MHC ii expression as a predictive biomarker for CD4 depletion anticancer immunotherapy

Abstract

Described herein are methods for treating cancer based with a CD4 depleting therapy based on MHC II expression level or a molecular surrogate level for expression MHC II level. Also described are methods of selecting a cancer treatment for a subject based on MHC II expression level or a molecular surrogate level for expression MHC II level.

Description

MHC II EXPRESSION AS A PREDICTIVE BIOMARKER FOR CD4 DEPLETION ANTICANCER IMMUNOTHERAPYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application includes a claim of priority under 35 U.S.C. §119(e) to U.S. provisional patent application No. 63 / 755,063, filed February 6, 2025, the entirety of which is hereby incorporated by reference.FIELD OF INVENTION

[0002] This invention relates to use of MHC II expression levels for the selection of cancer treatment and the treatment of cancer.BACKGROUND

[0003] All publications herein are 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. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] Immunotherapy is becoming a cornerstone for cancer treatment. Among promising approaches under investigation is the depletion of CD4+ lymphocytes as a strategy to target CD4+FoxP3+ regulatory T cells (Tregs). Tregs are a pivotal regulator of the immune response and function as a critical immune checkpoint. However, despite their significance, there is currently no FDA-approved cancer therapy that directly and specifically targets Tregs. Therefore, CD4 depletion has been proposed as a strategy to control Tregs. This strategy is based on the premise that by the time a malignancy is diagnosed, the immune system has been primed by CD4+ effector T cells and the vast majority of CD4+ T cells are immunosuppressive. Therefore, CD4+ T cells are no longer needed for antitumor immunity. In previously reported preclinical models and early phase clinical testing, CD4 depletion is effective in stimulating antitumor immunity.

[0005] However, the interplay between tumor and host immunity is heterogeneous, and CD4 lymphocytes have been reported to control tumor growth in some settings. As such, there remains a need in the art for methods to predict the response to CD4 depletion therapy, and therapies based on those predictions.14935-2291-41872065472-001010WOPTSUMMARY OF THE INVENTION

[0006] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.

[0007] Various embodiments provide for a method for treating cancer in a subject, comprising: administering a CD4 depleting therapy to a subject having tumor tissue determined to have an MHC II expression level that is lower than a reference level for the tumor type, or a level of a molecular surrogate level for expression MHC II level that is lower than its reference level for the tumor type.

[0008] In various embodiments, the method can further comprise selecting the subject having tumor tissue determined to have an MHC II expression level that is lower than a reference level for the tumor type, or a level of a molecular surrogate level for expression MHC II level that is lower than its reference level for the tumor type, prior to administering a CD4 depleting therapy to a subject.

[0009] Various embodiments provide for a method for treating cancer in a subject, comprising: detecting an MHC II expression level or a molecular surrogate level for MHC II expression level in tumor tissue obtained from the subject; and administering a CD4 depleting therapy to a subject who has been determined to have an MHC II expression level that is lower than a reference level for the tumor type, or a molecular surrogate level for MHC II expression level that is lower than its reference level for the tumor type.

[0010] Various embodiments provide for a method for treating cancer in a subject, comprising: obtaining or requesting the results of an analysis of an MHC II expression level or a molecular surrogate level for MHC II expression level in tumor tissue obtained from the subject; and administering a CD4 depleting therapy to a subject who has been determined to have an MHC II expression level that is lower than a reference level for the tumor type, or a molecular surrogate level for MHC II expression level that is lower than its reference level for the tumor type.

[0011] Various embodiments provide for a method of selecting a cancer treatment for a subject, comprising detecting an MHC II expression level or a molecular surrogate level for MHC II expression level in tumor tissue obtained from the subject; and selecting a CD4 depleting therapy for a subject who has been determined to have MHC II expression level that is lower than reference level for the tumor type, or a level of a molecular surrogate for MHC II level that is lower than its reference level for the tumor type, or avoiding CD424935-2291-41872065472-001010WOPTdepleting therapy for a subject who has been determined to have MHC II expression level that is higher than reference level for the tumor type, or a level of a molecular surrogate for MHC II level that is higher than its reference level for the tumor type.

[0012] In various embodiments, the method can further comprise administering the selected CD4 depleting therapy.

[0013] In various embodiments, the molecular surrogate for MHC II level can be CIITA, HLA-DP, HLA-DQ, HLA-DR, RFX5, or RFXAP.

[0014] In various embodiments, the CD4 depleting therapy can be selected from CC182, IT1208 (a defucosylated humanized anti-CD4 depleting antibody), daclizumab, zanolimumab, cM-T412, keliximab, ibalizumab, or combinations thereof. In various embodiments, the CD4 depleting therapy can be CC182.

[0015] In various embodiments, the molecular surrogate for MHC II level can be CIITA.

[0016] In various embodiments, the cancer can be selected from melanoma, renal cell carcinoma, or hepatocellular carcinoma. In various embodiments, the cancer can be selected from Acute Myeloid Leukemia (LAML), Adrenocortical Carcinoma (ACC), Bladder Urothelial Carcinoma (BLCA), Brain Lower Grade Glioma (LGG), Breast Invasive Carcinoma (BRCA), Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma (CESC), Cholangiocarcinoma (CHOL), Colon Adenocarcinoma (COAD), Esophageal Carcinoma (ESCA), Glioblastoma Multiforme (GBM), Head and Neck Squamous Cell Carcinoma (HNSC), Kidney Renal Clear Cell Carcinoma (KIRC), Kidney Renal Papillary Cell Carcinoma (KIRP), Liver Hepatocellular Carcinoma (LIHC), Lung Adenocarcinoma (LUAD), Lung Squamous Cell Carcinoma (LUSC), Lymphoid Neoplasm Diffuse Large B-cell Lymphoma (DLBC), Mesothelioma (MESO), Ovarian Serous Cystadenocarcinoma (OV), Pancreatic Adenocarcinoma (PAAD), Pheochromocytoma and Paraganglioma (PCPG), Prostate Adenocarcinoma (PRAD), Rectum Adenocarcinoma (READ), Sarcoma (SARC), Skin Cutaneous Melanoma (SKCM), Stomach Adenocarcinoma (STAD), Testicular Germ Cell Tumors (TGCT), Thyroid Carcinoma (THCA), Thymoma (THYM), Uterine Corpus Endometrial Carcinoma (UCEC), Uterine Carcinosarcoma (UCS), or Uveal Melanoma (UVM).

[0017] In various embodiments, the method can further comprise administering chemotherapy, radiotherapy or immunotherapy to the subject.

[0018] In various embodiments, the subject can be a human subject.34935-2291-41872065472-001010WQPT

[0019] Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention.BRIEF DESCRIPTION OF THE FIGURES

[0020] Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0021] Figure 1 (panels A-D) shows the effect of CD4 depletion across tumor models. A) Mice were injected s.c. with syngeneic tumor cells, and after Renca and Hepal-6 tumors were palpable, mice were treated with 2 doses of CD4 depleting antibody, 200pg / mouse (typically on days 9 and 11), and tumor growth was monitored. B) B16 melanoma cells were injected into B6 mice. C) Renca kidney cancer cells were injected into Balb / c mice. D) Hepal-6 hepatocellular cells were injected into B6 mice. Statistical significance determined by repeated measures ANOVA.

[0022] Figure 2 (panels A-D) shows the role of CD4+ and CD8+ lymphocytes in Hepal-6 tumor growth. A) Depleting antibodies against CD4, CD8, or both (200pg of each antibody / mouse) were administered 1 day after s.c. injection of Hepa 1-6 cells. B) Tumor growth was monitored. C) After Hepal-6 tumor were injected s.c., both CD4 and CD8 lymphocytes were depleted with antibodies as described for Figure A (recipient mice). From separate, non-tumor bearing mice, antitumor immunity was generated by treating with dendritic cells pulsed with Hepal-6 lysate (donor mice). CD4 or CD8 cells were enriched from donor mouse splenocytes and lymphocytes. CD4, CD8, or both cells were adoptive transferred into tumor-bearing recipient mice on day 2. D) Tumor growth was monitored. Statistical significance determined by repeated measures ANOVA. Representative results from duplicate experiments are shown.

[0023] Figure 3 (panels A-D) shows tumor MHC I and II levels were assessed in mouse models and humans. A) Hep 1-6, B16 and Renca cells were assessed for MHC I expression by flow cytometry. B) The same cell lines were assessed for MHC II expression. C) Expression levels for MHC I and D) MHC II of various cancer types using the TCGA pan-cancer dataset. Gene expressions were standardized using the z-score method. The box plot depicts the median as well as the upper and lower quartiles, and the whiskers depict 1.5 times the interquartile range.44935-2291-41872065472-001010WQPT

[0024] Figure 4 (panels A-D) shows CIITA expression was knocked down and overexpressed. A) CIITA is a transcriptional coactivator for MHC II. B) MHC II expression and CIITA expression are highly correlated in the TCGA pan-cancer dataset with Spearman’s Rho of 0.8395 and pO.OOOl. C) Wild type Hepal-6 cells have high MHC II expression, and shRNA knock down (KD) reduced MHC II expression as assessed by immunocytochemistry and flow cytometry. D) Wild type Renca cells have no MHC II expression, and transfection with a CIITA overexpression plasmid resulted in high MHC II expression as assessed by immunocytochemistry and flow cytometry.

[0025] Figure 5 (panels A-E) shows characterization of Hepal-6 with CIITA KD. A) In vitro growth rate was compared for CIITA knock down (KD) and WT Hepal-6 cells. B) KD and WT Hepal-6 cells were injected s.c. into B6 mice and tumor growth was assessed. Representative results from duplicate experiments are shown. C) For an immune-mediated tumor killing assay, lymphocytes were harvested from WT Hepal-6 tumor-bearing mice, which were co-cultured with WT or KD Hepal-6 cells. Hepal-6 cell death was detected by flow cytometry by gating on CD45 negative cells and counting propidium iodine (PI) positive cells. D) WT and E) KD cells were injected s.c. into B6 mice to establish tumor, and tumor growth with and without CD4 lymphocyte depletion was compared. CD4 depleting antibodies were administered on days 9 and 11.

[0026] Figure 6 (panels A-I) shows characterization of Renca with CIITA overexpression. A) In vitro growth rate was compared for CIITA overexpressing (OE) and WT Renca cells. B) OE and WT Renca cells were injected s.c. into Balb / c mice and tumor growth was assessed. Representative results from duplicate experiments are shown. C) Splenocytes were harvested from tumor-bearing mice. CD4+ and CD8+ cells were quantified as a percent of all splenocytes. D) To assess CD8+ cell activation, CD4+IFNY+ and CD8+ IFNY+ cells were quantified as a percent of splenocytes, after ex vivo stimulation with fresh DCs pulsed with tumor lysate. E) CD45+CD4+ and CD45+CD8+ cells were quantified from tumor-dissociated cells. F) WT Renca cells were injected s.c. into Balb / C mice to establish tumor, and tumor growth with and without CD4 lymphocyte depletion was compared. CD4 depleting antibodies were administered on days 9 and 11. G) From these mice, splenocytes were harvested and CD4+FoxP3 splenocytes were quantified. CD8+IFNy+ splenocytes were quantified as described for Figure 6D. H-I) The experiments in Figures 6F&G were repeated with CIITA OE Renca cells. All cell quantifications were by flow cytometry.54935-2291-41872065472-001010WOPT

[0027] Figure 7 (panels A-C) shows CD4+ status as a predictor of overall survival. A) Scatterplot based on z-score for MHC I and MHC II expression in all patients in the TCGA pan-cancer dataset. The red lines define the 33.3 and 66.6 percentiles for MHC I and MHC II expression levels. For the four groups defined by the highest and lowest expressions, forest plots show the mean hazard ratio (HR) and 95% confidence interval for the association between CD4 or CD8 T-cell scores and overall survival. Group B (top left comer) has high MHC II and low MHC I expressions. Group C (bottom right corner) has low MHC II and high MHC I expressions. B-C) Kaplan-Meier curves for overall (OS) and progression-free survival (PFS). Cohorts were divided into two groups using the median CD4 expression as the cutoff. B) In group B, the difference in OS approached significance (p=0.0770) while the difference in PFS was significant (p=0.0217), favoring high CD4 expression. C) In group C, the difference in OS was significant (p = 0.0101), while the difference in PFS approached significance (p=0.1497), favoring low CD4 expression.

[0028] Figure 8 shows an independent replicate of experiments shown in Figure 1, with tumor growth curves for each individual tumor shown. For the Hepal-6 experiment, the growth curve for the control group represents 5 tumors.

[0029] Figure 9 shows Hepal-6 cells were treated in vitro with PBS, IFNy and TNFa for 48 hours before staining with antibodies against mouse MHC I or MHC II and assessing by flow cytometry.

[0030] Figure 10 shows the control lentiviral transduction particle (shRNA control virus) was confirmed to have no effect on Hepal-6 proliferation in vitro (middle panel) or mouse Hepal-6 tumor growth (right panel) when compared to the wild type (WT) Hepal-6.

[0031] Figure 11 shows the control lentiviral transduction particle (Control Vims) was confirmed to have no effect on Renca proliferation in vitro (middle panel) or mouse Renca tumor growth (right panel) when compared to the wild type (WT) Renca.

[0032] Figure 12 shows flow cytometry showing that percent of mouse splenocytes that are CD4 or CD8 positive does not change whether they are first gated on CD3 or not.

[0033] Figure 13 shows flow cytometry showing the numbers of CD45+CD4+ cells and CD45+CD8+ cells from the same experiment described in Figure 6E.

[0034] Figure 14 shows CD4+ status as a predictor of overall survival.Scatterplot based on z-score for MHC I and MHC II expression in all patients in the TCGA pan-cancer dataset. The red lines define the 33.3 and 66.6 percentiles for MHC I and MHC II expression levels. For the subgroups highlighted, forest plots show the mean hazard ratio64935-2291-41872065472-001010WOPT(HR) and 95% confidence interval for the association between CD4 or CD8 T-cell scores and overall survival.DESCRIPTION OF THE INVENTION

[0035] All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton el al., Dictionary of Microbiology and Molecular Biology 3rded., Revised, J. Wiley & Sons (New York, NY 2006); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4thed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application.

[0036] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0037] As used herein the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 5% of that referenced numeric indication, unless otherwise specifically provided for herein. In various embodiments, the term “about” when used in connection with a referenced numeric indication can mean the referenced numeric indication plus or minus up to 4%, 3%, 2%, 1%, 0.5%, or 0.25% of that referenced numeric indication, if specifically provided for in the claims.

[0038] CD4 depleting therapy” as used herein refers to a therapy that suppresses or removes CD4+ T cells, including CD4+ regulatory T cells (Tregs), from a subject. CD4 depletion can occur directly or indirectly, and can be mediated through mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), activation-induced cell death, apoptosis, or immune-mediated clearance, or a combination of these mechanisms. Examples of CD4 depleting therapy include but are not limited to anti-CD4 antibodies. For example, CC182 (anti-CD4 monoclonal antibody developed by Crown Bioscience), IT1208 (a defucosylated humanized anti-CD4 depleting antibody developed by ID AC Theranostics, Inc. (Tokyo, Japan) and Ono Pharmaceutical Co., Ltd.), daclizumab, zanolimumab, cM-T412 (aka Priliximab), keliximab, ibalizumab. Additional examples include but are not limited to 74935-2291-41872065472-001010WOPT0KT4, HuMax-CD4, Tregalizumab, bispecific antibodies binding CD4, antibody drug conjugates targeting CD4, Alemtuzumab, polyclonal antibodies targeting CD4, Afucosylated anti-CD4, and anti-CD4 antibodies with Fey receptor variants

[0039] A “cancel’ or “tumor” as used herein refers to an uncontrolled growth of cells which interferes with the normal functioning of the bodily organs and systems, and / or all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. A subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject’s body. Included in this definition are benign and malignant cancers, as well as dormant tumors or micrometastasis. Cancers which migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs. As used herein, the term “invasive” refers to the ability to infiltrate and destroy surrounding tissue. In some embodiments, the tumor is a solid tumor.

[0040] Examples of cancer include but are not limited to breast cancer such as a ductal carcinoma in duct tissue in a mammary gland, medullary carcinomas, colloid carcinomas, tubular carcinomas, and inflammatory breast cancer; ovarian cancer, including epithelial ovarian tumors such as adenocarcinoma in the ovary and an adenocarcinoma that has migrated from the ovary into the abdominal cavity; cervical cancers such as adenocarcinoma in the cervix epithelial including squamous cell carcinoma and adenocarcinomas; prostate cancer, such as a prostate cancer selected from the following: an adenocarcinoma or an adenocarinoma that has migrated to the bone; pancreatic cancer such as epitheliod carcinoma in the pancreatic duct tissue and an adenocarcinoma in a pancreatic duct; bladder cancer such as a transitional cell carcinoma in urinary bladder, urothelial carcinomas (transitional cell carcinomas), tumors in the urothelial cells that line the bladder, squamous cell carcinomas, adenocarcinomas, and small cell cancers; acute myeloid leukemia (AML), preferably acute promyleocytic leukemia in peripheral blood; lung cancer such as non-small cell lung cancer (NSCLC), which is divided into squamous cell carcinomas, adenocarcinomas, and large cell undifferentiated carcinomas, and small cell lung cancer; skin cancer such as basal cell carcinoma, melanoma, squamous cell carcinoma and actinic keratosis, which is a skin condition that sometimes develops into squamous cell carcinoma; eye retinoblastoma; intraocular (eye) melanoma; primary liver cancer (cancer that begins in the liver); kidney cancer; thyroid cancer such as papillary, follicular, medullary and anaplastic; AIDS-related lymphoma such as diffuse large B-cell lymphoma, B-cell immunoblastic lymphoma and small non-cleaved cell lymphoma; Kaposi’s sarcoma; Ewing sarcoma; central nervous system cancers such as primary brain tumor, which includes gliomas 84935-2291-41872065472-001010WOPT(astrocytoma, anaplastic astrocytoma, or glioblastoma multiforme (GBM)), Oligodendroglioma, Ependymoma, Meningioma, Lymphoma, Schwannoma, and Medulloblastoma; peripheral nervous system (PNS) cancers such as acoustic neuromas and malignant peripheral nerve sheath tumor (MPNST) including neurofibromas and schwannomas; oral cavity and oropharyngeal cancer; stomach cancer such as lymphomas, gastric stromal tumors, and carcinoid tumors; testicular cancer such as germ cell tumors (GCTs), which include seminomas and nonseminomas; and gonadal stromal tumors, which include Leydig cell tumors and Sertoli cell tumors; head cancer; neck cancer; throat cancer; and thymus cancer, such as to thymomas, thymic carcinomas, Hodgkin disease, non-Hodgkin lymphomas carcinoids or carcinoid tumors. Also, the methods may be used to treat viral-induced cancers. The major virus-malignancy systems include hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatocellular carcinoma; human lymphotropic virus-type 1 (HTLV-1) and adult T-cell leukemia / lymphoma; and human papilloma virus (HPV) and cervical cancer.

[0041] Treatment” and “treating,” as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder even if the treatment is ultimately unsuccessful. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. For example, in tumor (e.g., cancer) treatment, a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents or by the subject’s own immune system.

[0042] Examples of biological samples include but are not limited to body fluids, whole blood, plasma, stool, intestinal fluids or aspirate, and stomach fluids or aspirate, serum, cerebral spinal fluid (CSF), urine, sweat, saliva, tears, pulmonary secretions, breast aspirate, prostate fluid, seminal fluid, cervical scraping, amniotic fluid, intraocular fluid, mucous, and moisture in breath. Additional examples of biological samples include but are not limited to tissue samples from healthy or diseased tissue (e.g., tumor tissue, cancer tissue), or cells from healthy or diseased tissue (e.g., tumor cells, cancer cells)

[0043] Described herein, we explored the efficacy and limitations of CD4 depletion in controlling tumor growth across various syngeneic cancer models. We observed that CD4 depletion suppressed the growth of B16 melanomas and Renca kidney cancers. In contrast, it accelerated the growth of Hepal-6 hepatocellular carcinomas. Amongst our tumor models, Hepal-6 had uniquely high MHC II expression, suggesting that MHC II determines how CD494935-2291-41872065472-001010WOPTdepletion affects tumor growth. We confirm the mechanistic relationship between MHC II expression and CD4+ cells by manipulating MHC II expressions in Hepal-6 and Renca cell lines and noting in vitro and in vivo changes in tumor growth and immune-mediated tumor killing.

[0044] Our observations in mouse tumor models indicate that MHC I and MHC II expression may identify human tumors where tumor growth suppression depends on CD4+ or CD8+ lymphocytes. This was supported by our analysis of survival outcomes from the TCGA pancancer dataset. Specifically, in tumors with high MHC I expression, higher CD8 expression predicted better survival and higher CD4 levels predicted worse survival. However, as expected, in tumors with low MHC I expression and high MHC II expression, higher tumor CD4 levels predicted survival in the opposite direction, predicting improved survival.

[0045] The findings described herein highlights the importance of patient selection for immunotherapies in general and for CD4 depletion more specifically, underscoring the complexity of the interplay between tumor and immunity. Our findings from various syngeneic mouse tumor models reveal that the effect of CD4 depletion is contingent on the tumor’s MHC expression profile. The suppression of B16 and Renca tumors upon CD4 depletion contrasts with the accelerated growth observed in Hepal-6 tumors. The causative relation between MHC II and CD4 cells in generating antitumor immunity was established by manipulating MHC II expression in tumors with varying baseline MHC II expressions. We report that in tumors with high MHC II, CD4+ lymphocytes suppress tumor growth while in tumors with low MHC II, CD4+ lymphocytes have the opposite effect on tumor growth.

[0046] These observations then led to predictions about how tumor CD4 levels will differentially predict survival in cancer patients with high vs. low tumor MHC II expressions. An analysis of the TCGA pan-cancer dataset bore out our prediction, showing that MHC II is a valuable biomarker for deployment of CD4 depletion as a novel checkpoint inhibitor. In tumors with high MHC I expression, higher CD8 expression predicted better survival as expected. However, in tumors with low MHC I expression and high MHC II expression, higher CD4 predicted improved survival. In tumors with the opposite profile, that is high MHC I and low MHC II, CD4 predicted survival in the oppositive direction, with higher CD4 predicting worse survival.

[0047] Our previous work in B16 and Renca models showed that CD4 depletion removes Tregs, which in turn results in a robust antitumor immune response. Zhang et al showed that CD4 depletion results in protective tumor-specific CD8 memory T-cells that 104935-2291-41872065472-001010WOPTpersisted for as long as 5 months following surgical excision of B16 tumors. Using a mouse fibrosarcoma model, Yu et al showed that CD4 depletion enhances antitumor immunity. These studies also showed that the timing of CD4 depletion is important. Optimal results occur with CD4 depletion performed after immune priming, when the majority of CD4+ cells are expected to be immunosuppressive Tregs rather than CD4+ effector cells. Consequently, CD4 depletion prior to immune priming can inhibit antitumor immunity. However, from a practical standpoint, this is a clinical scenario that is not encountered in patients; by the time a patient’s cancer is diagnosed, the tumor has already primed the immune system.

[0048] Based on these preclinical observations, Shitara et al conducted a phase I clinical trial using a defucosylated humanized anti-CD4 antibody in 11 patients with advanced solid tumors. CD8+ T cells increased and one patient with metastatic colon cancer achieved a durable partial response and two additional patients with gastric or esophageal cancer achieved stable disease lasting more than 3 months. In a follow up study, the authors concluded that CD4 depletion is accompanied by expansion of tumor-reactive T-cell clones that mediate antitumor immunity. The treatment was well tolerated with only grade I and II toxicities, which is consistent with prior studies of CD4 depletion for other indications. In several studies, chimeric aCD4 antibodies effectively depleted CD4 levels in cases of refractory cutaneous T-cell lymphoma, without causing serious infections or dose-related toxicities. A similar CD4 depleting chimeric monoclonal antibody was tested in two randomized, double-blind, placebo-controlled studies for rheumatoid arthritis. While it did not show therapeutic benefits for rheumatoid arthritis, there were no significant adverse events, and patients didn't experience opportunistic infections.

[0049] Tregs are the master regulators of immunity and FoxP3 is the most reliable Treg marker. FoxP3 is specific for Tregs and is required for its function. Unfortunately, there is no clinical strategy for targeting FoxP3 expressing cells in patients. Therefore, prior strategies have targeted CD25, which is expressed by the majority of Tregs. However, an important limitation of this approach is that some Tregs are CD25 negative. Furthermore, activated CD8+ T cells express CD25 and can be depleted by CD25-targeting strategies. In murine models, depleting CD25 expressing cells with aCD25 antibodies was effective in preventing tumor growth, but was not effective in treating established tumors and has been shown to restrict adoptive immunotherapy. It is also interesting that daclizumab, an aCD25 antibody, was approved by the FDA for immune suppression and prevention of acute rejection of kidney transplant, which is consistent with its limited role as an immune stimulating anticancer therapy.114935-2291-41872065472-001010WOPT

[0050] CD8+ cytotoxic T cells can kill tumor cells directly and have been the focus of much attention in cancer immunology. Tumor-infiltrating CD8+ lymphocytes are required to generate an effect antitumor immunity, and tumor CD8+ lymphocytes have been proposed as a predictive biomarker and as an early-response marker for cancer immunotherapy. However, this and other studies point to the importance of CD4+ T cells. For example, Ott et al reported that when administering personalized neoantigen vaccines for melanoma, the MHC II responses were higher than MHC I responses. In our TCGA analysis, CD4 and CD8 levels had varying prognostic significant in groups defined by MHC I and MHC II levels. In most groups, high CD4 correlated with worse survival since CD4+ cells are likely Tregs. However, when MHC II was high and MHC I was low, high tumor CD4 levels predicted better survival since CD4+ cells are likely effectors cells contributing to antitumor immunity.

[0051] Our findings have important implications for development of CD4 depletion therapy. Future clinical trials of CD4 depletion strategies should consider MHC status as a predictive biomarker, and MHC status should be used for patient selection. The ability to enrich for patients most likely to respond to a therapy makes a treatment highly attractive. Also, when planning for phase II testing, baseline MHC I and MHC II status can help prioritize tumor types for clinical testing.

[0052] These observations highlight the role of CD4+ lymphocytes in controlling or promoting cancer growth, and identify MHC status as a determinant of these divergent roles. In the TCGA pan-cancer dataset, we found corroborative evidence suggesting that MHC status can determine whether CD4 or CD8 may be the best tissue-based early-response marker for immunotherapy.

[0053] Various embodiments of the present invention are based, at least in part, on these findings.

[0054] Various embodiments provide for a method for treating cancer in a subject, comprising: administering a CD4 depleting therapy to a subject having tumor tissue determined to have an MHC II expression level that is lower than a reference level for the tumor type, or a level of a molecular surrogate level for expression MHC II level that is lower than its reference level for the tumor type.

[0055] In various embodiments, markers that co-express with MHC II can be detected in lieu of or in additional to MHC II or molecular surrogate level for expression MHC II. Examples of these co-expressed markers include but are not limited to CD45, CDllb, CDllc, CD68, CD80, CD86, and Ibal (in microglia).124935-2291-41872065472-001010WQPT

[0056] In various embodiments, the method further comprise selecting the subject having tumor tissue determined to have an MHC II expression level that is lower than a reference level for the tumor type, or a level of a molecular surrogate level for expression MHC II level that is lower than its reference level for the tumor type, based on the knowledge that an MHC II expression level that is lower than a reference level for the tumor type indicates an antitumor immunity that is CD8+ lymphocyte dependent and CD4 depletion decreases tumor growth, prior to administering the CD4 depleting therapy. Thus, the method comprises selecting a subject having tumor tissue determined to have an MHC II expression level that is lower than a reference level for the tumor type, or a level of a molecular surrogate level for expression MHC II level that is lower than its reference level for the tumor type, based on the knowledge that an MHC II expression level that is lower than a reference level for the tumor type indicates an antitumor immunity that is CD8+ lymphocyte dependent and CD4 depletion decreases tumor growth, and administering the CD4 depleting therapy to the subject.

[0057] In various embodiments, administering the CD4 depleting therapy to the subject comprises administering daily, weekly, bi-weekly, or monthly. In various embodiments, there can be a drug holiday, that is a break from the periodic administration. The drug holiday can be for 1 day, 3 days, 5 days, 7 days, 2 weeks, 3 weeks, or 1 month between each of the periodic administration. In various embodiments, the drug holiday can be for more than 1 month. In various embodiments, administering the CD4 depleting therapy to the subject comprises administering for a period of time. The period of time can be about 1 week, 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months. In other embodiments, the period of time can be about 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years or 5 years. In other embodiments, the period of time can be more than 5 years.

[0058] In various embodiments, the molecular surrogate for MHC II level is CIITA, HLA-DP, HLA-DQ, HLA-DR, RFX5, or RFXAP. In particular embodiments, the molecular surrogate for MHC II level is CIITA.

[0059] In various embodiments, the CD4 depleting therapy is selected from CC182, IT 1208 (a defucosylated humanized anti-CD4 depleting antibody), daclizumab, zanolimumab, cM-T412, keliximab, ibalizumab, or combinations thereof. In various embodiments, the CD4 depleting therapy is selected from CC182.

[0060] In various embodiments, the method further comprises administering chemotherapy, radiotherapy or immunotherapy to the subject.134935-2291-41872065472-001010WQPT

[0061] Examples of chemotherapeutic agents include cytotoxic agents (e.g., 5-fluorouracil, cisplatin, carboplatin, methotrexate, daunorubicin, doxorubicin (Adriamycin®), vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine (CCNU), cytarabine USP, cyclophosphamide, estramucine phosphate sodium, altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan, cyclophosphamide, mitoxantrone, carboplatin, cisplatin, interferon alfa-2a recombinant, paclitaxel, teniposide, and streptozoci), cytotoxic akylating agents (e.g., busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylesulfonic acid), alkylating agents (e.g., asaley, AZQ, BCNU, busulfan, bisulphan, carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, cisplatinum, clomesone, cyanomorpholinodoxorubicin, cyclodisone, cyclophosphamide, dianhydrogalactitol, fluorodopan, hepsulfam, hycanthone, iphosphamide, melphalan, methyl CCNU, mitomycin C, mitozolamide, nitrogen mustard, PCNU, piperazine, piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard, streptozotocin, teroxirone, tetraplatin, thiotepa, tri ethylenemelamine, uracil nitrogen mustard, and Yoshi-864), antimitotic agents (e.g., allocolchicine, Halichondrin M, colchicine, colchicine derivatives, dolastatin 10, maytansine, rhizoxin, paclitaxel derivatives, paclitaxel, thiocolchicine, trityl cysteine, vinblastine sulfate, and vincristine sulfate), plant alkaloids (e.g., actinomycin D, bleomycin, L-asparaginase, idarubicin, vinblastine sulfate, vincristine sulfate, mitramycin, mitomycin, daunorubicin, VP-16-213, VM-26, navelbine and taxotere), biologicals (e.g., alpha interferon, BCG, G-CSF, GM-CSF, and interleukin-2), topoisomerase I inhibitors (e.g., camptothecin, camptothecin derivatives, and morpholinodoxorubicin), topoisomerase II inhibitors (e.g., mitoxantron, amonafide, m-AMSA, anthrapyrazole derivatives, pyrazoloacridine, bisantrene HCL, daunorubicin, deoxydoxorubicin, menogaril, N,N-dibenzyl daunomycin, oxanthrazole, rubidazone, VM-26 and VP- 16), and synthetics (e.g., hydroxyurea, procarbazine, o,p'-DDD, dacarbazine, CCNU, BCNU, cis-diamminedichloroplatimun, mitoxantrone, CBDCA, levamisole, hexamethylmelamine, all-trans retinoic acid, gliadel and porfimer sodium).

[0062] Examples of immunotherapy include but are not limited to PD1 inhibitors or PDL1 inhibitors. In various embodiments, the PD1 inhibitor can be selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, AMP-224, AMP-514, spartalizumab, cemiplimab, penpulimab (AK105), prolgolimab (BCD-100), ezabenlimab (BI 754091), toripalimab (JS001), lipustobart (LZM009), retifanlimab (MGA012), Sym021, dostarlimab (TSR-042), tebotelimab (MGD013), cadonilimab (AK104), vudalimab (XmAb20717), tislelizumab, PF-06801591, anti-PDl antibody expressing pluripotent killer T lymphocytes (PIK-PD-1), autologous anti-EGFRvIII 4SCAR-IgT cells, and combinations thereof. In various 144935-2291-41872065472-001010WOPTembodiments, the PDL1 inhibitor can be selected from the group consisting of garivulimab (BGB-A333), cosibelimab (CK-301), FAZ053, envafolimab (KN035), MDX-1105, betifisolimab (MSB2311), adebrelimab (SHR-1316), atezolizumab, avelumab, durvalumab, BMS-936559, CK-301, M7824, and combinations thereof.

[0063] Additional examples of immunotherapy include but are not limited to anti-CTLA-4 antibodies. For example, ipilimumab, tremelimumab, zalifrelimab, quavonlimab, cadonilimab (a bispecific antibody targeting PD-1 and CTLA-4, erfonrilimab (a bispecific antibody targeting PD-L1 and CTLA-4), and ATOR-1015.

[0064] Additional examples of immunotherapy further include but are not limited to cancer vaccines. For example, Sipuleucel-T, Bacillus Calmette-Guerin (BCG), Talimogene laherparepvec , Nadofaragene firadonevec, GV AX (modified tumor cells, being studied for pancreatic and prostate cancers), DCVax-L (dendritic cell vaccine for glioblastoma (GBM), Neoantigen Vaccines (mRNA / Peptide), TG4010 (Viral vector vaccine for non-small cell lung cancer (NSCLC)), ISA-101 (Peptide vaccine targeting HPV-positive cancers).

[0065] In various embodiments, the effective amount of radiotherapy is any one or more of about 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 51-55, 56-60, 61-65, 66-70, 71-75, 76-80, 81-85, 86-90, 91-95, or 96-100 Gy. These doses may be given in one or more fractions; for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 fractions. In various embodiments the dose is about 0.5-1.0, 1.0-1.5, 1.5-2.0, or 2.0-2.5 Gy per fraction.

[0066] In various embodiments, the cancer is selected from melanoma, renal cell carcinoma, or hepatocellular carcinoma.

[0067] In various embodiments, the cancer is selected from Acute Myeloid Leukemia (LAML), Adrenocortical Carcinoma (ACC), Bladder Urothelial Carcinoma (BLCA), Brain Lower Grade Glioma (LGG), Breast Invasive Carcinoma (BRCA), Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma (CESC), Cholangiocarcinoma (CHOL), Colon Adenocarcinoma (COAD), Esophageal Carcinoma (ESCA), Glioblastoma Multiforme (GBM), Head and Neck Squamous Cell Carcinoma (HNSC), Kidney Renal Clear Cell Carcinoma (KIRC), Kidney Renal Papillary Cell Carcinoma (KIRP), Liver Hepatocellular Carcinoma (LIHC), Lung Adenocarcinoma (LUAD), Lung Squamous Cell Carcinoma (LUSC), Lymphoid Neoplasm Diffuse Large B-cell Lymphoma (DLBC), Mesothelioma (MESO), Ovarian Serous Cystadenocarcinoma (OV), Pancreatic Adenocarcinoma (PAAD), Pheochromocytoma and Paraganglioma (PCPG), Prostate Adenocarcinoma (PRAD), Rectum Adenocarcinoma (READ), Sarcoma (SARC), Skin Cutaneous Melanoma (SKCM), Stomach 154935-2291-41872065472-001010WOPTAdenocarcinoma (STAD), Testicular Germ Cell Tumors (TGCT), Thyroid Carcinoma (THCA), Thymoma (THYM), Uterine Corpus Endometrial Carcinoma (UCEC), Uterine Carcinosarcoma (UCS), or Uveal Melanoma (UVM).

[0068] Various embodiments provide for a method for treating cancer in a subject, comprising: detecting an MHC II expression level or a molecular surrogate level for MHC II expression level in tumor tissue obtained from the subject; and administering a CD4 depleting therapy to the subject who has been determined to have an MHC II expression level that is lower than a reference level for the tumor type, or a molecular surrogate level for MHC II expression level that is lower than its reference level for the tumor type.

[0069] Various embodiments provide for a method for predicting treatment response in a subject in need thereof, comprising: detecting an MHC II expression level or a molecular surrogate level for MHC II expression level in tumor tissue obtained from the subject, wherein an MHC II expression level that is lower than a reference level for the tumor type, or a molecular surrogate level for MHC II expression level that is lower than its reference level for the tumor type indicates that the subject is likely to respond to or benefit from a CD4 depleting therapy. While not wishing to be bound by any particular theory, the method is based, at least in part, on the knowledge that an MHC II expression level that is lower than a reference level for the tumor type indicates an antitumor immunity that is CD8+ lymphocyte dependent and CD4 depletion decreases tumor growth, prior to administering the CD4 depleting therapy.

[0070] In various embodiments, markers that co-express with MHC II can be detected in lieu of or in additional to MHC II or molecular surrogate level for expression MHC II. Examples of these co-expressed markers include but are not limited to CD45, CDllb, CDllc, CD68, CD80, CD86, and Ibal (in microglia).

[0071] In various embodiments, the molecular surrogate for MHC II level is CIITA, HLA-DP, HLA-DQ, HLA-DR, RFX5, or RFXAP.

[0072] In various embodiments, the CD4 depleting therapy is selected from CC182, ipilimumab, IT 1208 (a defucosylated humanized anti-CD4 depleting antibody), daclizumab, zanolimumab, cM-T412, keliximab, ibalizumab, or combinations thereof. In various embodiments, the CD4 depleting therapy is selected from CC182.

[0073] In various embodiments, administering the CD4 depleting therapy to the subject comprises administering daily, weekly, bi-weekly, or monthly. In various embodiments, there can be a drug holiday, that is a break from the periodic administration.164935-2291-41872065472-001010WOPTThe drug holiday can be for 1 day, 3 days, 5 days, 7 days, 2 weeks, 3 weeks, or 1 month between each of the periodic administration. In various embodiments, the drug holiday can be for more than 1 month. In various embodiments, administering the CD4 depleting therapy to the subject comprises administering for a period of time. The period of time can be about 1 week, 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months. In other embodiments, the period of time can be about 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years or 5 years. In other embodiments, the period of time can be more than 5 years.

[0074] In various embodiments, the method further comprises administering chemotherapy, radiotherapy or immunotherapy to the subject.

[0075] Examples of chemotherapeutic agents include cytotoxic agents (e.g., 5-fluorouracil, cisplatin, carboplatin, methotrexate, daunorubicin, doxorubicin (Adriamycin®), vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine (CCNU), cytarabine USP, cyclophosphamide, estramucine phosphate sodium, altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan, cyclophosphamide, mitoxantrone, carboplatin, cisplatin, interferon alfa-2a recombinant, paclitaxel, teniposide, and streptozoci), cytotoxic akylating agents (e.g., busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylesulfonic acid), alkylating agents (e.g., asaley, AZQ, BCNU, busulfan, bisulphan, carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, cisplatinum, clomesone, cyanomorpholinodoxorubicin, cyclodisone, cyclophosphamide, dianhydrogalactitol, fluorodopan, hepsulfam, hycanthone, iphosphamide, melphalan, methyl CCNU, mitomycin C, mitozolamide, nitrogen mustard, PCNU, piperazine, piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard, streptozotocin, teroxirone, tetraplatin, thiotepa, tri ethylenemelamine, uracil nitrogen mustard, and Yoshi-864), antimitotic agents (e.g., allocolchicine, Halichondrin M, colchicine, colchicine derivatives, dolastatin 10, maytansine, rhizoxin, paclitaxel derivatives, paclitaxel, thiocolchicine, trityl cysteine, vinblastine sulfate, and vincristine sulfate), plant alkaloids (e.g., actinomycin D, bleomycin, L-asparaginase, idarubicin, vinblastine sulfate, vincristine sulfate, mitramycin, mitomycin, daunorubicin, VP-16-213, VM-26, navelbine and taxotere), biologicals (e.g., alpha interferon, BCG, G-CSF, GM-CSF, and interleukin-2), topoisomerase I inhibitors (e.g., camptothecin, camptothecin derivatives, and morpholinodoxorubicin), topoisomerase II inhibitors (e.g., mitoxantron, amonafide, m-AMSA, anthrapyrazole derivatives, pyrazoloacridine, bisantrene HCL, daunorubicin, deoxydoxorubicin, menogaril, N,N-dibenzyl daunomycin, oxanthrazole, rubidazone, VM-26 and VP- 16), and synthetics (e.g., hydroxyurea, procarbazine, o,p'-DDD,174935-2291-41872065472-001010WOPTdacarbazine, CCNU, BCNU, cis-diamminedichloroplatimun, mitoxantrone, CBDCA, levamisole, hexamethylmelamine, all-trans retinoic acid, gliadel and porfimer sodium).

[0076] Examples of immunotherapy include but are not limited to PD1 inhibitors or PDL1 inhibitors. In various embodiments, the PD1 inhibitor can be selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, AMP-224, AMP-514, spartalizumab, cemiplimab, penpulimab (AK105), prolgolimab (BCD-100), ezabenlimab (BI 754091), toripalimab (JS001), lipustobart (LZM009), retifanlimab (MGA012), Sym021, dostarlimab (TSR-042), tebotelimab (MGD013), cadonilimab (AK104), vudalimab (XmAb20717), tislelizumab, PF-06801591, anti-PDl antibody expressing pluripotent killer T lymphocytes (PIK-PD-1), autologous anti-EGFRvIII 4SCAR-IgT cells, and combinations thereof. In various embodiments, the PDL1 inhibitor can be selected from the group consisting of garivulimab (BGB-A333), cosibelimab (CK-301), FAZ053, envafolimab (KN035), MDX-1105, betifisolimab (MSB2311), adebrelimab (SHR-1316), atezolizumab, avelumab, durvalumab, BMS-936559, CK-301, M7824, and combinations thereof.

[0077] Additional examples of immunotherapy include but are not limited to anti-CTLA-4 antibodies. For example, ipilimumab, tremelimumab, zalifrelimab, quavonlimab, cadonilimab (a bispecific antibody targeting PD-1 and CTLA-4, erfonrilimab (a bispecific antibody targeting PD-L1 and CTLA-4), and ATOR-1015.

[0078] Additional examples of immunotherapy further include but are not limited to cancer vaccines. For example, Sipuleucel-T, Bacillus Calmette-Guerin (BCG), Talimogene laherparepvec , Nadofaragene firadonevec, GV AX (modified tumor cells, being studied for pancreatic and prostate cancers), DCVax-L (dendritic cell vaccine for glioblastoma (GBM), Neoantigen Vaccines (mRNA / Peptide), TG4010 (Viral vector vaccine for non-small cell lung cancer (NSCLC)), ISA-101 (Peptide vaccine targeting HPV-positive cancers).

[0079] In various embodiments, the effective amount of radiotherapy is any one or more of about 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 51-55, 56-60, 61-65, 66-70, 71-75, 76-80, 81-85, 86-90, 91-95, or 96-100 Gy. These doses may be given in one or more fractions; for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 fractions. In various embodiments the dose is about 0.5-1.0, 1.0-1.5, 1.5-2.0, or 2.0-2.5 Gy per fraction.

[0080] In various embodiments, the cancer is selected from melanoma, renal cell carcinoma, or hepatocellular carcinoma,

[0081] In various embodiments, the cancer is selected from Acute Myeloid Leukemia (LAML), Adrenocortical Carcinoma (ACC), Bladder Urothelial Carcinoma (BLCA), Brain 184935-2291-41872065472-001010WOPTLower Grade Glioma (LGG), Breast Invasive Carcinoma (BRCA), Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma (CESC), Cholangiocarcinoma (CHOL), Colon Adenocarcinoma (COAD), Esophageal Carcinoma (ESCA), Glioblastoma Multiforme (GBM), Head and Neck Squamous Cell Carcinoma (HNSC), Kidney Renal Clear Cell Carcinoma (KIRC), Kidney Renal Papillary Cell Carcinoma (KIRP), Liver Hepatocellular Carcinoma (LIHC), Lung Adenocarcinoma (LUAD), Lung Squamous Cell Carcinoma (LUSC), Lymphoid Neoplasm Diffuse Large B-cell Lymphoma (DLBC), Mesothelioma (MESO), Ovarian Serous Cystadenocarcinoma (OV), Pancreatic Adenocarcinoma (PAAD), Pheochromocytoma and Paraganglioma (PCPG), Prostate Adenocarcinoma (PRAD), Rectum Adenocarcinoma (READ), Sarcoma (SARC), Skin Cutaneous Melanoma (SKCM), Stomach Adenocarcinoma (STAD), Testicular Germ Cell Tumors (TGCT), Thyroid Carcinoma (THCA), Thymoma (THYM), Uterine Corpus Endometrial Carcinoma (UCEC), Uterine Carcinosarcoma (UCS), or Uveal Melanoma (UVM).

[0082] Various embodiments provide for a method for treating cancer in a subject, comprising: obtaining or requesting the results of an analysis of an MHC II expression level or a molecular surrogate level for MHC II expression level in tumor tissue obtained from the subject; and administering a CD4 depleting therapy to the subject who has been determined to have an MHC II expression level that is lower than a reference level for the tumor type, or a molecular surrogate level for MHC II expression level that is lower than its reference level for the tumor type.

[0083] In various embodiments, markers that co-express with MHC II can be detected in lieu of or in additional to MHC II or molecular surrogate level for expression MHC II. Examples of these co-expressed markers include but are not limited to CD45, CDllb, CDllc, CD68, CD80, CD86, and Ibal (in microglia).

[0084] In various embodiments, the molecular surrogate for MHC II level is CIITA, HLA-DP, HLA-DQ, HLA-DR, RFX5, or RFXAP.

[0085] In various embodiments, the CD4 depleting therapy is selected from CC182, ipilimumab, IT 1208 (a defucosylated humanized anti-CD4 depleting antibody), daclizumab, zanolimumab, cM-T412, keliximab, ibalizumab, or combinations thereof. In various embodiments, the CD4 depleting therapy is selected from CC182.

[0086] In various embodiments, administering the CD4 depleting therapy to the subject comprises administering daily, weekly, bi-weekly, or monthly. In various embodiments, there can be a drug holiday, that is a break from the periodic administration.194935-2291-41872065472-001010WOPTThe drug holiday can be for 1 day, 3 days, 5 days, 7 days, 2 weeks, 3 weeks, or 1 month between each of the periodic administration. In various embodiments, the drug holiday can be for more than 1 month. In various embodiments, administering the CD4 depleting therapy to the subject comprises administering for a period of time. The period of time can be about 1 week, 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months. In other embodiments, the period of time can be about 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years or 5 years. In other embodiments, the period of time can be more than 5 years.

[0087] In various embodiments, the method further comprises administering chemotherapy, radiotherapy or immunotherapy to the subject.

[0088] Examples of chemotherapeutic agents include cytotoxic agents (e.g., 5-fluorouracil, cisplatin, carboplatin, methotrexate, daunorubicin, doxorubicin (Adriamycin®), vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine (CCNU), cytarabine USP, cyclophosphamide, estramucine phosphate sodium, altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan, cyclophosphamide, mitoxantrone, carboplatin, cisplatin, interferon alfa-2a recombinant, paclitaxel, teniposide, and streptozoci), cytotoxic akylating agents (e.g., busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylesulfonic acid), alkylating agents (e.g., asaley, AZQ, BCNU, busulfan, bisulphan, carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, cisplatinum, clomesone, cyanomorpholinodoxorubicin, cyclodisone, cyclophosphamide, dianhydrogalactitol, fluorodopan, hepsulfam, hycanthone, iphosphamide, melphalan, methyl CCNU, mitomycin C, mitozolamide, nitrogen mustard, PCNU, piperazine, piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard, streptozotocin, teroxirone, tetraplatin, thiotepa, tri ethylenemelamine, uracil nitrogen mustard, and Yoshi-864), antimitotic agents (e.g., allocolchicine, Halichondrin M, colchicine, colchicine derivatives, dolastatin 10, maytansine, rhizoxin, paclitaxel derivatives, paclitaxel, thiocolchicine, trityl cysteine, vinblastine sulfate, and vincristine sulfate), plant alkaloids (e.g., actinomycin D, bleomycin, L-asparaginase, idarubicin, vinblastine sulfate, vincristine sulfate, mitramycin, mitomycin, daunorubicin, VP-16-213, VM-26, navelbine and taxotere), biologicals (e.g., alpha interferon, BCG, G-CSF, GM-CSF, and interleukin-2), topoisomerase I inhibitors (e.g., camptothecin, camptothecin derivatives, and morpholinodoxorubicin), topoisomerase II inhibitors (e.g., mitoxantron, amonafide, m-AMSA, anthrapyrazole derivatives, pyrazoloacridine, bisantrene HCL, daunorubicin, deoxydoxorubicin, menogaril, N,N-dibenzyl daunomycin, oxanthrazole, rubidazone, VM-26 and VP- 16), and synthetics (e.g., hydroxyurea, procarbazine, o,p'-DDD,204935-2291-41872065472-001010WOPTdacarbazine, CCNU, BCNU, cis-diamminedichloroplatimun, mitoxantrone, CBDCA, levamisole, hexamethylmelamine, all-trans retinoic acid, gliadel and porfimer sodium).

[0089] Examples of immunotherapy include but are not limited to PD1 inhibitors or PDL1 inhibitors. In various embodiments, the PD1 inhibitor can be selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, AMP-224, AMP-514, spartalizumab, cemiplimab, penpulimab (AK105), prolgolimab (BCD-100), ezabenlimab (BI 754091), toripalimab (JS001), lipustobart (LZM009), retifanlimab (MGA012), Sym021, dostarlimab (TSR-042), tebotelimab (MGD013), cadonilimab (AK104), vudalimab (XmAb20717), tislelizumab, PF-06801591, anti-PDl antibody expressing pluripotent killer T lymphocytes (PIK-PD-1), autologous anti-EGFRvIII 4SCAR-IgT cells, and combinations thereof. In various embodiments, the PDL1 inhibitor can be selected from the group consisting of garivulimab (BGB-A333), cosibelimab (CK-301), FAZ053, envafolimab (KN035), MDX-1105, betifisolimab (MSB2311), adebrelimab (SHR-1316), atezolizumab, avelumab, durvalumab, BMS-936559, CK-301, M7824, and combinations thereof.

[0090] Additional examples of immunotherapy include but are not limited to anti-CTLA-4 antibodies. For example, ipilimumab, tremelimumab, zalifrelimab, quavonlimab, cadonilimab (a bispecific antibody targeting PD-1 and CTLA-4, erfonrilimab (a bispecific antibody targeting PD-L1 and CTLA-4), and ATOR-1015.

[0091] Additional examples of immunotherapy further include but are not limited to cancer vaccines. For example, Sipuleucel-T, Bacillus Calmette-Guerin (BCG), Talimogene laherparepvec , Nadofaragene firadonevec, GV AX (modified tumor cells, being studied for pancreatic and prostate cancers), DCVax-L (dendritic cell vaccine for glioblastoma (GBM), Neoantigen Vaccines (mRNA / Peptide), TG4010 (Viral vector vaccine for non-small cell lung cancer (NSCLC)), ISA-101 (Peptide vaccine targeting HPV-positive cancers).

[0092] In various embodiments, the effective amount of radiotherapy is any one or more of about 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 51-55, 56-60, 61-65, 66-70, 71-75, 76-80, 81-85, 86-90, 91-95, or 96-100 Gy. These doses may be given in one or more fractions; for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 fractions. In various embodiments the dose is about 0.5-1.0, 1.0-1.5, 1.5-2.0, or 2.0-2.5 Gy per fraction.

[0093] In various embodiments, the cancer is selected from melanoma, renal cell carcinoma, or hepatocellular carcinoma,

[0094] In various embodiments, the cancer is selected from Acute Myeloid Leukemia (LAML), Adrenocortical Carcinoma (ACC), Bladder Urothelial Carcinoma (BLCA), Brain 214935-2291-41872065472-001010WOPTLower Grade Glioma (LGG), Breast Invasive Carcinoma (BRCA), Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma (CESC), Cholangiocarcinoma (CHOL), Colon Adenocarcinoma (COAD), Esophageal Carcinoma (ESCA), Glioblastoma Multiforme (GBM), Head and Neck Squamous Cell Carcinoma (HNSC), Kidney Renal Clear Cell Carcinoma (KIRC), Kidney Renal Papillary Cell Carcinoma (KIRP), Liver Hepatocellular Carcinoma (LIHC), Lung Adenocarcinoma (LUAD), Lung Squamous Cell Carcinoma (LUSC), Lymphoid Neoplasm Diffuse Large B-cell Lymphoma (DLBC), Mesothelioma (MESO), Ovarian Serous Cystadenocarcinoma (OV), Pancreatic Adenocarcinoma (PAAD), Pheochromocytoma and Paraganglioma (PCPG), Prostate Adenocarcinoma (PRAD), Rectum Adenocarcinoma (READ), Sarcoma (SARC), Skin Cutaneous Melanoma (SKCM), Stomach Adenocarcinoma (STAD), Testicular Germ Cell Tumors (TGCT), Thyroid Carcinoma (THCA), Thymoma (THYM), Uterine Corpus Endometrial Carcinoma (UCEC), Uterine Carcinosarcoma (UCS), or Uveal Melanoma (UVM).

[0095] Various embodiments provide for a method of selecting a cancer treatment for a subject, comprising detecting an MHC II expression level or a molecular surrogate level for MHC II expression level in tumor tissue obtained from the subject; and selecting a CD4 depleting therapy for a subject who has been determined to have MHC II expression level that is lower than reference level for the tumor type, or a level of a molecular surrogate for MHC II level that is lower than its reference level for the tumor type, or avoiding CD4 depleting therapy for a subject who has been determined to have MHC II expression level that is higher than reference level for the tumor type, or a level of a molecular surrogate for MHC II level that is higher than its reference level for the tumor type.

[0096] Various embodiments provide for a method of monitoring treatment response in a subject in need thereof, comprising: detecting an MHC II expression level or a molecular surrogate level for MHC II expression level in tumor tissue obtained from the subject, wherein the subject is undergoing treatment for cancer; and continuing to administer a CD4 depleting therapy for a subject who has been determined to have MHC II expression level that is lower than a reference level for the tumor type, or a level of a molecular surrogate for MHC II level that is lower than its reference level for the tumor type, or discontinuing to administer the CD4 depleting therapy for a subject who has been determined to have MHC II expression level that is higher than reference level for the tumor type, or a level of a molecular surrogate for MHC II level that is higher than its reference level for the tumor type.224935-2291-41872065472-001010WOPT

[0097] In various embodiments, markers that co-express with MHC II can be detected in lieu of or in additional to MHC II or molecular surrogate level for expression MHC II. Examples of these co-expressed markers include but are not limited to CD45, CDllb, CDllc, CD68, CD80, CD86, and Ibal (in microglia).

[0098] In various embodiments, the molecular surrogate for MHC II level is CIITA, HLA-DP, HLA-DQ, HLA-DR, RFX5, or RFXAP.

[0099] In various embodiments, the method further comprises administering the selected CD4 depleting therapy.

[0100] In various embodiments, the CD4 depleting therapy is selected from CC182, ipilimumab, IT 1208 (a defucosylated humanized anti-CD4 depleting antibody), daclizumab, zanolimumab, cM-T412, keliximab, ibalizumab, or combinations thereof. In various embodiments, the CD4 depleting therapy is selected from CC182.

[0101] In various embodiments, administering the CD4 depleting therapy to the subject comprises administering daily, weekly, bi-weekly, or monthly. In various embodiments, there can be a drug holiday, that is a break from the periodic administration. The drug holiday can be for 1 day, 3 days, 5 days, 7 days, 2 weeks, 3 weeks, or 1 month between each of the periodic administration. In various embodiments, the drug holiday can be for more than 1 month. In various embodiments, administering the CD4 depleting therapy to the subject comprises administering for a period of time. The period of time can be about 1 week, 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months. In other embodiments, the period of time can be about 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years or 5 years. In other embodiments, the period of time can be more than 5 years.

[0102] In various embodiments, the method further comprises administering chemotherapy, radiotherapy or immunotherapy to the subject.

[0103] Examples of chemotherapeutic agents include cytotoxic agents (e.g., 5-fluorouracil, cisplatin, carboplatin, methotrexate, daunorubicin, doxorubicin (Adriamycin®), vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine (CCNU), cytarabine USP, cyclophosphamide, estramucine phosphate sodium, altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan, cyclophosphamide, mitoxantrone, carboplatin, cisplatin, interferon alfa-2a recombinant, paclitaxel, teniposide, and streptozoci), cytotoxic akylating agents (e.g., busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylesulfonic acid), alkylating agents (e.g., asaley, AZQ, BCNU, busulfan, bisulphan, carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, cis- 234935-2291-41872065472-001010WQPTplatinum, clomesone, cyanomorpholinodoxorubicin, cyclodisone, cyclophosphamide, dianhydrogalactitol, fluorodopan, hepsulfam, hycanthone, iphosphamide, melphalan, methyl CCNU, mitomycin C, mitozolamide, nitrogen mustard, PCNU, piperazine, piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard, streptozotocin, teroxirone, tetraplatin, thiotepa, tri ethylenemelamine, uracil nitrogen mustard, and Yoshi-864), antimitotic agents (e.g., allocolchicine, Halichondrin M, colchicine, colchicine derivatives, dolastatin 10, maytansine, rhizoxin, paclitaxel derivatives, paclitaxel, thiocolchicine, trityl cysteine, vinblastine sulfate, and vincristine sulfate), plant alkaloids (e.g., actinomycin D, bleomycin, L-asparaginase, idarubicin, vinblastine sulfate, vincristine sulfate, mitramycin, mitomycin, daunorubicin, VP-16-213, VM-26, navelbine and taxotere), biologicals (e.g., alpha interferon, BCG, G-CSF, GM-CSF, and interleukin-2), topoisomerase I inhibitors (e.g., camptothecin, camptothecin derivatives, and morpholinodoxorubicin), topoisomerase II inhibitors (e.g., mitoxantron, amonafide, m-AMSA, anthrapyrazole derivatives, pyrazoloacridine, bisantrene HCL, daunorubicin, deoxydoxorubicin, menogaril, N,N-dibenzyl daunomycin, oxanthrazole, rubidazone, VM-26 and VP- 16), and synthetics (e.g., hydroxyurea, procarbazine, o,p'-DDD, dacarbazine, CCNU, BCNU, cis-diamminedichloroplatimun, mitoxantrone, CBDCA, levamisole, hexamethylmelamine, all-trans retinoic acid, gliadel and porfimer sodium).

[0104] Examples of immunotherapy include but are not limited to PD1 inhibitors or PDL1 inhibitors. In various embodiments, the PD1 inhibitor can be selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, AMP-224, AMP-514, spartalizumab, cemiplimab, penpulimab (AK105), prolgolimab (BCD-100), ezabenlimab (BI 754091), toripalimab (JS001), lipustobart (LZM009), retifanlimab (MGA012), Sym021, dostarlimab (TSR-042), tebotelimab (MGD013), cadonilimab (AK104), vudalimab (XmAb20717), tislelizumab, PF-06801591, anti-PDl antibody expressing pluripotent killer T lymphocytes (PIK-PD-1), autologous anti-EGFRvIII 4SCAR-IgT cells, and combinations thereof. In various embodiments, the PDL1 inhibitor can be selected from the group consisting of garivulimab (BGB-A333), cosibelimab (CK-301), FAZ053, envafolimab (KN035), MDX-1105, betifisolimab (MSB2311), adebrelimab (SHR-1316), atezolizumab, avelumab, durvalumab, BMS-936559, CK-301, M7824, and combinations thereof.

[0105] Additional examples of immunotherapy include but are not limited to anti-CTLA-4 antibodies. For example, ipilimumab, tremelimumab, zalifrelimab, quavonlimab, cadonilimab (a bispecific antibody targeting PD-1 and CTLA-4, erfonrilimab (a bispecific antibody targeting PD-L1 and CTLA-4), and ATOR-1015.244935-2291-41872065472-001010WOPT

[0106] Additional examples of immunotherapy further include but are not limited to cancer vaccines. For example, Sipuleucel-T, Bacillus Calmette-Guerin (BCG), Talimogene laherparepvec , Nadofaragene firadonevec, GV AX (modified tumor cells, being studied for pancreatic and prostate cancers), DCVax-L (dendritic cell vaccine for glioblastoma (GBM), Neoantigen Vaccines (mRNA / Peptide), TG4010 (Viral vector vaccine for non-small cell lung cancer (NSCLC)), ISA-101 (Peptide vaccine targeting HPV-positive cancers).

[0107] In various embodiments, the effective amount of radiotherapy is any one or more of about 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 51-55, 56-60, 61-65, 66-70, 71-75, 76-80, 81-85, 86-90, 91-95, or 96-100 Gy. These doses may be given in one or more fractions; for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 fractions. In various embodiments the dose is about 0.5-1.0, 1.0-1.5, 1.5-2.0, or 2.0-2.5 Gy per fraction.

[0108] In various embodiments, the cancer is selected from melanoma, renal cell carcinoma, or hepatocellular carcinoma,

[0109] In various embodiments, the cancer is selected from Acute Myeloid Leukemia (LAML), Adrenocortical Carcinoma (ACC), Bladder Urothelial Carcinoma (BLCA), Brain Lower Grade Glioma (LGG), Breast Invasive Carcinoma (BRCA), Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma (CESC), Cholangiocarcinoma (CHOL), Colon Adenocarcinoma (COAD), Esophageal Carcinoma (ESCA), Glioblastoma Multiforme (GBM), Head and Neck Squamous Cell Carcinoma (HNSC), Kidney Renal Clear Cell Carcinoma (KIRC), Kidney Renal Papillary Cell Carcinoma (KIRP), Liver Hepatocellular Carcinoma (LIHC), Lung Adenocarcinoma (LUAD), Lung Squamous Cell Carcinoma (LUSC), Lymphoid Neoplasm Diffuse Large B-cell Lymphoma (DLBC), Mesothelioma (MESO), Ovarian Serous Cystadenocarcinoma (OV), Pancreatic Adenocarcinoma (PAAD), Pheochromocytoma and Paraganglioma (PCPG), Prostate Adenocarcinoma (PRAD), Rectum Adenocarcinoma (READ), Sarcoma (SARC), Skin Cutaneous Melanoma (SKCM), Stomach Adenocarcinoma (STAD), Testicular Germ Cell Tumors (TGCT), Thyroid Carcinoma (THCA), Thymoma (THYM), Uterine Corpus Endometrial Carcinoma (UCEC), Uterine Carcinosarcoma (UCS), or Uveal Melanoma (UVM).Reference Levels

[0110] The reference level for MHC II, as well as each of the surrogates (e.g., CIITA, HLA-DP, HLA-DQ, HLA-DR, RFX5, or RFXAP) is determined for each type of tumor. Different types of tumors may have different reference levels.254935-2291-41872065472-001010WQPT

[0111] In some embodiments, the reference level is established from a trial and a cutoff (e.g., reference level) is based on the clinical outcome of interest. One of ordinary skill in the art is able to readily conduct a trial and determine the cutoff (e.g., reference level). For example, during a clinical trial testing a CD4 depleting antibody, tumor tissue is collected. A cutoff for tumor MHC II expression is established that separates patients into those that respond to treatment with tumor shrinkage and those that do not. In some embodiments, this cutoff would have no prognostic value in patients who were not treated with CD4 depleting therapy.

[0112] In some embodiments, a mean MHC II expression level is the reference level. In various embodiments, the mean expression level of CIITA, HLA-DP, HLA-DQ, HLA-DR, RFX5, or RFXAP, are each their reference level for each surrogate.

[0113] In some embodiments, a median MHC II expression level is the reference level. In various embodiments, the median expression level of CIITA, HLA-DP, HLA-DQ, HLA-DR, RFX5, or RFXAP, are each their reference level for each surrogate.

[0114] The mean and median expression level for MHC II, or for CIITA, HLA-DP, HLA-DQ, HLA-DR, RFX5, or RFXAP for each type of tumor can be determined from a population of patients having the tumor type of interest. The population of patients can be; for example, 5-10 patients, 11-25 patients, 26-50 patients, 51-75 patients, 76-100 patients, 101-200 patients, or more than 200 patients.

[0115] In some embodiments, the reference level can be established from biological samples from a healthy subject (e.g., a subject who does not have cancer). The reference value can be obtained from the non-tumorous cells or non-tumorous tissue. In other embodiments, the reference level is the average level for the same type of biological sample from a population of healthy subjects (e.g., MHC II reference level for lung carcinoma is based on healthy lung cells or tissues, MHC II reference level for melanoma is based on healthy skin cells or tissues). In other embodiments, the reference level is the average plus one standard deviation of average level for the same type of biological sample from a population of healthy subjects. In other embodiments, the reference level is the average plus two standard deviations of average level for the same type of biological sample from a population of healthy subjects. In some embodiments, the population of healthy subjects can range from at least three healthy individuals to 25 healthy individuals, more than 50 healthy individuals, or even more than 100 healthy individuals.264935-2291-41872065472-001010WQPTDetection Methods and Assays

[0116] In various embodiments, detecting MHC II expression levels or detecting a molecular surrogate level for MHC II expression level can comprise using enzyme-linked immunosorbent assay (ELISA). In various embodiments, detecting can comprise using immunohistochemistry, flow cytometry, fluorescence in situ hybridization (FISH), radioimmuno assays, affinity purification, mass spectrometry, western blotting, RNAseq, or PCR.Effective amounts of CD4 depleting therapy

[0117] In various embodiments of the invention, the therapeutically effective amounts of one or more CD4 depleting therapy for use with the methods described herein may be in the range of 1-5 units / kg, 5-10 units / kg, 10-50 units / kg, 50-100 units / kg, 100-150 units / kg, 150-200 units / kg, 100-200 units / kg, 200-300 units / kg, 300-400 units / kg, or 400-500 units / kg. In some embodiments, the therapeutically effective amount of CD4 depleting therapy is about 25-50 units / kg, about 50-75 units / kg, about 75-100 units / kg or about 50 units / kg. In various embodiments, the therapeutically effective amount is about 50 units / kg. In various embodiments, the therapeutically effective amount is about 25-100 units / kg.

[0118] In some embodiments of the invention, the therapeutically effective amounts of the CD4 depleting therapy can be in the range of about 1-5 pg / day, 5-1 Opg / day, 10- 15pg / day, 15-20pg / day, 10-20pg / day, 20-30pg / day, 30-40pg / day, 40-50pg / day, 50-60pg / day, 60-70pg / day, 70-80pg / day, 80-90pg / day, 90-100pg / day, 100-1 lOpg / day, 110-120 pg / day, 120- 13 Opg / day, 130-140 pg / day, 140-150pg / day, 150-160 pg / day, 160- 170pg / day, 170-180pg / day, 180-190pg / day, 190-200pg / day, 200-210 pg / day, 210- 220pg / day, 220-23 Opg / day, 230-240 pg / day, 240-250pg / day, 250-260pg / day, 260- 270pg / day, 270-280pg / day, 280-290pg / day or 290-300pg / day.

[0119] In some embodiments of the invention, the therapeutically effective amounts of the CD4 depleting therapy can be in the range of about 10-50pg / day, 50-100pg / day, 100-150pg / day, 150-200 pg / day, 100-200pg / day, 200-300pg / day, 300-400pg / day, 400- 500pg / day, 500-600pg / day, 600-700pg / day, 700-800pg / day, 800-900pg / day, 900-lOOOpg / day, 1000-1100 pg / day, 1100- 1200 pg / day, 1200- 1300 pg / day, 1300- 1400 pg / day, 1400-1500pg / day, 1500- 1600 pg / day, 1600- 1700 pg / day, 1700-1800pg / day, 1800-1900pg / day, 1900-2000pg / day, 2000-2100 pg / day, 2100-2200 pg / day, 2200-2300 pg / day, 2300-2400pg / day, 2400-2500pg / day, 2500-2600pg / day, 2600-2700pg / day, 2700-2800pg / day, 2800-2900pg / day or 2900-3000pg / day.274935-2291-41872065472-001010WOPT

[0120] In some embodiments of the invention, the therapeutically effective amounts of one or more CD4 depleting therapy can be in the range of about 10-50mg / day, 50-lOOmg / day, 100-150mg / day, 150-200mg / day, 100-200mg / day, 200-3 OOmg / day, 300- 400mg / day, 400-500mg / day, 500-600mg / day, 600-700mg / day, 700-800mg / day, 800- 900mg / day, 900-1000mg / day, 1000-11 OOmg / day, 1100-1200mg / day, 1200- 13 OOmg / day, 1300-1400mg / day, 1400- 15 OOmg / day, 1500-1600mg / day, 1600-1700mg / day, 1700-1800mg / day, 1800-1900mg / day, 1900-2000mg / day, 2000-21 OOmg / day, 2100-2200mg / day, 2200-23 OOmg / day, 2300-2400mg / day, 2400-2500mg / day, 2500-2600mg / day, 2600-2700mg / day, 2700-2800mg / day, 2800-2900mg / day or 2900-3000mg / day.

[0121] In various embodiments, the effective amount of CD4 depleting therapy is any one or more of about 0.001-0.01, 0.01-0.1, 0.1-0.5, 0.5-5, 5-10, 10-20, 20-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 pg / kg / day, or a combination thereof. In various embodiments, the effective amount of CD4 depleting therapy is any one or more of about 0.001-0.01, 0.01-0.1, 0.1-0.5, 0.5-5, 5-10, 10-20, 20-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 pg / m2 / day, or a combination thereof. Here, “pg / kg / day” or “mg / kg / day” refers to pg or mg agent per kg body weight of the subject per day, and “pg / m2 / day” or “mg / m2 / day” refers to pg or mg agent per m2body surface area of the subject per day.Effective amounts of chemotherapy or immunotherapy

[0122] In various embodiments of the invention, the therapeutically effective amounts of one or more chemotherapy or immunotherapy for use with the methods described herein may be in the range of 1-5 units / kg, 5-10 units / kg, 10-50 units / kg, 50-100 units / kg, 100-150 units / kg, 150-200 units / kg, 100-200 units / kg, 200-300 units / kg, 300-400 units / kg, or 400-500 units / kg. In some embodiments, the therapeutically effective amount of chemotherapy or immunotherapy is about 25-50 units / kg, about 50-75 units / kg, about 75-100 units / kg or about 50 units / kg. In various embodiments, the therapeutically effective amount is about 50 units / kg. In various embodiments, the therapeutically effective amount is about 25-100 units / kg.

[0123] In some embodiments of the invention, the therapeutically effective amounts of the chemotherapy or immunotherapy can be in the range of about l-5pg / day, 5-10pg / day, 10-15pg / day, 15-20pg / day, 10-20pg / day, 20-30pg / day, 30-40pg / day, 40-50pg / day, 50-60pg / day, 60-70pg / day, 70-80pg / day, 80-90pg / day, 90-100pg / day, 100-1 lOpg / day, 110-120pg / day, 120-130pg / day, 130-140pg / day, 140-150pg / day, 150-160pg / day, 160- 284935-2291-41872065472-001010WQPT170pg / day, 170-180pg / day, 180-190pg / day, 190-200 gg / day, 200-210 pg / day, 210- 220pg / day, 220-230 pg / day, 230-240 pg / day, 240-250pg / day, 250-260pg / day, 260- 270pg / day, 270-280pg / day, 280-290pg / day or 290-300pg / day.

[0124] In some embodiments of the invention, the therapeutically effective amounts of the chemotherapy or immunotherapy can be in the range of about 10-50pg / day, 50-lOOpg / day, 100-150pg / day, 150-200 pg / day, 100-200pg / day, 200-300pg / day, 300- 400pg / day, 400-500pg / day, 500-600pg / day, 600-700pg / day, 700-800pg / day, 800- 900pg / day, 900-1000pg / day, 1000-1100 pg / day, 1100-1200pg / day, 1200- 1300 pg / day, 1300-1400pg / day, 1400-1500pg / day, 1500- 1600 pg / day, 1600-1700pg / day, 1700-1800pg / day, 1800-1900pg / day, 1900-2000pg / day, 2000-2100 pg / day, 2100-2200 pg / day, 2200-2300pg / day, 2300-2400pg / day, 2400-2500pg / day, 2500-2600pg / day, 2600-2700pg / day, 2700-2800pg / day, 2800-2900pg / day or 2900-3000pg / day.

[0125] In some embodiments of the invention, the therapeutically effective amounts of one or more chemotherapy or immunotherapy can be in the range of about 10-50mg / day, 50-100mg / day, 100-150mg / day, 150-200mg / day, 100-200mg / day, 200-3 OOmg / day, 300-400mg / day, 400-500mg / day, 500-600mg / day, 600-700mg / day, 700-800mg / day, 800-900mg / day, 900-1000mg / day, 1000-11 OOmg / day, 1100-1200mg / day, 1200- 13 OOmg / day, 1300-1400mg / day, 1400- 15 OOmg / day, 1500-1600mg / day, 1600-1700mg / day, 1700-1800mg / day, 1800-1900mg / day, 1900-2000mg / day, 2000-21 OOmg / day, 2100-2200mg / day, 2200-23 OOmg / day, 2300-2400mg / day, 2400-2500mg / day, 2500-2600mg / day, 2600-2700mg / day, 2700-2800mg / day, 2800-2900mg / day or 2900-3000mg / day.

[0126] In various embodiments, the effective amount of chemotherapy or immunotherapy is any one or more of about 0.001-0.01, 0.01-0.1, 0.1-0.5, 0.5-5, 5-10, 10-20, 20-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 pg / kg / day, or a combination thereof. In various embodiments, the effective amount of chemotherapy or immunotherapy is any one or more of about 0.001-0.01, 0.01-0.1, 0.1-0.5, 0.5-5, 5-10, 10-20, 20-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 pg / m2 / day, or a combination thereof. Here, “pg / kg / day” or “mg / kg / day” refers to pg or mg agent per kg body weight of the subject per day, and “pg / m2 / day” or “mg / m2 / day” refers to pg or mg agent per m2body surface area of the subject per day.294935-2291-41872065472-001010WOPTPharmaceutical Compositions

[0127] In various embodiments, the present invention provides pharmaceutical compositions including a pharmaceutically acceptable excipient along with a therapeutically effective amount of CD4 depleting therapy. “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.

[0128] In certain embodiments, the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term “pharmaceutically acceptable salts, esters, amides, and prodrugs” as used herein refers to those carboxylate salts, amino acid addition salts, esters, amides, and prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use of the compounds of the invention. The term “salts” refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. These may include cations based on the alkali and alkaline earth metals such as sodium, lithium, potassium, calcium, magnesium and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylanunonium, tetraethyl ammonium, methyl amine, dimethyl amine, trimethylamine, triethylamine, ethylamine, and the like (see, e.g., Berge S. M., et al. (1977) J. Pharm. Sci. 66, 1, which is incorporated herein by reference).

[0129] The term “pharmaceutically acceptable esters” refers to the relatively nontoxic, esterified products of the compounds of the present invention. These esters can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form or hydroxyl with a suitable esterifying agent. Carboxylic acids can be converted into esters via treatment with an alcohol in the presence of a catalyst. The term is further intended to include lower hydrocarbon groups capable of being solvated under physiological conditions, e.g., alkyl esters, methyl, ethyl and propyl esters.304935-2291-41872065472-001010WOPT

[0130] As used herein, “pharmaceutically acceptable salts or prodrugs” are salts or prodrugs that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subject without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use.Routes of Administrations

[0131] In various embodiments, the pharmaceutical compositions according to the invention may be formulated for delivery via any route of administration. “Route of administration” may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal or parenteral.

[0132] Transdermal” administration may be accomplished using a topical cream or ointment or by means of a transdermal patch.

[0133] “Parenteral” refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders.

[0134] Via the enteral route, the pharmaceutical compositions can be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlled release. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection.

[0135] Via the topical route, the pharmaceutical compositions based on compounds according to the invention may be formulated for treating the skin and mucous membranes and are in the form of ointments, creams, milks, salves, powders, impregnated pads, solutions, gels, sprays, lotions or suspensions. They can also be in the form of microspheres or nanospheres or lipid vesicles or polymer vesicles or polymer patches and hydrogels allowing controlled release. These topical-route compositions can be either in anhydrous form or in aqueous form depending on the clinical indication.

[0136] Via the ocular route, they may be in the form of eye drops.

[0137] The pharmaceutical compositions according to the invention can also contain any pharmaceutically acceptable carrier. “Pharmaceutically acceptable carrier” as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is 314935-2291-41872065472-001010WOPTinvolved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.

[0138] The pharmaceutical compositions according to the invention can also be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.

[0139] The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.

[0140] The pharmaceutical compositions according to the invention may be delivered in a therapeutically effective amount. The precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for instance, by monitoring a subject’s response to administration of a compound and adjusting the dosage 324935-2291-41872065472-001010WOPTaccordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

[0141] Typical dosages of an effective CD4 depleting therapy can be in the ranges recommended by the manufacturer where known therapeutic compounds are used, and also as indicated to the skilled artisan by the in vitro responses or responses in animal models. Such dosages typically can be reduced by up to about one order of magnitude in concentration or amount without losing the relevant biological activity. Thus, the actual dosage will depend upon the judgment of the physician, the condition of the patient, and the effectiveness of the therapeutic method based, for example, on the in vitro responsiveness of the relevant primary cultured cells or histocultured tissue sample, such as biopsied malignant tumors, or the responses observed in the appropriate animal models, as previously described.Kits

[0142] The present invention is also directed to a kit to treat cancer and a kit to detect MHC II expression level or MHC II surrogate expression level. The kits are useful for practicing the inventive method of treating cancer, or to detect MHC II expression level or MHC II surrogate expression level. The kit is an assemblage of materials or components, including at least one of the inventive compositions. Thus, in some embodiments the kit contains a composition including CD4 depleting therapy, as described above.

[0143] The exact nature of the components configured in the inventive kit depends on its intended purpose. For example, some embodiments are configured for the purpose of treating cancer; some embodiments are configured for the purpose of detecting MHC II expression level or MHC II surrogate expression level. In one embodiment, the kit is configured particularly for use in the mammalian subjects. In another embodiment, the kit is configured particularly for use in human subjects. In further embodiments, the kit is configured for veterinary applications, treating subjects such as, but not limited to, farm animals, domestic animals, and laboratory animals.

[0144] Instructions for use may be included in the kit. “Instructions for use” typically include a tangible expression describing the technique to be employed in using the components of the kit to effect a desired outcome. Optionally, the kit also contains other useful components, such as, diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandaging materials or other useful paraphernalia as will be readily recognized by those of skill in the art.334935-2291-41872065472-001010WOPT

[0145] The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase “packaging material” refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like. The packaging material is constructed by well known methods, preferably to provide a sterile, contaminant-free environment. As used herein, the term “package” refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components. The packaging material generally has an external label which indicates the contents and / or purpose of the kit and / or its components.EXAMPLES

[0146] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.Example 1MethodsTumor cell and Mice

[0147] Renca, B16F10, Hepal-6 cancer cell lines were purchased from ATCC (Manassas, Virginia). The cells were maintained in RPMI 1640 or DMEM medium supplemented with 10% heat-inactivated FBS (Gemcell, West Sacramento, CA), 2 mmol / L of L-glutamine, 100 units / mL of penicillin, 100 pg / mL of streptomycin, and 250 ng / mL of Amphotericin B (ThermoFisher, Waltham, MA). These cells were periodically authenticated by morphologic and histologic inspection and animal grafting to assess their ability to grow and metastasize. The cells were annually tested for mycoplasma using Myco Alert Kit (Lonza, Allendale NJ).

[0148] Balb / C or C57BL / 6 mice, 5-8 weeks old, were purchased from Jackson laboratory (Ellsworth, Maine) and housed under pathogen free conditions. All experiments344935-2291-41872065472-001010WOPTinvolving animals followed federal and state standards in the federal Animal Welfare Act and the NIH guide for the care and use of laboratory animals. The IACUC approved this study (IACUC010003) for all animal experiments. The Renca tumors were generated by subcutaneously injecting 2xl05tumor cells into flanks of Balb / C mice. Hepal-6 tumors were generated by subcutaneously injecting 2xl06tumor cells and B16 tumor were generated by subcutaneously injecting 2xl05tumor cells into flanks of B6 mice. Mouse CD8 cells were depleted with 200 pg aCD8 administered i.p. after injection of tumor cells, CD4 depletion was performed by i.p. injection of 200 pg aCD4 after tumor cells were injected. No mouse anesthesia was required, and euthanasia was performed by placing mice in a CO2 chamber (fill rate for 30-70% displacement of the chamber volume per minute with CO2) followed by cervical dislocation.

[0149] For all animal studies, 5 or more mice were used per group. Mice were genetically and phenotypically identical, they were randomly assigned to experimental groups, and no mice were excluded from analysis. After randomization, to minimize risk of treatment and measurement mistakes, investigators were not blinded to treatment allocation. Tumor volume was calculated using the following formula: V=W2xL / 2, and no tumor measurements were excluded for data analysis. ARRIVE reporting guidelines were followed.8Antibodies and reagents

[0150] The following monoclonal antibodies (mAb) with a fluorescent conjugate for flow cytometry were obtained from Biolegend (San Diego, CA): aMCH I H-2kb (AF6-88.5), H-2Kd (SF1-1.1), aMHCII I-A / I-E (M5 / 114.15.2) aCD4 (GK 1.5 and RM4-5), aCD8 (53-6.7), aCD45 (30-F11), alFN-y (XMG1.2), aTNFa (TN3-19.12), aCD107 (ID4B). aCD8 (2.43) and aCD4 (GK 1.5) antibodies for T cell depletion were purchased from BioXcell (West Lebanon, NH). aMHCII (PA5-116876) and goat anti-rabbit (A16101) antibody for immunofluorescence staining were purchased from Life Technologies (Carlsbad, CA). Brefeldin A was purchased from eBioscience / ThermoFisher Scientific (San Diego, CA), PMA was purchased from Selleck Chemicals LLC (Houston, TX). The following tumor dissociation reagents were purchased from Sigma-Aldrich (Saint Louis, MO): Collagenase type IV (Sigma, C5138), DNase type IV (20 U / mL, Sigma, D5025), and Hyaluronidase type V (0.1 mg / mL, Sigma, H6254).354935-2291-41872065472-001010WOPTCD4, CD8 isolation and adoptive transfer

[0151] To generate antitumor immunity, mice were treated with 2 doses of dendritic cell (DC) vaccine (generated with cell lysate) over 14 days. Spleen and lymph nodes were harvested from these mice, and lymphocytes were isolated using EasySep mouse CD4 or CD8 T cell isolation kit (STEMCELL technologies) and cultured in vitro with tumor lysate pulsed DC and IL-2 for 3 days. 2xl06activated CD4 or CD8+ cells were adoptively transferred by tail vein injection into recipient mice (5 mice per group), which were challenged with s.c. tumor cells. Tumor growth was measured every 2-3 days with caliper.

[0152] To generate the DC vaccine, 10 million Renca or Hepal-6 cells were lysed using three freeze and thaw cycles and then centrifuged at 10,000 rpm; ImL of the supernatant was added to 10 million fresh DCs with LPS (1 pg / mL) for 24 hours. DCs were harvested and washed 3 times with PBS and then used to stimulate lymphocytes.CIITA was knocked down (KD) in Hepa 1-6 cells and over-expressed (OE) in Renca cells

[0153] The CIITA KD cells were created using MISSION® shRNA lentiviral transduction particles (Sigma-Aldrich, SHCLNV_NM_007575), following the manufacturer’s instructions. Five predesigned shRNA clones (TRCN0000086448, TRCN0000086449, TRCN0000086450, TRCN0000086451, and TRCN0000086452) in vector pLKOl were purchased from Sigma-Aldrich. Hepal-6 cells were transduced in the presence of 5 pg / mL of polybrene and selected using 500 pg / mL G418 to create stable lines.

[0154] CIITA OE cells were generated by transducing CIITA (NM_007575.3) mouse Tagged ORF Clone lentiviral particle (GeneCopoeia, LPP-MmO1492-Lvl 58-100) into Renca cells. The CIITA tagged ORF was inserted into pReceiver-Lvl58-Neomycin vector. The Renca cells were transduced in the presence of 5 pg / mL polybrene and selected using 3 pg / mL puromycin to create stable lines.T cells activation and T cell cytotoxicity

[0155] To prepare DC vaccine, mouse bone marrow was harvested from C57BL / 6 mice and suspended in RPMI supplemented with 10% FBS. GM-CSF (10 ng / mL) was added to the medium and placed at 37°C with 5% CO2. RPMI medium was changed every other day. DCs were harvested on day 7 and pulsed with tumor cell lysate for 16 hours and activated with 10 pg / mL CpG for 4 hours.

[0156] To assess mouse T cell activation following in vivo treatment, lymph nodes and spleen were collected to generate single cell suspensions and activated ex vivo with DC364935-2291-41872065472-001010WOPTvaccine (fresh DC pulsed with tumor lysate) for 4 days. CD4, CD8, fFNy were analyzed by flow cytometry (LSR II, BD Biosciences, San Jose, CA 95131).

[0157] To assess T cell cytotoxicity in vitro, lymphocytes were harvested from WT Hepal-6 tumor-bearing mice and co-cultured with CIITA WT or KD Hepal-6 cells at various target: effector ratios for 18 hours. Hepal-6 cell death was quantified by flow cytometry by gating on CD45 negative cells and determining the percent of cells taking up propidium iodine.Tumor cell dissociation and flow cytometry

[0158] Mouse tumors were cut into small pieces using surgical scissors and placed in 2.5 ml RPMI with collagenase type IV (1 mg / mL, Sigma, C5138), DNase type IV (20 U / mL, Sigma, D5025), and hyaluronidase type V (0.1 mg / mL, Sigma, H6254). The samples were processed using a gentleMACS™ dissociator with heaters (Miltenyi Biotec), according to the manufacturer's protocol. Tumor-dissociated cells were harvested by centrifugation at 300*g for 7 min and stained with aCD45 and aCD8 (BioLegend). Flow cytometry data were collected using the Symphony Flow Cytometer (BD Biosciences) and were analyzed using FlowJo vlO (BD Biosciences).Immunofluorescence Staining

[0159] Hepa 1-6 (WT and CIITA KD) or Renca (WT and CIITA OE) cells were grown on cover slides. When cell confluency reached 80%, the cells were fixed with 4% paraformaldehyde. After blocking with 5% bovine serum albumin (BSA), the slides were stained with a rabbit polyclonal anti-MHC II antibody (Life Technologies, PA5-116876) and visualized with the goat anti-rabbit IgG (H+L) TRITC (Life Technologies, A16101). Mounting Medium with DAPI (Vector Laboratories, H-1200) was added to all slides before image capture using a Keyence BZ-X710 microscope.TCGA Pan-cancer data analysis

[0160] RNA-sequencing data and corresponding clinical information of TCGA Pancancer samples were downloaded from University of California Santa Cruz Xena Browser9using R package UCSCXenaTools. The gene set z-score method was used to summarize the expression of genes belonging to MHC I (HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, and HLA-H) and MHC II (HLA-DMA, HLA-DMB, HLA-DOA, HLA-DOB, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5). CD4 and CD8 T-cell score were estimated using EPIC method in R package immunedeconv (v2.1.0).374935-2291-41872065472-001010WQPTStatistical Analysis

[0161] The Kaplan-Meier method was used to present survival functions. The Cox proportional hazard regression was used to estimate survival association. Hazard ratios (HRs) and 95% confidence intervals (Cis) were reported, and statistical significance was assessed based on nonoverlapping 95% Cis and the log-rank test. Statistical analyses were performed using R statistical software (v4.0.2; R Foundation, Vienna, Austria) and MATLAB (Mathworks, Natick, MA, USA) with two-sided tests and a significance level of 0.05. Error bars represent standard error of the mean.Example 2ResultsCD4 depletion has varied effects on tumor growth among tumor models.

[0162] CD4 depletion, after tumor growth has already primed the immune system, has been shown to remove Tregs, stimulate antitumor immunity, and suppressive tumor growth in mouse models. However, we noted that this does not happen in all tumor models (Figure 1, Figure 8). Tumors were established by subcutaneous injection of syngeneic tumor cells into mice, and after immune priming was expected to have occurred, CD4 depleting antibodies were administered and tumor growth was monitored (Figure 1A). CD4 depletion suppressed the growth of B16 melanomas and Renca kidney cancers (Figure 1B-C). However, Hepal-6 hepatoma tumors, which normally do not grow in immunocompetent B6 mice, grew after CD4 depletion (Figure ID), suggesting CD4 cells suppress the growth of Hepal-6 tumors. Our Hepal-6 cells were newly purchased from ATCC, and injection of even IxlO7cells subcutaneously did not produce palpable tumor, which is consistent with a report by Hiotis et al, where only intrahepatic injection resulted in tumor formation.Suppression of Hepal-6 tumor growth is dependent on CD4+ lymphocytes.

[0163] To assess the role of CD4+ lymphocytes and CD8+ lymphocytes on Hepal-6 tumor growth, CD4+ cells, CD8+ cells, or both cells were depleted after tumor cell injection in mice (Figure 2A). As expected, CD4 depletion increased tumor growth while CD8 depletion did not significantly affect tumor growth (Figure 2B). Interestingly, depleting both CD4+ and CD8+ cells caused the tumor to grow the fastest, suggesting that in the presence of CD4 depletion, CD8+ cells may assume a more important antitumor role.

[0164] To confirm the importance of CD4+ cells, both lymphocyte populations were depleted and then replaced (Figure 2C). Adoptively transferred lymphocytes were harvested from nontumor-bearing (donor) mice stimulated with Hepal-6 tumor lysate. After depleting 384935-2291-41872065472-001010WOPTboth CD4+ cell and CD8+ cells in the tumor bearing (recipient) mice, replacing CD4+ cells or both CD4+ / CD8+ cells restored tumor growth-suppression (Figure 2D). However, replacing CD8+ cells did not, confirming the importance of CD4+ cells in suppressing Hepal-6 growth.Hepal-6 has high MHC II expression.

[0165] Since CD4 functions as a bridge between the T cell receptor and MHC II, which can be found on tumor cells15'17and antigen presenting cells, we assessed MHC I and MHC II expression in tumor cells. We hypothesized that MHC II expression on Hepal-6 may allow CD4+ lymphocytes to suppress tumor growth. Consistent with this hypothesis, MHC II was highly expressed in only Hepal-6 cells (Figure 3A-B) , which is consistent with previous reports of MHC II expression in Hepal-6 cell lines and tumors18. MHC II, but not MHC I, expression increased in Hepal-6 cells in the presence of immune activating cytokines, INFg and TNFa ( Figure 9). With respect to the various tumor models we examined, the effect of CD4 depletion on tumor growth correlated with MHC II expression but not with MHC I expression.

[0166] These results also suggest that MHC expressions may be useful as clinical biomarkers. However, any candidate biomarker should have heterogenous expression to have potential to reflect biological heterogeneity. Interrogation of the TCGA pan-cancer dataset indicates that both MHC I and MHC II have heterogeneous expression both within and between tumor types (Figure 3C).MHC II expression was manipulated in tumor cell lines.

[0167] Given these correlative observations, we sought to firmly establish MHC II expression as the mechanism that can produce differential responses to CD4 depletion. Therefore, we created cell lines where MHC II expression was either increased or decreased by manipulating CIITA, which is a transcriptional coactivator of MHCII (Figure 4A). We confirmed in the TCGA pan-cancer dataset that CIITA and MHC II expressions are highly correlated (Figure 4B). Hepal-6 cells have high MHC II expression and shRNA knock down (KD) of CIITA reduced MHC II expression (Figure 4C, Figure 10). Renca on the other hand has no MHC II expression and CIITA overexpression (OE) resulted in high MHC II expression (Figure 4D, Figure 11). Both manipulated cell lines were characterized in our mouse models, which were subjected to CD4 depletion. We predicted that CIITA manipulation would change whether CD4 depletion would suppress or stimulate tumor growth.394935-2291-41872065472-001010WOPTMHC II KD in Hepal-6 removed their dependence on CD4+ lymphocytes for tumor growth suppression.

[0168] Prior to evaluating the growth of KD Hepal-6 in mice, it was essential to confirm that genetic alteration did not impact their intrinsic growth rate. Therefore, the in vitro proliferation of WT and KD Hepal-6 cells was compared and was not significantly different (Figure 5A). However, when KD Hepal-6 cells were injected into mice, they grew rapidly while the WT cells did not, suggesting that CD4+ lymphocytes could no longer control the KD Hepal-6 tumors (Figure 5B). To confirm that the KD cells were less capable of generating lymphocyte-mediated immunity, a killing assay was performed (Figure 5C). In mice, the WT tumor cells generated a robust lymphocyte-mediated tumor-specific killing response while the KD tumor cells did not. Finally, we predicted that the KD Hepal-6 tumors with low MHC II expression would be sensitive to CD4 depletion, like B16 and Renca tumors. This was in fact the case; while the WT Hepal-6 tumors grew faster after CD4 depletion (Figure 5D), the KD Hepal-6 tumors were suppressed by CD4 depletion (Figure 5E).MHC II OE in Renca made them dependent on CD 4+ lymphocytes for tumor growth suppression.

[0169] The inverse experiments were performed with OE Renca cells. In vitro proliferation of WT and OE Renca cells were not significantly different (Figure 6A). However, as expected, the in vivo growth of OE Renca was significantly decreased when compared to WT Renca (Figure 6B). To better characterize the resulting immune response, splenocytes from these mice were compared. When compared to WT, OE Renca had significantly increased CD4+ cell and CD8+ splenocytes (Figure 6C, Figure 12), although this may not necessarily reflect an increase in tumor-specific lymphocytes. Therefore, tumorspecific activation of CD4+ and CD8+ cells was assessed by measuring fFNy expression following ex vivo restimulating with tumor-lysate pulsed DCs (Figure 6D). In addition, immune activation should result in lymphocyte infiltration into tumor, and both CD4+ and CD8+ lymphocyte infiltration was increased in OE Renca tumors when compared to WT tumors (Figure 6E, Figure 13).

[0170] As previously demonstrated, WT Renca tumor growth was suppressed by CD4 depletion (Figure 6F), which decreased Tregs and increased tumor-specific CD8+ lymphocyte activation (Figure 6G). This is consistent with CD4 depletion generating an antitumor immune response.1However, for OE Renca, CD4 depletion increased tumor404935-2291-41872065472-001010WOPTgrowth (Figure 6H) and suppressed the antitumor immune response. This happened despite decrease in Tregs, and tumor-specific CD8+ lymphocyte activation was decreased (Figure 61). This is consistent with CD4 depletion decreasing antitumor immunity. Therefore, the response to CD4 depletion was similar to what was seen for WT Hepal-6, which have high baseline MHC II expression.In TCGA, MHC status identifies tumors where CD4 levels predicts survival.

[0171] Based on our mechanistic studies, we predicted that patient MHC I and MHC II status will identify subgroups where CD4 levels will predict survival. CD8 levels from bulk tumor RNA sequencing serve as a surrogate for tumor-infiltration of CD8+ lymphocytes and immune activation, and high CD8 expression has been shown to predict better survival. However, we hypothesized that tumors with high MHC II expression will be more dependent on CD4+ lymphocytes for suppression of tumor growth and that for these tumors, CD4 expression will predict better survival.

[0172] An analysis of the TCGA pan-cancer dataset supported our hypothesis. We illustrate our observation by focusing on two subgroups where our predictions may be most pronounced (Figure 7). In patients with high MHC II and low MHC I, high CD4 correlated with better progression free survival (p=0.0217) and trended towards better overall survival (p=0.0770). Conversely, in patient with low MHC II and high MHC I, high CD4 predicted survival in the opposite direction; high CD4 correlated with worse overall survival (p=0.0101) and trended towards worse progression free survival (p=0.1497). Forest plots in Figure 7 and Figure 14 show how CD4 and CD8 predict overall survival in all the subgroups. The only two subgroups where high CD4 correlated with better survival had high or intermediate MHC II levels.

[0173] Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and / or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

[0174] The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the 414935-2291-41872065472-001010WOPTpurposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

[0175] While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention. As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of’ or “consisting essentially of.”

[0176] Unless stated otherwise, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment of the application (especially in the context of claims) may be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. The abbreviation, “e.g.” is derived from the 424935-2291-41872065472-001010WOPTLatin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g” is synonymous with the term “for example.” No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[0177] “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0178] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.434935-2291-41872065472-001010WOPT

Claims

WHAT IS CLAIMED IS:

1. A method for treating cancer in a subject, comprising:administering a CD4 depleting therapy to a subject having tumor tissue determined to have an MHC II expression level that is lower than a reference level for the tumor type, or a level of a molecular surrogate level for expression MHC II level that is lower than its reference level for the tumor type.

2. The method of claim 1, further comprising selecting the subject having tumor tissue determined to have an MHC II expression level that is lower than a reference level for the tumor type, or a level of a molecular surrogate level for expression MHC II level that is lower than its reference level for the tumor type, prior to administering a CD4 depleting therapy to a subject.

3. A method for treating cancer in a subject, comprising:detecting an MHC II expression level or a molecular surrogate level for MHC II expression level in tumor tissue obtained from the subject; andadministering a CD4 depleting therapy to a subject who has been determined to have an MHC II expression level that is lower than a reference level for the tumor type, or a molecular surrogate level for MHC II expression level that is lower than its reference level for the tumor type.

4. A method for treating cancer in a subject, comprising:obtaining or requesting the results of an analysis of an MHC II expression level or a molecular surrogate level for MHC II expression level in tumor tissue obtained from the subject; andadministering a CD4 depleting therapy to a subject who has been determined to have an MHC II expression level that is lower than a reference level for the tumor type, or a molecular surrogate level for MHC II expression level that is lower than its reference level for the tumor type.

5. A method of selecting a cancer treatment for a subject, comprisingdetecting an MHC II expression level or a molecular surrogate level for MHC II expression level in tumor tissue obtained from the subject; andselecting a CD4 depleting therapy for a subject who has been determined to have MHC II expression level that is lower than reference level for the tumor type, or a level of a molecular surrogate for MHC II level that is lower than its reference level for the tumor type, or444935-2291-41872065472-001010WOPTavoiding CD4 depleting therapy for a subject who has been determined to have MHC II expression level that is higher than reference level for the tumor type, or a level of a molecular surrogate for MHC II level that is higher than its reference level for the tumor type.

6. The method of claim 5, further comprising administering the selected CD4 depleting therapy.

7. A method of monitoring treatment response in a subject in need thereof, comprising:detecting an MHC II expression level or a molecular surrogate level for MHC II expression level in tumor tissue obtained from the subject, wherein the subject is undergoing treatment for cancer; andcontinuing to administer a CD4 depleting therapy for a subject who has been determined to have MHC II expression level that is lower than a reference level for the tumor type, or a level of a molecular surrogate for MHC II level that is lower than its reference level for the tumor type, ordiscontinuing to administer the CD4 depleting therapy for a subject who has been determined to have MHC II expression level that is higher than reference level for the tumor type, or a level of a molecular surrogate for MHC II level that is higher than its reference level for the tumor type.

8. The method of any one of claims 1-7, wherein the molecular surrogate for MHC II level is CIITA, HLA-DP, HLA-DQ, HLA-DR, RFX5, or RFXAP.

9. The method of any one of claims 1-8, wherein the CD4 depleting therapy is selected from CC182, IT 1208 (a defucosylated humanized anti-CD4 depleting antibody), daclizumab, zanolimumab, cM-T412, keliximab, ibalizumab, or combinations thereof.

10. The method of any one of claims 1-8, wherein the CD4 depleting therapy is CC182.

11. The method of any one of claims 1-8, wherein the molecular surrogate for MHC II level is CIITA.

12. The method of any one of claims 1-11, wherein the cancer is selected from melanoma, renal cell carcinoma, or hepatocellular carcinoma.

13. The method of any one of claims 1-11, wherein the cancer is selected from Acute Myeloid Leukemia (LAML), Adrenocortical Carcinoma (ACC), Bladder Urothelial Carcinoma (BLCA), Brain Lower Grade Glioma (LGG), Breast Invasive Carcinoma (BRCA), Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma (CESC), Cholangiocarcinoma (CHOL), Colon Adenocarcinoma (COAD), Esophageal Carcinoma (ESCA), Glioblastoma Multiforme (GBM), Head and Neck Squamous 454935-2291-41872065472-001010WOPTCell Carcinoma (HNSC), Kidney Renal Clear Cell Carcinoma (KIRC), Kidney Renal Papillary Cell Carcinoma (KIRP), Liver Hepatocellular Carcinoma (LIHC), Lung Adenocarcinoma (LU D), Lung Squamous Cell Carcinoma (LUSC), Lymphoid Neoplasm Diffuse Large B-cell Lymphoma (DLBC), Mesothelioma (MESO), Ovarian Serous Cystadenocarcinoma (OV), Pancreatic Adenocarcinoma (PAAD), Pheochromocytoma and Paraganglioma (PCPG), Prostate Adenocarcinoma (PRAD), Rectum Adenocarcinoma (READ), Sarcoma (SARC), Skin Cutaneous Melanoma (SKCM), Stomach Adenocarcinoma (STAD), Testicular Germ Cell Tumors (TGCT), Thyroid Carcinoma (THCA), Thymoma (THYM), Uterine Corpus Endometrial Carcinoma (UCEC), Uterine Carcinosarcoma (UCS), or Uveal Melanoma (UVM).

14. The method of any one of claims 1-13, further comprising administering chemotherapy, radiotherapy or immunotherapy to the subject.

15. The method of any one of claims 1-14, wherein the subject is a human subject.464935-2291-41872065472-001010WOPT

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