Cooperative Armoring of CAR and TCR T Cells by T Cell-Restricted IL15 and IL21 Universally Enhances Solid Tumor Efficacy.

PURPOSE: Chimeric antigen receptor (CAR) and T-cell receptor (TCR) T-cell therapies are effective in a subset of patients with solid tumors, but new approaches are needed to universally improve patient outcomes. Here, we developed a technology to leverage the cooperative effects of IL15 and IL21, two common cytokine-receptor gamma chain family members with distinct, pleiotropic effects on T cells and other lymphocytes, to enhance the efficacy of adoptive T cells. EXPERIMENTAL DESIGN: We designed vectors that induce the constitutive expression of either membrane-tethered IL15, IL21, or IL15/IL21. We used clinically relevant preclinical models of transgenic CARs and TCRs against pediatric and adult solid tumors to determine the effect of the membrane-tethered cytokines on engineered T cells for human administration. RESULTS: We found that self-delivery of these cytokines by CAR or TCR T cells prevents functional exhaustion by repeated stimulation and limits the emergence of dysfunctional natural killer (NK)-like T cells. Across different preclinical murine solid tumor models, we observed enhanced regression with each individual cytokine but the greatest antitumor efficacy when T cells were armored with both. CONCLUSIONS: The coexpression of membrane-tethered IL15 and IL21 represents a technology to enhance the resilience and function of engineered T cells against solid tumors and could be applicable to multiple therapy platforms and diseases. See related commentary by Ruffin et al., p. 1431.

CD3ζ ITAMs enable ligand discrimination and antagonism by inhibiting TCR signaling in response to low-affinity peptides.

The T cell antigen receptor (TCR) contains ten immunoreceptor tyrosine-based activation motif (ITAM) signaling sequences distributed within six CD3 subunits; however, the reason for such structural complexity and multiplicity is unclear. Here we evaluated the effect of inactivating the three CD3ζ chain ITAMs on TCR signaling and T cell effector responses using a conditional 'switch' mouse model. Unexpectedly, we found that T cells expressing TCRs containing inactivated (non-signaling) CD3ζ ITAMs (6F-CD3ζ) exhibited reduced ability to discriminate between low- and high-affinity ligands, resulting in enhanced signaling and cytokine responses to low-affinity ligands because of a previously undetected inhibitory function of CD3ζ ITAMs. Also, 6F-CD3ζ TCRs were refractory to antagonism, as predicted by a new in silico adaptive kinetic proofreading model that revises the role of ITAM multiplicity in TCR signaling. Finally, T cells expressing 6F-CD3ζ displayed enhanced cytolytic activity against solid tumors expressing low-affinity ligands, identifying a new counterintuitive approach to TCR-mediated cancer immunotherapy.

Multi-tiered approach to detect autoimmune cross-reactivity of therapeutic T cell receptors.

T cell receptor (TCR)-engineered T cell therapy using high-affinity TCRs is a promising treatment modality for cancer. Discovery of high-affinity TCRs especially against self-antigens can require approaches that circumvent central tolerance, which may increase the risk of cross-reactivity. Despite the potential for toxicity, no standardized approach to screen cross-reactivity has been established in the context of preclinical safety evaluation. Here, we describe a practical framework to prospectively detect clinically prohibitive cross-reactivity of therapeutic TCR candidates. Cross-reactivity screening consisted of multifaceted series of assays including assessment of p-MHC tetramer binding, cell line recognition, and reactivity against candidate peptide libraries. Peptide libraries were generated using conventional contact residue motif-guided search, amino acid substitution matrix-based search unguided by motif information, and combinatorial peptide library scan-guided search. We demonstrate the additive nature of a layered approach, which efficiently identifies unsafe cross-reactivity including one undetected by conventional motif-guided search. These findings have important implications for the safe development of TCR-based therapies.

Replication competent retrovirus testing (RCR) in the National Gene Vector Biorepository: No evidence of RCR in 1,595 post-treatment peripheral blood samples obtained from 60 clinical trials.

The clinical impact of any therapy requires the product be safe and effective. Gammaretroviral vectors pose several unique risks, including inadvertent exposure to replication competent retrovirus (RCR) that can arise during vector manufacture. The US FDA has required patient monitoring for RCR, and the National Gene Vector Biorepository is an NIH resource that has assisted eligible investigators in meeting this requirement. To date, we have found no evidence of RCR in 338 pre-treatment and 1,595 post-treatment blood samples from 737 patients associated with 60 clinical trials. Most samples (75%) were obtained within 1 year of treatment, and samples as far out as 9 years after treatment were analyzed. The majority of trials (93%) were cancer immunotherapy, and 90% of the trials used vector products produced with the PG13 packaging cell line. The data presented here provide further evidence that current manufacturing methods generate RCR-free products and support the overall safety profile of retroviral gene therapy.

Engineered T cell therapy for viral and non-viral epithelial cancers.

Engineered T cell therapy has shown remarkable efficacy in hematologic malignancies and has the potential for application to common epithelial cancers. Diverse T cell therapy strategies including adoptive transfer of tumor-infiltrating lymphocytes, chimeric antigen receptor (CAR)-T cells, and T cell receptor (TCR)-T cells have been studied in clinical trials. Recent research has established treatment of human papillomavirus (HPV)-associated cancers with TCR-T cells as a model for proof-of-principle studies in epithelial cancers. These studies and others have provided critical insight into mechanisms of tumor regression, therapeutic targets, treatment safety, treatment design, and barriers to curative cell therapies for common types of cancer. This perspective will review and consolidate understanding gained from clinical trials to treat viral and non-viral epithelial cancers with cell and gene therapy and will examine how past experience may guide future strategy in treatment and biomarker discovery.

Size-dependent activation of CAR-T cells.

As the targets of chimeric antigen receptor (CAR)-T cells expand to a variety of cancers, autoimmune diseases, viral infections, and fibrosis, there is an increasing demand for identifying new antigens and designing new CARs that can be effectively activated. However, the rational selection of antigens and the design of CARs are limited by a lack of knowledge regarding the molecular mechanism by which CARs are activated by antigens. Here, we present data supporting a "size exclusion" model explaining how antigen signals are transmitted across the plasma membrane to activate the intracellular domains of CARs. In this model, antigen engagement with CAR results in a narrow intermembrane space that physically excludes CD45, a bulky phosphatase, out of the CAR zone, thus favoring CAR phosphorylation by kinases, which further triggers downstream pathways leading to T cell activation. Aligned with this model, increasing the size of CAR extracellular domains diminished CAR-T activation both in vitro and in a mouse lymphoma model; membrane-proximal epitopes activated CAR-Ts better than membrane-distal epitopes. Moreover, increasing the size of CD45 by antibody conjugation enhanced the activation of CARs that recognize membrane-distal epitopes. Consistently, CAR-Ts expressing CD45RABC, the larger isoform, were activated to a higher level than those expressing a smaller isoform CD45RO. Together, our work revealed that CAR-T activation depends on the size difference between the CAR-antigen pair and CD45; the size of CAR, antigen, and CD45 can thus be targets for tuning CAR-T activation.

Non-synergy of PD-1 blockade with T-cell therapy in solid tumors.

BACKGROUND: Cell therapy has shown promise in the treatment of certain solid tumors, but its efficacy may be limited by inhibition of therapeutic T cells by the programmed cell death protein-1 (PD-1) receptor. Clinical trials are testing cell therapy in combination with PDCD1 disruption or PD-1-axis blockade. However, preclinical data to support these approaches and to guide the treatment design are lacking. METHODS: Mechanisms of tumor regression and interaction between cell therapy and PD-1 blockade were investigated in congenic murine tumor models based on targeting established, solid tumors with T-cell receptor T cells directed against tumor-restricted, non-self antigens (ie, tumor neoantigens). RESULTS: In solid tumor models of cell therapy, PD-1 blockade mediated a reproducible but non-synergistic increase in tumor regression following adoptive T-cell transfer. Tumor regression was associated with increased tumor infiltration by endogenous T cells but not by transferred T cells. The effect was independent of PD-1 receptor expression by transferred T cells and was dependent on the endogenous T-cell repertoire and on tumor antigenicity. PD-1 blockade primarily induced cell state changes in endogenous tumor-antigen-specific T cells rather than transferred T cells. CONCLUSIONS: Together, these findings support the concept that PD-1 blockade acts primarily through endogenous rather than transferred T cells to mediate a non-synergistic antitumor effect in solid tumor cell therapy. These findings have important implications for strategies to leverage PD-1 receptor disruption or blockade to enhance the efficacy of cell therapy.

Dual PD-L1 and TGF-b blockade in patients with recurrent respiratory papillomatosis.

BACKGROUND: Recurrent respiratory papillomatosis (RRP) is a human papillomavirus (HPV) driven neoplastic disorder of the upper aerodigestive tract that causes significant morbidity and can lead to fatal airway obstruction. Prior clinical study demonstrated clinical benefit with the programmed death-ligand 1 (PD-L1) monoclonal antibody avelumab. Bintrafusp alpha is a bifunctional inhibitor of PD-L1 and transforming growth factor-beta (TGF-b) that has shown clinical activity in several cancer types. METHODS: We conducted a phase II clinical trial evaluating bintrafusp alpha in adults with RRP. Papilloma samples before and after treatment with bintrafusp alpha were assessed for correlates of response with multiplex immunofluorescence as well as immunological and genomic analyses. Post hoc analyses of papilloma samples before and after treatment with avelumab were assessed for comparison. RESULTS: Dual PD-L1/TGF-b inhibition failed to abrogate papilloma growth in most subjects and increased the frequency of clinically indicated interventions after treatment in four of eight subjects based on each subject's own historical control. TGF-b neutralization consistently decreased pSMAD3 and p21 and increased Ki67 expression within the basal layers of papillomas, indicating that TGF-b restrained proliferation. These alterations were not observed in papillomas treated with PD-L1 blockade alone. Dual PD-L1/TGF-b inhibition did not enhance anti-HPV immunity within papillomas beyond that observed with PD-L1 blockade. Genomic alterations in TGF-b superfamily genes were infrequent in papillomas and normal mucosa but present in a significant fraction of head and neck carcinomas. CONCLUSIONS: Intact TGF-b signaling restrains proliferation within papillomas, and the use of clinical agents that abrogate this pathway should be avoided in patients with RRP. TRIAL REGISTRATION NUMBERS: NCT03707587 and NCT02859454.

Advances in Adoptive Cell Therapy for Head and Neck Cancer.

This article reviews the most recent literature describing clinical advances in adoptive cell therapy for patients with head and neck cancer. Clinical trials with tumor-infiltrating lymphocyte and gene-engineered T-cell receptor T-cell therapy are highlighted.

TCR-engineered T cells targeting E7 for patients with metastatic HPV-associated epithelial cancers.

Genetically engineered T cell therapy can induce remarkable tumor responses in hematologic malignancies. However, it is not known if this type of therapy can be applied effectively to epithelial cancers, which account for 80-90% of human malignancies. We have conducted a first-in-human, phase 1 clinical trial of T cells engineered with a T cell receptor targeting HPV-16 E7 for the treatment of metastatic human papilloma virus-associated epithelial cancers (NCT02858310). The primary endpoint was maximum tolerated dose. Cell dose was not limited by toxicity with a maximum dose of 1 × 1011 engineered T cells administered. Tumor responses following treatment were evaluated using RECIST (Response Evaluation Criteria in Solid Tumors) guidelines. Robust tumor regression was observed with objective clinical responses in 6 of 12 patients, including 4 of 8 patients with anti-PD-1 refractory disease. Responses included extensive regression of bulky tumors and complete regression of most tumors in some patients. Genomic studies, which included intra-patient tumors with dichotomous treatment responses, revealed resistance mechanisms from defects in critical components of the antigen presentation and interferon response pathways. These findings demonstrate that engineered T cells can mediate regression of common carcinomas, and they reveal immune editing as a constraint on the curative potential of cellular therapy and possibly other immunotherapies in advanced epithelial cancer.

Bintrafusp alfa, a bifunctional fusion protein targeting TGF-ß and PD-L1, in patients with human papillomavirus-associated malignancies.

BACKGROUND: Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of transforming growth factor (TGF)-ßRII (a TGF-ß 'trap') fused to a human IgG1 mAb blocking programmed cell death ligand 1. This is the largest analysis of patients with advanced, pretreated human papillomavirus (HPV)-associated malignancies treated with bintrafusp alfa. METHODS: In these phase 1 (NCT02517398) and phase 2 trials (NCT03427411), 59 patients with advanced, pretreated, checkpoint inhibitor-naive HPV-associated cancers received bintrafusp alfa intravenously every 2 weeks until progressive disease, unacceptable toxicity, or withdrawal. Primary endpoint was best overall response per Response Evaluation Criteria in Solid Tumors (RECIST) V.1.1; other endpoints included safety. RESULTS: As of April 17, 2019 (phase 1), and October 4, 2019 (phase 2), the confirmed objective response rate per RECIST V.1.1 in the checkpoint inhibitor-naive, full-analysis population was 30.5% (95% CI, 19.2% to 43.9%; five complete responses); eight patients had stable disease (disease control rate, 44.1% (95% CI, 31.2% to 57.6%)). In addition, three patients experienced a delayed partial response after initial disease progression, for a total clinical response rate of 35.6% (95% CI, 23.6% to 49.1%). An additional patient with vulvar cancer had an unconfirmed response. Forty-nine patients (83.1%) experienced treatment-related adverse events, which were grade 3/4 in 16 patients (27.1%). No treatment-related deaths occurred. CONCLUSION: Bintrafusp alfa showed clinical activity and manageable safety and is a promising treatment in HPV-associated cancers. These findings support further investigation of bintrafusp alfa in patients with advanced, pretreated HPV-associated cancers.

Enhanced efficacy and limited systemic cytokine exposure with membrane-anchored interleukin-12 T-cell therapy in murine tumor models.

BACKGROUND: Interleukin-12 (IL-12) is a potent, proinflammatory cytokine that holds promise for cancer immunotherapy, but its clinical use has been limited by its toxicity. To minimize systemic exposure and potential toxicity while maintaining the beneficial effects of IL-12, we developed a novel IL-12-based therapeutic system that combines tumor-specific T-cell-mediated delivery of IL-12 with membrane-restricted IL-12 localization and inducible IL-12 expression. METHODS: Therapeutic T cells targeting a tumor antigen were genetically engineered to express membrane-anchored IL-12 (aIL-12). Expression, function, and shedding of the aIL-12 molecule was assessed in vitro. Tumor treatment efficacy was assessed in vivo with T cell receptor (TCR) transgenic murine tumor models and a tumor xenograft model. Key outcomes were change in tumor size, circulating levels of IL-12 and other cytokines, and survival. Toxicity was assessed via change in body weight. Tumor growth curve measurements were compared using repeated-measures two-way analyses of variance. RESULTS: Retroviral gene transfer resulted in cell membrane expression of aIL-12 by transduced T cells. In each of two transgenic murine tumor models, tumor-specific T cells constitutively expressing aIL-12 demonstrated increased antitumor efficacy, low circulating IL-12 and interferon-γ, and no weight loss. Expression of aIL-12 via a NFAT-inducible promoter resulted in coordinate expression of aIL-12 with T cell activation. In an OT-I TCR transgenic murine tumor model, the NFAT-inducible aIL-12 molecule improved tumor treatment and did not result in detectable levels of IL-12 in serum or in weight loss. In a human tumor xenograft model, the NFAT-inducible aIL-12 molecule improved antitumor responses by human T cells coexpressing a tumor-specific engineered TCR. Serum IL-12 levels were undetectable with the NFAT-inducible construct in both models. CONCLUSION: Expression of aIL-12 by tumor-targeting therapeutic T cells demonstrated low systemic exposure and improved efficacy. This treatment strategy may have broad applications to cellular therapy with tumor-infiltrating lymphocytes, chimeric antigen receptor T cells, and TCR T cells.

T-Cell Receptor Gene Therapy for Human Papillomavirus-Associated Epithelial Cancers: A First-in-Human, Phase I/II Study.

PURPOSE: Genetically engineered T-cell therapy is an emerging treatment of hematologic cancers with potential utility in epithelial cancers. We investigated T-cell therapy for the treatment of metastatic human papillomavirus (HPV)-associated epithelial cancers. METHODS: This phase I/II, single-center trial enrolled patients with metastatic HPV16-positive cancer from any primary tumor site who had received prior platinum-based therapy. Treatment consisted of autologous genetically engineered T cells expressing a T-cell receptor directed against HPV16 E6 (E6 T-cell receptor T cells), a conditioning regimen, and systemic aldesleukin. RESULTS: Twelve patients were treated in the study. No dose-limiting toxicities were observed in the phase I portion. Two patients, both in the highest-dose cohort, experienced objective tumor responses. A patient with three lung metastases experienced complete regression of one tumor and partial regression of two tumors, which were subsequently resected; she has no evidence of disease 3 years after treatment. All patients demonstrated high levels of peripheral blood engraftment with E6 T-cell receptor T cells 1 month after treatment (median, 30%; range, 4% to 53%). One patient's resistant tumor demonstrated a frameshift deletion in interferon gamma receptor 1, which mediates response to interferon gamma, an essential molecule for T-cell-mediated antitumor activity. Another patient's resistant tumor demonstrated loss of HLA-A*02:01, the antigen presentation molecule required for this therapy. A tumor from a patient who responded to treatment did not demonstrate genetic defects in interferon gamma response or antigen presentation. CONCLUSION: Engineered T cells can induce regression of epithelial cancer. Tumor resistance was observed in the context of T-cell programmed death-1 expression and defects in interferon gamma and antigen presentation pathway components. These findings have important implications for development of cellular therapy in epithelial cancers.

Cancer targeting by TCR gene-engineered T cells directed against Kita-Kyushu Lung Cancer Antigen-1.

T cell receptor (TCR) gene-engineered T cells have shown promise in the treatment of melanoma and synovial cell sarcoma, but their application to epithelial cancers has been limited. The identification of novel therapeutic TCRs for the targeting of these tumors is important for the development of new treatments. Here, we describe the preclinical characterization of a TCR directed against Kita-Kyushu Lung Cancer Antigen-1 (KK-LC-1, encoded by CT83), a cancer germline antigen with frequent expression in human epithelial malignancies including gastric cancer, breast cancer, and lung cancer. Gene-engineered T cells expressing the KK-LC-1 TCR (KK-LC-1 TCR-Ts) demonstrated recognition of CT83+ tumor lines in vitro and mediated regression of established CT83+ xenograft tumors in immunodeficient mouse models. Cross-reactivity studies based on experimental determination of the recognition motifs for the target epitope did not demonstrate cross-reactivity against other human proteins. CT83 gene expression studies in 51 non-neural tissues and 24 neural tissues showed expression restricted exclusively to germ cells. CT83 was however expressed by a range of epithelial cancers, with the highest expression noted in gastric cancer. Collectively, these findings support the further investigation and clinical testing of KK-LC-1 TCR-Ts for gastric cancer and possibly other malignancies.

Safety and clinical activity of PD-L1 blockade in patients with aggressive recurrent respiratory papillomatosis.

BACKGROUND: Recurrent respiratory papillomatosis (RRP) is a human papillomavirus (HPV)-driven disorder that causes substantial morbidity and can lead to fatal distal airway obstruction and post-obstructive pneumonias. Patients require frequent surgical debridement of disease, and no approved systemic adjuvant therapies exist. METHODS: A phase II study was conducted to investigate the clinical activity and safety of programmed death-ligand 1 (PD-L1) blockade with avelumab in patients with RRP. RESULTS: Twelve patients were treated. All patients with laryngeal RRP displayed improvement in disease burden, and 5 of 9 (56%) displayed partial responses. None of 4 patients with pulmonary RRP displayed a response. Using each patient's surgical history as their own control, patients required fewer surgical interventions after avelumab treatment (p = 0.008). A subset of partial responders developed HPV-specific reactivity in papilloma-infiltrating T-cells that correlated with reduced HPV viral load and an increased Tissue Inflammation Signature. CONCLUSIONS: Avelumab demonstrated safety and clinical activity in patients with laryngeal RRP. Further study of immune checkpoint blockade for RRP, possibly with longer treatment duration or in combination with other immunotherapies aimed at activating antiviral immunity, is warranted. TRIAL REGISTRATION: NCT, number NCT02859454 , registered August 9, 2016.

A Phase II Study of Tumor-infiltrating Lymphocyte Therapy for Human Papillomavirus-associated Epithelial Cancers.

PURPOSE: Cellular therapy is an emerging cancer treatment modality, but its application to epithelial cancers has been limited. This clinical trial evaluated tumor-infiltrating lymphocyte (TIL) therapy for the treatment of patients with metastatic human papillomavirus (HPV)-associated carcinomas. PATIENTS AND METHODS: The trial was a phase II design with two cohorts, cervical cancers and noncervical cancers. Cell infusion was preceded by a lymphocyte-depleting conditioning regimen and followed by systemic high-dose aldesleukin. RESULTS: Objective tumor responses occurred in 5 of 18 (28%) patients in the cervical cancer cohort and 2 of 11 (18%) patients in the noncervical cancer cohort. Two of the responses in cervical cancer were complete and are ongoing 67 and 53 months after treatment. Responses in the noncervical cancer cohort were in anal cancer and oropharyngeal cancer. The HPV reactivity of the infused T cells correlated with clinical response. Peripheral blood repopulation with HPV-reactive T cells also correlated with clinical response. CONCLUSIONS: These findings support the concept that cellular therapy can mediate the regression of epithelial cancers, and they suggest the importance of predictive biomarkers and novel treatment platforms for more effective therapies.

Engineered T cells targeting E7 mediate regression of human papillomavirus cancers in a murine model.

T cell receptor (TCR) T cell therapy is a promising cancer treatment modality. However, its successful development for epithelial cancers may depend on the identification of high-avidity TCRs directed against tumor-restricted target antigens. The human papillomavirus (HPV) E7 antigen is an attractive therapeutic target that is constitutively expressed by HPV+ cancers but not by healthy tissues. It is unknown if genetically engineered TCR T cells that target E7 can mediate regression of HPV+ cancers. We identified an HPV-16 E7-specific, HLA-A*02:01-restricted TCR from a uterine cervix biopsy from a woman with cervical intraepithelial neoplasia. This TCR demonstrated high functional avidity, with CD8 coreceptor-independent tumor targeting. Human T cells transduced to express the TCR specifically recognized and killed HPV-16+ cervical and oropharyngeal cancer cell lines and mediated regression of established HPV-16+ human cervical cancer tumors in a mouse model. These findings support the therapeutic potential of this approach and established the basis for an E7 TCR gene therapy clinical trial in patients with metastatic HPV+ cancers (NCT02858310).

Cell-based molecularly targeted therapy: targeting oncoproteins with T cell receptor gene therapy.

As oncogenes drive carcinogenesis and promote cancer cell survival, they are highly attractive therapeutic targets, and oncogene-targeting small molecules have achieved some clinical success. While many oncogenes are presently considered to be "druggable," tumors often acquire treatment resistance, and patients are rarely cured in response to oncogene-specific treatment. In this issue of the JCI, Veatch and colleagues describe a patient with metastatic acral melanoma who experienced a complete tumor response following infusion of tumor-infiltrating T cells that targeted multiple tumor antigens, including a BRAFV600E driver mutation. T cells genetically engineered to express an anti-BRAFV600E T cell receptor (TCR) from the patient demonstrated recognition of an epitope that spanned the BRAFV600E mutation. These findings suggest that BRAFV600E might be targeted therapeutically with adoptive transfer of anti-BRAFV600E T cells. This research supports the emerging therapeutic paradigm of targeting oncogenic drivers with T cell immunotherapy.

Enhanced clinical-scale manufacturing of TCR transduced T-cells using closed culture system modules.

BACKGROUND: Genetic engineering of T-cells to express specific T cell receptors (TCR) has emerged as a novel strategy to treat various malignancies. More widespread utilization of these types of therapies has been somewhat constrained by the lack of closed culture processes capable of expanding sufficient numbers of T-cells for clinical application. Here, we evaluate a process for robust clinical grade manufacturing of TCR gene engineered T-cells. METHODS: TCRs that target human papillomavirus E6 and E7 were independently tested. A 21 day process was divided into a transduction phase (7 days) and a rapid expansion phase (14 days). This process was evaluated using two healthy donor samples and four samples obtained from patients with epithelial cancers. RESULTS: The process resulted in ~ 2000-fold increase in viable nucleated cells and high transduction efficiencies (64-92%). At the end of culture, functional assays demonstrated that these cells were potent and specific in their ability to kill tumor cells bearing target and secrete large quantities of interferon and tumor necrosis factor. Both phases of culture were contained within closed or semi-closed modules, which include automated density gradient separation and cell culture bags for the first phase and closed GREX culture devices and wash/concentrate systems for the second phase. CONCLUSION: Large-scale manufacturing using modular systems and semi-automated devices resulted in highly functional clinical-grade TCR transduced T-cells. This process is now in use in actively accruing clinical trials and the NIH Clinical Center and can be utilized at other cell therapy manufacturing sites that wish to scale-up and optimize their processing using closed systems.