Tumor Antigen-specific T-cell Receptor Research Articles

Acellular scaffold-based approach for in situ genetic engineering of host T-cells in solid tumor immunotherapy

BackgroundTargeted T-cell therapy has emerged as a promising strategy for the treatment of hematological malignancies. However, its application to solid tumors presents significant challenges due to the limited accessibility and heterogeneity. Localized delivery of tumor-specific T-cells using biomaterials has shown promise, however, procedures required for genetic modification and generation of a sufficient number of tumor-specific T-cells ex vivo remain major obstacles due to cost and time constraints.MethodsPolyethylene glycol (PEG)-based three-dimensional (3D) scaffolds were developed and conjugated with positively charged poly-L-lysine (PLL) using carbamide chemistry for efficient loading of lentiviruses (LVs) carrying tumor antigen-specific T-cell receptors (TCRs). The physical and biological properties of the scaffold were extensively characterized. Further, the scaffold loaded with OVA-TCR LVs was implanted in B16F10 cells expressing ovalbumin (B16-OVA) tumor model to evaluate the anti-tumor response and the presence of transduced T-cells.ResultsOur findings demonstrate that the scaffolds do not induce any systemic inflammation upon subcutaneous implantation and effectively recruit T-cells to the site. In B16-OVA melanoma tumor-bearing mice, the scaffolds efficiently transduce host T-cells with OVA-specific TCRs. These genetically modified T-cells exhibit homing capability towards the tumor and secondary lymphoid organs, resulting in a significant reduction of tumor size and systemic increase in anti-tumor cytokines. Immune cell profiling revealed a significantly high percentage of transduced T-cells and a notable reduction in suppressor immune cells within the tumors of mice implanted with these scaffolds.ConclusionOur scaffold-based T-cell therapy presents an innovative in situ localized approach for programming T-cells to target solid tumors. This approach offers a viable alternative to in vitro manipulation of T-cells, circumventing the need for large-scale in vitro generation and culture of tumor-specific T-cells. It offers an off-the-shelf alternative that facilitates the use of host cells instead of allogeneic cells, thereby, overcoming a major hurdle.

Abstract 2756: Multiplexed-engineered, iPSC-derived T cells expressing three unique targeting modalities address tumor heterogeneity and antigen escape

Abstract Adoptive T-cell therapy with chimeric antigen receptor (CAR) has shown promising results in cancer treatment, however, antigen escape and tumor heterogeneity are major causes for disease relapse. While CARs are known to trigger an effective immune response through surface antigen recognition many solid tumor cancer antigens are intracellular and presented by MHC molecules recognized by T cell receptors (TCRs). In addition, many therapeutic antibodies have shown clinical efficacy in solid tumor settings. However, antibody-dependent cellular cytotoxicity (ADCC) is mediated by the CD16 Fc receptor naturally expressed on NK cells although its application in T cells is yet not fully appreciated. Utilizing our proprietary induced pluripotent stem cell (iPSC) platform to engineer multiple modalities into a clonal iPSC line, which can serve as the starting cell source for mass production of off-the-shelf, iPSC-derived CAR-T cells (CAR-iT cells), we aimed to study the combination of these three targeting modalities, CAR, TCR, and CD16, to determine whether challenges associated with the treatment of solid tumors, which are heterogeneous and challenging to treat, may be overcome.To test the base line activity of CAR-iT cells in the solid tumor setting, we selected our anti-MICA/B CAR, previously shown to effectively target stress ligands found on transformed cells, to demonstrate effective anti-tumor activity against multiple solid tumor cell lines (72 hrs cytotoxicity: A2058 = 99%; 786-O = 98%; versus non-specific CAR-iT cells: A258 = 13%; 786-O = 17%). To test compatibility of TCR in our iT cell platform, we engineered MR1-TCR in iT cells to show increased cytokine release and degranulation upon stimulated with MR1 positive lung carcinoma epithelial cells line A549 (fold change compared to un-stimulated: IFNg = 210, p = 0.0032; TNFa = 76.9, p = 0.0005; CD107ab = 115.0, p=0.0013). Notably, with the engineering of tumor antigen specific TCR in TCR-less CAR-iT cells, CD3 complex can be re-established to provide an opportunity to combine with bispecific T cell engagers. Lastly, combining CAR-iT cells with MR1-TCR and hnCD16 uniquely demonstrated synergistic tumor growth inhibition and validated our approach to target multiple antigens at once for an effective anti-tumor response (A549 cytotoxicity: tumor only = 3.68±2.04%; effector+TCR = 41.31±2.27%; effector+TCR+ADCC = 90.28±1.87%). In summary, using the unique approach to engineer iPSCs at the clonal level to create a distinct population of engineered iT cells, we successfully demonstrated the compatibility between CAR, TCR, and hnCD16 to mitigate tumor heterogeneity. This approach is an ideal strategy to create off-the-shelf cellular immunotherapy for a promising therapeutic approach to combat heterogeneous and difficult to treat solid tumors, including those that are resistant due to antigen escape. Citation Format: Chia-Wei Chang, Bi-Huei Yang, Eason Lin, Soheila Shirinbak, Wen-I Yeh, Mochtar Pribadi, Helen Chu, Alma Gutierrez, Earl Avramis, Jason ORourke, Tom Lee, Alec Witty, Eigen Peralta, Martin Hosking, Bahram Valamehr. Multiplexed-engineered, iPSC-derived T cells expressing three unique targeting modalities address tumor heterogeneity and antigen escape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2756.

Enhanced Antitumor Responses of Tumor Antigen-Specific TCR T Cells Genetically Engineered to Produce IL7 and CCL19.

Although adoptive transfer of T cells genetically engineered to express chimeric antigen receptor (CAR) or T-cell receptor (TCR) has been actively developed and applied into clinic recently, further improvement of these modalities is highly demanded, especially in terms of its efficacy. Because we previously revealed the profound enhancement of antitumor effects of CAR T cells by concomitant expression of IL7 and CCL19, this study further explored a potential of IL7/CCL19 production technology to augment antitumor effects of TCR T cells. IL7/CCL19-producing P1A tumor antigen-specific TCR T cells (7 × 19 P1A T cells) demonstrated significantly improved antitumor effects, compared with those without IL7/CCL19 production, and generated long-term memory responses. The antitumor effects of 7×19 P1A T cells were further upregulated by combination with anti-PD-1 antibody, in which blockade of PD-1 signal in both 7×19 P1A T cells and endogenous T cells plays an important role. Taken together, our study demonstrated that concomitant production of IL7 and CCL19 by genetically engineered tumor-reactive T cells could synergize with PD-1 blockade therapy to generate potent and long-lasting antitumor immunity.

Sensitive identification of neoantigens and cognate TCRs in human solid tumors

The identification of patient-specific tumor antigens is complicated by the low frequency of T cells specific for each tumor antigen. Here we describe NeoScreen, a method that enables the sensitive identification of rare tumor (neo)antigens and of cognate T cell receptors (TCRs) expressed by tumor-infiltrating lymphocytes. T cells transduced with tumor antigen-specific TCRs identified by NeoScreen mediate regression of established tumors in patient-derived xenograft mice.

Evolution of CD8+ T Cell Receptor (TCR) Engineered Therapies for the Treatment of Cancer.

Engineered T cell receptor T (TCR-T) cell therapy has facilitated the generation of increasingly reliable tumor antigen-specific adaptable cellular products for the treatment of human cancer. TCR-T cell therapies were initially focused on targeting shared tumor-associated peptide targets, including melanoma differentiation and cancer-testis antigens. With recent technological developments, it has become feasible to target neoantigens derived from tumor somatic mutations, which represents a highly personalized therapy, since most neoantigens are patient-specific and are rarely shared between patients. TCR-T therapies have been tested for clinical efficacy in treating solid tumors in many preclinical studies and clinical trials all over the world. However, the efficacy of TCR-T therapy for the treatment of solid tumors has been limited by a number of factors, including low TCR avidity, off-target toxicities, and target antigen loss leading to tumor escape. In this review, we discuss the process of deriving tumor antigen-specific TCRs, including the identification of appropriate tumor antigen targets, expansion of antigen-specific T cells, and TCR cloning and validation, including techniques and tools for TCR-T cell vector construction and expression. We highlight the achievements of recent clinical trials of engineered TCR-T cell therapies and discuss the current challenges and potential solutions for improving their safety and efficacy, insights that may help guide future TCR-T studies in cancer.

T Cell Receptor Engineered Lymphocytes for Cancer Therapy.

T lymphocytes are capable of specific recognition and elimination of target cells. Physiological antigen recognition is mediated by the T cell receptor (TCR), which is an alpha beta heterodimer comprising the products of randomly rearranged V, D, and J genes. The exquisite specificity and functionality of T cells can be leveraged for cancer therapy: specifically, the adoptive transfer of T cells that express tumor-reactive TCRs can induce regression of solid tumors in patients with advanced cancer. However, the isolation and expression of a tumor antigen-specific TCRs is a highly involved process that requires identifying an immunogenic epitope, ensuring human cells are of the correct haplotype, performing a laborious T cell expansion process, and carrying out downstream TCR sequencing and cloning. Recent advances in single-cell sequencing have begun to streamline this process. This protocol synthesizes and expands upon methodologies to generate, isolate, and engineer human T cells with tumor-reactive TCRs for adoptive cell therapy. Though this process is perhaps more arduous than the alternative strategy of using chimeric antigen receptors (CARs) for engineering, the ability to target intracellular proteins using TCRs substantially increases the types of antigens that can be safely targeted. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of human autologous dendritic cells from monocytes Basic Protocol 2: In vitro priming and expansion of human antigen-specific T cells Basic Protocol 3: Cloning of antigen-specific T cell receptors based on single-cell VDJ sequencing data Basic Protocol 4: Validation of T cell receptor expression and functionality in vitro Basic Protocol 5: Rapid expansion of T cell receptor-transduced T cells and human T cell clones.

T-cells with a single tumor antigen-specific T-cell receptor can be generated in vitro from clinically relevant stem cell sources

ABSTRACT Chimeric antigen receptor (CAR) T-cells have shown great promise in the treatment of B-cell malignancies. For acute myeloid leukemia (AML), however, the optimal target surface antigen has yet to be discovered. Alternatively, T-cell receptor (TCR)-redirected T-cells target intracellular antigens, marking a broader territory of available target antigens. Currently, adoptive TCR T-cell therapy uses peripheral blood lymphocytes for the introduction of a transgenic TCR. However, this can cause graft-versus-host disease, due to mispairing of introduced and endogenous TCR chains. Therefore, we started from hematopoietic stem and progenitor cells (HSPC), that do not express a TCR yet, isolated from healthy donors, patients in remission after chemotherapy and AML patients at diagnosis. Using the OP9-DL1 in vitro co-culture system and agonist selection, TCR-transduced HSPC develop into mature tumor antigen-specific T-cells with only one TCR. We show here that this approach is feasible with adult HSPC from clinically relevant sources, albeit with slower maturation and lower cell yield compared to cord blood HSPC. Moreover, cryopreservation of HSPC does not have an effect on cell numbers or functionality of the generated T-cells. In conclusion, we show here that it is feasible to generate TA-specific T-cells from HSPC from adult healthy donors and patients and we believe these T-cells could be of use as a very valuable form of patient-tailored T-cell immunotherapy.

Efficient identification of neoantigen-specific T-cell responses in advanced human ovarian cancer

BackgroundEfficient identification of neoantigen-specific T-cell responses in epithelial ovarian cancer (EOC) remains a challenge. Existing investigations of spontaneous T-cell response to tumor neoepitope in EOC have taken the approach of comprehensive screening all neoantigen candidates, with a validation rate of 0.5–2%.MethodsWhole-exome and transcriptome sequencing analysis of treatment-naive EOC patients were performed to identify neoantigen candidates, and the immunogenicity of prioritized neoantigens was evaluated by analyzing spontaneous neoantigen-specfic CD4+ and CD8+ T-cell responses in the tumor and/or peripheral blood. The biological relevance of neoantigen-specific T-cell lines and clones were analyzed by evaluating the capacity of autologous ovarian tumor recognition. Genetic transfer of T-cell receptor (TCR) from these neoantigen-specific T-cell clones into peripheral blood T-cells was conducted to generate neoepitope-specific T-cells. The molecular signature associated with positive neoantigen T-cell responses was investigated, and the impacts of expression level and lymphocyte source on neoantigen identification were explored.ResultsUsing a small subset of prioritized neoantigen candidates, we were able to detect spontaneous CD4+ and/or CD8+ T-cell responses against neoepitopes from autologous lymphocytes in half of treatment-naïve EOC patients, with a significantly improved validation rate of 19%. Tumors from patients exhibiting neoantigen-specific T-cell responses exhibited a signature of upregulated antigen processing and presentation machinery, which was also associated with favorable patient survival in the TCGA ovarian cohort. T-cells specific against two mutated cancer-associated genes, NUP214 and JAK1, recognized autologous tumors. Gene-engineering with TCR from these neoantigen-specific T-cell clones conferred neoantigen-reactivity to peripheral T-cells.ConclusionsOur study demonstrated the feasibility of efficiently identifying both CD4+ and CD8+ neoantigen-specific T-cells in EOC. Autologous lymphocytes genetically engineered with tumor antigen-specific TCR can be used to generate cells for use in the personalized adoptive T-cell transfer immunotherapy.

A rare population of tumor antigen-specific CD4+CD8+ double-positive \u03b1\u03b2 T lymphocytes uniquely provide CD8-independent TCR genes for engineering therapeutic T cells

BackgroundHigh-affinity tumor antigen-specific T-cell receptor (TCR) gene is required to engineer potent T cells for therapeutic treatment of cancer patients. However, discovery of suitable therapeutic TCR genes is hampered by the fact that naturally occurring tumor antigen-specific TCRs are generally of low-affinity, and artificial modification of TCRs can mediate cross-reactivity to other antigens expressed in normal tissues. Here, we discovered a naturally occurring T-cell clone which expressed high-affinity HLA-A*02:01 (A*02)-restricted TCR against NY-ESO-1 from a patient who had NY-ESO-1-expressing ovarian tumor.MethodsA*02-restricted NY-ESO-1-specific T-cell clones were established from peripheral blood of patients who had NY-ESO-1-expressing ovarian tumors. TCR α and β chain genes were retrovirally transduced into polyclonally activated T cells. Phenotype and function of the parental and TCR-transduced T cells were analyzed by flow cytometry, ELISA and cytotoxicity assay. In vivo therapeutic efficacy was investigated in a xenograft model using NOD/SCID/IL-2Rγ-deficient (NSG) mice.ResultsA rare population of NY-ESO-1-specific T cells, which we named 19305DP, expressed cell surface CD4, CD8α, and CD8β but not CD56 and recognized A*02+NY-ESO-1+ cancer cell lines in a CD4- and CD8-independent manner. 19305DP showed a gene expression profile that is consistent with a mixed profile of CD4+ and CD8+ single-positive T cells. Both CD4+ and CD8+ T cells that were retrovirally transduced with 19305DP-derived TCR gene (19305DP-TCR) showed strong reactivity against A*02+NY-ESO-1+ cancer cells, whereas TCR genes from the conventional A*02-restricted NY-ESO-1-specific CD8+ single-positive T-cell clones functioned only in CD8+ T cells. Both 19305DP-TCR gene-engineered CD4+ and CD8+ T cells eliminated A*02+NY-ESO-1+ tumor xenografts in NSG mice. Finally, based on reactivity against a series of alanine-substituted peptides and a panel of normal human tissue-derived primary cells, 19305DP-TCR was predicted to have no cross-reactivity against any human non-NY-ESO-1 proteins.ConclusionTogether, our results indicate that the naturally occurring 19305DP-TCR derived from CD4+CD8+ double-positive αβ T cells, is a promising therapeutic TCR gene for effective and safe adoptive T-cell therapy in A*02+ patients with NY-ESO-1-expressing tumor.

Rapid Construction of Antitumor T-cell Receptor Vectors from Frozen Tumors for Engineered T-cell Therapy.

T cells genetically engineered with tumor antigen-specific T-cell receptor (TCR) genes have demonstrated therapeutic potential in patients with solid tumors. In order to achieve broader application, an efficient method to identify TCR genes for an array of tumor antigens and HLA restriction elements is required. Here, we have developed a method to construct a TCR-expression library from specimens, including frozen tumor biopsies, that contain antigen-specific T cells. TCR-expressing cassettes were constructed and cloned in a retroviral plasmid vector within 24 hours by unbiased PCR amplification of TCR α and β chain variable regions assembled with TCR constant regions. The method was validated by constructing TCR-expressing vectors from tumor antigen-specific T-cell clones and functionally assessing TCR gene-transduced T cells. We applied this method to frozen ovarian tumor specimens that were infiltrated by tumor antigen-specific T cells. The tumor-derived TCR libraries were expressed in peripheral T cells from healthy volunteers and screened for tumor antigen-specific TCR pairs with the use of an MHC/peptide tetramer reagent. Tumor antigen-specific TCR-expressing transgenes were recovered from isolated tetramer-positive T cells. Peripheral T cells that were engineered with library-derived TCR gene showed potent therapeutic antitumor effect in a tumor xenograft model. Our method can efficiently and rapidly provide tumor-specific TCR-expressing viral vectors for the manufacture of therapeutic and personalized antitumor T-cell products. Cancer Immunol Res; 6(5); 594-604. ©2018 AACR.

Characterization of Postinfusion Phenotypic Differences in Fresh Versus Cryopreserved TCR Engineered Adoptive Cell Therapy Products

Adoptive cell therapy (ACT) consisting of genetically engineered T cells expressing tumor antigen-specific T-cell receptors displays robust initial antitumor activity, followed by loss of T-cell activity/persistence and frequent disease relapse. We characterized baseline and longitudinal T-cell phenotype variations resulting from different manufacturing and administration protocols in patients who received ACT. Patients with melanoma who enrolled in the F5-MART-1 clinical trial (NCT00910650) received infusions of MART-1 T-cell receptors transgenic T cells with MART-1 peptide-pulsed dendritic cell vaccination. Patients were divided into cohorts based on several manufacturing changes in the generation and administration of the transgenic T cells: decreasing ex vivo stimulation/expansion time, increased cell dose, and receiving fresh instead of cryopreserved cells. T-cell phenotypes were analyzed by flow cytometry at baseline and longitudinally in peripheral blood. Transgenic T cells with shorter ex vivo culture/expansion periods displayed significantly increased expression of markers associated with less differentiated naive/memory populations, as well as significantly decreased expression of the inhibitory receptor programmed death 1 (PD1). Patients receiving fresh infusions of transgenic cells demonstrated expansion of central memory T cells and delayed acquisition of PD1 expression compared with patients who received cryopreserved products. Freshly infused transgenic T cells showed persistence and expansion of naive and memory T-cell populations and delayed acquisition of PD1 expression, which correlated with this cohort’s superior persistence of transgenic cells and response to dendritic cell vaccines. These results may be useful in designing future ACT protocols.

Inducible MyD88/CD40 (iMC) Enhances Proliferation and Survival of Tumor-Specific TCR-Modified T Cells and Improves Anti-Tumor Efficacy in Myeloma

Introduction: Use of T cells engineered to express antigen-specific T cell receptors (TCRs) has shown promise as a cancer immunotherapy treatment; however, durable responses have been limited by poor T cell persistence and expansion in vivo. Additionally, MHC class I downregulation on tumor cells further reduces therapeutic efficacy. Therefore, we co-expressed in human T cells a novel, small molecule dimerizer (rimiducid)-dependent T cell "activation switch", called inducible MyD88/CD40 (iMC), along with tumor antigen-specific TCRs to regulate T cell activation and expansion, while upregulating MHC class I expression on tumor cells.Methods: Human T cells were activated with anti-CD3/CD28 and transduced with g-retroviruses encoding TCR α and β chains recognizing either the cancer-testes antigen PRAME (HLA-A*201-restricted SLLQHLIGL) or the B cell-specific transcriptional co-activator, Bob1/OBF-1 (HLA-B*702-restricted APAPTAVVL). Parallel "GoTCR" vectors co-expressed the αβ TCR and iMC, comprising signaling domains from MyD88 and CD40 fused in-frame with tandem rimiducid-binding FKBP12v36 domains. Proliferation, cytokine production and cytotoxicity of modified T cells was assessed using peptide-pulsed EGFPluc-expressing T2 cells (PRAME only) or PRAME+/Bob1+, HLA-A2+ HLA-B7+ EGFPluc-expressing U266 myeloma cells ± rimiducid (10 nM). MHC class I upregulation on tumor cells was measured using transwell assays and flow cytometry. In vitro tumor killing and T cell proliferation were analyzed using T cell and tumor coculture assays by either measuring loss of luciferase activity overnight or by flow cytometry over a period of 4-7 days. Finally, in vivo efficacy was determined using immune-deficient NSG mice engrafted i.v. with U266 cells and treated i.v. with 5x106-1x107 transduced T cells. iMC was activated in vivo by weekly or biweekly i.p. rimiducid injections (1-5 mg/kg). Tumor size and T cell expansion was measured using in vivo bioluminescence imaging and flow cytometry, respectively.Results: All vectors efficiently (~85%) transduced activated T cells and showed antigen-specific IFN-g production and cytolytic function against peptide-pulsed T2 cells and/or PRAME+Bob1+ U266 myeloma cells. However, both TCR ligation and rimiducid-dependent iMC costimulation were required for IL-2 production against PRAME peptide-pulsed T2 cells. Coculture assays against U266 cells showed that tumor elimination was optimized with concurrent rimiducid-driven iMC activation in both "GoPRAME" and "GoBob1" constructs, and this was accompanied by greatly increased IL-2 secretion and robust T cell proliferation (~ 50-fold vs PRAME or Bob1-specific TCRs alone). Further, iMC activation produced IFN-g independently of TCR ligation, which significantly increased MHC class I expression on tumor cells (~ 7-fold) relative to PRAME TCR-transduced T cells. In NSG mice engrafted with PRAME+ U266 myeloma tumors, GoPRAME TCR-modified T cells persisted for at least 81 days post-injection and prevented tumor growth, unlike any of the other T cell groups. Importantly, weekly rimiducid injection dramatically expanded iMC-PRAME TCR-expressing T cell numbers by ~1000-fold on day 81 post-injection compared to T cells expressing only the PRAME TCR (p < 0.001).Summary: iMC is a novel "Go" switch that utilizes rimiducid, a small molecule dimerizer, to provide costimulation to PRAME and Bob1-specific TCR-engineered T cells while sensitizing tumors to TCR-mediated recognition via cytokine-induced MHC I upregulation. These iMC-enhanced TCRs are prototypes of novel "GoTCR" engineered T cell therapies that may increase efficacy, safety and durability of adoptive T cell therapies. DisclosuresPentcheva-Hoang:Bellicum Pharmaceuticals: Employment. Torres:Bellicum Pharmaceuticals: Employment. Rodriguez:Bellicum Pharmaceuticals: Employment. Korngold:Bellicum Pharmaceuticals: Employment. Lu:Bellicum Pharmaceuticals: Employment. Crisostomo:Bellicum Pharmaceuticals: Employment. Moseley:Bellicum Pharmaceuticals: Employment, Membership on an entity's Board of Directors or advisory committees. Slawin:Bellicum Pharmaceuticals: Employment, Equity Ownership. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership. Foster:Bellicum Pharmaceuticals: Employment.

Abstract B078: GoTCR: Inducible MyD88/CD40 (iMC) enhances proliferation and survival of tumor-specific TCR-modified T cells and improves antitumor efficacy in myeloma

Abstract Introduction: Use of T cells engineered to express antigen-specific T cell receptors (TCRs) has shown promise as a cancer immunotherapy treatment; however, durable responses have been limited by poor T cell persistence and expansion in vivo. Additionally, MHC class I downregulation on tumor cells further reduces therapeutic efficacy. Therefore, we co-expressed in human T cells a novel, small molecule dimerizer (rimiducid)-dependent T cell “activation switch”, called inducible MyD88/CD40 (iMC), along with tumor antigen-specific TCRs to regulate T cell activation and expansion, while upregulating MHC class I expression on tumor cells. Methods: Human T cells were activated with anti-CD3/CD28 and transduced with γ-retroviruses encoding TCR α and β chains recognizing either the cancer-testes antigen PRAME (HLA-A*201-restricted SLLQHLIGL) or the B cell-specific transcriptional co-activator, Bob1/OBF-1 (HLA-B*702-restricted APAPTAVVL). Parallel “GoTCR” vectors co-expressed the αβ TCR and iMC, comprising signaling domains from MyD88 and CD40 fused in frame with tandem rimiducid-binding FKBP12v36 domains. Proliferation, cytokine production and cytotoxicity of modified T cells was assessed using peptide-pulsed EGFPluc-expressing T2 cells (PRAME only) or PRAME+/Bob1+, HLA-A2+ HLA-B7+ EGFPluc-expressing U266 myeloma cells ± rimiducid (10 nM). MHC class I upregulation on tumor cells was measured using transwell assays and flow cytometry. In vitro tumor killing and T cell proliferation were analyzed using T cell and tumor coculture assays by either measuring loss of luciferase activity overnight or by flow cytometry over a period of 4-7 days. Finally, in vivo efficacy was determined using immune-deficient NSG mice engrafted i.v. with U266 cells and treated i.v. with 5×106-1×107 transduced T cells. iMC was activated in vivo by weekly or biweekly i.p. rimiducid injections (1-5 mg/kg). Tumor size and T cell expansion was measured using in vivo bioluminescence imaging and flow cytometry, respectively. Results: All vectors efficiently (∼85%) transduced activated T cells and showed antigen-specific IFN-γ production and cytolytic function against peptide-pulsed T2 cells and/or PRAME+Bob1+ U266 myeloma cells. However, both TCR ligation and rimiducid-dependent iMC costimulation were required for IL-2 production against PRAME peptide-pulsed T2 cells. Coculture assays against U266 cells showed that tumor elimination was optimized with concurrent rimiducid-driven iMC activation in both “GoPRAME” and “GoBob1” constructs, and this was accompanied by greatly increased IL-2 secretion and robust T cell proliferation (∼ 50-fold vs PRAME or Bob1-specific TCRs alone). Further, iMC activation produced IFN-γ independently of TCR ligation, which significantly increased MHC class I expression on tumor cells (∼ 7-fold) relative to PRAME TCR-transduced T cells. In NSG mice engrafted with PRAME+ U266 myeloma tumors, GoPRAME TCR-modified T cells persisted for 81 days post-injection and prevented tumor growth, unlike any of the other T cell groups. Importantly, weekly rimiducid injection dramatically expanded iMC-PRAME TCR-expressing T cell numbers by ∼1000-fold on day 81 post-injection compared to T cells expressing only the PRAME TCR (p &amp;lt; 0.001). Summary: iMC is a novel “Go” switch that utilizes rimiducid, a small molecule dimerizer, to provide costimulation to PRAME and Bob1-specific TCR-engineered T cells while sensitizing tumors to TCR-mediated recognition via cytokine-induced MHC I upregulation. These iMC-enhanced TCRs are prototypes of novel “GoTCR” engineered T cell therapies that may increase efficacy, safety and durability of adoptive T cell therapies. Citation Format: Tsvetelina Pentcheva-Hoang, David Torres, Tania Rodriguez, Ana Korngold, An Lu, Jeannette Crisostomo, Annemarie Moseley, Lorenz Jahn, Mirjam H.M. Heemskerk, Kevin Slawin, David Spencer, Aaron Foster. GoTCR: Inducible MyD88/CD40 (iMC) enhances proliferation and survival of tumor-specific TCR-modified T cells and improves antitumor efficacy in myeloma [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B078.

641. Highly Efficient, ZFN-Driven Knockout of Surface Expression of the T-Cell Receptor and HLA Class I Proteins in Human T-Cells for Enhancing Allogeneic Adoptive Cell Therapies

While adoptive transfer of T-cells modified to express a chimeric antigen receptor or tumor antigen specific T-cell receptor (TCR) has shown great promise for the treatment of malignant cancers, most current clinical applications are limited by the use of autologous T-cell products. Targeting of the TCR and HLA Class I genes in primary T-cells thus represent attractive targets for genome editing in order to produce universal T cells from allogeneic donors. Elimination of the native TCR and HLA class I proteins on T-cells would, respectively, reduce the risk of graft-versus-host disease and host-versus-graft clearance mediated by the adaptive immune system. We have developed clinical grade zinc finger nuclease (ZFN) reagents that can efficiently target the T-cell receptor alpha constant (TRAC) and beta-2-microglobulin (B2M) loci. ZFN encoding mRNAs were introduced into purified T-cells by electroporation. Without selection, T-cells treated with TRAC specific ZFNs showed modification of up to 89% of alleles as gauged by deep sequencing. Flow analysis showed 91% of treated cells were negative for CD3 expression. Results from ZFN mRNA dose titration studies showed that the level of TRAC gene modification by deep sequencing was highly correlated with the percentage of cells that stain negative for CD3 expression by FACS (Spearman rho = 0.96; p < 0.0001). B2M is a subunit in all HLA class I molecules, and represents a conserved target for eliminating HLA class I presentation in cells from different donors. Analysis of T-cells transfected with mRNAs encoding B2M specific ZFNs showed up to 94% of alleles were modified, as determined by deep sequencing. FACS analysis showed that 89% of treated cells were negative for expression of HLA-A, B, C. Similar to TRAC modified cells, a marked correlation was observed between the percentage of B2M alleles modified and percentage of cells lacking surface expression of HLA-A,B, and C (Spearman rho = 0.57; p < 0.0001). ZFN mediated genome editing was well tolerated. T-cells treated with TRAC or B2M specific ZFNs showed similar viability and growth characteristics as mock transfected cells. Together, these highly efficient ZFN reagents permit the highly efficient double knockout of TCR and HLA class I surface expression in primary human T-cells for potential use in the development of allogeneic cell therapies.

Functional Characterization of a T Cell Stimulation Reagent for the Production of Therapeutic Chimeric Antigen Receptor T Cells

Adoptive cell therapy using gene-modified T cells has demonstrated promising clinical outcomes in hematologic malignancies. Production of gene-modified T cells involves the selection of patient T cells, activation via stimulation through the endogenous T cell receptor (TCR) complex and a costimulatory domain, followed by introduction of a tumor antigen-specific TCR or chimeric antigen receptor (CAR) through gene modification. Here we characterize a soluble T cell stimulation reagent, known as an ExpamerTM reagent, in the production of therapeutic CAR T cells.The Expamer reagent used in these studies is designed to be a late-stage clinical and commercial manufacturing ancillary material with two important attributes that make it highly attractive from a manufacturing and regulatory standpoint; it is a soluble and dissociable reagent. These attributes increase the ease of both introduction and removal from the manufacturing process, giving products manufactured with this reagent consistent product quality and purity. This reagent activates T cells through the simultaneous engagement of the TCR-CD3 complex and the costimulatory receptor CD28 and is compatible with manufacturing of both current and next-generation therapeutics.Purified healthy donor T cells cultured in the presence of the Expamer reagent rapidly fluxed Ca2+, demonstrating the capacity to induce early TCR signaling. Activation through this reagent additionally promotes upregulation of the cell surface activation marker CD25 and proliferation as measured by CFSE dilution. Following stimulation with this reagent, T cells are readily transduced with a CD19-specific CAR construct. The function of CAR T cells generated with this reagent was measured by effector cytokine production, proliferation, and cytolytic activity in the presence of CD19 expressing and control target cells in vitro. CAR T cells robustly produced IFN-ɣ and IL-2 after activation with a CAR specific antigen. In addition, proliferation in the presence of CD19 expressing target cells was observed as measured by CFSE dilution. Finally, significant cytolytic activity against CD19-expressing target cells was observed.Collectively, these data provide evidence that functional engineered T cells can be manufactured using the Expamer reagent and support implementation into the production of both current and next-generation therapeutic gene-modified T cells.The first two authors contributed equally to this work. DisclosuresBashour:Juno Therapeutics: Employment. Larson:Juno Therapeutics: Employment. Graef:Juno Therapeutics: Employment. Stemberger:Juno Therapeutics: Employment. Lothar:Juno Therapeutics: Employment. Odegard:Juno Therapeutics: Employment. Ramsborg:Juno Therapeutics: Employment.

Engineered T-Cells Expressing An HLA-Restricted Affinity-Enhanced TCR In Advanced Multiple Myeloma Patients Post Auto-SCT Engraft and Are Associated With Encouraging Post Auto-SCT Responses

▪ BackgroundDespite recent therapeutic advances, multiple myeloma (MM) remains primarily an incurable cancer. Patients experiencing rapid recovery of T cells post autologous stem cell transplant (auto-SCT) may have improved outcomes, and spontaneous cellular responses to tumor can occur, suggesting immune mediated control of tumor is possible. We and others have investigated therapeutic cancer vaccines that have shown promise in pilot studies, in particular following post-transplant infusion of activated autologous T cells. However, efficacy of these approaches may be limited by thymic selection which restricts the repertoire of T cell receptors (TCRs) to low affinity TCRs that cannot recognize the low level of antigen present on most tumor cells. We hypothesized that incorporation of affinity-enhanced tumor antigen-specific TCRs into autologous T cells infused post-transplant would overcome this limitation and improve response rates in the post auto-SCT setting. MethodsWe report interim results of a Phase II clinical trial (NCT01352286) to evaluate the safety and activity of autologous T cells genetically engineered to express an affinity-enhanced TCR that recognizes the NY-ESO-1/LAGE-1 peptide complex HLA‐A*0201‐SLLMWITQC; these cells are infused in the setting of profound lymphodepletion that accompanies high dose chemotherapy administered during auto-SCT. Patients with high risk or relapsed MM, who are HLA‐A*0201 positive, and whose tumor is positive for NY-ESO-1 and/or LAGE-1 by RT-PCR are eligible. CD25 depleted CD4 and CD8 T cells are activated and expanded using anti-CD3/CD28 antibody conjugated microbeads, and genetically modified with a lentiviral vector containing the TCR construct at a multiplicity of infection of 1. Engineered T cells are administered four days after high dose melphalan and two days following auto-SCT, at a dose range of 1-10 billion total cells with a minimum gene modification requirement of 10%. Patients are evaluated for MM responses in accordance with the IMWG criteria at 6 weeks, and 3 and 6 months post infusion. At 3 months, patients start lenalidomide maintenance. The initial 6 patient phase is complete and a 20 patient extension phase is ongoing. ResultsPrior to enrollment on study, patients had received a median of 3 prior therapies including 6 with prior transplant. 50% of tumors contained high risk chromosomal abnormalities, and NY-ESO-1 expression is correlated with adverse prognosis. 20 patients (average age of 57) have been infused with an average of 2.3 X 109 engineered T cells (range 4.5 X 108-3.9 X 109); this reflects an average clinical scale transduction efficiency of 34% (range 18% – 49%). Infusions have been well tolerated, and the majority of adverse events were related to the high dose melphalan. Possibly related SAEs were neutropenia and thrombocytopenia, and GI and metabolism disorders including diarrhea, colitis, hyponatremia and hypomagnesemia. 10, 4, 2, and 2 patients have reached the 1 year, 9 month, 6 month and 3 month assessment timepoints, respectively, and 17/20 patients are alive. Best response by day 100 is sCR/CR in 2/15 (13%), nCR in 10/15 (67%), and PR in 3/15% (20%), which compares favorably to historic responses in patients undergoing first or second transplant. Engineered T cells expanded and persisted in blood and marrow at 180 days by Q-PCR and flow-cytometry in all but one case (Figure). 7 patients progressed after day 100, which was accompanied either by loss of engineered T cells or loss of tumor antigen. Detailed phenotyping and functional analysis of engineered T cells, and correlates with clinical responses, is underway. SummaryThis is the first clinical evaluation of engineered T cells in the MM setting. Infusions are safe, well tolerated, and are associated with encouraging responses in a high risk myeloma population. A study evaluating the engineered T cells in a non-transplant study is underway. Disclosures:Stadtmauer:Celgene: Consultancy. Binder-Scholl:Adaptimmune: Employment. Smethurst:Adaptimmune: Employment. Brewer:Adaptimmune: Employment. Bennett:Adaptimmune: Employment. Gerry:Adaptimmune: Employment. Pumphrey:Adaptimmune: Employment. Tayton-Martin:Adaptimmune: Employment. Ribeiro:Adaptimmune: Employment. Levine:Novartis: cell and gene therapy IP, cell and gene therapy IP Patents & Royalties. Jakobsen:Adaptimmune: Employment. Kalos:Novartis corporation: CART19 technology, CART19 technology Patents & Royalties; Adaptive biotechnologies: Member scientific advisory board , Member scientific advisory board Other. June:Novartis: Patents & Royalties, Research Funding. [Display omitted]

A new cloning and expression system yields and validates TCRs from blood lymphocytes of patients with cancer within 10 days

Antigen-specific T cell therapy, or T cell receptor (TCR) gene therapy, is a promising immunotherapy for infectious diseases and cancers. However, a suitable rapid and direct screening system for antigen-specific TCRs is not available. Here, we report an efficient cloning and functional evaluation system to determine the antigen specificity of TCR cDNAs derived from single antigen-specific human T cells within 10 d. Using this system, we cloned and analyzed 380 Epstein-Barr virus-specific TCRs from ten healthy donors with latent Epstein-Barr virus infection and assessed the activity of cytotoxic T lymphocytes (CTLs) carrying these TCRs against antigenic peptide-bearing target cells. We also used this system to clone tumor antigen-specific TCRs from peptide-vaccinated patients with cancer. We obtained 210 tumor-associated antigen-specific TCRs and demonstrated the cytotoxic activity of CTLs carrying these TCRs against peptide-bearing cells. This system may provide a fast and powerful approach for TCR gene therapy for infectious diseases and cancers.

Influenza virus-specific TCR-transduced T cells as a model for adoptive immunotherapy

Adoptive transfer of T lymphocytes equipped with tumor-antigen specific T-cell receptors (TCRs) represents a promising strategy in cancer immunotherapy, but the approach remains technically demanding. Using influenza virus (Flu)-specific T-cell responses as a model system we compared different methods for the generation of T-cell clones and isolation of antigen-specific TCRs. Altogether, we generated 12 CD8+ T-cell clones reacting to the Flu matrix protein (Flu-M) and 6 CD4+ T-cell clones reacting to the Flu nucleoprotein (Flu-NP) from 4 healthy donors. IFN-γ-secretion-based enrichment of antigen-specific cells, optionally combined with tetramer staining, was the most efficient way for generating T-cell clones. In contrast, the commonly used limiting dilution approach was least efficient. TCR genes were isolated from T-cell clones and cloned into both a previously used gammaretroviral LTR-vector, MP91 and the novel lentiviral self-inactivating vector LeGO-MP that contains MP91-derived promotor and regulatory elements. To directly compare their functional efficiencies, we in parallel transduced T-cell lines and primary T cells with the two vectors encoding identical TCRs. Transduction efficiencies were approximately twice higher with the gammaretroviral vector. Secretion of high amounts of IFN-γ, IL-2 and TNF-α by transduced cells after exposure to the respective influenza target epitope proved efficient specificity transfer of the isolated TCRs to primary T-cells for both vectors, at the same time indicating superior functionality of MP91-transduced cells. In conclusion, we have developed optimized strategies to obtain and transfer antigen-specific TCRs as well as designed a novel lentiviral vector for TCR-gene transfer. Our data may help to improve adoptive T-cell therapies.

Introduction of Exogenous T-cell Receptors Into Human Hematopoietic Progenitors Results in Exclusion of Endogenous T-cell Receptor Expression

Current tumor immunotherapy approaches include the genetic modification of peripheral T cells to express tumor antigen-specific T-cell receptors (TCRs). The approach, tested in melanoma, has led to some limited success of tumor regression in patients. Yet, the introduction of exogenous TCRs into mature T cells entails an underlying risk; the generation of autoreactive clones due to potential TCR mispairing, and the lack of effective negative selection, as these peripheral cells do not undergo thymic selection following introduction of the exogenous TCR. We have successfully generated MART-1-specific CD8 T cells from genetically modified human hematopoietic stem cells (hHSC) in a humanized mouse model. The advantages of this approach include a long-term source of antigen specific T cells and proper T-cell selection due to thymopoiesis following expression of the TCR. In this report, we examine the molecular processes occurring on endogenous TCR expression and demonstrate that this approach results in exclusive cell surface expression of the newly introduced TCR, and the exclusion of endogenous TCR cell surface expression. This suggests that this stem cell based approach can provide a potentially safer approach for anticancer immunotherapy due to the involvement of thymic selection.

Adoptive Transfer of Gene-Modified T-Cells Engineered to Express High-Affinity TCRs for Cancer-Testis Antigens (CTAs) NY-ESO-1 or Lage-1, in MM Patients Post Auto-SCT

Abstract 472 Background:Despite recent therapeutic advances, multiple myeloma (MM) remains primarily an incurable cancer. Patients experiencing rapid recovery of T cells post auto-SCT may have improved outcomes, and spontaneous cellular responses to tumor can occur, suggesting immune mediated control of tumor is possible. We and others have investigated therapeutic cancer vaccines that have shown promise in pilot studies. However, vaccine efficacy may be limited by thymic selection which restricts the repertoire of T cell receptors (TCRs) to low affinity TCRs that cannot recognize the reduced level of antigen present on most tumor cells. We hypothesized that incorporation of affinity-enhanced tumor antigen specific TCRs into autologous T cells would overcome this limitation. Methods:We report interim results of a Phase II clinical trial (NCT01352286) to evaluate the safety and activity of autologous T cells genetically engineered to express an HLA-A201 restricted, affinity-enhanced TCR targeting an epitope shared by the NY-ESO-1 and LAGE-1 tumor antigens (gene engineered cells, or GEC). Inclusion criteria include: 1) eligibility for auto-SCT, 2) PS of 0–2, 3) high risk MM or relapse after prior therapy (prior allo-SCT excluded), 4) HLA-A201 positive, and 5) NY-ESO-1 and/or LAGE-1 positive tumor. GEC are manufactured from monocyte and T regulatory cell depleted apheresis, followed by lentivector gene transfer and T cell expansion by bead-based artificial antigen presenting cells. GEC are administered four days after high dose melphalan and two days following auto-SCT, at a dose range of 1–10 billion cells. Patients are evaluated for MM responses in accordance with the IMWG criteria at 6 weeks, and 3 and 6 months post infusion. At 3 months, patients start lenalidomide maintenance. The initial 6 patient phase is complete and a 20 patient extension phase is ongoing. Results:19 patients have been enrolled, 12 have been infused, 3 are pending infusion, and 4 were taken off study prior to infusion due to disease progression. The average TCR transfer efficiency was 31.9% (range 18.2%-45%). 6, 4, and 1 patients have reached the 6 month, 3 month, and 6 week post infusion MM assessment time points, respectively. All treated patients are alive. 3/11 (27%) and 7/11 (64%) patients have a stringent CR or best response of VGPR or better, respectively; 3/11 (27%) have stable or partial responses, and 1/11 (9%) had progressive disease. In patients with VGPR, oligoclonal banding was common, marrow showed no expansion of clonal plasma cells, and all had normalized light chain ratios, suggesting at most minimal residual disease. These results are encouraging, especially considering that 3/11 patients had prior ASCT and 8/11 had adverse cytogenetics. GEC infusions have been well tolerated. The majority of adverse events are related to the high dose melphalan. Several patients had a transient skin rash with lymphocytosis and others had a diarrheal syndrome that occurred later than expected for melphalan-induced mucositis. In all cases GI toxicity has been self-limited or treatable with a short dose of corticosteroids. Possibly related SAEs were cytopenias such as neutropenia and thrombocytopenia, and GI and metabolism disorders such as diarrhea, colitis, hyponatremia and hypomagnesemia. GEC have been detected for up to 1 year at 1% in the circulation and bone marrow. Detailed correlative analysis is reported elsewhere in this meeting (Kalos, M. et al). Summary:This is the first test of GEC in the MM setting. Infusion of GEC administered post auto-SCT is safe, well tolerated, and leads to high response rate in a high risk myeloma population. Phase II accrual continues and a study evaluating GEC outside of auto-SCT is planned. Disclosures:Stadtmauer:celgene: Consultancy; millenium: Consultancy. Binder-Scholl:Adaptimmune : Employment. Brewer:Adaptimmune Ltd: Employment. Bennett:Adaptimmune Ltd: Employment. Gerry:Adaptimmune Ltd: Employment. Pumphrey:Adaptimmune Ltd: Employment. Smethurst:Adaptimmune Ltd: Employment. Tayton-Martin:Adaptimmune Ltd: Employment. Lilliam:Adaptimmune: Employment. Levine:TxCell: Consultancy, Membership on an entity’s Board of Directors or advisory committees; University of Pennsylvania: financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight, financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight Patents & Royalties. Jakobsen:Adaptimmune Ltc: Employment. June:Novartis: Research Funding, entitled to receive royalties from patents licensed to Novartis, entitled to receive royalties from patents licensed to Novartis Patents & Royalties.