Tumor-specific T-cell Receptor Research Articles

Abstract 5569: Discovery of tumor-specific T cell receptors (TCRs) by reverse genetic engineering platform

Abstract TCR-T therapy is an ideal cell therapeutic strategy against solid tumors. However, the traditional method of TCR cloning has two major problems. First, we could only develop TCRs against previously discovered tumor antigen epitopes. Second, the affinity maturation of TCR sometimes leads to mortal toxicities due to the unexpected cross-reactivities. Therefore we hereby present a reverse genetic engineering platform to clone tumor specific TCRs from immunotherapies benefit cancer patients. Multi-antigen stimulating cell therapy (MASCT) is a cell-based immunotherapy combining therapeutic DC vaccine and adoptive T cell transfer, the IND application of which was approved in China. We observed dynamic T cell responses against multiple tumor-associated antigens used in MASCT-treated HCC patients, which showed a significant correlation with clinical outcome. Based on this observation, we propose that expanded tumor-specific T cells in MASCT-benefit cancer patients could be good sources to isolate clinical relevant TCRs. A HPV positive metastatic cervical cancer patient has shown partially response after MASCT treatment alone, and survived over 5 years. Moreover, a metastatic lung cancer patient has also shown partially response and survived for longer than 2 years after combination treatment of MASCT and anti-PD1 antibody. Enhanced and persist T cell responses against several TAAs as well as HPV antigens were detected in PBMCs of both patients. We successfully expended and isolated tumor antigen-specific T cells by IFNγ-secreting cell enrichment, and obtained alpha/beta chains paired TCR sequences using single T cell NGS technology. We selected 14 pairs of TCRs isolated from cervical cancer patient for further validation. TCRs from lung cancer patient were currently under synthesis or sequencing. Among 14 TCRs, a TAA-specific TCR and a HPV18E7-specific TCR showed superior antigen specificity and optimum avidity, and would be applied for TCR-T development. Of notes, our reverse genetic engineering of TCR-T (ReGET) platform.can not only identify both HLA class I and II-restricted TCRs, but also identify novel epitopes of tumor antigens, which may provide new targets for TCR-T therapy against solid tumors. Citation Format: Yanyan Han, Xiaoling Liang, Shudan Ou, Xihe Chen, Minjun Ma, Jun Li, Yifan Ma, Xiangjun Zhou. Discovery of tumor-specific T cell receptors (TCRs) by reverse genetic engineering platform [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5569.

Structural basis for oligoclonal T cell recognition of a shared p53 cancer neoantigen

Adoptive cell therapy (ACT) with tumor-specific T cells can mediate cancer regression. The main target of tumor-specific T cells are neoantigens arising from mutations in self-proteins. Although the majority of cancer neoantigens are unique to each patient, and therefore not broadly useful for ACT, some are shared. We studied oligoclonal T-cell receptors (TCRs) that recognize a shared neoepitope arising from a driver mutation in the p53 oncogene (p53R175H) presented by HLA-A2. Here we report structures of wild-type and mutant p53–HLA-A2 ligands, as well as structures of three tumor-specific TCRs bound to p53R175H–HLA-A2. These structures reveal how a driver mutation in p53 rendered a self-peptide visible to T cells. The TCRs employ structurally distinct strategies that are highly focused on the mutation to discriminate between mutant and wild-type p53. The TCR–p53R175H–HLA-A2 complexes provide a framework for designing TCRs to improve potency for ACT without sacrificing specificity.

Massively parallel interrogation and mining of natively paired human TCRαβ repertoires.

T cells engineered to express antigen-specific T cell receptors (TCRs) are potent therapies for viral infections and cancer. However, efficient identification of clinical candidate TCRs is complicated by the size and complexity of T cell repertoires and the challenges of working with primary T cells. Here, we present a high-throughput method to identify TCRs with high functional avidity from diverse human T cell repertoires. The approach uses massively parallel microfluidics to generate libraries of natively paired, full-length TCRαβ clones, from millions of primary T cells, which are then expressed in Jurkat cells. The TCRαβ-Jurkat libraries enable repeated screening and panning for antigen-reactive TCRs using peptide:MHC binding and cellular activation. We captured >2.9 million natively paired TCRαβ clonotypes from six healthy human donors and identified rare (<0.001% frequency) viral antigen–reactive TCRs. We also mined a tumor-infiltrating lymphocyte (TIL) sample from a melanoma patient and identified several tumor-specific TCRs, which, after expression in primary T cells, led to tumor cell killing.

Abstract 3221: T-cell receptor sequencing for pharmacodynamic and response biomarkers of checkpoint blockade

Abstract Background: Immunomodulatory cancer drugs such as anti-programmed death 1 (PD-1) and anti-cytotoxic T-lymphocyte antigen 4 (CTLA4) antibodies have shown significant clinical benefits across multiple indications. However, not all patients benefit from checkpoint blockade (CB) treatment strategies, highlighting the need for predictive biomarkers of response. Features of the T-cell receptor (TCR) repertoire have been reported to correlate with CB treatment and response (1). Here, we performed a comprehensive, retrospective analysis of TCR repertoires from different tissues (blood and tumor) at different time points (baseline and on treatment). To the best of our knowledge, our study, comprising a total of 558 samples across 3 tumor types (non-small cell lung cancer [NSCLC], renal cell carcinoma [RCC], and melanoma), is the largest analysis of TCR-sequencing data in CB clinical trials to date. Methods: We performed TCR sequencing on formalin-fixed paraffin-embedded tissue samples or peripheral blood samples using immunoSEQ Assays (Adaptive Biotechnologies Corp.) to profile the immune repertoire of patients from 3 clinical trials of nivolumab (anti-PD-1) or nivolumab + ipilimumab (anti-CTLA4) (NCT02041533, NCT01358721, NCT01621490). Repertoire clonality and clonal expansion statistics were computed, compared, and tested for their association with clinical activity. Results: We characterized the TCR repertoire from peripheral blood at baseline for 263 patients (209 NSCLC, 54 RCC) with matched on-treatment profiles for 49 patients (49 RCC). In addition, we profiled tumor TCR repertoires at baseline and on treatment for 127 patients (54 RCC, 73 melanoma) at time points predefined in each study’s protocol. Our analyses show that clonal expansion was accompanied by a comparable contraction of other clones and thus was not directionally enriched upon CB treatment. Moreover, comparison of baseline and on-treatment repertoire clonality did not reveal a significant shift following CB treatment. Results were consistent in blood and tumor tissue and across response groups. Baseline peripheral blood TCR clonality was not associated with best overall response or progression-free survival. Conclusions: Our results highlight that TCR repertoires are highly dynamic and that the currently established metrics are difficult to interpret or implement clinically as pharmacodynamic or response biomarkers. The variability in TCR clonality modulation upon CB treatment underscores the challenge of selecting the adequate on-treatment time point. Thus, our comprehensive analysis highlights the need for a better understanding of the TCR repertoire dynamics and development of methods to identify and functionally annotate tumor-specific TCRs. Reference: 1. Riaz N et al. Cell 2017;171:934-49 Citation Format: Simon Papillon-Cavanagh, Alice M. Walsh, Zhenhao Qi, Megan Wind-Rotolo, Abdel Saci, Parul Doshi, Radu Dobrin, Joseph Szustakowski. T-cell receptor sequencing for pharmacodynamic and response biomarkers of checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3221.

T-cell receptor gene therapy targeting melanoma-associated antigen-A4 by silencing of endogenous TCR inhibits tumor growth in mice and human.

Genetically engineered T cells expressing a T-cell receptor (TCR) are powerful tools for cancer treatment and have shown significant clinical effects in sarcoma patients. However, mismatch of the introduced TCR α/β chains with endogenous TCR may impair the expression of transduced TCR, resulting in an insufficient antitumor capacity of modified T cells. Here, we report the development of immunotherapy using human lymphocytes transduced with a codon-optimized melanoma-associated antigen (MAGE)-A4 and HLA-A*2402-restricted TCR, which specifically downregulate endogenous TCR by small interfering RNA (si-TCR). We evaluated the efficacy of this immunotherapy in both NOD-SCID mice and uterine leiomyosarcoma patients. Our results revealed that transduced human lymphocytes exhibited high surface expression of the introduced tumor-specific TCR, enhanced cytotoxic activity against antigen-expressing tumor cells, and increased interferon-γ production by specific MAGE-A4 peptide stimulation. Retarded tumor growth was also observed in NOD-SCID mice inoculated with human tumor cell lines expressing both MAGE-A4 and HLA-A*2402. Furthermore, we report the successful management of a case of uterine leiomyosarcoma treated with MAGE-A4 si-TCR/HLA-A*2402 gene-modified T cells. Our results indicate that the TCR-modified T cell therapy is a promising novel strategy for cancer treatment.

Gene editing: Towards the third generation of adoptive T-cell transfer therapies.

First-generation adoptive T-cell transfer (ACT) administering tumor-infiltrating lymphocytes (TILs), and second-generation ACT using autologous T cells genetically modified to express tumor-specific T-cell receptors (TCRs) or chimeric antigen receptors (CARs) have both shown promise for the treatment of several cancers, including melanoma, leukemia and lymphoma. However, these treatments require labor-intensive manufacturing of the cell product for each patient, frequently utilize lentiviral or retroviral vectors to genetically modify the T cells, and have limited antitumor efficacy in solid tumors. Gene editing is revolutionizing the field of gene therapy, and ACT is at the forefront of this revolution. Gene-editing technologies can be used to re-engineer the phenotype of T cells to increase their antitumor potency, to generate off-the-shelf ACT products, and to replace endogenous TCRs with tumor-specific TCRs or CARs using homology-directed repair (HDR) donor templates. Adeno-associated viral vectors or linear DNA have been used as HDR donor templates. Of note, non-viral delivery substantially reduces the time required to generate clinical-grade reagents for manufacture of T-cell products—a critical step for the translation of personalized T-cell therapies. These technological advances in the field using gene editing open the door to the third generation of ACT therapies.

Efficient and Non-genotoxic RNA-Based Engineering of Human T Cells Using Tumor-Specific T Cell Receptors With Minimal TCR Mispairing.

Genetic engineering of T cells with tumor specific T-cell receptors (TCR) is a promising strategy to redirect their specificity against cancer cells in adoptive T cell therapy protocols. Most studies are exploiting integrating retro- or lentiviral vectors to permanently introduce the therapeutic TCR, which can pose serious safety issues when treatment-related toxicities would occur. Therefore, we developed a versatile, non-genotoxic transfection method for human unstimulated CD8+ T cells. We describe an optimized double sequential electroporation platform whereby Dicer-substrate small interfering RNAs (DsiRNA) are first introduced to suppress endogenous TCR α and β expression, followed by electroporation with DsiRNA-resistant tumor-specific TCR mRNA. We demonstrate that double sequential electroporation of human primary unstimulated T cells with DsiRNA and TCR mRNA leads to unprecedented levels of transgene TCR expression due to a strongly reduced degree of TCR mispairing. Importantly, superior transgenic TCR expression boosts epitope-specific CD8+ T cell activation and killing activity. Altogether, DsiRNA and TCR mRNA double sequential electroporation is a rapid, non-integrating and highly efficient approach with an enhanced biosafety profile to engineer T cells with antigen-specific TCRs for use in early phase clinical trials.

Two Weeks' Notice from Allogeneic Sources

A novel pipeline for neoantigen-specific T-cell receptor (TCR) identification has been validated in ovarian cancer, making use of HLA-matched allogeneic healthy donor T cells. This workflow allows for the identification of tumor-specific TCRs 2 weeks after antigen-specific stimulation and eliminates problematic patient-to-patient variation in the selection of neoantigen-specific TCRs. Clin Cancer Res; 24(21); 5195-7. ©2018 AACR See related article by Matsuda et al., p. 5357.

Isolation and characterization of NY-ESO-1–specific T cell receptors restricted on various MHC molecules

Tumor-specific T cell receptor (TCR) gene transfer enables specific and potent immune targeting of tumor antigens. Due to the prevalence of the HLA-A2 MHC class I supertype in most human populations, the majority of TCR gene therapy trials targeting public antigens have employed HLA-A2-restricted TCRs, limiting this approach to those patients expressing this allele. For these patients, TCR gene therapy trials have resulted in both tantalizing successes and lethal adverse events, underscoring the need for careful selection of antigenic targets. Broad and safe application of public antigen-targeted TCR gene therapies will require (i) selecting public antigens that are highly tumor-specific and (ii) targeting multiple epitopes derived from these antigens by obtaining an assortment of TCRs restricted by multiple common MHC alleles. The canonical cancer-testis antigen, NY-ESO-1, is not expressed in normal tissues but is aberrantly expressed across a broad array of cancer types. It has also been targeted with A2-restricted TCR gene therapy without adverse events or notable side effects. To enable the targeting of NY-ESO-1 in a broader array of HLA haplotypes, we isolated TCRs specific for NY-ESO-1 epitopes presented by four MHC molecules: HLA-A2, -B07, -B18, and -C03. Using these TCRs, we pilot an approach to extend TCR gene therapies targeting NY-ESO-1 to patient populations beyond those expressing HLA-A2.

Bluebird and Gritstone team up

Gene therapy specialist Bluebird Bio and immunotherapy drug development firm Gritstone Oncology have agreed to collaborate on research, development, and commercialization of cell therapy treatments for cancer. Gritstone, based in Emeryville, Calif., will use its artificial intelligence tumor-analysis platform to identify tumor-specific targets and natural T-cell receptors, to which Bluebird will apply its cell therapy approach. Bluebird, based in Cambridge, Mass., will conduct all development, manufacturing, and commercialization activities, while Gritstone will apply its technology to support patient selection for clinical development of therapies.

Abstract 2554: To clone tumor-specific TCRs from a cell-based immunotherapy-benefit cervical cancer patient

Abstract Adoptive transfer of tumor-specific T cell receptor (TCR)-engineered T cells, TCR-T therapy, has shown great efficacy in clinical trials against solid tumors. A cervical cancer patient with bone metastasis was treated with a cell-based immunotherapy, named Multiple-Antigens Stimulating Cellular Therapy (MASCT), which is a combination of multiple tumor antigen peptides loaded dendritic cells (DCs) and autologous T cells stimulated by these DCs. After repeated treatment, the patient showed partially response and remained stable disease for 20 months. Moreover, boosted specific immune responses were detected in patient's periphery blood by IFNγ-Elispot assay against various tumor antigens, such as CEA, RGS5 and HPV18/58. The clinical benefits of this patient indicated that tumor-specific T cells were expanded in vivo and played an important role to control tumor progression. These T cells may be the good sources to isolate tumor-specific TCRs for TCR-T therapies. Given that, patient's PBMCs were stimulated in vitro with selected tumor peptides respectively, such as CEA, RGS5 and HPV18 E7. Tumor-specific T cells were defined as IFNγ-secreting T cells upon peptide stimulation and were further enriched by using magnetic beads. Part of the enriched T cells were analyzed for TCR alpha/beta repertoire via NGS, the other part were sorted into single T cells and analyzed for TCR alpha/beta pairing. The TCR repertoire NGS data revealed that the tumor-specific T cells harbor several hundred unique TCR alpha/beta clonotypes. Some unique TCR alpha/beta clonotypes were considered as tumor-specific TCR candidates, since the copy numbersof these clonotypes were significantly and repeatedly increased after stimulation with CEA, RGS5 or HPV18 E7, and further increased after IFNγ enrichment. After TCR alpha/beta pairing confirmation by single T cell sequencing, alpha and beta chains of these TCRs were sent to synthesis for further investigation. In conclusion we have successfully identified specific TCR clonotypes targeting selected tumor antigens from a MASCT-benefited cervical cancer patient. After functional validations, including epitope specificity, HLA restriction as well as tumor recognition, these tumor-specific TCRs may be good candidates for developing safer and more effective TCR-T therapy targeting multiple tumor antigens. Citation Format: Xiaoling Liang, Shudan Ou, Minjun Ma, Xihe Chen, Xiangjun Zhou, Yanyan Han. To clone tumor-specific TCRs from a cell-based immunotherapy-benefit cervical cancer patient [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2554.

Introduction of Genetically Modified CD3ζ Improves Proliferation and Persistence of Antigen-Specific CTLs.

The clinical efficacy of T-cell therapies based on T cells transduced with genes encoding tumor-specific T-cell receptors (TCR-T) is related to the in vivo persistence of the T cells. To improve persistence without modifying TCR affinity, we instead modified intracellular signaling, using artificial T cell-activating adapter molecules (ATAM), generated by inserting the intracellular domain (ICD) of activating T-cell signaling moieties into CD3ζ. ATAMs with the ICD of either CD28 or 4-1BB were generated, assembled into the TCR complex as a part of CD3ζ, and enhanced downstream signaling from the supramolecular activation cluster. ATAMs were retrovirally introduced into human CMV-specific or NY-ESO-1-specific TCR-transduced CD8+ T lymphocytes, and downstream functionality was then examined. ATAM-transduced NY-ESO-1 TCR-T cells were also investigated using the U266-xenograft mouse model. ATAMs were successfully transduced and localized to the cell membrane. ATAM-transduced CMV-specific T cells retained their cytotoxic activity and cytokine production against peptide-pulsed target cells without altering antigen-specificity and showed resistance to activation-induced cell death. Upon both single and repeated stimulation, CD3ζ/4-1BB-transduced T cells had superior proliferation to the CD3ζ-transduced T cells in both the CMV-specific and the NY-ESO-1 TCR-T models and significantly improved antitumor activity compared with untransduced T cells both in vitro and in a mouse xenograft model. ATAM-transduced TCR-T cells demonstrated improved proliferation and persistence in vitro and in vivo This strategy to control the intracellular signaling of TCR-T cells by ATAM transduction in combination with various tumor-specific TCRs may improve the efficacy of TCR-T therapy. Cancer Immunol Res; 6(6); 733-44. ©2018 AACR.

RNA-transfection of \u03b3/\u03b4 T cells with a chimeric antigen receptor or an \u03b1/\u03b2 T-cell receptor: a safer alternative to genetically engineered \u03b1/\u03b2 T cells for the immunotherapy of melanoma

BackgroundAdoptive T-cell therapy relying on conventional T cells transduced with T-cell receptors (TCRs) or chimeric antigen receptors (CARs) has caused substantial tumor regression in several clinical trials. However, genetically engineered T cells have been associated with serious side-effects due to off-target toxicities and massive cytokine release. To obviate these concerns, we established a protocol adaptable to GMP to expand and transiently transfect γ/δ T cells with mRNA.MethodsPBMC from healthy donors were stimulated using zoledronic-acid or OKT3 to expand γ/δ T cells and bulk T cells, respectively. Additionally, CD8+ T cells and γ/δ T cells were MACS-isolated from PBMC and expanded with OKT3. Next, these four populations were electroporated with RNA encoding a gp100/HLA-A2-specific TCR or a CAR specific for MCSP. Thereafter, receptor expression, antigen-specific cytokine secretion, specific cytotoxicity, and killing of the endogenous γ/δ T cell-target Daudi were analyzed.ResultsUsing zoledronic-acid in average 6 million of γ/δ T cells with a purity of 85% were generated from one million PBMC. MACS-isolation and OKT3-mediated expansion of γ/δ T cells yielded approximately ten times less cells. OKT3-expanded and CD8+ MACS-isolated conventional T cells behaved correspondingly similar. All employed T cells were efficiently transfected with the TCR or the CAR. Upon respective stimulation, γ/δ T cells produced IFNγ and TNF, but little IL-2 and the zoledronic-acid expanded T cells exceeded MACS-γ/δ T cells in antigen-specific cytokine secretion. While the cytokine production of γ/δ T cells was in general lower than that of conventional T cells, specific cytotoxicity against melanoma cell lines was similar. In contrast to OKT3-expanded and MACS-CD8+ T cells, mock-electroporated γ/δ T cells also lysed tumor cells reflecting the γ/δ T cell-intrinsic anti-tumor activity. After transfection, γ/δ T cells were still able to kill MHC-deficient Daudi cells.ConclusionWe present a protocol adaptable to GMP for the expansion of γ/δ T cells and their subsequent RNA-transfection with tumor-specific TCRs or CARs. Given the transient receptor expression, the reduced cytokine release, and the equivalent cytotoxicity, these γ/δ T cells may represent a safer complementation to genetically engineered conventional T cells in the immunotherapy of melanoma (Exper Dermatol 26: 157, 2017, J Investig Dermatol 136: A173, 2016).

NY-ESO-1 TCR single edited stem and central memory T cells to treat multiple myeloma without graft-versus-host disease

AbstractTransfer of T-cell receptors (TCRs) specific for tumor-associated antigens is a promising approach for cancer immunotherapy. We developed the TCR gene editing technology that is based on the knockout of the endogenous TCR α and β genes, followed by the introduction of tumor-specific TCR genes, and that proved safer and more effective than conventional TCR gene transfer. Although successful, complete editing requires extensive cell manipulation and 4 transduction procedures. Here we propose a novel and clinically feasible TCR “single editing” (SE) approach, based on the disruption of the endogenous TCR α chain only, followed by the transfer of genes encoding for a tumor-specific TCR. We validated SE with the clinical grade HLA-A2 restricted NY-ESO-1157-165–specific TCR. SE allowed the rapid production of high numbers of tumor-specific T cells, with optimal TCR expression and preferential stem memory and central memory phenotype. Similarly to unedited T cells redirected by TCR gene transfer (TCR transferred [TR]), SE T cells efficiently killed NY-ESO-1pos targets; however, although TR cells proved highly alloreactive, SE cells showed a favorable safety profile. Accordingly, when infused in NSG mice previously engrafted with myeloma, SE cells mediated tumor rejection without inducing xenogeneic graft-versus-host disease, thus resulting in significantly higher survival than that observed in mice treated with TR cells. Overall, single TCR gene editing represents a clinically feasible approach that is able to increase the safety and efficacy of cancer adoptive immunotherapy.

Abstract 2671: Antitumor activity of NKTR-214 in combination with pmel-1 ACT in an aggressive murine melanoma model

Abstract The adoptive cell transfer (ACT) of genetically engineered T cells expressing cancer-specific T-cell receptors (TCR) has been shown to induce effective anti-tumor response. However, tumors frequently relapse after an initial response. Another strategy towards stimulating the immune system is the use of high-dose interleukin-2 (IL-2) to target the IL-2 receptor (IL2R), leading to immune cell expansion. However, clinically approved IL-2 expands both tumor-killing CD8+ effector T cells (CD8T) as well as regulatory T cells (Tregs) through binding the IL-2Rβγ and IL-2Rαβγ complexes, respectively. Tregs in the tumor lead to immune suppression, which hampers the antitumoral response. NKTR-214 is a CD122-biased cytokine agonist conjugated with multiple releasable chains of polyethylene glycol and designed to provide sustained signaling through the heterodimeric IL-2 receptor pathway (IL-2Rβγ) to preferentially activate and expand effector CD8+ T and NK cells over Tregs. We used the pmel-1 ACT/B16 melanoma tumor model to test the anti-tumor activity of NKTR-214 and evaluate its effects on tumor-specific TCR transgenic T cells. On Day 0 (D0) C57BL/6 mice were implanted with B16-F10 mouse melanoma cells and lymphodepleted with 500 cGy on D6. On D7, mice were treated with either the combination of ACT (T lymphocytes activated in vitro with 1 μg/ml gp100) plus NKTR-214 (0.8 mg/kg, q9dx3, i.v.) or with C57/B6 T cells plus PBS (vehicle control). The tumors of the vehicle control mice (n=12) rapidly grew to the 1500mm3 endpoint in 12 days post-treatment, versus 35 days for the NKTR-214 group (n=12) with only 1 out of 12 mice reaching endpoint. Bioluminescence imaging was used to visualize the in vivo distribution and tumor-homing of antigen-specific T cells. Interestingly, the reporter T cells were retained in the spleen until D7 and could be seen migrating to the tumor at D9 reaching peak of bioluminescence at day 12, a delayed time point compared to the 5 days usually observed in mice treated with standard IL-2. The signal persisted in the NKTR-214 + ACT group until D20 versus D7 in the vehicle-control animals. These data suggest that NKTR-214 + ACT is well tolerated and provides a robust anti-tumor response in the aggressive B16F10 model. Treatment with NKTR-214 + ACT robustly mobilizes T cells into the tumor where they durably persist. The robust and long-lasting effect of NKTR214 supports its potential use in combination with cell-based therapeutics. Citation Format: Giulia Parisi, Justin Saco, Siwen Hu-Lieskovan, Ruixue Zhang, Paige Krystofinski, Cristina Puig Saus, Deborah H. Charych, Antoni Ribas. Antitumor activity of NKTR-214 in combination with pmel-1 ACT in an aggressive murine melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2671. doi:10.1158/1538-7445.AM2017-2671

Identification of patient-specific and tumor-shared T cell receptor sequences in renal cell carcinoma patients.

A major requirement for cancer immunotherapy is the development of biomarkers for prognosis and for monitoring therapy response. In an attempt to evaluate the immune response of renal cell carcinoma (RCC) patients, tumor lesions and / or blood samples from 12 RCC patients underwent deep T cell receptor (TCR) sequencing. Despite the low number of samples, different TCR distribution patterns could be detected. Most of the RCC patients presented “patient-specific” TCR sequences, and those clonotypes were present at higher frequency in tumor lesions suggesting a specific extravasation from the blood. Comparison among the tumor samples revealed also “patient-shared” TCR patterns. Indeed, a central core of 16 different TCRs were shared by 3 patients, whereas other 6 patients shared between 4 and 6 TCR sequences, with two sub-groups sharing 12 to 17 different clonotypes. The relative frequencies of shared clonotypes were very different varying from < 1% to a maximum of 37% of the total TCR repertoire. These data confirm the presence of tumor-specific TCR within the cancer tissue and suggest the existence of shared epitopes among different patients that might be used as targets for tumor immunotherapy.

Driving gene-engineered T cell immunotherapy of cancer.

Chimeric antigen receptor (CAR) gene-engineered T cell therapy holds the potential to make a meaningful difference in the lives of patients with terminal cancers. For decades, cancer therapy was based on biophysical parameters, with surgical resection to debulk, followed by radiation and chemotherapy to target the rapidly growing tumor cells, while mostly sparing quiescent normal tissues. One breakthrough occurred with allogeneic bone-marrow transplant for patients with leukemia, which provided a sometimes curative therapy. The field of adoptive cell therapy for solid tumors was established with the discovery that tumor-infiltrating lymphocytes could be expanded and used to treat and even cure patients with metastatic melanoma. Tumor-specific T-cell receptors (TCRs) were identified and engineered into patient peripheral blood lymphocytes, which were also found to treat tumors. However, these were limited by patient HLA-restriction. Close behind came generation of CAR, combining the exquisite recognition of an antibody with the effector function of a T cell. The advent of CD19-targeted CARs for treating patients with multiple forms of advanced B-cell malignancies met with great success, with up to 95% response rates. Applying CAR treatment to solid tumors, however, has just begun, but already certain factors have been made clear: the tumor target is of utmost importance for clinicians to do no harm; and solid tumors respond differently to CAR therapy compared with hematologic ones. Here we review the state of clinical gene-engineered T cell immunotherapy, its successes, challenges, and future.

TCR-Ligand Dissociation-Rate, a Reliable and Determining Parameter, Identifying Tumor-Reactive CD8 T Cells of High Avidity and Functional Potency

Background & Aims: Kinetics of T-cell receptor (TCR) binding to peptide-MHC (pMHC) contribute to T-cell activation and various cellular responses. However, due to technical reasons, the characterization of T-cells is usually only done with functional assays, without assessment of TCR-pMHC binding kinetics, thus limiting the information on the overall quality and clinical relevance of TCR-ligand binding properties. With the aim to broadly evaluate possible correlations between T-cell function and TCR binding kinetics in humans, we undertook a large-scale analysis of combined multiple functional readouts (i.e. CD107a degranulation, cytokine production and proliferation) and off-rate measurements by NTAmers (ref. 1) to characterize tumor- and virus-specific CD8 T-cell clones (n=400) isolated from melanoma patients and healthy donors.Results: Our results show that, within an antigen-specific repertoire, TCR-ligand off-rates accurately predicted single-functional and poly-functional avidities (i.e. ligand potency) as well as co-receptors expression/modulation of CD8 T-cells. Importantly, the TCR-pMHC dissociation-rate represented a constant and highly reliable bio-physical parameter, contrasting to functional avidity which depended on the activation status of the T-cells following re-stimulation. Remarkably, we found that the TCR-ligand off-rate repertoire depended on the antigenic-specificity, as CD8 T-cells specific for the cancer-testis antigen NY-ESO-1157-165 displayed longer TCR-ligand interactions than T-cells specific for the tumor/differentiation antigen Melan-A26-35, and both these tumor-specific TCRs exhibited significantly lower avidities than those specific for persistent herpes virus antigens (CMV/pp65495-503, EBV/BMFL1259-267).Conclusions: Our data from a large library of human T-cell clones demonstrate that the TCR-ligand dissociation-rate is a stable/reliable and determining parameter of T-cell responsiveness, arguing that it should be systematically assessed for meaningful monitoring of human T-cell responses. Furthermore, our TCR-ligand off-rate comparisons highlighted strong binding differences between non-self/virus- and self/tumor-specific repertoires. Our data from individual clones suggest that the tumor-specific repertoires contains rare but nevertheless promising TCRs with sufficiently slow off-rate and high functional avidity to potentially confer protective immune responses against cancer.(ref. 1) Hebeisen et al., Cancer Res, 75(10):1983, 2015 DisclosuresNo relevant conflicts of interest to declare.

Personalization of T-Cell Therapy Using a High-Throughput Platform to Identify Tumor-Specific T-Cell Receptors

Background T-cell receptors (TCRs) can be used to redirect the specificity of T-cells for human application. This has particular appeal for the targeting of neoantigens. However, efficient identification, cloning, and characterization of antigen (Ag)-specific TCRs is needed to enable the timely adoptive transfer of T-cells genetically modified to express therapeutic TCRs. We have harnessed next generation sequencing (NGS) to identify desirable TCRs. This approach enables us to simultaneously identify hundreds of Ag-specific TCRs, along with the expression of genes to characterize functional and phenotypical values of individual Ag-specific T-cells (e.g. related to cytotoxicity, exhaustion, etc...). To take advantage of NGS technology, a sister high-throughput technology was developed to evaluate harvested TCRs. A reporter system was implemented using (immortalized) Jurkat T-ALL cells genetically modified to (i) enforce expression of CD8aβ, (ii) conditionally express GFP under minimal elements of NR4A1 promoter, and (iii) prevent expression of both endogenous TCRα and β chains. Sequenced CDR3 regions were coded within DNA plasmids from Sleeping Beauty (SB) transposons as TCR Vα and Vβ libraries that were expressed on the reporter cell to identify both TCR specificity as well as TCR avidity. Thus, we implemented (i) a TCR cloning system based on CDR3 sequencing of Ag-specific T-cells identified by NGS and (ii) a novel reporter cell based on the TCR-mediated induction of GFP expression. As a proof-of-concept to evaluate the entire platform, we used HLA-A2-restricted CMV peptide (NLVPMVATV: CMV/A2) and NY-ESO-1 peptide (SLLMWITQC: NY-ESO-1/A2) as model Ags.Results The reporter cell was initially genetically modified with either high- or low-avidity TCRs against a NY-ESO-1/A2. Upon stimulation with HLA-A2+ 721.221 immortalized B cells, it was found that the expression of GFP positively correlated with the avidity of TCRs (Figure). Next, we isolated naïve Ag-specific T-cells from umbilical cord blood using CMV/A2 tetramer. Single CMV/A2-specific CD8+ T-cells were sorted and their TCRαβ CDR3 sequences were amplified by reverse transcription and PCR with bar-coded probes. The pooled PCR products were sequenced in MiSeq Sequencer (illumina) to obtain TCRαβ CDR3 regions and analyzed in silico using IMGT (International Immunogenetics Information System). The efficiency of identifying individual TCRαβ pairs was 65% to 76% (using 96-well plate or 384-well plates, respectively). Individual SB-derived DNA transposons expressing TCRαβ constructs were synthesized by Gibson assembly and electroporated, with SB transposase, into the reporter cell. These cells were co-cultured with HLA-A2+ 721.221 stimulator cells loaded with graded doses of cognate peptide. The percentage and intensity of GFP expression was evaluated by high-throughput flow-cytometer (IntelliCyt) which revealed high-avidity Ag-specific TCRαβs.Conclusion Our new high-throughput system can identify and characterize, based on specificity and avidity, Ag-specific TCRαβs within a week. This system will be used to generate neoantigen-specific TCRαβs for human application. [Display omitted] DisclosuresZong:ZIOPHARM Oncology, Inc.: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties; Immatics US, Inc: Equity Ownership, Patents & Royalties. Cooper:Ziopharm Oncology: Employment, Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership; City of Hope: Patents & Royalties; Targazyme, Inc.,: Equity Ownership; Immatics: Equity Ownership; Sangamo BioSciences: Patents & Royalties; MD Anderson Cancer Center: Employment; Miltenyi Biotec: Honoraria. McNamara:GeoMcNamara: Consultancy, Other: Consultant in immuno-oncology field; ZIOPHARM Oncology, Inc: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Torikai:intrexon: Equity Ownership, Patents & Royalties; ZIOPHARM Oncology, Inc.: Equity Ownership, Patents & Royalties; Immatics US, Inc: Equity Ownership.

288. Development of Cocal Glycoprotein Envelope Producer Cell Lines for Robust Lentiviral Gene Transfer into Hematopoietic Stem Cells and T Cells

Lentiviral vectors (LVs) are routinely used for stable gene transfer and have demonstrated great promise in hematopoietic stem cell gene therapy and also immunotherapy using genetically modified T cells. LVs are commonly pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G), which confers broad tropism to the vector and allows for vector concentration by centrifugation. However, the use of VSV-G has several limitations, such as susceptibility to inactivation by human serum complement making it unsuitable for in vivo delivery. Furthermore, VSV-G is toxic when constitutively expressed, which has impeded efforts to generate stable producer cell lines. In this study, we first validate the use of cocal vesiculovirus envelope to pseudotype LVs by demonstrating that cocal LVs transduce hematopoietic stem cells and CD4+ T cells more efficiently than VSV-G LVs. We also provide evidence that cocal and VSV-G envelopes use the same receptor for cell entry. We then describe the development of two high-titer, cocal-pseudotyped, LV producer cell lines for a GFP reporter and for a WT1 tumor-specific T cell receptor (TCR). The different 3rd generation lentiviral helper genes were sequentially introduced in HEK293T cells by co-transfection with plasmids encoding antibiotic resistance genes followed by selection to allow for stable protein expression. Cells expressing the cocal envelope produced over 10-times more infectious LV particles as compared to VSV-G expressing cells. High-titer cocal producer cells were isolated by screening for best single clones, which were capable of generating concentrated titers above 108 infectious units per mL. We found that these producer cells were stable after serial passages for over 3 months, with no drop in titer detected over time. The resulting GFP and WT1-TCR vectors performed at least as well as identical vectors made with our standard transient transfection protocol for the transduction of CD34+ and CD4+ T cells, respectively. Cocal LV producer cells were also adapted for growth in suspension, serum-free culture, which will facilitate efforts for the scaling up of vector production. In summary, we have successfully developed two independent LV producer cells lines with clinically usable titers. The broad applicability of our cocal packaging cell line offers a promising tool toward the generation of large-scale, clinical grade LV.