PI: Dr. James Cripps
Mentor: Dr. Kavitha Yaddanapudi
Project Title: Enhancement of Anti-Melanoma Tumor Targeting Utilizing CARs and TILS
Project Summary:

Treatments for solid cancers mainly rely on traditional non-specific targeted therapies. In the past several decades the ability of harnessing and engineering of our own immune system to better combat cancer has went from a dream to a reality. This field termed immunotherapy is focused on enhancement of our own immune system in the battle against cancer. Specifically, the area of cellular immunotherapy utilizes cells from the patient’s body which are removed, enhanced, and re-introduced via a process known as adoptive cell therapy (ACT). The two most successful forms of ACT are the use of tumor infiltrating lymphocytes (TILs) and chimeric antigen receptor T cells (CAR-T cells). TILs are successfully utilized to treat multiple cancers including Melanoma. CAR-T cells are widely used successfully in the treatment of blood cancers such as acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL). However, approximately half of melanoma patients don’t respond to TIL therapy and CAR-T cells have yet to be effective in treating solid tumors. This study will determine if we can enhance existing Melanoma therapies by combining TILs and CAR-T cells. We have developed a novel CAR construct that recognizes MUC-18, an antigen expressed on Melanoma cells but not regular melanocytes or skin cells. The construct also produces a PD-1 antibody, which can counter the immune systems down regulation of T cells. Based on this we propose the following aims.

Aim 1: Can the MUC-18 and anti-PD-1 containing construct be successfully expressed in T cells from peripheral blood mononuclear cells from healthy donors? Aim 2: Can the MUC-18 and anti-PD-1 containing construct be successfully expressed in TILs and sentinel lymph node T cells isolated from melanoma patients? If any of these aims prove successful or demonstrate superior efficacy to current treatment paradigms, we would have a substantial step forward in utilizing Immunotherapy to treat Melanoma and other solid malignancies.  

PI: Dr. Chi Li
Project Title: A Lung Cancer Vaccine Using Exosomes for Induced Pluripotent Stem Cells.
Project Summary:

A critical challenge in lung cancer research is to develop innovative vaccines to protect against

pulmonary malignancy. Recent efforts to develop lung cancer vaccines have largely failed, probablydue to their focus on inducing immune responses against individual lung cancer-associated antigens.If a lung cancer vaccine targets multiple antigens present only in lung tumors, but not in normal adulttissues, the chance of success will be greatly improved. Induced pluripotent stem cells (iPSCs) are reprogrammed from somatic cells and have the capacity of self-renewing and developing into all cell types of the adult body. It is known that iPSCs and tumor cells share carcinoembryonic antigens which could be classified as neoantigens due to their absence in normal adult tissues. Lung tumors also contain a subpopulation of tumor-initiating cells (TICs) with high tumorigenicity and self-renewal capability that contribute to the resistance to conventional therapies. To exploit the antigenic similarity between tumor cells and iPSCs, we propose to thoroughly investigate efficacy of an anti-lung cancer vaccine composed of exosomes from murine iPSCs expressing granulocyte-macrophage colony stimulating factor (GM-CSF) as an immunostimulatory adjuvant (iPSC-exo). We hypothesize that iPSC-exo vaccination prevents lung tumorigenesis by triggering CD8-dependent immune responses and eradicating lung TICs. In this grant, we will investigate this hypothesis in a clinically relevant primary lung adenocarcinoma model of immunocompetent mice. Two specific aims are envisioned: 1) Investigate the translational potential of iPSC-exo vaccination against primary lung adenocarcinoma in mice; 2) Elucidate the mechanism by which iPSC-exo vaccine prevents lung adenocarcinoma. We believe that the success of proposed experiments will lead to clinical trials of a similar vaccine against human pulmonary malignancy.  

PI: Dr. Nobuyuki Matoba
Project Title: Development of Lectikines for Immunotherapy Against Ovarian Cancer
Project Summary:

The goal of this project is to create a novel immunotherapeutic drug candidate against ovarian cancer (OVCA). OVCA is the leading cause of death from gynecological malignancy in the United States, with poor survival rates due to late diagnosis and chemo-resistant, fatal recurrent disease after the first-line debulking surgery and chemotherapies. Immune checkpoint inhibitors have thus far failed to show any significant clinical benefit due to highly immunosuppressive tumor microenvironment and poor infiltration of T cells. Thus, the development of effective OVCA immunotherapy requires novel strategies. To address this problem, the proposed project led by Dr. Matoba (biopharmaceutical scientist, PI), in close collaboration with Drs. Yaddanapudi (tumor immunologist, co-I) and Kakar (ovarian cancer research expert, co-I), will create novel immunotherapeutic proteins based on Avaren lectin. It is a small recombinant lectin originally engineered in the PI’s laboratory as an antiviral agent based on its ability to selectively recognize high-mannose glycans overexpressed on the surface of enveloped viruses. A fusion protein consisting of Avaren lectin and human IgG1 Fc (AvFc) was previously shown to exhibit potent anti-HIV and anti-HCV activities without toxicity in in vitro and in vivo animal models. Meanwhile, growingevidence suggests that the aberrant accumulation of high-mannose glycans also occurs in the cell surface glycome of various malignancies, including OVCA. In fact, our preliminary data show that AvFc efficiently binds to OVCA but not to adjacent normal tissues. Furthermore, AvFc elicits potent antibody-dependent cell-mediated cytotoxicity (ADCC) against OVCA cell lines. Based on these data, we hypothesize that immunostimulatory variants of Avaren lectin will exhibit potent immunotherapeutic activity by transforming OVCA to more immunoactive tumors. Specifically, we will engineer translational fusion proteins consisting of Avaren lectin and an antitumor cytokine (“lectikines”). In Aim 1, we will generate lectikines by fusing IL-2 to AvFc and TNF to Avaren monomer, which will be produced in Nicotiana benthamiana plants using a transient overexpression system. After thorough purification, we will assess their molecular properties, high-mannose-binding affinity and cytokine functions in a battery of biochemical, biophysical and cell-based assays. In Aim 2, we will evaluate the therapeutic effects of lectikines in a syngeneic, orthotopic murine OVCA model using the ID8 cell line inoculated into the peritoneal cavity of immunocompetent C57bl/6 mice. Disease progression will be monitored through abdomen circumference, body weight and live animal bioluminescence imaging. Immunophenotyping of peritoneal cells will be performed by flow cytometry. We anticipate that lectikines will elicit significant efficacy through increased immune activation in the tumor microenvironment. Successful completion of this pilot project will establish an initial POC for a first-in-class OVCA immunotherapy and generate compelling preliminary data in a future R01 application for further investigation and optimization of MOA of top candidate lectikines.

PI: Dr. Melissa Smith
Project Title: Characterization of the IgG4 Repertoire and Tolerance-Associated Variants in Late Stage Melanoma
Project Summary:

It is widely accepted that T cells play a crucial role in the tumor microenvironment and anti-cancer immunity; however, the role of B cells has not well characterized. Further investigation and definition of the role B cells play in tumor environments creates a major opportunity for the development of new B-cell based immunotherapies for melanoma. Although trafficking of B cells to the tumor microenvironment has been described in melanoma, it is unknown whether this increase in B cells is antigen-specific or due to a general inflammatory response. 

Recent studies have speculated that B cells in the melanoma tumor microenvironment induce a tolerizing effect through the production of Th2-type cytokines and chemokines, as well as elevated secretion of IgG4 antibodies, resulting in a qualitatively weaker humoral response. In general, IgG4 is thought to be a weak immune activator compared to anti-tumor IgG1 antibodies, resulting in decreased antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP). Overall, it remains unknown whether antigen specific clonotypes are associated with melanoma disease severity and/or if IgG4-specific variants can be identified that confer the decreased effector functions observed in late stage disease.

We hypothesize that circulating IgG4 and Th2-like cytokine levels will be elevated in advanced melanoma disease and that the IgG4-specific repertoire will contain polymorphisms in key residues and posttranslational modification motifs impacting effector function. Addressing this hypothesis with current immune profiling methods would be impossible, as they provide only limited resolution of isotype identify and no definition of the Fc domain, which modulates function. The utilization of a novel IgG repertoire profiling method, full length (FL) RepSeq, will resolve the Fc domain, including unique variation in Fc that may impact antibody effector function. Definition of critical variants impacting antibody effector function, and identification of inflammatory analytes associated with this skewed response, will aid in our effort to understand the role of humoral immunity in melanoma and advance our ability to leverage IgG antibodies as part of immunotherapy approaches to treat this disease.