Chimeric Immune Receptor Research Articles

A Novel Approach for Treatment of T-Cell Malignancies: Targeting T-Cell Receptor Vβ Families

Background: Peripheral T-cell lymphomas (PTCL) encompass a highly heterogeneous group of T-cell malignancies and are generally associated with a poor prognosis. Combination chemotherapy results in consistently poorer outcomes for T-cell lymphomas compared with B-cell lymphomas.1 There is an urgent clinical need to develop novel approaches to treatment of PTCL. While CD19- and CD20-directed immunotherapies have been successful in the treatment of B-cell malignancies, T-cell malignancies lack suitable immunotherapeutic targets. Brentuximab Vedotin, a CD30 antibody-drug conjugate, is not applicable to PTCL subtypes which do not express CD30.2 Broadly targeting pan-T cell markers is predicted to result in extensive T-cell depletion and clinically significant immune deficiency; therefore, a more tumor-specific antigen that primarily targets the malignant T-cell clone is needed. We reasoned that since malignant T cells are clonal and express the same T-cell receptor (TCR) in a given patient, and since the TCR β chain in human α/β TCRs can be grouped into 24 functional Vβ families targetable by monoclonal antibodies, immunotherapeutic targeting of TCR Vβ families would be an attractive strategy for the treatment of T-cell malignancies. Methods: We developed a flexible approach for targeting TCR Vβ families by engineering T cells to express a CD64 chimeric immune receptor (CD64-CIR), comprising a CD3ζ T cell signaling endodomain, CD28 costimulatory domain, and the high-affinity Fc gamma receptor I, CD64. T cells expressing CD64-CIR are predicted to be directed to tumor cells by Vβ-specific monoclonal antibodies that target tumor cell TCR, leading to T cell activation and induction of tumor cell death by T cell-mediated cytotoxicity. Results: This concept was first evaluated in vitro using cell lines. SupT1 T-cell lymphoblasts, which do not express a native functioning TCR, were stably transduced to express a Vβ12+ MART-1 specific TCR, resulting in a Vβ12 TCR expressing target T cell line.3 Vβ family specific cytolysis was confirmed by chromium release assays using co-culture of CD64 CIR transduced T cells with the engineered SupT1-Vβ12 cell line in the presence of Vβ12 monoclonal antibody. Percent specific lysis was calculated as (experimental - spontaneous lysis / maximal - spontaneous lysis) x 100. Controls using no antibody, Vβ8 antibody, and untransduced T cells did not show significant cytolysis (figure A). Next, the Jurkat T cell leukemic cell line, which expresses a native Vβ8 TCR, was used as targets in co-culture. Again, Vβ family target specific cytolysis was achieved in the presence of CD64 CIR T cells and Vβ8, but not Vβ12 control antibody. Having demonstrated Vβ family specific cytolysis in vitro using target T cell lines, we next evaluated TCR Vβ family targeting in vivo. Immunodeficient mice were injected with SupT1-Vβ12 or Jurkat T cells with the appropriate targeting Vβ antibody, and either CD64 CIR T cells or control untransduced T cells. The cell lines were transfected with firefly luciferase and tumor growth was measured by bioluminescence. The CD64 CIR T cells, but not untransduced T cells, in conjunction with the appropriate Vβ antibody, successfully controlled tumor growth (figure B). Our results provide proof-of-concept that TCR Vβ family specific T cell-mediated cytolysis is feasible, and informs the development of novel immunotherapies that target TCR Vβ families in T-cell malignancies. Unlike approaches that target pan-T cell antigens, this approach is not expected to cause substantial immune deficiency and could lead to a significant advance in the treatment of T-cell malignancies including PTCL.

206. Follicle-Stimulating Hormone Receptor as a Target in the Redirected T-Cell Therapy for Cancer

Adoptive transfer of T-cells engineered to express chimeric immune receptors is an effective strategy to treat hematological cancers, however, such therapy for solid cancers such as ovarian cancer remains challenging because a safe and effective immunotherapeutic target has not yet been identified. Here, we constructed and evaluated a novel redirected T-cell-based immunotherapy targeting human follicle-stimulating hormone receptor (FSHR), a highly conserved molecule in vertebrate animals with expression limited to gonadal tissues, ovarian cancer and cancer-associated vasculature. Receptor ligand-based anti-FSHR immune receptors were constructed that contained small binding fragments from the ligand for FSHR, follicle stimulating hormone (FSH), fused to T-cell transmembrane and T-cell signaling domains. Human T-cells transduced to express anti-FSHR immune receptors were specifically immunoreactive against FSHR-expressing human and mouse ovarian cancer cell lines in a MHC-non-restricted manner, and produced more than one Th1 type of proinflammatory cytokine. This observation is of special interest, as it provides the opportunity to address potential, but unpredicted, toxicity issues associated with targeting of FSHR in preclinical in vivo studies. Moreover, anti-FSHR-IR T-cells specifically upregulated surface expression of the activation marker CD69 in the presence of FSHR positive targets, but not FSHR-deficient the 293T cell line. Additionally, specific activation of anti-FSHR-IR T-cells by FSHR expressed on tumor cell surface led to efficient tumor cell killing in vitro. Furthermore, to assess the anti-tumor effect of anti-FSHR T-cells, we established human OvCa xenografts in NSG mice by subcutaneous flank injection of CaOV3 cancer cell line followed by two intravenous redirected T-cell administrations. As shown by caliper measurements, FSHR-redirected T-cells elicited anti-tumor potential with suppressed outgrowth of established tumor. Tumor growth was statistically reduced in comparison to control, GFP engineered T-cell treated group or PBS treated animals. These data suggest that systemic delivery of anti-FSHR T-cells to mice bearing FSHR-expressing tumors may result in tumor trafficking, activation, and lysis at location of tumor.In summary, we have successfully exploited for the first time a novel therapeutic antigen, the FSHR protein, expressed on tumor cell surface, to deliver T-cell based immune-therapy of cancer. Given its highly restricted expression in cancer, cancer-associated vessels and gonadal tissues, the rationale for FSHR targeting is strong and supports further exploration of FSHR-targeted immune therapy approaches for cancer patients.

Anti-KIT designer T cells for the treatment of gastrointestinal stromal tumor

BackgroundImatinib mesylate is an effective treatment for metastatic gastrointestinal stromal tumor (GIST). However, most patients eventually develop resistance and there are few other treatment options. Immunotherapy using genetically modified or designer T cells (dTc) has gained increased attention for several malignancies in recent years. The aims of this study were to develop and test novel anti-KIT dTc engineered to target GIST cells.MethodsHuman anti-KIT dTc were created by retroviral transduction with novel chimeric immune receptors (CIR). The gene for stem cell factor (SCF), the natural ligand for KIT, was cloned into 1st generation (SCF-CD3ζ, 1st gen) and 2nd generation (SCF-CD28-CD3ζ, 2nd gen) CIR constructs. In vitro dTc proliferation and tumoricidal capacity in the presence of KIT+ tumor cells were measured. In vivo assessment of dTc anti-tumor efficacy was performed by treating immunodeficient mice harboring subcutaneous GIST xenografts with dTc tail vein infusions.ResultsWe successfully produced the 1st and 2nd gen anti-KIT CIR and transduced murine and human T cells. Average transduction efficiencies for human 1st and 2nd gen dTc were 50% and 42%. When co-cultured with KIT+ tumor cells, both 1st and 2nd gen dTc proliferated and produced IFNγ. Human anti-KIT dTc were efficient at lysing GIST in vitro compared to untransduced T cells. In mice with established GIST xenografts, treatment with either 1st or 2nd gen human anti-KIT dTc led to significant reductions in tumor growth rates.ConclusionsWe have constructed a novel anti-KIT CIR for production of dTc that possess specific activity against KIT+ GIST in vitro and in vivo. Further studies are warranted to evaluate the therapeutic potential and safety of anti-KIT dTc.

Abstract LB-156: Improved T cell survival, tumor localization and tumor eradication by folate receptor-alpha redirected T cells is achieved by provision of CD137 costimulatory signaling

Abstract Human T cells engineered to express a chimeric immune receptor (CIR) specific for folate receptor-alpha (FRα) have shown robust anti-tumor activity against epithelial cancers in vitro but not in the clinic due to their inability to persist and home to tumor in vivo. To address these issues, we constructed CIRs containing an FRα-specific scFv (MOv19) coupled to the T cell receptor CD3ζ chain signaling module alone or in combination with the CD137 (4–1BB) costimulatory motif in tandem. Primary human T cells transduced to express conventional MOv19-ζ or costimulated MOv19-BBζ CIRs secreted various proinflammatory cytokines and exerted cytotoxic function when cocultured with FRα+ tumor cells in vitro. However, only transfer of human T cells expressing the costimulated MOv19-BBζ CIR mediated tumor regression in immunodeficient mice bearing large, established FRα+ human cancer. MOv19-BBζ CIR T cell infusion mediated tumor regression in models of metastatic intraperitoneal, subcutaneous, and lung-involved human ovarian cancer. Importantly, tumor response was associated with the selective survival and tumor localization of human T cells in vivo and was only observed in mice receiving costimulated MOv19-BBζ CIR T cells. T cell persistence and anti-tumor activity were primarily antigen-driven, however, antigen-independent CD137 signaling by CIR did improve T cell persistence but not anti-tumor activity in vivo. Our results show that anti-FRα CIR outfitted with CD137 costimulatory signaling in tandem overcome issues of T cell persistence and tumor localization that limit the conventional FRα T cell targeting strategy to provide potent antitumor activity in vivo. Based upon these encouraging findings, we are preparing to open an anti-FRα CIR protocol for ovarian cancer in mid-2011, pending FDA approval. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-156. doi:10.1158/1538-7445.AM2011-LB-156

Direct and indirect antitumor effects by human peripheral blood lymphocytes expressing both chimeric immune receptor and interleukin-2 in ovarian cancer xenograft model

Human peripheral blood lymphocytes (PBLs) electroporated with RNA encoding anti-Her-2/neu-specific chimeric immune receptor (CIR) have been reported to elicit potent immune responses against SKOV3 tumors in a nude mouse model. However, CIR-electroporated PBL (CIR-PBL) did not proliferate, and the cell number rapidly decreased in the absence of exogenous interleukin-2 (IL-2). In this study, PBLs electroporated with both CIR and IL-2 RNA (CIR/IL-2-PBL) were studied to determine whether antitumor effects could be improved by adoptive immunotherapy. CIR and IL-2 were expressed in CIR/IL-2-PBL at levels similar to PBLs electroporated, with IL-2 RNA (IL-2-PBL) or CIR-PBL. Transfer of IL-2 RNA induced proliferation and prolonged survival of PBLs in vitro. In a xenograft model, both IL-2-PBL and CIR/IL-2-PBL showed significantly higher antitumor effects than CIR-PBL. The number of tumor-infiltrating natural killer (NK) cells was significantly increased in IL-2-PBL and CIR/IL-2-PBL. After NK cell depletion, IL-2-PBL showed significantly lower antitumor effects than CIR/IL-2-PBL. These results suggest that transfer of IL-2 RNA to CIR-PBL can promote NK cell infiltration of tumors and prolong survival of infused PBLs in vivo. RNA electroporated PBLs may represent efficient tools for delivery of functional molecules to tumors by multiple gene transfer.

Abstract 4775: HER-2 is expressed by all ovarian cancer cells and invariably recognized by genetically engineered HER-2 specific T cells

Abstract Background: HER-2/neu protein has been repetitively reported to be overexpressed in 20-30% of epithelial ovarian cancer and is associated with relapsing after chemotherapy and poor survival. Thus, HER-2 represents an attractive candidate antigen for immunotherapy of cancer patients. While HER-2 specific T cells can be induced by vaccination or generated de novo by genetic engineering, it remains uncertain to what extent T cell-based “HER-2 directed” immunotherapy can be utilized for the treatment of advanced ovarian cancer. Objective: To investigate the role of HER-2 as the resolute tumor antigen for T cell based adoptive immunotherapy of ovarian cancer. Methods: HER-2 expression was analysed by Real Time PCR, flow cytometry and western blot in a large number of both establish and short-term cultured cancer cell lines and tumor cells derived from malignant ascites and solid tumor harvested from ovarian cancer patients. T cells with specificity against HER-2 were generated by two different ways: either by stimulating CD8+ T cells enriched from PBMC with monocyte-derived mature DC from patients vaccinated against HER-2 peptides; or by genetic engineering of CD4+ and CD8+T lymphocytes to express HER-2 specific chimeric immune receptors (CIRs) capable of redirecting potent non-MHC-dependent cytotoxicity to tumor cells expressing HER-2. HER-2 specific T cells were tested for their capacity to recognize and kill HER-2 expressing tumors. Results: HER-2 overexpression was observed in all (13/13) established ovarian cancer cell lines and all 7/7 (100 %) short-term cultured cell lines. Consistent with these results, all primary ascites (n=22) and solid tumor (n=12) derived tumor cells tested expressed detectable levels of cell surface HER-2. To validate the role of HER-2 as a strong T cell target, natural HER-2 peptide-specific T cells with high functional avidity and CIR redirected T cells were tested for tumor reactivity. Genetically redirected T cells recognized all tested ovarian cancer cell targets (n=16), and were superior to naturally occurring HER-2 specific T cells which were functionally restricted by MHC haplotype and impaired antigen processing by tumor cells. Conclusions: HER-2 is expressed by all ovarian cancer cells and invariably recognized by genetically engineered HER-2 specific T cells suggesting that targeting HER-2 may be more efficacious in patients with ovarian carcinoma than previously suggested by reports of 20-30% overexpression of HER-2 in primary ovarian tumors. These findings provide the rationale for the development of HER-2 redirected T cell-based immunotherapeutic approaches for ovarian cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4775.

Abstract 1936: Anti CD30 chimeric immune receptor mRNA for therapy of CD30+ lymphomas

Abstract CD30 is a transmembrane receptor which is overexpressed on Hodgkin lymphoma and large cell anaplastic lymphoma and represents an attractive target for therapy. Current methods of immunotherapy of CD30+ lymphomas include antibody based therapy (immunotoxins, bispecific molecules, radioimmunotherapy), and cellular immunotherapy. Here we describe an approach to cellular immunotherapy of CD30+ lymphomas using a synthetic mRNA of an anti CD30 chimeric immune receptor (anti CD30-CIR) introduced into activated T cells by electroporation. Anti-CD30 CIR consists of a single chain antibody directed against CD30, a transmembrane anchor to localize the protein in the cellular membrane, and the cytoplasmic domain of the TcR receptor chain. In these studies, the cDNA coding sequence was used as a template to produce anti CD30-CIR mRNA. Normal T cells expanded in vitro were electroporated with CIR mRNA and expression of anti CD30-CIR was detected by biotin-conjugated F(ab’)2 followed with Streptavidin-APC. To measure cytotoxicity of the CIR, target cells (HH, K562, Jurkat) were pulsed with carboxyfluorescein diacetate succinimidyl ester (CFSE) and then were cultured with T cells transfected with anti-CD30 CIR mRNA or mock transfected T cells. Cytotoxicity was evaluated at different target::effector (T: E) ratios and normalized by standard algorithm. Twenty-four hours after electroporation almost all viable T cell (>90%) expressed anti-CD30 CIR and were able to specifically kill CD30+ targets in a 4h in vitro assay. These results demonstrate that anti-CD30-CIR mRNA modified T cells can be generated and are capable of killing CD30-expressing cells in vitro and therefore may be an effective strategy for the therapy of refractory CD30-expressing lymphomas. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1936.

2nd generation anti-HIV designer T cells cure HIV-infected cultures and preferentially survive versus 1st generation designer T cells (52.19)

Abstract Immunotherapies that have the potential to eradicate reservoirs of HIV infected cells may be useful to suppress or cure HIV/AIDS. T cells expressing chimeric immune receptors (CIR) through gene modification (designer T cells) can be redirected to kill HIV-infected cells. Previously, an anti-HIV CIR, CD4ζ (1st generation), was created to kill HIV+ cells. However, the adoptive transfer of autologous CD4ζ T cells failed to control the viral infection in clinical trials. We created a 2nd generation CIR for enhanced potency by providing co-stimulatory signal (CD4CD28ζ). Our data show that both 1st and 2nd generation CIRs expressed well on retrovirally transduced human T cells and kill HIV+ targets with high efficiency, whereas CD28 in the CIR confers a distinct advantage in the expansion potential and cytokine IL2 secretion. Interestingly, our preliminary data show that all 1st generation designer T cells died in 7 days when co-cultured with HIV+ cells, whereas 2nd generation designer T cells survived with cure of the infected cultures. This stunning result shows dramatic benefit in the new 2nd generation CIR versus the 1st generation that was used in human clinical trials. We are presently exploring whether this cell death in the 1st generation designer T cells is due to heightened sensitivity to AICD and/or due to HIV infection. Either way, these data show a striking benefit to our new 2nd generation designer T cells as a promising therapy for HIV/AIDS.

Lymphocyte Display: A Novel Antibody Selection Platform Based on T Cell Activation

Since their onset, display technologies have proven useful for the selection of antibodies against a variety of targets; however, most of the antibodies selected with the currently available platforms need to be further modified for their use in humans, and are restricted to accessible antigens. Furthermore, these platforms are not well suited for in vivo selections. We present here a novel cell based antibody display platform, which takes advantage of the functional capabilities of T lymphocytes. The display of antibodies on the surface of T lymphocytes, as a part of a chimeric-immune receptor (CIR) mediating signaling, may ideally link the antigen-antibody interaction to a demonstrable change in T cell phenotype, due to subsequent expression of the early T cell activation marker CD69. In this proof-of-concept, an in vitro selection was carried out using a human T cell line lentiviral-transduced to express a tumor-specific CIR on the surface, against a human tumor cell line expressing the carcinoembryonic antigen. Based on an effective interaction between the CIR and the tumor antigen, we demonstrated that combining CIR-mediated activation with FACS sorting of CD69+ T cells, it is possible to isolate binders to tumor specific cell surface antigen, with an enrichment factor of at least 103-fold after two rounds, resulting in a homogeneous population of T cells expressing tumor-specific CIRs.

Transfer of Her-2/neu Specificity into Cytokine-Induced Killer (CIK) Cells with RNA Encoding Chimeric Immune Receptor (CIR)

Efficient RNA transfer to dendritic cell and T cells by electroporation have been successfully applied for immunotherapy. Herein, RNA electroporation was used to transfer antigen-specific receptor (scFv) gene to cytokine-induced killer cells (CIK). CIK was generated from peripheral blood mononuclear cells with anti-CD3 antibody, interleukin-2, and interferon (IFN)-gamma for 14 days and showed typical characteristics of CIK expressing both CD3+ and CD56+ markers and NKG2D+. CIK could lyse K562 cells, but not SKOV3 and MCF7/Her-2/neu. After RNA encoding anti-Her-2/neu chimeric immune receptor (CIR) with signaling portion of CD28 and CD3zeta was electroporated to CIK, more than 95% of CIK expressed anti-Her-2/neu CIR (CIR-CIK). CIR-CIK was able to produce cytokines including IFN-gamma, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha, and show cytotoxicity specific to tumor cell lines expressing Her-2/neu, SKOV3, and MCF7/Her-2/neu. Adoptive transfer of CIR-CIK in SKOV3 xenograft nude mice model led to significant inhibition of tumor growth compared with transfer of mock-transduced CIK and showed higher inhibition than that of Herceptin, humanized monoclonal antibody specific for Her-2/neu. These results suggest that RNA transfer is the convenient and efficient strategy to introduce antigen-specificity into CIK and provide potential therapeutic value of CIR-CIK in the treatment of tumors.

569. Engineering of “3rd Generation” Chimeric Immune Receptor Expressing T Cells for Improved Adoptive Immunotherapy Against Glioblastoma

569. Engineering of “3rd Generation” Chimeric Immune Receptor Expressing T Cells for Improved Adoptive Immunotherapy Against Glioblastoma

Genetically engineered T cells to target EGFRvIII expressing glioblastoma

Glioblastoma remains a significant therapeutic challenge, warranting further investigation of novel therapies. We describe an immunotherapeutic strategy to treat glioblastoma based on adoptive transfer of genetically modified T-lymphocytes (T cells) redirected to kill EGFRvIII expressing gliomas. We constructed a chimeric immune receptor (CIR) specific to EGFRvIII, (MR1-zeta). After in vitro selection and expansion, MR1-zeta genetically modified primary human T-cells specifically recognized EGFRvIII-positive tumor cells as demonstrated by IFN-gamma secretion and efficient tumor lysis compared to control CIRs defective in EGFRvIII binding (MRB-zeta) or signaling (MR1-delzeta). MR1-zeta expressing T cells also inhibited EGFRvIII-positive tumor growth in vivo in a xenografted mouse model. Successful targeting of EGFRvIII-positive tumors via adoptive transfer of genetically modified T cells may represent a new immunotherapy strategy with great potential for clinical applications.

Enhancing potency of anti-HIV chimeric immune receptor (CIR) (128.5)

Abstract Immunotherapies that have the potential to eradicate reservoirs of HIV infected cells may be useful to suppress or cure HIV/AIDS. in combination with current antiviral drug therapies. T cells expressing chimeric immune receptors (CIR) through gene modification ("designer T cells") can be redirected to kill HIV-infected cells. Previously, an anti-HIV CIR, CD4-ζ (1st generation), was created to bind to HIV-1 gp120 antigen through its CD4 extracellular domain and induction of signaling through cytoplasmic CD3ζ chain. However, the adoptive transfer of autologous CD4-ζ T cells to HIV/AIDS patients failed to control the viral infection in clinical trials. To improve potency, our laboratory has created 2nd generation anti-HIV designer T cells that incorporate CD28 costimulation (CD4-CD28-ζ). 2nd generation designer T cells kill HIV-Env expressing Jurkat cells and HIV-1 infected CEM-SS cells as well or better than 1st generation designer T cells. Stimulation with anti-CD4 mAb induces more IL2 production in 2nd generation than 1st generation designer T cells. In addition, 2nd generation designer T cells showed significantly better proliferative response upon culture with HIV-Env expressing HeLa cells or HIV-1 infected CEM-SS cells. These data suggest that gene modified CD8+ T cells could be an effective therapy to eradicate HIV+ cells during and after anti-retroviral therapy. We hypothesize that CD28 domain in the CIR will confer a benefit of longer T cell survival after infusion into HIV patients. Research supported by NIH, R21.

Eradication of Established B-cell Lymphoma by CD19-specific Murine T Cells is Dependent on Host Lymphopenic Environment and Can be Mediated by CD4+ and CD8+ T Cells

B-cell malignancies seem to be particularly amenable to immunotherapy and as such make particularly attractive targets for adoptive T-cell therapy. Murine T cells gene-modified to express a chimeric immune receptor specific for CD19+ (aCD19z) efficiently kill CD19 B-cell lymphoma cells in vitro. aCD19z T cells also secrete high levels of interleukin-2 during culture with target cells in a CD86 independent manner. aCD19z T cells proved effective at eradicating established B-cell lymphoma in a syngeneic model system when combined with a lymphodepleting preconditioning regimen. In mice deficient of T, B, and natural killer cells (severe combined immunodeficient/Beige), aCD19z T cells efficiently eradicated long-term (13 d) established tumors with 100% of treated animals remaining tumor free for greater than 77 days. Although gene-modified CD4+ and CD8+ were both active in this setting, poor engraftment by CD8+ T cells coupled with the rigorous expansion of CD4+ cells in the Balb/c background suggests that CD4+ T cells may be playing a predominant role in lymphoma rejection in this model. Taken together, the therapeutic effectiveness of aCD19z T cells in this model supports a recently opened phase 1 trial of this receptor in non-Hodgkin lymphoma.

Amelioration of Colitis by Genetically Engineered Murine Regulatory T Cells Redirected by Antigen-Specific Chimeric Receptor

The therapeutic application of regulatory T cells (Tregs) for the treatment of inflammatory diseases is limited by the scarcity of antigen-specific Tregs. A preferred approach to endow effector T cells (Teff) with a desired specificity uses chimeric immune receptors with antibody-type specificity. Accordingly, employing such chimeric immune receptors to redirect Tregs to sites of inflammation may be a useful therapeutic approach to alleviate a broad scope of diseases in which an uncontrolled inflammatory response plays a major role. To enable application of the approach in clinical setting, which requires the genetic modification of the patient's own Tregs, we describe here a novel protocol that allows the efficient retroviral transduction and 2,4,6-trinitrophenol-specific expansion of murine naturally occurring regulatory T cells (nTregs), with a 2,4,6-trinitrophenol-specific tripartite chimeric receptor. Transduced Tregs maintained their Foxp3 level, could undergo repeated expansion upon ex vivo encounter with their cognate antigen in a major histocompatibility complex-independent, costimulation-independent, and contact-dependent manner and specifically suppressed Teff cells. Adoptive transfer of small numbers of the transduced nTregs was associated with antigen-specific, dose-dependent amelioration of trinitrobenzenesulphonic acid colitis. This study demonstrates that nTregs can be efficiently transduced to express functional, antigen-specific chimeric receptors that enable the specific suppression of effector T cells both in vitro and in vivo. This approach may enable future cell-based therapeutic application in inflammatory bowel disease, as well as other inflammatory disorders.

Adoptive immunotherapy using human peripheral blood lymphocytes transferred with RNA encoding Her-2/neu-specific chimeric immune receptor in ovarian cancer xenograft model

The current gene transfer technology for single chain (scFv)-based chimeric immune receptor (CIR) has relied on retrovirus and lentivirus vectors which require a long time to obtain sufficient number of transduced cells and stably incorporate into genome. To ameliorate these limitations, we applied RNA electroporation to human peripheral blood lymphocytes (PBLs) activated with anti-CD3 antibody and interleukin-2 (IL-2) for 3 days and assessed that PBL transiently expressing anti-Her-2/neu CIR (CIR-PBL) containing signaling portion of CD28 and CD3zeta could elicit strong cytotoxicity in vitro and antitumor responses in vivo. The CIR-PBL expressed high level of CIR in CD4+, CD8+ and CD56+ cells. Her-2/neu-specific stimulation induced secretion of type-I cytokines including interferon-gamma (IFN-gamma), IL-8 and granulocyte-macrophage colony-stimulating factor, and IFN-gamma secretion was mainly mediated by CD8+ T cells. CIR-PBL specifically killed SKOV3 cell line expressing Her-2/neu. Adoptive transfer of CIR-PBL in SKOV3 xenograft model led to significant inhibition of tumor growth compared with transfer of mock-transduced PBL and showed higher inhibition than those with Herceptin, humanized monoclonal antibody specific for Her-2/neu. These results provided evidence that electroporation of CIR RNA to human PBLs could be used for rapid generation and high number of therapeutic antigen-specific T cells for adoptive immunotherapy.

Second-generation anti-carcinoembryonic antigen designer T cells resist activation-induced cell death, proliferate on tumor contact, secrete cytokines, and exhibit superior antitumor activity in vivo: a preclinical evaluation.

This report describes the development and preclinical qualification tests of second-generation anti-carcinoembryonic (CEA) designer T cells for use in human trials. The progenitor first-generation immunoglobulin-T-cell receptor (IgTCR) that transmits Signal 1-only effectively mediated chimeric immune receptor (CIR)-directed cytotoxicity, but expressor T cells succumbed to activation-induced cell death (AICD). The second-generation CIR (termed "Tandem" for two signals) was designed to transmit TCR Signal 1 and CD28 Signal 2 to render T cells resistant to AICD and provide prolonged antitumor effect in vivo. A CIR was created that combines portions of CD28, TCRzeta, and a single chain antibody domain (sFv) specific for CEA into a single molecule (IgCD28TCR). As designed, the gene-modified Tandem T cells exhibit the new property of being resistant to AICD, showing instead an accelerated proliferation on tumor contact. Tandem T cells are more potent than first generation in targeting and lysing CEA+ tumor. Tandem T cells secrete high levels of interleukin-2 and IFNgamma on tumor contact that first-generation T cells lacked, but secretion was exhaustible, suggesting a need for interleukin-2 supplementation in therapy even for these second-generation agents. Finally, second-generation T cells were more effective in suppressing tumor in animal models. An advanced generation of anti-CEA designer T cells is described with features that promise a more potent and enduring antitumor immune response in vivo. These preclinical data qualify the human use of this agent that is currently undergoing trial in patients with CEA+ cancers.

Chimeric receptor mRNA transfection as a tool to generate antineoplastic lymphocytes.

mRNA transfection is a useful approach for temporal cell reprogramming with minimal risk of transgene-mediated mutagenesis. We applied this to redirect lymphocyte cytotoxicity toward malignant cells. Using the chimeric immune receptor (CIR) constructs anti-CD19 CIR and 8H9 CIR, we achieved uniform expression of CIRs on virtually the entire population of lymphocytes. We reprogrammed CD3+ CD8+, CD3+ CD4+, and natural killer (NK ) cells toward autologous and allogeneic targets such as B cells, Daudi lymphoma, primary melanoma, breast ductal carcinoma, breast adenocarcinoma, and rhabdomyosarcoma. The reprogramming procedure is fast. Although most of the experiments were performed on lymphocytes obtained after 7-day activation, only 1-day activation of T cells with anti-CD3, anti-CD28 antibodies, and interleukin-2 is sufficient to develop both lymphocyte cytotoxicity and competence for mRNA transfer. The entire procedure, which includes lymphocyte activation and reprogramming, can be completed in 2 days. The efficiency of mRNA-modified human T cells was tested in a murine xenograft model. Human CD3+CD8+ lymphocytes expressing anti-CD19 CIR mRNA inhibited Daudi lymphoma growth in NOD=SCID mice. These results demonstrate that a mixed population of cytotoxic lymphocytes, including T cells together with NK cells, can be quickly and simultaneously reprogrammed by mRNA against autologous malignancies. With relatively minor modifications the described method of lymphocyte reprogramming can be scaled up for cancer therapy.

Anti‐JCV TCR‐based chimeric immune receptors create functional designer T cells for PML therapy

Progressive Multifocal Leukoencephalopathy (PML) is a lethal brain disease caused by JC polyomavirus (JCV) that arises in immunodepressed HIV+ patients and in rare individuals who are treated with natalizumab (Tysabri) for multiple sclerosis or Crohn's disease. Chimeric immune receptors (CIRs) are engineered from JCV specific TCR á and â chains cloned from a PML survivor and fused to the transmembrane (TM) and cytoplasmic (cyt) domains of CD3æ providing Signal 1. These form functional á/â chain heterodimers, induce retrovirally transduced expressor T cells to secrete cytokines and exhibit cytotoxicity against JCV+ cells. In further experiments, these TCR‐based CIRs were engineered to create 2nd generation products with added CD28, providing Signal 1+2, containing an extracellular (EC) region and TM and cyt domains of CD28 and cyt of CD3æ. The 2nd generation anti‐JCV designer T cells are intended to resist AICD and proliferate on antigen contact, with resistance to regulatory T cell and other suppressions, and function in the absence of CD4 T cell help. Here, we demonstrate that under the hypothesis of several variations of constructs with either CD28, CD3æ or hybrid TM domains that removal of the CD28 EC region is required for effective expression, heterodimerization and designer T cell function. These products are being prepared for clinical application in HIV+ or natilizumab treated PML patients.

Redirected Activity of Human Antitumor Chimeric Immune Receptors is Governed by Antigen and Receptor Expression Levels and Affinity of Interaction

Novel Ab-based immunotherapeutic strategies have exploited T-cell receptor-like chimeric immune receptors (CIR) expressed on the surface of transduced human peripheral blood mononuclear cell (PBMC) to redirect potent non-major histocompatibility complex-dependent cytotoxicity to tumor cells expressing a tumor-associated antigens. We transduced human PBMC with 2 fully human CIRs that trigger through the zeta-chain of CD3 and contain either one of two human scFv specific for the same epitope on the extracellular domain of HER2 but with distinctly different affinities (KD 1616 and 1 nM) for this antigen. Potent direct CIR-mediated killing and in vitro tumor growth inhibition mediated by transduced PBMC were observed against targets expressing different levels of HER2. High-affinity CIR showed stronger ability to bind Ag and retain binding than low-affinity CIR. When lytic potential of the 2 CIRs was evaluated, their efficiency was comparable under conditions of high CIR and Ag expression, whereas low-affinity CIR was more efficient than high-affinity CIR in conditions of limiting Ag and CIR expression levels. When tumor growth inhibition was evaluated, Ag and CIR levels, rather than CIR affinity appeared relevant. Ag-driven CIR activation resulted in the production of soluble factors mediating efficient bystander effect. By carefully defining CIR surface expression and increasing affinity for a specific target antigen, it may be possible to selectively exclude CIR-mediated activity against targets expressing low levels of antigen, as normal cells. On the contrary, low antigen-expressing tumor variants could be eliminated by decreasing CIR affinity. Tuning CIR expression and affinity might help in discriminating different biologic contexts.