Following are public and technical abstracts for the CAR T-Cells project funded by the Department of Defense Kidney Cancer Research Program (KCRP) for 2017.
Principal Investigator: Wayne Marasco
Institution: Dana-Farber Cancer Institute
Funding Mechanism: Idea Development Award — Established Investigator
Award Amount: $698,272
Public Abstract
Background: A modified T cell receptor called Chimeric Antigen Receptor (CAR) T cell therapy (CART) is a new form of cellular immune therapy to treat cancer. It has proven to be a powerful, clinically translatable immunotherapy for hematologic malignancies. This new therapy is made by extracting immune system T cells from an individual patient, altering their DNA to sharpen their ability to spot and kill cancer cells, and infusing them back into the same patient. On August 30, 2017, the first CART cell therapy was approved for treatment of certain children and young adults with B-cell precursor acute lymphoblastic leukemia (ALL) that is refractory or in second or later relapses. Subsequently, FDA approved the second CART cell therapy on October 18, 2017, for patients with large-B-cell lymphomas who had received at least two prior treatment regimens. However, this promising treatment has not been translatable to solid tumors. There are several reasons why CART cell immunotherapy has not been successful for solid tumors. These include inefficient homing of CART cells to tumor locations, low persistence of CART cells in vivo, and the strong ability of tumors to suppress our immune system. There are also safety concerns since protein targets that are expressed on the surface of solid tumors are often also expressed on normal tissues, which can cause the CART therapy to have adverse side effects that are sometimes severe. The goal of our translational proposal is to engineer improvements in CART cells so that they can be safely delivered to patients with clear cell renal cell carcinoma (ccRCC) with the goal of achieving cancer cures.
Objectives: In solid tumors, a normal process by which our immune system limits damage after an infection or injury, called checkpoint blockade, is highjacked by the cancer cells. Recently, new drugs called monoclonal antibodies that are based on proteins that our immune system produces have been developed to invigorate our immune systems to fight cancer. We have generated remarkable preclinical data demonstrating that we can engineer CART cells to produce these types of antibody drugs at the tumor site, which has shown a profound ability to restore anti-cancer immunity. We demonstrated that this was possible by engineering CART cells to produce a human antibody drug called anti-PDL1. We call this new therapy CART cell factories and the goal of our proposal is to make safer CART cell factories to treat RCC.
Areas of Emphasis: Immunotherapies
Innovative Aspects of Proposal Research Project: We are biomedical engineers with years of experience in therapeutic human antibody drug development. We will use our tools to make CART cells recognize two proteins on the RCC surface instead of just one protein. We believe that this engineering advance will make the CART factories safer and decrease the likelihood that they will inadvertently bind to and injure healthy non-cancer cells in the body.
Short-Term Impact of Proposed Research: We will perform discovery research to identify antibodies to a second RCC target protein called CD70; we already have antibodies to the first target called CAIX. We have other technology that allows us to tether two antibodies together and our plan is to engineer CART cells to recognize both CAIX and CD70. Once we accomplish this we will test the therapy in tissue culture and in humanized mice; the latter refers to a weakened strain of mice that can be made to carry a primitive human immune system. We will test these humanized mice carrying human RCC tumors for the ability of the CART factories to kill the tumor cells and restore anti-cancer immunity without causing adverse side effects.
Long-Term Impact of Proposed Research on Service Members, Their Families, Veterans, and the American Public: Our long-term goal is to move this innovative therapy into the clinic. Our studies will allow us to understand how to use CART cell factories to reverse the suppression of the immune system to fight kidney cancer. We believe that no one should die of kidney cancer. We are also warriors, but our battle is not on the field with weapons; it is rather in the laboratory, where we can win the battle for our Service members, their families, Veterans, and the American people.
Technical Abstract
Background: Chimeric Antigen Receptor (CAR) T cells have proven to be a powerful, clinically translatable immunotherapy for hematologic malignancies. In 2017, the first two CAR T cell therapies were approved by the FDA, the first for the treatment of children and young adults with B-cell precursor acute lymphoblastic leukemia (ALL) and the second for patients with large-B-cell. However, this promising treatment targeting CD19+ malignant B cells has not been translatable to solid tumors due, in large part, to limitations in efficiency and safety. Inefficient homing of CART cells to tumors, due to physical and metabolic barriers, low persistence of CART cells in vivo, and the profound immunosuppressive microenvironment of solid tumors all contribute to limiting clinical efficacy. Safety limitations such as on-target off-tumor toxicities are due to the sharing of CART targeting epitopes on normal tissues, a limitation that is not seen with anti-CD19 CART cells.
We have generated remarkable preclinical data on restoring efficacy of CART therapy for clear cell (cc) renal cell carcinoma (RCC), which forms the basis of this proposal. Our strategy, called CART factories, is designed to modulate the tumor microenvironment through the ability of CART cells to secrete human anti-immune checkpoint inhibitor monoclonal antibodies (mAbs) locally at the tumor site to reverse T cell exhaustion and restore effective anti-cancer immunity. We established proof-in-principle by engineering anti-CAIX CART cell factories to secrete human anti-PDL1 mAb. Our results show a dramatic improvement in CART killing of ccRCC in vitro and in vivo by reversing CART cell and tumor infiltrating lymphocyte (TIL) exhaustion. Moreover, this combination immunotherapy is formulated so that it can be administered as a single agent.
Areas of Emphasis: Immunotherapies
Hypothesis/Objective: Increased safety (restricted on-target on-tumor killing) and efficacy of 2nd generation CART factories to achieve ccRCC cures can be accomplished through the engineering of bispecific anti-CAIX/CD70 CART factories with optimized co-stimulatory domains.
Specific Aims
Aim 1. Develop bispecific tandem CART cell factories that recognize CAIX and CD70, both of which are overexpressed on ccRCC. We will use our proprietary tetravalent bispecific human antibody format as the targeting moiety and compare targeting specificity to either single-targeted CAR. To improve in vivo persistence of CART factories, both 2nd generation 4-1BB and 3rd generation tandem CD28/4-1BB costimulatory domains will be tested and compared to CD28 domain encoding CAR vectors.
Aim 2. Perform validation studies in vitro and in vivo that the bispecific anti-CAIX/CD70 CART cell factories with optimal signal transducing activity have greater killing specificity and potency for ccRCC cells than single-target CART cells. This will be validated in an orthotopic humanized mouse-ccRCC model.
Study Design
Aim 1. Our 27-billion-member human scFv-phage display library will be used to pan against the soluble extracellular domain of CD70 to obtain candidate anti-CD70 scFv targeting moieties; further selection and characterization of individual anti-CD70 scFvs will be performed sequentially through scFv-yeast display. Unique anti-CD70 scFvs will be characterized for binding kinetics (Kon/Koff), affinity (Kd) studies, and epitope mapping. Studies will be performed to ensure that the anti-CAIX/CD70 CART cells remain in basal state and free of exhaustion in the absence of ccRCC engagement.
Aim 2. In vitro killing assays and in vivo therapeutic studies will be performed using our novel neonatal humanized NSG-SGM3 mouse-RCC model comparing single-targeted versus dual-targeted CART factories. Innate and adaptive tumor immune infiltrates will be analyzed by multiparameter FACS and IHC staining. The tumor microenvironment will also be analyzed by single-cell RNA sequencing using 10X Genomics Chromium Single Cell 5′ platform. The new 5′ platform will allow us to quantitate individual cellular transcripts but also to capture B cell V(D)J and T cell VJ recombination so complete immune repertoire analysis before and after CART factory therapy can be obtained.
Innovation: CART factories represent a highly innovative advance to the field of CART therapy. We will apply our proprietary tetravalent bispecific antibody technology to improve the safety of this cellular therapy.
Impact: Our bispecific anti-CAIX/CD70 CART factories should result in a new cellular therapy for ccRCC that steadily advances us toward a cure.