Overall Goals The primary objective of this MIRA is to provide cancer patients with a cell therapy that is safe and effective, avoiding the harsh side effects typically associated with surgeries and standard chemo-radiotherapies used in cancer treatment. Our therapy utilizes T-lymphocytes, a type of immune cell, engineered to carry proteins called chimeric antigen receptors (CARs). These CARs are designed to specifically recognize and eliminate cancer cells. The normal role of T-lymphocytes is to search and destroy abnormal cells, including cancerous or virus-infected cells. When equipped with CARs, these modified T-lymphocytes (referred to as CARTs) can precisely locate and kill tumor cell...

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Overall Goals The primary objective of this MIRA is to provide cancer patients with a cell therapy that is safe and effective, avoiding the harsh side effects typically associated with surgeries and standard chemo-radiotherapies used in cancer treatment. Our therapy utilizes T-lymphocytes, a type of immune cell, engineered to carry proteins called chimeric antigen receptors (CARs). These CARs are designed to specifically recognize and eliminate cancer cells. The normal role of T-lymphocytes is to search and destroy abnormal cells, including cancerous or virus-infected cells. When equipped with CARs, these modified T-lymphocytes (referred to as CARTs) can precisely locate and kill tumor cells, sparing healthy surrounding cells. This approach reduces the risk of long-term adverse effects like infertility, developmental issues, heart complications, or secondary malignancies often seen with conventional therapies. Although some CART therapies may cause short-term, reversible side effects, they are far less severe than caused by traditional treatments. Challenges with CART Therapies While CART therapies have shown great success in treating certain blood cancers, they have been less effective for solid tumors like liver and breast cancer, which are a focus of this research. The main problem is that solid tumors lack the necessary signals that CARTs need to grow and effectively destroy all cancer cells. Additionally, tumor cells have various mechanisms to evade or weaken T-cells. They produce proteins that deactivate T-cells and attract other inhibitory cell types that guard the tumor, preventing CARTs from entering the tumor site and performing their cancer-killing function. A second challenge with CART therapies is that they need to be custom-made from each patient's T-lymphocytes. This process is time-consuming, often taking several weeks, and requires skilled personnel and specialized, costly facilities. Additionally, patients who have undergone extensive chemotherapy may have damaged T-cells, making them hard to cultivate in the laboratory. One proposed solution is to create "off-the-shelf" (OTS) CARTs from healthy donors. These OTS CARTs can treat numerous patients and are available immediately, making the therapy more accessible and affordable. However, the challenge with OTS CARTs is that they carry foreign tissue antigens (allo antigens) that the patient's immune system recognizes. This recognition activates "alloreactive" T-cells (ARTs) in the patient, which then destroy the infused CARTs. Moreover, the CARTs themselves may contain ARTs that can attack the patient, causing a condition known as graft-versus-host disease (GVHD). The solutions: To overcome the first challenge, Projects 1 to 3 have developed clinical trials using enhanced CARTs, which are "armored" with additional genes. These genes provide two critical functions: (1) they supply the signals needed for the CARTs to continue growing and attacking tumor cells for as long as these cells exist, and (2) they equip CARTs with molecules that help them survive in the hostile environment of the tumor (the TME) and potentially combat the tumor and its protective cells. These innovative approaches are crucial for creating effective CART therapies for tumors that are currently difficult to treat. Project 4 focuses on research using mice to understand the factors that determine the success or failure of CART therapies in treating tumors. This research aims to identify logical modifications to enhance the effectiveness of CARTs, based on their performance in these animal studies. Project 4 introduces an element called CAdVEC in the fight against cancer. CAdVEC is a combination of two types of adenoviruses: one, known as OAd, specifically targets and kills only cancer cells, and the other, HDAd, has been modified to carry genes that support T-cell growth and transform the TME from one that suppresses CARTs to one that aids them. This dual action of CAdVEC not only destroys tumor cells but also enhances the effectiveness of CARTs. CAdVEC has been safe in a clinical study and, when used in combination with CARTs, has shown promising results in treating patients with head and neck cancer. Building on this success, Project 4 is now applying CAdVEC to triple negative breast cancer (TNBC), pairing it with CARTs that target B7H3, a protein found in about 60% of TNBC cases. This approach represents a significant advance in the treatment of this challenging cancer type. Projects 3 and 4 are focused on solving the issues related to the rejection of off-the-shelf (OTS) CARTs and graft-versus-host disease (GVHD). Their innovative approach to prevent GVHD involves creating CARTs from T-lymphocytes that only recognize the Epstein-Barr virus (EBV), thus lacking alloreactive T-cells (ARTs) that cause GVHD. To counteract the rejection of these CAR-EBV-specific T-cells (CAR-EBVSTs), they explore CARs that target ARTs once they become activated. These CARs are termed "ADRs" (alloimmune defense receptors). Remarkably, Project 3 discovered that a CAR targeting CD30 on lymphoma cells also functions as an ADR. In clinical trials, they found that CD30.CAR-EBVSTs were effective in treating lymphoma, with 78% of patients showing tumor responses, including 39% complete responses. However, these CAR-EBVSTs did not remain in the patients' blood for more than a week, suggesting that not all ARTs were eliminated. To enhance their effectiveness, they are now adding a growth-promoting gene to the 30.CAR.EBVSTs to boost their numbers after-infusion, thereby increasing their potential to eradicate ARTs and, consequently, tumors. A clinical trial for lymphoma patients who have experienced multiple relapses will test whether this strategy prolongs the presence of CAR.EBVSTs in patients and improves their capacity for sustained tumor elimination. In a parallel approach, both Projects 3 and 4 are testing ADRs designed to prevent the growth of ARTs from the outset, rather than eliminating them after activation, as is the case with the CD30.CAR. This complementary strategy aims to further reduce the risk of rejection and enhance the effectiveness of CART therapies. Enhancing CART Safety with "Suicide" Genes Our projects have enhanced CARTs with potent genes to boost their effectiveness. However, to control the risk of excessive CART growth, we have incorporated a safety feature: a "suicide" gene, known as inducible caspase 9 (iC9). This gene is dormant in the CARTs until activated by a specific small molecule called CID, which triggers the iC9 to eliminate the CARTs if they start behaving undesirably. This safety mechanism has shown remarkable success in clinical trials. It can rapidly alleviate clinical symptoms (within 2 hours) and decrease the number of modified T lymphocytes in the blood. An additional advantage of this system is its precision. By administering lower doses of CID, we can selectively eliminate the most excessively growing CARTs while preserving enough to continue controlling and killing tumor cells. The iC9 suicide gene is a critical component, allowing us to safely expand the limits of cell therapy. This is essential for achieving significant therapeutic outcomes in difficult-to-treat cancers, providing both efficacy and safety in these advanced treatments. Types of Cancer Targeted Our research is aimed at treating several types of cancer, focusing on lymphoma, liver cancer, and breast cancer, including the particularly challenging triple-negative breast cancer (TNBC). These cancers have worse outcomes in disadvantaged populations, who may lack sufficient insurance and access to healthcare. Consequently, these patients frequently receive late-stage diagnoses when the cancer is more advanced and harder to treat. Our patient catchment area has a notably young demographic, predominantly consisting of underrepresented minorities, including Hispanic and African American communities. Notably, TNBC is more common among African American women than other racial groups. Additionally, our Cancer Center has identified a significant prevalence of obesity-related cancers, including liver cancer, in our area. Addressing these cancers, particularly in populations that are typically underserved in healthcare, is a key focus of our efforts. Potential Significance and Impact Our research introduces groundbreaking combination therapies that tackle the main challenges in current CART treatments. By incorporating the iC9 suicide gene, we can safely enhance CARTs' potency, mitigating the risk of uncontrolled growth in patients. Our projects work interactively, allowing advancements in one area to benefit others. This collaboration could lead to significant strides, especially if Projects 3 and 4 successfully develop an off-the-shelf (OTS) CART approach that prevents graft-versus-host disease and immune rejection. This OTS strategy could be adapted across all projects to target different cancers, potentially transforming cancer treatment by making CART therapies more accessible and affordable, significantly impacting the field.

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