Stable, Nonviral Expression of Mutated Tumor Neoantigen-specific T-cell Receptors Using the Sleeping Beauty Transposon/Transposase System.

Neoantigens unique to each patient's tumor can be recognized by autologous T cells through their T-cell receptor (TCR) but the low frequency and/or terminal differentiation of mutation-specific T cells in tumors can limit their utility as adoptive T-cell therapies. Transfer of TCR genes into younger T cells from peripheral blood with a high proliferative potential could obviate this problem. We generated a rapid, cost-effective strategy to genetically engineer cancer patient T cells with TCRs using the clinical Sleeping Beauty transposon/transposase system. Patient-specific TCRs reactive against HLA-A*0201-restriced neoantigens AHNAK(S2580F) or ERBB2(H473Y) or the HLA-DQB*0601-restricted neoantigen ERBB2IP(E805G) were assembled with murine constant chains and cloned into Sleeping Beauty transposons. Patient peripheral blood lymphocytes were coelectroporated with SB11 transposase and Sleeping Beauty transposon, and transposed T cells were enriched by sorting on murine TCRß (mTCRß) expression. Rapid expansion of mTCRß(+) T cells with irradiated allogeneic peripheral blood lymphocytes feeders, OKT3, interleukin-2 (IL-2), IL-15, and IL-21 resulted in a preponderance of effector (CD27(-)CD45RA(-)) and less-differentiated (CD27(+)CD45RA(+)) T cells. Transposed T cells specifically mounted a polyfunctional response against cognate mutated neoantigens and tumor cell lines. Thus, Sleeping Beauty transposition of mutation-specific TCRs can facilitate the use of personalized T-cell therapy targeting unique neoantigens.

Bispecific T-cells expressing polyclonal repertoire of endogenous γδ T-cell receptors and introduced CD19-specific chimeric antigen receptor.

Even though other γδ T-cell subsets exhibit antitumor activity, adoptive transfer of γδ Tcells is currently limited to one subset (expressing Vγ9Vδ2 T-cell receptor (TCR)) due to dependence on aminobisphosphonates as the only clinically appealing reagent for propagating γδ T cells. Therefore, we developed an approach to propagate polyclonal γδ T cells and rendered them bispecific through expression of a CD19-specific chimeric antigen receptor (CAR). Peripheral blood mononuclear cells (PBMC) were electroporated with Sleeping Beauty (SB) transposon and transposase to enforce expression of CAR in multiple γδ T-cell subsets. CAR(+)γδ T cells were expanded on CD19(+) artificial antigen-presenting cells (aAPC), which resulted in >10(9) CAR(+)γδ T cells from <10(6) total cells. Digital multiplex assay detected TCR mRNA coding for Vδ1, Vδ2, and Vδ3 with Vγ2, Vγ7, Vγ8, Vγ9, and Vγ10 alleles. Polyclonal CAR(+)γδ T cells were functional when TCRγδ and CAR were stimulated and displayed enhanced killing of CD19(+) tumor cell lines compared with CAR(neg)γδ T cells. CD19(+) leukemia xenografts in mice were reduced with CAR(+)γδ T cells compared with control mice. Since CAR, SB, and aAPC have been adapted for human application, clinical trials can now focus on the therapeutic potential of polyclonal γδ T cells.

Cancer genes disfavoring T cell immunity identified via integrated systems approach.

Adoptive T cell therapies (ACT) have been curative for a limited number of cancer patients. The sensitization of cancer cells to T cell killing may expand the benefit of these therapies for more patients. To this end, we use a three-step approach to identify cancer genes that disfavor T cell immunity. First, we profile gene transcripts upregulated by cancer under selection pressure from T cell killing. Second, we identify potential tumor gene targets and pathways that disfavor T cell killing using signaling pathway activation libraries and genome-wide loss-of-function CRISPR-Cas9 screens. Finally, we implement pharmacological perturbation screens to validate these targets and identify BIRC2, ITGAV, DNPEP, BCL2, and ERRα as potential ACT-drug combination candidates. Here, we establish that BIRC2 limits antigen presentation and T cell recognition of tumor cells by suppressing IRF1 activity and provide evidence that BIRC2 inhibition in combination with ACT is an effective strategy to increase efficacy.

Adoptive Cellular Therapy with Autologous Tumor-Infiltrating Lymphocytes and T-cell Receptor-Engineered T Cells Targeting Common p53 Neoantigens in Human Solid Tumors.

Adoptive cellular therapy (ACT) targeting neoantigens can achieve durable clinical responses in patients with cancer. Most neoantigens arise from patient-specific mutations, requiring highly individualized treatments. To broaden the applicability of ACT targeting neoantigens, we focused on TP53 mutations commonly shared across different cancer types. We performed whole-exome sequencing on 163 patients with metastatic solid cancers, identified 78 who had TP53 missense mutations, and through immunologic screening, identified 21 unique T-cell reactivities. Here, we report a library of 39 T-cell receptors (TCR) targeting TP53 mutations shared among 7.3% of patients with solid tumors. These TCRs recognized tumor cells in a TP53 mutation- and human leucocyte antigen (HLA)-specific manner in vitro and in vivo. Twelve patients with chemorefractory epithelial cancers were treated with ex vivo-expanded autologous tumor-infiltrating lymphocytes (TIL) that were naturally reactive against TP53 mutations. However, limited clinical responses (2 partial responses among 12 patients) were seen. These infusions contained low frequencies of mutant p53-reactive TILs that had exhausted phenotypes and showed poor persistence. We also treated one patient who had chemorefractory breast cancer with ACT comprising autologous peripheral blood lymphocytes transduced with an allogeneic HLA-A*02-restricted TCR specific for p53R175H. The infused cells exhibited an improved immunophenotype and prolonged persistence compared with TIL ACT and the patient experienced an objective tumor regression (-55%) that lasted 6 months. Collectively, these proof-of-concept data suggest that the library of TCRs targeting shared p53 neoantigens should be further evaluated for the treatment of patients with advanced human cancers. See related Spotlight by Klebanoff, p. 919.

Internal checkpoint regulates T cell neoantigen reactivity and susceptibility to PD1 blockade.

BACKGROUND: Adoptive transfer of tumor-infiltrating lymphocytes (TIL) fails to consistently elicit tumor rejection. Manipulation of intrinsic factors that inhibit T cell effector function and neoantigen recognition may therefore improve TIL therapy outcomes. We previously identified the cytokine-induced SH2 protein (CISH) as a key regulator of T cell functional avidity in mice. Here, we investigate the mechanistic role of CISH in regulating human T cell effector function in solid tumors and demonstrate that CRISPR/Cas9 disruption of CISH enhances TIL neoantigen recognition and response to checkpoint blockade. METHODS: Single-cell gene expression profiling was used to identify a negative correlation between high CISH expression and TIL activation in patient-derived TIL. A GMP-compliant CRISPR/Cas9 gene editing process was developed to assess the impact of CISH disruption on the molecular and functional phenotype of human peripheral blood T cells and TIL. Tumor-specific T cells with disrupted Cish function were adoptively transferred into tumor-bearing mice and evaluated for efficacy with or without checkpoint blockade. FINDINGS: CISH expression was associated with T cell dysfunction. CISH deletion using CRISPR/Cas9 resulted in hyper-activation and improved functional avidity against tumor-derived neoantigens without perturbing T cell maturation. Cish knockout resulted in increased susceptibility to checkpoint blockade in vivo. CONCLUSIONS: CISH negatively regulates human T cell effector function, and its genetic disruption offers a novel avenue to improve the therapeutic efficacy of adoptive TIL therapy. FUNDING: This study was funded by Intima Bioscience, U.S. and in part through the Intramural program CCR at the National Cancer Institute.

Rapid Identification and Evaluation of Neoantigen-reactive T-Cell Receptors From Single Cells.

Engineered T cells expressing tumor-specific T-cell receptors (TCRs) are emerging as a mode of personalized cancer immunotherapy that requires identification of TCRs against the products of known driver mutations and novel mutations in a timely fashion. We present a nonviral and non-next-generation sequencing platform for rapid, and efficient neoantigen-specific TCR identification and evaluation that does not require the use of recombinant cloning techniques. The platform includes an innovative method of TCRα detection using Sanger sequencing, TCR pairings and the use of TCRα/ß gene fragments for putative TCR evaluation. Using patients' samples, we validated and compared our new methods head-to-head with conventional approaches used for TCR discovery. Development of a unique demultiplexing method for identification of TCRα, adaptation of synthetic TCRs for gene transfer, and a reliable reporter system significantly shortens TCR discovery time over conventional methods and increases throughput to facilitate testing prospective personalized TCRs for adoptive cell therapy.

Antigen Experienced T Cells from Peripheral Blood Recognize p53 Neoantigens.

PURPOSE: The purpose of this study was to evaluate antigen experienced T cells in peripheral blood lymphocytes (PBL) for responses to p53 neoantigens. EXPERIMENTAL DESIGN: PBLs from patients with a mutated TP53 tumor were sorted for antigen-experienced T cells and in vitro stimulation (IVS) was performed with p53 neoantigens. The IVS cultures were stimulated with antigen-presenting cells expressing p53 neoantigens, enriched for 41BB/OX40 and grown with rapid expansion protocol. RESULTS: T-cell responses were not observed in the PBLs of 4 patients who did not have tumor-infiltrating lymphocyte (TIL) responses to mutated TP53. In contrast, 5 patients with TIL responses to mutated TP53 also had similar T-cell responses in their PBLs, indicating that the PBLs and TILs were congruent in p53 neoantigen reactivity. CD4+ and CD8+ T cells were specific for p53R175H, p53Y220C, or p53R248W neoantigens, including a 78% reactive T-cell culture against p53R175H and HLA-A*02:01. Tracking TCRB clonotypes (clonality, top ranked, and TP53 mutation-specific) supported the enrichment of p53 neoantigen-reactive T cells from PBLs. The same T-cell receptor (TCR) from the TIL was found in the IVS cultures in three cases and multiple unique TCRs were found in another patient. TP53 mutation-specific T cells also recognized tumor cell lines bearing the appropriate human leukocyte antigen restriction element and TP53 mutation, indicating these T cells could recognize processed and presented p53 neoantigens. CONCLUSIONS: PBL was a noninvasive source of T cells targeting TP53 mutations for cell therapy and can provide a window into intratumoral p53 neoantigen immune responses.See related commentary by Olivera et al., p. 1203.

Tumor-infiltrating human CD4+ regulatory T cells display a distinct TCR repertoire and exhibit tumor and neoantigen reactivity.

CD4+ regulatory T (Treg) cells have an essential function in maintaining self-tolerance; however, they may also play a detrimental role in antitumor immune responses. The presence of elevated frequencies of Treg cells in tumors correlates with disease progression and poor survival in patients with cancer. The antigen specificity of Treg cells that have expanded in the tumor microenvironment is poorly understood; answering this question may provide important insights for immunotherapeutic approaches. To address this, we used a novel combinatorial approach to characterizing the T cell receptor (TCR) profiles of intratumoral Treg cells from patients with metastatic melanoma, gastrointestinal, and ovarian cancers and elucidated their antigen specificities. The TCR repertoires of tumor-resident Treg cells were diverse yet displayed significant overlap with circulating Treg cells but not with conventional T cells in tumor or blood. TCRs isolated from Treg cells displayed specific reactivity against autologous tumors and mutated neoantigens, suggesting that intratumoral Treg cells act in a tumor antigen-selective manner leading to their activation and clonal expansion in the tumor microenvironment. Tumor antigen-specific Treg-derived TCRs resided in the tumor and in the circulation, suggesting that both Treg cell compartments may serve as a source for tumor-specific TCRs. These findings provide insights into the TCR specificity of tumor-infiltrating human Treg cells that may have potential implications for cancer immunotherapy.

Memory T cells targeting oncogenic mutations detected in peripheral blood of epithelial cancer patients.

T cells targeting shared oncogenic mutations can induce durable tumor regression in epithelial cancer patients. Such T cells can be detected in tumor infiltrating lymphocytes, but whether such cells can be detected in the peripheral blood of patients with the common metastatic epithelial cancer patients is unknown. Using a highly sensitive in vitro stimulation and cell enrichment of peripheral memory T cells from six metastatic cancer patients, we identified and isolated CD4+, and CD8+ memory T cells targeting the mutated KRASG12D and KRASG12V variants, respectively, in three patients. In an additional two metastatic colon cancer patients, we detected CD8+ neoantigen-specific cells targeting the mutated SMAD5 and MUC4 proteins. Therefore, memory T cells targeting unique as well as shared somatic mutations can be detected in the peripheral blood of epithelial cancer patients and can potentially be used for the development of effective personalized T cell-based cancer immunotherapy across multiple patients.

Unique Neoantigens Arise from Somatic Mutations in Patients with Gastrointestinal Cancers.

Immunotherapies can mediate regression of human tumors with high mutation rates, but responses are rarely observed in patients with common epithelial cancers. This raises the question of whether patients with these common cancers harbor T lymphocytes that recognize mutant proteins expressed by autologous tumors that may represent ideal targets for immunotherapy. Using high-throughput immunologic screening of mutant gene products identified via whole-exome sequencing, we identified neoantigen-reactive tumor-infiltrating lymphocytes (TIL) from 62 of 75 (83%) patients with common gastrointestinal cancers. In total, 124 neoantigen-reactive TIL populations were identified, and all but one of the neoantigenic determinants were unique. The results of in vitro T-cell recognition assays demonstrated that 1.6% of the gene products encoded by somatic nonsynonymous mutations were immunogenic. These findings demonstrate that the majority of common epithelial cancers elicit immune recognition and open possibilities for cell-based immunotherapies for patients bearing these cancers. SIGNIFICANCE: TILs cultured from 62 of 75 (83%) patients with gastrointestinal cancers recognized neoantigens encoded by 1.6% of somatic mutations expressed by autologous tumor cells, and 99% of the neoantigenic determinants appeared to be unique and not shared between patients.This article is highlighted in the In This Issue feature, p. 983.

Enhanced detection of neoantigen-reactive T cells targeting unique and shared oncogenes for personalized cancer immunotherapy.

Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TILs) targeting neoantigens can mediate tumor regression in selected patients with metastatic epithelial cancer. However, effectively identifying and harnessing neoantigen-reactive T cells for patient treatment remains a challenge and it is unknown whether current methods to detect neoantigen-reactive T cells are missing potentially clinically relevant neoantigen reactivities. We thus investigated whether the detection of neoantigen-reactive TILs could be enhanced by enriching T cells that express PD-1 and/or T cell activation markers followed by microwell culturing to avoid overgrowth of nonreactive T cells. In 6 patients with metastatic epithelial cancer, this method led to the detection of CD4+ and CD8+ T cells targeting 18 and 1 neoantigens, respectively, compared with 6 and 2 neoantigens recognized by CD4+ and CD8+ T cells, respectively, when using our standard TIL fragment screening approach. In 2 patients, no recognition of mutated peptides was observed using our conventional screen, while our high-throughput approach led to the identification of 5 neoantigen-reactive T cell receptors (TCRs) against 5 different mutations from one patient and a highly potent MHC class II-restricted KRASG12V-reactive TCR from a second patient. In addition, in a metastatic tumor sample from a patient with serous ovarian cancer, we isolated 3 MHC class II-restricted TCRs targeting the TP53G245S hot-spot mutation. In conclusion, this approach provides a highly sensitive platform to isolate clinically relevant neoantigen-reactive T cells or their TCRs for cancer treatment.

T-cell Responses to TP53 "Hotspot" Mutations and Unique Neoantigens Expressed by Human Ovarian Cancers.

Purpose: This was a study prospectively evaluating intratumoral T-cell responses to autologous somatic mutated neoepitopes expressed by human metastatic ovarian cancers.Patients and Methods: Tumor-infiltrating lymphocytes (TIL) were expanded from resected ovarian cancer metastases, which were analyzed by whole-exome and transcriptome sequencing to identify autologous somatic mutations. All mutated neoepitopes, independent of prediction algorithms, were expressed in autologous antigen-presenting cells and then cocultured with TIL fragment cultures. Secretion of IFNγ or upregulation of 41BB indicated a T-cell response.Results: Seven women with metastatic ovarian cancer were evaluated, and 5 patients had clear, dominant T-cell responses to mutated neoantigens, which were corroborated by comparison with the wild-type sequence, identification of the minimal epitope, human leukocyte antigen (HLA) restriction element(s), and neoantigen-specific T-cell receptor(s). Mutated neoantigens were restricted by HLA-B, -C, -DP, -DQ, and/or -DR alleles and appeared to principally arise from random, somatic mutations unique to each patient. We established that TP53 "hotspot" mutations (c.659A>G; p.Y220C and c.733G>A; p.G245S) expressed by two different patients' tumors were both immunogenic in the context of HLA-DRB3*02:02.Conclusions: Mutation-reactive T cells infiltrated ovarian cancer metastases at sufficient frequencies to warrant their investigation as adoptive cell therapy. In addition, transfer of TP53 "hotspot" mutation-reactive T-cell receptors into peripheral blood T cells could be evaluated as a gene therapy for a diverse range of tumor histologies. Clin Cancer Res; 24(22); 5562-73. ©2018 AACR See related commentary by McNeish, p. 5493.

A Pilot Trial of the Combination of Vemurafenib with Adoptive Cell Therapy in Patients with Metastatic Melanoma.

PURPOSE: This pilot feasibility clinical trial evaluated the coadministration of vemurafenib, a small-molecule antagonist of BRAFV600 mutations, and tumor-infiltrating lymphocytes (TIL) for the treatment of metastatic melanoma. EXPERIMENTAL DESIGN: A metastatic tumor was resected for growth of TILs, and patients were treated with vemurafenib for 2 weeks, followed by resection of a second lesion. Patients then received a nonmyeloablative preconditioning regimen, infusion of autologous TILs, and high-dose interleukin-2 administration. Vemurafenib was restarted at the time of TIL infusion and was continued for 2 years or until disease progression. Clinical responses were evaluated by Response Evaluation Criteria in Solid Tumors (RECIST) 1.0. Metastases resected prior to and after 2 weeks of vemurafenib were compared using TCRB deep sequencing, immunohistochemistry, proliferation, and recognition of autologous tumor. RESULTS: The treatment was well tolerated and had a safety profile similar to that of TIL or vemurafenib alone. Seven of 11 patients (64%) experienced an objective clinical response, and 2 patients (18%) had a complete response for 3 years (one response is ongoing at 46 months). Proliferation and viability of infusion bag TILs and peripheral blood T cells were inhibited in vitro by research-grade vemurafenib (PLX4032) when approaching the maximum serum concentration of vemurafenib. TCRB repertoire (clonotypes numbers, clonality, and frequency) did not significantly change between pre- and post-vemurafenib lesions. Recognition of autologous tumor by T cells was similar between TILs grown from pre- and post-vemurafenib metastases. CONCLUSIONS: Coadministration of vemurafenib and TILs was safe and feasible and generated objective clinical responses in this small pilot clinical trial. Clin Cancer Res; 23(2); 351-62. ©2016 AACRSee related commentary by Cogdill et al., p. 327.

Tumor- and Neoantigen-Reactive T-cell Receptors Can Be Identified Based on Their Frequency in Fresh Tumor.

Adoptive transfer of T cells with engineered T-cell receptor (TCR) genes that target tumor-specific antigens can mediate cancer regression. Accumulating evidence suggests that the clinical success of many immunotherapies is mediated by T cells targeting mutated neoantigens unique to the patient. We hypothesized that the most frequent TCR clonotypes infiltrating the tumor were reactive against tumor antigens. To test this hypothesis, we developed a multistep strategy that involved TCRB deep sequencing of the CD8(+)PD-1(+) T-cell subset, matching of TCRA-TCRB pairs by pairSEQ and single-cell RT-PCR, followed by testing of the TCRs for tumor-antigen specificity. Analysis of 12 fresh metastatic melanomas revealed that in 11 samples, up to 5 tumor-reactive TCRs were present in the 5 most frequently occurring clonotypes, which included reactivity against neoantigens. These data show the feasibility of developing a rapid, personalized TCR-gene therapy approach that targets the unique set of antigens presented by the autologous tumor without the need to identify their immunologic reactivity. Cancer Immunol Res; 4(9); 734-43. ©2016 AACR.

Sleeping Beauty Transposition of Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like Orphan Receptor-1 (ROR1) into Diverse Memory T-Cell Populations.

T cells modified with chimeric antigen receptors (CARs) targeting CD19 demonstrated clinical activity against some B-cell malignancies. However, this is often accompanied by a loss of normal CD19+ B cells and humoral immunity. Receptor tyrosine kinase-like orphan receptor-1 (ROR1) is expressed on sub-populations of B-cell malignancies and solid tumors, but not by healthy B cells or normal post-partum tissues. Thus, adoptive transfer of T cells specific for ROR1 has potential to eliminate tumor cells and spare healthy tissues. To test this hypothesis, we developed CARs targeting ROR1 in order to generate T cells specific for malignant cells. Two Sleeping Beauty transposons were constructed with 2nd generation ROR1-specific CARs signaling through CD3ζ and either CD28 (designated ROR1RCD28) or CD137 (designated ROR1RCD137) and were introduced into T cells. We selected for T cells expressing CAR through co-culture with γ-irradiated activating and propagating cells (AaPC), which co-expressed ROR1 and co-stimulatory molecules. Numeric expansion over one month of co-culture on AaPC in presence of soluble interleukin (IL)-2 and IL-21 occurred and resulted in a diverse memory phenotype of CAR+ T cells as measured by non-enzymatic digital array (NanoString) and multi-panel flow cytometry. Such T cells produced interferon-γ and had specific cytotoxic activity against ROR1+ tumors. Moreover, such cells could eliminate ROR1+ tumor xenografts, especially T cells expressing ROR1RCD137. Clinical trials will investigate the ability of ROR1-specific CAR+ T cells to specifically eliminate tumor cells while maintaining normal B-cell repertoire.

Clinical applications of gamma delta T cells with multivalent immunity.

γδ T cells hold promise for adoptive immunotherapy because of their reactivity to bacteria, viruses, and tumors. However, these cells represent a small fraction (1-5%) of the peripheral T-cell pool and require activation and propagation to achieve clinical benefit. Aminobisphosphonates specifically expand the Vγ9Vδ2 subset of γδ T cells and have been used in clinical trials of cancer where objective responses were detected. The Vγ9Vδ2 T cell receptor (TCR) heterodimer binds multiple ligands and results in a multivalent attack by a monoclonal T cell population. Alternatively, populations of γδ T cells with oligoclonal or polyclonal TCR repertoire could be infused for broad-range specificity. However, this goal has been restricted by a lack of applicable expansion protocols for non-Vγ9Vδ2 cells. Recent advances using immobilized antigens, agonistic monoclonal antibodies (mAbs), tumor-derived artificial antigen presenting cells (aAPC), or combinations of activating mAbs and aAPC have been successful in expanding gamma delta T cells with oligoclonal or polyclonal TCR repertoires. Immobilized major histocompatibility complex Class-I chain-related A was a stimulus for γδ T cells expressing TCRδ1 isotypes, and plate-bound activating antibodies have expanded Vδ1 and Vδ2 cells ex vivo. Clinically sufficient quantities of TCRδ1, TCRδ2, and TCRδ1(neg)TCRδ2(neg) have been produced following co-culture on aAPC, and these subsets displayed differences in memory phenotype and reactivity to tumors in vitro and in vivo. Gamma delta T cells are also amenable to genetic modification as evidenced by introduction of αß TCRs, chimeric antigen receptors, and drug-resistance genes. This represents a promising future for the clinical application of oligoclonal or polyclonal γδ T cells in autologous and allogeneic settings that builds on current trials testing the safety and efficacy of Vγ9Vδ2 T cells.

Activating and propagating polyclonal gamma delta T cells with broad specificity for malignancies.

PURPOSE: To activate and propagate populations of γδ T cells expressing polyclonal repertoire of γ and δ T-cell receptor (TCR) chains for adoptive immunotherapy of cancer, which has yet to be achieved. EXPERIMENTAL DESIGN: Clinical-grade artificial antigen-presenting cells (aAPC) derived from K562 tumor cells were used as irradiated feeders to activate and expand human γδ T cells to clinical scale. These cells were tested for proliferation, TCR expression, memory phenotype, cytokine secretion, and tumor killing. RESULTS: γδ T-cell proliferation was dependent upon CD137L expression on aAPC and addition of exogenous IL2 and IL21. Propagated γδ T cells were polyclonal as they expressed TRDV1, TRDV2-2, TRDV3, TRDV5, TRDV7, and TRDV8 with TRGV2, TRGV3F, TRGV7, TRGV8, TRGV9*A1, TRGV10*A1, and TRGV11 TCR chains. IFNγ production by Vδ1, Vδ2, and Vδ1(neg)Vδ2(neg) subsets was inhibited by pan-TCRγδ antibody when added to cocultures of polyclonal γδ T cells and tumor cell lines. Polyclonal γδ T cells killed acute and chronic leukemia, colon, pancreatic, and ovarian cancer cell lines, but not healthy autologous or allogeneic normal B cells. Blocking antibodies demonstrated that polyclonal γδ T cells mediated tumor cell lysis through combination of DNAM1, NKG2D, and TCRγδ. The adoptive transfer of activated and propagated γδ T cells expressing polyclonal versus defined Vδ TCR chains imparted a hierarchy (polyclonal>Vδ1>Vδ1(neg)Vδ2(neg)>Vδ2) of survival of mice with ovarian cancer xenografts. CONCLUSIONS: Polyclonal γδ T cells can be activated and propagated with clinical-grade aAPCs and demonstrate broad antitumor activities, which will facilitate the implementation of γδ T-cell cancer immunotherapies in humans.

Targeting and penetration of virus receptor bearing cells by nanoparticles coated with envelope proteins of Moloney murine leukemia virus.

One of the most important steps in a productive viral infection is when the virus fuses to a cell membrane and delivers its genome into the cell cytosol. This dynamic event is mediated by interactions between specific virus envelope proteins with their cell-bound receptors. This process is exemplified by Moloney murine leukemia virus (Mo-MLV) where envelope protein interaction with its receptor, mCAT-1, leads to virus-cell membrane fusion and infection of cells. Here, fluorescent nanoparticles (NPs) were coated with Mo-MLV derived membranes (Mo-NPs) by extrusion. Electron microscopy and biochemical analysis showed tight association of the virus membranes and NPs. The coated NPs mimic native virus by binding and entering only cells expressing the virus receptor. Confocal microscopy revealed that the coated NPs were taken up into endocytic compartments containing receptor and were also seen associated with caveolin, a marker of caveolae. To demonstrate that the Mo-NPs could escape endosomes and deliver a protein cargo into the cell cytosol, beta-lactamase (betalac) was covalently coupled to the Mo-NP cores and incubated with cells. betalac activity was only detected in the cytosol of mCAT-1-expressing cells. This is the first time that virus proteins have been used to specifically target NPs to receptor-bearing cells as well as penetration into the cell cytosol. Extrusion provides a rapid, detergent-free method to couple virus membranes to NPs and should be readily applicable for many other virus and NP types.