ALBERT

Description: Central nervous system lymphoma (CNSL) is a lymphoid malignancy in which tumors from lymph tissue start in the brain, spinal cord, eyes, and/or meninges (primary CNSL) or present as a result of metastasis from initial systemic sites to the CNS (secondary CNSL). The incidence of primary CNS lymphoma has been increasing over the past 20 years. CNS lymphomas carry a worse prognosis than systemic lymphoma, and therefore, effective treatment is urgently needed for CNS disease. T cells that are genetically engineered with chimeric antigen receptors (CAR) targeting CD19 have broad applications in adoptive therapy of B cell malignancies and have shown tremendous potential in the treatment of systemic lymphoma. During the early phase of CD19-CAR T cell studies, most if not all protocols excluded patients with active CNS involvement. In all CD19-CAR T cell trials, T cell products are administrated intravenously. Systemic CD19-CAR T administration for ALL and DLBCL has resulted in complete remission of concurrent CNS disease. CD19-CAR T cell trafficking to the cerebrospinal fluid (CSF) is frequently reported; however, there has been no evidence thus far to indicate that CAR T cells in CSF are related to neurotoxicity. Therefore, an increasing number of CD19-CAR T cell trial protocols no longer exclude patients with active CNS lymphoma involvement. Based on the success of CD19-CAR T cell therapy in ALL and lymphoma, we aimed to translate this strategy toward a more effective therapy for CNS B cell disease. Methods and Results: Isolated naïve and central memory T cells were genetically modified with CD19-CAR lentivirus and expanded in vitro for 14 days. A mouse model with both CNS and systemic lymphoma in the same animal was established by simultaneously engrafting Daudi cells (human B cell lymphoma) intracranially and subcutaneously into NSG mice. We then administered 2x10^6 CD19-CAR T cells via two delivery routes: intracerebroventricular (i.c.v.) to bypass the blood-brain barrier and target tumor throughout the entire CNS, and intravenous injection (i.v.). We repeatedly observed that a single i.c.v. infusion was capable of completely eradicating CNS lymphoma and systemic lymphoma in all mice by day 14 post CAR T cell infusion and 100% of mice remained tumor free for 300 days until the termination of the experiment. In contrast, a single delivery of CD19-CAR T cells via i.v. infusion resulted in a noticeably delayed antitumor activity with complete remissions only observed approximately 40 days post CAR T cell treatment. Eventually, the tumors relapsed and all i.v. treated mice died before day 180 (Figure 1). T cell trafficking experiments demonstrated that i.c.v. CAR T cells are able to efficiently migrate to the periphery, home to systemic tumor locations, and dramatically expand outside the CNS. We were able to detect CAR T cells in the blood, bone marrow, and spleens of mice that received i.c.v. therapy at 300 days post CAR T cell treatment. These persisting T cells are CD4 dominant and express high levels of CD28 with a broad TCR repertoire. The persisting T cells also maintain anti-tumor functionality and are able to resist tumor re-challenge. Further mechanistic studies indicate that factors within the CSF are able to reprogram i.c.v. infused CAR T cells and upregulate genes that are related to memory function. In conclusion, our studies suggest that CAR T cells administrated via i.c.v. and nurtured by CSF exhibit better efficacy, expansion, and persistence, resulting in disease elimination. More interestingly, i.c.v. delivered CAR T cells efficiently traffic beyond the CNS to the periphery and completely eradicate systemic tumors in the same mouse. This study is the first to demonstrate that locally delivered CAR T cells are capable of efficiently treating both systemic lymphoma and concurrent CNS disease, which can lower the risk of cytokine release syndrome and avoid toxicities derived from lymphodepletion and systemic infusion of CAR T cells. Disclosures Wang: Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding. Budde:Mustang Therapeutics: Consultancy, Other: Licensing Agreement, Patents & Royalties, Research Funding. Brown:Mustang Therapeutics: Consultancy, Other: Licensing Agreement, Patents & Royalties, Research Funding. Forman:Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding.

Description: Multiple myeloma (MM) is an incurable malignancy of plasma cells even with great advances in treatment. Chimeric Antigen Receptor (CAR) directed T cell therapy, which can specifically recognize tumor associated antigens and kill tumor cells in an MHC independent manner, is a promising approach for hematological malignancy. There are several candidate antigens for CAR T cell targeting of multiple myeloma, including BCMA and CS1. Our goal is to develop novel CARs for the treatment of MM and explore the potential benefits of combinatorial therapy of CAR T cells and immunomodulatory drugs (IMiDs) such as lenalidomide. In the present study, we redirected central memory T cells to express second-generation CARs specific for either CS1 or BCMA that incorporate CD28 signaling moieties. Central memory T cells were activated by CD3/CD28 bead stimulation, transduced with lentivirus encoding the CAR construct, and expanded ex vivo. The engineered and expanded CS1 and BCMA CAR T cells exhibited similar phenotypes and comparable in vitro effector function. However, once adoptively transferred into MM tumor-bearing NOD/Scid IL2RγCnull (NSG) mice by intravenous injection of 1x10^6 CAR T cells, CS1 CAR T cells exhibited superior antitumor activity over BCMA CART cells and significantly prolonged mouse survival (P

Description: We have successfully established a clinical platform for CD19 chimeric antigen receptor (CAR) T cells and evaluated its safety and efficacy in a series of pilot clinical trials following autologous hematopoietic cell transplantation (HCT) for treatment of high-risk non-Hodgkin lymphoma (NHL) and acute lymphoblastic leukemia (ALL). However, the full potential of this therapy has been limited by the lack of engraftment and persistence of CAR T cells in patients, most likely due to the inadequate antigen drive to stimulate robust expansion and long-term persistence of the infused CAR T cells. Additionally, CD19+ NHL seems less responsive to current CAR T cell technology than does CD19+ ALL that might be explained by lower engraftment and persistence of CAR T cells in NHL. To improve the efficacy and durability of CAR T cells in these disease settings, we sought to examine a novel approach based on properties of cytomegalovirus (CMV)-specific T cells. Specifically, we select CMV pp65-specific T cells for ex vivo modification with a CAR targeting CD19. After in vitro expansion, CMV-CD19 bi-specific T cells will be infused into the patient. A second round of expansion will be done in vivo, using a CMV vaccine (Triplex), developed and clinically evaluated multiple clinical sites including City of Hope. Triplex is a multi-antigen recombinant modified vaccinia Ankara (MVA) virus vaccine expressing pp65, IE1 and IE2, that has proven safe and immunogenic in Phase I trials in healthy volunteers and transplant patients and was highly tolerable and efficacious in a completed Phase II vaccine trial in allogeneic HCT recipients. We hypothesis that shorter ex vivo expansion time will prevent CAR T cell exhaustion that results in better in vivo expansion, especially after Triplex vaccination. Thus far, we have performed six large-scale manufacturing process development (PD) runs of CMV-CD19 CAR T cells using T cells from healthy donors. Briefly, peripheral blood mononuclear cells (PBMCs) were collected and processed in the CliniMACS Prodigy® system, in which PBMCs were first stimulated with a GMP-grade PepTivator® overlapping CMV pp65 peptide pool, then enriched for CMV-responsive IFNɣ+ T cells using the IFNγ Catchmatrix reagent (Miltenyi Biotec Inc.). CMV-responsive IFNɣ+ T cells were next transduced with a lentiviral vector encoding EGFRt/CAR, and finally expanded for approximately 15 days in vitro. As summarized in Table 1, we consistently recovered 4.8%+/-1.4 ×106 CMV specific T cells with 78.3%±2.9 purity from 1×109 PBMC input, which is the maximum input number suggested by the CCS program associated with the Prodigy system (Miltenyi Biotec Inc.). During the early stage PD runs, we noticed that red blood cell contamination in the PBMC layer after ficoll separation could negatively impact the yield of CMV-specific T cells. Thus, we optimized the separation process by performing a second ficoll whenever a red buffy coat was observed, and increased the yield of CMV-specific T cells from 2.8 ×106 to 9.25×106 (Table 1, HD 187.2 PBMCs underwent 2 subsequent ficolls). Phenotypic analysis demonstrated that freshly isolated CMV-specific T cells consisted of four different memory subsets (TEMRA, Tscm, Tcm and effector memory T cells). After in vitro expansion for 15 days, CMV-CAR bi-specific T cell expansion was significantly improved from 60×106 cells in the early stage runs to 200×106 in the late stage optimized runs (Figure 1). The CMV-CD19CAR T cells expressed CD62L, though not exhaustion markers such as PD1. Bispecific T cells exhibited bi-functionality upon stimulation with CD19+ tumor cells or CMVpp65 antigen, as indicated by secretion of IFNγ. Interestingly, we detected stronger effector function against CD19+ tumor cells from CMV-CD19CAR T cells compared to conventional CD19CAR T cells that were derived from the same donor (Figure 2). To validate our manufacturing process, we are currently conducting IND-enabling studies using patient-derived PBMC as the starting population and will initiate our first clinical trial in early 2020 for patients with intermediate/advanced-grade B cell NHL post lymphodepletion or autologous/allogeneic HCT followed by Triplex vaccination 28 days after T cell infusion for in vivo expansion of bi-specific CAR T cells. The primary objectives of these two trials are to examine safety and persistence/expansion of CMV-CD19CAR T cell before and after Triplex vaccine boost. Disclosures Nakamura: Merck: Membership on an entity's Board of Directors or advisory committees; Celgene: Other: support for an academic seminar in a university in Japan; Alexion: Other: support to a lecture at a Japan Society of Transfusion/Cellular Therapy meeting ; Kirin Kyowa: Other: support for an academic seminar in a university in Japan.