Chimeric immunoglobulin-T Cell Receptor Research Articles

Advanced generation anti‐prostate specific membrane antigen designer T Cells for prostate cancer immunotherapy

Adoptive immunotherapy by infusion of designer T cells (dTc) engineered with chimeric antigen receptors (CARs) for tumoricidal activity represents a potentially highly specific modality for the treatment of cancer. In this study, 2nd generation (gen) anti-prostate specific membrane antigen (PSMA) dTc were developed for improving the efficacy of previously developed 1st gen dTc for prostate cancer immunotherapy. The 1st gen dTc are modified with chimeric immunoglobulin-T cell receptor (IgTCR) while the 2nd gen dTc are engineered with an immunoglobulin-CD28-T cell receptor (IgCD28TCR), which incorporates a CD28 costimulatory signal for optimal T cell activation. A 2nd gen anti-PSMA IgCD28TCR CAR was constructed by inserting the CD28 signal domain into the 1st gen CAR. 1st and 2nd gen anti-PSMA dTc were created by transducing human T cells with anti-PSMA CARs and their antitumor efficacy was compared for specific activation on PSMA-expressing tumor contact, cytotoxicity against PSMA-expressing tumor cells in vitro, and suppression of tumor growth in an animal model. The 2nd gen dTc can be optimally activated to secrete larger amounts of cytokines such as IL2 and IFNγ than 1st gen and to proliferate more vigorously on PSMA-expressing tumor contact. More importantly, the 2nd gen dTc preserve the PSMA-specific cytotoxicity in vitro and suppress tumor growth in animal models with significant higher potency. Our results demonstrate that 2nd gen anti-PSMA designer T cells exhibit superior antitumor functions versus 1st gen, providing a rationale for advancing this improved agent toward clinical application in prostate cancer immunotherapy.

Phase I trial of anti-PSMA designer T cells in advanced prostate cancer.

130 Background: We created chimeric immunoglobulin-T cell receptors (IgTCR) specific for prostate specific membrane antigen (PSMA). When expressed in patient T cells, these “designer T cells” (dTc) specifically kill prostate cancer cells in vitro and in vivo in animal models, with 5/9 (55%) of xenografted mice experiencing complete remissions (Ma et al. Prostate 2004:61:12–25). A phase I clinical trial was approved by the FDA. Methods: Patient T cells are retrovirally transduced and expanded ex vivo to span dose levels of 10^9 to 10^11 T cells. Patients undergo prior non-myeloablative (NMA) conditioning to create “hematologic space” into which designer T cells are infused for stable engraftment and prolonged in vivo efficacy. Patients are co-administered continuous infusion IL2. Outcomes include Phase Ia goals of safety and Phase Ib goals of establishing an optimal biologic dose in terms of designer T cell engraftment. Results: Five patients have been treated, three at 10^9 and two at 10^10 cell dose levels. Excellent T cell modifications of 30%-60% were obtained. After NMA conditioning, T cells were infused. Stable engraftments of 1%-20% post-recovery at one month after T cell infusion, thus affirming one of the study end-points. Patients experienced neutropenic fever after conditioning, but no designer T cell-related toxicities. Two patients had partial responses with PSA reductions of 50 and 70% in the two months following treatment with projected benefits of 4 months gain of time to return to starting PSA levels. Results will be updated at the conference. Conclusions: A new approach to adoptive immune therapy in metastatic prostate cancer has been devised with encouraging early results. Patients are being actively recruited. This clinical trial received partial funding from the US Army/DOD. Preclinical work was supported by the Prostate Cancer Foundation. No significant financial relationships to disclose.

Phase I trial of anti-PSMA designer T cells in advanced prostate cancer.

e13614 Background: We created chimeric immunoglobulin-T cell receptors (IgTCR) specific for prostate specific membrane antigen (PSMA). When expressed in patient T cells, these “designer T cells” specifically kill prostate cancer cells in vitro and in vivo in animal models (Ma et al. Prostate 2004:61:12-25). A phase I clinical trial was approved by the FDA in metastatic prostate cancers. Methods: Patient T cells are retrovirally transduced and expanded ex vivo to span dose levels of 10^9 to 10^11 T cells. Patients undergo prior non-myeloablative (NMA) conditioning to create a “hematologic space” into which the designer T cells are infused to expand and stably engraft for prolonged in vivo efficacy. Patients are co-administered continuous infusion outpatient IL2 at 75,000 IU/kg/d ×28d. Outcomes include phase Ia goals of safety and toxicity and phase Ib goals of establishing an optimal biologic dose in terms of designer T cell engraftment and tumor response. Results: Three patients were treated at the 10^9 cell dose and one at the 10^10 cell dose. All patients experienced neutropenia and fever post conditioning, but no designer T cell-related toxicities. T cells were infused and engraftments of 1-20% were observed one-month post recovery, thus affirming one of the study end-points. Two patients (both at the 10^9 cell level) had PSA reductions of 50 and 70% in the two months following treatment, whereas two had no response (<20%) (one at 10^9 and one at 10^10 cells). Systemic IL2 was more than 10-fold lower in the two non-responders than in the two responders (2 IU/ml versus 30 IU/ml), suggesting need for adequate IL2 to realize designer T cell therapeutic activity. Patient enrollments are ongoing; data will be updated at conference time. Conclusions: A new approach to adoptive immune therapy in metastatic prostate cancer has been devised with encouraging early results. Patients are being actively recruited. This clinical trial received partial funding from the US Army/DOD. No significant financial relationships to disclose.

Phase I trial of anti-PSMA designer T-cell autografting in prostate cancer

e16132 Background: We created chimeric immunoglobulin-T cell receptors (IgTCR) specific for prostate specific membrane antigen (PSMA). When expressed in patient T cells, these “designer T cells” specifically kill prostate cancer cells in vitro and in vivo in animal models, with 5/9 (55%) of xenografted mice experiencing complete remissions (Ma et al. Prostate 2004:61:12–25). A Phase I clinical trial was approved by the FDA in metastatic prostate cancers. Methods: Patient T cells are retrovirally transduced and expanded ex vivo to span dose levels of 10^9 to 10^11 T cells. Adapting methods of Dudley, Rosenberg and colleagues, patients undergo prior non-myeloablative (NMA) conditioning to create a “hematologic space” into which the infused designer T cells will stably engraft for prolonged in vivo efficacy. Patients are co-administered continuous infusion IL2. Outcomes will include Phase Ia goals of safety and toxicity and Phase Ib goals of establishing an optimal biologic dose in terms of designer T cell engraftment and tumor response. Results: For the first two patients, excellent T cell modifications of 50–60% were obtained. After NMA conditioning, T cells were infused and stable engraftments of 1–5% were observed post recovery, even at this lowest 10^9 T cell dose level, thus affirming one of the study end-points. The patients had PSA reductions of 50 and 70% in the two months following treatment. Patients experienced neutropenia and lymphopenia after conditioning, but no designer T cell-related toxicities. Results with additional patients will be described in terms of safety, engraftment efficiency and tumor responses. Conclusions: A new approach to adoptive immune therapy in metastatic prostate cancer has been devised. This clinical trial is funded by the US Army/DOD. No significant financial relationships to disclose.

Phase I trial of advanced generation anti-CEA designer T cells in gastrointestinal and breast cancers

e15123 Background: We created “designer T cells” by retroviral gene therapy to express chimeric immunoglobulin-T cell receptors (IgTCR) with specificity for carcinoembryonic antigen (CEA). Our previous Phase I trial with 1st generation (1st gen) designer T cells was well tolerated with proof-of-principle “biologic responses”, but of limited duration. Lab correlates showed modified T cells repeatedly kill tumor targets over 4–7 days but then undergo activation-induced cell death (AICD). We created 2nd gen designer T cells that incorporate CD28 co-stimulation into the IgTCR (IgCD28TCR), suppressing AICD and promoting T cell proliferation on tumor contact with superior tumor responses in vivo (Emtage et al. Clin Cancer Res 2008;14:8112). A Phase I clinical trial was approved under FDA BB-IND 10791. Methods: Patient T cells are modified ex vivo, expanded and then administered in a Phase Ia dose escalation, spanning doses of 10^9 to 10^11 cells. Patients are monitored for safety, pharmacokinetics and response. Results: To date, three subjects enrolled with doses prepared and two were treated. T cells were transduced with equal modification of CD4 helper and CD8 cytotoxic T cells and good ex vivo expansions of 30-fold or more. Cells were infused over 15–30 minutes. Blood clearance was rapid. Dosing was without toxicity but also without responses at this lowest T cell dose level. Results will be updated to include new patients at conference time. Conclusions: The safety of 2nd generation designer T cells is supported in two patients at the lowest T cell dose level. Higher planned doses are 10- to 100-fold more T cells, to be observed for toxicity and where responses may begin to be observed. Funding is from the Office of Orphan Products Development of the FDA. No significant financial relationships to disclose.

Antitumor activity of chimeric immunoreceptor gene‐modified Tc1 and Th1 cells against autologous carcinoembryonic antigen‐expressing colon cancer cells

To generate tumor-specific and interferon (IFN)-gamma-producing Tc1 and Th1 cells applicable for many cancer patients, we previously developed a protocol for generating carcinoembryonic antigen (CEA)-specific Tc1 and Th1 cells from healthy human T cells by transduction with a lentivirus containing a chimeric immunoglobulin T-cell receptor (cIgTCR) gene composed of single-chain variable fragments from an anti-CEA-specific monoclonal antibody fused to an intracellular signaling domain of CD28 and CD3zeta. These cells, designated Tc1-T and Th1-T bodies, respectively, showed strong antitumor activity against CEA-expressing tumor cells in RAG2-/- mice when both of them were transferred. However, it remains unclear whether it is possible to generate Tc1-T and Th1-T bodies from cancer patients with defective T-cell function because of significant immunosuppression. Here, we prepared Tc1-T and Th1-T bodies from T cells of a colon cancer patient, and asked whether these T bodies can exert effective T-cell function against autologous tumor cells. These T bodies showed high cytotoxicity and produced IFN-gamma in response to CEA-expressing autologous tumor cells, even in the presence of soluble CEA. It was also demonstrated that Th1-T bodies supported the survival of Tc1-T bodies through cell-to-cell interactions. Furthermore, our protocol utilized retrovirus for cIgTCR transduction to achieve better induction efficiency compared to lentivirus-mediated transduction. Taken together, our findings here indicate that retrovirally transduced Tc1-T and Th1-T bodies will become a promising strategy for adoptive immunotherapy of human cancer.

Generation and Targeting of Human Tumor-Specific Tc1 and Th1 Cells Transduced with a Lentivirus Containing a Chimeric Immunoglobulin T-Cell Receptor

CD4+ Th cells, in particular IFN-gamma-producing Th1 cells, play a critical role in the activation and maintenance of Tc1 cells that are essential for tumor eradication. Here, we report the generation of artificial tumor-specific Th1 and Tc1 cells from nonspecifically activated T cells using a lentiviral transduction system. Anti-CD3-activated T cells from healthy human donors were transduced with a lentivirus containing a chimeric immunoglobulin T-cell receptor gene composed of single-chain variable fragments derived from an anticarcinoembryonic antigen (CEA)-specific monoclonal antibody fused to an intracellular signaling domain derived from the cytoplasmic portions of membrane-bound CD28 and CD3zeta. These artificial tumor-specific Tc1 and Th1 cells, termed Tc1- and Th1-T bodies, respectively, could be targeted to CEA+ tumor cells independently of MHC restriction. Specifically, Tc1-T bodies demonstrated high cytotoxicity and produced IFN-gamma in response to CEA+ tumor cell lines but not CEA- tumors. Although Th1-T bodies exhibited low cytotoxicity, they secreted high levels of IFN-gamma and interleukin-2 in response to CEA+ tumor cells. Such CEA+ tumor-specific activation was not observed in mock gene-transduced nonspecific Tc1 and Th1 cells. Moreover, Tc1- and Th1-T bodies exhibited strong antitumor activities against CEA+ human lung cancer cells implanted into RAG2(-/-) mice. Furthermore, combined therapy with Tc1- and Th1-T bodies resulted in enhanced antitumor activities in vivo. Taken together, our findings demonstrate that Tc1- and Th1-T bodies represent a promising alternative to current methods for the development of effective adoptive immunotherapies.

APPLICATION OF GENETICALLY ENGINEERED TUMOR-SPECIFIC TH1 CELLS TO ADOPTIVE TUMOR IMMUNOTHERAPY

CD4+ Th cells, in particular IFN-γ-producing Th1 cells, play a critical role in the activation and maintenance of CD8+ Tc1 cells that are essential for tumor eradication. Recent identification of MHC class II binding peptides derived from tumor antigens theoretically made possible to tumor-specific CD4+ T cells in addition to CD8+ T cells induced by MHC class I binding tumor antigen peptides. However, the generation of tumor-specific T cells is still a practically challenging and time-consuming procedure. This fact makes difficult the application of tumor-specific T cells to adoptive immunotherapy of cancer patients. Recently, novel methods have been developed to prepare tumor-specific T cells from nonspecifically activated T cells by gene transfer. Specifically, two types of genes were shown to be successful when they were introduced into nonspecific-T cells to generate genetically engineered tumor-specific T cells. One is the TCR α and β chain genes obtained from tumor-specific T cells, and the other is the chimeric immunoglobulin-T cell receptor (cIgTCR) gene which contains a single chain variable fragment from antitumor monoclonal antibody. In this review, we overviewed these methods briefly, and showed our recent results generating genetically engineered tumor-specific Th1 cells successfully by both these methods. In both mouse and human systems, non-specifically activated Th1 cells transduced with TCR genes specific for tumor antigen-derived peptide showed potent antitumor activity against tumor cells when they recognized tumor antigen peptide presented on MHC molecules. On the other hand, Th1 cells transduced with a cIgTCR gene specific for carcinoembryonic antigen, CEA, were shown to be reactive with cell surface CEA in an MHC independent manner. Taken together, these results strongly suggest that it is possible to prepare tumor-specific human Th1 cells by gene transfer technique that is applicable to adoptive tumor immunotherapy for cancer patients.

Human natural killer cell line modified with a chimeric immunoglobulin T-cell receptor gene leads to tumor growth inhibition in vivo.

The gene transfer of tumor-specific chimeric immunoglobulin T-cell receptors (cIgTCRs) combining antibody-like specificity with the effector cell function could be an attractive tool in immunotherapy. In this study, we directed the human natural killer (NK) cell line YT to tumor cells by gene transfer of a cIgTCR with specificity against the human carcinoembryonic antigen (CEA). The cIgTCR was constructed of a CEA-specific humanized single-chain Fv antibody fragment fused to the IgG1 Fc domain and the CD3 zeta chain. YT cells were transfected with the cIgTCR gene by electroporation and cIgTCR-expressing cells were enriched by immunoaffinity purification. cIgTCR-expressing YT cells specifically lysed CEA(+) colon carcinoma cell lines, which were resistant to the parental YT cell line. The lysis was not inhibited in the presence of soluble CEA. Receptor gene-modified YT cells retained their CEA-specific cytolytic activity after gamma-irradiation in vitro and inhibited the tumor growth in vivo after adoptive transfer into NOD/SCID mice. This gene-modified NK cell line available in unlimited source might be useful in clinical immunotherapy of CEA(+) cancer.

Dynamics of tumor cell killing by human T lymphocytes armed with an anti-carcinoembryonic antigen chimeric immunoglobulin T-cell receptor.

Chimeric immunoglobulin T-cell receptors (IgTCR) join the antigen-binding portion of an antibody to one of the signaling chains of the TCR. A previous report described the construction and functional testing of an IgTCR gene directed against the carcinoembryonic tumor antigen (CEA). These preclinical studies showed the proper assembly and cell surface expression of anti-CEA IgTCR molecules, specific target antigen binding, and activation of T-cell effector functions. Although IgTCR-modified T cells function well in vitro, therapeutic applications in humans may be complicated by various factors, such as the availability of appropriate T-cell cytokines, high systemic levels of antagonistic soluble CEA, and antigenic diversity in tumor cell populations. The current study analyzes tumor cell killing by IgTCR-modified human T cells under conditions that more closely model those that may be encountered in persons with cancer. This analysis shows that 1) depriving IgTCR-modified T cells of interleukin-2 does not diminish anti-CEA cytotoxic T lymphocyte activity, but does eliminate killing by lymphokine-activated killer cells; 2) high levels of soluble CEA do not significantly inhibit tumor cell killing even when approximately 80% of the chimeric receptors are blocked; and 3) CEA+ tumor cells that can down-regulate cell surface CEA evade immune destruction by IgTCR-modified T cells. These results have important implications for application strategies and protocol design considerations for early clinical testing of IgTCR anti-tumor therapies.

Chimeric immunoglobulin-T cell receptor proteins form functional receptors: Implications for T cell receptor complex formation and activation

We constructed chimeric receptor chains in which an immunoglobulin heavy chain variable region (V H) from a phosphorylcholine-specific antibody is substituted for T cell receptor (Tcr) α and β V regions. We demonstrate that the V H region joined to either the C α or the C β region can form stable chimeric proteins in EL4 T cells. Both chimeric receptor chains associate with CD3 polypeptides in functional receptor complexes and respond to phosphorylcholine coupled to Sepharose beads. The V H-C α chimeric chain associates with the EL4 β chain, while the V H-C β chimeric protein appears to form either a homodimer or a heterodimer with the native EL4 β chain. Thus, functional receptor complexes can be formed using two C β regions, and the C α region may not be required for CD3 association and surface expression of Tcr complexes.