HK40112946A - Anti-cd36 antibodies and uses thereof - Google Patents

Description

Anti-CD36 antibodies and their uses

This application claims priority to U.S. Provisional Patent Application No. 63/317,160, filed March 7, 2022, the entire contents of which are incorporated herein by reference.

Technical Field

This disclosure relates to antibodies that bind to CD36 and methods of using such antibodies.

References to sequence lists

An official copy of the sequence list, filed with the specification as an XML file in WIPO standard ST.26 format, is named “09793-005WO1.xml”, created on February 26, 2023, and is 75,330 bytes in size. The sequence list, filed through the USPTO Patent Center, is part of the specification, and its entire contents are incorporated herein by reference.

Background Technology

Macrophages and other myeloid cells play a prominent role in creating and maintaining the immunosuppressive environment of tumors and exist in distinct microenvironments in which tumor-associated macrophages (TAMs) suppress immune responses and promote other processes, including angiogenesis, cancer cell migration, and metastasis (Lewis & Pollard, 2006; Pollard, 2008; Wu et al., 2020). About two decades ago, for example, vascular endothelial growth factor produced by TAMs was proposed to play a role in lymphatic metastasis of several human cancers (e.g., Pepper et al., 2003), and in 2006, clophosphate liposome-mediated TAM depletion was demonstrated to inhibit tumor growth in experimental models via mechanisms believed to include reduced tumor angiogenesis (Zeisberger et al., 2006). Furthermore, macrophages not only facilitate the outflow of tumor cells from primary tumors but are also thought to aid in the inoculation of distant metastatic sites (Joyce & Pollard, 2009; Psaila & Lyden, 2009).

While our understanding of various pro-tumorigenic (commonly referred to as M2) and anti-tumorigenic M1 macrophages, as well as other myeloid cells, is still evolving (e.g., see Wu et al., 2020), the association between high TAM numbers and reduced patient survival (with only a few significant exceptions) and their status as an independent prognostic factor in many cancers has long been recognized (Lewis & Pollard, 2006). Furthermore, myeloid cells known as myeloid-derived suppressor cells (MDSCs) have been found in the peripheral blood and tumor tissues of cancer patients, and depletion or other targeting of MDSCs in mouse models leads to improved immune responses and delayed tumor growth (Goedegebuure et al., 2011). MDSCs are reportedly heterogeneous but are still described as belonging to a subset of monocytes and granulocytes with different immunosuppressive mechanisms. Among other roles, tumor MDSCs have been involved in the recruitment and maintenance of immunosuppressive regulatory T cells, as well as promoting angiogenesis and metastasis (Goedegebuure et al., 2011; Oh et al., 2013). Similar to MDSC, at least as we now know, a subset of human peripheral blood cells that are CD45 positive but CD14 negative was described by Barrett et al. (2007) as expressing CD36 and the immunosuppressive cytokine interleukin-10.

CD36 is a transmembrane cell surface protein, also known by other names such as platelet glycoprotein 4, fatty acid translocase (FAT), and scavenger receptor class B member 3 (SCARB3). CD36 binds to and transports fatty acids into the cell, but also binds to a wide range of other ligands, such as apoptotic cells, low-density lipoproteins, phospholipids, and their oxidized forms (Pepino et al., 2014; Wang & Li, 2019). CD36 also possesses a separate platelet-reactive protein-binding domain (Pepino et al., 2014; Wang & Li, 2019). Both of these distinct functional events—platelet-reactive protein binding to fatty acids—can lead to a variety of downstream signaling events that can vary depending on cell type and various other CD36 and/or ligand interacting partners (Pepino et al., 2014; Wang & Li, 2019). Fatty acid transport via CD36, if not required for cell differentiation or survival, involves various cellular metabolic "wiring." For example, it has been shown that fatty acid transport via CD36 is required for M2 macrophage differentiation, which is achieved through mechanisms related to endoplasmic reticulum stress levels or states (Oh et al., 2012).

CD36 has also been shown to be crucial for M2 macrophage activation, as measured by immunosuppressive capacity markers such as PDL-2 expression (Huang et al., 2014). CD36-mediated lipid transport is also involved in the acquisition of immunosuppressive effectors during MDSC differentiation (Al-Khami et al., 2017). CD36-deficient mice exhibited a reduced number of tumor-resident MDSCs in all tested (lung and colon) tumor models compared to tumor-bearing wild-type mice, and this was reflected in tumors from wild-type chimeric mice, which were CD36-deficient in their bone marrow (Al-Khami et al., 2017). Recently, CD36-deficient mice have been shown to have significantly reduced TAMs in melanoma and myeloma models compared to wild-type mice, and these TAMs in wild-type mice exhibit elevated CD36 expression and lipid accumulation compared to normal macrophages (Su et al., 2020).

Given the importance of CD36-mediated fatty acid or other ligand uptake to the biology of M2 TAMs and MDSCs, it is expected that blocking CD36 ligand transport would reduce tumor growth by leading to decreased immunosuppression, angiogenesis, and/or metastasis, which 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 mice lacking CD36, and this is due to the loss of CD36 in bone marrow-derived cells (Al-Khami et al., 2017).

In addition to myeloid cells, CD36 has recently been reported to be expressed on certain tumor-resident regulatory T cells, and blocking CD36 with antibodies that inhibit CD36-mediated fatty acid transport leads to reduced melanoma growth in experimental models (Wang et al., 2020). Furthermore, it has been shown that CD36 deficiency on mouse regulatory T cells alone is sufficient to cause reduced melanoma growth (Wang et al., 2020). Recently, CD36 expression on tumor-resident CD8 T cells has been shown to impair their anti-tumor capabilities and lead to increased tumor CD8 T cell death via lipid peroxidation that causes ferroptosis (Ma et al., 2021). A previous report also described CD36 on tumor-infiltrating CD8 T cells and its adverse consequences on such CD8 T cells (Xu et al., 2020), which was later published in peer-reviewed form (Xu et al., 2021).

Numerous reports have also documented the role of ligand transport via CD36 expressed on tumor cells. For example, CD36 is involved in 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, a screening of doxorubicin-resistant sublines of the human myeloid leukemia line K562 revealed an association between CD36 and doxorubicin resistance, although overexpression of CD36 in doxorubicin-sensitive parental cell lines does not inherently confer resistance (Sugimoto et al., 1993). CD36 is also involved in increased stem cell self-renewal, survival, and/or proliferation in glioblastoma (Hale et al., 2014) and leukemia (Ye et al., 2016). In other words, CD36 appears to be involved in chemotherapy resistance in the adipose tissue microenvironment via leukemia stem cells (Ye et al., 2016). When CD36 in stem cells was “knocked down” via RNA interference, glioblastomas formed from patient-derived glioblastoma stem cells in xenograft mouse models were significantly reduced, and glioblastoma stem cell proliferation increased in a CD36-dependent manner upon exposure to oxidized low-density lipoprotein (Hale et al., 2014). CD36 has also been shown to be an informative biomarker for brain malignancies and is negatively correlated with patient prognosis (Hale et al., 2014).

Recently, CD36 has been 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 other methods of blocking CD36-mediated lipid transport with antibodies reduces cell migration and tumor growth in mouse models (Watt et al., 2019).

CD36 has also been proposed as a prognostic biomarker for metastasis in various cancers (Reviewed by Enciu et al., 2018) and has been shown to promote the metastasis of oral squamous cell carcinoma (OSCC) cells, breast cancer cells, and melanoma cells in mouse models (PCT/EP2016/073208; Pascual et al., 2017). OSCC metastasis was observed after cell injection into the orthotopic site (tongue), and OSCC metastasis was reduced by blocking fatty acid transport via CD36 with antibodies. Injection of melanoma and breast cancer cells into the bloodstream of mice to allow them to potentially colonize a metastatic microenvironment reduced this metastasis when such cells were “knocked out” of CD36 via RNA interference (PCT/EP2016/073208; Pascual et al., 2017). As mentioned above, CD36 is also expected to be involved, primarily in the invasion of tumor cells from the primary tumor site into the lymphatic and/or bloodstream, through its role in myeloid cells of the immune system. Fatty acid transport via CD36 in hepatocellular carcinoma cells has also been shown to lead to increased epithelial-mesenchymal transition, while chemoinhibition of CD36-mediated fatty acid transport reduces this phenotype and hepatocellular carcinoma cell migration (Nath et al., 2015).

All of the above paints a picture in which the transport of fatty acids or other ligands into cells via CD36 involves metabolic adaptation or differentiation across various cell types, but it should also be emphasized that CD36 signaling leads to more direct immunosuppressive effects. Some of these have been mentioned above, but it should be stressed that apoptotic cell binding via CD36 has been proposed to generally promote homeostatic anti-inflammatory processes, and that apoptotic cell binding via CD36 at least partially leads to the production of IL-10 by macrophages (and other immunosuppressive effects on macrophages) after apoptotic cell binding (Chung et al., 2007). CD36-mediated ligand transport may also lead to the production of immunosuppressive metabolites. That is, the increased production of arachidonic acid due to CD36 ligand-induced intracellular signaling provides a substrate for prostaglandin production (e.g., Kuda et al., 2011), some of which are known to have broad immunomodulatory effects (e.g., see Wang and Dubois, 2006; Mizuno et al., 2019). Of course, immunosuppressive prostaglandin E2 is produced by tumor cells as well as immune system cells in many cancers, including myeloid cells and others (Wang and Dubois, 2006; Mizuno et al., 2019), such as inducible regulatory T cells (Whiteside and Jackson, 2013). Furthermore, mouse macrophages have at least been shown to produce prostaglandin E2 in a CD36-dependent manner, although this is not the case in cancer models (Almeida et al., 2014).

In addition to the many roles of CD36 in cancer, CD36 ligand transport apparently plays a significant 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. Therefore, antibodies that can prevent apoptotic cells or other ligands from binding/transporting via CD36 have several potential applications.

Summary of the Invention

This disclosure provides anti-CD36 antibodies that specifically bind to human CD36 with high affinity. These antibodies are capable of reducing, inhibiting, and/or completely blocking CD36-mediated immunomodulatory effects, including CD36-mediated fatty acid transport (e.g., cellular uptake of oxidized low-density lipoprotein or "oxLDL").

In at least one embodiment, this disclosure provides an anti-CD36 antibody 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), wherein:

(a) CDR-H1 contains the amino acid sequence of SEQ ID NO: 3, 21, 24 or 27;

(b) CDR-H2 contains the amino acid sequence of SEQ ID NO: 4, 28, 31, 34, 37, 40 or 43;

(c) CDR-H3 contains the amino acid sequence of SEQ ID NO:5;

(d) CDR-L1 contains an amino acid sequence selected from SEQ ID NO:7;

(e) CDR-L2 contains an amino acid sequence selected from SEQ ID NO: 8, 12, or 15; and

(f) CDR-L3 contains an amino acid sequence selected from SEQ ID NO:9, 13 or 18.

In at least one embodiment of the anti-CD36 antibody disclosed herein:

(a) CDR-H1 contains the amino acid sequence of SEQ ID NO:3, CDR-H2 contains the amino acid sequence of SEQ ID NO:4, CDR-H3 contains the amino acid sequence of SEQ ID NO:5, CDR-L1 contains the amino acid sequence of SEQ ID NO:7, CDR-L2 contains the amino acid sequence of SEQ ID NO:8, and CDR-L3 contains the amino acid sequence of SEQ ID NO:9;

(b) CDR-H1 contains the amino acid sequence of SEQ ID NO:3, CDR-H2 contains the amino acid sequence of SEQ ID NO:4, CDR-H3 contains the amino acid sequence of SEQ ID NO:5, CDR-L1 contains the amino acid sequence of SEQ ID NO:7, CDR-L2 contains the amino acid sequence of SEQ ID NO:12, and CDR-L3 contains the amino acid sequence of SEQ ID NO:13;

(c) CDR-H1 contains the amino acid sequence of SEQ ID NO:3, CDR-H2 contains the amino acid sequence of SEQ ID NO:4, CDR-H3 contains the amino acid sequence of SEQ ID NO:5, CDR-L1 contains the amino acid sequence of SEQ ID NO:7, CDR-L2 contains the amino acid sequence of SEQ ID NO:15, and CDR-L3 contains the amino acid sequence of SEQ ID NO:13;

(d) CDR-H1 contains the amino acid sequence of SEQ ID NO:3, CDR-H2 contains the amino acid sequence of SEQ ID NO:4, CDR-H3 contains the amino acid sequence of SEQ ID NO:5, CDR-L1 contains the amino acid sequence of SEQ ID NO:7, CDR-L2 contains the amino acid sequence of SEQ ID NO:15, and CDR-L3 contains the amino acid sequence of SEQ ID NO:18;

(e) CDR-H1 contains the amino acid sequence of SEQ ID NO:21, CDR-H2 contains the amino acid sequence of SEQ ID NO:4, CDR-H3 contains the amino acid sequence of SEQ ID NO:5, CDR-L1 contains the amino acid sequence of SEQ ID NO:7, CDR-L2 contains the amino acid sequence of SEQ ID NO:15, and CDR-L3 contains the amino acid sequence of SEQ ID NO:18;

(f) CDR-H1 contains the amino acid sequence of SEQ ID NO:24, CDR-H2 contains the amino acid sequence of SEQ ID NO:4, CDR-H3 contains the amino acid sequence of SEQ ID NO:5, CDR-L1 contains the amino acid sequence of SEQ ID NO:7, CDR-L2 contains the amino acid sequence of SEQ ID NO:15, and CDR-L3 contains the amino acid sequence of SEQ ID NO:18;

(g) CDR-H1 contains the amino acid sequence of SEQ ID NO:27, CDR-H2 contains the amino acid sequence of SEQ ID NO:28, CDR-H3 contains the amino acid sequence of SEQ ID NO:5, CDR-L1 contains the amino acid sequence of SEQ ID NO:7, CDR-L2 contains the amino acid sequence of SEQ ID NO:15, and CDR-L3 contains the amino acid sequence of SEQ ID NO:18;

(h) CDR-H1 contains the amino acid sequence of SEQ ID NO:3, CDR-H2 contains the amino acid sequence of SEQ ID NO:31, CDR-H3 contains the amino acid sequence of SEQ ID NO:5, CDR-L1 contains the amino acid sequence of SEQ ID NO:7, CDR-L2 contains the amino acid sequence of SEQ ID NO:15, and CDR-L3 contains the amino acid sequence of SEQ ID NO:18;

(i) CDR-H1 contains the amino acid sequence of SEQ ID NO:3, CDR-H2 contains the amino acid sequence of SEQ ID NO:34, CDR-H3 contains the amino acid sequence of SEQ ID NO:5, CDR-L1 contains the amino acid sequence of SEQ ID NO:7, CDR-L2 contains the amino acid sequence of SEQ ID NO:15, and CDR-L3 contains the amino acid sequence of SEQ ID NO:18;

(j) CDR-H1 contains the amino acid sequence of SEQ ID NO:3, CDR-H2 contains the amino acid sequence of SEQ ID NO:37, CDR-H3 contains the amino acid sequence of ammonia SEQ ID NO:5, CDR-L1 contains the amino acid sequence of SEQ ID NO:7, CDR-L2 contains the amino acid sequence of ammonia SEQ ID NO:15, and CDR-L3 contains the amino acid sequence of SEQ ID NO:18;

(k) CDR-H1 contains the amino acid sequence of SEQ ID NO:3, CDR-H2 contains the amino acid sequence of SEQ ID NO:40, CDR-H3 contains the amino acid sequence of SEQ ID NO:5, CDR-L1 contains the amino acid sequence of SEQ ID NO:7, CDR-L2 contains the amino acid sequence of SEQ ID NO:15, and CDR-L3 contains the amino acid sequence of SEQ ID NO:18; or

(l) CDR-H1 contains the amino acid sequence of SEQ ID NO:24, CDR-H2 contains the amino acid sequence of SEQ ID NO:43, CDR-H3 contains the amino acid sequence of SEQ ID NO:5, CDR-L1 contains the amino acid sequence of SEQ ID NO:7, CDR-L2 contains the amino acid sequence of SEQ ID NO:15, and CDR-L3 contains the amino acid sequence of SEQ ID NO:18.

In at least one embodiment, this disclosure provides an anti-CD36 antibody comprising: (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), wherein the sequences of CDR-H1, CDR-H2, and CDR-H3 are derived from a VH region having an amino acid sequence selected from SEQ ID NO: 2, 20, 23, 26, 30, 33, 36, 39, and 42; and (ii) The 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 wherein the sequences of CDR-L1, CDR-L2, and CDR-L3 are derived from the VL region, which has an amino acid sequence selected from SEQ ID NO: 6, 11, 14, and 17; wherein CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 are numbered according to Kabat.

In at least one embodiment of the anti-CD36 antibody:

(a) The amino acid sequence of VH is SEQ ID NO:2 and the amino acid sequence of VL is SEQ ID NO:6;

(b) The amino acid sequence of VH is SEQ ID NO:2 and the amino acid sequence of VL is SEQ ID NO:11;

(c) The amino acid sequence of VH is SEQ ID NO:2 and the amino acid sequence of VL is SEQ ID NO:14;

(d) The amino acid sequence of VH is SEQ ID NO:2 and the amino acid sequence of VL is SEQ ID NO:17;

(e) The amino acid sequence of VH is SEQ ID NO:20 and the amino acid sequence of VL is SEQ ID NO:17;

(f) The amino acid sequence of VH is SEQ ID NO:23 and the amino acid sequence of VL is SEQ ID NO:17;

(g) The amino acid sequence of VH is SEQ ID NO:26 and the amino acid sequence of VL is SEQ ID NO:17;

(h) The amino acid sequence of VH is SEQ ID NO:30 and the amino acid sequence of VL is SEQ ID NO:17;

(i) The amino acid sequence of VH is SEQ ID NO:33 and the amino acid sequence of VL is SEQ ID NO:17;

(j) The amino acid sequence of VH is SEQ ID NO:36 and the amino acid sequence of VL is SEQ ID NO:17;

(k) The amino acid sequence of VH is SEQ ID NO:39 and the amino acid sequence of VL is SEQ ID NO:17; or

(l) The amino acid sequence of VH is SEQ ID NO:42 and the amino acid sequence of VL is SEQ ID NO:17.

In at least one embodiment of the anti-CD36 antibody disclosed herein, the antibody comprises a heavy chain variable domain ( _VH ) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO:2, 20, 23, 26, 30, 33, 36, 39 or 42; and/or a light chain variable domain ( _VL ) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO:6, 11, 14 or 17.

In at least one embodiment of the anti-CD36 antibody disclosed herein:

(a) The antibody comprises a V _H amino acid sequence having at least 90% identity with SEQ ID NO:2 and a V _L amino acid sequence having at least 90% identity with SEQ ID NO:6;

(b) The antibody comprises a V _H amino acid sequence having at least 90% identity with SEQ ID NO:2 and a V _L amino acid sequence having at least 90% identity with SEQ ID NO:11;

(c) The antibody comprises a V _H amino acid sequence having at least 90% identity with SEQ ID NO:2 and a V _L amino acid sequence having at least 90% identity with SEQ ID NO:14;

(d) The antibody comprises a V _H amino acid sequence having at least 90% identity with SEQ ID NO:2 and a V _L amino acid sequence having at least 90% identity with SEQ ID NO:17;

(e) The antibody comprises a V _H amino acid sequence having at least 90% identity with SEQ ID NO:20 and a V _L amino acid sequence having at least 90% identity with SEQ ID NO:17;

(f) The antibody comprises a V _H amino acid sequence having at least 90% identity with SEQ ID NO:23 and a V _L amino acid sequence having at least 90% identity with SEQ ID NO:17;

(g) The antibody comprises a V _H amino acid sequence having at least 90% identity with SEQ ID NO:26 and a V _L amino acid sequence having at least 90% identity with SEQ ID NO:17;

(h) The antibody comprises a V _H amino acid sequence having at least 90% identity with SEQ ID NO:30 and a V _L amino acid sequence having at least 90% identity with SEQ ID NO:17;

(i) The antibody comprises a V _H amino acid sequence having at least 90% identity with SEQ ID NO:33 and a V _L amino acid sequence having at least 90% identity with SEQ ID NO:17;

(j) The antibody comprises a V _H amino acid sequence having at least 90% identity with SEQ ID NO:36 and a V _L amino acid sequence having at least 90% identity with SEQ ID NO:17;

(k) The antibody comprises a V _H amino acid sequence having at least 90% identity with SEQ ID NO:39 and a V _L amino acid sequence having at least 90% identity with SEQ ID NO:17; or

(l) The antibody comprises a V _H amino acid sequence having at least 90% identity with SEQ ID NO:42 and a V _L amino acid sequence having at least 90% identity with SEQ ID NO:17.

In at least one embodiment of the anti-CD36 antibody of this disclosure, the antibody comprises a heavy chain (HC) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO: 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 with a sequence selected from SEQ ID NO: 45, 46, 47 or 49.

In at least one embodiment of the anti-CD36 antibody of this disclosure, the antibody comprises:

(a) An HC amino acid sequence having at least 90% identity with SEQ ID NO:44 and an LC amino acid sequence having at least 90% identity with SEQ ID NO:45;

(b) The HC amino acid sequence having at least 90% identity with SEQ ID NO:44 and the LC amino acid sequence having at least 90% identity with SEQ ID NO:46;

(c) The HC amino acid sequence having at least 90% identity with SEQ ID NO:44 and the LC amino acid sequence having at least 90% identity with SEQ ID NO:47;

(d) An HC amino acid sequence having at least 90% identity with SEQ ID NO:44, and an LC amino acid sequence having at least 90% identity with SEQ ID NO:49;

(e) an HC amino acid sequence having at least 90% identity with SEQ ID NO:48, and an LC amino acid sequence having at least 90% identity with SEQ ID NO:49;

(f) HC amino acid sequences having at least 90% identity with SEQ ID NO:50 and LC amino acid sequences having at least 90% identity with SEQ ID NO:49;

(g) HC amino acid sequences having at least 90% identity with SEQ ID NO:51 and LC amino acid sequences having at least 90% identity with SEQ ID NO:49;

(h) HC amino acid sequences having at least 90% identity with SEQ ID NO:52 and LC amino acid sequences having at least 90% identity with SEQ ID NO:49;

(i) an HC amino acid sequence having at least 90% identity with SEQ ID NO:53 and an LC amino acid sequence having at least 90% identity with SEQ ID NO:49;

(j) HC amino acid sequences having at least 90% identity with SEQ ID NO:54 and LC amino acid sequences having at least 90% identity with SEQ ID NO:49;

(k) HC amino acid sequences having at least 90% identity with SEQ ID NO:55 and LC amino acid sequences having at least 90% identity with SEQ ID NO:49;

(l) an HC amino acid sequence having at least 90% identity with SEQ ID NO:56 and an LC amino acid sequence having at least 90% identity with SEQ ID NO:49; or

(m) HC amino acid sequence having at least 90% identity with SEQ ID NO:57 and LC amino acid sequence having at least 90% identity with SEQ ID NO:49.

In at least one embodiment of the anti-CD36 antibody disclosed herein:

(a) The antibody binds to human CD36 with a binding affinity of 1× ^10⁻⁸ M or less, 1× ^10⁻⁹ M or less, or 1× ^10⁻¹⁰ M or less; optionally, the binding affinity is measured by the equilibrium dissociation constant ( _KD ) of the hCD36 peptide with SEQ ID NO: 58 or 59.

(b) The antibody binds to mouse CD36 with a binding affinity of 1× ^10⁻⁸ M or less, 1× ^10⁻⁹ M or less, or 1× ^10⁻¹⁰ M or less; optionally, the binding affinity is measured by the equilibrium dissociation constant ( _KD ) of the mCD36 peptide with SEQ ID NO: 60 or 61.

(c) The antibody binds to rhesus CD36 with a binding affinity of 1× ^10⁻⁸ M or less, 1× ^10⁻⁹ M or less, or 1× ^10⁻¹⁰ M or less; optionally, the binding affinity is measured by the equilibrium dissociation constant ( _KD ) of the rhesus CD36 peptide with SEQ ID NO: 62 or 63.

(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 human CD36; optionally, the antibody has an _IC50 of 5 nM or less, 1 nM or less, 0.5 nM or less, or 0.1 nM or less at a concentration of 1-10 μg/mL of oxLDL.

(e) 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 U937 cells; optionally, wherein the antibody has an IC50 of 5 nM or less, 1 nM or less, 0.5 nM or less, or 0.1 nM or less at a concentration of _{1-10 μg} /mL of oxLDL; and/or

(f) 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 mouse CD45+TIL; optionally, the antibody has an _IC50 of 5 nM or less, 1 nM or less, 0.5 nM or less, or 0.1 nM or less at a concentration of 1-10 μg/mL of oxLDL.

This disclosure also provides embodiments of the anti-CD36 antibody disclosed herein, including embodiments in which: (i) the antibody is a human antibody, a humanized antibody, or a chimeric antibody; (ii) the antibody comprises a fusion with a protein; optionally, a fusion with an immunostimulatory cytokine (e.g., IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, or IFN-α); (iii) the antibody is a full-length IgG antibody, optionally wherein the IgG antibody has an isotype selected from IgG1, IgG2, IgG3, and IgG4; (iv) the antibody comprises an Fc region variant, optionally an Fc region variant that alters effector function, and/or a variant that alters antibody half-life; (v) the antibody is an antibody fragment, optionally selected from F(ab') _2. The antibody comprises 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 CD36-mediated diseases or conditions; or (vii) the antibody is a multispecific antibody; optionally, it is a bispecific antibody.

In at least one embodiment, this disclosure provides an isolated polynucleotide or a vector containing a polynucleotide, wherein the sequence of said polynucleotide encodes the anti-CD36 antibody of this disclosure or a polypeptide chain of the anti-CD36 antibody of this disclosure. In at least one embodiment, said isolated polynucleotide or vector contains a sequence encoding a polypeptide chain encoding the anti-CD36 antibody of this disclosure. In at least one embodiment of said isolated polynucleotide or vector, the encoded polypeptide chain has an amino acid sequence comprising the following:

(a) CDR-H1 contains the amino acid sequence of SEQ ID NO:3, 21, 24 or 27; CDR-H2 contains the amino acid sequence of SEQ ID NO:4, 28, 31, 34, 37, 40 or 43; and CDR-H3 contains the amino acid sequence of SEQ ID NO:5;

(b) CDR-L1 contains an amino acid sequence selected from SEQ ID NO:7; CDR-L2 contains an amino acid sequence selected from SEQ ID NO:8, 12 or 15; and CDR-L3 contains an amino acid sequence selected from SEQ ID NO:9, 13 or 18.

(c) A heavy chain variable domain ( _VH ) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO:2, 20, 23, 26, 30, 33, 36, 39 or 42;

(d) A light chain variable domain ( _VL ) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO: 6, 11, 14 or 17;

(e) a heavy chain (HC) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO: 44, 48, 50, 51, 52, 53, 54, 55, 56 or 57; and/or

(f) A light chain (LC) amino acid sequence that has at least 90% identity with a sequence selected from SEQ ID NO:45, 46, 47 or 49.

In at least one embodiment, this disclosure also provides an isolated host cell containing a polynucleotide or vector encoding a polypeptide chain of the anti-CD36 antibody or an anti-CD36 antibody of this disclosure. In at least one embodiment, this disclosure also provides a method for generating the anti-CD36 antibody of this disclosure, comprising culturing a host cell containing a polynucleotide or vector encoding an anti-CD36 antibody, thereby generating the antibody.

In at least one embodiment, this disclosure provides a pharmaceutical composition comprising the anti-CD36 antibody of this disclosure and a pharmaceutically acceptable carrier; optionally, said composition further comprises a chemotherapeutic agent and/or an antibody specific to an immune checkpoint molecule.

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

In at least one embodiment, this 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 containing a specific antibody against 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.

Attached Figure Description

A better understanding of the novel features and advantages of this disclosure will be obtained by referring to the following detailed description, which illustrates illustrative embodiments utilizing the principles of this disclosure, and the accompanying drawings (also referred to herein as “Figures”), wherein:

Figures 1A, 1B, 1C, and 1D depict the ELISA results of the exemplary anti-CD36 antibody (12P109, A8A) of this disclosure binding to recombinant human CD36.ECD (Figure 1A, Figure 1C) or mouse CD36.ECD (Figure 1B, Figure 1D) in the form of full-length human IgG1. The ELISA was performed as described in Example 1.

Figures 2A, 2B, 2C, 2D, 2E, and 2F depict the ELISA results of the exemplary anti-CD36 antibody (12P109 and 117 variants) of this disclosure binding to recombinant human CD36.ECD. The ELISA was performed as described in Example 1.

Figures 3A, 3B, 3C, 3D, and 3E depict the results of SEC-UPLC analysis of the anti-CD36 antibodies (12P109, A8A, and 117 variants) as described in Example 1.

Figures 4A, 4B, 4C, 4D, 4E, and 4F depict the results of cell surface binding analysis of the exemplary anti-CD36 antibody (117 variant) of this disclosure with stable F293 cells overexpressing human CD36 (“hCD36”) (Figures 4A and 4B), rhesus monkey CD36 (Figures 4C and 4D), or mouse CD36 (“mCD36”) (Figures 4E and 4F) by flow cytometry, as described in Example 2.

Figures 5A and 5B depict graphs of extracted flow cytometry data, showing the activity of the anti-CD36 antibody of this disclosure in blocking oxLDL binding (Figure 5A) and oxLDL uptake (Figure 5B) in U937 cells, as described in Example 3.

Figures 6A, 6B, and 6C depict plots extracted from flow cytometry data, illustrating the oxLDL uptake blocking activity exhibited by anti-CD36 antibodies (12P109 and 117 variants) in F293/hCD36 or F293/mCD36 cells, as described in Example 3.

Figures 7A, 7B, 7C, 7D, and 7E depict data showing the ability of the anti-CD36 antibodies (12P109, A8A, and 117 variants) of this disclosure to inhibit oxLDL uptake in TILs of mouse models of colon cancer (CT26, MC38), liver cancer (BNL 1MEA.7R.1), lung cancer (LL2), and melanoma (B16F10), as described in Example 4.

Figures 8A, 8B, 8C, 8D, 8E, and 8F depict graphs illustrating data demonstrating the ability of the anti-CD36 antibodies (12P109 and 117 variants) of this disclosure to inhibit M2 macrophage polarization (Figures 8A and 8B) and oxLDL-induced M2 macrophage activation (Figures 8C, 8D, 8E, and 8F), as described in Example 5.

Figure 9 depicts the tumor growth curve results measured by bioluminescence in MYC ^OE /p53 ^KO HCC model mice after treatment with the anti-CD36 antibody 117_30DA as described in Example 6.

Figures 10A, 10B, and 10C depict the results measured in β-catenin ^OE /MYC ^OE HCC model mice after treatment with the anti-CD36 antibody 117_DA57E as described in Example 6. Figure 10A shows the tumor growth curve results measured by bioluminescence. Figure 10B shows the endpoint liver weight results. Figure 10C shows the plasma ALT (alanine aminotransferase) activity analysis results.

Detailed Implementation

This 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, particularly the function of CD36 in 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 of tumor cells, tumor-associated macrophages (TAMs), MDSCs, regulatory T cells, and CD8 T cells, and in maintaining or surviving (or dying, as appropriate).

Therefore, any composition or formulation containing the anti-CD36 antibody of this disclosure is considered as a therapeutic agent for treating diseases mediated by the function of CD36 in fatty acid transport (e.g., cancer). Furthermore, the anti-CD36 antibody of this disclosure is considered as a therapeutic agent in combination with other therapeutic agents, such as cell therapy, cytokines, and other biological agents or drugs that alter the tumor microenvironment and/or increase the immune response, antibody-drug conjugates, antibodies that regulate 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, and ICOS.

Terminology and Technical Overview

In the description herein and the appended claims, unless the context clearly indicates otherwise, the singular form “a” includes plural references. Thus, for example, “a protein” refers to more than one protein, and “a compound” refers to more than one compound. It should be further noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended as a prior basis for using exclusive terms such as “only” or “just” or using negative terms when reciting elements of a claim. The use of “comprising” and “including” is interchangeable and not intended to be restrictive. It should also be understood that when the term “comprising” is used in the description of various embodiments, those skilled in the art will understand that in certain specific cases, embodiments may also be described alternatively using the language “consistently of…” or “composed of…”.

When a range of values is provided, unless the context explicitly specifies otherwise, it should be understood that every intermediate integer between the upper and lower limits of the range, and one-tenth of every intermediate integer, unless the context explicitly specifies otherwise, as well as any other stated or intermediate value within the range, are covered within this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and are also covered within this invention, except for any specifically excluded limitations within the range. When the range includes one or both of the limitations, the range excluding (i) one or (ii) both of the included limitations is also included within this invention. For example, “1 to 50” includes “2 to 25”, “5 to 20”, “25 to 50”, “1 to 10”, etc.

Generally, the nomenclature used herein, as well as the techniques and procedures described herein, include those that are well understood and commonly used by one of ordinary skill in the art, such as the following common techniques and methodologies: Sambrook et al., Molecular Cloning : A Laboratory Manual (2nd Edition), Volumes 1–3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 (hereinafter “Sambrook”); Existing Protocols in Molecular Biology , edited by FMAusubel et al., Current Protocols, Greene Publishing Co., Ltd. A joint venture between Associates, Inc. and John Wiley & Sons, Inc. (2011 Supplement) (hereinafter referred to as “Ausubel”); Antibody Engineering , Volumes 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 edition, Humana Press (2014); Therapeutic Antibodies: From Laboratory to Clinic , edited by Z. An, John Wiley & Sons, Hoboken, NJ (2009); and Phage Display , edited by Tim Clackson and Henry B. Lowman, Oxford University Press, UK (2004).

All publications, patents, patent applications and other documents cited in this disclosure are incorporated herein by reference in their entirety for all purposes, to the extent that each individual publication, patent, patent application or other document is individually identified as incorporated herein by reference for all purposes.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. For the purposes of this disclosure, the following description of terminology will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

As used herein, “CD36” refers to the CD36 protein, and as used herein, it encompasses the CD36 protein in humans, cynomolgus monkeys, mice, and any isotypes 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 the appended sequence listing.

The terms "CD36-mediated condition" or "CD36-mediated disease" as used herein encompass any medical condition related to the specific binding of ligands to the cell surface protein CD36. For example, the specific binding of CD36 to lipids and/or fatty acids plays a role in modulating or increasing the immunosuppressive capacity of TAMs and regulatory T cells in the tumor microenvironment. Therefore, CD36-mediated disease can include, but is not limited to, any disease or condition mediated by and/or responsive to CD36 antagonists or inhibitors, including but not limited to cancer.

As used herein, “antibody” refers to a molecule comprising one or more polypeptide chains that specifically binds to or is immunoreactive with a specific antigen. Exemplary antibodies disclosed herein 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 mimics).

"Anti-CD36 antibody" or "CD36-binding antibody" refers to an antibody that binds to CD36 with sufficient affinity such that the antibody can be used as a therapeutic and/or diagnostic agent targeting CD36. In some embodiments, the anti-CD36-specific antibody binds to unrelated non-CD36 antigens to less than about 20%, about 15%, about 10%, or about 5% of the antibody's binding to CD36, as measured by, for example, radioimmunoassay (RIA) or surface plasmon resonance (SPR). In some embodiments, the anti-CD36 antibody of this disclosure has a dissociation constant (K_D) of <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., from 10 ^{^-8} M to 10 ^{^-13} M, e.g., from 10 ^{^-9} M to 10 ^{^-13} _M ). The amino acid sequence of an exemplary CD36 protein of this disclosure is provided in Table 2 below and in the attached sequence listing.

The terms "full-length antibody," "complete antibody," or "whole antibody" are used interchangeably in this article and refer to antibodies that have a structure substantially similar to that of natural antibodies or that contain a heavy chain with an Fc region as defined herein.

"Antibody fusion" refers to an antibody covalently conjugated (or fused) to a polypeptide or protein, typically via a linker to the end of the antibody light chain (LC) or heavy chain (HC). Exemplary antibody fusions contemplated in this disclosure may include an anti-CD36 antibody fused to a protein via a linker, said protein being a T-cell activating or immunostimulatory cytokine, such as IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, or IFN-α.

An "antibody fragment" is a portion of a full-length antibody that can bind 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 ; biantibodies; linear antibodies; monovalent or single-arm antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are respectively named α, δ, ε, γ, and μ.

"Variable regions" or "variable domains" refer to domains of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains ( _VH and _VL , respectively) of the heavy and light chains of natural antibodies typically have similar structures, each containing four conserved frame regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman and colleagues, p. 91). A single _VH or _VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies binding to specific antigens can be isolated using the _VH or _VL domains of antibodies binding to antigens to screen libraries of complementary _VL or _VH domains, respectively (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

As used herein, “hypervariant region” or “HVR” refers to each region of the antibody variable domain that is hypervariable in the sequence and/or forms a structurally defined loop (“hypervariant loop”). Typically, natural antibodies contain four chains with six HVRs: three in the heavy chain variable domain, V _H (HVR-H1, HVR-H2, HVR-H3), and three in the light chain variable domain, V _L (HVR-L1, HVR-L2, HVR-L3). The HVRs typically contain amino acid residues from the hypervariant loop and/or from the “complementarity-determining region” (CDR). Numerous hypervariant region descriptions are in use and are covered herein. The Kabat complementarity-determining region (CDR) is based on sequence variability and is the most commonly used (Kabat et al., Immunologically Significant Protein Sequences, 5th Edition, Department of Public Health, National Institutes of Health, Bethesda, MD (1991)). Chothia refers to the location of the structural ring (Chothia and Lesk, J.Mol.Biol.196:901-917 (1987)). The AbM hypervariable region represents a compromise between the Kabat CDR and the Chothia structural ring and is used through Oxford Molecular's AbM antibody modeling software. The “Contact” hypervariable region is based on the analysis of available complex crystal structures. The residue ranges of the hypervariable region defined under these systems are shown in the table below.

In addition to the systems mentioned above, the international ImMunoGeneTics information system (referred to as IMGT/V-Quest) can be used to identify HVR and CDR. This system is described in Brochet, X. et al., Nucl. Acids Res. 36, W503-508 (2008). PMID: 18503082; and is available online at www.imgt.org/IMGT_vquest/input. IMGT/V-Quest uses unique IMGT identifiers to analyze alignments with the nearest germline V gene variable region nucleotide sequence to identify HVR and CDR.

As used herein, a hypervariable region (HVR) may include the following extended or alternative hypervariable regions: 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); 26-33, 26-35, or 31-35 (HVR-H1); 51-58, 50-61, or 50-66 (H2); and 97-110, 97-112, 99-110, or 99-112 (H3) within the V- _H domain. For each of these definitions, the variable domain residues are numbered as per the method described by Kabat et al. (above).

As used herein, the “complementarity-determining region” or “CDR” refers to the region within the HVR of a variable domain that has the highest sequence variability and/or is involved in antigen recognition. Typically, a natural antibody contains four chains with six HVRs; three in the heavy chain variable domain, 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 appear at the positions of amino acid residues in the variable domain: 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).

"Frame" or "FR" refers to the variable domain residues other than the HVR residues. A variable domain FR typically consists of four domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences usually appear in V _H (or V _L ) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

Unless otherwise stated, the positions of residues in HVR, CDR, FR and other residues in the variable domain are numbered in this paper as per Kabat et al. (above).

"Natural antibodies" refer to naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. Each heavy chain has a variable region (V_{_H}), also called a variable heavy domain or heavy chain variable domain, from the N-terminus to the C-terminus, followed by three constant domains (CH₁, CH₂, and CH₃). Similarly, each light chain has a variable region (V_{_L} ), also called a variable light domain or light chain variable domain, from the N-terminus to the C-terminus, followed by a constant light (CL) domain. Based on the amino acid sequence of their constant domains, the light chains of antibodies can be classified into one of two types: kappa (κ) and lambda (λ).

As used herein, “monoclonal antibody” means an antibody obtained from a substantially homogeneous group of antibodies, i.e., individual antibodies comprising said group are identical and/or bind to the same epitopes, except for possible variant antibodies (e.g., variant antibodies contain mutations that occur naturally or during monoclonal antibody production and are typically present in small amounts). In contrast to polyclonal antibody preparations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody prepared targets a single determinant on an antigen. Therefore, the term “monoclonal” indicates that the antibody’s properties are obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies can be prepared using 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, such methods for preparing monoclonal antibodies described herein, and other exemplary methods.

"Chimeric antibody" refers to an antibody in which part of the heavy chain and/or light chain originates from a specific source or species, while the rest of the heavy chain and/or light chain originates from antibodies from different sources or species.

"Humanized antibody" refers to a chimeric antibody comprising an amino acid sequence from a non-human CDR and an amino acid sequence from a human FR. In some embodiments, a humanized antibody may comprise substantially all of at least one (and typically two) variable domains, wherein all or substantially all of the CDRs correspond to those CDRs of the non-human antibody, and all or substantially all of the FRs correspond to those FRs of the human antibody. Optionally, a humanized antibody may comprise at least a portion of the antibody constant region derived from a human antibody. The "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization.

"Human antibody" refers to an antibody that has an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or an antibody derived from a non-human source using a human antibody library or other human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues.

The “human consensus framework” represents the framework of amino acid residues that most frequently occur in the selection of the human immunoglobulin _VL or _VH framework sequence. Typically, the selection of the human immunoglobulin _VL or _VH sequence is derived from a subgroup of variable domain sequences. Typically, the subgroup is the one described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for _VL , the subgroup is subgroup kappa I as described in Kabat et al. (see above). In one embodiment, for _VH , the subgroup is subgroup III as described in Kabat et al. (see above).

The term "recipient human framework" as used herein refers to a framework comprising an amino acid sequence of a light chain variable domain ( _VL ) framework or a heavy chain variable domain ( _VH ) framework derived from the human immunoglobulin framework or the human consensus framework. A recipient human framework "derived from" the human immunoglobulin framework or the human consensus framework may contain the same amino acid sequence, or it may contain amino acid sequence variations. In some embodiments, the number of amino acid variations is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer. In some embodiments, the _VL recipient human framework is sequence-identical to the _VL human immunoglobulin framework sequence or the human consensus framework sequence.

The “Fc region” refers to a dimeric complex containing the C-terminal polypeptide sequence of the immunoglobulin heavy chain, which is obtained by digesting the intact antibody with papain. The Fc region can contain native or variant Fc sequences. Although the boundaries of the Fc sequence of the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc sequence is generally defined as extending from an amino acid residue at approximately Cys226 or approximately Pro230 to the C-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 immunoglobulins typically contains two constant domains, a CH2 domain and a CH3 domain, and optionally includes a CH4 domain.

"Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a natural human FcR. In some embodiments, the FcR is a receptor (γ receptor) that binds to IgG antibodies and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splice forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an activation motif (ITAM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an inhibitory motif (ITIM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain (see, for example, Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). As used in this article, FcR also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and the regulation of immunoglobulin homeostasis. FcR has been reviewed in, for example, 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).

A "multispecific antibody" is an antibody with at least two different binding sites, each with different binding specificity. Multispecific antibodies can be full-length antibodies or antibody fragments, and the different binding sites can allow each binding site to bind to a different antigen, or the different binding sites can bind to two different epitopes of the same antigen.

"Fv fragments" refer to antibody fragments containing complete antigen recognition and binding sites. This region consists of a dimer of a tightly bound heavy-chain variable domain and a light-chain variable domain, and its properties can be covalent, as in scFv. In this configuration, the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the _VH - _VL dimer. Generally, six CDRs or a subset thereof confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specifically targeting the antigen) has the ability to recognize and bind antigens, although typically with an affinity lower than that of the entire binding site.

"Fab fragment" refers to an antibody fragment containing both variable and constant domains of the light chain and both variable and first constant domains (CH1) of the heavy chain. "F(ab') ₂ fragment" comprises a pair of Fab fragments, typically covalently linked near their carboxyl terms by a hinge cysteine residue between them. Other chemical conjugations of antibody fragments are also known in the art.

As used herein, an "antigen-binding arm" refers to an antibody component that has the ability to specifically bind to a target molecule. Typically, the antigen-binding arm is a complex of immunoglobulin polypeptide sequences, such as CDR and/or variable domain sequences of the immunoglobulin light and heavy chains.

"Single-chain Fv" or "scFv" refers to an antibody fragment containing the _VH and _VL domains of the antibody, where these domains are contained within a single polypeptide chain. Typically, Fv polypeptides also include a polypeptide linker between the _VH and _VL domains, which allows the scFv to form the desired antigen-binding structure.

"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 mate (e.g., an antigen). "Binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its mate Y is typically expressed by the equilibrium dissociation constant (K_{_D} ). Affinity can be measured using methods commonly known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

"Specific binding" or "specific binding" refers to an antibody binding to an antigen with an affinity value not exceeding about 1 × ^10⁻⁷ M. In some embodiments, the antibody may have a secondary affinity for antigens other than those it specifically binds to, where "secondary affinity" typically refers to the antibody binding to a secondary antigen with an affinity value exceeding about 10 nM, as described elsewhere herein. When an antibody may have a secondary affinity for a secondary antigen, such antibodies will still specifically bind to the primary antigen.

"Isolated antibody" refers to an antibody that has been isolated from components of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, such as by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods used to assess antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87.

"Effective functions" refer to the biological activities attributed to the Fc region of an antibody, which vary across antibody isotypes. Examples of antibody effector functions include: Clq binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

"Immune conjugates" refer to antibodies conjugated to one or more heterologous molecules (including but not limited to cytotoxic agents).

"Treatment," "treat," or "treating" refers to a clinical intervention that attempts to alter the natural course of a disease in an individual to be treated, and may be used for prevention or administered during the clinicopathological process. The desired outcomes of treatment may include, but are not limited to, preventing the onset or recurrence of the disease, relieving symptoms, mitigating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of progression, improving or alleviating the disease state, and mitigating or improving prognosis. For example, treatment may include administering to a subject a therapeutically effective amount of a pharmaceutical preparation containing an anti-CD36 antibody to delay or slow the development of a disease or condition mediated by CD36 and/or its binding to a ligand, or a disease or condition in which CD36 may play a role in pathogenesis and/or progression.

"Pharmaceutical formulation" means a preparation in a form that allows the bioactivity of the active ingredient to be effective and that does not contain any additional components that are toxic to a subject administered the formulation. A pharmaceutical formulation may include one or more active agents. For example, a pharmaceutical formulation may include an anti-CD36 antibody as the sole active agent of the formulation, or it may include an anti-CD36 antibody and one or more additional active agents, such as inhibitors of immune checkpoint molecules.

As used herein, "sole active agent" means an active agent in a pharmaceutical formulation that is the only active agent present in the formulation and provides, or is expected to provide, a relevant pharmacological effect to treat the condition to be treated in the subject. A pharmaceutical formulation containing a sole active agent does not exclude the presence of one or more inactive agents, such as pharmaceutically acceptable carriers. "Inactive agent" is an agent that is not expected to provide or otherwise significantly enhance the relevant pharmacological effect intended to treat the condition in the subject.

"Pharmaceutically acceptable carriers" refer to components in a pharmaceutical preparation other than the active ingredient that are non-toxic to the subject to which the drug is administered. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

As used herein, “immune checkpoint molecules” refer to molecules that regulate immune system pathways and thereby prevent them from unnecessarily attacking cells. Many immune checkpoint molecules (both inhibitory and co-stimulatory) are targets for immunotherapy in the treatment of cancer and viral infections (e.g., using blocking antibodies to block immunosuppression or using agonists to promote immune stimulation). Exemplary immune checkpoint molecules for cancer immunotherapy include, but are 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, and ICOS.

"Therapeutic effective amount" refers to the amount of an active ingredient or agent (e.g., a pharmaceutical preparation) that achieves the desired therapeutic or preventative outcome, such as treating or preventing a subject's disease, symptom, or condition. In the case of CD36-mediated diseases or conditions, a therapeutically effective amount of a therapeutic agent is an amount that reduces, prevents, inhibits, and/or alleviates one or more symptoms associated with the disease, symptom, or condition to a certain extent. For cancer therapies, in vivo efficacy can be measured, for example, by assessing the growth of the primary tumor, the occurrence and/or growth of secondary tumors, the occurrence and/or number of metastases, the duration, severity, and/or recurrence of symptoms, the response rate (RR), the duration of response, and/or quality of life.

As used herein, "simultaneous" means the administration of two or more therapeutic agents, wherein at least a portion of the administration overlaps in time. Therefore, simultaneous administration includes a dosing regimen in which administration of one or more other agents continues after the discontinuation of administration of those other agents.

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

Detailed description of various implementation schemes

I.CD36

CD36 is a multifunctional transmembrane glycoprotein that serves as a cell surface receptor with a wide range of ligands. Typically, CD36 possesses two distinct binding domains for binding platelet-reactive proteins to other lipid-based ligands, such as oxidized low-density lipoprotein (oxLDL), anionic phospholipids, long-chain fatty acids, and bacterial diacytized lipopeptides. Cellular responses mediated by CD36 binding to these ligands are believed to include fatty acid metabolism, dietary fat processing, angiogenesis, and inflammatory responses. CD36 also acts as a co-receptor for the TLR4:TLR6 heterodimer, thereby promoting inflammation in monocytes/macrophages. It is believed that upon binding with ligands (e.g., oxidized LDL (“oxLDL”)), CD36 interacts with the TLR4:TLR6 heterodimer, and the complex is internalized, thereby triggering an inflammatory response that leads to the production of NF-kappa-B-dependent CXCL1, CXCL2, and CCL9 cytokines via the MyD88 signaling pathway, the production of CCL5 cytokines via the TICAM1 signaling pathway, and the secretion of IL-1B through the initiation and activation of the NLRP3 inflammasome. Other co-receptors that interact with CD36 have also been described.

The sequence and annotation of human CD36 (also referred to herein as “hCD36”) can be found at UniProt entry P16671, and the full-length 472-amino acid sequence of isotype 1 is shown herein as SEQ ID NO:58. The sequence and annotation of mouse CD36 (also referred to herein as “mCD36”) can be found at UniProt entry Q08857, and the full-length 472-amino acid sequence is shown herein as SEQ ID NO:60. Table 1 below provides an overview of the sequences of human and mouse CD36 peptides used in this disclosure and their sequence identifiers. These sequences are also included in the accompanying sequence listing.

Table 1: Human and mouse CD36 polypeptides

II. Anti-CD36 antibody

In some embodiments, this disclosure provides the structure of anti-CD36 antibodies in terms of the amino acid and encoding nucleotide sequences of various well-known immunoglobulin features, such as CDR, FR, _VH , _VL domains, and full-length heavy and light chains. Table 2 below provides an overview of the anti-CD36 antibodies of this disclosure generated and functionally characterized as described in the examples. The relevant sequence and sequence identifier for each antibody are provided in Table 2 and are also included in the accompanying sequence listing.

Table 2: Anti-CD36 antibody sequences

1. Binding affinity and functional properties of anti-CD36 antibodies

In some implementations, the equilibrium dissociation constant (K_{_D} ) for the binding of the anti-CD36 antibody to CD36 provided herein is <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 ^{^-8} M or lower, from 10 ^{^-8} M to 10 ^{^-13} M, e.g., from 10 ^{^-9} M to 10 ^{^-13} M).

Considering the various anti-CD36 antibodies disclosed herein, including antibodies capable of binding with high affinity 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 this disclosure bind to hCD36 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× ^10⁻¹¹ M or less. In some embodiments, said binding affinity is measured as the equilibrium dissociation constant (K_{_D}) of binding to the hCD36 peptide of SEQ ID NO:58 or SEQ ID NO:59. In some embodiments, the anti-CD36 antibody of this disclosure binds to 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× ^10⁻¹¹ M or less. In some embodiments, the binding affinity is measured as the equilibrium dissociation constant ( _KD ) of binding to the mCD36 peptide of SEQ ID NO:60 or SEQ ID NO:61. In some embodiments, the anti-CD36 antibody of this disclosure binds to rhesus macaque CD36 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× ^10⁻¹¹ M or less. In some embodiments, the binding affinity is measured as the equilibrium dissociation constant ( _KD ) of binding to the rhesus macaque CD36 peptide of SEQ ID NO:62 or SEQ ID NO:63. In some embodiments, the anti-CD36 antibody of this disclosure binds 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× ^10⁻¹¹ M or less.

Typically, the binding affinity between a ligand and its receptor can be determined using any of a variety of assays and expressed in a variety of quantitative values. Specific CD36 binding assays for determining antibody affinity are disclosed in the embodiments 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 assays using techniques such as Western blotting, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), sandwich immunoassay, surface plasmon resonance-based assays (e.g., the BIAcore assay as described in WO2005/012359), immunoprecipitation assays, fluorescence immunoassays, protein A immunoassays, flow cytometry, and fluorescence activated cell sorting (FACS) assays, etc.

Therefore, in some embodiments, binding affinity is expressed as a K_{_D} value and reflects intrinsic binding affinity (e.g., having a minimized affinity effect). The anti-CD36 antibody of this disclosure exhibits strong binding affinity to the hCD36 peptide of SEQ ID NO:58, for example, exhibiting a K_{_D} value between 10 nM and 1 pM. Therefore, the anti-CD36 antibody of this disclosure can compete with antibodies that have lower affinity for the same or overlapping epitopes of CD36.

In some embodiments, the anti-CD36 antibodies provided herein reduce, inhibit, and/or completely block the binding of the ligand to CD36, as well as the immunomodulation and/or immune signaling mediated by ligand-CD36 binding, including maintaining TAM in the tumor microenvironment. The ability of the antibody to inhibit these immunomodulatory and immune signaling pathways mediated by ligand-CD36 binding can be measured in vitro using known cell-based assays, including those described in the embodiments of this disclosure.

Therefore, in some embodiments, the CD36 antibody of this disclosure is characterized by one or more of the following functional properties, which are based on the ability of CD36-mediated pathways to reduce, inhibit, and/or completely block intracellular signal transduction.

In at least one embodiment, the anti-CD36 antibody binds to human CD36 with a binding affinity of 1× ^10⁻⁸ M or less, 1× ^10⁻⁹ M or less, or 1 ^{×10⁻¹⁰ M} or less; optionally, said binding affinity is measured by the equilibrium dissociation constant ( _KD ) with the hCD36 peptide of SEQ ID NO: 58 or 59.

In at least one embodiment of the anti-CD36 antibody, the antibody binds to mouse CD36 with a binding affinity of 1× ^10⁻⁸ M or less, 1× ^10⁻⁹ M or less, or 1× ^10⁻¹⁰ M or less; optionally, the binding affinity is measured by the equilibrium dissociation constant ( _KD ) of the mCD36 peptide with SEQ ID NO: 60 or 61.

In at least one embodiment of the anti-CD36 antibody, the antibody binds to rhesus monkey CD36 with a binding affinity of 1× ^10⁻⁸ M or less, 1× ^10⁻⁹ M or less, or 1× ^10⁻¹⁰ M or less; optionally, the binding affinity is measured by the equilibrium dissociation constant ( _KD ) of the rhesus monkey CD36 peptide of SEQ ID NO: 62 or 63.

In at least one embodiment of the anti-CD36 antibody, the antibody inhibits at least 65%, at least 75%, at least 85%, at least 95%, or at least 100% of the uptake of CD36-dependent oxidized LDL in F293 cells overexpressing human CD36; wherein the antibody has an _IC50 of 5 nM or less, 1 nM or less, 0.5 nM or less, or 0.1 nM or less at a concentration of 1-10 μg/mL of oxLDL.

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%; wherein, at an oxLDL concentration of 1-10 μg/mL, the antibody has an _IC50 of 5 nM or less, 1 nM or less, 0.5 nM or less, or 0.1 nM or less.

In at least one embodiment of the anti-CD36 antibody, the antibody inhibits at least 65%, at least 75%, at least 85%, at least 95%, or at least 100% of the uptake of CD36-dependent oxidized LDL in mouse CD45+TIL; optionally, the antibody has an _IC50 of 5 nM or less, 1 nM or less, 0.5 nM or less, or 0.1 nM or less at a concentration of 1-10 μg/mL of oxLDL.

2. Anti-CD36 antibody fragment

In some embodiments, the anti-CD36 antibody of this disclosure may be an antibody fragment. Antibody fragments that can be used with the binding determinants of this disclosure include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , Fv, scFv fragments, monovalent, single-domain antibodies, one-arm or single-arm antibodies, and other fragments described herein and known in the art. Therefore, in some embodiments of the anti-CD36 antibody of this 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.

For reviews of various antibody fragments, see, for example, Hudson et al., Nat. Med. 9:129-134 (2003). For reviews of scFv fragments, see, for example, Pluckthun, Monoclonal Antibody Pharmacology, Vol. 113, edited by Rosenburg and Moore (Springer, New York), pp. 269-315 (1994); see also WO93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For descriptions of Fab and F(ab') ₂ fragments containing salvage receptor-binding epitope residues and having an increased in vivo half-life, see U.S. Patent No. 5,869,046. Other monovalent antibody forms are described, for example, in WO2007/048037, WO2008/145137, WO2008/145138, and WO2007/059782. Monovalent single-arm antibodies are described, for example, in WO2005/063816. Biantibodies are antibody fragments having two antigen-binding sites, which can 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)).

In some embodiments, the antibody fragment is a single-domain antibody that contains all or part of the heavy chain variable domain or all or part of the light chain variable domain. In some embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent No. 6,248,516).

Antibody fragments can be produced by a variety of techniques, including but not limited to the proteolytic digestion of intact antibodies and the production of them through recombinant host cells (such as Escherichia coli or bacteriophages), as described herein.

3. Chimeric, humanized, and human anti-CD36 antibodies

In some embodiments, the anti-CD36 antibody of this disclosure may be a chimeric antibody. (See, for example, chimeric antibodies described in U.S. Patent No. 4,816,567; and 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 mouse, rat, hamster, rabbit, or non-human primate (e.g., a monkey)) and a human constant region. In some embodiments, the chimeric antibody is a “class-switching” antibody, wherein the class or subclass has been changed from that of the parent antibody. Consideration may be given to chimeric antibodies that may include their antigen-binding fragments.

In some embodiments, the anti-CD36 antibody of this disclosure is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. Typically, humanized antibodies contain one or more variable domains, wherein the HVR, CDR (or portions thereof) are derived from the non-human antibody, and the FR (or portions thereof) are derived from the human antibody sequence. Optionally, humanized antibodies may also contain at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., an antibody from which the CDR residues are derived) to restore or improve antibody specificity or affinity.

Humanized antibodies and their preparation methods are reviewed in, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989); US patents 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., M Methods 36:25-34 (2005) (describes SDR(a-HVR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describes “surface reshaping”); Dall’Acqua et al., Methods 36:43-60 (2005) (describes “FR reshaping”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describes a “guided selection” approach to FR reshaping).

Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using a “best fit” method (see, for example, Sims et al., J. Immunol. 151:2296 (1993)); frame regions of consensus sequences of human antibodies derived from specific subgroups of light or heavy chain variable regions (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); and Presta et al., J. Immunol. 151:2623 (1993)). 3)); human mature (somatic mutation) frame regions or human germline frame regions (see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frame regions derived from screening FR libraries (see, for example, Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

In some embodiments, the anti-CD36 antibody of this disclosure may be a human antibody. Human antibodies can be produced using various 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 transgenic animals that have been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of human immunoglobulin loci, which replace endogenous immunoglobulin loci, or are present extrachromosomally or randomly integrated into the chromosome of the animal. In such transgenic mice, endogenous immunoglobulin loci are typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, the XENOMOUSE ^™ technology in U.S. Patent Nos. 6,075,181 and 6,150,584; the technology in U.S. Patent No. 5,770,429; the KM technology in U.S. Patent No. 7,041,870; and the technology in U.S. Patent Application Publication No. US2007/0061900. The human variable region of an intact antibody derived from such animals can be further modified, for example, by combining it with different human constant regions.

Human antibodies can also be produced using hybridoma-based methods. Human myeloma and mouse-human allogeneic myeloma cell lines for producing human monoclonal antibodies have been described. See, for example, Kozbor J. Immunol, 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Technology and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol. 147:86 (1991). Human antibodies produced via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006). Other methods include, for example, those described in U.S. Patent No. 7,189,826 (Description of Monoclonal Human IgM Antibodies from Hybridoma Cell Lines). Human trioma technology has also been 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).

Human antibodies can also be generated by isolating variable domain sequences of Fv clones selected from human phage display libraries. These variable domain sequences can then be combined with desired human constant domains. The techniques for selecting human antibodies from antibody libraries are described below.

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 to find antibodies with the desired improved functional properties (e.g., binding affinity or cross-reactivity). For example, various methods are known in the art for generating phage display libraries and screening such libraries to find variant antibodies with improved binding properties. Other methods for generating such library-derived antibodies can be found, for example, Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., 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, 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):119-132 (2004).

5. Multispecific antibodies and antibody fusions

In at least one embodiment, the anti-CD36 antibody of this disclosure may be a multispecific antibody, such as a bispecific antibody. In some embodiments, the multispecific antibody has at least two distinct binding sites, each binding site having binding specificity against a different antigen, wherein at least one specifically binds to CD36. In at least one embodiment, the multispecific antibody is considered to be a bispecific antibody, which includes specificity against CD36 and specificity against another antigen mediating immune regulation, immune signaling, and/or expressed on cancer or tumor cells. For example, the other specificity may be against immune checkpoint molecules 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.

Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of heavy-light chain pairs of two immunoglobulins with different specificities (see, for example, Milstein and Cuello, Nature, 305:537 (1983), WO93/08829, and Traunecker et al., EMBO J.10:3655 (1991)). "Knob-in-hole" engineering can also be used to produce bispecific antibodies for use with the anti-CD36 antibody of this disclosure. Techniques for knob-in-hole engineering are known in the art and are described, for example, in U.S. Patent No. 5,731,168.

Multispecific antibodies can also be produced through engineered "electrostatic redirection" effects, which favor the formation of Fc-heterodimeric antibody molecules rather than homodimers (WO2009/089004A1); crosslinking two or more antibodies or fragments (see, for example, US Patent No. 4,676,980 and Brennan et al., Science 229:81 (1985)); and bispecific antibodies can be produced using leucine zippers (see, for example, Kostelny et al., J. Immunol. 148(5):1547-1553(19)). 92)); using “double antibody” technology to prepare bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); using single-chain Fv (scFv) dimers (see, for example, Gruber et al., J. Immunol. 152:5368 (1994)); or trispecific antibodies (see, for example, Tutt et al., J. Immunol. 147:60 (1991)).

In at least one embodiment, the anti-CD36 antibody provided herein may comprise an antibody fusion with a protein. Methods for preparing and using antibody fusions or fusion proteins are well known in the art. Typically, antibodies are covalently conjugated (or fused) to proteins, usually via a linker polypeptide. The conjugation may occur via the terminal of the light chain (LC) or heavy chain (HC) of the antibody. Antibody fusions may also be prepared from antibody fragments. In one exemplary embodiment of an antibody fusion contemplated by this disclosure, the fusion comprises a full-length anti-CD36 antibody fused to a T-cell activating or immunostimulatory cytokine via a linker located at the terminal of the light or heavy chain. The cytokine may include, but is not limited to, IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, or IFN-α. Such anti-CD36 antibody fusions may block CD36 signaling-mediated activity and provide an immunostimulatory cytokine effect. The ability of such anti-CD36 antibody fusions to provide an immunostimulatory cytokine effect can be determined in vitro using known cell-based assays associated with the cytokine.

6. Variants of anti-CD36 antibodies

In some embodiments, variants of the anti-CD36 antibody disclosed herein are intended to possess improved properties, such as antibody binding affinity and/or other biological properties. 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 and/or insertions into the amino acid sequence of the antibody and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct possesses the desired CD36 antigen-binding properties.

A. Variations of substitution, insertion, and deletion

In some embodiments, anti-CD36 antibody variants are provided having one or more amino acid substitutions other than those described herein. Sites for mutagenesis may include CDR, HVR, and FR. Typical “conserved” amino acid substitutions and/or substitutions based on general side chain categories or properties are well known in the art and may be used in embodiments of this disclosure. Variants based on non-conserved amino acid substitutions are also contemplated, wherein a member of one of the amino acid side chain categories is exchanged for an amino acid from another category. Amino acid side chains are generally grouped according to the following categories or general properties: (1) hydrophobic: Met, Ala, Val, Leu, Ile, leucine; (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. The technique of replacing amino acids into antibodies and then screening for desired functions (e.g., preserving/improving antigen binding, reducing immunogenicity, or improving ADCC or CDC) is well known in the art.

Amino acid substitution variants may also include variants with one or more substitutions in the hypervariable region of the parent antibody. Typically, the resulting variants selected for further research will have modifications to certain biological properties relative to the parent antibody (e.g., increased affinity, decreased immunogenicity) and/or will retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity-matured antibodies, which can be conveniently generated using phage display-based affinity maturation techniques. In short, one or more HVR residues are mutated, and the variant antibody is displayed on a phage and screened for specific biological activities (e.g., binding affinity).

A useful method for identifying antibody residues or regions that may be targeted for mutagenesis is “alanine scan mutagenesis” (see, for example, Cunningham and Wells, Science 244:1081-1085 (1989)). In this method, a residue or a target group of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and substituted with a neutral or negatively charged amino acid (e.g., Ala or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that have been shown to be functionally sensitive to the initial substitution. Alternatively, or additionally, the crystal structure of the antigen-antibody complex, which identifies the contact point between the antibody and the antigen, can be determined. Such contacting residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

Amino acid sequence insertions that can be prepared include amino and/or carboxyl-terminal fusions of peptides ranging in length from one residue to one hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionine residue. Other insertion variants of antibody molecules may include fusions of the N-terminus or C-terminus of the antibody with an enzyme or peptide, which increases the antibody's serum half-life.

Other residue substitutions can be made in HVR to improve antibody affinity. Such changes can be made in “hotspots,” which are codon-encoded residues that undergo high-frequency mutations during somatic cell maturation (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and binding affinity can be tested on the resulting variants VH or VL. In one implementation, affinity maturation can be performed by constructing and reselecting from a secondary library (see, for example, Hoogenboom et al., Methods in Molecular Biology 178:1-37 (edited by O’Brien et al., Human Press, Totowa, NJ, (2001)). Another approach to introducing diversity involves HVR-directed methods, 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 scan mutagenesis or modeling. HVR-H3 and HVR-L3 are often specifically targeted. Typically, substitutions, insertions, or deletions can be made within one or more HVRs, as long as such changes do not substantially reduce the antibody’s ability to bind to the antigen. For example, conserved changes that do not substantially reduce binding affinity (e.g., conserved substitutions as presented herein) can be made in HVRs. Such changes may occur outside of HVR “hotspots.”

In some embodiments, the anti-CD36 antibody described herein may be substituted with a cysteine residue at a specific non-HVR position to create a reactive thiol group. Such engineered “thiomonoclonal antibodies” can be used to conjugate antibodies to, for example, drug moieties or linker-drug moieties to create immunoconjugates, as described elsewhere herein. Cysteine-engineered antibodies may be produced as described, for example, in U.S. Patent No. 7,521,541. In some embodiments, any one or more of the following antibody residues may be substituted with cysteine: V205 (Kabat number) of the light chain; A118 (EU number) of the heavy chain; and S400 (EU number) of the Fc region of the heavy chain.

B. Glycosylation variants

In some embodiments, the anti-CD36 antibody of this disclosure is modified to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites on the antibody can be performed by altering the amino acid sequence, thereby creating or removing one or more glycosylation sites. In embodiments where the antibody contains an Fc region, the carbohydrates attached to the Fc region can be modified. Typically, naturally occurring antibodies produced by mammalian cells contain a branched diantennaroic oligosaccharide of asparagine attached to approximately position 297 (“N297”) of the CH2 domain of the Fc region via an N-link (see, e.g., Wright et al., TIBTECH 15:26-32 (1997)). The oligosaccharide can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the “stem” of the diantennaroic oligosaccharide structure. In some embodiments, oligosaccharide modification of the antibody Fc region can create variants with certain improved properties.

In some embodiments, the anti-CD36 antibody of this disclosure may be a variant comprising a carbohydrate structure lacking (directly or indirectly) fucose attached to the Fc region. For example, the amount of fucose in such antibodies may be from about 1% to about 80%, from about 1% to about 65%, from about 5% to about 65%, or from about 20% to about 40%. The amount of fucose can be determined by calculating the average amount of fucose within the glycan chain attached to residue N297 relative to the sum of all sugar structures attached to N297 (e.g., complex, mixed, and high-mannose structures), as measured by MALDI-TOF mass spectrometry (see, for example, WO2008/077546).

In some embodiments, fucosylated variants can provide improved ADCC function of the variant antibody. See, for example, U.S. Patent Publications US2003/0157108 or US2004/0093621. Examples of “defucosylated” or “fucose-deficient” antibodies and related methods for preparing them are disclosed, for example, in US2003/0157108; US2003/0115614; US2002/0164328; US2004/0093621; US2004/0132140; US2004/0110704; US2004/0110282; US2004/0109865; WO2000/61739; WO200 1/29246; WO2003/085119; WO2003/084570; WO2005/035586; WO2005/035778; WO2005/053742; WO2002/031 140; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004). Cell lines used to generate defucosylation antibodies include Led 3CHO cells lacking protein fucosylation (see, for example, Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US2003/0157108 and WO2004/056312) and knockout cell lines, such as α-1,6-fucosylation gene FUT8 knockout CHO cells (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng. 94(4):680-688 (2006) and WO2003/085107).

C.Fc region variant

In some embodiments, the anti-CD36 antibody of this disclosure may contain one or more amino acid modifications (i.e., Fc region variants) in its Fc region. The Fc region variants may contain a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid substitutions at one or more amino acid residue positions. A wide range of Fc region variants known in the art that can be used with the anti-CD36 antibody of this disclosure are described below.

In some embodiments, the anti-CD36 antibody is an Fc region variant with altered effector function. In some embodiments, the antibody with altered effector function has some (but not all) of the parent antibody's effector function, reduced effector function, or no effector function (e.g., no effector). For certain applications where effector function (such as ADCC) is unnecessary or detrimental, and/or the antibody's in vivo half-life is important, an Fc region variant with no effector function is more desirable. Fc region variant antibodies with reduced or no effector function can be caused by amino acid substitutions at one or more of the following Fc region positions: 238, 265, 269, 270, 297, 327, and 329 (see, for example, U.S. Patent No. 6,737,056). Such Fc region variants may include amino acid substitutions at two or more of positions 265, 269, 270, 297, and 327. Such Fc region variants may also include replacing both residues 265 and 297 with alanine (see, for example, U.S. Patent No. 7,332,581).

Some Fc region variants are capable of providing enhanced or weakened binding to FcR (see, for example, U.S. Patent No. 6,737,056; WO2004/056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001)). Some Fc region variants capable of providing improved ADCC contain one or more amino acid substitutions located, for example, at positions 298, 333, and/or 334 of the Fc region (based on EU numbering). Fc region variants with altered (i.e., enhanced or weakened) Clq binding and/or complement-dependent cytotoxicity (CDC) are described, for example, in U.S. Patent Nos. 6,194,551, WO99/51642, and Idusogie et al., J. Immunol. 164:4178-4184(2000).

Several Fc region variants, such as those disclosed in US2005/0014934A1 (Hinton et al.), offer increased half-life and improved binding to the neonatal Fc receptor (FcRn). These 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 increased half-life include the YTE mutant group (i.e., M252Y/S254T/T256E) located at positions 252, 254, and 256, as described in US7658921B2 (Dall’Acqua et al.). Further examples of Fc region variants can be found, for example, U.S. Patent Nos. 5,648,260 and 5,624,821; and WO94/29351.

Typically, 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, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity) but retains FcRn binding capacity. NK cells, the primary cells mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Non-limiting examples of in vitro assays for evaluating the ADCC activity of target molecules are described in U.S. Patent Nos. 5,500,362 (see, for example, Hellstrom et al., Proc. Natl. Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom et al., Proc. Natl. Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, for example, the ACTI ^™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA); and non-radioactive cytotoxicity assays (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the target molecule can be assessed in vivo, for example in animal models (e.g., in Clynes et al., Proc. Natl. Acad. Sci. USA). Animal models disclosed in 95:652-656 (1998). Clq binding assays can also be performed to confirm that the antibody cannot bind Clq and therefore lacks CDC activity. See, for example, Clq and C3c binding ELISAs in WO2006/029879 and WO2005/100402. To assess complement activation, CDC assays can be performed (see, for example, 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, Blood...). 103:2738-2743(2004)). FcRn binding and in vivo clearance/half-life determination can be performed using methods known in the art (see, for example, Petkova et al., Intl. Immunol. 18(12):1759-1769(2006)).

D. Non-protein antibody derivatives-immunoclavable conjugates

In some embodiments, the anti-CD36 antibody of this disclosure may be further modified (i.e., derivatized) with a non-protein portion. Suitable non-protein portions for antibody derivatization include, but are not limited to, water-soluble polymers such as: polyethylene glycol (PEG), copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, 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 homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. In some embodiments, methoxylated polyethylene glycol propionaldehyde may be used for antibody modification. 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 if more than one polymer is attached, they can be the same or different molecules. Typically, the quantity and/or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody, such as whether the antibody derivative will be used in therapy under specific conditions.

In some embodiments, the anti-CD36 antibody of this disclosure may also be an immunoconjugate, wherein the immunoconjugate comprises an anti-CD36 antibody conjugated to one or more cytotoxic agents. Suitable cytotoxic agents contemplated by this disclosure include chemotherapeutic agents, pharmaceuticals, growth inhibitors, toxins (e.g., protein toxins, bacterial, fungal, plant or animal-derived enzyme-active toxins, or fragments thereof), or radioisotopes. In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC), wherein the anti-CD36 antibody as described herein is conjugated to one or more pharmaceuticals. In some embodiments, the immunoconjugate of this disclosure comprises an anti-CD36 antibody as described herein conjugated to a pharmaceutical or therapeutic agent for treating CD36-mediated diseases or conditions.

In some embodiments, the anti-CD36 antibody described herein may be conjugated to an enzyme-active toxin or a fragment thereof, including but not limited to diphtheria toxin A chain, a non-conjugated active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, saccharin A chain, α-saccharin, tung oil protein, carnation toxin protein, phytolacca americana protein, bitter melon inhibitor, jatropha toxin, croton toxin, soapwort inhibitor, white tree toxin, mitogellin, localized aspergillin, phenolmycin, enoxacin, and trichosporine.

In some embodiments, the immunoconjugates of this disclosure comprise an anti-CD36 antibody (i.e., a radioconjugate) conjugated with a radioisotope as described herein. A variety of radioisotopes are available for producing such radioconjugates. Examples include radioisotopes of ^211At , ^131I , ^125I , ^90Y , ^186Re , ^188Re , ^153Sm , ^212Bi , ^32P , ^212Pb , and Lu. In some embodiments, the immunoconjugate may comprise a radioisotope for scintillation detection or a spin label for NMR detection or MRI. Suitable radioisotopes or spin labels may include various isotopes such as ^123I , ^131I , ^111In , ^13C , ^19F , ^15N , ^17O , Gd, Mn, and Fe.

Immunoconjugates for anti-CD36 antibodies and cytotoxic agents can be prepared using a variety of well-known bifunctional reagents and chemicals suitable for protein conjugation. Such reagents include, but are not limited to: N-succinimide-3-(2-pyridyl dithio)propionate (SPDP), succinimide-4-(N-maleimide methyl)cyclohexane-1-carboxylate (SMCC), iminothiones (IT), bifunctional derivatives of imine esters (e.g., dimethyl adipamide HQ), active esters (e.g., disuccinimide octanoate), aldehydes (e.g., glutaraldehyde), diazid compounds (e.g., bis-(p-azidobenzoyl)-hexamethylenediamine), diazide derivatives (e.g., bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (e.g., toluene-2,6-diisocyanate), and bifunctional fluorinated compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). Reagents used to prepare the immunoconjugates of this disclosure may also include commercially available “crosslinking” reagents, such as: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfon-EMCS, sulfon-GMBS, sulfon-KMUS, sulfon-MBS, sulfon-SIAB, sulfon-SMCC and sulfon-SMPB, and SVSB (succinimide-(4-vinyl sulfone)benzoate) (see, for example, Pierce Biotechnology, Inc., Rockford, IL., U.S.A.).

III. Recombination Methods and Compositions

The anti-CD36 antibody of this disclosure can be produced using recombinant methods and materials well known in the field of antibody production. In some embodiments, this disclosure provides isolated polynucleotides encoding fragments or domains of the anti-CD36 antibody of this disclosure. For example, the isolated polynucleotide may encode an amino acid sequence comprising the CDR or HVR disclosed herein, an amino acid sequence comprising the _VL domain and/or _VH domain of an antibody, or an amino acid sequence comprising the complete light chain 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 of any anti-CD36 antibody disclosed herein, or an amino acid sequence comprising the CDR-L1, CDR-L2, and CDR-L3 sequences. Similarly, the isolated polynucleotide may encode an amino acid sequence comprising the _VL domain or _VH domain or the complete heavy chain (HC) or light chain (LC) of the anti-CD36 antibody of this disclosure.

In some embodiments, this disclosure also provides vectors (e.g., expression vectors) comprising a polynucleotide sequence (as described above) encoding a fragment or domain of the anti-CD36 antibody or anti-CD36 antibody of this disclosure. Such vector constructs comprising polynucleotides generated for antibody recombinant production are well known in the art. Furthermore, in some embodiments, a host cell comprising a polynucleotide or vector having a sequence encoding a fragment or domain of the anti-CD36 antibody or anti-CD36 antibody of this disclosure is provided. In at least one embodiment, the host cell is a cell transformed with a vector comprising a polynucleotide sequence encoding an amino acid sequence comprising a _VL domain of an antibody and/or an amino acid sequence comprising a _VH domain of the anti-CD36 antibody of this disclosure. In another embodiment, the host cell has been transformed with a first vector and a second vector, the first vector comprising a polynucleotide sequence encoding an amino acid sequence comprising a _VL domain of an antibody, and the second vector comprising a nucleic acid encoding an amino acid sequence comprising a _VH domain of an antibody.

In some embodiments of the recombinant method, the host cells used are eukaryotic cells, such as Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NSO, Sp20). In one embodiment, a method for preparing an anti-CD36 antibody is provided, wherein the method includes culturing a host cell containing nucleic acid encoding the antibody (as described above) under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium).

In short, the recombinant generation of anti-CD36 antibodies is carried out by isolating the nucleic acid encoding the antibody (e.g., as described herein) and inserting that nucleic acid into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids can be readily 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 the vector encoding the antibody are well-known in the art and include prokaryotic or eukaryotic cells. Typically, after expression, the antibody can be separated from the cytoplasm in a soluble fraction and further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for vectors encoding antibodies, including fungal and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of antibodies with partial or complete human glycosylation patterns (see, for example, Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006)).

Suitable host cells for expressing the glycosylated anti-CD36 antibody of this disclosure can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that can be used in combination with insect cells, particularly for transfecting fall armyworm 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).

Examples of mammalian host cell lines that can be used to generate the anti-CD36 antibodies of this disclosure include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (see, for example, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); myeloma cell lines such as Y0, NSO, and Sp2/0; monkey kidney CV1 line (COS-7) transformed from SV40; human embryonic kidney cell lines (293 or 293 cells, as described, for example, in Graham et al., J. Gen. Virol. 36:59 (1977)); young hamster kidney cells (BHK); mouse Cetori cells (TM4 cells, as described, for example, in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); and 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, for example, Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982) and US 6,235,498); Medical Research Council 5 (MRC 5) cells (e.g., available from ATCC and also known as CCL-1). 71 cells); and foreskin 4 (FS-4) cells (see, for example, Vilcek et al., Ann. N.Y. Acad. Sci. 284:703-710 (1977), Gardner and Vilcek, J. Gen. Virol. 44:161-168 (1979), and Pang et al., Proc. Natl. Acad. Sci. U.S.A. 77:5341-5345 (1980)). For a general review of useful mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

IV. Pharmaceutical compositions and formulations of anti-CD36 antibodies

This disclosure also provides pharmaceutical compositions and pharmaceutical formulations comprising anti-CD36 antibodies. In some embodiments, this disclosure provides pharmaceutical formulations comprising an anti-CD36 antibody as described herein and a pharmaceutically acceptable carrier. In some embodiments, the anti-CD36 antibody is the sole active agent in the pharmaceutical composition. Such pharmaceutical formulations can be prepared by mixing an anti-CD36 antibody having the desired purity with one or more pharmaceutically acceptable carriers. Typically, such antibody formulations can be prepared as aqueous solutions (see, for example, U.S. Patent Nos. 6,171,586 and WO2006/044908) or as lyophilized formulations (see, for example, U.S. Patent No. 6,267,958).

Pharmaceutically acceptable carriers are generally non-toxic to recipients at the doses and concentrations used. A wide range of such pharmaceutically acceptable carriers are well known in the art (see, for example, Remington’s Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)). Exemplary pharmaceutically acceptable carriers that can be used in formulations disclosed herein may include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride; benzyl chloride; phenol; butanol or benzyl alcohol; alkyl parabens, such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) peptides; Proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including 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 nonionic surfactants, such as polyethylene glycol (PEG).

Pharmaceutically acceptable carriers that can be used in formulations of this disclosure may also include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP) (see, for example, U.S. Patent Publications 2005/0260186 and 2006/0104968), such as human soluble PH-20 hyaluronidase glycoprotein (e.g., rHuPH20 or Baxter International, Inc.).

Furthermore, considering that the formulations disclosed herein may contain active ingredients in addition to anti-CD36, this is necessary for the specific indication being treated in the subjects receiving the formulation. Preferably, any additional active ingredient has an activity complementary to the anti-CD36 antibody activity, and these activities do not adversely affect each other.

In some embodiments, the pharmaceutical composition comprises an anti-CD36 antibody and an additional active agent for cancer treatment, such as an immune checkpoint inhibitor. Checkpoint inhibitors that can be used in such embodiments include, but are not limited to, a second antibody containing specificity against an antigen that is an immune checkpoint molecule. In some embodiments, the second antibody contains specificity against 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, and ICOS. In at least one embodiment, the pharmaceutical composition comprises an anti-CD36 antibody and other active agents, wherein said other active agents are antibodies containing specificity against the immune checkpoint molecule PD1. Exemplary antibodies containing specificity against PD1 that can be used in the pharmaceutical composition embodiments disclosed herein include, but are not limited to, dostalimab, pembrolizumab, nivolumab, and pildizumab.

Furthermore, in some embodiments, this disclosure provides pharmaceutical compositions or formulations for use in therapy, wherein said compositions further comprise T-cell activating cytokines or immunostimulatory cytokines. 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 cytokines may be provided in the composition as a fusion of an anti-CD36 antibody.

The active ingredient can be encapsulated in microcapsules, for example, prepared by coagulation techniques or by interfacial polymerization, such as in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions in hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules. Such techniques are disclosed in Remington’s Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980).

In some embodiments, the formulation may be a sustained-release preparation of an antibody and/or other active ingredient. Suitable examples of sustained-release preparations include a semi-permeable matrix of a solid hydrophobic polymer containing the antibody, the matrix being in the form of a shaped article, such as a film or microcapsule.

Typically, the formulations of this disclosure intended for administration to a subject are sterile. Sterile formulations can be readily prepared using well-known techniques, such as filtration through a sterile filter membrane.

V. Uses and Treatments

Any composition or formulation containing the anti-CD36 antibody of this disclosure is intended for use in any method or application, such as in therapeutics utilizing its 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 signaling, 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 death, as appropriate) of tumor cells, tumor-associated macrophages (TAMs), myeloid-derived suppressor 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 can deplete regulatory T cells while increasing CD8 T cell survival and function, thereby inducing an increased anti-tumor T cell response.

A range of diseases, conditions, and illnesses can be potentially treated by inhibiting, reducing, and/or completely blocking the immunomodulatory and/or immune signaling activity of CD36 (particularly the effect of CD36 on TAM). These diseases, conditions, and illnesses include, but are not limited to, cancers, including but not limited to, colon cancer, pancreatic cancer, ovarian cancer, HCC, kidney cancer, breast cancer, lung cancer, gastric cancer, melanoma, head and neck cancer, or oral cancer. Any composition or formulation comprising the anti-CD36 antibody of this disclosure is considered for use in treating any of the cancers listed above. In some embodiments, the cancer is selected from colon cancer, pancreatic cancer, ovarian cancer, HCC, kidney cancer, breast cancer, lung cancer, gastric cancer, melanoma, head and neck cancer, or oral cancer. In some embodiments, this disclosure provides a method of treating a subject with cancer, the method comprising administering to a subject in need a therapeutically effective amount of the anti-CD36 antibody of this disclosure or administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising the anti-CD36 antibody of this disclosure and a pharmaceutically acceptable carrier.

As disclosed herein, including in the following embodiments, the anti-CD36 antibody of this disclosure has the ability to reduce, inhibit, and/or block the binding of a ligand to CD36, thereby altering CD36-mediated immune signaling pathways. Therefore, in some embodiments, this disclosure provides a method of treating a CD36-mediated disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of the anti-CD36 antibody of this disclosure or administering to a subject in need a therapeutically effective amount of a pharmaceutical composition comprising the anti-CD36 antibody of this disclosure and a pharmaceutically acceptable carrier. Similarly, in some embodiments, this disclosure provides a method of treating a disease in a subject mediated by binding to CD36 expressed on cells, the method comprising administering to the subject a therapeutically effective amount of the anti-CD36 antibody of this disclosure or administering to a subject in need a therapeutically effective amount of a pharmaceutical composition comprising the anti-CD36 antibody of this disclosure and a pharmaceutically acceptable carrier.

According to the treatment method, administration of an anti-CD36 antibody, composition, or pharmaceutical formulation provides an antibody-induced therapeutic effect that protects the subject from and/or treats the progression of CD36-mediated disease in the subject. In some embodiments, the treatment method may further include administration of one or more additional therapeutic agents or treatments known to those skilled in the art to prevent and/or treat CD36-mediated disease or conditions. Such methods including the administration of one or more additional pharmaceutical agents may cover combination administration (where two or more therapeutic agents are included in the same or separate formulations) and separate administration, in which case the administration of the antibody composition or formulation may be performed before, simultaneously with, and/or after the administration of additional therapeutic agents.

In some embodiments of the treatment methods disclosed herein, an anti-CD36 antibody or a pharmaceutical preparation containing an anti-CD36 antibody is administered to a subject by any administration method, either systemic delivery or delivery to a desired target tissue. Systemic administration generally refers to any method of administering the antibody to a site in the subject rather than directly to a desired target site, tissue, or organ, thereby allowing the antibody or its preparation to enter the subject's circulatory system and undergo metabolism and other similar processes.

Therefore, the administration methods that can be used for the treatment methods disclosed herein may include, but are not limited to, injection, infusion, drip, and inhalation. Injection administration may include intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, intracerebral, and intrasternal injections, as well as infusions.

In some embodiments, the pharmaceutical formulation of the anti-CD36 antibody is formulated to prevent the antibody from being inactivated in the intestine. Therefore, treatment methods may include oral administration of the formulation.

In some embodiments, the use of compositions or formulations comprising the anti-CD36 antibody of this disclosure as medicaments is also provided. Furthermore, in some embodiments, this disclosure also provides the use of compositions or formulations comprising the anti-CD36 antibody in the manufacture or preparation of medicaments, particularly medicaments for treating, preventing, or inhibiting CD36-mediated diseases. In further embodiments, the medicament is a method for treating, preventing, or inhibiting CD36-mediated diseases, the method comprising administering an effective amount of the medicament to an individual suffering from a CD36-mediated disease. In some embodiments, the medicament further comprises an effective amount of at least one additional therapeutic agent or treatment.

In at least one embodiment, the adjunctive therapeutic agent or treatment that can be used in conjunction with the anti-CD36 antibody of this disclosure in such a medicament may include, but is not limited to, therapeutic antibodies containing specificity to immune checkpoint molecules, such as PD1, PD-L1, LAG3, CTLA-4, A2AR, TIM-3, BTLA, CD276, CD328, VTCN1, IDO, KIR, NOX2, VISTA, OX40, CD27, CD28, CD40, CD122, CD137, GITR, and ICOS. Exemplary antibodies containing specificity to immune checkpoint molecules include, but are not limited to, anti-PD1 antibodies selected from dostalimab, pembrolizumab, nivolumab, and pildizumab.

In a further embodiment, the drug is used to treat, inhibit, or prevent CD36-mediated diseases, such as cancer, in a subject, and the method includes administering an effective amount of the drug to the subject to treat, inhibit, or prevent CD36-mediated diseases.

The appropriate dose of the anti-CD36 antibody contained in the compositions and formulations disclosed herein (when used alone or in combination with one or more other additional therapeutic agents) will depend on the specific disease or condition being treated, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, the patient's prior therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The anti-CD36 antibody contained in the compositions and formulations described herein may be appropriately administered to the patient once or in a series of treatments. Various dosing regimens are considered herein, including but not limited to single or multiple administrations at different time points, bolus administration, and pulsatile infusion.

Depending on the type and severity of the disease, the anti-CD36 antibody in the formulation disclosed herein, ranging from about 1 μg/kg to 15 mg/kg, is the initial candidate dose for administration to human subjects, whether, for example, by single or multiple separate administrations or by continuous infusion. Typically, the antibody is administered at doses ranging 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) may be administered to the patient.

Dosing can be maintained for several days or longer, depending on the subject's condition; for example, administration may continue until CD36-mediated disease is adequately treated, as determined by methods known in the art. In some embodiments, an initial high loading dose may be administered, followed by one or more lower doses. However, other dosing regimens may be useful. The progression of therapeutic effects during dosing can be monitored using conventional techniques and assays.

Therefore, in some embodiments of the methods disclosed herein, administration of the anti-CD36 antibody comprises a daily dose from about 1 mg/kg to about 100 mg/kg. In some embodiments, the dose of the anti-CD36 antibody comprises a daily dose 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.

Example

Various features and embodiments of this disclosure are illustrated in the following representative examples, which are intended to be illustrative and not restrictive. Those skilled in the art will readily understand that the specific embodiments are merely illustrative of the invention, as more fully described in the following claims. Each embodiment and feature described in this application should be understood to be interchangeable and combined with each embodiment contained herein.

Example 1: Analysis of anti-CD36 antibody production and CD36 binding

This embodiment illustrates the use of phage display antibody library technology to generate exemplary anti-CD36 antibodies of this disclosure that specifically bind to human and mouse CD36.

A. Select anti-CD36 scFv conjugates from a phage display antibody library.

The screening procedure is briefly described below. First, human CD36.ECD antigen (5 μg per well, Sino Biological) in PBS buffer (pH 7.4) was coated onto the wells of a 96-well plate (NUNC Maxisorb immunoassay plate) overnight at 4°C, and then blocked for 1 hour with PBST [0.1% (v/v) Tween 20] containing 5% skim milk. After blocking, 100 μL of concentrated phage library (10¹³ cfu/mL in PBS buffer) was mixed with 100 μL of blocking buffer and added to each well with gentle shaking for 1 hour. The plate was washed 12 times with PBST and 3 times with PBS. The bound phages were eluted with 100 μL of 0.1 M HCl/glycine (pH 2.2)/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 (A600 nm = 0.6) at 37°C for 30 minutes; uninfected bacteria were removed by adding ampicillin. After 30 minutes of ampicillin treatment, the bacterial culture was infected for 1 hour at 37°C with 100 μL of M13KO7 helper phage (total ~10¹¹ CFU), and then added to 50 mL of 2X YT medium containing 50 μg/mL kanamycin and 100 μg/mL ampicillin, and incubated overnight 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 screening.

After 3–4 rounds of selection-amplification cycles, single colonies were randomly selected into 96-well deep-well plates (plate A; secretory scFv); each well contained 950 μL of 2YT (100 μg/mL ampicillin). After incubation with shaking at 37°C for 3 hours, 50 μL of bacterial culture was transferred to the corresponding wells of a fresh 96-well deep-well plate (plate B; phage form); each well contained 0.8 mL of 2YT with 100 μg/mL ampicillin. Simultaneously, 50 μL of M13KO7 (total ~5 × 10¹⁰ CFU) was added to each well of plate B. After incubation for 1 hour, 100 μL of 2YT containing IPTG (10 mM) was added to each well of plate A; and 100 μL of 2YT containing kanamycin (500 μg/mL) was added to each well of plate B. After incubating overnight with vigorous shaking at 37°C, the culture was centrifuged at 3000g for 10 minutes at 4°C. Plate B was stored for further sequencing. For secretory scFv culture plates (plate A), 100 μL of culture medium and 100 μL of 5% PBST were added to the 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. The plates were blocked with 5% PBST. After incubation at room temperature for 1 hour, the plates were washed three times with PBST. 100 μL of protein A-HRP (Thermo Scientific) was added to each well of the protein L-coated immunosorbent assay plate; 100 μL of anti-E-tag HRP (ICL Inc.) was added to each well of the human CD36 antigen-coated and BSA-coated plates. After incubation for 1 hour, the plate was washed three times with PBST buffer and twice with PBS. It was then developed with 3,3',5,5'-tetramethylbenzidine peroxidase substrate (Kirkegaard & Perry Laboratories) for 3 minutes, quenched with 1.0M HCl, and the spectrophotometric reading was taken at 450 nm.

Positive clones were selected according to the following criteria: wells coated with human CD36 antigen (antigen binding positive), ELISA OD450 >0.2; wells coated with BSA (non-specific binding negative), OD450 <0.05; wells coated with protein L (soluble scFv binds to both protein L and protein A to ensure proper folding in solution), OD450 > 0.5, followed by DNA sequencing. The polynucleotide sequence of the scFv of the exemplary anti-CD36 antibody 12P109 (SEQ ID NO: 1) obtained from phage display screening is provided in Table 2 and the accompanying sequence listing. Further screening of phage display libraries consisting of “reorganized” LC sequences derived from different anti-CD36 antibodies and the HC sequence of 12P109 provided the scFv of the exemplary anti-CD36 antibody A8A (SEQ ID NO: 10), which is also listed in Table 2 and the accompanying sequence listing. The CDR of the A8A antibody _VL domain (SEQ ID NO:11) was further subjected to standard PCR-based mutagenesis to provide the exemplary anti-CD36 antibody VL domain (SEQ ID NO:14) of A8A-N52T, which is also listed in Table 2 and the accompanying sequence listing. B. Generation of anti-CD36 antibody in full-length IgG form

scFv reformatting and cloning : The CD36 binding determinant of scFv selected from phage display screening was reformatted into full-length IgG antibodies by cloning the _VH and _VL domains of the fragment into human IgG1-N297A heavy chain vector and human kappa light chain vector using restriction enzyme sites MluI/NheI and BsiWI/DraIII, respectively. The following forward and reverse oligonucleotide primer pairs were used to amplify the _VH and _VL domains: (1) PhageLib_VL_Fw : 5'-AATCACgATgTgATATTCAAATgACCCAgAgCCCgAgC-3' (SEQ ID NO: 64), (2) PhageLib_VL_Rv: 5'-AATCgTACgTTTgATTTCCACTTTggTgCCTTg-3' (SEQ ID NO: 65), (3) PhageLib_VH_Fw: 5'-AATACgCgTgTCCTgTCCgAAgTgCAgCTggTggAATCg-3' (SEQ ID NO: 66), and (4) PhageLib_VH_Rv: 5'-AATgCTAgCCgAgCTCACggTAACAAg-3' (SEQ ID NO: 67).

Expression and purification of the full-length antibody : Transient expression was performed in ExpiCHO-S cells (Thermo Scientific) using a reformulated vector containing the anti-CD36 antibody gene clone. During the exponential growth phase, ExpiCHO-S cells were diluted to a final density of 6 × ^10⁶ cells/mL with ExpiCHO expression medium. The ExpiFectamine CHO/plasmid DNA complex was prepared using a cold reagent according to the manual, incubated at room temperature for 1–5 minutes, and then slowly added to the cells. One day after transfection, the culture was supplemented with ExpiFectamine CHO enhancer and incubated for another 7 days. The transfected cell supernatant was centrifuged and then filtered through a 0.22 μm filter. The antibody was 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) to 1/10 volume of 1 M Tris buffer (pH 9.0). The eluent was buffer-exchanged and concentrated using PBS (pH 7.4) containing 0.1 M arginine. The mass of the purified anti-CD36 antibody was determined by SDS-PAGE with and without a reducing agent.

Results : Examination of SDS-PAGE images showed that expression and purification yielded purified full-length anti-CD36 antibody.

C. Optimization of anti-CD36 antibodies

To further improve the druggability of the anti-CD36 antibody A8A, two phage display libraries composed of mutations in N52, H91, and L94 were used for screening. The scFv clone 117 selected from the phage display screening was reformulated into a full-length IgG antibody by cloning the _VH and _VL domains of the fragment into the human IgG1-N297A heavy chain vector and the human kappa light chain vector using restriction enzyme sites MluI/NheI and BsiWI/DraIII, respectively. The following forward and reverse oligonucleotide primer pairs were used to amplify the _VH and _VL domains: (1) PhageLib_VL_Fw : 5'-AATCACgATgTgATATTCAAATgACCCAgAgCCCgAgC-3' (SEQ ID NO: 64), (2) PhageLib_VL_Rv: 5'-AATCgTACgTTTgATTTCCACTTTggTgCCTTg-3' (SEQ ID NO: 65), (3) PhageLib_VH_Fw: 5'-AATACgCgTgTCCTgTCCgAAgTgCAgCTggTggAATCg-3' (SEQ ID NO: 66), and (4) PhageLib_VH_Rv: 5'-AATgCTAgCCgAgCTCACggTAACAAg-3' (SEQ ID NO: 67).

The CDR-H1, H2, H3, and CDR-L1 sequences of antibody 117 are identical to those of A8A, and an N52T mutation is present in CDR-L2, while an L94A mutation is present in CDR-L3. The Fab fragment of antibody 117 was also constructed into the human IgG4-S228P/F234A/L235A heavy chain vector using the restriction enzyme sites NheI and BamHI. Further PCR-based mutagenesis of the CDRs of the VH and VL domains of antibody 117 was performed to generate a series of variants, summarized in Table 3 below.

Table 3

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

D. ELISA for specific binding of anti-CD36 antibody to CD36

ELISA Materials and Methods : Recombinant His-labeled human CD36.ECD protein or His-labeled mouse CD36.ECD protein (both from Sino Biological) were immobilized at a concentration of 1 μg/mL in coating solution (SeraCare) on 96-well microtiter plates and incubated overnight at 4°C. Wells were washed with washing buffer (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 incubation at 37°C for 1 hour, the wells were washed with washing buffer. Peroxidase-conjugated goat anti-human kappa light chain antibody (Sigma) was applied to each well and incubated at 37°C for 1 hour. After washing, the wells were developed with TMB substrate at room temperature for 5–10 minutes, then terminated with 1N HCl. Absorbance was then measured at 450 nm and 650 nm. EC50 values were calculated using a GraphPad Prism7.

Results : Figures 1A and 1B depict ELISA data showing the binding of the full-length IgG anti-CD36 antibody 12P109 and other positive clones 12P102, 12P103, 12P104, 12P105, 12P106, 12P107, 12P110, and 12P212 to human or mouse CD36.ECD, respectively. The binding curves of the antibodies labeled 12P109, 12P103, and 12P212 show the highest affinity for human CD36 (hCD36, see Figure 1A). 12P109 also shows high binding affinity for mouse CD36 (mCD36, see Figure 1B). The EC50 values calculated from the ELISA data are shown in Table 4 below.

Table 4

Figures 1C and 1D depict ELISA data showing the binding of anti-CD36 antibodies to recombinant human CD36.ECD or mouse CD36.ECD coated on ELISA plates, respectively. The ELISA used anti-CD36 antibodies 12P109 and A8A in the full-length human IgG1 form, which specifically bind to CD36, and the commercial mouse IgA antibody D2712 (clone CRF D-2712, BD Biosciences, US). The EC50 values from the curves in Figures 1C and 1D are shown in Table 5 below.

Table 5

Figures 2A, 2B, 2C, 2D, 2E, and 2F depict ELISA data showing the binding of full-length IgG anti-CD36 antibodies 12P109, 117, and the various 117 variants listed in Table 3 to human CD36.ECD. The competitor, anti-CD36 antibody ONA, is derived from the clone "ONA-0-v1" in the form of hIgG4-S228P-FALA, as disclosed in WO2021176424A1. The EC50 values calculated from the ELISA data in Figures 2A-2F are shown in Table 7 below.

Table 7

F.SEC-UPLC Analysis

Prolonged retention time (RT) of antibodies, as determined by SEC-UPLC analysis, may be associated with nonspecific hydrophobic interactions, which could pose a risk to the development of said antibodies for therapeutic use. Therefore, increased RT in SEC-UPLC analysis of anti-CD36 antibodies 12P109, A8A, 117, and 117 variants, as well as IgG standards (“STD”, BEH200 SEC protein standard mixture) and control monoclonal antibodies (“ctl Ab”), indicates the formation of protein aggregates.

Materials and Methods : After purification of protein A, protein aggregates and retention times of anti-CD36 antibody 12P109, A8A, 117, and 117 variants were analyzed by standard size exclusion UPLC (SEC-UPLC). 3 μg of antibody was applied to UPLC (Waters ARC UPLC), and separation was performed using PBS as the mobile phase on a gel filter column (Waters, Xbridge BEH450 SEC 4.6 × 150). The UV absorbance of the antibody peak was monitored at 280 nm, and the peak area was determined using Empower software.

Results : The SEC-UPLC results are illustrated in Figures 3A, 3B, 3C, 3D, and 3E, along with the measured RT values. The control antibody showed an RT value of 7.243 minutes, while both anti-CD36 antibodies 12P109 and 117 showed relatively increased RT values. However, the increased RT value of 117 was significantly reduced due to mutations present in variants 117_30DA, 117_31AD, 117_57D, 117_57E, 117_57EE, 117_101D, and 117_DA57E. The RT values of anti-CD36 antibodies measured in the SEC-UPLC experiments are provided in Table 8 below. Furthermore, some aggregates were found in 117_DA57E by UPLC analysis. These aggregates may be related to the relatively poor stability of 117_DA57E under acidic elution conditions. The R409K mutation in IgG4 has been shown to increase the strength of CH3 domain interactions and reduce the tendency to aggregate at low pH. SEC-UPLC analysis showed that adding R409K to 117_DA57E reduced aggregation induced by acidic conditions.

Table 8

BLI analysis of G.CD36 combined with 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) (ForteBio Octet RED96). BLI assays were 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 offset. The biosensor was then immersed in varying concentrations (i.e., 0, 1.5625, 3.125, 6.25, 4.94, 12.5, 25, 50, and 100 nM) of recombinant His-labeled human CD36.ECD protein (Sino Biological) in a running buffer containing 0.1% BSA, 0.1% Tween-20, and 250 mM NaCl in PBS. The rate constants were calculated by curve fitting analysis of the binding reaction (1:1 Langmuir model), representing a binding interaction time of 2.5 min and a dissociation interaction time of 5 min.

Results : Table 10 below provides the dissociation constants KD and kinetic rate constants ka and kd for the determination of binding of anti-CD36 antibodies 12P109, A8A, 117 and various 117 variants (in IgG1 or IgG4 format) to human CD36, as determined in separate BLI assays.

Table 10

Example 2: Binding of anti-CD36 antibody to CD36 expressed on the cell surface

This embodiment illustrates the preparation of a stable F293 cell line overexpressing hCD36 or mCD36 on the cell surface, and the study of determining the binding affinity of the exemplary anti-CD36 antibody of this disclosure to human, rhesus monkey or mouse CD36 expressed on the cell surface.

A. Generation of stable F293 cell lines overexpressing CD36

Materials and Methods : Gene fragments encoding full-length human CD36-Flag (Sino Biological), rhesus monkey CD36-His (Sino Biological), and mouse CD36-His (Sino Biological) were subcloned into the pcDNA3.4 topological vector using XbaI/HindIII. Freestyle 293-F cells (Thermo Scientific) were transfected with plasmids encoding CD36 using the polyethyleneimine (PEI) method and selected with Henmycin (Thermo Scientific) to establish a stable CD36 cell pool. Human CD36 expression was verified by surface staining with anti-CD36 (clone 5-271, Biolegend) or intracellular staining with anti-FLAG. Rhesus monkey CD36 expression was verified by surface staining with anti-CD36 clone 117 or intracellular staining with anti-His. Mouse CD36 expression was verified by surface staining with anti-CD36 antibody D2712 (clone CRF D-2712; BD Biosciences, US) or by intracellular staining with anti-His. To enrich stable cell lines expressing CD36, F293 cell pools overexpressing hCD36, rhesus monkey CD36, or mCD36 were stained with anti-CD36 (clone 5-271, Biolegend), anti-CD36 clone 117, or anti-CD36 D2712 (clone CRF D-2712, BD Biosciences). High CD36 cell populations were selected using FACSAriaIIIu (BD).

Results : Analysis of FACS data confirmed that stable F293 cell overexpression of F293/hCD36, F293/rhesus monkey CD36 and F293/mCD36 can bind to surface CD36 with anti-CD36 antibody.

B. Cell surface CD36 binding activity of anti-CD36 antibody

Materials and Methods : F293 cells with high CD36 expression, as described in Section A above, were incubated at 4°C for 30 min with anti-CD36 antibody 117 and several variants of 117 (see Table 3). After washing with FACS buffer (PBS solution of 2% FBS), cells were stained with anti-human IgG-Alexa Fluor 647 and analyzed by Attune NxT flow cytometry (Thermo Scientific).

Results : Analysis of flow cytometry data expressed as geometric mean (MFI) confirmed that the anti-CD36 antibody specifically binds to cells expressing hCD36 and mCD36 on their surface.

Figures 4A, 4B, 4C, 4D, and 4E depict data showing the binding of full-length IgG anti-CD36 antibody 117 and several 117 variants to the surface of F293 cells expressing human, rhesus monkey, or mouse CD36. _EC50 values from the curves in Figures 4A-4E are shown in Table 11 below.

Table 11

Example 3: Blocking activity of anti-CD36 antibody against oxidized LDL uptake

This embodiment illustrates a study on the ability of an exemplary anti-CD36 antibody of this disclosure to block the uptake of oxidative LDL by CD36-expressing cells.

A. Inhibition of oxidized LDL binding and uptake in U937 cells by anti-CD36 antibody

U937 cells expressing high levels of CD36 were pre-incubated with control IgG or anti-CD36 antibody at 4°C for 30 minutes. To measure oxLDL binding or uptake, Dil-oxLDL (5–10 μg/mL) (catalog number 770232-9; Kalen Biomedical) (a purified human LDL oxidized with copper(II) sulfate and labeled “Dil” (1,1’-bis(octadecyl-3,3,3’,3’-tetramethylindocyanine perchlorate)) was added to serum-free medium and incubated at 4°C for 2 hours or at 37°C for 5 minutes, respectively. After washing with PBS, cells were analyzed by Attune NxT flow cytometry (Thermo Scientific).

The binding of the anti-CD36 antibody disclosed herein to U937 cells resulted in a characteristic shift, confirming the endogenous expression of CD36 on the surface of U937 cells. Analysis of the extracted U937 flow cytometry data confirmed that, when incubated with the anti-CD36 antibodies 12P103, 12P110, or 12P109 disclosed herein, U937 cells showed reduced oxLDL binding (Fig. 5A) and oxLDL uptake (Fig. 5B) compared to the IgG isotype control.

B. Inhibition of oxidized LDL uptake in F293/CD36 cells by anti-CD36 antibody: F293/hCD36 cells were pre-incubated with control IgG or anti-CD36 antibody at 4°C for 30 minutes. To measure oxLDL binding or uptake, Dil-oxLDL (5 μg/mL) (Kalen Biomedical) was added to serum-free medium and incubated at 4°C for 2 hours to measure "oxLDL binding", or at 37°C for 5 minutes to measure "oxLDL uptake". After washing with PBS, cell preparations were analyzed using an Attune NxT flow cytometer (Thermo Scientific).

Results : As shown in the flow cytometry data plots depicted 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 oxidative LDL uptake in F293 cells overexpressing human CD36 on their surface. Furthermore, as shown in Figure 6C, the presence of anti-CD36 antibodies 12P109, 117_30DA, 117_57D, or 117_57E effectively blocked oxLDL uptake in F293 cells overexpressing mouse CD36 on their surface. The _IC50 values for these anti-CD36 antibodies that identified inhibition of oxLDL uptake in F293/hCD36 or F293/mCD36 cells are shown in Table 13 below.

Table 13

Example 4: Activity of anti-CD36 antibody in blocking oxLDL uptake by TIL

This embodiment illustrates a study on the blocking of oxidized LDL uptake by anti-CD36 antibody in mouse tumor-infiltrating lymphocytes (TILs).

Materials and Methods : Mouse colon cancer cell line CT-26 (2 × ^10⁵ cells, ATCC number: CRL-2638) or mouse liver cancer cell line BNL 1MEA.7R.1 (5 × ^10⁶ cells, ATCC number: TIB-75) were subcutaneously injected into the right ventral side of BALB/c mice. Mouse colon cancer cell line MC38 (1 × ^10⁶ cells, Kerafast#ENH204-FP), mouse melanoma cell line B16-F10 (1 × ^10⁶ cells, BCRC#60031), or mouse lung cancer cell line LL/2 (2 × ^10⁵ cells, BCRC#60050) were subcutaneously injected into the right ventral side of C57BL/6 mice. CT-26 tumors were harvested after 4 weeks. B16-F10, BNL1MEA.7R.1, MC38, and LL/2 tumors were harvested after 2 weeks. According to the manufacturer's instructions, a suspension of tumor cells was prepared from solid tumors using a mouse tumor isolation kit (Miltenyi Biotec). CD45+ TILs were isolated from the tumor cell suspension using CD45 microbeads (Miltenyi Biotec).

Isolated CD45+ TILs were pre-incubated with control IgG or anti-CD36 antibody (5-10 μg/mL) at 4°C for 30 min. Dil-oxLDL (5-10 μg/mL) was added to RPMI medium containing 1% fatty acid-free BSA and incubated at 37°C for 15 min. After washing with PBS, cells were analyzed using an Attune NxT flow cytometer (Thermo Scientific). oxLDL binding and uptake were analyzed by flow cytometry and expressed as a percentage (%) of positive cells based on histogram gating on negative control cells.

Data are expressed as mean ± SD. *p<0.05, **p<0.005, one-way ANOVA.

Results : As shown in Figure 7A, the anti-CD36 antibodies 12P109 and A8A, which specifically bind hCD36 and mCD36, provided approximately 35% inhibition of oxLDL uptake in CD45+ TILs isolated from CT26 tumors injected into BALB/c mice. This level of inhibition is equivalent to that observed with the commercially available antibody D2712, which specifically binds to mouse CD36.

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

Example 5: Inhibition of M2 macrophage polarization and activation

This study was conducted to determine the ability of the anti-CD36 antibody disclosed herein to inhibit M2 macrophage polarization and activation.

Materials and Methods : To generate monocyte-derived macrophages, human CD14+ monocytes were isolated from PBMCs and cultured for 6 days at 2 × ^10⁶ cells/ml in RPMI 1640 supplemented with 10% FBS and 20 ng/ml CSF1. Adhering macrophages were then collected by EDTA and seeded in 24-well plates for further polarization. For M0 macrophages, cells were cultured in medium for 2 days. To polarize M2 macrophages, macrophages were cultured for another 2 days in medium containing 50 ng/mL IL-4 and 50 ng/mL IL-13. 10 μg/mL control IgG or anti-CD36 antibody was added during M2 macrophage polarization. For oxLDL activation, macrophages were pre-incubated with control IgG or anti-CD36 antibody for 10 min and then cultured for another 2 days in M2 medium containing 10 μg/mL oxLDL (Kalen Biomedical). To detect M2 macrophage activation, macrophages were harvested and stained with antibodies against CD206, CD301, and PDL2. The levels of surface markers were analyzed using Attune NxT flow cytometry (ThermoScientific).

Results : As shown 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 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 and 8D) and CD206+/PDL2+ (Figures 8E and 8F) double-positive cell populations. As shown in the data plots depicted in Figures 8D and 8F, 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”. “ONA” is the Fab clone “ONA-0-v1” with the hIgG4-S228P-FALA Fc region disclosed in WO2021176424A1.

Example 6: Activity of anti-CD36 antibody in a mouse hepatocellular carcinoma model

This embodiment illustrates the activity of anti-CD36 antibody in inhibiting tumor growth in mouse models of two gene-induced hepatocellular carcinoma (HCC): (1) HCC induced by transgenes of MYC-luc-ova overexpression and p53 knockout mediated by the Sleeping Beauty transposon (SB100x) system; and (2) HCC induced by transgenes of MYC-luc-ova and β-catenin ^N90 mediated by the Sleeping Beauty transposon (SB100x) system.

Materials and methods

Six-week-old mice were restrained and subjected to hydrodynamic delivery of endotoxin-free plasmid DNA via a lateral tail vein injection equivalent to 10% of their body weight over 5–7 seconds. For the MYC ^OE /p53 ^KO HCC model, pT3-Myc-luc-ova plasmid (Addgene#129776), p53 gRNA plasmid (Addgene#59910), and SB100x (a transposase-containing plasmid; Addgene#34879) were injected into mice. For the β-catenin/Myc (β-catenin ^OE /MYC ^OE ) HCC model, pT3-bcatenin (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.

To monitor tumor growth, fluorescein (150 mg/kg) was injected into mice, and its bioluminescent activity was analyzed using an IVIS imaging system. Mice with persistent tumor growth were randomly assigned to either anti-CD36 antibody (10 mg/kg) or PBS (control) via intraperitoneal injection as described below.

Two weeks later, MYC ^OE /p53 ^KO HCC model mice were intraperitoneally injected with four doses of anti-CD36 antibody (10 mg/kg; clone: 117_30DA) or control antibody every three days, and bioluminescence imaging of the mice was measured in the IVIS system every six days (n=5 per group).

Three weeks later, 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 in the IVIS system.

In the β-catenin ^OE /MYC ^OE HCC model, tumors exhibited aggressive progression, and tumor-bearing mice typically died 38 days after hydrodynamic injection. Therefore, tumors were harvested and weighed 35–38 days after hydrodynamic tail vein injection.

result

Figure 9 shows the tumor growth curves measured by bioluminescence in MYC ^OE /p53 ^KO HCC model mice after treatment with anti-CD36 antibody 117_30DA. From day 14 to day 38 post-HCC induction, tumor growth was significantly inhibited in the treated mice compared to the control group. P = 0.0114, t-test, two-tailed.

Figure 10A shows the tumor growth curves measured by bioluminescence in β-catenin ^OE /MYC ^OE HCC model mice after treatment with anti-CD36 antibody 117_DA57E. From day 21 to day 36 post-HCC induction, tumor growth was significantly inhibited in the treated mice compared to the control group. P = 0.0104, t-test, two-tailed. Figure 10B shows a significant reduction in endpoint liver weight in mice treated with anti-CD36. Furthermore, as shown in Figure 10C, plasma ALT (alanine aminotransferase) activity analysis indicated that anti-CD36 treatment also alleviated liver damage caused by cancer development.

Although the invention has been described in detail above by way of examples and illustrations for clarity and understanding, this disclosure, including the embodiments, descriptions, and implementations described herein, is for illustrative purposes and is intended as an example, and should not be construed as limiting the scope of this disclosure. Those skilled in the art will appreciate that various modifications or changes can be made to the embodiments, descriptions, and implementations described herein, and that such modifications or changes should be included within the spirit and scope of this disclosure and the appended claims. Furthermore, those skilled in the art will recognize numerous methods and procedures equivalent to those described herein. All such equivalents should be understood to be within the scope of this disclosure and covered by the appended claims.

Other embodiments of the present invention are set forth in the following claims.

All publications, patent applications, patents or other documents mentioned herein are expressly incorporated herein by reference in their entirety for all purposes, to the same extent that each such individual publication, patent, patent application or other document is individually and specifically indicated to be incorporated herein by reference in its entirety for all purposes and is listed herein in its entirety. In case of any conflict, this specification (including designated terminology) shall prevail.

References

Al-Khami,A.A.et al.(2017).Exogenous lipid uptake induces metabolicand functional reprogramming of tumor-associated myeloid-derived suppressorcells.Oncoimmunology 6:e1344804,doi:10.1080/2162402X.2017.1344804.

Almeida,P.E.et al.(2014).Differential TLR2 downstream signalingregulates lipid metabolism and cytokine production triggered by Mycobacteriumbovis BCG infection.Biochim.Biophys.Acta.1841:97-107,doi:10.1016/j.bbalip.2013.10.008.

Aloia,A.et al.(2019).A fatty acid oxidation-dependent metabolic shiftregulates the adaptation of BRAF-mutated melanoma to MAPKinhibitors.Clin.Cancer Res.25:6852,doi:10.1158/1078-0432.CCR-19-0253.

Barrett,L.et al.(2007).Circulating CD14-CD36+peripheral bloodmononuclear cells constitutively produce interleukin-10.J.Leukoc.Biol.82:152-60,doi:10.1189/jlb.0806521.

Chung,E.Y.et al.(2007).Interleukin-10 expression in macrophagesduring phagocytosis of apoptotic cells i s mediated by homeodomain proteinsPbx1 and Prep-1. Immunity 27:952-964,doi:10.1016/j.immuni.2007.11.014.

Enciu,A.-M.et al.(2018).Targeting CD36 as biomarker for metastasisprognostic: how far from translation into clinical practice? Biomed.Res.Int.2018:7801202,doi:10.1155/2018/7801202.

Feng,W.W.et al.(2019).CD36-mediated metabolic rewiring of breastcancer cells promotes resistance to HER2-targeted therapeutics. Cell Reports 29:3405-20,doi:10.1016/j.celrep.2019.11.008.

Goedegebuure,P.et al.(2011).Myeloid-derived suppressor cells:generalcharacteristics and relevance to clinical management of pancreaticcancer.Curr.Cancer Drug Targets 11:734-51,doi:10.2174/156800911796191024.

Hale,J.S.et al.(2014).Cancer stem cell-specific scavenger receptorCD36 drives glioblastoma progression.Stem Cells 32:1746-58,doi:10.1002/stem.1716.

Huang,S.C.et al.(2014).Cell-intrinsic lysosomal lipolysis isessential for alternative activation of macrophages.Nature Immunol.15:846-55,doi:10.1038/ni.2956.

Joyce,J.A.&Pollard,J.W.(2009).Microenvironmental regulation ofmetastasis.Nature Reviews Cancer 9:239-52,doi:10.1038/nrc2618.

Kuda,O.et al.(2011).CD36 protein is involved in store-operatedcalcium flux,phospholipase A2 activation,and production of prostaglandinE2.J.Biol.Chem.286:17785-95,doi:10.1074/jbc.M111.232975.

Lewis,C.E.&Pollard,J.W.(2006).Distinct role of macrophages indifferent tumor microenvironments.Cancer Res.66:605-12,doi:10.1158/0008-5472.CAN-05-4005.

Liang,Y.et al.(2018).CD36 plays a critical role in proliferation,migration and tamoxifen-inhibited growth of ER-positive breast cancer cells.Oncogenesis 7:98,doi:10.1038/s41389-018-0107-x.

Ma,

Mizuno,R.et al.(2019).Prostaglandin E2/EP signaling in the tumormicroenvironment of colorectal cancer.Int.J.Mol.Sci.20:6254,doi:10.3390/ijms20246254.

Nath,A.et al.(2015).Elevated free fatty acid uptake via CD36 promotesepithelial-mesenchymal transition in hepatocellular carcinoma.Sci.Rep.5:14752,doi:10.1038/srep14752.

Oh,J.et al.(2012).Endoplasmic reticulum stress controls M2 macrophagedifferentiation and foam cell formation.J.Biol.Chem.287:11629-41,doi:10.1074/jbc.M111.338673.

Oh,K.et al.(2013).A mutual activation loop between breast cancercells and myeloid-derived suppressor cells facilitates spontaneous metastasisthrough IL-6 trans-signaling in a murine model.Breast Cancer Research 15:R79,doi:10.1186/bcr3473.

Pascual,G.et al.(2017).Targeting metastasis-initiating cells through the fatty acid receptor CD36.Nature 541:41-45,doi:10.1038/nature20791.

Pepino,Y.M.et al.(2014).Structure-function of CD36 and importance offatty acid signal transduction in fat metabolism.Annu.Rev.Nutr.34:281-303,doi:10.1146/annurev-nutr-071812-161220.

Pepper,M.S.et al.(2003).Lymphangiogenesis and tumor metastasis.CellTissue Res 314:167–77,doi:10.1007/s00441-003-0748-7.

Pollard,J.W.(2008).Macrophages define the invasive microenvironmentin breast cancer.J.Leukoc.Biol.84:623–30,doi:10.1189/jlb.1107762.

Psaila, B. & Lyden, D. (2009). The metastatic niche: adapting the foreign oil. Nature Reviews Cancer 9:285–93, doi:10.1038/nrc2621.

Rada,P.et al.(2020).Understanding lipotoxicity in NAFLD pathogenesis: is CD36 a key driver? Cell Death and Disease 11:802,doi:10.1038/s41419-020-03003-w.

Su,P.et al.(2020).Enhanced lipid accumulation and metabolism are required for the differentiation and activation of tumor-associated macrophages.Cancer Res.80:1438-50,doi:10.1158/0008-5472.CAN-19-2994.

Sugimoto,Y.et al.(1993).Molecular cloning and characterization of the complementary DNA for the Mr 85,000 protein overexpressed in adriamycin-resistant human tumor cells.Cancer Res.53:2538-43.

Wang,D.&Dubois,R.N.(2006).Prostaglandins and cancer.Gut 55:115-22,doi:10.1136/gut.2004.047100.

Wang,J.&Li,Y.(2019).CD36 tango in cancer: signaling pathways and functions.Theranostics 9:4893-908,doi:10.7150/thno.36037.

Wang,H.et al.(2020).CD36-mediated metabolic adaptation supportsregulatory T cell survival and function in tumors.Nat.Immunol.21:298–308,doi:10.1038/s41590-019-0589-5.

Watt,M.J.et al.(2019).Suppressing fatty acid uptake has therapeutic effects in preclinical models of prostate cancer.Sci.Transl.Med.11:eaau5758,doi:10.1126/scitranslmed.aau5758.

Whiteside,T.L.&Jackson,E.K.(2013). Adenosine and prostaglandin e2production by human inducible regulatory T cells in health and disease.Frontiers Immunol.4:212,doi:10.3389/fimmu.2013.00212.

Wu,K.et al.(2020).Redefining tumor-associated macrophagesubpopulations and functions in the tumor microenvironment.Front.Immunol.11:1731,doi:10.3389/fimmu.2020.01731.

Xu,S.et al.(2020).Oxidized lipids and CD36-mediated lipidperoxidation in CD8 T cells suppress anti-tumor immune responses.BioRxivpreprint doi:10.1101/2020.09.03.281691.

Xu,S.et al.(2021).Uptake of oxidized lipids by the scavenger receptorCD36 promotes lipid peroxidation and dysfunction in CD8+T cells intumors.Immunity 54(7):1561-1577.e7,doi:10.1016/j.immuni.2021.05.003.

Ye,H.et al.(2016).Leukemic stem cells evade chemotherapy by metabolicadaptation to an adipose tissue niche.Cell Stem Cell 19:23–37,doi:10.1016/j.stem.2016.06.001.

Zeisberger,S.M.et al.(2006).Clodronate-liposome-mediated depletion oftumour-associated macrophages:a new and highly effective antiangiogenictherapy approach.British J.Cancer 95:272-81,doi:10.1038/sj.bjc.6603240.

Zhao,L.et al.(2018).CD36 and lipid metabolism in the evolution ofatherosclerosis.British Medical Bulletin 126:101–112,10.1093/bmb/ldy006.

Claims (20)

1. An anti-CD36 antibody comprising (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), wherein: (a) CDR-H1 contains the amino acid sequence of SEQ ID NO: 24, 3, 21 or 27; (b) CDR-H2 contains the amino acid sequence of SEQ ID NO: 43, 4, 28, 31, 34, 37 or 40; (c) CDR-H3 contains the amino acid sequence of SEQ ID NO:5; (d) CDR-L1 contains an amino acid sequence selected from SEQ ID NO:7; (e) CDR-L2 contains an amino acid sequence selected from SEQ ID NO: 15, 8, or 12; and (f) CDR-L3 contains an amino acid sequence selected from SEQ ID NO:18, 9 or 13. 2. The antibody according to claim 1, wherein: (a) CDR-H1 contains the amino acid sequence of SEQ ID NO:24, CDR-H2 contains the amino acid sequence of SEQ ID NO:43, CDR-H3 contains the amino acid sequence of SEQ ID NO:5, CDR-L1 contains the amino acid sequence of SEQ ID NO:7, CDR-L2 contains the amino acid sequence of SEQ ID NO:15, and CDR-L3 contains the amino acid sequence of SEQ ID NO:18; (b) CDR-H1 contains the amino acid sequence of SEQ ID NO:3, CDR-H2 contains the amino acid sequence of SEQ ID NO:4, CDR-H3 contains the amino acid sequence of SEQ ID NO:5, CDR-L1 contains the amino acid sequence of SEQ ID NO:7, CDR-L2 contains the amino acid sequence of SEQ ID NO:8, and CDR-L3 contains the amino acid sequence of SEQ ID NO:9; (c) CDR-H1 contains the amino acid sequence of SEQ ID NO:3, CDR-H2 contains the amino acid sequence of SEQ ID NO:4, CDR-H3 contains the amino acid sequence of SEQ ID NO:5, CDR-L1 contains the amino acid sequence of SEQ ID NO:7, CDR-L2 contains the amino acid sequence of SEQ ID NO:12, and CDR-L3 contains the amino acid sequence of SEQ ID NO:13; (d) CDR-H1 contains the amino acid sequence of SEQ ID NO:3, CDR-H2 contains the amino acid sequence of SEQ ID NO:4, CDR-H3 contains the amino acid sequence of SEQ ID NO:5, CDR-L1 contains the amino acid sequence of SEQ ID NO:7, CDR-L2 contains the amino acid sequence of SEQ ID NO:15, and CDR-L3 contains the amino acid sequence of SEQ ID NO:13; (e) CDR-H1 contains the amino acid sequence of SEQ ID NO:3, CDR-H2 contains the amino acid sequence of SEQ ID NO:4, CDR-H3 contains the amino acid sequence of SEQ ID NO:5, CDR-L1 contains the amino acid sequence of SEQ ID NO:7, CDR-L2 contains the amino acid sequence of SEQ ID NO:15, and CDR-L3 contains the amino acid sequence of SEQ ID NO:18; (f) CDR-H1 contains the amino acid sequence of SEQ ID NO:21, CDR-H2 contains the amino acid sequence of SEQ ID NO:4, CDR-H3 contains the amino acid sequence of SEQ ID NO:5, CDR-L1 contains the amino acid sequence of SEQ ID NO:7, CDR-L2 contains the amino acid sequence of SEQ ID NO:15, and CDR-L3 contains the amino acid sequence of SEQ ID NO:18; (g) CDR-H1 contains the amino acid sequence of SEQ ID NO:24, CDR-H2 contains the amino acid sequence of SEQ ID NO:4, CDR-H3 contains the amino acid sequence of SEQ ID NO:5, CDR-L1 contains the amino acid sequence of SEQ ID NO:7, CDR-L2 contains the amino acid sequence of SEQ ID NO:15, and CDR-L3 contains the amino acid sequence of SEQ ID NO:18; (h) CDR-H1 contains the amino acid sequence of SEQ ID NO:27, CDR-H2 contains the amino acid sequence of SEQ ID NO:28, CDR-H3 contains the amino acid sequence of SEQ ID NO:5, CDR-L1 contains the amino acid sequence of SEQ ID NO:7, CDR-L2 contains the amino acid sequence of SEQ ID NO:15, and CDR-L3 contains the amino acid sequence of SEQ ID NO:18; (i) CDR-H1 contains the amino acid sequence of SEQ ID NO:3, CDR-H2 contains the amino acid sequence of SEQ ID NO:31, CDR-H3 contains the amino acid sequence of SEQ ID NO:5, CDR-L1 contains the amino acid sequence of SEQ ID NO:7, CDR-L2 contains the amino acid sequence of SEQ ID NO:15, and CDR-L3 contains the amino acid sequence of SEQ ID NO:18; (j) CDR-H1 contains the amino acid sequence of SEQ ID NO:3, CDR-H2 contains the amino acid sequence of SEQ ID NO:34, CDR-H3 contains the amino acid sequence of SEQ ID NO:5, CDR-L1 contains the amino acid sequence of SEQ ID NO:7, CDR-L2 contains the amino acid sequence of SEQ ID NO:15, and CDR-L3 contains the amino acid sequence of SEQ ID NO:18; (k) CDR-H1 contains the amino acid sequence of SEQ ID NO:3, CDR-H2 contains the amino acid sequence of SEQ ID NO:37, CDR-H3 contains the amino acid sequence of SEQ ID NO:5, CDR-L1 contains the amino acid sequence of SEQ ID NO:7, CDR-L2 contains the amino acid sequence of SEQ ID NO:15, and CDR-L3 contains the amino acid sequence of SEQ ID NO:18; or (l) CDR-H1 contains the amino acid sequence of SEQ ID NO:3, CDR-H2 contains the amino acid sequence of SEQ ID NO:40, CDR-H3 contains the amino acid sequence of SEQ ID NO:5, CDR-L1 contains the amino acid sequence of SEQ ID NO:7, CDR-L2 contains the amino acid sequence of SEQ ID NO:15, and CDR-L3 contains the amino acid sequence of SEQ ID NO:18. 3. An anti-CD36 antibody comprising: (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), wherein the sequences of CDR-H1, CDR-H2, and CDR-H3 originate from the VH region, the VH region having an amino acid sequence selected from SEQ ID NO: 42, 2, 20, 23, 26, 30, 33, 36, and 39; (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), wherein the sequences of CDR-L1, CDR-L2, and CDR-L3 originate from the VL region, the VL region having an amino acid sequence selected from SEQ ID NO: 17, 6, 11, and 14, and The CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 are numbered according to Kabat. 4. The antibody according to claim 3, wherein: (a) The amino acid sequence of VH is SEQ ID NO:42 and the amino acid sequence of VL is SEQ ID NO:17; (b) The amino acid sequence of VH is SEQ ID NO:2 and the amino acid sequence of VL is SEQ ID NO:6; (c) The amino acid sequence of VH is SEQ ID NO:2 and the amino acid sequence of VL is SEQ ID NO:11; (d) The amino acid sequence of VH is SEQ ID NO:2 and the amino acid sequence of VL is SEQ ID NO:14; (e) The amino acid sequence of VH is SEQ ID NO:2 and the amino acid sequence of VL is SEQ ID NO:17; (f) The amino acid sequence of VH is SEQ ID NO:20 and the amino acid sequence of VL is SEQ ID NO:17; (g) The amino acid sequence of VH is SEQ ID NO:23 and the amino acid sequence of VL is SEQ ID NO:17; (h) The amino acid sequence of VH is SEQ ID NO:26 and the amino acid sequence of VL is SEQ ID NO:17; (i) The amino acid sequence of VH is SEQ ID NO:30 and the amino acid sequence of VL is SEQ ID NO:17; (j) The amino acid sequence of VH is SEQ ID NO:33 and the amino acid sequence of VL is SEQ ID NO:17; (k) The amino acid sequence of VH is SEQ ID NO:36 and the amino acid sequence of VL is SEQ ID NO:17; or (l) The amino acid sequence of VH is SEQ ID NO:39 and the amino acid sequence of VL is SEQ ID NO:17. 5. The antibody according to any one of claims 1-4, wherein the antibody comprises a heavy chain variable domain ( _VH ) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO:42, 2, 20, 23, 26, 30, 33, 36 or 39; and a light chain variable domain ( _VL ) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO:17, 6, 11 or 14. 6. The antibody according to any one of claims 1-4, wherein: (a) The antibody comprises a V _H amino acid sequence having at least 90% identity with SEQ ID NO:42 and a V _L amino acid sequence having at least 90% identity with SEQ ID NO:17; (b) The antibody comprises a V _H amino acid sequence having at least 90% identity with SEQ ID NO:2 and a V _L amino acid sequence having at least 90% identity with SEQ ID NO:6; (c) The antibody comprises a V _H amino acid sequence having at least 90% identity with SEQ ID NO:2 and a V _L amino acid sequence having at least 90% identity with SEQ ID NO:11; (d) The antibody comprises a V _H amino acid sequence having at least 90% identity with SEQ ID NO:2 and a V _L amino acid sequence having at least 90% identity with SEQ ID NO:14; (e) The antibody comprises a V _H amino acid sequence having at least 90% identity with SEQ ID NO:2 and a V _L amino acid sequence having at least 90% identity with SEQ ID NO:17; (f) The antibody comprises a V _H amino acid sequence having at least 90% identity with SEQ ID NO:20 and a V _L amino acid sequence having at least 90% identity with SEQ ID NO:17; (g) The antibody comprises a V _H amino acid sequence having at least 90% identity with SEQ ID NO:23 and a V _L amino acid sequence having at least 90% identity with SEQ ID NO:17; (h) The antibody comprises a V _H amino acid sequence having at least 90% identity with SEQ ID NO:26 and a V _L amino acid sequence having at least 90% identity with SEQ ID NO:17; (i) The antibody comprises a V _H amino acid sequence having at least 90% identity with SEQ ID NO:30 and a V _L amino acid sequence having at least 90% identity with SEQ ID NO:17; (j) The antibody comprises a VH amino acid sequence having at least 90% identity with SEQ ID NO:33 and a _VL amino acid sequence having at least 90% identity with SEQ ID NO:17; (k) The antibody comprises a V _H amino acid sequence having at least 90% identity with SEQ ID NO:36 and a V _L amino acid sequence having at least 90% identity with SEQ ID NO:17; or (l) The antibody comprises a V _H amino acid sequence having at least 90% identity with SEQ ID NO:39 and a V _L amino acid sequence having at least 90% identity with SEQ ID NO:17. 7. The antibody according to any one of claims 1-4, wherein the antibody comprises a heavy chain (HC) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO: 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 with a sequence selected from SEQ ID NO: 49, 45, 46 or 47. 8. The antibody according to any one of claims 1-4, wherein the antibody comprises: (a) An HC amino acid sequence having at least 90% identity with SEQ ID NO:57 and an LC amino acid sequence having at least 90% identity with SEQ ID NO:49; (b) an HC amino acid sequence having at least 90% identity with SEQ ID NO:44 and an LC amino acid sequence having at least 90% identity with SEQ ID NO:45; (c) An HC amino acid sequence having at least 90% identity with SEQ ID NO:44 and an LC amino acid sequence having at least 90% identity with SEQ ID NO:46; (d) An HC amino acid sequence having at least 90% identity with SEQ ID NO:44, and an LC amino acid sequence having at least 90% identity with SEQ ID NO:47; (e) an HC amino acid sequence having at least 90% identity with SEQ ID NO:44 and an LC amino acid sequence having at least 90% identity with SEQ ID NO:49; (f) HC amino acid sequences having at least 90% identity with SEQ ID NO:48 and LC amino acid sequences having at least 90% identity with SEQ ID NO:49; (g) an HC amino acid sequence having at least 90% identity with SEQ ID NO:50 and an LC amino acid sequence having at least 90% identity with SEQ ID NO:49; (h) an HC amino acid sequence having at least 90% identity with SEQ ID NO:51 and an LC amino acid sequence having at least 90% identity with SEQ ID NO:49; (i) an HC amino acid sequence having at least 90% identity with SEQ ID NO:52 and an LC amino acid sequence having at least 90% identity with SEQ ID NO:49; (j) an HC amino acid sequence having at least 90% identity with SEQ ID NO:53 and an LC amino acid sequence having at least 90% identity with SEQ ID NO:49; (k) an HC amino acid sequence having at least 90% identity with SEQ ID NO:54, and an LC amino acid sequence having at least 90% identity with SEQ ID NO:49; (l) an HC amino acid sequence having at least 90% identity with SEQ ID NO:55, and an LC amino acid sequence having at least 90% identity with SEQ ID NO:49; or (m) an HC amino acid sequence having at least 90% identity with SEQ ID NO:56 and an LC amino acid sequence having at least 90% identity with SEQ ID NO:49. 9. The antibody according to any one of claims 1-4, wherein the antibody: (a) is an antibody fragment, optionally selected from the group consisting of F(ab') ₂ , Fab', Fab, Fv, single-domain antibody (VHH) and scFv; (b) Contains a fusion with a protein; optionally, said protein is an immunostimulatory cytokine selected from IL-2, IL-7, IL-10, IL-12, IL-15, IL-21 or IFN-α. (c) Human antibodies, humanized antibodies, or chimeric antibodies; (d) is a full-length antibody of the IgG class, optionally wherein the IgG antibody has an isotype selected from IgG1, IgG2, IgG3 and IgG4; (e) Contains Fc region variants, optionally including Fc region variants that alter effector function and/or variants that alter antibody half-life; (f) Containing an immunoconjugate, optionally, said immunoconjugate contains a therapeutic agent for treating CD36-mediated diseases or conditions; and/or (g) is a multispecific antibody; optionally, it is a bispecific antibody. 10. The antibody according to any one of claims 1-9, wherein: (a) The antibody binds to human CD36 with a binding affinity of 1× ^10⁻⁸ M or less, 1× ^10⁻⁹ M or less, or 1× ^10⁻¹⁰ M or less; optionally, the binding affinity is measured by the equilibrium dissociation constant ( _KD ) of the hCD36 peptide with SEQ ID NO: 58 or 59. (b) The antibody binds to mouse CD36 with a binding affinity of 1× ^10⁻⁸ M or less, 1× ^10⁻⁹ M or less, or 1× ^10⁻¹⁰ M or less; optionally, the binding affinity is measured by the equilibrium dissociation constant ( _KD ) of the mCD36 peptide with SEQ ID NO: 60 or 61. (c) The antibody binds to rhesus CD36 with a binding affinity of 1× ^10⁻⁸ M or less, 1× ^10⁻⁹ M or less, or 1× ^10⁻¹⁰ M or less; optionally, the binding affinity is measured by the equilibrium dissociation constant ( _KD ) of the rhesus CD36 peptide with SEQ ID NO: 62 or 63. (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 human CD36; optionally, the antibody has an _IC50 of 5 nM or less, 1 nM or less, 0.5 nM or less, or 0.1 nM or less at a concentration of 1-10 μg/mL of oxLDL. (e) 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 U937 cells; optionally, wherein the antibody has an IC50 of 5 nM or less, 1 nM or less, 0.5 nM or less, or 0.1 nM or less at a concentration of _{1-10 μg} /mL of oxLDL; and/or (f) 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 mouse CD45+TIL; optionally, the antibody has an _IC50 of 5 nM or less, 1 nM or less, 0.5 nM or less, or 0.1 nM or less at a concentration of 1-10 μg/mL of oxLDL. 11. An isolated polynucleotide or vector comprising a sequence encoding a polypeptide of any one of claims 1-10 against an anti-CD36 antibody. 12. The isolated polynucleotide or vector according to claim 11, wherein the polypeptide comprises an amino acid sequence, the amino acid sequence comprising: (a) CDR-H1 contains the amino acid sequence of SEQ ID NO:3, 21, 24 or 27; CDR-H2 contains the amino acid sequence of SEQ ID NO:4, 28, 31, 34, 37, 40 or 43; and CDR-H3 contains the amino acid sequence of SEQ ID NO:5; (b) CDR-L1 contains an amino acid sequence selected from SEQ ID NO:7; CDR-L2 contains an amino acid sequence selected from SEQ ID NO:8, 12 or 15; and CDR-L3 contains an amino acid sequence selected from SEQ ID NO:9, 13 or 18. (c) A heavy chain variable domain ( _VH ) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO:2, 20, 23, 26, 30, 33, 36, 39 or 42; (d) A light chain variable domain ( _VL ) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO: 6, 11, 14 or 17; (e) a heavy chain (HC) amino acid sequence having at least 90% identity with a sequence selected from SEQ ID NO: 44, 48, 50, 51, 52, 53, 54, 55, 56 or 57; and/or (f) A light chain (LC) amino acid sequence that has at least 90% identity with a sequence selected from SEQ ID NO:45, 46, 47 or 49. 13. An isolated host cell comprising a polynucleotide or vector according to any one of claims 11-12; optionally, wherein the host cell is selected from Chinese hamster ovary (CHO) cells, myeloma cells (e.g., Y0, NSO, Sp2/O), monkey kidney cells (COS-7), human embryonic kidney cell line (293), young hamster kidney cells (BHK), mouse serratus cells (e.g., TM4), African green monkey kidney cells (VERO-76), human cervical cancer 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. A method for producing antibodies, comprising culturing host cells according to claim 13 to produce antibodies. 15. A pharmaceutical composition comprising an antibody according to any one of claims 1-10 and a pharmaceutically acceptable carrier. 16. The composition of claim 15, wherein the composition further comprises a chemotherapeutic agent. 17. The composition of claim 15, wherein the composition further comprises an antibody specific to 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, and ICOS. 18. The composition of claim 15, wherein the composition further comprises 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 of treating a CD36-mediated disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody according to any one of claims 1-10, or administering to the subject a therapeutically effective amount of a pharmaceutical composition according to any one of claims 15-18; 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. The method of claim 19, wherein the cancer is colon cancer, pancreatic cancer, ovarian cancer, HCC, kidney cancer, breast cancer, lung cancer, gastric cancer, melanoma, head and neck cancer, or oral cancer.