Anti-CD36 antibody for treatment of cancer

Abstract

This disclosure provides compositions and methods for treating liver cancer and liver metastasis, including in patients having tumors that are particularly challenging to treat, such as cold tumors and liver metastasis characterized with increased fatty acid content, Treg or CD8+ T cell concentrations.

Description

Attorney Docket No.: 92EW-385670-WO ANTI-CD36 ANTIBODY FOR TREATMENT OF CANCER CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of the United States Provisional Application Serial Nos. 63 / 573,374 filed April 2, 2024 and 63 / 708,092 filed October 16, 2024, the content of which is hereby incorporated by reference in its entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (385670.xml; Size: 12,660 bytes; and Date of Creation: March 26, 2025) is herein incorporated by reference in its entirety. BACKGROUND

[0003] Accumulation of lipid metabolic products in tumor microenvironment (TME) has been documented to augment the malignancy of cancer by dampening anti-tumor immunity, including increased immunosuppressive features in various immune cells and development of exhausted phenotypes in T cells, which further orchestrates hurdles for immunotherapies. CD36, a fatty acid transporter, is frequently up-regulated in both malignant cells and tumor-associated immune cells, including regulatory T cells, tumor-associated macrophages and CD8+T cells, to adjust the metabolic preference, which allow the cells to adapt in the lipid-enriched TME. This CD36- mediated adaptation not only alters metabolic regulations, but also impacts immune cell properties to construct an immunosuppressive TME, which is composed of enriched regulatory T cells, exhausted CD8 T cells and other suppressive cell populations. SUMMARY

[0004] This disclosure provides compositions and methods for treating liver cancer and liver metastasis, including in patients having tumors that are particularly challenging to treat, such as cold tumors and liver metastasis characterized with increased fatty acid content, Treg or CD8+ T cell concentrations.

[0005] In accordance with one embodiment of the present disclosure, provided is a method for treating, in a subject, liver metastasis originated from a cancer in a tissue that is not liver,Attorney Docket No.: 92EW-385670-WO comprising administering to the subject an antibody or antigen-binding fragment comprising a heavy chain variable region (VH) comprising a VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) comprising a VL CDR1, VL CDR2, and VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprise, respectively, the amino acid sequences of SEQ ID NO:3-8.

[0006] Also provided is a method for treating a cancer in a subject having a body mass index (BMI) of 25 kg / m² or higher or having an increased fatty acid concentration in the liver as compared to a reference healthy subject, comprising administering to the subject an antibody or antigen-binding fragment comprising a heavy chain variable region (VH) comprising a VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) comprising a VL CDR1, VL CDR2, and VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprise, respectively, the amino acid sequences of SEQ ID NO:3-8.

[0007] In some embodiments, the subject has an increased fatty acid concentration in the liver as compared to a reference healthy subject. In some embodiments, the subject has hepatic steatosis. In some embodiments, the subject has alcoholic fatty liver disease (AFLD) or nonalcoholic fatty liver disease (NAFLD). In some embodiments, the subject has nonalcoholic steatohepatitis (NASH).

[0008] In some embodiments, the subject has a body mass index (BMI) of 25 kg / m² or higher. In some embodiments, the subject has a BMI of 28 kg / m² or higher, or 30 kg / m² or higher.

[0009] In some embodiments, the subject has a decreased Treg concentration in the liver as compared to an average cancer patient having liver metastasis.

[0010] In some embodiments, the subject has an increased CD8+ T cell infiltration in the liver as compared to an average cancer patient having liver metastasis. In some embodiments, the liver metastasis is from a cold tumor. In some embodiments, the cold tumor is characterized with one or more p53 mutation or β-catenin mutation, high microsatellite instability (MSI-high), or microsatellite stable (MSS) with CD36 expression.

[0011] In some embodiments, the subject has been treated with a PD-L1 or PD-1 inhibitor or is resistant to a PD-L1 or PD-1 inhibitor.Attorney Docket No.: 92EW-385670-WO

[0012] In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is hepatocellular carcinoma (HCC), colorectal cancer (CRC), or colorectal liver metastasis (CRLM).

[0013] In some embodiments, the method further comprises administering to the subject an inhibitor of PD-L1 or PD-1.

[0014] In some embodiments, the method further comprises administering to the subject an inhibitor of VEGFα.

[0015] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:1 and the VL comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the antibody comprises a human IgG4 Fc fragment. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:11 and a light chain comprising the amino acid sequence of SEQ ID NO:12. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 shows that PLT001 interacts with Domains 1 and 2 of the extracellular domain of the human CD36 protein. Mutations in either of these domains reduced PLT001 binding and those of both almost completely eliminated the binding.

[0017] FIG. 2 shows that CD36 was upregulated in tumor-associated Treg, CD8 T and NK cells in HCC-bearing mice.

[0018] FIG. 3 shows that, upon administration, the retention of PLT001 was prolonged in livers from tumor-bearing mice.

[0019] FIG. 4A-B show that single-agent PLT001 eliminated the majority of tumors in a hydrodynamic injection-induced HCC “hot tumor” model.

[0020] FIG. 5A-B show that single-agent PLT001 efficiently controlled tumor growth with boosted anti-tumor immunity in a HCC “cold tumor” model.Attorney Docket No.: 92EW-385670-WO

[0021] FIG. 6A-B demonstrate the synergism between PLT001 and anti-PD-L1, thereby showing that PLT001 can overcome the irresponsiveness of anti-PDL1 in cold HCC tumors.

[0022] FIG. 7 shows that PLT001 had remarkable anti-tumor responses when combined with the current standard of care (SoC) in treating “cold” HCC (anti-VEGFα and anti-PD-L1).

[0023] FIG. 8 shows PLT001 eliminated liver metastasis to re-sensitize immune checkpoint blocking (ICB) treatment in a MSS / pMMR CRC mouse model.

[0024] FIG. 9A-D show that CD36 expression correlated with poorer clinical outcome of HCC associated with hepatitis B viral (HBV) infection.

[0025] FIG. 10A-D show that CD36 expression correlated with poorer clinical outcome of HCC associated with hepatitis C viral (HCV) infection.

[0026] FIG. 11 shows a positive correlation between CD36 expression and both the percentage of regulatory T cells (Tregs) and the Exhaustion score in TCGA cohort in MSS type CRC patients.

[0027] FIG. 12 shows that higher levels of CD36 expression were linked to poorer clinical outcomes, encompassing overall survival (OS), progression-free survival (PFS), and disease- specific survival (DSS) MSS type CRC patients.

[0028] FIG. 13 shows impacts of PLT001 on HCC in a lipid-dense milieu. A, Experiment design and procedure, n=12-13 / group. B-D, Tumor growth (B), tumor mass (C), and ex vivo tumor BLI (D) of MYCOE / CTNNB1N90HCC tumors was assessed in indicated groups. E-I, Proportions of Tregs (E), CD8+T cells (F), granzyme B+CD8 T cells (G), Prog Tex (G), and Term Tex (H) within the CD45+cell population were quantified using FACS. Data are the cumulative results from three independent experiments, with each symbol representing one individual. Data are means ± s.e.m. and analyzed by One-way ANOVA.

[0029] FIG. 14 shows characterization of the progression and immune profile of the MYCOE / CTNNB1N90liver cancer mouse model in the context of lipid-rich condition. A, Temporal progression of mouse body weight in response to the specified treatment, as indicated in Figure 5B. B-D, Tumor growth (B) as assessed by bioluminescence imaging (BLI), ex vivo BLI of theAttorney Docket No.: 92EW-385670-WO liver (C), and the final tumor weight (D) in mice subjected to either a Western diet (WD) or a standard chow diet, n=12 / group. E and F, Percentages of Tregs (E) and CD8+T cells (F) were quantified using FACS for each group, n=12 / group. G and H, CD36 expression levels in Treg, CD8+T cells, progenitor-exhausted T cells (Prog Tex), and terminal-exhausted T cells (Term Tex) from indicated groups were measured by staining with AF647-labelled PLT001 (G) and a commercial anti-CD36 antibody, HM36 (H), n=4 / group. Each symbol represents one individual. Data are means ± s.e.m. and analyzed by two-tailed, unpaired Student’s t-test.

[0030] FIG. 15 shows that PLT001 treatment suppresses primary and metastasis in a mouse model of colon cancer. A and B, An in silico analysis of POG570 cohort for expression level of CD36 and fatty acid metabolism score, as well as relative percentages for Treg and CD8 T cells across various metastatic sites (A) and aggressive tumor types (B). C, Schematic representation of PLT001 treatment protocol utilized in the MC38 colon cancer metastasis model. D and E, Growth curve of MC38 liver metastases (D) and subcutaneous tumors (E) in mice treated with vehicle control (Ctrl), anti-PD-1, PLT001, or combination (combo). F and G, Analysis of the percentages of CD8 T cells and Tregs percentages within CD45+cells in liver metastasis (F) and primary tumors (G). H and I, Representative plots (H) and quantitative results (I) for population of M1 and M2 macrophages among CD45+cells from the indicated groups. Data are the cumulative results from two independent experiments. Each symbol represents one individual. Data are means ± s.e.m. and analyzed by One-way ANOVA.

[0031] FIG. 16 shows an in silico analysis on POG570 to evaluate the potential of PLT001 in targeting metastatic diseases. A, A workflow starting with the selection of samples from the POG570 cohort, containing advanced and metastatic tumors with a subset of 438 samples. The Sankey plot illustrates the original cancer types associated with the biopsy sites within this subset. Subsequent analyses involve the examination of CD36 expression and the distribution of fatty acid (FA) scores, as well as the evaluation of FA scores in metastatic and recurrent sites across various cancer types. Additionally, the analysis assesses the correlation between CD36 expression and FA scores, exhaustion (EX) scores, and the relative proportions of CD8 T cells compared to regulatory T cells (Tregs). B, Evaluation of fatty acid (FA) scores for tumors in metastatic and recurrent sites of breast cancer (BRCA), colon cancer (COAD), pancreatic cancer (PAAD), stomach cancer (STAD), and non-small cell lung cancer (NSCLC). C, The PearsonAttorney Docket No.: 92EW-385670-WO coefficient assessed the correlation between CD36 expression and FA (Left) or EX (exhaustion; Right) scores in metastatic and recurrent sites of specific cancer types.

[0032] FIG. 17 shows the evaluation of PLT001’s efficacy in inhibiting both primary and metastatic growth of MC38 colon cancer. A, Subcutaneous MC38 tumor growth curve treated with vehicle or PD-1 inhibitor in the presence and absence of liver metastases from colon cancer. B, Representative BLI from indicated groups to FIG. 15D. C, Quantitative ex vivo BLI results for liver tumors from indicated groups, along with representative BLI images. D and E, Quantitative data and representative gross tumor images for liver metastases mass (D) and primary tumor mass (E) for indicated groups. F, Number of CD8+T cells per gram of metastatic tumor tissues (Left) and primary tumor samples (Right) from indicated groups. G, Gating Strategy employed for FACS analysis of tumor-associated macrophages in colon cancer metastasis mouse models. H and I, Representative plots (H) and quantitative results (I) for proportion of M1 and M2 macrophage among total tumor-infiltrating CD45+T cells of primary colon tumors from the indicated mice. J and K, Representative plots and quantitative results for CD206 staining of both primary tumor and metastatic tumor in the indicated treatment. Scale bars: 50 µm. Data are the cumulative results from two independent experiments. Each symbol represents one individual. Data are means ± s.e.m. and analyzed by ordinary one-way ANOVA.

[0033] FIG. 18 shows the results of the safety and efficacy study of PLT001 evaluated involving repeated doses in non-human primates and human HCC patient ex vivo tissue fragment. A and B, FACS analysis of CD36 expression levels in red blood cells (RBCs) and platelets (A), as well as in CD4, CD8, B cells, and monocytes from PBMC samples of humans, cynomolgus monkeys, and mice (B). C, Diagram depicting the procedure for the dose range finding (DRF) study of PLT001, involving 5 repeated doses at concentrations of 0, 10, 60, and 200 mg / kg. D and E, Serum concentrations of ALT and AST (D) and number of circulating RBC and platelets (E) were measured over the study period at the indicated doses. F, Proportions of CD4, CD8, NK cells, and monocytes within CD45+cells were assessed on Day 1 and Day 29 in cynomolgus monkeys receiving PLT001 treatment. G, Proportion of CD36 positive CD8 T cells and Tregs identified in human HCC tissues fragments through FACS, following the indicated treatment (n=11). H, FACS analysis was conducted to evaluate the pharmacodynamic immune profiles of indicated TILs in ex vivo human HCC tissues fragments after a two-day cultureAttorney Docket No.: 92EW-385670-WO period with either control or PLT001 antibodies (n=11). I, Response rates based on pharmacodynamic markers into three classifications: SR (Strong Response, defined as a fold change greater than 2), MR (Moderate Response, defined as a fold change between 1 and 2), and NR (No Response). Data are presented as mean ± sd in panels A-F, with a sample size of n = 4 animals, comprising both male and female subjects, for each treatment group. DETAILED DESCRIPTION Definitions

[0034] It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “an antibody,” is understood to represent one or more antibodies. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0035] As used herein, an “antibody” or “antigen-binding polypeptide” refers to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen binding fragment or a single chain thereof. Thus the term “antibody” includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to the antigen. Examples of such include, but are not limited to a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein.

[0036] The terms “antibody fragment” or “antigen-binding fragment,” as used herein, is a portion of an antibody such as F(ab’)2, F(ab)2, Fab’, Fab, Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. The term “antibody fragment” includes aptamers, spiegelmers, and diabodies. The term “antibody fragment” also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0037] The term antibody encompasses various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains areAttorney Docket No.: 92EW-385670-WO classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε) with some subclasses among them (e.g., γl-γ4). It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively.

[0038] The immunoglobulin subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant disclosure. All immunoglobulin classes are clearly within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000. The four chains are typically joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region.

[0039] Antibodies, antigen-binding polypeptides, variants, or derivatives thereof of the disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab’ and F(ab’)2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VK or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti- Id antibodies to the antibodies disclosed herein). Immunoglobulin or antibody molecules of the disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.

[0040] As used herein, the term “chimeric antibody” will be held to mean any antibody wherein the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial or modified in accordance with the instant disclosure) is obtained from a second species. In certain embodiments the target binding region or site will be from a non-human source (e.g. mouse or primate) and the constant region is human.Attorney Docket No.: 92EW-385670-WO

[0041] Antibodies disclosed herein can be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In some embodiments, the variable region may be condricthoid in origin (e.g., from sharks).

[0042] As used herein, the term “recombinant” as it pertains to polypeptides or polynucleotides intends a form of the polypeptide or polynucleotide that does not exist naturally, a non-limiting example of which can be created by combining polynucleotides that would not normally occur together.

[0043] Hybridoma technology can be performed under conditions of different “stringency.” In general, a low stringency hybridization reaction is carried out at about 40°C in about 10 x SSC or a solution of equivalent ionic strength / temperature. A moderate stringency hybridization is typically performed at about 50°C in about 6 x SSC, and a high stringency hybridization reaction is generally performed at about 60°C in about 1 x SSC. Hybridization reactions can also be performed under “physiological conditions” which is well known to one of skill in the art. A nonlimiting example of a physiological condition is the temperature, ionic strength, pH and concentration of Mg2+normally found in a cell. Cancer Treatments

[0044] PLT001 is an antibody specific to the CD36 protein, which is upregulated in several tumor-infiltrating immune cell populations to allow them to adapt in a lipid-enriched tumor microenvironment (TME). In the accompanying experimental examples, the instant inventors discovered that PLT001 binds to both Domain 1 (residues 153 to 160) and Domain 2 (residues 191 to AA197). These domains, structural analysis shows, are important for oxidized low-density lipoprotein (OxLDL) uptake. Accordingly, it is contemplated that PLT001 can effectively block fatty acid uptake by the target cells, including target tumor cells, which may contribute to metastasis of the tumor.

[0045] Also discovered, in the examples, is that PLT001 was also effective in tumors and tumor metastasis (e.g., metastasis in liver) with reduced intratumoral Tregs and boosted CD8+ TILs. In particular, PLT001 treatment resulted in increased levels of progenitor exhausted CD8 T cells,Attorney Docket No.: 92EW-385670-WO which are the major responders to anti-PD1 / PD-L1 treatment. Therefore, PLT001 can sensitize tumors for the anti-PD1 / PD-L1 treatment. Indeed, as reported in the examples, the combination of PLT001 and PD-L1 antibody exhibited significant synergism. Such synergism extended to the standard of care for HCC (anti-VEGFα + anti-PD-L1).

[0046] In accordance with one embodiment of the present disclosure, therefore, provided is a method for treating liver cancer or liver metastasis that is originated from a cancer in a tissue that is not liver. In some embodiments, the method entails administration of an antibody or antigen- binding fragment of the present disclosure.

[0047] In another embodiment, provided is a method for treating a cancer in a patient that has an increased amount of fatty acid or fat concentration in the liver or in the body. In some embodiments, the increase is as compared to an average healthy subject not having cancer. In some embodiments, the increase is as compared to an average cancer patient that does not have increased fat content. Also provided, in one embodiment, is a method for treating a cancer in a subject having a body mass index (BMI) of 25 kg / m² or higher.

[0048] In one aspect of any one of the above embodiments, the subject has a BMI that is 25 kg / m², 26 kg / m², 27 kg / m², 28 kg / m², 29 kg / m², 30 kg / m², 31 kg / m², 32 kg / m², 33 kg / m², 34 kg / m², 35 kg / m², or 40 kg / m².

[0049] In some embodiments, wherein the subject is on a high-fat diet or a high-sugar diet. The term “on a high-fat diet” or “on a high-sugar diet” refers to a subject’s exposure to such diets and does not require that such a diet is consumed by the subject every day or in every meal. For instance, the subject may consume such a diet daily, once every two days, once every three days, once every four days, once every five days, once every six days, once a week, once every two weeks, without limitation.

[0050] A high-fat diet (HFD), in some embodiment, refers to a diet or meal with at least 25% of total calories from fats, unsaturated or saturated. In some embodiments, the diet or meal has at least 30%, 35% or 40% of total calories from fats.

[0051] A high-sugar diet (HSD), in some embodiment, refers to a diet or meal with at least 5% of total calories from carbohydrates, including monosaccharides and disaccharides. In someAttorney Docket No.: 92EW-385670-WO embodiments, the diet or meal has at least 10%, 15%, 20%, 25%, 30%, 35% or 40% of total calories from carbohydrates.

[0052] In some embodiments, the subject has hepatic steatosis. In some embodiments, the subject has alcoholic fatty liver disease (AFLD) or nonalcoholic fatty liver disease (NAFLD). In some embodiments, the subject has nonalcoholic steatohepatitis (NASH).

[0053] Hepatic steatosis, also known as fatty liver, refers to an accumulation of fat within the liver tissue. Steatosis occurs when the liver contains more than 5-10% of its weight as fat. When fat accumulates excessively in the liver, it leads to a fatty liver or hepatic steatosis. This condition can be a precursor to nonalcoholic fatty liver disease (NAFLD). In a person that drinks alcohol, this can lead to alcoholic fatty liver disease (AFLD). For NAFLD, if there’s no inflammation or other complications, the condition is known as simple NAFLD. When the buildup of excess fat in the liver is accompanied by inflammation, a diagnosis of nonalcoholic steatohepatitis (NASH) may be made.

[0054] Patients that can be treated with the instant methods may have one or more other characteristics that render the patients harder to treat by conventional treatments, including immune checkpoint blockade (ICB) therapies. Sometimes, such tumors are referred to as “cold tumors” or “nonimmunogenic tumors.”

[0055] In some embodiments, a nonimmunogenic tumor is one that is not infiltrated with T cells, or that is deficient in T cell filtration, in antigen presenting cells (APCs), or in T cell activation, or has deficit in T cell homing into the tumor bed. All of prostate cancer, pancreatic cancer, and leukemia are nonimmunogenic. The vast majority of breast cancer (95%), colorectal cancer (95%), gastric cancer (87%), head and neck cancer (84%), liver cancer (83%), esophageal cancer (86%), cervical cancer (87%), and thyroid cancer (87%) are also nonimmunogenic. In addition, 83% of lung cancer, 79% of bladder cancer, 77% of kidney cancer, 70% uterus cancer, and 66% melanoma are also nonimmunogenic.

[0056] Identification of nonimmunogenic, or cold tumors can also be made with measurements of type, density and location of immune cells within the tumors. For instance, Galon and Bruni (Nature Reviews Drug Discovery volume 18, pages 197–218 (2019)) describes a standardizedAttorney Docket No.: 92EW-385670-WO scoring system, Immunoscore, based on the quantification of two lymphocyte populations (CD3 and CD8), e.g., in resected tissues, for guided stratification of hot and cold tumors. The Immunoscore ranges from Immunoscore 0 (I0, for low densities, such as absence of both cell types in both regions) to I4 (high immune cell densities in both locations). By classifying cancers according to their immune infiltration, the scoring system provides an immune-based classification of tumors, including a definition of “hot” (highly infiltrated, Immunoscore I4) and “cold” (non-infiltrated, Immunoscore I0) tumors.

[0057] In some embodiments, the patient has an I0 immunoscore. In some embodiments, the patient has an I1 immunoscore. In some embodiments, the patient has an I2 immunoscore. In some embodiments, the patient has an I3 immunoscore.

[0058] In some embodiments, the patient has a decreased Treg concentration in the liver as compared to an average cancer patient having liver metastasis. In some embodiments, the patient has an increased CD8+ T cell infiltration in the liver as compared to an average cancer patient having liver metastasis.

[0059] In some embodiments, the patient has one or more p53 mutation or β-catenin mutation, or high microsatellite instability (MSI-high). MSI-High (MSI-H) is a classification used in cancer diagnosis. It refers to tumors with significant microsatellite instability. Around 15% of all colon cancers fit into the MSI high category.

[0060] It is herein demonstrated that some MSS (microsatellite stable) patients can be CD36 positive. Such patients have higher percentage of regulatory T cells and correlate to poorer clinical outcomes. These CD36-positive MSS patients, however, are suitable patients for the instantly disclosed therapies, even thought they may not be responsive to immune checkpoint inhibitors.

[0061] In some embodiments, the patient has been treated with an anti-PD-L1 or anti-PD-1 treatment. In some embodiments, the patient is resistant to such a treatment.

[0062] Combination treatments are also provided in this disclosure. In some embodiments, the subject that is administered an antibody or antigen-binding fragment of the present disclosure is also administered a PD-L1 or PD-1 inhibitor.Attorney Docket No.: 92EW-385670-WO

[0063] A PD-1 inhibitor is a molecule that binds to and inhibits the biological activity of the PD-1 protein. Programmed cell death protein 1, also known as PD-1 and CD279 (cluster of differentiation 279), is a protein on the surface of cells that has a role in regulating the immune system’s response to the cells of the human body by down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. Examples are anti-PD-1 antibodies and fragments thereof, such as those described below.

[0064] Pembrolizumab (formerly MK-3475 or lambrolizumab, Keytruda) is an anti-PD-1 monoclonal antibody developed by Merck and first approved by the Food and Drug Administration in 2014 for the treatment of melanoma. It was later approved for metastatic non- small cell lung cancer and head and neck squamous cell carcinoma.

[0065] Nivolumab (Opdivo) is an anti-PD-1 monoclonal antibody developed by Bristol-Myers Squibb and first approved by the FDA in 2014 for the treatment of melanoma. It was later approved for squamous cell lung cancer, renal cell carcinoma, and Hodgkin’s lymphoma.

[0066] Cemiplimab (Libtayo) is an anti-PD-1 monoclonal antibody developed by Regeneron Pharmaceuticals and first approved by the FDA in 2018 for the treatment of cutaneous squamous cell carcinoma (CSCC) or locally advanced CSCC who are not candidates for curative surgery or curative radiation.

[0067] Spartalizumab (PDR001) is an anti-PD-1 monoclonal antibody developed by Novartis to treat both solid tumors and lymphomas.

[0068] Camrelizumab (SHR1210) is an anti-PD-1 monoclonal antibody introduced by Jiangsu HengRui Medicine Co., Ltd. that recently received conditional approval in China for the treatment of relapsed or refractory classical Hodgkin lymphoma.

[0069] Sintilimab (IBI308) is an anti-PD-1 monoclonal antibody developed by Innovent and Eli Lilly for patients with non-small cell lung cancer (NSCLC).

[0070] Tislelizumab (BGB-A317) is a humanized IgG4 anti–PD-1 monoclonal antibody developed by BeiGene for solid tumors and hematologic cancers.Attorney Docket No.: 92EW-385670-WO

[0071] Dostarlimab (TSR-042, WBP-285) is a humanized monoclonal antibody against PD-1 under investigation by GlaxoSmithKline.

[0072] Toripalimab (Loqtorzi) is a monoclonal antibody used for the treatment of melanoma and nasopharyngeal carcinoma. It has been approved in China for the treatment of unresectable or metastatic melanoma that has failed previous systemic therapy and in the US for the first-line treatment of adults with metastatic or recurrent, locally advanced nasopharyngeal carcinoma when used with cisplatin and gemcitabine.

[0073] INCMGA00012 (MGA012) is a humanized IgG4 monoclonal antibody developed by Incyte and MacroGenics.

[0074] AMP-224 is an anti-PD-1 monoclonal antibody by AstraZeneca / MedImmune and GlaxoSmithKline.

[0075] AMP-514 (MEDI0680) is an anti-PD-1 monoclonal antibody by AstraZeneca.

[0076] A PD-L1 inhibitor is a molecule that binds to and inhibits the biological activity of the PD-L1 protein. Programmed death-ligand 1 (PD-L1) also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1) is a protein that in humans is encoded by the CD274 gene. Examples are anti-PD-L1 antibodies and fragments thereof, such as those described below.

[0077] Atezolizumab (Tecentriq) is a humanized anti-PD-L1 IgG1 antibody developed by Roche Genentech. It has been approved by the FDA for urothelial carcinoma and non-small cell lung cancer.

[0078] Avelumab (Bavencio) is a human anti-PD-L1 IgG1 antibody developed by Merck Serono and Pfizer. Avelumab has been approved by the FDA for the treatment of metastatic Merkel-cell carcinoma.

[0079] Durvalumab (Imfinzi) is a human anti-PD-L1 IgG1 antibody developed by AstraZeneca. Durvalumab has been approved by the FDA for the treatment of urothelial carcinoma and unresectable non-small cell lung cancer after chemoradiation.Attorney Docket No.: 92EW-385670-WO

[0080] KN035 is an anti-PD-L1 antibody with subcutaneous formulation currently under clinical evaluations in the US, China, and Japan.

[0081] CK-301 is an anti-PD-L1 antibody being developed by Checkpoint Therapeutics.

[0082] Some small peptide and small molecule inhibitors are also being developed. Examples are shown below.

[0083] AUNP12 is a 29-mer peptide as the first peptic PD-1 / PD-L1 inhibitor developed by Aurigene and Laboratoires Pierre Fabre that is being evaluated in clinical trial, following promising in vitro results.

[0084] CA-170, discovered by Aurigene / Curis as the PD-L1 and VISTA antagonist, was indicated as a potent small molecule inhibitor in vitro. The compound is currently under phase I clinical trial over mesothelioma patients.

[0085] BMS-986189 is a macrocyclic peptide discovered by Bristol-Myers Squibb of which the pharmacokinetics, safety and tolerability are currently being studied on healthy subjects.

[0086] In some embodiments, the subject that is administered an antibody or antigen-binding fragment of the present disclosure is also administered a PD-L1 or PD-1 inhibitor, and an inhibitor to VEGFα.

[0087] Inhibitors of VEGFα include small molecule drugs and antibodies. Non-limiting examples of antibodies include bevacizumab and ranibizumab (Lucentis). Small molecule inhibitors include, without limitation, sunitinib, sorafenib, axitinib, and pazopanib.

[0088] In some embodiments, the anti-CD36 antibody or antigen-binding fragment of the present disclosure and the PD-L1 / PD-1 inhibitor, or further with the VEGFα inhibitors, are administered concurrently. In some embodiments, they are administered separately or sequentially.

[0089] The anti-CD36 antibody or antigen-binding fragment of the present disclosure, in some embodiments, includes those that have the same CDRs or VH / VL as PLT001. As shown in Table 1, PLT001 has a VH of SEQ ID NO:1 and VL of SEQ ID NO:2. The VH CDR1-3 are providedAttorney Docket No.: 92EW-385670-WO in SEQ ID NO:3-5, and the VL CDR1-3 are provided in SEQ ID NO:6-8. In some embodiments, the antibody is humanized. In some embodiments, the antibody is of IgG4 type with a human IgG4 Fc fragment.

[0090] The instant methods can be used for treating various cancer types. In some embodiments, the cancer is hepatocellular carcinoma (HCC), colorectal cancer (CRC), or colorectal liver metastasis (CRLM). In some embodiments, the treatment is for liver metastasis from a cancer of a different type.

[0091] Non-limiting examples of cancers include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer. In some embodiments, the cancer being treated is colorectal cancer.

[0092] Additional diseases or conditions associated with increased cell survival, that may be treated, prevented, diagnosed and / or prognosed with the antibodies or variants, or derivatives thereof of the disclosure include, but are not limited to, progression, and / or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin’s disease and non-Hodgkin’s disease), multiple myeloma, Waldenstrom’s macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyo sarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm’s tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lungAttorney Docket No.: 92EW-385670-WO carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma.

[0093] A specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the particular antibodies, variant or derivative thereof used, the patient’s age, body weight, general health, sex, and diet, and the time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated. Judgment of such factors by medical caregivers is within the ordinary skill in the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0094] Methods of administration of the antibody, fragment, or antibody-drug conjugate include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antigen-binding polypeptides or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Thus, pharmaceutical compositions containing the antigen-binding polypeptides of the disclosure may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), buccally, or as an oral or nasal spray.

[0095] The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intra-articular injection and infusion.

[0096] Administration can be systemic or local. In addition, it may be desirable to introduce the antibodies of the disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.Attorney Docket No.: 92EW-385670-WO Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

[0097] It may be desirable to administer the antigen-binding polypeptides or compositions of the disclosure locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction, with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the disclosure, care must be taken to use materials to which the protein does not absorb.

[0098] The amount of the antibodies, fragments, or antibody-drug conjugates of the disclosure which will be effective in the treatment, inhibition and prevention of an inflammatory, immune or malignant disease, disorder or condition can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease, disorder or condition, and should be decided according to the judgment of the practitioner and each patient’s circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0099] As a general proposition, the dosage administered to a patient of the antibodies, fragments, or antibody-drug conjugates of the present disclosure is typically 0.001 mg / kg to 100 mg / kg of the patient’s body weight, between 0.01 mg / kg and 20 mg / kg of the patient’s body weight, or 0.5 mg / kg to 10 mg / kg of the patient’s body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration are often possible. Further, the dosage and frequency of administration of antibodies of the disclosure may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.

[0100] In additional embodiments, the compositions of the disclosure are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiationAttorney Docket No.: 92EW-385670-WO therapy. In some embodiments, the composition includes one or more pharmaceutically acceptable excipients.

[0101] In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Further, a “pharmaceutically acceptable carrier” will generally be a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0102] The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington’s Pharmaceutical Sciences by E. W. Martin, incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.Attorney Docket No.: 92EW-385670-WO The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0103] In an embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachets indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0104] The compounds of the disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. EXAMPLES Example 1: Identification of PLT001’s Binding Domains

[0105] This example examined the binding domains on the target CD36 protein for the anti- CD36 antibody PLT001, which has the VH and VL sequences as provided in Table 1.

[0106] The Fab domain of PLT001 (PLT001-Fab) was incubated with the extracellular domain of CD36 (CD36-ECD) in vitro, followed by structural analysis. Two binding domains on CD36, AA153-AA160 (Domain 1) and AA191-AA197 (Domain 2), were identified through CryoEM examination. The interaction between PLT001-Fab and CD36-ECD was further confirmed byAttorney Docket No.: 92EW-385670-WO FACS-based binding assays. Point mutations in Domain 1 (M156F, S160F, numbering according to the whole CD36 protein which further includes a signal peptide (SEQ ID NO:9) before the ECD (SEQ ID NO:10)) reduced the interaction between PLT001-Fab and CD36-ECD by nearly 50%, whereas mutations in Domain 2 (Y192R, P193R, F194R) only slightly affected the interaction at lower concentrations of PLT001 (FIG. 1, upper panel). Importantly, the interaction was completely abolished when mutations were introduced in both Domains 1 and 2, thereby confirming the findings from structural analyses. FIG. 2, lower panel, illustrates the interaction between the PLT001 Fab and the CD36 ECD. Domains 1 and 2 are responsible for fatty acid / oxLDL uptake through CD36. Table 1. Antibody and Target Sequences Name Sequence SEQ ID NO: PLT001 VHEVQLVESGGGLVQPGGSLRLSCAASGFTIDAFGIHWVRQAPGKGLEWVAW 1 IAPYGGETYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSF FGYFDYWGQGTLVTVSS PLT001 VL DIQMTQSPSSLSASVGDRVTITCRASQDVSNWVAWYQQKPGKAPKLLISY 2 ATSLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHSNAPLTFGQ GTKVEIKR PLT001 VH CDR1 AASGFTIDAFGIH 3 VH CDR2 WIAPYGGETY 4 VH CDR3 ARSFFGYFDY 5 VL CDR1 RASQDVSNWVA 6 VL CDR2 SYATSLYS 7 VL CDR3 HSNAPLTF 8 Human CD36 SP MGCDRNCGLIAGAVIGAVLAVFGGILMPV 9 Human CD36 GDLLIQKTIKKQVVLEEGTIAFKNWVKTGTEVYRQFWIFDVQNPQEVMMN 10 ECD SSNIQVKQRGPYTYRVRFLAKENVTQDAEDNTVSFLQPNGAIFEPSLSVG TEADNFTVLNLAVAAASHIYQNQFVQMILNSLINKSKSSMFQVRTLRELL WGYRDPFLSLVPYPVTTTVGLFYPYNNTADGVYKVFNGKDNISKVAIIDT YKGKRNLSYWESHCDMINGTDAASFPPFVEKSQVLQFFSSDICRSIYAVF ESDVNLKGIPVYRFVLPSKAFASPVENPDNYCFCTEKIISKNCTSYGVLD ISKCKEGRPVYISLPHFLYASPDVSEPIDGLNPNEEEHRTYLDIEPITGF TLQFAKRLQVNLLVKPSEKIQVLKNLKRNYIVPILWLNETGTIGDEKANM FRSQVTGKINAttorney Docket No.: 92EW-385670-WO PLT001 Heavy EVQLVESGGGLVQPGGSLRLSCAASGFTIDAFGIHWVRQAPGKGLEWVAW 11 Chain IAPYGGETYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSF FGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC NVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG PLT001 Light DIQMTQSPSSLSASVGDRVTITCRASQDVSNWVAWYQQKPGKAPKLLISY 12 Chain ATSLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHSNAPLTFGQ GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC Example 2. Expression Patterns of CD36 in Tumors

[0107] This example examined the expression of the CD36 protein in various tissue types.

[0108] Tumor-associated immune cells were isolated from hepatocellular carcinoma (HCC)- bearing mice, which were induced by oncogene-containing plasmids delivered via hydrodynamic injections. CD36 expression was examined for various tissues extracted from the mice.

[0109] As shown in FIG. 2, CD36 expression was upregulated in tumor-associated Treg, CD8+ T and NK Cells in the HCC-bearing mice. Example 3. Retention of PLT001 in vivo

[0110] This example checked the concentrations of the antibody PLT001 in various tissues at different time points after its administration, and shows that PLT001 targets CD36-expressing tumor-associated immune cells in murine HCC.

[0111] The expression level of CD36 was examined in regulatory T cells (Tregs), CD8+ T cells, and NK cells isolated from the livers, spleens, and peripheral blood of naïve and HCC- bearing mice. CD36 was specifically upregulated in tumor-associated Tregs, CD8+ T cells, and NK cells isolated from HCC compared to cells isolated from other organs (FIG. 3).Attorney Docket No.: 92EW-385670-WO Biodistribution assays with fluorescence-conjugated PLT001 revealed that PLT001 was retained in the livers with HCC and bound to target cells, including Tregs and CD8+ cells, in the tumor microenvironment. Example 4. In vivo Antitumor Efficacy of Single-Agent PLT001

[0112] This example tested the in vivo antitumor efficacy of single-agent PLT-012 and demonstrated that the single reagent had remarkable anti-tumor efficacy in both inflamed and cold HCC murine models.

[0113] Inflamed and cold HCC murine models were established by hydrodynamic injection of plasmids containing Trp53mut / cMycOE and Ctnnb1mut / cMycOE, respectively. Tumor progression was monitored via luciferase-based bioluminescence imaging using the IVIS imaging system. PLT001 exhibited striking tumor control efficacy in both inflamed (FIG. 4A) and cold (2) HCC models (FIG. 5A), with reduced intratumoral Tregs and boosted CD8+ TILs. Notably, in the inflamed HCC model, 31.6% of tumor-bearing mice became tumor-free upon PLT001 treatment. Moreover, progenitor exhausted CD8 T cells, which are the major responders to anti-PD1 / PD-L1 treatment, also significantly increased upon PLT001 treatment (FIG. 4B and FIG. 5B), highlighting the potential synergistic effect of PLT001 with ICB (immune checkpoint blocking). Example 5. Synergism of Combination Treatments

[0114] This example tested the combination of PLT001 and an anti-PD-L1 antibody, and demonstrated their synergism, which suggests that PLT001 can reprogram the tumor microenvironment of cold HCC to sensitize the effect of PD-L1 inhibition.

[0115] As reported, anti-PD-L1 antibodies have failed to control tumor progression in cold HCC with an activated β-catenin signaling pathway. By contrast, single treatment with PLT001 already resulted in nearly an 80% reduction in tumors.

[0116] Interestingly, it was observed herein that the combination therapy with PLT001 and an anti-PD-L1 antibody resulted in dramatic anti-tumor efficacy, with a 31.25% tumor-free rate (FIG. 6A). Consistent with the effects of single treatment with PLT001, the combination therapyAttorney Docket No.: 92EW-385670-WO also increased the CD8 / Treg ratio, boosted CD8 effector functions, and augmented progenitor exhausted T cells (FIG. 6B).

[0117] The current standard-of-care (SoC) of advanced HCC is the combo therapy of anti- VEGFα and anti-PD-L1, which has promising tumor control ability in the cold HCC model compared to control and monotherapy of anti-VEGFα. In this example, the combination of PLT001 with SoC was tested in the cold tumor model, and exhibited strikingly higher tumor response, with a 41.6% complete response rate (FIG. 7). Example 6. Antitumor Efficacy for Colorectal Cancer

[0118] This example tested the antitumor efficacy of the PLT001 / PD1 combination in a colorectal liver metastasis (CRLM) murine model, and shows that such a combination exhibited marvelous in vivo anti-tumor efficacy.

[0119] The colorectal liver metastasis (CRLM) murine model was established by orthotopically injecting SL4 CRC cells into both the cecum and liver of recipient mice. Treatment with PLT001 alone significantly reduced tumor burdens in the cecum, while combination therapy of PLT001 and anti-PD1 exhibited a dramatic anti-tumor effect, resulting in a 70% tumor-free rate in the cecum (FIG. 8). Also importantly, 100% of the animals became tumor-free in the liver. Example 7. CD36 Expression Correlated with Survival in HCC Patients

[0120] This example examined the CD36 expression levels in hepatocarcinoma (HCC) patients, and compared the expression levels to the survival duration of the patients.

[0121] In patients of HCC with either hepatitis B viral (HBV, FIG. 9A-D) or hepatitis C viral (HCV, FIG. 10A-D) infections, elevated CD36 expression was associated with adverse clinical outcomes, including overall survival (OS), progression-free survival (PFS), and disease-free survival (DFS).Attorney Docket No.: 92EW-385670-WO Example 8. CD36 Expression Correlated with Survival in CRC-MSS Patients

[0122] Cancer immunotherapy has been successfully used to treat colorectal (CRC) cancers characterized as MSI-high. By contrast, cancer immunotherapy has not been effective in microsatellite stable (MSS)-type CRC. This example examined the CD36 expression in MSS CRC patients.

[0123] In patients with MSS-type colorectal cancer (CRC), a positive correlation between CD36 expression and both the percentage of regulatory T cells (Tregs) and the Exhaustion score in TCGA cohort was observed (FIG. 11). Further, in these patients, higher levels of CD36 expression were linked to poorer clinical outcomes, encompassing overall survival (OS), progression-free survival (PFS), and disease-specific survival (DSS) (FIG. 12).

[0124] These data, therefore, suggest that anti-CD36 antibodies can be suitably used to treat MSS CRC patients that are CD36-positive. Example 9. PLT001 was Effective for Unleashing Anti-Tumor Immunity against Liver Cancer and Liver Metastasis

[0125] This example provides a more detailed description of certain of the foregoing examples.

[0126] PLT001, a humanized IgG4 antibody against lipid-binding pocket of CD36, was developed with reactivity against multiple species and a superior safety profile in monkeys through phage display techniques followed by affinity maturation and optimization for enhanced developability. Using bioinformatic analysis to delineate human tumor types characterized by CD36-mediated immune regulations, the inventors unveiled that PLT001 antibody effectively augmented anti-hepatocellular carcinoma responses, including reduction of intratumoral Tregs, enhancement of CD8+T cell infiltration, and improved cytotoxic functions in CD8+T cells, in both hot and cold HCC types. Of note, the inventors further uncovered that PLT001 can promote the abundance of progenitor-exhausted T cells, a critical cell subset responsible for responsiveness of immune checkpoint blockade treatment, in tumors.Attorney Docket No.: 92EW-385670-WO

[0127] In line with this, this example demonstrated that PLT001 used in conjunction with the PD-L1 antibody or in combination with standard liver cancer therapies (anti-VEGF plus anti- PD-L1 agents) can elicit superior anti-tumor responses in murine with cold HCCs and mice fed a high-fat diet- a condition, which is typically associated with reduced responsiveness to anti- PD-L1 therapy in HCCs. The inventors further revealed that PLT001 can be utilized for restricting liver metastasis of colon cancer and restoring PD-1 responsiveness against primary colon cancer in mice harboring liver metastasis. The mechanism of action (MOA) of PLT001 was further validated in human HCC samples using an ex vivo culture platform. These findings highlight that blocking CD36-mediated metabolic alterations in Treg and CD8+TILs with PLT001 treatment can elicit robust tumor growth inhibition and shift the immunosuppressive nature within the TME toward an immune-supportive TME. Materials and Methods Assays for affinity binding of antibody, oxLDL binding, and oxLDL uptake

[0128] For the evaluation of PLT001 binding affinity, human CD36-expressing F293 cell lines were incubated with PLT001 at concentrations ranging from 0.002 to 10 μg / mL at 4 °C for 30 minutes, followed by staining with commercial PE-conjugated anti-CD36 antibodies (0.4 μg / mL), clone D-2712 (BD Biosciences Cat# 552544, RRID: AB_2072646), at 4 °C for 20 minutes. For the assessment of oxLDL binding in tumor-infiltrating lymphocytes (TILs), CD45+TILs were isolated from MC38 tumors two weeks post-inoculation using the mouse Tumor Dissociation Kit and CD45 microbeads (Miltenyi Biotec), in accordance with manufacturers’ protocols. To evaluate the inhibitory effect of PLT001 on oxLDL binding and uptake, human CD36-expressing F293 cells or indicated TILs were initially pre-incubated with either control IgG or anti-CD36 antibodies (5 µg / mL) at 4 °C for 30 minutes. Subsequently, the cells were treated with oxLDL-Dil (5 µg / mL, Kalen Biomedical #770232-9) in RPMI medium containing 1% fatty acid-free BSA at 4°C for 2 hours to measure oxLDL binding or at 37°C for 5-15 minutes to measure oxLDL uptake, respectively. Data collection was done using an Attune NxT Flow Cytometer (Thermo Fisher Scientific). The uptake of oxLDL was analyzed by flow cytometry and expressed as a percentage (%) of positive cells based on a histogram gate placed on negative control cells.Attorney Docket No.: 92EW-385670-WO CryoEM structural analysis of the CD36(ECD)-PLT001(Fab) complex

[0129] The CD36 extracellular domain complexed with the Fab domain of a specific protein was reconstituted and then loaded into a Chameleon system (SPT Labtech) by spraying a nanoliter onto a self-wicking 1.2 / 1.3 grid (SPT Labtech). The single particle cryo-EM data was collected using an automated process on a Titan Krios G4 TEM operating at 300 kV (Thermofisher Scientific). Raw EER movies were imported to cryoSPARCv4.3, drift-corrected and dose-weighted with Patch Motion Correction, and estimated for contrast transfer function (CTF) with Patch CTF Estimation. After initial processing and selection, a total of 21,008 images were used for further analysis. Particles were picked and further processed to obtain a 3D reconstruction. The resulting 3D reconstruction was used to generate templates for particle picking, and a total of 16,529,240 particles were extracted. Following 3 rounds of 2D classification and refinement, the final 3D class with 84,937 particles reached a resolution of 3.75 Å. To account for flexibility in the Fab region, a Flexible 3D refinement was performed, resulting in a 3.45 Å reconstruction (B-factor -127).

[0130] To compensate for artifacts and improve the interpretability of the model building, the quality of 3D reconstruction was enhanced with EMReady. The initial model was obtained from AlphaFold, fitting into the cryo-EM density using a rigid body fit in Chimera. Atomic model of the Fab region was manually inspected and adjusted in Coot, refined with Servalcat and the quality was assessed with MolProbity. Atomic model of CD36 region was fit into the cryo-EM density using rigid body fit in Chimera and manually inspected and real-space refined with Phenix. Raw cryo-EM images were deposited to the Electron Microscopy Public Image Archive (EMPIAR) with accession code EMPIAR-X. The cryo-EM map of CD36-Fab was deposited to the Electron Microscopy Data Bank (EMDB) with accession code EMD-X. Built atomic model of Fab region was deposited to the Protein Data Bank (PDB) with accession code PDB-X. Cell lines

[0131] YUMM1.7 melanoma cell line (RRID:CVCL_JK16) was kindly provided by Marcus Bosenberg. The MC38 cells (RRID:CVCL_B288) were provided by P. Romero. Mycoplasma testing is routinely conducted using PCR as a standard procedure for cell culture. YUMM1.7 and MC38 cells were cultured in DMEM with 10% fetal bovine serum and 1% penicillin-Attorney Docket No.: 92EW-385670-WO streptomycin and used for experiments when they were in the exponential growth phase. Luciferase-expressing MC38 cells (MC38-luc) were established by transfection with a pLV- EF1a-IRES-Blast (RRID: Addgene_85133) expressing luciferase. In vivo experiment

[0132] In subcutaneous engraftment tumor models, Yumm1.7-gp33 (8 × 105 cells) and MC38- OVA cells (5 × 105 cells) were administered via subcutaneous injection into 6-week-old C57BL / 6 mice. Tumor dimensions were assessed every 2-3 days post-engraftment, with tumor volume calculated using the formula: volume = (length × width2) / 2. MC38 cells were injected subcutaneously (1 × 106) and intrahepatically (5 × 105) for experimental liver metastasis models as previously described.

[0133] HCC mouse models are generated by hydrodynamic delivery of endotoxin-free plasmid DNA through the lateral tail vein within 5-7 seconds, as previously described. In the MYCOE / p53KOHCC model, a total of 12 µg of pT3-Myc plasmid (RRID:Addgene_129776), 13.2 µg of p53 gRNA plasmid (RRID:Addgene_59910), and 4 µg of SB100x (RRID:Addgene_34879) were injected into the mice. For the β-catenin / Myc (CTNNB1N90 / MYCOE) HCC model, each mouse received 12 µg of pT3-β-catenin (RRID:Addgene_31785), 12 µg of pT3-Myc plasmid, and 8 µg of SB100x. Tumor growth was monitored weekly through bioluminescence imaging starting from week 2. Mice exhibiting persistent tumor growth were randomly assigned to specific treatment groups to ensure objectivity. Tumor samples were collected 35-38 days post-hydrodynamic injection for subsequent weight measurement and immune profiling via flow cytometry. The bioluminescence activity of the mice was evaluated using an IVIS imaging system following luciferin administration (150 mg / Kg). Flow cytometric analysis

[0134] Tumors were finely minced and digested in RPMI medium with 2% FBS, 1% penicillin- streptomycin, DNase I (1 µg ml-1; Sigma-Aldrich), and collagenase (0.5 mg ml-1; Sigma-Aldrich) at 37°C for 40 minutes. The resulting mixture was filtered through a 70-μm cell strainer, and the filtered cells were treated with RBC Lysing Buffer (BioLegend), followed by washing with FACS buffer (phosphate-buffered saline with heat-inactivated 2% FBS and 0.1% sodium azide).Attorney Docket No.: 92EW-385670-WO Tumor-infiltrating leukocytes were then enriched using Percoll density gradient centrifugation (800g for 20 min) at room temperature. Single-cell suspensions were incubated with anti- CD16 / 32 (BioLegend Cat# 101320, RRID:AB_1574975) Fc receptor-blocker on ice for 10 min before staining. Viable cells were identified by staining with BD Horizon Fixable Viability Stain 450 (BD Biosciences Cat# 562247, RRID:AB_2869405) at room temperature for 15 min. Cells were processed for surface marker staining for 1 hour at 4 °C and then intracellular staining. Intracellular staining was performed according to the instruction of Transcription Factor Fixation / Permeabilization Buffer Set (BioLegend). Briefly, cells were fixed and permeabilized in True-Nuclear Fix solution for 20 min and then stained by indicated antibody for 1 hour. Samples were analyzed on Cytek Northern Lights flow cytometer, and data were analyzed with FlowJo v10 (RRID:SCR_008520). The following antibodies were used for flow cytometry: anti-CD45 (BioLegend Cat# 103136, RRID:AB_2562612), anti-CD19 (BioLegend Cat# 115530, RRID:AB_830707), anti-CD3ε (BD Biosciences Cat# 564378, RRID:AB_2738779), anti-CD4 (BD Biosciences Cat# 566939, RRID:AB_2869957), anti-FoxP3 (BD Biosciences Cat# 560401, RRID:AB_1645201), anti-CD25 (BioLegend Cat# 102048, RRID:AB_2564124), anti-CD8a (BD Biosciences Cat# 747134, RRID:AB_2871881), anti-CD44 (BD Biosciences Cat# 566506, RRID:AB_2744396), anti-granzyme B (BD Biosciences Cat# 563388, RRID:AB_2738174), anti-Tim 3 (BD Biosciences Cat# 747622, RRID:AB_2744188), anti-PD-1 (BioLegend Cat# 109110, RRID:AB_572017), anti-CD11b (BioLegend Cat# 101217, RRID:AB_389305), anti- CD206 (BioLegend Cat# 141732, RRID:AB_2565932), anti-MHC class II I-Ab / I-E (BioLegend Cat# 107639, RRID:AB_2565894), anti-F4 / 80 (BD Biosciences Cat# 746070, RRID:AB_2743450), anti-Ly6C (BD Biosciences Cat# 566987, RRID:AB_2869991), anti-Ly6G (BD Biosciences Cat# 740554, RRID:AB_2740255), anti-Tim 4 (BioLegend Cat# 130010, RRID:AB_2565719), and anti-NK1.1 (BioLegend Cat# 108724, RRID:AB_830871),. All antibodies were purchased from Biolegend or BD Biosciences. Immunohistochemistry analysis

[0135] Paraffin sections at a thickness of 4 μm were deparaffinized and stained with antibodies against CD8α (Abcam Cat# ab217344, RRID: AB_2890649), FoxP3 (Cell Signaling Technology Cat# 12653, RRID: AB_2797979), cleavage caspase 3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), granzyme B (Cell Signaling Technology Cat# 44153, RRID:Attorney Docket No.: 92EW-385670-WO AB_2857976), and CD31 (Cell Signaling Technology Cat# 77699, RRID: AB_2722705), followed by PolyTek Polymerized Imaging System (ScyTek Laboratories Inc). Chromogenic staining was determined in the Zeiss Axioscan 7 slide scanner. Six random regions were selected to quantify positive levels for each tumor tissue section, and the value for each image was quantified using ImageJ (RRID:SCR_003070). In silico analysis on TCGA and POG570 cohorts

[0136] Data from the Cancer Genome Atlas (TCGA) cohort was acquired from UCSC Xena (xena.ucsc.edu). The gene expression quantification was conducted using the UCSC Xena Toil RNA-seq pipeline. Furthermore, information from the POG570 cohort was obtained from cBioPortal. The RPKM gene expression values were transformed to TPM using the method suggested by Wagner et al.

[0137] The Fatty Acid Metabolism signature was obtained from the Molecular Signatures Database (MSigDB; HALLMARK_FATTY_ACID_METABOLISM), and the T Cell Exhaustion signature was sourced from Bao X et al. The enrichment levels of these signatures in each sample were quantified using the “GSVA” R package (version 1.48.3) and the single-sample gene set enrichment analysis (ssGSEA) method.

[0138] Tumor Infiltrating Lymphocytes Fraction Analysis: CIBERSORT, a deconvolution algorithm, was used to estimate cell type proportions within bulk cancer samples based on gene expression data. The proportions of 22 types of infiltrating immune cells were estimated using the CIBERSORT method. For the TCGA cohort, the immune infiltration proportions were obtained from TIMER2.0 (timer.cistrome.org). For the POG570 cohort, the “immunedeconv” R package (version 2.1.0) was used with CIBERSORT (absolute mode), and the CIBERSORT source code was downloaded under a licensed agreement. The proportions of T regulatory (Treg) cells and CD8 T cells were extracted with the default parameters.

[0139] Cancer types with a mean of Fatty Acid (FA) enrichment score (ES) greater than zero and a CD36 expression level higher than the median of the mean CD36 expression among 33 cancer types, minus one standard deviation (SD), were selected. A higher gene set enrichment score indicates a higher activity level of the associated biological process or pathway.Attorney Docket No.: 92EW-385670-WO scRNA-seq analysis

[0140] The cell multiplexing (CellPlex reagents, 10 × Genomics) and single-cell library preparation were conducted using Single Cell 3’ v3.1 kit (10 × Genomics) according to the manufacturer’s protocol. The libraries were sequenced on the Illumina NovaSeq 6000 platform with NovaSeq 6000 S4 Reagent Kit (300 cycles). The scRNA-seq data was processed, and the matrix data containing gene counts for each cell per sample was generated using Cell Ranger (V3.0.2, support.10xgenomics.com).

[0141] CellBender was used to minimize ambient RNA content. Subsequent downstream analyses and data normalization were performed using the Seurat v5 pipeline. For quality control, cells were filtered based on specific criteria: cells with fewer than 400 or more than 10,000 detected genes, fewer than 1,000 or more than 40,000 counts, a ribosomal gene proportion below 0.04 or above 0.50, a mitochondrial gene proportion above 0.2, log10 genes per UMI ratio below 0.65, or a hemoglobin-related gene proportion exceeding 0.03 were excluded. After this step, 9,422 cells from condition PLT001 and 12,322 cells from the Ctrl condition were retained for further analysis.

[0142] Cell types were annotated using scGate, and doublets were removed using DoubletFinder, assuming a doublet rate of 10%. The doublet-free dataset was re-annotated with scGate, and cell type classifications were further refined through unsupervised clustering and marker definition. This process allowed to remove additional doublets missed by DoubletFinder and to better define clusters of unconventional T cells, such as γδT and iNKT cells. Subsequent analyses were performed on 2,227 T cells from condition PLT001 and 4,385 T cells from the Ctrl condition.

[0143] Conventional T cells (CD4+and CD8+T cells) were further subclassified using ProjecTILs, with a murine reference atlas for tumor-infiltrating conventional T cells, including CD8+Tex, CD8+Tpex, and Treg (doi.org / 10.6084 / m9.figshare.12478571.v3). Differential gene expression was performed for each T cell subtype between PLT001 and Ctrl using `FindMarkers` function from Seurat, with default parameters (Wilcoxon Rank Sum test). Differential expression analysis was performed only when at least 10 cells were present in at least one condition.Attorney Docket No.: 92EW-385670-WO

[0144] Gene Set Enrichment Analysis (GSEA) focused on T cell activation, effector functions, and cellular metabolism pathways. These pathways were derived by filtering the mouse MSigDB database. The custom combined list of pathways was used for GSEA analysis using ClusterProfiler, using 2000 permutations. The enrichment analysis was based on genes ranked by -log10 of the adjusted p-value plus the absolute value of log2 fold change and its sign, obtained from the differential expression analysis between conditions. Cynomolgus monkey toxicity study

[0145] Cynomolgus monkeys (Macaca fascicularis) received 5 once-weekly IV doses of PLT001 at concentrations of 0, 10, 60, or 200 mg / kg on Day 1, 8, 15, 22 and 29. Each group was comprised of two males and two females, with one animal of each sex exhibiting low expression and one exhibiting high expression of CD36 in RBC. The animals were monitored for clinical signs, including injection site reactions, body weight, and ophthalmoscopy. Clinical pathology (hematology, serum chemistry, and coagulation) was evaluated before the study commenced and on days 2, 8, 16, and 30 (before the scheduled necropsy). Immunotoxicology (immunophenotyping) was conducted on blood samples collected before the study began and 4, 24, and 168 hours after the 1st and 4th administrations. Upon completion of the 30-day study, all cynomolgus monkeys underwent necropsy with comprehensive macroscopic postmortem examination and organ weight recording. A complete list of tissues was obtained for histopathological examination.

[0146] The experiments involving cynomolgus monkeys were carried out at Wuxi AppTec (Suzhou, China). The protocol, along with any amendments or procedures concerning the care or use of animals in this study, was reviewed and approved by the WuXi AppTec Institutional Animal Care and Use Committee (IACUC) before the study commenced. Human sample ex vivo culture platform

[0147] Human HCC samples were collected from Centre Hospitalier Universitaire Vaudois (CHUV, Switzerland), Clinic Favoriten (Vienna), Chang Gung Memorial Hospital (Taiwan), and New Taipei City Tucheng Hospital (Taiwan) in accordance with the approval of the institutional review board (IRB) at each institution. The detailed culture procedures were described previously. In short, cryopreserved vials containing minced tumor fragments were thawed,Attorney Docket No.: 92EW-385670-WO embedded in Matrigel-containing matrix (Matrix High Concentration, Phenol Red-Free, 4 mg / ml final concentration; BD Biosciences), and cultured for two days with either 10 μg / mL of control human anti-β-Gal-hIgG4 (S228P; InvivoGen) or PLT001. Tumor-associated cell suspensions were then collected from pooled tumor fragments. These suspensions underwent FACS analysis of the immune profile using Aurora spectral flow cytometry (Cytek) with the following antibodies: anti-CD3 APC-F750 (BioLegend Cat# 981006, RRID:AB_2894549), anti-CD4 NovaFB585 (Thermo Fisher Scientific Cat# H001T03B04-A, RRID:AB_3097881), anti-CD8 PerCP (BioLegend Cat# 344708, RRID:AB_1967149), anti-CD14 PerCP-eF710 (Thermo Fisher Scientific Cat# 46-0149-42, RRID:AB_10671405), anti-CD19 PerCP-eF710 (Thermo Fisher Scientific Cat# 46-0199-42, RRID:AB_2866432), anti-CD36 BUV805 (BD Biosciences Cat# 748645, RRID:AB_2873052), anti-CD45RA BV785 (BioLegend Cat# 304140, RRID:AB_2563816), anti-CCR7 PE-Cy7 (BioLegend Cat# 353226, RRID:AB_11126145), anti- FOXP3 PE-Cy5 (Thermo Fisher Scientific Cat# 15-4777-42, RRID:AB_2811750), anti-GzmB BV510 (BD Biosciences Cat# 563388, RRID:AB_2738174), anti-KI-67 AF700 (BD Biosciences Cat# 561277, RRID:AB_10611571), anti-PD-1 BV421 (BioLegend Cat# 329920, RRID:AB_10960742), and ZombieNIR (BioLegend). Quantification and statistical analysis

[0148] Statistical analyses for multiple groups were performed using one-way ANOVA, and two-tailed, unpaired Student t-tests were conducted for two-group comparisons. The data are presented as means ± s.e.m., and each point represents a biological replicate. The Box and Whisker plot displayed the distribution of immune cells, with the Whisker indicating minimum to maximum values. Results Generation and characterization of PLT001, a cross-reactive anti-CD36 antibody

[0149] The development of anti-human CD36 monoclonal antibodies (mAbs), which can block lipid loading ability controlled by CD36, is largely hampered due to two major obstacles: a. the hydrophobic nature of the lipid-binding pocket of CD36 can lead to identification of candidates with weak and non-specific binding; b. the high frequency of false-negative hits on applying antigen capture assay to identify antibody candidate. To overcome these bottlenecks, thisAttorney Docket No.: 92EW-385670-WO example applied the synthetic human scFv phage-display antibody libraries, followed by affinity maturation processes, to identify antibodies recognizing human CD36. Given that CD36 can be expressed in numerous cell types, this example then engineered antibody candidates into the IgG4 format to reduce antibody-dependent immune responses and optimized Fc domain to minimize aggregates during antibody production.

[0150] This example identified PLT001, a full-length human monoclonal IgG4 antibody, displaying reactivity against CD36 across various species, including rodents, monkeys, and humans. Of note, due to the reactivity across species, this example can examine biochemical properties and anti-tumor function of PLT001 without using murine surrogate antibodies. Next, this example tested whether PLT001 can compete with D2712, a commercially available antibody against murine CD36 lipid-binding pocket. The result showed that PLT001 can compete with D2712 on binding with CD36, indicating that PLT001 may bind on a comparable epitope of CD36 and inhibit the uptake of fatty acids and oxidized low-density lipoprotein (oxLDL). Indeed, this example found that PLT001 markedly inhibited the binding (IC50: 1.798nM) and uptake (IC50: 1.357nM) of fluorochrome-labeled oxLDL in F293 cells that overexpress human CD36. In addition, this example confirmed that PLT001 treatment significantly suppressed oxLDL uptake compared to control IgG in primary tumor-infiltrating immune cells, including myeloid cells, CD3+T lymphocytes, and intratumoral Tregs isolated from MC38 tumor-bearing mice. Then, this example examined whether PLT001 treatment can suppress tumor growth in syngeneic mouse melanoma and colorectal cancer models. In alignment with prior reports showing that blocking lipid transport activity of CD36 can reduce tumor growth cells, PLT001 exhibited favorable anti-tumor responses in both Yumm1.7 melanoma and MC38 colorectal cancer models. Altogether, the results reveal that PLT001, an anti-CD36 monoclonal antibody with reactivities against multiple species, can effectively inhibit CD36-mediated lipid uptake and induce effective anti-tumor responses. Identification of the binding epitopes of PLT001 on CD36

[0151] The precise interacting regions between PLT001 and CD36 molecule remain unclear. To confirm the binding property and examine whether this interacting region is within lipid-binding pocket of CD36, this example conducted a structural analysis by utilizing Cryo-Electron Microscopy (Cryo-EM) to determine the binding epitopes of PLT001 on CD36. The Cryo-EMAttorney Docket No.: 92EW-385670-WO analysis identified two interaction domains of PLT001 on the extracellular domain (ECD) of CD36, specifically located within the amino acid regions AA153-AA160 (Domain 1) and AA191-AA197 (Domain 2). These domains are situated within the fatty acid- and oxLDL- responsive regions of CD36, as corroborated by prior domain mapping studies. To further investigate the impacts of these domains in the interaction between PLT001 and CD36, this example introduced point mutations to alter the charge and hydrophobicity of CD36 and then examined PLT001 binding efficiency. Flow cytometry-based binding assays showed that point mutations in Domain 1 resulted in a nearly 50% reduction in the interaction between PLT001 and the ECD of CD36, while mutations in Domain 2 did not affect the interaction. Notably, the interaction was almost completely abolished when both domains were mutated. These results unveil that PLT001 predominantly interacts with domain 1 of CD36, thereby limiting lipid entry, while domain 2 provides supplementary support. PLT001 displays superior distribution and targeting for hepatocellular carcinomas

[0152] Since CD36-mediated lipid uptake plays a critical role in orchestrating the immunosuppressive tumor microenvironment by modulating survival and functions of intratumoral Tregs and CD8+T cells, it was speculated that PLT001 may elicit more robust anti- tumor responses in tumor types enriched with lipids. This example conducted in silico analyses to examine which tumor types simultaneously displayed higher expression of CD36 and gene enrichment score related to fatty acid metabolism by utilizing data from The Cancer Genome Atlas (TCGA). The top 9 tumor types with high CD36 expression and fatty acid metabolism score were further subjected to the analysis for deconvoluting the abundance of CD8+T cells and Tregs by CIBERSORT method. Based on these sequential analyses, the result highlighted that liver hepatocellular carcinoma (LIHC) may be a tumor type that can be sensitive to anti-CD36 antibody treatment based on the characteristics, including enriched fatty acid metabolism scores, a high CD36 expression profile, and high CD8+T cells and Tregs tumor infiltrations. However, this example did not pursue cholangiocarcinoma (CHOL) due to the potential biases stemming from a limited sample size. To confirm if tumor-infiltrating lymphocytes (TILs) increased CD36 expression in the context of liver cancer, this example measured CD36 expression in Tregs and CD8+T cells from peripheral blood mononuclear cells (PBMCs), spleens, and liver of hepatocellular carcinoma (HCC)-bearing mice. To better mimic the pathological progression ofAttorney Docket No.: 92EW-385670-WO hepatocellular carcinomas, this example utilized a hydrodynamic injection system to initiate liver cancer by overexpressing constitutive activation mutant of β-catenin and Myc in hepatocytes (CTNNB1N90 / MYCOEHCC). By staining single-cell suspension with Alexa Fluor 647- conjugated PLT001, this example found that both Tregs (characterized by CD45+CD3+CD4+FoxP3+) and CD8+T cells (characterized by CD45+CD3+CD8+) in HCC upregulated CD36 compared to their counterparts obtained from normal liver, spleen, or peripheral blood of both normal and HCC-bearing mice. Of note, this example also observed that NK cells (characterized by CD45+CD3-NK1.1+) in tumors also increased CD36 expression, which has been reported to impair NK cell cytotoxicity. These findings align with previous studies reporting that the TME promotes CD36 expression in tumor-infiltrating immune cells.

[0153] To further examine the distribution and kinetics of PLT001 treatment in mice with or without HCC, this example intraperitoneally injected Alexa Fluor 647-conjugated PLT001 (10mg per kg body weight) into control mice or CTNNB1N90 / MYCOEHCC-bearing mice and then collected samples from various organs at 4, 24, and 48-hour intervals. The results showed that PLT001 predominantly accumulates in liver, lung, intestine, epididymal white adipose tissue, and pancreas, as evidenced by fluorescent intensity measured by the In Vivo Bioluminescent Imaging (IVIS). This accumulation exhibited similar intensity levels in both normal and tumor-bearing mice, which may be attributed to the presence of adipocytes and endothelial cells, which highly express CD36. Importantly, PLT001 level was significantly increased in livers from HCC- bearing mice compared to normal liver 24 hours post-injection, suggesting that the HCC microenvironment may facilitate the recruitment of CD36-expressing cells. To validate if PLT001 injection can target tumor-infiltrating lymphocytes, this example determined the labeling of PLT001 on Tregs, CD8+T cells, and NKs from livers of normal and HCC-bearing mice followed with one dose of intraperitoneal administration of Alexa Fluor 647-conjugated PLT001. This example observed that both Tregs and CD8+T cells from HCC-bearing mice displayed a higher percentage of labeled cells compared with their counterparts from normal mice. Notably, these differences persisted for up to 48 hours following administration. In addition, NK cells from tumor-bearing mice exhibited higher levels of PLT001 binding at the 24-hour time point, but not 48-hour time point, compared to NK cells from normal livers. Taken together, these results reveal that HCC microenvironment could boost CD36 expression in infiltrating immuneAttorney Docket No.: 92EW-385670-WO cells, including Tregs, CD8+T cells, and NK cells, and PLT001 may have the potential to restore anti-tumor immunity against HCC by targeting CD36-guided immune regulations. PLT001 elicits superior anti-tumor responses in murine HCCs

[0154] This example next examined the therapeutic effects of PLT001 on restoring anti-tumor responses in HCC. To do so, this example first assessed the impacts of PLT001 treatment on T- cell activity and tumor growth in two murine HCC models. The first model was driven by Myc overexpression and p53 deletion in hepatocytes via hydrodynamic injection (referred to MYCOE / p53KOHCC), and the second model was driven by Myc and mutated β-catenin (CTNNB1N90) overexpression via same approach (referred to CTNNB1N90 / MYCOEHCC). MYCOE / p53KOHCC is known to have an inflamed tumor (hot tumor) microenvironment, characterized by high infiltration of CD103+dendritic cells and CD8+T cells, and exhibits a slower progression rate compared to CTNNB1N90 / MYCOEHCC. This example found that PLT001 treatment robustly restrained MYCOE / p53KOHCC growth compared to control treatment and resulted in 32% of HCC-bearing mice being tumor-free. Measurements of tumor weight and ex vivo liver BLI further corroborated these findings. Of note, PLT001 treatment also mitigated liver injury associated with tumor progression, as indicated by the reduced serum alanine transaminase (ALT) activity. Furthermore, PLT001 treatment led to a reduction in Tregs but an increase of CD8+T cells within the TME. Consequently, the ratios of CD8+T cells to Tregs in HCC from PLT001-treated mice were higher compared to the control group). Since targeting CD36 can mitigate exhaustion and maintain the effector function in CD8+T cells, this example next examined whether PLT001 can ameliorate T cell exhaustion by determining the expression of exhaustion markers as well as granzyme B (a key molecule for cytolytic activity) in tumor-infiltrating CD8+T cells. Strikingly, PLT001 enhanced the abundance of granzyme B+CD8+T cells. Moreover, PLT001 treatment resulted in increases in both progenitor-exhausted T cells (Prog Tex; characterized by CD44+PD-1+TCF1+Tim3-) and terminally exhausted T cells (Term Tex; characterized by CD44+PD-1+TCF1- Tim3+). Given that the abundance and presence of progenitor-exhausted cells have been shown to contribute to better responsiveness to PD-1 blockade treatment, these results indicate that PLT001 is capable of restoring anti-tumor responses in HCC and might also improve responsiveness to PD-1 blockade.Attorney Docket No.: 92EW-385670-WO

[0155] More than 35% of HCC patients harbor genetic mutations leading to aberrant activation of Wnt / β-catenin signaling. These types of HCC have a cold tumor microenvironment with extremely low responsive rates to immune checkpoint blockade, in part due to a scarcity of TILs. To elucidate if CD36 blockade with PLT001 can overcome β-catenin-driven immune tolerance in HCC, this example treated CTNNB1N90 / MYCOEHCC-bearing mice with PLT001. The results showed that, similar to the outcome in MYCOE / p53KOHCC model, PLT001 also significantly inhibited tumor growth and markedly decreased circulating ALT levels compared to the control treatment. PLT001 treatment also increased the abundance of CD8+T cells while concurrently decreased the proportion of Tregs in the tumors, thereby resulting in increased ratios of CD8+T cells to Tregs within individual tumors. Similar to the treatment in MYCOE / p53KOHCC model, PLT001 also promoted the abundance of granzyme B-expressing CD8+T cells as well as the population of Prog Tex and Term Tex CD8+T cells in CTNNB1N90 / MYCOEHCC. To confirm the compositional changes in immune subsets, activation of T cells, and tumor cell death within tumors, this example conducted immunohistochemical staining to detect FoxP3, CD8, granzyme B, and cleaved caspase-3. The results corroborated the findings that PLT001 treatment significantly transformed the tumor microenvironment from an immune-suppressive to an immune-supportive state, characterized by elevated tumor infiltration of CD8+T cells but decreased abundance of intratumoral Tregs. This example also observed more granzyme B staining and cleaved caspase-3 in tumors from PLT001-treated mice, suggesting that CD8+T cells in PLT001-treated tumors exhibited enhanced cytotoxic activity that led to tumor cell death.

[0156] To comprehensively explore the overall immune response following PLT001 treatment, this example isolated CD45+tumor-infiltrating cells and then subjected them to single-cell RNA sequencing. This example next focused on the changes in T cell populations by annotating T cell types with scGate by in silico filtering T cells using scGate. Following this, this example annotated T cell subsets, identifying various CD8+T cells subsets, Tregs, T helper 1 (Th1) cells, T follicular helper (Tfh) cells, and unconventional T cells based on their lineage-specific markers. In line with flow cytometry-based analyses, the scRNA-seq compositional analysis also showed a reduction in Tregs but increases in both total terminally and progenitor-exhausted T cells. Of note, PLT001 treatment robustly induced changes to the transcriptome of terminally exhausted T cells compared to their counterpart from control group. Among the differentiallyAttorney Docket No.: 92EW-385670-WO expressed gene signatures, those associated with T cell activation and effector function were significantly enriched in terminally exhausted T cells from PLT001-treated tumors, indicating that PLT001 treatment can restore effector functions in exhausted T cells. Furthermore, this example noted that PLT001 also changed expression of genes involved in lipid metabolism pathways in terminally exhausted T cells. Together, these results unveil that PLT001 can induce robust anti-HCC immune responses in both hot and cold tumor microenvironment, characterized by increased CD8+TIL abundance, restored effector functions in CD8+TIL, and diminished intratumoral Treg populations. PLT001 synergizes with standard-of-care immune therapy for combating HCCs

[0157] Given that the presence of Prog Tex cells is responsible for anti-tumor responses induced by PD-1 / PD-L1 blockade therapy and the findings that PLT001 enhances Prog Tex populations in HCCs, it was then speculated that PLT001 may augment the therapeutic efficacy of PD-1 / PD-L1 blockade therapy. To test this, this example treated CTNNB1N90 / MYCOEHCC- bearing mice with either anti-PD-L1 mAb, PLT001, or the combination of PLT001 and anti-PD- L1 mAb. As expected, anti-PD-L1 mAb only induced mild anti-tumor responses in this cod HCC; however, the combination treatment of PLT001 and anti-PD-L1 mAb exhibited a more profound anti-tumor effect than monotherapy. Of note, the combinatorial treatment further boosted PLT001-mediated reprogramming of the immune state in tumors, including a reduction of Treg percentages, but increases of total CD8+T cells, Term Tex, and Prog Tex, while concurrently resulting in elevation of granzyme B-expressing CD8+T cells. Next, it was wondered if the incorporation of PLT001 into the standard-of-care (SoC) regimen for human HCC, which encompasses anti-VEGF and anti-PD-L1 mAbs, can induce more superior beneficial outcomes. This example found that the incorporation of PLT001 with SoC regimen resulted in a significant improvement in tumor suppression. Notably, the overall response rate for the triple treatment (PLT001 / anti-PD-L1 / anti-VEGF combined therapy) exceeded 70%; however, there was a mere 27% response rate for the SoC alone. Importantly, while no subjects achieved tumor-free status with vehicle, anti-VEGF, or SoC, the combination of SoC and PLT001 led to tumor-free status in 45% of CTNNB1N90 / MYCOEHCC-bearing mice. In conclusion, targeting CD36 with PLT001 presents a promising strategy when combined with immune checkpointAttorney Docket No.: 92EW-385670-WO inhibitors to reprogram the tumor-immune microenvironment in the management of liver cancers. PLT001 restrains HCC progression under elevated dietary lipid uptake

[0158] High dietary fat intake and deregulated lipid profile in livers have been reported to modulate fatty acid biosynthesis in HCC and promote aggressiveness in human and murine HCC. HCC subtypes characterized by increased fatty acid biosynthesis are also associated with more aggressive phenotypes and reduced survival rates; however, an effective treatment to restrict HCC progression under high dietary fat intake remains ambiguous due to the complications of auto-reactive T cells and changes in other immune parameters. Since CD36 expression can be induced by lipid-enriched conditions in Tregs and CD8+T cells, it was then speculated that HCC-bearing mice under high dietary fats may promote CD36 expression in these cell types and enhance the immunosuppressive states in tumors. To test this, twenty days post hydrodynamic injection, CTNNB1N90 / MYCOEHCC-bearing mice were fed either a chow diet or a Western diet enriched with sugars and fats (FIG. 13A). This dietary modification increased body weight, expedited the progression of liver cancer, and fostered an immune- suppressive tumor microenvironment, as evidenced by the increase in intratumoral Tregs and the reduction in CD8+T cells (FIG. 14A-F). Importantly, by using both PLT001 and one commercially available anti-CD36 antibody (HM36), this example found that western diet feeding promoted CD36 expression in intratumoral Tregs and tumor-infiltrating CD8+T cells, including both progenitor and terminally exhausted CD8+T cells (FIG. 14G-H), indicating that increase dietary lipid uptake may strengthen CD36-mediated immune modulations in the TME. Next, this example tested the anti-tumor responses of PLT12 and anti-PD-L1 mAb in this setting. In alignment with a previous report, the administration of anti-PD-L1 antibody only induced limited effects on curtailing tumor growth; however, PLT001 consistently showed a greater reduction in tumor growth when the mice were maintained on a chow diet (FIG. 13B-D). Notably, PLT001 treatment remained effective in restricting tumor growth even under a Western diet (FIG. 13E). In contrast to increased intratumoral Treg under anti-PD-L1 mAb treatment, PLT001 resulted in lower intratumoral Treg abundance in HCC from both chow diet and Western diet groups. Furthermore, PLT001 treatment robustly promoted the infiltration of CD8+T cells, including progenitor and terminally exhausted CD8+T cells, and enhanced granzyme B+Attorney Docket No.: 92EW-385670-WO populations (FIG.13F-I), underscoring more robust anti-tumor immune responses elicited by PLT001 in liver cancers with aberrant dietary lipid uptake. Collectively, these results highlight that targeting CD36 with PLT001 represents a promising strategy to restore immune responsiveness in HCCs with or without excessive metabolic challenges due to dietary lipid uptake. PLT001 treatment restricts liver metastasis and restores systemic anti-tumor immunity

[0159] The immune tolerance mechanisms presenting in the liver, particularly the development of Tregs, significantly impair immunosurveillance, rendering the liver a frequent site for metastases from various malignancies. The notably low response rates to immunotherapy in cancer patients with liver metastases highlights a critical unmet medical need for restoring effective therapeutic strategies by targeting liver metastasis. Leveraging the unique attributes of the POG570 cohort, which encompasses extensive bulk RNA analyses of biopsy samples from various metastatic sites in patients with advanced malignancies, this example initially assessed the metabolic properties of tumor microenvironment (FIG. 16A). Following the exclusion of metastatic samples with inadequate sample sizes, the analysis revealed that liver metastases across multiple cancer types exhibited TME features marked by heightened CD36 expression and increased fatty acid metabolism scores, alongside a high Treg abundance but reduced infiltration of CD8+T cells, when compared to other metastatic locations (FIG. 15A). To explore which primary tumor types exhibited high fatty acid scores and CD36 expression in liver colonization, this example applied the same analytical methodology to samples derived from liver metastasis of various cancer type in the same cohort. The analysis identified liver metastasis of breast cancer (BRCA), colon adenocarcinoma (COAD), pancreatic cancer (PAAD), and stomach cancer (STAD) exhibited significant fatty acid characteristics relative to other metastatic disease or other metastatic sites within same cancer type (FIG. 15B). Notably, the tissue microenvironment is not the sole determinant of fatty acid metabolism activation. For instance, liver metastases from non-small cell lung carcinoma (NSCLC) did not display significant fatty acid characteristics in comparison to other metastatic sites of NSCLC, suggesting that both intrinsic factors and the surrounding environment collaboratively influence metabolic features (FIG. 15B and 16B). Consequently, this example further assessed the correlation between CD36 expression level, FA score, and exhaustion score to identify the tumor types with liver metastasisAttorney Docket No.: 92EW-385670-WO for CD36 blockade treatment (FIG. 16C). The data showed that liver metastases originating from colon adenocarcinoma exhibit strong correlations with both fatty acid (FA) score and exhaustion score with CD36 levels. This suggests that the growth of liver metastases may be significantly influenced by CD36-mediated immune regulation, highlighting the potential therapeutic implications of targeting CD36 in this specific context.

[0160] A prior investigation reported that antigen-specific CD8+T cells undergo apoptosis following interaction with CD11b+F4 / 80+monocyte-derived macrophages in the context of colon cancer liver metastasis. Consequently, liver metastases eliminate tumor-specific T cells within the body, leading to acquired resistance to immune checkpoint blockade. Indeed, by using a concurrent tumor engraftment model to establish subcutaneous and liver metastases in the same mouse, this example found that PD-1 blockade became less potent on limiting subcutaneous MC38 colorectal cancer growth when liver metastases were present (FIG. 17A). Since this example found that the majority of liver metastases of human cancers elevated CD36 expression and other tumor-immune microenvironmental features controlled by CD36, it was postulated that PLT001 could also effectively target liver metastases and re-instate systemic antitumor immunity. To do so, this example treated mice concurrently engrafted with subcutaneous and liver metastases of MC38 with either control vehicle, anti-PD-1 mAb, PLT001, or anti-PD-1 plus PLT001 (FIG. 15C). PLT001 exhibited superior antitumor effects on both liver metastatic (FIG. 15D) and local tumors (FIG. 15E) compared to control and anti-PD-1 monotherapy, as assessed by BLI and endpoint tumor weight measurements (FIG. 17B-E). Most importantly, the combination of PLT001 with anti-PD-1 therapy significantly improved tumor suppression in primary tumors and the abundance of CD8+TILs in both primary and metastatic tumors compared to monotherapy (FIG. 15D-G and 17F). In addition, PLT001 treatment alone and combination with anti-PD-1 effectively reduced intratumoral Treg abundance in both primary and metastatic tumors (FIG. 15F and 15G). Next, this example examined whether PLT001 treatment can influence tumor-associated macrophage (TAM) phenotypes in both subcutaneous tumors and liver metastases (FIG. 17G). The findings showed that PLT001 alone drives an increase of M1-like macrophage population, characterized by CD45+Ly6G-CD11b+F4 / 80+Tim4- Clec4f+MHCII+CD206-, but a decrease of M2-like population, characterized by CD45+Ly6G- CD11b+F4 / 80+Tim4-Clec4f+MHCII-CD206+, in liver metastases (FIG. 15H-I). Similar to liver metastases, PLT001 alone induced a remarkable increase of M1-like TAMs (characterized byAttorney Docket No.: 92EW-385670-WO CD45+Ly6G-CD11b+F4 / 80+MHCII+CD206-), but a reduction of M2-like TAMs (characterized by CD45+Ly6G-CD11b+F4 / 80+MHCII-CD206+) in primary tumors (FIG. 17H-I). Of note, treatment of PLT001 combined with anti-PD-1 further amplified these changes in subcutaneous tumors (FIG. 17H-I). By staining with CD206, this example further confirmed that PLT001 and combination treatment groups robustly decreased CD206+population in tumors, whereas tumors treated with anti-PD-1 failed to achieve this beneficial outcome (FIG. 17J-K). Taken together, the results imply that PLT001 treatment could reprogram the tumor-immune microenvironment for hampering liver metastasis of colon cancer and improving sensitivity to PD-1 blockade in mice with liver metastasis. PLT001 is well-tolerated in non-human primates

[0161] To better understand how one can deploy PLT001 for the potential treatment in human patients, this example next assessed the safety profile of PLT001. This example first tested CD36 expression in red blood cells (RBCs) and platelets from mice, monkeys, and humans. This result showed that RBCs and platelets from monkeys and humans have a more similar CD36 expression pattern, characterized by low expression levels in RBCs but higher expression levels in platelets (FIG. 18A). Of note, monkeys had high and low CD36 expression groups. Conversely, similar expression levels of CD36 were noted in peripheral immune cell populations, including T cells, B cells, and monocytes, particularly between monkeys and humans (FIG. 18B). Consequently, cynomolgus monkeys were chosen for the subsequent safety assessments of PLT001 in a repeated-dose study (FIG. 18C). The administration of PLT001 was well tolerated in monkeys receiving three different dose levels (10mg / kg, 60mg / kg, and 200mg / kg), with no treatment-related mortality, clinical signs, or alterations in body weight, food intake, hematological parameters, or coagulation metrics (data not shown). Furthermore, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were comparable between these three dosing groups and control groups, with the exception of a transient increase in the 200 mg / kg group at 24 hours post-injection, indicating PLT001 treatment did not affect normal liver functions throughout the study (FIG. 18D). Moreover, PLT001 treatment did not affect overall RBC and platelet counts, irrespective of the CD36 expression profile on monkey RBCs and platelets (FIG. 18E). FACS-based immunophenotyping further indicated a favorable tolerance to PLT001 characterized by a stable immune profile, including CD4+T cells, CD8+TAttorney Docket No.: 92EW-385670-WO cells, NK cells, and monocytes, maintained throughout the study (FIG. 18F). Consistent with these findings, a phenotypic assay also indicated a limited effect on normal hematopoiesis and similar T-cell phenotypes between Cd36-KO and wild-type mice. Altogether, these results affirm that PLT001 did not elicit any adverse effects and exhibited an exceptional safety profile, with no observed adverse effect level (NOAEL) established even at 200 mg / kg. PLT001 triggers desirable immune responses in human HCC tumors

[0162] The pharmacodynamic effects, anti-tumor activity, and safety profile observed in pre- clinical studies underscore the therapeutic potential of PLT001. Interestingly, a newly established ex vivo culture of human tumors has been demonstrated to preserve stromal compartments, including immune infiltrates, and tumor micro-structure from patients and enable screening of immune profile changes in response to PD-1 blockade. To further translate these pre-clinical findings into a clinical-relevant condition, this example assessed the efficacy of PLT001 on modulating abundance and functions of intratumoral CD8+T cells and Tregs by utilizing human hepatocellular carcinoma (HCC) samples within this ex vivo culture platform. This example obtained 11 HCC patient samples that represented a spectrum of disease stages and conducted ex vivo cultures with control IgG or PLT001 for 2 days. Flow cytometry analysis was utilized to identify intratumoral CD8+T cells and Tregs. This example first assessed CD36 expression levels on intratumoral Tregs and CD8+T cells prior to treatment, as well as the immune profile following treatment with either control IgG or PLT001 in the human HCC samples. Despite notable variability in the percentage of CD36-positive intratumoral Tregs and CD8+T cells, which ranged from 10% to 80% among patients (FIG. 18G), nearly all patients displayed responses to PLT001 treatment, characterized by an increased proportion of CD8+T cells, a decreased proportion of Tregs, increased granzyme B+ population among CD8+T cells, and increased ratios of CD8+T cells to Tregs (FIG. 18H). Importantly, assessments of Treg-related pharmacodynamic markers, such as reduced Treg percentages and increased CD8 / Treg ratios, indicated an 81.8% response rate following PLT001 treatment. Furthermore, the PLT001-induced increase in CD8+T cell percentage and GzmB expression exhibited a 45.4% response rate (FIG. 18I). Of note, no definitive correlation between CD36 expression on Tregs and CD8 T cells and the level of response was identified, suggesting a feed-forward effect of PLT001 in promoting an immune-supportive microenvironment. These findings highlight the potential of PLT001 toAttorney Docket No.: 92EW-385670-WO effectively modulate the tumor microenvironment in HCC by targeting CD36. The efficacy on modulating immune profiles in HCC patient sample ex vivo cultures and superior safety profile of PLT001 in non-human primates emphasize its promise as a novel therapeutic agent for enhancing beneficial outcomes in HCC patients.

[0163] Here, this example demonstrated that modulation of CD36-mediated lipid signaling with PLT001 antibody treatment leads to improved therapeutic outcomes for liver cancer and colon cancer that metastasize to the liver. PLT001 has the potential to enhance the tumoricidal efficacy of immune cells and to modify the tumor microenvironment towards a more immunosupportive state, characterized by an increased M1 / M2 macrophage ratio and a higher CD8+T cell to Treg ratio. Moreover, PLT001 can provide superior anti-HCC immune responses in mice under a high- fat diet and elicit anti-liver metastasis responses in murine models. In addition, PLT001 can be utilized to restore PD-1 responsiveness against primary colon cancer in mice harboring liver metastasis. Importantly, PLT001 targets the fatty-acid binding region but exhibits limited antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) effects due to its IgG4 serotype. These rationale designs render PLT001 a superior capacity for tumor suppression while displaying a negligible impact on normal immune homeostasis and vascular balance. Notably, these findings offer significant implications for the translation of fundamental research into clinical oncology, as evidenced by evaluations conducted in human hepatocellular carcinoma tumor ex vivo cultures and cynomolgus monkeys for safety and pharmacokinetic studies. In summary, this example introduces a new perspective in tumor immunology, providing valuable insights for the development of metabolism-targeting therapies that could significantly influence the management of liver cancer and other metastatic conditions affecting the liver. * * *

[0164] The present disclosure is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the disclosure, and any compositions or methods which are functionally equivalent are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present disclosure without departing from the spirit or scope of the disclosure. Thus, it is intended that the present disclosure cover theAttorney Docket No.: 92EW-385670-WO modifications and variations of this disclosure provided they come within the scope of the appended claims and their equivalents.

[0165] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

Attorney Docket No.: 92EW-385670-WO CLAIMS What is claimed is:

1. A method for treating, in a subject, liver metastasis originated from a cancer in a tissue that is not liver, comprising administering to the subject an anti-CD36 antibody or antigen- binding fragment comprising a heavy chain variable region (VH) comprising a VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) comprising a VL CDR1, VL CDR2, and VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprise, respectively, the amino acid sequences of SEQ ID NO:3-8.

2. The method of claim 1, wherein the subject has an increased fatty acid concentration in the liver as compared to a reference healthy subject.

3. The method of claim 2, wherein the subject has hepatic steatosis.

4. The method of claim 2, wherein the subject has alcoholic fatty liver disease (AFLD) or nonalcoholic fatty liver disease (NAFLD).

5. The method of claim 4, wherein the subject has nonalcoholic steatohepatitis (NASH).

6. The method of any preceding claim, wherein the subject has a body mass index (BMI) of 25 kg / m² or higher.

7. The method of claim 6, wherein the subject has a BMI of 28 kg / m² or higher, or 30 kg / m² or higher.Attorney Docket No.: 92EW-385670-WO 8. The method of any preceding claim, wherein the subject is on a high-fat diet or a high- sugar diet.

9. The method of claim 8, wherein the high-fat diet (HFD) comprises at least 25% of total calories from fats, preferably at least 30%, 35% or 40% of total calories from fats, unsaturated or saturated.

10. A method for treating a cancer in a subject having a body mass index (BMI) of 25 kg / m² or higher or having an increased fatty acid concentration in the liver as compared to a reference healthy subject, comprising administering to the subject an anti-CD36 antibody or antigen- binding fragment comprising a heavy chain variable region (VH) comprising a VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) comprising a VL CDR1, VL CDR2, and VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprise, respectively, the amino acid sequences of SEQ ID NO:3-8.

11. The method of claim 10, wherein the subject has hepatic steatosis.

12. The method of claim 10, wherein the subject has alcoholic fatty liver disease (AFLD) or nonalcoholic fatty liver disease (NAFLD).

13. The method of claim 12, wherein the subject has nonalcoholic steatohepatitis (NASH).

14. The method of any one of claims 10-13, wherein the subject has a BMI of 28 kg / m² or higher, or 30 kg / m² or higher.Attorney Docket No.: 92EW-385670-WO 15. The method of any preceding claim, wherein the subject has a decreased Treg concentration in the liver as compared to an average cancer patient having liver metastasis.

16. The method of any preceding claim, wherein the subject has an increased CD8+ T cell infiltration in the liver as compared to an average cancer patient having liver metastasis.

17. The method of any preceding claim, wherein the liver metastasis is from a cold tumor.

18. The method of claim 17, wherein the cold tumor is characterized with one or more p53 mutation or β-catenin mutation, high microsatellite instability (MSI-high), or microsatellite stable (MSS) with CD36 expression.

19. The method of any preceding claim, wherein the subject has been treated with a PD-L1 or PD-1 inhibitor or is resistant to a PD-L1 or PD-1 inhibitor.

20. The method of any preceding claim, wherein the cancer is colorectal cancer.

21. The method of any one of claims 10-19, wherein the cancer is hepatocellular carcinoma (HCC), colorectal cancer (CRC), or colorectal liver metastasis (CRLM).

22. The method of any preceding claim, further comprising administering to the subject an inhibitor of PD-L1 or PD-1.

23. The method of claim 22, further comprising administering to the subject an inhibitor of VEGFα.Attorney Docket No.: 92EW-385670-WO 24. The method of any preceding claim, wherein the VH comprises the amino acid sequence of SEQ ID NO:1 and the VL comprises the amino acid sequence of SEQ ID NO:

2.

25. The method of any preceding claim, wherein the antibody comprises a human IgG4 Fc fragment.

26. The method of claim 25, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:11 and a light chain comprising the amino acid sequence of SEQ ID NO:12.

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