Artificial receptors provide a promising approach to target T lymphocytes to tumor antigens. However, the receptors described thus far produce either an activation or a co-stimulatory signal alone, thus limiting the spectrum of functions accomplished by the genetically modified cells. Here we show that human primary T lymphocytes expressing fusion receptors directed to prostate-specific membrane antigen (PSMA) and containing combined T-cell receptor-zeta (TCRzeta), and CD28 signaling elements, effectively lyse tumor cells expressing PSMA. When stimulated by cell-surface PSMA, retrovirally transduced lymphocytes undergo robust proliferation, expanding by more than 2 logs in three weeks, and produce large amounts of interleukin-2 (IL-2). Importantly, the amplified cell populations retain their antigen-specific cytolytic activity. These data demonstrate that fusion receptors containing both TCR and CD28 signaling moieties are potent molecules able to redirect and amplify human T-cell responses. These findings have important implications for adoptive immunotherapy of cancer, especially in the context of tumor cells that fail to express major histocompatibility complex antigens and co-stimulatory molecules.

Summary T cell engineering is a powerful means to rapidly generate anti-tumor T cells. The costimulatory properties of second-generation chimeric antigen receptors (CARs) determine the overall potency of adoptively transferred T cells. Utilizing an in vivo “stress test” to challenge CD19-targeted T cells, we studied the functionality and persistence imparted by 7 different CAR structures providing CD28 and/or 4-1BB costimulation. One configuration, which utilizes two signaling domains (CD28 and CD3ζ) and the 4-1BB ligand, provided the highest therapeutic efficacy, showing balanced tumoricidal function and increased T cell persistence accompanied by an elevated CD8/CD4 ratio and decreased exhaustion. Remarkably, induction of the IRF7/IFNβ pathway was required for optimal anti-tumor activity. Thus, 1928z-41BBL T cells possess strikingly potent intrinsic and immunomodulatory qualities.

Although many adults with B cell acute lymphoblastic leukemia (B-ALL) are induced into remission, most will relapse, underscoring the dire need for novel therapies for this disease. We developed murine CD19-specific chimeric antigen receptors (CARs) and an immunocompetent mouse model of B-ALL that recapitulates the disease at genetic, cellular, and pathologic levels. Mouse T cells transduced with an all-murine CD3ζ/CD28-based CAR that is equivalent to the one being used in our clinical trials, eradicate B-ALL in mice and mediate long-term B cell aplasias. In this model, we find that increasing conditioning chemotherapy increases tumor eradication, B cell aplasia, and CAR-modified T cell persistence. Quantification of recipient B lineage cells allowed us to estimate an in vivo effector to endogenous target ratio for B cell aplasia maintenance. In mice exhibiting a dramatic B cell reduction we identified a small population of progenitor B cells in the bone marrow that may serve as a reservoir for long-term CAR-modified T cell stimulation. Lastly, we determine that infusion of CD8+ CAR-modified T cells alone is sufficient to maintain long-term B cell eradication. The mouse model we report here should prove valuable for investigating CAR-based and other therapies for adult B-ALL.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for hematological malignancies. However, graft-versus-host disease (GVHD) and relapse after allo-HSCT remain major impediments. Chimeric antigen receptors (CARs) direct tumor cell recognition of adoptively transferred T cells.1–5 CD19 is an attractive CAR target, expressed in most B cell malignancies as well as normal B cells.6,7 Clinical trails using autologous CD19-targeted T cells have shown remarkable outcomes in various B cell malignancies8–15. The use of allogeneic CAR T cells poses a concern of increased GVHD, which however has not been reported in selected patients infused with donor-derived CD19-CAR T cells after allo-HSCT.16,17 To understand the mechanism whereby allogeneic CD19-CAR T cells may mediate anti-lymphoma activity without significant GVHD, we studied donor-derived CD19-CAR T cells in allo-HSCT and lymphoma models in mice. We demonstrate that alloreactive T cells expressing CD28-costimulated CD19-CARs experienced enhanced T cell stimulation, resulting in progressive loss of effector function and proliferative potential, clonal deletion, and significantly decreased GVHD. Concurrently, other CAR T cells present in bulk donor T cell populations retained their anti-lymphoma activity consistent with the requirement for engaging both the TCR and the CAR to accelerate T cell exhaustion. In contrast, first generation and 4-1BB-costimulated CARs increased GVHD. These findings could explain reduced risk of GVHD with cumulative TCR and CAR signaling.

The genetic transfer of antigen receptors is a powerful approach to rapidly generate tumor-specific T lymphocytes. Unlike the physiologic T-cell receptor, chimeric antigen receptors (CARs) encompass immunoglobulin variable regions or receptor ligands as their antigen recognition moiety, thus permitting T cells to recognize tumor antigens in the absence of human leukocyte antigen expression. CARs encompassing the CD3Z chain as their activating domain induce T-cell proliferation in vitro, but limited survival. The requirements for genetically targeted T cells to function in vivo are less well understood. We have, therefore, established animal models to assess the therapeutic efficacy of human peripheral blood T lymphocytes targeted to prostate-specific membrane antigen (PSMA), an antigen expressed in prostate cancer cells and the neovasculature of various solid tumors. In vivo specificity and antitumor activity were assessed in mice bearing established prostate adenocarcinomas, using serum prostate-secreted antigen, magnetic resonance, computed tomography, and bioluminescence imaging to investigate the response to therapy. In three tumor models, orthotopic, s.c., and pulmonary, we show that PSMA-targeted T cells effectively eliminate prostate cancer. Tumor eradication was directly proportional to the in vivo effector-to-tumor cell ratio. Serial imaging further reveals that the T cells must survive for at least 1 week to induce durable remissions. The eradication of xenogeneic tumors in a murine environment shows that the adoptively transferred T cells do not absolutely require in vivo costimulation to function. These results thus provide a strong rationale for undertaking phase I clinical studies to assess PSMA-targeted T cells in patients with metastatic prostate cancer. (Cancer Res 2005; 65(19): 9080-8)

Noninvasive imaging technologies have the potential to enhance the monitoring and improvement of adoptive therapy with tumortargeted T lymphocytes. We established an imaging methodology for the assessment of spatial and temporal distributions of adoptively transferred genetically modified human T cells in vivo for treatment monitoring and prediction of tumor response in a systemic prostate cancer model. Methods: RM1 murine prostate carcinoma tumors transduced with human prostatespecific membrane antigen (hPSMA) and a Renilla luciferase reporter gene were established in SCID/beige mice. Human T lymphocytes were transduced with chimeric antigen receptors (CAR) specific for either hPSMA or human carcinoembryonic antigen (hCEA) and with a fusion reporter gene for herpes simplex virus type 1 thymidine kinase (HSV1tk) and green fluorescent protein, with or without click beetle red luciferase. The localization of adoptively transferred T cells in tumor-bearing mice was monitored with 29-18 F-fluoro-29-deoxy-1-b-D-arabinofuranosyl-5-ethyluracil ( 18 F-FEAU) small-animal PET and bioluminescence imaging (BLI). Results: Cotransduction of CAR-expressing T cells with the reporter gene did not affect CAR-mediated cytotoxicity. BLI of Renilla and click beetle red luciferase expression enabled concurrent imaging of adoptively transferred T cells and systemic tumors in the same animal. hPSMA-specific T lymphocytes persisted longer than control hCEA-targeted T cells in lung hPSMA-positive tumors, as indicated by both PET and BLI. Precise quantification of T-cell distributions at tumor sites by PET revealed that delayed tumor progression was positively correlated with the levels of 18 F-FEAU accumulation in tumor foci in treated animals. Conclusion: Quantitative noninvasive monitoring of genetically engineered human T lymphocytes by PET provides spatial and temporal information on T-cell trafficking and persistence. PET may be useful for predicting tumor response and for guiding adoptive T-cell therapy.

Adoptive T cell therapy represents a promising treatment for cancer. Human T cells engineered to express a chimeric antigen receptor (CAR) recognize and kill tumor cells in a MHC-unrestricted manner and persist in vivo when the CAR includes a CD28 costimulatory domain. However, the intensity of the CAR-mediated CD28 activation signal and its regulation by the CTLA-4 checkpoint are unknown. We investigated whether T cells expressing an anti-CD19, CD3 zeta and CD28-based CAR (19-28z) displayed the same proliferation and anti-tumor abilities than T cells expressing a CD3 zeta-based CAR (19z1) costimulated through the CD80/CD28, ligand/receptor pathway. Repeated in vitro antigen-specific stimulations indicated that 19-28z+ T cells secreted higher levels of Th1 cytokines and showed enhanced proliferation compared to those of 19z1+ or 19z1-CD80+ T cells. In an aggressive pre-B cell leukemia model, mice treated with 19-28z+ T cells had 10-fold reduced tumor progression compared to those treated with 19z1+ or 19z1-CD80+ T cells. shRNA-mediated CTLA-4 down-regulation in 19z1-CD80+ T cells significantly increased their in vivo expansion and anti-tumor properties, but had no effect in 19-28z+ T cells. Our results establish that CTLA-4 down-regulation may benefit human adoptive T cell therapy and demonstrate that CAR design can elude negative checkpoints to better sustain T cell function.

We have developed an adoptive T cell therapy strategy for treating multiple myeloma using chimeric antigen receptors targeting CD56. CD56 is strongly expressed by malignant plasma cells in 70% of patients with myeloma and represents a potential immunotherapy target. CD56 is also expressed at lower levels on normal tissue types including neuronal cells, NK cells and a subset of activated T cells. A second generation CAR was constructed containing the scFv of the murine monoclonal antibody against human CD56 (N901) as well as the CD28 transmembrane and cytoplasmic signaling domains. CD56 CAR cells were generated by retroviral transduction of human T cells and showed antigen dependent proliferation and cytokine secretion in vitro when stimulated with CD56 positive myeloma cells. In vitro cytotoxicity assays showed significant lysis (40-50% lysis at effector to target ratios > 5:1) of CD56 positive myeloma cell lines compared with a control prostate specific membrane antigen (PSMA) targeted CAR. To further assess the antitumor activity of CD56 CARs in vivo we developed a systemic xenograft model of myeloma by injecting the OPM2 myeloma cell line, modified to express the firefly luciferase gene, intravenously into NOD/SCID Il2rαnull mice. Bioluminescence imaging showed tumor progression predominantly within the bone marrow recapitulating the human disease phenotype. If untreated hind limb paralysis occurred at approximately 35 days following injection of 3x106 tumor cells. Cohorts of mice were then treated by intravenous injection of either 1 or 5 x106 CD56 CAR cells or control PSMA CAR cells at 7 days following tumor injection, when disease was firmly established. At the 1x106 T cell dose tumor development was significantly delayed compared to controls (median survival of 49 days compared with 34 days respectively, p=0.02) but 7/8 mice eventually progressed and had to be euthanised. In contrast all mice receiving 5x106 cells (n=8) showed complete tumor regression and remained tumor free at 3 months. Interferon-α secreting CD56 CAR cells were detected in the peripheral blood of these mice and correlated with tumor bulk with numbers eventually declining to low levels that persisted even at 3 months. These results demonstrate for the first time the impressive anti-tumor efficacy of a CD56 targeted chimeric antigen receptor in a systemic xenograft model of myeloma. CD56 CAR therapy therefore represents an attractive immunotherapy option and its use in patients with relapsed refractory myeloma should be considered. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3499. doi:1538-7445.AM2012-3499