Center for Cancer Immunology and Immunotherapy / COBRE

The Administrative Core oversees all CCII activities and provides the framework for selecting and mentoring junior investigators. The center is supported by a strong network of senior institutional mentors, an Internal Advisory Committee (IAC), and an External Advisory Committee (EAC) comprised of internationally recognized experts in cancer immunology and immunotherapy.

The newly established Functional Immunomics (FI) Core provides high-end molecular and cellular immune profiling for both mouse and human samples. By integrating institutional investments—such as mass cytometry and cell sorting—with existing facilities (genomics, biorespository, and tissue processing) and broader COBRE/INBRE resources like bioinformatics, the core provides vital capabilities for all CCII research projects.

The CCII provides financial backing, core facility access, mentorship, and networking opportunities for innovative researchers who have not yet secured R01-type funding. The inaugural cohort of CCII mentees spans multiple departments, unified by a shared focus on defining mechanisms of cancer immune evasion and translating discoveries into novel biomarkers and treatments. 

The center is designed for lasting impact through talent retention, faculty recruitment, and cross-disciplinary collaborations that build on institutional strengths. A structured pipeline ensures that incoming junior investigators continually refresh the program as project PIs transition, backed by ongoing institutional pilot project support. 

Conventional chemotherapy has been thought to act through the direct killing of tumor cells. However, accumulating evidence indicates that immune competence is crucially required for chemotherapy efficacy. It is expected that loss of this immunity during chemotherapy has a negative effect on its efficacy. Therefore, the impact of chemotherapy on anti-tumor immunity needs further investigation in order to rationally design combinatorial regimens for conventional chemotherapy. 

In the preliminary studies, we have demonstrated that repeated chemodrug gemcitabine (GEM) treatment promoted the expansion and differentiation of immunosuppressive Ly6C^high monocytic-MDSC (M-MDSC). Tumor-derived soluble factors, such as GM-CSF and soluble ICAM-1(sICAM-1), were up-regulated upon chemo-drug treatment. In tumor-free naïve mice, GEM treatment induced the immunosuppressive activity of Ly6C^high myeloid cells in the bone marrow (BM). The chemokine CX3CL1 and its receptor CX3CR1 expression levels were elevated in the BM. Higher levels of mitochondrial reactive oxygen species (mtROS) were also observed in tumor cells and BM stromal cells following chemotherapy. Furthermore, chemotherapy induced NF-κB activation leading to the hyperproduction of GM-CSF by tumor cells. Based on these preliminary data, we hypothesize that chemotherapy enhances the production of mtROS in tumor cells and BM stromal cells, which increases the expressions of GM-CSF and sICAM-1 as well as CX3CL1 leading to the enhanced immunosuppression of M-MDSC in the TME. These hypotheses will be addressed by two Specific Aims. Aim 1 defines the roles of GM-CSF and sICAM-1 in chemotherapy-induced differentiation of immunosuppressive Ly6C^high myeloid cells and the mechanisms underlying mtROS and upregulation of GM-CSF and sICAM-1 in tumor cells; Aim 2 determines the mechanisms by which host cell-derived chemokine CX3CL1 regulates the accumulation and immunosuppressive function of inflammatory Ly6C^highmyeloid cells in the BM. The findings from these studies will allow us to gain a better understanding the underlying mechanisms by which ongoing inflammation following multi-dose clinical regimens of chemotherapy modulates anti-tumor immunity, and rationally design a novel therapeutic approach by combining chemotherapy with mitochondria-targeted antioxidants for cancer treatment. 

Lung cancer is a prevalent disease and consumes many lives every year. Cancer immunotherapy using immune checkpoint inhibitors (ICIs; e.g. anti-PD-1, anti-CTLA-4 antibodies) has revolutionized the treatment of metastatic lung cancer, resulting in long-term complete responses for many patients. Nevertheless, there remains an urgent need for new strategies because not all patients respond to ICIs; moreover, resistance can occur in those that do. Myeloid-derived suppressor cells (MDSCs), in particular, monocytic MDSCs (M-MDSCs) are potent immunosuppressive innate immune cells that actively inhibit CD8⁺ T cell tumor homing and activation. Since M-MDSC levels are elevated in multiple human cancers and correlate with decreased patient survival, we postulate that these cells contribute to anti-PD-1 resistance. The purine nucleoside, adenosine, is produced in copious amounts within the tumor microenvironment (TME), where it serves to suppress the immune system and promote tumor growth. There is evidence to suggest that overproduction of adenosine can mediate resistance to ICIs. Immune suppressive adenosine is produced via the enzymatic conversion of extracellular AMP by the cell surface enzyme, CD73 (ecto-5¢-nucleotidase; AMP ® adenosine). Our preliminary studies demonstrate that tumor cell-derived prostaglandin E₂ (PGE₂) maintains M-MDSC suppressive activity, in large part, by directly inducing cell surface CD73 expression via a novel PGE₂→cAMP→CREB/STAT3 pathway leading to increased immune suppressive adenosine within the TME. The overall hypothesis of this proposal is that tumor cell-derived PGE₂ dictates CD73 expression in M-MDSCs leading to substantial increases in adenosine-dependent inhibition of anti-tumor CD8⁺ T cell activation resulting, ultimately, in anti-PD-1 immunotherapeutic resistanceA major goal of this proposal is to test the anti-tumor efficacy of a novel cancer immunotherapy involving systemic administration of adenosine deaminase (ADA)—an enzyme that irreversibly converts adenosine into inosine, a non-immunosuppresive nucleoside. A pegylated version of bovine ADA (PEG-ADA) is already FDA-approved for use as an enzyme replacement therapy in children with ADA-associated severe combined immunodeficiency (ADA-SCID). We hypothesize that depletion of adenosine-mediated T cell immune suppression by PEG-ADA sensitizes tumors to PD-1 inhibitor therapy and improves clinical outcomes for NSCLC patients. To fulfill the stated objectives, the following aims are proposed: 1) Delineate the signaling pathway and molecular mechanisms by which PGE₂ induces CD73 expression in M-MDSCs; 2)Determine whether inhibition of the PGE₂→cAMP→CREB/STAT3→CD73→adenosine pathway attenuates anti-PD-1 resistance in a mouse model of lung cancer; and 3) Validate PGE₂→ CD73⁺ M-MDSC → adenosine mediated anti-PD-1 resistance pathway in lung cancer patients receiving pembrolizumab therapy. Our proposed study will provide important insights towards developing a safe and novel immunotherapy to attenuate ICI resistance in lung cancer patients. 

Lung cancers are the most common malignancies and are the leading cause of cancer-related deaths worldwide. Immunotherapies are transforming treatment regimes, and trials conducted in lung cancer have already demonstrated improvements in long-term survival outcomes. However, existing therapies are not effective in all patients, stressing a need to better characterize the complex molecular and immunological processes associated with treatment responsiveness and disease progression. Although T cells have been the primary target of immunotherapies in lung cancer, there is a growing appreciation that T cell-mediated anti-tumor immunity can be influenced by actions of other immune cells within the tumor microenvironment (TME). Tumor-infiltrating (TI) B cells have been shown to have dual roles in tumor progression generally, but in non-small cell lung cancers (NSCLC), specifically adenocarcinomas (LUAD), particular B cell subsets have been shown to both promote T cell anti-tumor responses and positively impact long-term survival outcomes. Indications of a role for specific antibody (Ab) responses in LUAD have also recently been reported, including evidence of focused tumor antigen-specific responses. However, characterizations of anti-tumor B cells and their functional signatures have been limited by the study of only a small number of pre-selected surface markers in relatively small numbers of patients. Nonetheless, it is evident that B cell-mediated roles in LUAD tumor progression are variable among patients, and indicates that a more comprehensive and integrated characterization of TI B cell phenotypic variation and the associated effects on the anti-tumor T cell response has great potential to inform our understanding of disease progression, as well as the development of novel, potentially personalized, therapies. The overall goal of this proposal is to define TI B cell signatures that are associated with the T cell-mediated anti-tumor response in human lung cancer. To achieve this goal, we will leverage several cutting edge techniques to first comprehensively profile lymphocytes (B and T cells) and myeloid cells in tumor and paired normal tissues from a large cohort of early-stage LUAD patients, including the first pairing of this type of data with Ab repertoire sequencing as a means to interrogate critical functional Ab response signatures in LUAD B cells. Importantly, the single-cell profiling approach proposed will leverage B cell markers already determined to be associated with long-term LUAD survival outcomes, so that distinct functional B cell profiles and their associations with TI T cells and myeloid cells can be identified specifically in the context of disease progression (Aim 1). These large-scale statistical associations will be validated and then serve as a framework for conducting functional in vitro culture and serum-based analyses to assess whether prognostically favorable B cell subsets identified have distinct functional phenotypes, associate with tumor antigen-specific Ab responses, and have differential effects on T cell activation and effector function (Aim 2). Successful completion of this project will facilitate novel discovery and biomarkers critical for the development of novel therapeutics in human lung cancer. 

Despite having been approved as first and second line therapy for non-small cell lung cancer (NSCLC), anti-PD-1 antibodies still fail in a substantial proportion of lung cancer patients. The mechanism that underlies the failure of anti-PD-1 therapy in the majority of NSCLC patients is not yet fully understood. We have discovered that, in the anti-PD-1-resistant LSL-Kras^G12D murine lung adenocarcinoma mouse model, treatment induces a T-cell activation profile that favors Th17/γδT17 reinvigoration over CD8+ T cell activation. In contrast, when administered in conjunction with an anti-IL-17 neutralizing antibody, anti-PD-1 treatment results in a dramatic enhancement of CD8+ T-cell cytotoxicity with near-complete eradication of established disease.  These findings provide the premise for our central hypothesis that in NSCLC, the failure of anti-PD-1 is, at least in part, due to reinvigoration of PD-1⁺ type 17 T cells (Th17/γδT17), which actively undermine anti-PD-1-mediated restoration of cytotoxic function in CD8⁺ T-cells.  Based on additional murine data, we are also advancing the extended hypothesis that the severity of pre-existing T17 activity in the neoplastic lung is determined by commensal bacteria and that the lung microbiota signature can ultimately predict responsiveness to anti-PD-1 therapy.  The goal of this proposal is to demonstrate the relevance of these findings to human prior to initiating an R01 application. Specifically, in Aim 1, we will establish whether intrinsic lung T17/CTL ratio is predictive of anti-PD-1 responsiveness in NSCLC patients independent of neoantigen burden. In Aim 2 we will determine whether specific human lung microbiota, individually or in defined combinations, drive the ontogeny of intrinsic T17 immunity and ultimately resistance to ICI therapy.  The proposed study is conceptually impactful as it addresses an important clinical conundrum; is mechanistically novel; and has translational relevance since it introduces therapeutic/prognostic approaches that can rapidly move to the clinic. 

Oncolytic viral therapy shows promise for high-grade solid tumors, but none have been shown to provide curative potential. This study explores the potential of harnessing the natural tropism of Zika virus (ZIKV) to target glioblastoma stem cells (GSCs) and reprogram the typically immune suppressive GBM tumor environment to an inflammatory, antigen-presenting environment that induces GBM-specific CD8+ T cell responses. The first goal of this proposal is to identify the cellular mechanisms that drive anti-tumor T cell responses following ZIKV treatment. Our research will examine the functional roles of two distinct myeloid cell subsets induced following ZIKV treatment - CCR2+ monocytes in the tumor microenvironment (TME) and dendritic cell subset-2 (DC-2s) in draining lymph nodes (dLN) - in driving anti-tumor T cell responses against GBM. Additionally, our study will examine the synergistic therapeutic potential of a highly novel Flt3 ligand therapy and ZIKV combination therapy as a strategy to activate both dendritic cell subset-1 (DC-1s) and DC-2, to maximize anti-tumor CD8+ T cell responses and subsequent GBM rejection. This research is significant because 1) it addresses the potential of ZIKV as an effective oncolytic virus for GBM treatment, 2) identifies key cellular mechanisms that contribute to its anti-tumor effects and 3) addresses the current failure of immunotherapy and maps a therapeutic path forward. 

Recent studies reveal that immunosuppressive regions vary substantially throughout glioblastoma (GBM) tumors. These immune-restricted niches appear to correspond to specific extracellular matrix (ECM) components that are thought to drive regional mesenchymal transition. We hypothesize that these distinct GBM mesenchymal regions both coincide with, and are responsible for, monocytic myeloid-derived suppressor cell (M-MDSC) infiltration. Our project hypothesis predicts that changes in regional hyaluronic acid (HA) extracellular matrix (ECM) stiffness induces the activation of proneural GBM cell surface CD44 that, in turn, drives simultaneous mesenchymal transition and increases an M-MDSC recruiting/differentiation transcriptional program. Studies proposed in this application will rigorously delineate the mechanistic pathway and effectors involved by focusing on a hypothesized HA→CD44→STAT3/ZEB1-dependent pathway and (2) delineate whether/how regional activation of this pathway results in a STAT3/ZEB1-dependent induction of IL-6/IL-17-mediated regional accumulation of M-MDSC-dependent immune suppression in GBM. We anticipate that results from this proposal will identify a novel targetable axis and address a critical gap in our understanding of the impact of biophysical changes in GBM TME. 

Recent studies from our laboratory revealed that age-related anti-viral immune dysfunction in the respiratory tract is driven by the increased expression of an inducible phospholipase A2 group IID (PLA 2G2D) enzyme and the prostaglandin D 2 receptor, DP1. Specifically, we discovered that age-related mortality in SARs-CoV2-infected mice were largely reversed in both PLA2G2D-/- and DP1 -/- mice as a consequence of enhanced lung dendritic cell (DC) mediated anti-viral-specific T cell responses. These survival benefits were subsequently confirmed in mice treated with a highly specific DP1 receptor antagonist, Asapiprant, which is currently being tested in phase II clinical trials. In our new preliminary studies, we have confirmed this age-related immune dysfunction pathway also exists in metastatic lung tumor-bearing mice. Specifically, we have found that PLA2G2D or DP1 deficiency, very effectively restricts the growth of melanoma lung metastasis in a manner that correlates with a substantial early enrichment of -T cells. In this phase 2 CCII COBRE project, we hypothesize that PLA2G2D/PGD2-DP1 signaling contributes to primary and metastatic tumor growth in the lung and acts by inhibiting age-associated inflammasome activation in lung DCs. To test this hypothesis, we will: 1) Determine the mechanisms and signaling effectors underlying PLA 2G2D/PGD2-DP1 signaling in lung DCs from aged, tumor-bearing mice, and 2) Investigate the potential of the DP1-targeting small molecule, Asapiprant, as a potential mono-immunotherapy in treating clinically relevant lung primary and metastatic disease models. Finally, these studies will evaluate whether DP1 targeting in immune checkpoint blockade (ICB) resistant implantable and spontaneous tumor models is sufficient to reduce resistance/increase efficacy in combination with anti-PD-1. 

Immunotherapy has been gaining increased attention in cancer treatment. Considering the versatile mechanisms of tumor evasion from the host immune system, the RNAi approach can be utilized targeting different molecules to enhance the immune response against tumors. In most cancer cells PD-L1 binds to PD1 on the surface of lymphocytes and allows the immune system to escape. The interaction between PD-L1 and PD-1 on CD8+ T cells results in upregulation of Cbl E3 ubiquitin ligases in activated T cells and subsequent downmodulation of T cell receptors. shRNA-mediated PD-L1 silencing leads to hyperactivated proinflammatory T cell receptor CD8+ T cells with considerably accelerated anti-tumor immune responses. The knockdown of PD-L1/PD-L2 improves T-cell proliferation and cytokine production. Anti PD-1/PD-L1- based therapeutics (atezolizumab, avelumab and durvalumab) have significantly improved the survival of lung cancer patients. Yet, the overall response rate remains unsatisfactory (<20%) owing to the fact that cancer cells release PD-L1 in soluble form into the tumor microenvironment, interfering with antibody treatment efficacy. With siRNA therapy, reduced PD-L1 production on the cancer cell surface allows T-cells to detect cancer cells more efficiently and prevent tumor progression.  

Thus, the limitations of cancer immunotherapy to solid tumors due to issues such as immunosuppressive tumor microenvironment, ineffective trafficking, tumor antigen heterogeneity and other severe adverse effects can be addressed and overcome using siRNA therapy.   

The goal of this project is to use novel nanoplatform of exosome-polyethyleneimine (PEI) matrix (EPM) to deliver siRNA. I hypothesize that siPD-L1 embedded in EPM is protected from nuclease degradation and functions to knockdown tumor surface bound PD-L1 leading to lung cancer cells inhibition. The strategy will be tested against lung cancer as a model with wide applications. The formulation will be functionalized with folic acid (FA) to target tumor cells which have higher expression of folate receptors. 

The recent development of adoptive cell transfer immunotherapies, such as chimeric antigen receptor-modified T cells (CAR T), is revolutionizing treatment of cancer and other diseases. Recent clinical trials in blood cancers (i.e., lymphoma and leukemia) have achieved impressive remission rates, but there are still significant challenges that limit the safety and availability of CAR T therapies. Life-threatening side effects are common, including cytokine release syndrome (>70% of patients) and neurotoxicity (>50% of patients) with delirium, seizures, and coma occurring in some patients. In addition, T cell processing techniques are currently very complex, inefficient, costly, and time-consuming. There is a significant need for improved methods to rapidly and precisely transfect T cells for adoptive cell transfer treatments such as CAR T. Current techniques to engineer T cells generally involve retroviral or lentiviral vectors which raises safety concerns, such as sustained transgene expression, overactivation of T cells, risk of insertional mutagenesis, and risk of adverse residual impurities from host cells. In addition, retrovirus and lentivirus production is very expensive and time-consuming, which can limit availability of this treatment. Therefore, there is a significant unmet need for effective nonviral transfection techniques that can increase safety, throughput, and scalability of cell therapy manufacturing. To address this unmet need, we are developing a novel 3D-printed acoustofluidic system which combines ultrasound waves with a continuous flow chamber to rapidly load biomolecules into T cells via “sonoporation.” Similar to electroporation, sonoporation is a phenomenon in which microbubble cavitation is driven by ultrasound waves and induces transient pores in cell membranes, enabling rapid delivery of compounds, such as nucleic acids or proteins, directly into the cytoplasm within seconds. The acoustofluidic system enables sequential transfection of T cells with high precision for adoptive cell transfer applications such as CAR T. We hypothesize that the acoustofluidic system can enable sustained CAR expression (using CRISPR/cas9) or transient CAR expression (using mRNA) in human T cells, which could potentially lead to future point-of-care treatments. Therefore, the objective of this proposal is to assess acoustofluidic delivery of CRISPR/cas9 or mRNA to human T cells for sustained or transient CAR expression to increase the safety and availability of CAR T therapy.

The introduction of immune checkpoint inhibitors (ICIs) which act through modulation of the host cytotoxic immune responses have dramatically improved durable response in the treatment of advanced melanoma. However, a majority of patients develop ICI resistance (1) . The literature reflects two divergent hypotheses regarding the development of secondary ICI resistance (2). One emphasizes changes in the localized interaction between the tumor and responding immune cells, while the other emphasizes immunophenotypic exhaustion, measured as increased expression of co-inhibitory modulators (LAG3, B2M, Tim-3 etc.) (3-5). In preliminary studies of patients with multiple distant metastatic lesions, who progressed while receiving anti-PD-1 therapy, we observed simultaneous progression of each independent lesion. These data support the hypothesis that secondary resistance to anti-PD-1 therapy is driven by changes in the host immune response. Prior studies to date have revealed evidence of immunophenotypic traits (e.g. expression of LAG3, B2M, Tim-3 by CD8+ PD1+ Ki67 + T cell sub-populations)(6) associated with ICI resistance (7-10). However, these studies have failed to demonstrate conclusive evidence of causal immunophenotypic changes due to overlapping expression patterns of discriminatory factors between radiographically defined responders and non-responders. We anticipate that these studies have been compromised by multiple confounding factors including: a) failure to distinguish between primary versus secondary ICI resistance, b) inclusion of both PD-1 and CTLA-4 directed therapies which differ in their mechanism of action, c) focusing on a limited number of immunophenotypic features on restricted immune cell lineages, and most importantly d) reliance on radiographically defined response (11). Our group and multiple others (12-17) demonstrate that increasing plasma circulating tumor DNA (ctDNA) concentrations reflect biological tumor progression weeks to months prior to radiographical progression. Consequently, longitudinal studies to identify causal immunophenotypic features associated with acquired resistance, that rely on radiographic response classification, are subject to misclassification of response status. Most notably, misclassification of status as responsive to immunotherapy, when in fact biological therapeutic non-response (not detected by imaging) has already developed. We hypothesize that secondary anti-PD-1 resistance is driven by compensatory host immune factors that diminish the effectiveness of PD-1/PD-L1 blockade. Profiling of plasma ctDNA to define anti-PD-1 resistance will enable a longitudinal study design which avoids confounding due to mis-classification of response status.

Ultraviolet (UV) radiation is a primary cause of skin cancer. Platelet-activating factor (PAF) is one of the lipid mediators in UV-induced immunosuppression signaling. Sentinel lymph nodes (SLNs) are the first organs to receive lymphatic drainage from the primary melanoma. Given the increasing importance of anti-tumor immunity in SLNs, it is critical to understand how PAF may alter the regional immune environment to favor melanoma nodal metastasis. Our rationale is to find the connection between PAF and the immunosuppression status in SLNs in an effort to restore immune function and to control melanoma progression.  

To identify the metabolites that might be involved in governing immune microenvironments from melanoma patients, we compared the metabolic changes in lymphatic fluid samples from high-risk patients with nodal metastasis (SLN+) versus low-risk melanoma patients without nodal metastasis (SLN-). The results showed that lymphatic fluid PAF is 2.97- fold greater in high-risk SLN+ melanoma patients compared with low-risk SLN- patients (p=0.0039). Previous studies have shown that PAF can activate numerous pro-inflammatory chemokines. We observed that CCL17 and CCL23 chemokine levels in serum samples from high-risk SLN+ patients are significantly greater than those in low-risk SLN- patients. CCL17 and CCL23 is believed to contribute to the immunosuppressive microenvironment in SLNs of melanoma patients; we have observed elevated levels of these chemokines in patients who undergo recurrence of their melanoma. Based on these data, we hypothesize that upregulation of PAF can activate the downstream chemokines, resulting in an immunosuppressed environment in SLNs that permits nodal metastasis and melanoma progression. We further hypothesize that reducing PAF levels will restore anti-tumor immunity to better control melanoma progression. The objective of this study is to define the role of PAF in immunosuppression of SLN in melanoma patients.  

We propose a single specific aim: To delineate how the upregulation of lipid metabolite PAF results in immunosuppression status of SLNs. We will further identify metabolic signatures, especially lipid metabolic profiling in lymph fluid, serum, and SLN samples from high-risk versus low-risk melanoma patients by mass spectrometry (MS). Cytokine and chemokine levels in the lymph fluid and blood samples from the above-matched patients will be compared. Since PAF exerts its biological function by binding to a single specific G-protein-coupled receptor (PAFR), we will observe the expression of PAF and PAFR in different types of immune cells in SLN. CD8+ T cell function from SLN samples will be tested after adding PAF. Tregs isolated from SLN samples will be treated with PAFR agonist carbamoyl-PAF (CPAF), and the function of the Tregs will be examined after CPAF treatment. Further studies will use a mouse model to determine whether PAF administration results in SLN immunosuppression and CPAF can restore immune competency.  

The significance of this research is to provide further understanding of lipid metabolite PAF in reduced adaptive anti-tumor immunity in melanoma SLN. Controlling PAF might present a strategy to reconstitute SLN immune function, prevent melanoma progression, and increase the overall survival rate.

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.   

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.   

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. 

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. 

Immune checkpoint blockade has revolutionized cancer therapy. Yet, its clinical benefit is limited to a subset of patients. This complexity is compounded by tumor heterogeneity, which generates distinct immunological architectures and context-dependent vulnerabilities. A key gap remains in understanding how to target these evolving landscapes. We propose a strategy focused on oncogenic drivers that create tumor-specific dependencies, enabling alignment of therapies with responsive populations within the same drivers and tissue type. In triple-negative, basal-like breast cancer (TNBC), TP53 mutations are present in approximately 84% of cases, with ~60% being missense mutations. These mutant p53 proteins are frequently overexpressed and confer oncogenic properties. Though mutant p53 itself is undruggable, its downstream signaling offers promising avenues for precision combinatorial therapies. To investigate these mechanisms, we developed two novel autochthonous somatic mouse models expressing p53R172H or p53R245W hotspot mutations. These models uniquely allow for precise expression of mutant p53 in cancer cells while maintaining wild-type p53 in the surrounding stroma and immune microenvironment, enabling physiologically relevant studies of tumor-immune interactions. Genetic deletion of mutant p53 in vivo significantly reduced tumor growth and prolonged survival, establishing mutant p53 as a therapeutic vulnerability. Using single-cell transcriptomics, we discovered that mutant p53 protects cancer cells from ferroptotic cell death. This ferroptosis resistance was conserved across six human breast cancer cell lines harboring p53 hotspot mutations. Deletion of mutant p53 also led to increased CD8+ T cell infiltration and IFNγ production, implicating mutant p53 in immune evasion. Because ferroptosis activators represent a therapeutic avenue in these cancers, ferroptosis may either enhance immune activation or drive immune suppression, depending on context—underscoring a complex interplay that requires direct experimental testing. To explore these findings, we hypothesize that mutant p53 drives immune suppression/resistance. Our specific aims are: (1) define the impact of ferroptotic injury in spontaneous Trp53-mutant TNBCs by deleting Gpx4 in cancer cells, and (2) perform a targeted CRISPR genetic screen to identify mutant p53- driven factors that render cancer cells resistant to killing under varying immunological conditions. 

Only about 10% of patients with microsatellite stable (MSS) colorectal cancer (CRC) respond to immunotherapy. Patients with locally advanced or metastatic MSS CRC are currently treated with conventional cytotoxic chemotherapy with or without targeted therapy, but 5-year overall survival remains between 15 and 50% and treatment has significant side effects. Identifying a strategy to overcome limited effectiveness of immunotherapy to expand its use in MSS CRC patients would have significant clinical value. To overcome the lack of efficacy of MSS CRC to immunotherapy, the goals of this project are 1) to determine if IL-17 limits the efficacy of immunotherapy in MSS CRC and the source of IL-17 production and 2) to determine the role of γδT cells and neutrophils in limiting efficacy of immunotherapy in MSS CRC. Building upon our previous experience using combination cytokine-based therapy to effectively treat CRC, we noted a lack of α-PD-1 monotherapy that mirrors clinical experience as well as the presence of IL-17 producing cells in the lamina propria and mesenteric lymph nodes, which are present in human CRC. The role of IL-17 in immunotherapy response and its mechanisms remain unclear and limit potential therapeutic strategies. In this proposal, we will overcome these limitations through systematic evaluation of IL-17 production and cellular function in CRC. We will do this through focused cellular evaluation of the colon lamina propria and mesenteric lymph nodes in mice with established CRC. These strategies will allow for determination of potential therapeutic targets to improve immunotherapy efficacy in CRC. We hypothesize that 1) IL-17 produced by γδT cells diminishes the effect of αPD-1 in MSS CRC and 2) the effects of IL-17 on αPD-1 efficacy are mediated by neutrophil interaction with cytotoxic T effector cells. Our long-term goal is to develop a rational immunotherapy strategy to improve outcomes in patients with MSS CRC.