Gertrude Gunset

Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta /CD28 receptor

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-ζ (TCRζ), 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.

Targeted Elimination of Prostate Cancer by Genetically Directed Human T Lymphocytes

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 CD3zeta 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.

CD19 CAR-targeted T cells induce long-term remission and B Cell Aplasia in an immunocompetent mouse model of B cell acute lymphoblastic leukemia.

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.

Monitoring the Efficacy of Adoptively Transferred Prostate Cancer–Targeted Human T Lymphocytes with PET and Bioluminescence Imaging

Noninvasive imaging technologies have the potential to enhance the monitoring and improvement of adoptive therapy with tumor-targeted 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 prostate-specific 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 2′-18F-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-ethyluracil (18F-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 18F-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.

Donor CD19 CAR T cells exert potent graft-versus-lymphoma activity with diminished graft-versus-host activity

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 to the success of allo-HSCT. Chimeric antigen receptors (CARs) direct tumor cell recognition of adoptively transferred T cells. CD19 is an attractive CAR target, which is expressed in most B cell malignancies, as well as in healthy B cells. Clinical trials using autologous CD19-targeted T cells have shown remarkable promise in various B cell malignancies. However, the use of allogeneic CAR T cells poses a concern in that it may increase risk of the occurrence of GVHD, although this has not been reported in selected patients infused with donor-derived CD19 CAR T cells after allo-HSCT. To understand the mechanism whereby allogeneic CD19 CAR T cells may mediate anti-lymphoma activity without causing a significant increase in the incidence of 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 experience enhanced stimulation, resulting in the progressive loss of both their effector function and proliferative potential, clonal deletion, and significantly decreased occurrence of GVHD. Concurrently, the other CAR T cells that were present in bulk donor T cell populations retained their anti-lymphoma activity in accordance with the requirement that both the T cell receptor (TCR) and CAR be engaged to accelerate T cell exhaustion. In contrast, first-generation and 4-1BB-costimulated CAR T cells increased the occurrence of GVHD. These findings could explain the reduced risk of GVHD occurring with cumulative TCR and CAR signaling.

Abstract 3499: CD56 targeted chimeric antigen receptors for immunotherapy of multiple myeloma

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

Tumor-Targeted Human T Cells Expressing CD28-Based Chimeric Antigen Receptors Circumvent CTLA-4 Inhibition

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.

207. Optimal Activation of Both CD28 and 4-1BB in CAR-Targeted T Cells Results in Enhanced Tumor Eradication

Chimeric Antigen Receptors (CARs) are a novel drug class, consisting of engineered receptors for antigen that redirect and reprogram T-cell function. We have shown in both xenogeneic tumor models as well as in leukemia patients, that CD19-targeted T-cells are capable of inducing complete remission of relapsed or refractory B cell malignancies. The inclusion of appropriate co-stimulation is essential for the potency, quality and durability of the CAR T cell response. Here we show that the co-stimulatory domain incorporated within the CAR dictates the kinetics of in vivo anti-tumor response and T-cell accumulation. Additionally, expression of co-stimulatory ligands complementary to the second-generation CAR provides an optimal balance of cytolytic activity and T-cell accumulation resulting in superior anti-tumor function. The combination of 19-28z CAR with 4-1BBL was associated with activation of the endogenous Interferon-β (IFNβ) pathway in an IRF7-dependent manner. This enabled very small T-cell doses to eradicate systemic established leukemia in a xenogeneic model. The unexpected role of the IRF7/IFNβ pathway provides insight into the synergy behind co-stimulatory molecules in T cell activation and a novel mechanism through which engineered T cells can increase tumor control.

763. Multimodality Imaging of Genetically Modified Lymphocytes In Vivo

Background: the lack of specific trafficking of genetically engineered T cells to tumor sites is one of the major limitations in adoptive immunotherapy. Multimodality imaging represents a powerful tool for repetitive non-invasive assessment of migration, activation and survival of immune effectors in living subjects in pre-clinical models and in patients. Methods: to establish a cancer model for adoptive immunotherapy we challenged immunodeficient mice with RM1 murine prostate carcinoma cells transduced with human prostate-specific membrane antigen (PSMA). The tumor cells were also co-transduced with dual fusion (red fluorescent protein and Renilla lusiferase) reporter gene for imaging of tumor progression. Human peripheral blood T-lymphocytes were transduced with chimeric receptors specific either for PSMA or CEA (control) and with reporter vector bearing HSV1-thymidine kinase (HSV1-tk), green fluorescent protein (GFP) and Click Beetle luciferase (CBR). GFP was used for assessment of transduction efficiency of T-lymphocytes and T-cell tumor infiltration. After ex vivo expansion genetically modified PSMA-specific or CEA-specific (control) lymphocytes were adoptively transferred into tumor bearing mice. Metastatic lung tumors were detected using in vivo bioluminescent imaging in all animals on the day of lymphocyte injection and thereafter. All mice underwent biolumincescent and microPET imaging with [18F]FEAU. Results: on day 1 after T-cell injection distinct luciferase and PET signals were detected in both PSMA-specific and CEA-specific (control) lymphocyte groups. On day 3 clear signal was obtained only in PSMA-specific lymphocyte group. PET signal was not detected on day 6 in either group. The %ID/g differed significantly on day 3 between two groups (Table 1). The bioluminescence data corroborated PET data. Tumor growth delay was observed using bioluminescent signal dynamic in the group, injected with PSMA-specific lymphocytes. Statistically significant survival advantage (p=0.01) was also observed in the same group. The presence of human genetically modified lymphocytes as well as PSMA antigen in lung tumor was confirmed by immunofluorescence and immunohistochemistry. Conclusion: multimodality imaging strategy is feasible and can be applied for longitudinal visualization of genetically modified immune cells and prediction of adoptive immunotherapy outcome.