a binding density of about 1 × 10 -7 It refers to the binding of an antibody to an antigen with an affinity value of M or less. In some embodiments, an antibody has a secondary affinity for an antigen other than the antigen to which it specifically binds, where "secondary affinity" generally refers to the binding of an antibody to a secondary antigen with an affinity value of greater than about 10 nM, as described elsewhere herein. Although an antibody may have a secondary affinity for a secondary antigen, such an antibody will nevertheless specifically bind to the primary antigen.

[0074] An "isolated antibody" refers to an antibody that is separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or greater than 99% purity, for example, as determined by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic methods (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87.

[0075] "Effector functions" refer to biological activities attributable to the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include: Clq binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cellular cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and activation of B cells.

[0076] "Immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent.

[0077] "Treatment", "treat" or "treating" refers to a clinical intervention that attempts to alter the natural history of a disorder in an individual being treated, whether it is performed to prevent onset or during the course of clinical pathology. Desired outcomes of treatment include, but are not limited to, preventing the onset or recurrence of the disorder, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disorder, preventing metastasis, slowing the rate of progression, remission or palliating the disease state, and improving remission or prognosis. For example, treatment can include administering to a subject a therapeutically effective amount of a pharmaceutical formulation comprising an anti-CD36 antibody to delay the onset or slow the progression of a disease or condition mediated by CD36 and / or its binding to a ligand, or in which CD36 may play a role in the etiology and / or progression.

[0078] "Pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and does not contain any additional ingredients that are toxic to the subject to which the formulation is administered. A pharmaceutical formulation can include one or more active agents. For example, a pharmaceutical formulation can include an anti-CD36 antibody as the sole active agent of the formulation, or can include an anti-CD36 antibody and one or more additional active agents, such as an inhibitor of an immune checkpoint molecule.

[0079] "Sole active agent" as used herein refers to an active agent in a pharmaceutical formulation that is the only active agent present in the formulation that provides or is expected to provide a relevant pharmacological effect to treat the subject against the condition being treated. A pharmaceutical formulation that includes a single active agent does not exclude the presence of one or more non-active agents in the pharmaceutical formulation, such as pharma- ceutically acceptable carriers. A "non-active agent" is an agent that would not be expected to provide or otherwise significantly contribute to the relevant pharmacological effect intended to treat the subject against the condition.

[0080] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject to which it is administered. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0081] "Immune checkpoint molecule" as used herein refers to a molecule that functions to regulate immune system pathways, thereby preventing the pathways from unnecessarily attacking cells. Many immune checkpoint molecules, both inhibitory and costimulatory, are targets of immunotherapy (e.g., by blocking antibodies to block immune inhibition or by agonists to promote immune stimulation) in the treatment of cancer and viral infections. Exemplary immune checkpoint molecules targeted by cancer immunotherapy include, but are not limited to, the following: PD1, CD36, LAG3, CTLA-4, A2AR, TIM-3, BTLA, CD276, CD328, VTCN1, IDO, KIR, NOX2, VISTA, OX40, CD27, CD28, CD40, CD122, CD137, GITR, ICOS.

[0082] "Therapeutically effective amount" refers to an amount of an active ingredient or active agent (e.g., a pharmaceutical formulation) to achieve a desired therapeutic or prophylactic result, e.g., to treat or prevent a disease, disorder, or condition in a subject. In the case of a CD36-mediated disease or condition, a therapeutically effective amount of a therapeutic agent is an amount that reduces, prevents, inhibits, and / or alleviates to some extent one or more symptoms associated with the disease, disorder, or condition. In cancer therapy, efficacy in vivo can be measured, for example, by assessing primary tumor growth, occurrence and / or growth of secondary tumors, occurrence and / or number of metastases, duration, severity, and / or recurrence of symptoms, response rate (RR), duration of response, and / or quality of life.

[0083] "Concurrently," as used herein, refers to the administration of two or more therapeutic agents, where at least a portion of the administration overlaps in time. Thus, concurrent administration includes dosing regimens where administration of one or more agents continues after administration of one or more other agents is discontinued.

[0084] "Individual" or "subject" refers to mammals, including, but not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0085] DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS I. CD36 CD36 is a multifunctional transmembrane glycoprotein that functions as a cell surface receptor for a wide range of ligands. In general, CD36 has two distinct binding domains, one for the binding of thrombospondin versus other ligands of lipid nature, such as oxidized low density lipoprotein (oxLDL), anionic phospholipids, long chain fatty acids, and bacterial diacylated lipopeptides. Cellular responses mediated by CD36 binding of these ligands are believed to include fatty acid metabolism, processing of dietary fat, angiogenesis, and inflammatory responses. CD36 functions as a co-receptor for the TLR4:TLR6 heterodimer, thereby promoting inflammation in monocytes / macrophages. Upon binding of ligands such as oxidized LDL ("oxLDL"), CD36 is believed to interact with the TLR4:TLR6 heterodimer and internalize the complex, thereby inducing an inflammatory response leading to NF-kappa-B-dependent production of CXCL1, CXCL2, and CCL9 cytokines via the MyD88 signaling pathway and CCL5 cytokine via the TICAM1 signaling pathway, as well as IL1B secretion through priming and activation of the NLRP3 inflammasome. Other CD36-interacting coreceptors have also been described.

[0086] The sequence and annotation of human CD36 (also referred to herein as "hCD36") can be found in UniProt entry P16671, with the full-length 472 amino acid sequence of isoform 1 set forth herein as SEQ ID NO: 58. The sequence and annotation of mouse CD36 (also referred to herein as "mCD36") can be found in UniProt entry Q08857, with the full-length 472 amino acid sequence set forth herein as SEQ ID NO: 60. A summary description of the human and mouse CD36 polypeptide sequences and their sequence identifiers used in this disclosure is provided in Table 1 below. The sequences are also included in the attached sequence listing.

[0087] [Table 2A]

[0088] [Table 2B]

[0089] II. Anti-CD36 antibody In some embodiments, the present disclosure provides a method for identifying a polypeptide that is a member of a group of polypeptides that are similar to those described herein, including a polypeptide that is a member of a group of polypeptides that are similar to those described herein. H , V L The structures of the anti-CD36 antibodies are provided by the amino acid and encoding nucleotide sequences of each domain of the heavy and light chains (as well as the full length heavy and light chains). A summary description of the anti-CD36 antibodies of the disclosure, generated and functionally characterized as described in the Examples, is provided in Table 2 below. The associated sequences and sequence identifiers for each of the antibodies are provided in Table 2 and are also included in the attached sequence listing.

[0090] [Table 3A]

[0091] [Table 3B]

[0092] [Table 3C]

[0093] [Table 3D]

[0094] [Table 3E]

[0095] [Table 3F]

[0096] [Table 3G]

[0097] [Table 3H]

[0098] 1. Anti-CD36 Antibody Binding Affinity and Functional Characteristics In some embodiments, the anti-CD36 antibodies provided herein have an equilibrium dissociation constant (K D ), <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 -8 M or less, 10 -8 M to 10 -13 Up to M, for example, 10 -9 M to 10 -13 M).

[0099] It is contemplated that the various anti-CD36 antibodies generated as disclosed herein include antibodies capable of high affinity binding to hCD36, mCD36, rhesus CD36, both hCD36 and mCD36, and / or hCD36, mCD36, and rhesus CD36. More specifically, in some embodiments, the anti-CD36 antibodies of the present disclosure are capable of binding to 1×10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less, or 1 x 10 -11 In some embodiments, the binding affinity is determined by the equilibrium dissociation constant (K D In some embodiments, the anti-CD36 antibody of the disclosure is measured at 1×10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less, or 1 x 10 -11In some embodiments, the binding affinity is determined by the equilibrium dissociation constant (K D In some embodiments, the anti-CD36 antibody of the disclosure is measured at 1×10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less, or 1 x 10 -11 In some embodiments, the binding affinity is determined by the equilibrium dissociation constant (K D In some embodiments, the anti-CD36 antibody of the disclosure is measured at 1×10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less, or 1 x 10 -11 In some embodiments, the anti-CD36 antibodies of the present disclosure bind to both hCD36 and mCD36 with a binding affinity of 1×10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less, or 1 x 10 -11 It binds to both hCD36 and rhesus CD36 with a binding affinity of less than or equal to M.

[0100] In general, the binding affinity of a ligand to its receptor can be determined using any of a variety of assays and expressed as a variety of quantitative values. A specific CD36 binding assay useful for determining the affinity of an antibody is disclosed in the Examples herein. Furthermore, antigen binding assays are known in the art and can be used herein, including, but not limited to, any direct or competitive binding assay using the following techniques: Western blot, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), "sandwich" immunoassay, surface plasmon resonance-based assay (e.g., BIAcore assay as described in WO2005 / 012359), immunoprecipitation assay, fluorescent immunoassay, protein A immunoassay, flow cytometry, and fluorescence-activated cell sorting (FACS) assay, etc.

[0101] Thus, in some embodiments, the binding affinity is K D The binding affinity is expressed as a K value and reflects the intrinsic binding affinity (e.g., with minimal avidity effects). Anti-CD36 antibodies of the present disclosure exhibit strong binding affinity to the hCD36 polypeptide of SEQ ID NO:58, e.g., a K between 10 nM and 1 pM. D Thus, the anti-CD36 antibodies of the present disclosure can compete with antibodies having lower affinity for the same or overlapping epitope of CD36.

[0102] In some embodiments, the anti-CD36 antibodies provided herein reduce, inhibit, and / or completely block ligand binding to CD36 and immune regulation and / or immune signaling mediated by ligand binding to CD36, including maintenance of TAMs in the tumor microenvironment. The ability of the antibodies to inhibit these immune regulation and immune signaling pathways mediated by ligand binding to CD36 can be assayed in vitro using known cell-based assays, including such assays described in the Examples of the present disclosure.

[0103] Thus, in some embodiments, the CD36 antibodies of the present disclosure are characterized by one or more of the following functional properties based on their ability to reduce, inhibit, and / or completely block intracellular signaling through CD36-mediated pathways.

[0104] In at least one embodiment, the anti-CD36 antibody is administered at a concentration of 1×10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less; optionally, wherein the binding affinity is equal to or less than the equilibrium dissociation constant (K D ) is measured.

[0105] In at least one embodiment of the anti-CD36 antibody, the antibody is at least 1×10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less; optionally, wherein the binding affinity is equal to or less than the equilibrium dissociation constant (K D ) is measured.

[0106] In at least one embodiment of the anti-CD36 antibody, the antibody is at least 1×10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less; optionally, wherein the binding affinity is equal to or less than the equilibrium dissociation constant (K D ) is measured.

[0107] In at least one embodiment of the anti-CD36 antibody, the antibody inhibits CD36-dependent oxidized LDL uptake by at least 65%, at least 75%, at least 85%, at least 95%, or at least 100% in F293 cells overexpressing surface human CD36; optionally, where at an oxLDL concentration of 1-10 μg / mL, the antibody has an IC of 5 nM or less, 1 nM or less, 0.5 nM or less, or 0.1 nM or less.50 has.

[0108] In at least one embodiment of the anti-CD36 antibody, the antibody inhibits CD36-dependent oxidized LDL uptake in U937 cells by at least 65%, at least 75%, at least 85%, at least 95%, or at least 100%; optionally, where at an oxLDL concentration of 1-10 μg / mL, the antibody has an IC of 5 nM or less, 1 nM or less, 0.5 nM or less, or 0.1 nM or less. 50 has.

[0109] In at least one embodiment of the anti-CD36 antibody, the antibody inhibits CD36-dependent oxidized LDL uptake in murine CD45+ TILs by at least 65%, at least 75%, at least 85%, at least 95%, or at least 100%; optionally, where at an oxLDL concentration of 1-10 μg / mL, the antibody has an IC of 5 nM or less, 1 nM or less, 0.5 nM or less, or 0.1 nM or less. 50 has.

[0110] 2. Anti-CD36 antibody fragment In some embodiments, the anti-CD36 antibodies of the present disclosure may be antibody fragments. Useful antibody fragments having a binding determinant of the present disclosure include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv fragments, monovalent, single domain antibodies, one-armed or single-arm antibodies, and other fragments described herein and known in the art. Thus, in some embodiments of the anti-CD36 antibodies of the present disclosure, the antibody is an antibody fragment selected from the group consisting of F(ab')2, Fab', Fab, Fv, single domain antibodies (VHH), single arm antibodies, and scFv.

[0111] For a review of various antibody fragments, see, e.g., Hudson et al. Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York) pp. 269-315 (1994); WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a description of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have extended in vivo half-lives, see U.S. Patent No. 5,869,046. Other monovalent antibody formats are described, for example, in WO2007 / 048037, WO2008 / 145137, WO2008 / 145138, and WO2007 / 059782. Monovalent single-armed antibodies are described, for example, in WO2005 / 063816. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific (see, for example, EP0404097; WO93 / 01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)).

[0112] In some embodiments, an antibody fragment is a single domain antibody that comprises all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody, hi some embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Patent No. 6,248,516).

[0113] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phages), as described herein.

[0114] 3. Chimeric, Humanized, and Human Anti-CD36 Antibodies In some embodiments, the anti-CD36 antibody of the present disclosure may be a chimeric antibody. (See, e.g., U.S. Pat. No. 4,816,567; and the chimeric antibodies described in Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one embodiment, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate, such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In some embodiments, the chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. It is contemplated that the chimeric antibody may include antigen-binding fragments thereof.

[0115] In some embodiments, the anti-CD36 antibodies of the present disclosure are humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In general, a humanized antibody comprises one or more variable domains, in which the HVRs, CDRs (or portions thereof) are derived from a non-human antibody, and the FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody will also optionally comprise at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with the corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues are derived) to restore or improve the specificity or affinity of the antibody.

[0116] Humanized antibodies and methods for making them are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13: 1619-1633 2008) and further described, for example, in Riechmann et al., Nature 332: 323-329 1988; Queen et al., Proc. Nat'l Acad. Sci. USA 86: 10029-10033 1989; U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36: 25-34 2005) (describing SDR (a-HVR) grafting); Padlan, Mol. Immunol. 28: 489-498 1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 2005) and Klimka et al., Br. J. Cancer, 83:252-260 2000) (describing a "guide selection" approach to FR shuffling).

[0117] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from human antibody consensus sequences of particular subgroups of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol, 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al. J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0118] In some embodiments, the anti-CD36 antibodies of the present disclosure may be human antibodies. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008). Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been engineered to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals usually contain all or part of a human immunoglobulin locus, which either replaces an endogenous immunoglobulin locus or is extrachromosomally present or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., the XENOMOUSE™ technology of U.S. Pat. Nos. 6,075,181 and 6,150,584; the HUMAB® technology of U.S. Pat. No. 5,770,429; the KM MOUSE® technology of U.S. Pat. No. 7,041,870; and the VELOCIMOUSE® technology of U.S. Patent Application Publication No. US 2007 / 0061900. The human variable regions from intact antibodies produced by such animals can be further modified, e.g., by combining with different human constant regions.

[0119] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. See, for example, Kozbor J. Immunol, 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991). Human antibodies produced by human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0120] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0121] 4. Library-derived variants of anti-CD36 antibodies In at least one embodiment, improved variants of anti-CD36 antibodies can be isolated by screening combinatorial libraries for antibodies with desired improved functional characteristics, such as binding affinity, cross-reactivity, etc. For example, various methods are known in the art for generating phage display libraries and screening such libraries for variant antibodies possessing improved binding characteristics. Other methods for producing such library-derived antibodies are described, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001); McCafferty et al., Nature 348: 552-554 (1990); Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, m Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 1 19-132 (2004) can be found in.

[0122] 5. Multispecific Antibodies and Antibody Fusions In at least one embodiment, it is contemplated that the anti-CD36 antibody of the present disclosure may be a multispecific antibody, e.g., a bispecific antibody. In some embodiments, a multispecific antibody has at least two different binding sites, each with binding specificity for a different antigen, at least one of which specifically binds to CD36. In at least one embodiment, it is contemplated that the multispecific antibody is a bispecific antibody that includes a specificity for CD36 and a specificity for another antigen that mediates immune regulation, immune signaling, and / or is expressed on cancer or tumor cells. For example, the other specificity may be for an immune checkpoint molecule, such as PD1, LAG3, CTLA-4, A2AR, TIM-3, BTLA, CD276, CD328, VTCN1, IDO, KIR, NOX2, VISTA, OX40, CD27, CD28, CD40, CD122, CD137, GITR, or ICOS.

[0123] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see, e.g., Milstein and Cuello, Nature 305: 537 (1983), WO 93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)). "Knobs-in-holes" engineering can also be used to generate bispecific antibodies useful in the anti-CD36 antibodies of the present disclosure. The technique of knobs-in-holes engineering is known in the art and is described, for example, in U.S. Pat. No. 5,731,168.

[0124] Multispecific antibodies can also be generated by: engineering the "electrostatic steering" effect, which favors the formation of Fc-heterodimeric antibody molecules rather than homodimers (WO 2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980; and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol, 148(5): 1547-1553 (1992)); using "diabody" technology to generate bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); using single-chain Fv (scFv) dimers (see, e.g., Gruber et al. J. Immunol, 1999, 229: 81 (1993)). 152:5368 (1994); or trispecific antibodies (see, e.g., Tutt et al., J. Immunol. 147:60 (1991)).

[0125] In at least one embodiment, the anti-CD36 antibody provided herein can comprise an antibody fusion with a protein. Methods for preparing and using antibody fusions or fusion proteins are well known in the art. Typically, the antibody is covalently conjugated (or fused) to the protein, typically via a linker polypeptide. Conjugation can occur via the light chain (LC) or heavy chain (HC) terminus of the antibody. Antibody fusions can also be prepared using antibody fragments. In an exemplary embodiment of an antibody fusion contemplated by the present disclosure, the fusion comprises a full-length anti-CD36 antibody fused via a linker at the light chain terminus or heavy chain terminus to a T cell activating or immune stimulating cytokine. The cytokines can include, but are not limited to, IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, or IFN-α. Such anti-CD36 antibody fusions can block activities mediated by CD36 signaling and provide immune stimulating cytokine effects. The ability of such anti-CD36 antibody fusions to produce an immunostimulatory cytokine effect can be assayed in vitro using known cell-based assays associated with cytokines.

[0126] 6. Anti-CD36 Antibody Variants In some embodiments, variants of the anti-CD36 antibodies of the present disclosure are contemplated to have improved characteristics, such as the binding affinity and / or other biological properties of the antibody. Variants can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct retains the desired characteristics of CD36 antigen binding.

[0127] A. Substitution, Insertion, and Deletion Variants In some embodiments, anti-CD36 antibody variants are provided that have one or more amino acid substitutions in addition to those described herein. Sites of mutagenesis may include CDRs, HVRs, and FRs. Conventional "conservative" amino acid substitutions and / or substitutions based on common side chain classes or properties are well known in the art and can be used in the disclosed embodiments. The disclosure also contemplates variants based on non-conservative amino acid substitutions, in which a member of one amino acid side chain class is replaced with an amino acid from another class. Amino acid side chains are typically grouped according to the following classes or common properties: (1) hydrophobic: Met, Ala, Val, Leu, Ile, norleucine; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. Techniques for making amino acid substitutions into antibodies and subsequent screening for the desired functionality, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC, are well known in the art.

[0128] Amino acid substitution variants may also include variants with one or more substitutions in the hypervariable regions of the parent antibody. Generally, the resulting variants selected for further study will have certain altered biological properties compared to the parent antibody (e.g., improved affinity, reduced immunogenicity) and / or will retain certain biological properties of the parent antibody. An exemplary substitution variant is an affinity matured antibody, which can be conveniently generated using phage display-based affinity maturation techniques. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a certain biological activity (e.g., binding affinity).

[0129] One useful method for identifying residues or regions of an antibody that may be targeted for mutagenesis is "alanine scanning mutagenesis" (see, e.g., Cunningham and Wells Science, 244:1081-1085 (1989)). In this method, residues or groups among the target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antigen with the antibody is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or additionally, a crystal structure of the antigen-antibody complex can be determined to identify contact points between the antibody and antigen. Such contact and adjacent residues can be targeted or eliminated as candidates for substitution. The variants can be screened to determine whether they contain the desired properties.

[0130] Amino acid sequence insertions which can be prepared include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion includes an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule can include the fusion of the N- or C-terminus of the antibody to an enzyme or a polypeptide which extends the serum half-life of the antibody.

[0131] Other residue substitutions can be made in HVRs to improve antibody affinity. Such changes can be made at "hot spots," i.e., residues encoded by codons that undergo high frequency mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), in which case the resulting variant V H Or V Lare tested for binding affinity. In one embodiment, affinity maturation can be performed by constructing and reselecting from a secondary library (see, e.g., Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, (2001).)). Another method of introducing diversity involves an HVR-directed approach in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. HVR-H3 and HVR-L3 in particular are often targeted. In general, substitutions, insertions, or deletions can be made in one or more HVRs so long as such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity can be made in HVRs. Such changes may be outside the "hot spots" of the HVR.

[0132] In some embodiments, it is contemplated that the anti-CD36 antibodies described herein may be substituted with cysteine ​​residues at certain non-HVR positions to create reactive thiol groups. Such engineered "ThioMAbs" may be used to conjugate the antibodies, for example, to drug moieties or linker-drug moieties, thereby creating immunoconjugates, as described elsewhere herein. Cysteine ​​engineered antibodies may be generated, for example, as described in U.S. Pat. No. 7,521,541. In some embodiments, any one or more of the following antibody residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region.

[0133] B. Glycosylation variants In some embodiments, the anti-CD36 antibodies of the present disclosure are altered to increase or decrease the extent to which the antibody is glycosylated. Glycosylation sites can be added or deleted from the antibody by altering the amino acid sequence to create or remove one or more glycosylation sites. In embodiments in which the antibody comprises an Fc region, the carbohydrate attached to the Fc region can be altered. Typically, natural antibodies produced by mammalian cells contain biantennary oligosaccharides that are branches attached by N-linkage to an asparagine at about position 297 ("N297") in the CH2 domain of the Fc region (see, e.g., Wright et al. TIBTECH 15:26-32 (1997)). The oligosaccharides can include a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, sialic acid, as well as fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modification of the oligosaccharides in the Fc region of an antibody can create variants with certain improved properties.

[0134] In some embodiments, the anti-CD36 antibodies of the present disclosure may be variants that contain carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies may be about 1% to about 80%, about 1% to about 65%, about 5% to about 65%, or about 20% to about 40%. The amount of fucose can be determined by calculating the average amount of fucose in the glycan attached to residue N297 relative to the sum of all glycostructures (e.g., complex, hybrid, and high mannose structures) attached to N297 as measured by MALDI-TOF mass spectrometry (see, e.g., WO 2008 / 077546).

[0135] In some embodiments, fucosylation variants can provide improved ADCC function of the variant antibody, see, e.g., U.S. Patent Application Publication Nos. 2003 / 0157108 or 2004 / 0093621. Examples of "defucosylated" or "fucose-deficient" antibodies and related methods for their preparation are disclosed, for example, in: US2003 / 0157108; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO2000 / 61739; WO2001 / 29246; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Useful cell lines for producing defucosylated antibodies include Led 3 CHO cells, which are deficient in protein fucosylation (see, e.g., Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US2003 / 0157108, and WO2004 / 056312), and knockout cell lines such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0136] C. Fc region variants In some embodiments, the anti-CD36 antibodies of the present disclosure can comprise one or more amino acid modifications in the Fc region (i.e., Fc region variants). Fc region variants can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that comprises an amino acid substitution at one or more amino acid residues. A wide range of Fc region variants known in the art that are useful in the anti-CD36 antibodies of the present disclosure are described below.

[0137] In some embodiments, the anti-CD36 antibody is an Fc region variant with altered effector function. In some embodiments, an antibody with altered effector function retains some (but not all), reduced, or no effector function of the parent antibody (e.g., effector-less). Effector-less Fc region variants are more desirable for certain applications where effector function (e.g., ADCC) is unnecessary or deleterious and / or the in vivo half-life of the antibody is important. Fc region variant antibodies with reduced or no effector function may result from amino acid substitutions at one or more of the following Fc region positions: 238, 265, 269, 270, 297, 327, and 329. (See, e.g., U.S. Patent No. 6,737,056). Such Fc region variants may include amino acid substitutions at two or more of the following positions: 265, 269, 270, 297, and 327. Such an Fc region variant can also include a substitution of both residues 265 and 297 to alanine (see, eg, US Pat. No. 7,332,581).

[0138] Some Fc region variants can provide improved or attenuated binding to FcR (see, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)). Some Fc region variants that can provide improved ADCC include, for example, one or more amino acid substitutions at positions 298, 333, and / or 334 (based on EU numbering) of the Fc region. Fc region variants with altered (i.e., improved or attenuated) Clq binding and / or complement dependent cytotoxicity (CDC), as described, for example, in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

[0139] Some Fc region variants can provide extended half-life and improved binding to the neonatal Fc receptor (FcRn) are disclosed, for example, in US2005 / 0014934A1 (Hinton et al.). Such Fc region variants contain amino acid substitutions at one or more of the following positions: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, and 434. Other Fc region variants with extended half-lives include the set of YTE mutations at positions 252, 254, and 256 (i.e., M252Y / S254T / T256E), as described, for example, in US 7658921B2 (Dall'Acqua et al.). Further examples of Fc region variants can be found, for example, in U.S. Patent Nos. 5,648,260 and 5,624,821; and WO 94 / 29351.

[0140] Generally, in vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity in Fc region variants. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus potentially lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom et al., Proc. Nat'l Acad. Sci. USA 82: 1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, e.g., Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assay methods can be used (see, e.g., ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA); and CytoTox96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Clq binding assays can also be performed to confirm that the antibody is unable to bind Clq and thus lacks CDC activity. See, for example, the Clq and C3c binding ELISAs of WO2006 / 029879 and WO2005 / 100402.To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, SW 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can be performed using methods known in the art (see, e.g., Petkova et al., Intl. Immunol. 18(12): 1759-1769 (2006)).

[0141] D. Non-Protein Antibody Derivatives - Immunoconjugates In some embodiments, the anti-CD36 antibodies of the present disclosure can be further modified (i.e., derivatized) with non-proteinaceous moieties. Non-proteinaceous moieties suitable for derivatization of the antibody include, but are not limited to, for example, the following water-soluble polymers: polyethylene glycol (PEG), copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acid homopolymers or random copolymers, and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. In some embodiments, modification of the antibody can be performed using methoxy-polyethylene glycol propionaldehyde. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and when multiple polymers are attached, the polymers can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, a particular property or function of the antibody, for example, whether the antibody derivative will be used therapeutically under defined conditions.

[0142] In some embodiments, the anti-CD36 antibody of the present disclosure may also be an immunoconjugate, where the immunoconjugate comprises an anti-CD36 antibody conjugated to one or more cytotoxic drugs. Suitable cytotoxic drugs contemplated by the present disclosure include chemotherapeutic agents, drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioisotopes. In some embodiments, the immunoconjugate is an antibody drug conjugate (ADC) in which the anti-CD36 antibody is conjugated to one or more drugs as described herein. In some embodiments, the immunoconjugate of the present disclosure comprises an anti-CD36 antibody as described herein conjugated to a drug or therapeutic agent for the treatment of a CD36-mediated disease or condition.

[0143] In some embodiments, the anti-CD36 antibodies described herein may be conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein, Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0144] In some embodiments, the immunoconjugates of the present disclosure comprise an anti-CD36 antibody described herein conjugated to a radioisotope (i.e., a radioconjugate). A variety of radioisotopes are available for the production of such radioconjugates. For example, 211 At, 131 I, 125 I, 90 Y, 186Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 Radioisotopes include Pb, and Lu. In some embodiments, the immunoconjugate can include a radioisotope for scintigraphic detection, or a spin label for NMR detection or MRI. Suitable radioisotopes or spin labels include, for example: 123 I, 131 I, 111 In, 13 C. 19 F, 15 N, 17 Various isotopes of O, Gd, Mn, and Fe may be included.

[0145] Immunoconjugates of anti-CD36 antibodies and cytotoxic drugs can be made using a variety of well-known bifunctional reagents and chemistries suitable for conjugation to proteins. Such reagents include, but are not limited to, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyladipimidate HQ), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis-(p-azidobenzoyl)-hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene-2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). Reagents for preparing the immunoconjugates of the present disclosure may also include the following commercially available "cross-linking" reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) (see, e.g., Pierce Biotechnology, Inc., Rockford, IL, USA).

[0146] III. Recombinant Methods and Compositions The anti-CD36 antibodies of the present disclosure can be produced using recombinant methods and materials well known in the art of antibody production. In some embodiments, the present disclosure provides an isolated polynucleotide encoding an anti-CD36 antibody of the present disclosure, or a fragment or domain of an anti-CD36 antibody. For example, the isolated polynucleotide may encode an amino acid sequence that includes the CDRs or HVRs disclosed herein, but does not encode the V domain of the antibody. L Domain and / or VH The isolated polynucleotide may encode an amino acid sequence comprising the V domain or may encode an amino acid sequence comprising the complete light and / or heavy chain of an anti-CD36 antibody. In at least one embodiment, the isolated polynucleotide may encode an amino acid sequence comprising the CDR-H1, CDR-H2, and CDR-H3 sequences or may encode an amino acid sequence comprising the CDR-L1, CDR-L2, and CDR-L3 sequences of any of the anti-CD36 antibodies disclosed herein. Similarly, the isolated polynucleotide may encode an amino acid sequence comprising the V domain or the complete light and / or heavy chain of an anti-CD36 antibody. L Domain or V H It may encode an amino acid sequence comprising a domain, or it may encode an amino acid sequence comprising a complete heavy chain (HC) or light chain (LC).

[0147] In some embodiments, the present disclosure also provides a vector (e.g., an expression vector) comprising a polynucleotide sequence (described above) encoding an anti-CD36 antibody, or an anti-CD36 antibody fragment, or domain of the present disclosure. Such vector constructs comprising polynucleotides for recombinant production of antibodies are well known in the art. Additionally, in some embodiments, a host cell is provided comprising a polynucleotide or vector having a sequence encoding an anti-CD36 antibody, or an anti-CD36 antibody fragment, or domain of the present disclosure. In at least one embodiment, the host cell comprises a polynucleotide or vector having a sequence encoding an anti-CD36 antibody, or an anti-CD36 antibody fragment, or domain of the present disclosure. L Amino acid sequence comprising domain and / or V of anti-CD36 antibody H In another embodiment, the host cell is a cell transformed with a vector comprising a polynucleotide sequence encoding an amino acid sequence comprising an antibody V domain. L A first vector comprising a polynucleotide sequence encoding an amino acid sequence comprising the V H The vector is transformed with a second vector, which comprises a nucleic acid encoding an amino acid sequence comprising:

[0148] In some embodiments of the recombinant methods, the host cells used are eukaryotic cells, such as Chinese Hamster Ovary (CHO) cells, or lymphoid cells (e.g., Y0, NS0, Sp20). In one embodiment, a method of making an anti-CD36 antibody is provided, the method comprising culturing a host cell comprising nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0149] Briefly, recombinant production of anti-CD36 antibodies is carried out by isolating nucleic acid encoding the antibody (e.g., as described herein) and inserting the nucleic acid into one or more vectors for further cloning and / or expression in host cells. Such nucleic acid is easily isolated and sequenced using conventional procedures well known in the art (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the desired antibody). Suitable host cells and culture methods for cloning or expressing vectors encoding the antibody are well known in the art and include prokaryotic or eukaryotic cells. Typically, after expression, the antibody is isolated from the cell paste in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast, including fungal and yeast strains whose glycosylation pathways have been "humanized" resulting in the production of antibodies with partially or fully human glycosylation patterns, are suitable cloning or expression hosts for antibody-encoding vectors (see, e.g., Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006)).

[0150] Host cells suitable for expressing the glycosylated anti-CD36 antibody of the present disclosure may also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts (see, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, and 7,125,978).

[0151] Examples of mammalian host cell lines useful for producing the anti-CD36 antibodies of the disclosure include: Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (see, e.g., Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); myeloma cell lines such as Y0, NS0, Sp2 / 0; monkey kidney CV1 lines transformed with SV40 (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TR1 cells (see, e.g., Mather et al., Annals N Y. Acad. Sci. 383:44-68 (1982) and US 6,235,498); Medical Research Council 5 (MRC 5) cells (such as those available from ATCC and also referred to as CCL-171); and Foreskin 4 (FS-4) cells (see, e.g., Vilcek et al. Ann. NY Acad. Sci. 284:703-710 (1977); Gardner & Vilcek. J. Gen. Virol. 44:161-168 (1979), and Pang et al. Proc. Natl. Acad. Sci. USA 77:5341-5345 (1980). For a general review of useful mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (ed. BKC Lo, Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0152] IV. PHARMACEUTICAL COMPOSITIONS AND FORMULATIONS OF ANTI-CD36 ANTIBODIES The present disclosure also provides pharmaceutical compositions and pharmaceutical formulations comprising anti-CD36 antibodies. In some embodiments, the present disclosure provides pharmaceutical formulations comprising the anti-CD36 antibodies described herein and a pharma- ceutically acceptable carrier. In some embodiments, the anti-CD36 antibody is the sole active agent of the pharmaceutical composition. Such pharmaceutical formulations can be prepared by mixing an anti-CD36 antibody having a desired degree of purity with one or more pharma- ceutically acceptable carriers. Typically, such antibody formulations can be prepared as aqueous solutions (see, e.g., U.S. Pat. No. 6,171,586 and WO2006 / 044908) or as lyophilized formulations (see, e.g., U.S. Pat. No. 6,267,958).

[0153] Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed. A wide range of such pharma-ceutically acceptable carriers is well known in the art (see, for example, Remington's Pharmaceutical Sciences 16th ed., Osol, A. Ed. (1980)). Exemplary pharma-ceutically acceptable carriers useful in the formulations of the present disclosure may include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin, immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, for example, glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0154] Pharmaceutically acceptable carriers useful in the formulations of the present disclosure can also include interstitial drug dispersing agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs) (see, e.g., U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968), such as human soluble PH-20 hyaluronidase glycoproteins (e.g., rHuPH20 or HYLENEX®, Baxter International, Inc.).

[0155] It is also contemplated that the formulations disclosed herein can optionally contain active ingredients in addition to anti-CD36 for the particular indication being treated in the subject to whom the formulation is administered. Preferably, the additional optional active ingredient has an activity complementary to the active ingredient of anti-CD36 antibody activity, and the activities do not adversely affect each other.

[0156] In some embodiments, the pharmaceutical composition comprises an anti-CD36 antibody and a further active agent for the treatment of cancer, such as an immune checkpoint inhibitor. Checkpoint inhibitors useful in such embodiments include secondary antibodies that comprise specificity for an antigen, including, but not limited to, immune checkpoint molecules. In some embodiments, the secondary antibody comprises specificity for an immune checkpoint molecule selected from PD1, PD-L1, LAG3, CTLA-4, A2AR, TIM-3, BTLA, CD276, CD328, VTCN1, IDO, KIR, NOX2, VISTA, OX40, CD27, CD28, CD40, CD122, CD137, GITR, ICOS. In at least one embodiment, the pharmaceutical composition comprises an anti-CD36 antibody and a further active agent, where the further active agent is an antibody that comprises specificity for the immune checkpoint molecule PD1. Exemplary antibodies with specificity for PD1 that are useful in embodiments of the pharmaceutical compositions disclosed herein include, but are not limited to, dostarimab, pembrolizumab, nivolumab, and pidilizumab.

[0157] In some embodiments, the present disclosure provides a pharmaceutical composition or formulation for use in therapy, where it is also contemplated that the composition further comprises a T cell activating cytokine or an immunostimulatory cytokine. Such cytokines are well known in the field of immunotherapy and include, but are not limited to, IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, and IFN-α. In at least one embodiment, the immunostimulatory cytokine can be provided in the composition as a fusion of an anti-CD36 antibody.

[0158] For example, the active ingredient can be encapsulated in microcapsules prepared by coacervation techniques or by interfacial polymerization, for example, in hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th Edition, edited by Osol, A. (1980).

[0159] In some embodiments, the formulation may be a sustained-release preparation of the antibody and / or other active ingredient. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0160] Typically, the formulations of the present disclosure that are administered to a subject are sterile. Sterile formulations can be readily prepared using well-known techniques, for example, by filtration through sterile filtration membranes.

[0161] V. Methods of Use and Treatment It is contemplated that any of the compositions or formulations comprising the anti-CD36 antibodies of the present disclosure may be used in any method or use, such as in a therapeutic method, to utilize their ability to specifically bind to CD36, thereby inhibiting, reducing, and / or completely blocking the function of CD36 as a cell surface protein involved in immune regulation or signal transduction, particularly the function of CD36 in regulating the uptake of lipoproteins, fatty acids, and other ligands involved in the survival and maintenance or survival (or in some cases, death) of tumor cells, tumor-associated macrophages (TAMs), myeloid-derived immunosuppressive cells (MDSCs), regulatory T cells, and CD8 T cells. For example, regulatory T cells are a major cellular component of the tumor microenvironment (TME) and contribute significantly to tumor growth and progression, while CD8 T cells help control tumor growth by killing tumor cells. Inhibition of CD36 binding results in the depletion of regulatory T cells, while at the same time improving the survival and function of CD8 T cells, which can induce improved anti-tumor T cell responses.

[0162] There are various diseases, disorders, and conditions that can potentially be treated by inhibiting, reducing, and / or completely blocking the immunomodulatory and / or immune signaling activity of CD36, particularly the effect of CD36 on TAMs. Diseases, disorders, and conditions include, but are not limited to, cancer, including colon cancer, pancreatic cancer, ovarian cancer, HCC, renal cancer, breast cancer, lung cancer, gastric cancer, melanoma, head and neck cancer, or oral cancer. It is contemplated that any of the compositions or formulations comprising the anti-CD36 antibody of the present disclosure can be used for the method or use for the treatment of any of the cancers listed above. In some embodiments, the cancer is selected from colon cancer, pancreatic cancer, ovarian cancer, HCC, renal cancer, breast cancer, lung cancer, gastric cancer, melanoma, head and neck cancer, or oral cancer. In some embodiments, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of an anti-CD36 antibody of the present disclosure, or administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an anti-CD36 antibody of the present disclosure and a pharma- ceutical acceptable carrier.

[0163] As disclosed herein, including in the Examples below, the anti-CD36 antibodies of the present disclosure have the ability to reduce, inhibit, and / or block binding of ligands to CD36, thereby altering immune signaling pathways mediated by CD36. Thus, in some embodiments, the present disclosure provides a method of treating a CD36-mediated disease or condition in a subject, comprising administering to the subject a therapeutically effective amount of an anti-CD36 antibody of the present disclosure, or administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an anti-CD36 antibody of the present disclosure and a pharmaceutically acceptable carrier. Similarly, in some embodiments, the present disclosure provides a method of treating a disease mediated by binding to CD36 expressed on cells in a subject, comprising administering to a subject a therapeutically effective amount of an anti-CD36 antibody of the present disclosure, or administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an anti-CD36 antibody of the present disclosure and a pharmaceutically acceptable carrier.

[0164] Administering an anti-CD36 antibody, composition, or pharmaceutical formulation according to the present treatment method provides an antibody-induced therapeutic effect that protects the subject from and / or treats the progression of a CD36-mediated disease in the subject. In some embodiments, the present treatment method can further include administration of one or more additional therapeutic agents or treatment methods known to those skilled in the art for preventing and / or treating a CD36-mediated disease or condition. Such methods that include administration of one or more additional agents can include combined administration (where two or more therapeutic agents are included in the same formulation or separate formulations) and separate administration, where administration of the antibody composition or formulation can occur before, simultaneously with, and / or after administration of the additional therapeutic agent.

[0165] In some embodiments of the treatment methods of the present disclosure, an anti-CD36 antibody or a pharmaceutical formulation comprising an anti-CD36 antibody is administered to a subject by any mode of administration that delivers the agent systemically or to a desired target tissue. Systemic administration generally refers to any mode of administration of an antibody to a subject at a site other than directly to the desired target site, tissue, or organ such that the antibody or a formulation thereof enters the subject's circulatory system and is therefore susceptible to metabolism and other similar processes.

[0166] Thus, the modes of administration useful in the treatment methods of the present disclosure may include, but are not limited to, injection, infusion, instillation, and inhalation. Administration by injection may include intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, and intrasternal injections and infusions.

[0167] In some embodiments, the pharmaceutical formulation of the anti-CD36 antibody is formulated to protect the antibody from inactivation in the intestine. Thus, the method of treatment can include oral administration of the formulation.

[0168] In some embodiments, the use of a composition or formulation comprising an anti-CD36 antibody of the present disclosure as a medicament is also provided. Furthermore, in some embodiments, the present disclosure also provides the use of a composition or formulation comprising an anti-CD36 antibody in the manufacture or preparation of a medicament, particularly a medicament for treating, preventing or inhibiting a CD36-mediated disease. In further embodiments, the medicament is for use in a method for treating, preventing or inhibiting a CD36-mediated disease, comprising administering an effective amount of the medicament to an individual having a CD36-mediated disease. In certain embodiments, the medicament further comprises an effective amount of at least one additional therapeutic agent or treatment.

[0169] In at least one embodiment, it is contemplated that additional therapeutic agents or treatments that may be used in such medicaments comprising the anti-CD36 antibodies of the present disclosure may include therapeutic antibodies comprising specificity for immune checkpoint molecules, such as, but not limited to, PD1, PD-L1, LAG3, CTLA-4, A2AR, TIM-3, BTLA, CD276, CD328, VTCN1, IDO, KIR, NOX2, VISTA, OX40, CD27, CD28, CD40, CD122, CD137, GITR, ICOS. Exemplary antibodies comprising specificity for immune checkpoint molecules include, but are not limited to, anti-PD1 antibodies selected from dostarimab, pembrolizumab, nivolumab, and pidilizumab.

[0170] In a further embodiment, the medicament is for use in treating, inhibiting or preventing a CD36-mediated disease, such as cancer, in a subject, comprising administering to the subject an effective amount of the medicament to treat, inhibit or prevent the CD36-mediated disease.

[0171] The appropriate dosage of the anti-CD36 antibody contained in the compositions and formulations of the present disclosure (when used alone or in combination with one or more other additional therapeutic agents) will depend on the particular disease or condition being treated, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous treatments administered to the patient, the patient's medical history and response to the antibody, and the discretion of the attending physician. The anti-CD36 antibody contained in the compositions and formulations described herein may be suitably administered to the patient at one time or over a series of treatments. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations over various time points, bolus administration, and pulse infusion.

[0172] Depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg of anti-CD36 antibody in the formulation of the present disclosure is an initial candidate dosage for administration to a human subject, whether by one or more separate administrations or by continuous infusion. In general, the administration dosage of the antibody will range from about 0.05 mg / kg to about 10 mg / kg. In some embodiments, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) can be administered to the patient.

[0173] Dose administration can be maintained for several days or more depending on the condition of the subject, and can be continued until CD36-mediated disease is sufficiently treated, for example, as determined by methods known in the art. In some embodiments, an initial higher loading dose can be administered, followed by one or more lower doses. However, other dosing regimens may be useful. The progress of therapeutic effect of dose administration can be monitored by conventional techniques and assays.

[0174] Thus, in some embodiments of the disclosed methods, administration of the anti-CD36 antibody comprises a daily dosage of about 1 mg / kg to about 100 mg / kg, hi some embodiments, the dosage of the anti-CD36 antibody comprises a daily dosage of at least about 1 mg / kg, at least about 5 mg / kg, at least about 10 mg / kg, at least about 20 mg / kg, or at least about 30 mg / kg. EXAMPLES

[0175] Various features and embodiments of the present disclosure are illustrated in the following representative examples, which are intended to be illustrative and not limiting. Those skilled in the art will readily appreciate that the specific examples are merely illustrative of the invention as more fully described in the claims that follow. It should be understood that all embodiments and features described in this application are compatible and combinable with all embodiments contained therein.

[0176] Example 1 Generation of anti-CD36 antibodies and CD36 binding analysis This example illustrates the use of phage display antibody library technology to generate exemplary anti-CD36 antibodies of the disclosure that specifically bind to human and mouse CD36.

[0177] A. Selection of anti-CD36 scFv binders from a phage-displayed antibody library The panning procedure is briefly described below. First, human CD36.ECD antigen (5 μg per well, Sino Biological Co., Ltd.) in PBS buffer (pH 7.4) was coated onto a 96-well plate (NUNC Maxisorb Immunoplate) overnight at 4° C. and blocked with 5% skim milk in PBST [0.1% (v / v) Tween 20] for 1 h. After blocking, 100 μL of enriched phage library (1013 cfu / mL in PBS buffer) was mixed with 100 μL of blocking buffer, then added to each well under gentle shaking for 1 h. The plate was washed 12 times with PBST and 3 times with PBS. Bound phages were eluted with 100 μL of 0.1 M HCl / glycine (pH 2.2) per well, and then immediately neutralized with 20 μL of 1 M Tris base buffer (pH 9.0). The eluted phages were mixed with 1 mL of E. coli ER2738 (A600nm=0.6) for 30 min at 37°C; non-infected bacteria were eliminated by adding ampicillin. After 30 min of ampicillin treatment, the bacterial culture was infected with 100 μL of M13K07 helper phages (approximately 1011 CFU total) for 1 h at 37°C, and then added to 50 mL of 2xYT medium containing 50 μg / mL kanamycin and 100 μg / mL ampicillin overnight at 37°C with vigorous shaking. The rescued phage library was precipitated with 20% polyethylene glycol / NaCl and resuspended in PBS. The concentrated phage solution was used for the next round of panning.

[0178] After 3-4 rounds of selection-amplification cycles, single colonies were randomly selected into a deep 96-well culture plate (Plate A; secreted scFv); each well contained 950 μL of 2YT (100 μg / mL ampicillin). After 3 h of incubation at 37 °C with shaking, 50 μL of bacterial culture was transferred to the corresponding well of a fresh deep 96-well plate (Plate B; phage morphology); each well contained 0.8 mL of 2YT with 100 μg / mL ampicillin. At the same time, 50 μL of M13K07 (approximately 5 × 1010 CFU total) was added to each well of Plate B. After 1 h of incubation, 100 μL of 2YT containing IPTG (10 mM) was added to each well of Plate A; 100 μL of 2YT containing kanamycin (500 μg / mL) was added to each well of Plate B. After overnight incubation at 37 °C with vigorous shaking, the cultures were centrifuged at 3000 g for 10 min at 4 °C. Plate B was kept for further sequencing determination. For the secreted scFv culture plate (plate A), 100 μL of culture medium and 100 μL of 5% PBST milk were added to corresponding wells of three 96-well plates pre-coated with protein L (0.1 μg / well), human CD36 (0.5-1 μg / well) and bovine serum albumin (BSA) (2 μg / well), respectively, and blocked with 5% PBST milk. After 1 h incubation at room temperature, the plates were washed three times with PBST. 100 μL of Protein A-HRP (Thermo Scientific) was added to each well of Protein L-coated immunoplates; 100 μL of anti-E-tag-HRP (ICL Inc.) was added to each well of human CD36 antigen-coated and BSA-coated plates. After 1 h of incubation, plates were washed three times with PBST buffer and twice with PBS, developed with 3,3',5,5'-tetramethylbenzidine peroxidase substrate (Kirkegaard & Perry Laboratories) for 3 min, quenched with 1.0 M HCl, and read spectrophotometrically at 450 nm.

[0179] Positive clones were selected by the following criteria: ELISA OD450>0.2 for human CD36 antigen-coated wells (positive for antigen binding); OD450<0.05 for BSA-coated wells (negative for non-specific binding); OD450>0.5 for Protein L-coated wells (soluble scFv that bind both Protein L and Protein A to ensure proper folding in solution) and then subjected to DNA sequencing. The polynucleotide sequence of the scFv of the exemplary anti-CD36 antibody 12P109 (SEQ ID NO: 1) obtained from phage display panning is provided in Table 2 and the attached sequence listing. Further panning of a phage display library composed of "shuffled" LC sequences from different anti-CD36 antibodies and the HC sequence of 12P109 resulted in the scFv of the exemplary anti-CD36 antibody A8A (SEQ ID NO: 10), which is also listed in Table 2 and the attached sequence listing. The A8A antibody V was further subjected to standard PCR-based mutagenesis. L Domain (SEQ ID NO: 11) on the CDRs to yield an exemplary anti-CD36 antibody VL domain of A8A-N52T (SEQ ID NO: 14), which is also listed in Table 2 and the attached sequence listing.

[0180] B. Generation of anti-CD36 antibodies in full-length IgG format Reformatting and cloning of scFvs: The CD36 binding determinants of scFvs selected from phage display panning were amplified by cloning the V and V fragments of the scFvs using the restriction sites MluI / NheI and BsiWI / DraIII, respectively. H Domain and V L The domains were reformatted into a full-length IgG antibody by cloning into a human IgG1-N297A heavy chain vector and a human kappa light chain vector. H and V L The domain was amplified using the following forward and reverse oligonucleotide primer pairs: (1) PhageLib_VL_Fw: 5'-AATCACgATgTgATATTCAAATgACCCAgAgCCCgAgC-3' (sequence number 64), (2) PhageLib_VL_Rv: 5'-AATCgTACgTTTgATTTCCACTTTggTgCCTTg-3' (sequence number 65), (3) PhageLib_VH_Fw: 5'-AATACgCgTgTCCTgTCCgAAgTgCAgCTggTggAATCg-3' (sequence number 66), and 4) PhageLib_VH_Rv: 5'-AATgCTAgCCgAgCTCACggTAACAAg-3' (sequence number 67).

[0181] Full-length antibody expression and purification: The vector cloned with the reformatted anti-CD36 antibody gene was transiently expressed in ExpiCHO-S cells (Thermo Scientific). During the exponential growth phase, ExpiCHO-S cells were cultured at a final concentration of 6 × 10 with ExpiCHO Expression Medium. 6 The cells were diluted to 1000 cells / mL. ExpiFectamine CHO / plasmid DNA complexes were prepared using chilled reagents according to the manual, incubated at room temperature for 1-5 min, and then slowly added to the cells. One day after transfection, the cultures were supplemented with ExpiFectamine CHO Enhancer and incubated for another 7 days. The transfected cell supernatant was centrifuged and subsequently filtered through a 0.22 μm filter. Antibodies were purified from the transfected cell supernatant using Protein A beads (Cytiva, 17127903). The antibody-loaded column was washed with 50 column volumes of PBS and then eluted directly with 10 bead volumes of 0.1 M glycine (pH 3) into 1 / 10 volume of 1 M Tris buffer (pH 9.0). The eluate was buffer exchanged and concentrated with PBS pH 7.4 containing 0.1 M arginine. The quality of the purified anti-CD36 antibody was assessed using SDS-PAGE in the presence or absence of reducing agents.

[0182] Results: Examination of the SDS-PAGE images showed that expression and purification resulted in purified full-length anti-CD36 antibody.

[0183] C. Anti-CD36 Antibody Optimization To further improve the druggability of anti-CD36 antibody A8A, two phage display libraries consisting of mutations N52, H91, and L94 were used for panning. The scFv clone 117 selected from phage display panning was cloned into the V and VF fragments using the restriction sites MluI / NheI and BsiWI / DraIII, respectively. H Domain and V L The domains were reformatted into a full-length IgG antibody by cloning into a human IgG1-N297A heavy chain vector and a human kappa light chain vector. H and V L The domain was amplified using the following forward and reverse oligonucleotide primer pairs: (1) PhageLib_VL_Fw: 5'-AATCACgATgTgATATTCAAATgACCCAgAgCCCgAgC-3' (sequence number 64), (2) PhageLib_VL_Rv: 5'-AATCgTACgTTTgATTTCCACTTTggTgCCTTg-3' (sequence number 65), (3) PhageLib_VH_Fw: 5'-AATACgCgTgTCCTgTCCgAAgTgCAgCTggTggAATCg-3' (sequence number 66), and (4) PhageLib_VH_Rv: 5'-AATgCTAgCCgAgCTCACggTAACAAg-3' (sequence number 67).

[0184] The 117 antibody shares the same CDR-H1, H2, H3, and CDR-L1 sequences as A8A, and has an N52T mutation in CDR-L2 and an L94A mutation in CDR-L3. The Fab fragment of the 117 antibody was also constructed into a human IgG4-S228P / F234A / L235A heavy chain vector using the restriction sites NheI and BamHI. The CDRs of the VH and VL domains of the 117 antibody were further subjected to PCR-based mutagenesis to generate a series of variants as summarized in Table 3 below.

[0185] [Table 4A]

[0186] [Table 4B]

[0187] The 117 antibody and variants were expressed as described above and purified as full-length antibodies. Examination of the SDS-PAGE images showed that expression and purification resulted in purified full-length anti-CD36 antibodies. The amino acid sequences of the CDR, VH, VL, HC, and LC regions of the 117 antibody and each of the exemplary variants 117_30AA, 117_30DA, 117_57D, 117_57E, and 117_DA57E are also listed in Table 2 and the attached sequence listing.

[0188] D. ELISA for CD36-specific binding by anti-CD36 antibody Materials and Methods for ELISA: Recombinant His-tagged human CD36.ECD protein or His-tagged mouse CD36.ECD protein (both from Sino Biological) was immobilized on 96-well microplates at a concentration of 1 μg / ml in coating solution (SeraCare) overnight at 4°C. The wells were washed with washing solution (0.05% Tween 20 in PBS) and blocked with 1% BSA in PBS. Serial dilutions of anti-CD36 antibody were added to the wells. After 1 h incubation at 37°C, the wells were washed with washing solution. Peroxidase-conjugated goat anti-human kappa light chain antibody (Sigma) was applied to each well for 1 h incubation at 37°C. After washing, the wells were developed with TMB substrate for 5-10 min at room temperature and then stopped with 1N HCl. The absorbance was then measured at 450 nm and 650 nm. EC50 values ​​were calculated using GraphPad Prism7.

[0189] Results: ELISA data plots showing binding to either human CD36.ECD or mouse CD36.ECD are presented in Figures 1A and 1B for full length IgG anti-CD36 antibody 12P109 and additional positive clones 12P102, 12P103, 12P104, 12P105, 12P106, 12P107, 12P110, and 12P212, respectively. Binding curves for labeled 12P109, 12P103, and 12P212 antibodies showed the highest affinity for human CD36 (hCD36, see Figure 1A). 12P109 also showed high binding affinity for mouse CD36 (mCD36, see Figure 1B). EC calculated from ELISA data 50 The values ​​are shown in Table 4 below.

[0190] [Table 5]

[0191] ELISA data plots showing binding of anti-CD36 antibodies to recombinant human CD36.ECD or mouse CD36.ECD coated onto ELISA plates are shown in Figures 1C and 1D, respectively, using the anti-CD36 antibodies 12P109 and A8A in full-length human IgG1 format, and the commercially available mouse IgA antibody D2712 (clone CRF D-2712, BD Biosciences, USA), which specifically binds to CD36. The EC values ​​derived from the curves in Figures 1C and 1D were 50 The values ​​are shown in Table 5 below.

[0192] [Table 6]

[0193] ELISA data plots showing binding to human CD36.ECD are presented in Figures 2A, 2B, 2C, 2D, 2E, and 2F for full-length IgG anti-CD36 antibodies, 12P109, 117, and various 117 variants listed in Table 3. Competitor anti-CD36 antibody ONA was derived from clone "ONA-0-v1" disclosed in WO2021176424A1 in the hIgG4-S228P-FALA format. EC50 values ​​calculated from the ELISA data in Figures 2A-F are shown below in Table 7.

[0194] [Table 7]

[0195] F. SEC-UPLC analysis The increased retention time (RT) of antibodies determined by SEC-UPLC analysis may be related to non-specific hydrophobic interactions that pose a risk for the development of antibodies for therapeutic use. Therefore, anti-CD36 antibodies, 12P109, A8A, 117, and 117 variants, as well as an IgG standard ("STD", BEH200 SEC Protein Standard Mix) and a control monoclonal antibody ("ctlAb") were analyzed by SEC-UPLC for increased RT, indicative of the formation of protein aggregates.

[0196] Materials and Methods: After Protein A purification, anti-CD36 antibodies 12, P109, A8A, 117, and 117 variants were analyzed for protein aggregates and retention time by standard size exclusion UPLC (SEC-UPLC). 3ug of antibody was applied to UPLC (Waters, ARC UPLC) and separation was performed on a gel filtration column (Waters, XBridge BEH450 SEC 4.6x150) with a mobile phase of PBS. Antibody peaks were monitored by UV absorbance at 280 and peak areas were determined using Empower software.

[0197] Results: Plots of the SEC-UPLC results are shown in Figures 3A, 3B, 3C, 3D, and 3E along with the determined RT values. The control antibody exhibited an RT value of 7.243 minutes, while both anti-CD36 antibodies 12P109 and 117 exhibited relatively increased RT values. However, the increased RT value of 117 was significantly reduced by the mutations present in variants 117_30DA, 117_31AD, 117_57D, 117_57E, 117_57EE, 117_101D, and 117_DA57E. The RT values ​​of the anti-CD36 antibodies determined in the SEC-UPLC experiments are provided in Table 8 below. In addition, some aggregates were found in 117_DA57E by UPLC analysis. The aggregates may be related to the fact that 117_DA57E is more unstable to acidic elution conditions. The R409K mutation in IgG4 has been shown to increase the interaction strength of the CH3 domain and reduce the tendency to aggregate at low pH. SEC-UPLC analysis showed that the addition of R409K to 117_DA57E reduced aggregation caused by acidic conditions.

[0198] [Table 8]

[0199] G. BLI analysis of CD36 binding kinetics Materials and Methods for BLI Assay: The kinetic rate constants ka and kd for the binding of anti-CD36 antibodies to human CD36 were measured by Biolayer Interferometry (BLI) (Octet RED96, ForteBio). The BLI assay was performed using an AHC (Anti-hIgG Fc Capture) biosensor (ForteBio) to capture each anti-CD36 antibody (5 μg / mL) to obtain a 0.5 nm shift, and then the biosensor was immersed in various concentrations (i.e., 0, 1.5625, 3.125, 6.25, 4.94, 12.5, 25, 50, and 100 nM) of recombinant His-tagged human CD36.ECD protein (Sino Biological) in running buffer containing 0.1% BSA, 0.1% Tween-20, 250 mM NaCl in PBS. Rate constants were calculated by curve-fitting analysis (1:1 Langmuir model) of the binding responses with interaction times of 2.5 min association and 5 min dissociation.

[0200] Results: The determined dissociation constants KD, and kinetic rate constants ka and kd for the binding of anti-CD36 antibodies 12P109, A8A, 117, and various 117 variants (in either IgG1 and IgG4 formats) to human CD36, as determined by separate BLI analysis experiments, are provided in Table 10 below.

[0201] [Table 9]

[0202] Example 2 Binding of cell surface-expressed CD36 by anti-CD36 antibodies This example illustrates the preparation of stable F293 cell lines overexpressing hCD36 or mCD36 on the cell surface and studies to determine the binding affinity of exemplary anti-CD36 antibodies of the disclosure to cell surface-expressed human, rhesus or mouse CD36, respectively.

[0203] Generation of stable F293 cell lines overexpressing CD36 Materials and Methods: Gene segments encoding full-length human CD36-Flag (Sino Biological), rhesus CD36-His (Sino Biological), and mouse CD36-His (Sino Biological) were subcloned into pcDNA3.4 topo vector using XbaI / HindIII. Freestyle 293-F cells (Thermo Scientific) were transfected with plasmids encoding CD36 by the polyethyleneimine (PEI) method and selected with Geneticin (Thermo Scientific) to establish a CD36 stable cell pool. Human CD36 expression was confirmed by surface staining with anti-CD36 (clone 5-271, Biolegend) or intracellular staining with anti-FLAG. Rhesus CD36 expression was confirmed by surface staining with anti-CD36 (clone 117) or intracellular staining with anti-His. Expression of mouse CD36 was confirmed by surface staining with anti-CD36 antibody D2712 (clone CRF D-2712; BD Biosciences, USA) or by intracellular staining with anti-His. To enrich for stable cell lines expressing CD36, hCD36, rhesus CD36 or mCD36 overexpressing F293 cell pools were stained with anti-CD36 (clone 5-271, Biolegend), anti-CD36 clone 117 or anti-CD36 D2712 (clone CRFD-2712, BD Biosciences), respectively. CD36 high cell populations were sorted by FACSAria IIIu (BD).

[0204] Results: Analysis of FACS data confirmed that stable F293 cells, F293 / hCD36, F293 / rhesus CD36 and F293 / mCD36, overexpressed surface CD36 to which anti-CD36 antibodies could bind.

[0205] B. Cell surface CD36 binding activity of anti-CD36 antibodies Materials and Methods: CD36-high expressing F293 cells generated as described in Part A above were incubated with serial dilutions of anti-CD36 antibody, 117, and several variants of 117 (see Table 3) for 30 min at 4° C. After washing with FACS buffer (2% FBS in PBS), cells were stained with anti-human IgG-Alexa Fluor 647 and analyzed by an Attune NxT Flow Cytometer (Thermo Scientific).

[0206] Results: Analysis of flow cytometry data, expressed as geometric mean intensity (MFI), confirmed that anti-CD36 antibodies specifically bound to cells expressing hCD36 and mCD36 on their surface.

[0207] Data plots showing surface binding to F293 cells expressing human, rhesus or mouse CD36 for the full-length IgG anti-CD36 antibody, 117, and several 117 variants are presented in Figures 4A, 4B, 4C, 4D, and 4E. EC values ​​derived from the curves in Figures 4A-4E. 50 The values ​​are shown in Table 11 below.

[0208] [Table 10]

[0209] Example 3 Blocking activity of oxidized LDL uptake by anti-CD36 antibody This example illustrates studies to determine the ability of exemplary anti-CD36 antibodies of the disclosure to block oxidized LDL uptake by CD36-expressing cells.

[0210] A. Inhibition of oxidized LDL binding and uptake in U937 cells by anti-CD36 antibodies U937 cells, which highly express CD36, were preincubated with control IgG or anti-CD36 antibody for 30 min at 4°C. To measure oxLDL binding or uptake, purified human LDL oxidized with copper(II) sulfate and labeled with “Dil” (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate), Dil-oxLDL (Cat. No. 770232-9; Kalen Biomedical) (5–10 μg / mL) was added to serum-free medium and incubated at 4°C for 2 h or at 37°C for 5 min, respectively. After washing with PBS, cells were analyzed using an Attune NxT Flow Cytometer (Thermo Scientific).

[0211] We observed that binding of anti-CD36 antibodies of the present disclosure to U937 cells resulted in a characteristic shift in surface CD36 that demonstrated endogenous expression by U937. Analysis of extracted U937 flow cytometry data confirmed a decrease in oxLDL binding (FIG. 5A) and oxLDL uptake (FIG. 5B) by U937 cells when incubated with anti-CD36 antibodies of the present disclosure, 12P103, 12P110, or 12P109, compared to IgG isotype control.

[0212] B. Inhibition of oxidized LDL uptake in F293 / CD36 cells by anti-CD36 antibodies F293 / hCD36 cells were preincubated with control IgG or anti-CD36 antibody for 30 min at 4°C. To measure oxLDL binding or uptake, Dil-oxLDL (5 μg / mL) (Kalen Biomedical) was added to serum-free medium and incubated for 2 h at 4°C to measure "oxLDL binding" or for 5 min at 37°C to measure "oxLDL uptake", respectively. After washing with PBS, cells were analyzed by an Attune NxT Flow Cytometer (Thermo Scientific).

[0213] Results: As shown by the plots of flow cytometry data presented in Figures 6A, 6B, and 6C, the presence of anti-CD36 antibodies 12P109, 117, 117_30DA, 117_57D, 117_57E, 117_30DE, 117_57EE, 117_DA57D, or 117_DA57E effectively blocked uptake of oxidized LDL by F293 cells overexpressing human CD36 on their surface. Furthermore, as shown by the plots in Figure 6C, the presence of anti-CD36 antibodies 12P109, 117_30DA, 117_57D, or 117_57E effectively blocked uptake of oxidized LDL by F293 cells overexpressing mouse CD36 on their surface. IC for inhibition of oxLDL uptake by F293 / hCD36 or F293 / mCD36 cells determined for these anti-CD36 antibodies 50 The values ​​are shown in Table 13 below.

[0214] [Table 11]

[0215] Example 4 Activity of anti-CD36 antibody in blocking the uptake of oxLDL by TILs This example illustrates the study of anti-CD36 antibodies in blocking the uptake of oxidized LDL in tumor-infiltrating lymphocytes (TIL) in mice.

[0216] Materials and Methods: Mouse colon cancer cell line CT-26 (2 × 10 5 cells, ATCC number: CRL-2638) or mouse hepatoma cell line BNL 1MEA.7R.1 (5 × 10 6 Cells, ATCC number: TIB-75) were injected subcutaneously into the right flank of BALB / c mice. Mouse colon cancer cell line MC38 (1 × 10 6 Cells, Kerafast #ENH204-FP), mouse melanoma cell line B16-F10 (1 × 10 6 cells, BCRC#60031), or the mouse lung cancer cell line LL / 2 (2 × 10 5Cells (BCRC#60050) were injected subcutaneously into the right flank of C57BL / 6 mice. CT-26 tumors were harvested 4 weeks later. B16-F10, BNL 1MEA.7R.1, MC38, and LL / 2 tumors were harvested 2 weeks later. Tumor cell suspensions were prepared from solid tumors using a Mouse Tumor Dissociation Kit (Miltenyi Biotec) according to the manufacturer's instructions. CD45+ TILs were isolated from tumor cell suspensions using CD45 microbeads (Miltenyi Biotec).

[0217] Isolated CD45+ TILs were preincubated with control IgG or anti-CD36 antibody (5–10 μg / mL) for 30 min at 4°C. Dil-oxLDL (5–10 μg / mL) was added in RPMI medium containing 1% fatty acid-free BSA and incubated at 37°C for 15 min. After washing with PBS, cells were analyzed by an Attune NxT Flow Cytometer (Thermo Scientific). Binding and uptake of oxLDL were analyzed by flow cytometry and expressed as the percentage (%) of positive cells based on a histogram gate placed on the negative control cells.

[0218] Data represent the mean ± SD. *p<0.05, **p<0.005, one-way ANOVA.

[0219] Results: As shown by the result plot depicted in Figure 7A, the anti-CD36 antibodies of the present disclosure, 12P109 and A8A, which specifically bind hCD36 and mCD36, caused approximately 35% inhibition of oxLDL uptake in CD45+ TILs isolated from CT26 tumors injected into BALB / c mice. The level of inhibition was comparable to that observed for the commercially available antibody D2712, which specifically binds mouse CD36.

[0220] As shown by the result plots presented in Figures 7B, 7C, 7D, and 7E, anti-CD36 antibodies 117_57E, 117_30DA, 117_57DE, and 117_DA57E showed inhibitory activity against oxLDL uptake in CD45+ TILs from B16-F10, BNL 1MEA.7R.1, MC38, and LL / 2 tumors.

[0221] Example 5 Polarization and activation blocking activity of M2 macrophages This study was conducted to determine the ability of the anti-CD36 antibodies of the present disclosure to inhibit M2 macrophage polarization and activation.

[0222] Materials and Methods: To generate monocyte-derived macrophages, human CD14+ monocytes were isolated from PBMCs and cultured at 2 × 10 6 The cells were cultured at 1000 cells / ml in RPMI1640 supplemented with 10% FBS and CSF1 20ng / ml for 6 days. Adherent macrophages were then collected by EDTA detachment and seeded in 24-well plates for further polarization. For M0 macrophages, cells were cultured in culture medium for 2 days. To polarize M2 macrophages, macrophages were cultured in medium containing IL-4 50ng / mL and IL-13 50ng / mL for an additional 2 days. Control IgG or anti-CD36 antibody 10μg / mL was added during M2 macrophage polarization. For oxLDL activation, macrophages were preincubated with control IgG or anti-CD36 antibody for 10 min and then cultured in M2 medium containing oxLDL (Kalen Biomedical) 10μg / mL for an additional 2 days. To test M2 macrophage activation, macrophages were harvested and stained with antibodies against CD206, CD301 and PDL2. Surface marker levels were analyzed using an Attune NxT Flow Cytometer (Thermo Scientific).

[0223] Results: As shown by the plots of data presented in Figures 8A and 8B, anti-CD36 antibody 117_30AA exhibited very strong inhibition of M2 macrophage polarization in both CD206+ / CD301+ (Figure 8A) and CD206+ / PDL2+ (Figure 8B) double positive cell populations. As shown by the plots of data presented in Figures 8C, 8D, 8E, and 8F, anti-CD36 antibodies 117_30DA, 117_57D, 117_57E, and 117_DA57E exhibited very strong inhibition of oxLDL-induced M2 macrophage activation in both CD206+ / CD301+ (Figures 8C, 8D) and CD206+ / PDL2+ (Figures 8E, 8F) double positive cell populations. As shown by the plots of the data presented in Figures 8D and 8F, the anti-CD36 antibodies 117_30DA and 117_DA57E exhibited very strong inhibition of oxLDL-induced M2 macrophage activation compared to the IgG control and the competitor anti-CD36 antibody "ONA" (which is the Fab clone "ONA-0-v1" with hIgG4-S228P-FALA Fc region disclosed in WO2021176424A1).

[0224] Example 6 Activity of anti-CD36 antibody in a mouse hepatocellular carcinoma model This example illustrates the study of the activity of anti-CD36 antibody in suppressing tumor growth in two genetically induced mouse models of hepatocellular carcinoma (HCC): (1) HCC induced by Sleeping Beauty transposon (SB100x) system-mediated transgene with MYC-luc-ova overexpression and p53 knockout; and (2) HCC induced by MYC-luc-ova and β-catenin N90 HCC induced by the Sleeping Beauty transposon (SB100x) system-mediated transgene.

[0225] Materials and Methods Restrain 6-week-old mice and perform hydrodynamic delivery of endotoxin-free plasmid DNA in a volume equivalent to 10% of body weight through a lateral tail vein injection within 5-7 s. OE / p53 KO For the HCC model, mice were injected with pT3-Myc-luc-ova plasmid (Addgene #129776), p53gRNA plasmid (Addgene #59910) and SB100x (transposase-containing plasmid; Addgene #34879). OE / MYC OE For the HCC model, pT3-β-catenin (Addgene #31785), pT3-Myc-luc-ova plasmid, and SB100x were injected into each mouse. β-catenin-driven liver tumors represent more aggressive HCC with a cold tumor phenotype.

[0226] To monitor tumor growth, mice were injected with luciferin (150 mg / Kg) and bioluminescence activity was analyzed with an IVIS imaging system. Mice with continued tumor growth were randomly divided into groups and administered anti-CD36 antibody (10 mg / kg) or PBS (control) by intraperitoneal injection as follows:

[0227] Two weeks later, MYC OE / p53 KO HCC model mice were intraperitoneally injected with 4 doses of anti-CD36 antibody (10 mg / kg, clone: ​​117_30DA) or control antibody every 3 days, and bioluminescence imaging of the mice was measured every 6 days with an IVIS system (n = 5 per group).

[0228] After 3 weeks, β-catenin OE / MYC OE HCC model mice were intraperitoneally injected with anti-CD36 antibody (10 mg / mL, clone: ​​117_DA57E) or control antibody (n=6 per group), and bioluminescence imaging of the mice was measured every 5 days with an IVIS system.

[0229] β-cateninOE / MYC OE In the HCC model, tumors exhibit malignant progression and tumor-bearing mice usually die 38 days after hydrodynamic injection. Therefore, tumors were harvested and masses were measured 35–38 days after hydrodynamic injection of the tail vein.

[0230] result Figure 9 shows the MYC expression levels after treatment with anti-CD36 antibody 117_30DA. OE / p53 KO 1 shows tumor growth curves measured by bioluminescence in HCC model mice. Starting from day 14 after HCC induction and continuing up to day 38, tumor growth was observed to be significantly suppressed in treated mice compared to the control group, P value=0.0114, t test, two-tailed.

[0231] FIG. 10A shows the uptake of β-catenin following treatment with anti-CD36 antibody 117_DA57E. OE / MYC OE Figure 1 shows the tumor growth curve measured by bioluminescence in HCC model mice. Starting from 21 days after HCC induction and continuing up to 36 days, tumor growth was observed to be significantly suppressed in treated mice compared to the control group. P value = 0.0104, t-test, two-tailed. Figure 10B shows that end-point liver mass was significantly reduced in anti-CD36 treated mice, and plasma ALT (alanine transaminase) activity analysis indicated that anti-CD36 treatment also reduced liver damage caused by cancer development, as shown in Figure 10C.

[0232] Although the above disclosure of the present invention has been described in some detail as examples and illustrations for purposes of clarity and understanding, the present disclosure, including the examples, explanations and embodiments described herein, are intended to be illustrative and exemplary and should not be construed as limiting the present disclosure. It will be apparent to those skilled in the art that various modifications or changes to the examples, explanations and embodiments described herein can be made and are within the spirit and scope of the present disclosure and the appended claims. Moreover, those skilled in the art will recognize several methods and procedures equivalent to those described herein. All such equivalents should be understood to be within the scope of the present disclosure and are encompassed by the appended claims.

[0233] Further embodiments of the invention are set out in the following claims.

[0234] The disclosures of all publications, patent applications, patents, or other documents mentioned in this specification are expressly incorporated by reference in their entirety for all purposes to the same extent as if each such individual publication, patent, patent application, or other document was individually and specifically indicated to be incorporated by reference in its entirety for all purposes and was set forth in its entirety herein. In case of conflict, the present specification, including specific language, will control. (References) TIFF2025509380000022.tif198156TIFF2025509380000023.tif225156TIFF2025509380000024.tif234156TIFF2025509380000025.tif131156

Claims

1. (i) a first heavy chain complementarity determining region (CDR-H1), a second heavy chain complementarity determining region (CDR-H2), and a third heavy chain complementarity determining region (CDR-H3), and (ii) a first light chain complementarity determining region (CDR-L1), a second light chain complementarity determining region (CDR-L2), and a third light chain complementarity determining region (CDR-L3); 1. An anti-CD36 antibody comprising: (a) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 24, 3, 21, or 27; (b) CDR-H2 comprises the amino acid sequence of SEQ ID NO: 43, 4, 28, 31, 34, 37, or 40; (c) CDR-H3 comprises the amino acid sequence of SEQ ID NO:5; (d) CDR-L1 comprises an amino acid sequence selected from SEQ ID NO:7; (e) CDR-L2 comprises an amino acid sequence selected from SEQ ID NO: 15, 8, or 12; and (f) CDR-L3 comprises an amino acid sequence selected from SEQ ID NO: 18, 9, or 13; Anti-CD36 antibody.

2. (a) CDR-H1 comprises the amino acid sequence of SEQ ID NO:24, CDR-H2 comprises the amino acid sequence of SEQ ID NO:43, CDR-H3 comprises the amino acid sequence of SEQ ID NO:5, CDR-L1 comprises the amino acid sequence of SEQ ID NO:7, CDR-L2 comprises the amino acid sequence of SEQ ID NO:15, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:18; (b) CDR-H1 comprises the amino acid sequence of SEQ ID NO:3, CDR-H2 comprises the amino acid sequence of SEQ ID NO:4, CDR-H3 comprises the amino acid sequence of SEQ ID NO:5, CDR-L1 comprises the amino acid sequence of SEQ ID NO:7, CDR-L2 comprises the amino acid sequence of SEQ ID NO:8, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:9; (c) CDR-H1 comprises the amino acid sequence of SEQ ID NO:3, CDR-H2 comprises the amino acid sequence of SEQ ID NO:4, CDR-H3 comprises the amino acid sequence of SEQ ID NO:5, CDR-L1 comprises the amino acid sequence of SEQ ID NO:7, CDR-L2 comprises the amino acid sequence of SEQ ID NO:12, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:13; (d) CDR-H1 comprises the amino acid sequence of SEQ ID NO:3, CDR-H2 comprises the amino acid sequence of SEQ ID NO:4, CDR-H3 comprises the amino acid sequence of SEQ ID NO:5, CDR-L1 comprises the amino acid sequence of SEQ ID NO:7, CDR-L2 comprises the amino acid sequence of SEQ ID NO:15, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:13; (e) CDR-H1 comprises the amino acid sequence of SEQ ID NO:3, CDR-H2 comprises the amino acid sequence of SEQ ID NO:4, CDR-H3 comprises the amino acid sequence of SEQ ID NO:5, CDR-L1 comprises the amino acid sequence of SEQ ID NO:7, CDR-L2 comprises the amino acid sequence of SEQ ID NO:15, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:18; (f) CDR-H1 comprises the amino acid sequence of SEQ ID NO:21, CDR-H2 comprises the amino acid sequence of SEQ ID NO:4, CDR-H3 comprises the amino acid sequence of SEQ ID NO:5, CDR-L1 comprises the amino acid sequence of SEQ ID NO:7, CDR-L2 comprises the amino acid sequence of SEQ ID NO:15, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:18; (g) CDR-H1 comprises the amino acid sequence of SEQ ID NO:24, CDR-H2 comprises the amino acid sequence of SEQ ID NO:4, CDR-H3 comprises the amino acid sequence of SEQ ID NO:5, CDR-L1 comprises the amino acid sequence of SEQ ID NO:7, CDR-L2 comprises the amino acid sequence of SEQ ID NO:15, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:18; (h) CDR-H1 comprises the amino acid sequence of SEQ ID NO:27, CDR-H2 comprises the amino acid sequence of SEQ ID NO:28, CDR-H3 comprises the amino acid sequence of SEQ ID NO:5, CDR-L1 comprises the amino acid sequence of SEQ ID NO:7, CDR-L2 comprises the amino acid sequence of SEQ ID NO:15, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:18; (i) CDR-H1 comprises the amino acid sequence of SEQ ID NO:3, CDR-H2 comprises the amino acid sequence of SEQ ID NO:31, CDR-H3 comprises the amino acid sequence of SEQ ID NO:5, CDR-L1 comprises the amino acid sequence of SEQ ID NO:7, CDR-L2 comprises the amino acid sequence of SEQ ID NO:15, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:18; (j) CDR-H1 comprises the amino acid sequence of SEQ ID NO:3, CDR-H2 comprises the amino acid sequence of SEQ ID NO:34, CDR-H3 comprises the amino acid sequence of SEQ ID NO:5, CDR-L1 comprises the amino acid sequence of SEQ ID NO:7, CDR-L2 comprises the amino acid sequence of SEQ ID NO:15, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:18; (k) CDR-H1 comprises the amino acid sequence of SEQ ID NO:3, CDR-H2 comprises the amino acid sequence of SEQ ID NO:37, CDR-H3 comprises the amino acid sequence of SEQ ID NO:5, CDR-L1 comprises the amino acid sequence of SEQ ID NO:7, CDR-L2 comprises the amino acid sequence of SEQ ID NO:15, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:18; or (l) CDR-H1 comprises the amino acid sequence of SEQ ID NO:3, CDR-H2 comprises the amino acid sequence of SEQ ID NO:40, CDR-H3 comprises the amino acid sequence of SEQ ID NO:5, CDR-L1 comprises the amino acid sequence of SEQ ID NO:7, CDR-L2 comprises the amino acid sequence of SEQ ID NO:15, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:18; The antibody of claim 1.

3. (i) CDR-H1, CDR-H2, and CDR-H3, wherein each sequence of the first heavy chain complementarity determining region (CDR-H1), the second heavy chain complementarity determining region (CDR-H2), and the third heavy chain complementarity determining region (CDR-H3) is from a VH region having an amino acid sequence selected from SEQ ID NOs: 42, 2, 20, 23, 26, 30, 33, 36, and 39; (ii) CDR-L1, CDR-L2, and CDR-L3, each of which has an amino acid sequence selected from SEQ ID NOs: 17, 6, 11, and 14, is from a VL region, and each of the sequences of the first light chain complementarity determining region (CDR-L1), the second light chain complementarity determining region (CDR-L2), and the third light chain complementarity determining region (CDR-L3) is from a VL region having an amino acid sequence selected from SEQ ID NOs: 17, 6, 11, and 14. and CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 are according to the Kabat numbering. Anti-CD36 antibody.

4. (a) the VH amino acid sequence is SEQ ID NO:42 and the VL amino acid sequence is SEQ ID NO:17; (b) the VH amino acid sequence is SEQ ID NO:2 and the VL amino acid sequence is SEQ ID NO:6; (c) the VH amino acid sequence is SEQ ID NO:2 and the VL amino acid sequence is SEQ ID NO:11: (d) the VH amino acid sequence is SEQ ID NO:2 and the VL amino acid sequence is SEQ ID NO:14; (e) the VH amino acid sequence is SEQ ID NO:2 and the VL amino acid sequence is SEQ ID NO:17; (f) the VH amino acid sequence is SEQ ID NO:20 and the VL amino acid sequence is SEQ ID NO:17; (g) the VH amino acid sequence is SEQ ID NO:23 and the VL amino acid sequence is SEQ ID NO:17; (h) the VH amino acid sequence is SEQ ID NO:26 and the VL amino acid sequence is SEQ ID NO:17; (i) the VH amino acid sequence is SEQ ID NO:30 and the VL amino acid sequence is SEQ ID NO:17; (j) the VH amino acid sequence is SEQ ID NO: 33 and the VL amino acid sequence is SEQ ID NO: 17; (k) the VH amino acid sequence is SEQ ID NO:36 and the VL amino acid sequence is SEQ ID NO:17; or (l) the VH amino acid sequence is SEQ ID NO: 39 and the VL amino acid sequence is SEQ ID NO: 17; The antibody of claim 3.

5. A heavy chain variable domain (V) having at least 90% identity to a sequence selected from SEQ ID NO: 42, 2, 20, 23, 26, 30, 33, 36, or 39. H ) amino acid sequence; and a light chain variable domain (V) having at least 90% identity to a sequence selected from SEQ ID NO: 17, 6, 11, or 14. L ) the amino acid sequence, An antibody described in any one of claims 1 to 4.

6. (a) a V having at least 90% identity to SEQ ID NO: 42 H V having an amino acid sequence and at least 90% identity with SEQ ID NO:17 L Contains an amino acid sequence; (b) a V having at least 90% identity to SEQ ID NO:2 H V having an amino acid sequence and at least 90% identity with SEQ ID NO:6 L Contains an amino acid sequence; (c) a V having at least 90% identity to SEQ ID NO:2 H V having an amino acid sequence and at least 90% identity with SEQ ID NO:11 L Contains an amino acid sequence; (d) a V having at least 90% identity to SEQ ID NO:2 H V having an amino acid sequence and at least 90% identity with SEQ ID NO:14 L Contains an amino acid sequence; (e) a V having at least 90% identity to SEQ ID NO:2 H V having an amino acid sequence and at least 90% identity with SEQ ID NO:17 L Contains an amino acid sequence; (f) a V having at least 90% identity to SEQ ID NO: 20 H V having an amino acid sequence and at least 90% identity with SEQ ID NO:17 L Contains an amino acid sequence; (g) a V having at least 90% identity to SEQ ID NO: 23 H V having an amino acid sequence and at least 90% identity with SEQ ID NO:17 L Contains an amino acid sequence; (h) comprises a VH amino acid sequence having at least 90% identity to SEQ ID NO: 26 and a VL amino acid sequence having at least 90% identity to SEQ ID NO: 17; (i) a V having at least 90% identity to SEQ ID NO: 30 H V having an amino acid sequence and at least 90% identity with SEQ ID NO:17 L Contains an amino acid sequence; (j) a V having at least 90% identity to SEQ ID NO: 33 H V having an amino acid sequence and at least 90% identity with SEQ ID NO:17 L Contains an amino acid sequence; (k) comprises a VH amino acid sequence having at least 90% identity to SEQ ID NO: 36 and a VL amino acid sequence having at least 90% identity to SEQ ID NO: 17; or (l) a VH amino acid sequence having at least 90% identity to SEQ ID NO: 39 and a VL amino acid sequence having at least 90% identity to SEQ ID NO: 17; 5. An antibody described in any one of claims 1 to 4.

7. comprising a heavy chain (HC) amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 57, 44, 48, 50, 51, 52, 53, 54, 55, or 56, and / or a light chain (LC) amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 49, 45, 46, or 47; An antibody described in any one of claims 1 to 4.

8. (a) an HC amino acid sequence having at least 90% identity to SEQ ID NO:57, and an LC amino acid sequence having at least 90% identity to SEQ ID NO:49; (b) an HC amino acid sequence having at least 90% identity to SEQ ID NO: 44, and an LC amino acid sequence having at least 90% identity to SEQ ID NO: 45; (c) an HC amino acid sequence having at least 90% identity to SEQ ID NO: 44, and an LC amino acid sequence having at least 90% identity to SEQ ID NO: 46; (d) an HC amino acid sequence having at least 90% identity to SEQ ID NO: 44, and an LC amino acid sequence having at least 90% identity to SEQ ID NO: 47; (e) an HC amino acid sequence having at least 90% identity to SEQ ID NO: 44, and an LC amino acid sequence having at least 90% identity to SEQ ID NO: 49; (f) an HC amino acid sequence having at least 90% identity to SEQ ID NO: 48, and an LC amino acid sequence having at least 90% identity to SEQ ID NO: 49; (g) an HC amino acid sequence having at least 90% identity to SEQ ID NO:50, and an LC amino acid sequence having at least 90% identity to SEQ ID NO:49; (h) an HC amino acid sequence having at least 90% identity to SEQ ID NO:51, and an LC amino acid sequence having at least 90% identity to SEQ ID NO:49; (i) an HC amino acid sequence having at least 90% identity to SEQ ID NO:52, and an LC amino acid sequence having at least 90% identity to SEQ ID NO:49; (j) an HC amino acid sequence having at least 90% identity to SEQ ID NO:53, and an LC amino acid sequence having at least 90% identity to SEQ ID NO:49; (k) an HC amino acid sequence having at least 90% identity to SEQ ID NO:54, and an LC amino acid sequence having at least 90% identity to SEQ ID NO:49; (l) an HC amino acid sequence having at least 90% identity to SEQ ID NO:55, and an LC amino acid sequence having at least 90% identity to SEQ ID NO:49; or (m) an HC amino acid sequence having at least 90% identity to SEQ ID NO:56, and an LC amino acid sequence having at least 90% identity to SEQ ID NO:

49. The antibody of any one of claims 1 to 4, comprising:

9. (a) The antibody is an antibody fragment, F(ab') 2 , Fab', Fab, Fv, single domain antibody (VHH), and scFv; (b) the antibody comprises a fusion to a protein; optionally, where the protein is an immunostimulatory cytokine selected from IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, or IFN-α; (c) the antibody is human, humanized, or chimeric; (d) the antibody is a full-length antibody of the class IgG, optionally where the class IgG antibody has an isotype selected from IgG1, IgG2, IgG3, and IgG4; (e) the antibody comprises an Fc region variant, optionally an Fc region variant that alters effector function and / or a variant that alters the antibody half-life; (f) the antibody comprises an immunoconjugate, optionally where the immunoconjugate comprises a therapeutic agent for treating a CD36-mediated disease or condition; and / or (g) the antibody is a multispecific antibody; optionally a bispecific antibody; An antibody described in any one of claims 1 to 4.

10. (a) Antibody: 1 × 10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less; optionally, wherein the binding affinity is equal to or less than the equilibrium dissociation constant (K D ) measured by; (b) Antibody, 1 × 10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less; optionally, wherein the binding affinity is equal to or less than the equilibrium dissociation constant (K D ) measured by; (c) Antibody, 1 × 10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less; optionally, wherein the binding affinity is equal to or less than the equilibrium dissociation constant (K D ) measured by; (d) the antibody inhibits CD36-dependent oxidized LDL uptake by at least 65%, at least 75%, at least 85%, at least 95%, or at least 100% in F293 cells overexpressing surface human CD36; optionally, where at an oxLDL concentration of 1-10 μg / mL, the antibody has an IC of 5 nM or less, 1 nM or less, 0.5 nM or less, or 0.1 nM or less. 50 having; (e) the antibody inhibits CD36-dependent oxidized LDL uptake in U937 cells by at least 65%, at least 75%, at least 85%, at least 95%, or at least 100%; optionally, where at an oxLDL concentration of 1-10 μg / mL, the antibody has an IC of 5 nM or less, 1 nM or less, 0.5 nM or less, or 0.1 nM or less. 50 and / or (f) the antibody inhibits CD36-dependent oxidized LDL uptake in murine CD45+ TILs by at least 65%, at least 75%, at least 85%, at least 95%, or at least 100%; optionally, where at an oxLDL concentration of 1-10 μg / mL, the antibody has an IC of 5 nM or less, 1 nM or less, 0.5 nM or less, or 0.1 nM or less. 50 having 10. An antibody according to any one of claims 1 to 9.

11. 11. An isolated polynucleotide or vector comprising a sequence encoding an anti-CD36 antibody polypeptide according to any one of claims 1 to 10.

12. The polypeptide is: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, 21, 24, or 27; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 4, 28, 31, 34, 37, 40, or 43; and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5; (b) a CDR-L1 comprising an amino acid sequence selected from SEQ ID NO: 7; a CDR-L2 comprising an amino acid sequence selected from SEQ ID NO: 8, 12, or 15; and a CDR-L3 comprising an amino acid sequence selected from SEQ ID NO: 9, 13, or 18; (c) a heavy chain variable domain (V) having at least 90% identity to a sequence selected from SEQ ID NO: 2, 20, 23, 26, 30, 33, 36, 39, or 42. H ) amino acid sequence; (d) a light chain variable domain (V) having at least 90% identity to a sequence selected from SEQ ID NO: 6, 11, 14, or 17. L ) amino acid sequence; (e) a heavy chain (HC) amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 44, 48, 50, 51, 52, 53, 54, 55, 56, or 57; and / or (f) a light chain (LC) amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 45, 46, 47, or 49.

12. The isolated polynucleotide or vector of claim 11, comprising an amino acid sequence comprising:

13. 13. An isolated host cell comprising the polynucleotide or vector of claim 11 or 12, optionally selected from Chinese hamster ovary (CHO) cells, myeloma cells (e.g., Y0, NS0, Sp2 / 0), monkey kidney cells (COS-7), human embryonic kidney line (293), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells, human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor cells, TR1 cells, Medical Research Council 5 (MRC 5) cells, and FS4 cells.

14. 14. A method for producing an antibody, comprising culturing the host cell of claim 13 so that the antibody is produced.

15. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 10 and a pharma- ceutically acceptable carrier.

16. 16. The composition of claim 15, further comprising a chemotherapeutic agent.

17. 16. The composition of claim 15, further comprising an antibody comprising specificity for an immune checkpoint molecule; optionally, wherein the immune checkpoint molecule is selected from PD1, PD-L1, LAG3, CTLA-4, A2AR, TIM-3, BTLA, CD276, CD328, VTCN1, IDO, KIR, NOX2, VISTA, OX40, CD27, CD28, CD40, CD122, CD137, GITR, ICOS.

18. 16. The composition of claim 15, further comprising an immunostimulatory cytokine; optionally, wherein the immunostimulatory cytokine is selected from IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, and IFN-α.

19. A method for treating a CD36-mediated disease in a subject, comprising administering to the subject a therapeutically effective amount of an antibody described in any one of claims 1 to 10, or administering to the subject a therapeutically effective amount of a pharmaceutical composition described in any one of claims 15 to 18; optionally, wherein the disease is cancer; optionally, wherein the cancer is colon cancer, pancreatic cancer, ovarian cancer, HCC, renal cancer, breast cancer, lung cancer, gastric cancer, melanoma, head and neck cancer, or oral cancer.

20. 20. The method of claim 19, wherein the cancer is colon cancer, pancreatic cancer, ovarian cancer, HCC, renal cancer, breast cancer, lung cancer, gastric cancer, melanoma, head and neck cancer, or oral cancer.

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