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Adoptive cell transfer of tumor-infiltrating lymphocytes (TIL) can mediate durable complete responses in some patients with common epithelial cancers but does so infrequently. A better understanding of T-cell responses to neoantigens and tumor-related immune evasion mechanisms requires having the autologous tumor as a reagent. We investigated the ability of patient-derived tumor organoids (PDTO) to fulfill this need and evaluated their utility as a tool for selecting T-cells for adoptive cell therapy. PDTO established from metastases from patients with colorectal, breast, pancreatic, bile duct, esophageal, lung, and kidney cancers underwent whole exomic sequencing (WES), to define mutations. Organoids were then evaluated for recognition by autologous TIL or T-cells transduced with cloned T-cell receptors recognizing defined neoantigens. PDTO were also used to identify and clone TCRs from TIL targeting private neoantigens and define those tumor-specific targets. PDTO were successfully established in 38/47 attempts. 75% were available within 2 months, a timeframe compatible with screening TIL for clinical administration. These lines exhibited good genetic fidelity with their parental tumors, especially for mutations with higher clonality. Immunologic recognition assays demonstrated instances of HLA allelic loss not found by pan-HLA immunohistochemistry and in some cases WES of fresh tumor. PDTO could also be used to show differences between TCRs recognizing the same antigen and to find and clone TCRs recognizing private neoantigens. PDTO can detect tumor-specific defects blocking T-cell recognition and may have a role as a selection tool for TCRs and TIL used in adoptive cell therapy., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

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., Competing Interests: Declaration of interests M.C. is a co-founder of Intima Bioscience. B.R.W., S.A.R., and B.S.M. have received sponsored research support from Intima Bioscience. D.C.P., B.R.W., M.C., S.A.R., B.S.M., and N.P.R. have patents filed based on the findings described here., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The accurate identification of antitumor T cell receptors (TCRs) represents a major challenge for the engineering of cell-based cancer immunotherapies. By mapping 55 neoantigen-specific TCR clonotypes (NeoTCRs) from 10 metastatic human tumors to their single-cell transcriptomes, we identified signatures of CD8 + and CD4 + neoantigen-reactive tumor-infiltrating lymphocytes (TILs). Neoantigen-specific TILs exhibited tumor-specific expansion with dysfunctional phenotypes, distinct from blood-emigrant bystanders and regulatory TILs. Prospective prediction and testing of 73 NeoTCR signature-derived clonotypes demonstrated that half of the tested TCRs recognized tumor antigens or autologous tumors. NeoTCR signatures identified TCRs that target driver neoantigens and nonmutated viral or tumor-associated antigens, suggesting a common metastatic TIL exhaustion program. NeoTCR signatures delineate the landscape of TILs across metastatic tumors, enabling successful TCR prediction based purely on TIL transcriptomic states for use in cancer immunotherapy.

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/β 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.

BACKGROUNDTherapeutic vaccinations against cancer have mainly targeted differentiation antigens, cancer-testis antigens, and overexpressed antigens and have thus far resulted in little clinical benefit. Studies conducted by multiple groups have demonstrated that T cells recognizing neoantigens are present in most cancers and offer a specific and highly immunogenic target for personalized vaccination.METHODSWe recently developed a process using tumor-infiltrating lymphocytes to identify the specific immunogenic mutations expressed in patients' tumors. Here, validated, defined neoantigens, predicted neoepitopes, and mutations of driver genes were concatenated into a single mRNA construct to vaccinate patients with metastatic gastrointestinal cancer.RESULTSThe vaccine was safe and elicited mutation-specific T cell responses against predicted neoepitopes not detected before vaccination. Furthermore, we were able to isolate and verify T cell receptors targeting KRASG12D mutation. We observed no objective clinical responses in the 4 patients treated in this trial.CONCLUSIONThis vaccine was safe, and potential future combination of such vaccines with checkpoint inhibitors or adoptive T cell therapy should be evaluated for possible clinical benefit in patients with common epithelial cancers.TRIAL REGISTRATIONPhase I/II protocol (NCT03480152) was approved by the IRB committee of the NIH and the FDA.FUNDINGCenter for Clinical Research, NCI, NIH.

Cancer elimination in humans can be achieved with immunotherapy that relies on T lymphocyte-mediated recognition of tumor antigens. Several types of these antigens have been recognized based on their cellular origins and expression patterns, while their detection has been greatly facilitated by recent achievements in next-generation sequencing and immunopeptidomics. Some of them have been targeted in clinical trials with various immunotherapy approaches, while many others remain untested. Here, we discuss molecular identification of different tumor antigen types, and the clinical safety and efficacy of targeting them with immunotherapy. Additionally, we suggest strategies to increase the efficacy and availability of antigen-directed immunotherapies for treatment of patients with metastatic cancer., (Published by Elsevier Inc.)

Complete cancer regression occurs in a subset of patients following adoptive T cell therapy (ACT) of ex vivo expanded tumor-infiltrating lymphocytes (TILs). However, the low success rate presents a great challenge to broader clinical application. To provide insight into TIL-based immunotherapy, we studied a successful case of ACT where regression was observed against tumors carrying the hotspot mutation G12D in the KRAS oncogene. Four T cell receptors (TCRs) made up the TIL infusion and recognized two KRAS-G12D neoantigens, a nonamer and a decamer, all restricted by human leukocyte antigen (HLA) C*08:02. Three of them (TCR9a, 9b, and 9c) were nonamer-specific, while one was decamer-specific (TCR10). We show that only mutant G12D but not the wild-type peptides stabilized HLA-C*08:02 due to the formation of a critical anchor salt bridge to HLA-C. Therapeutic TCRs exhibited high affinities, ranging from nanomolar to low micromolar. Intriguingly, TCR binding affinities to HLA-C inversely correlated with their persistence in vivo, suggesting the importance of antigenic affinity in the function of therapeutic T cells. Crystal structures of TCR-HLA-C complexes revealed that TCR9a to 9c recognized G12D nonamer with multiple conserved contacts through shared CDR2β and CDR3α. This allowed CDR3β variation to confer different affinities via a variable HLA-C contact, generating an oligoclonal response. TCR10 recognized an induced and distinct G12D decamer conformation. Thus, this successful case of ACT included oligoclonal TCRs of high affinity recognizing distinct conformations of neoantigens. Our study revealed the potential of a structural approach to inform clinical efforts in targeting KRAS-G12D tumors by immunotherapy and has general implications for T cell-based immunotherapies., Competing Interests: The authors declare no competing interest.

A deletion variant of epidermal growth factor receptor (EGFRvIII) is a known driver mutation in a subset of primary and secondary glioblastoma multiforme. Adoptive transfer of genetically modified chimeric antigen receptor (CAR) lymphocytes has demonstrated efficacy in hematologic malignancies but is still early in development for solid cancers. The surface expression of the truncated extracellular ligand domain created by EGFRvIII makes it an attractive target for a CAR-based cancer treatment. Patients with recurrent glioblastoma expressing EGFRvIII were enrolled in a dose escalation phase I trial, using a third-generation CAR construct derived from a human antibody. Transduced cells were administered after lymphodepleting chemotherapy and supported posttransfer with intravenous interleukin-2. The dose escalation proceeded at half-log increments from 10 to >10 cells. Primary endpoints were safety and progression-free survival. Eighteen patients were treated with final infusion products ranging from 6.3×10 to 2.6×10 anti-EGFRvIII CAR T cells. Median progression-free survival was 1.3 months (interquartile range: 1.1-1.9), with a single outlier of 12.5 months. Two patients experienced severe hypoxia, including one treatment-related mortality after cell administration at the highest dose level. All patients developed expected transient hematologic toxicities from preparative chemotherapy. Median overall survival was 6.9 months (interquartile range: 2.8-10). Two patients survived over 1 year, and a third patient was alive at 59 months. Persistence of CAR cells correlated with cell dose, but there were no objective responses. Administration of anti-EGFRvIII CAR-transduced T cells did not demonstrate clinically meaningful effect in patients with glioblastoma multiforme in this phase I pilot trial.

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 ., (©2018 American Association for Cancer Research.)

The quest for tumor-associated antigens (TAA) and neoantigens is a major focus of cancer immunotherapy. Here, we combine a neoantigen prediction pipeline and human leukocyte antigen (HLA) peptidomics to identify TAAs and neoantigens in 16 tumors derived from seven patients with melanoma and characterize their interactions with their tumor-infiltrating lymphocytes (TIL). Our investigation of the antigenic and T-cell landscapes encompassing the TAA and neoantigen signatures, their immune reactivity, and their corresponding T-cell identities provides the first comprehensive analysis of cancer cell T-cell cosignatures, allowing us to discover remarkable antigenic and TIL similarities between metastases from the same patient. Furthermore, we reveal that two neoantigen-specific clonotypes killed 90% of autologous melanoma cells, both in vitro and in vivo , showing that a limited set of neoantigen-specific T cells may play a central role in melanoma tumor rejection. Our findings indicate that combining HLA peptidomics with neoantigen predictions allows robust identification of targetable neoantigens, which could successfully guide personalized cancer immunotherapies. Significance: As neoantigen targeting is becoming more established as a powerful therapeutic approach, investigating these molecules has taken center stage. Here, we show that a limited set of neoantigen-specific T cells mediates tumor rejection, suggesting that identifying just a few antigens and their corresponding T-cell clones could guide personalized immunotherapy. Cancer Discov; 8(11); 1366-75. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1333 ., (©2018 American Association for Cancer Research.)

The adoptive transfer of neoantigen-reactive tumor-infiltrating lymphocytes (TILs) can result in tumor regression in patients with metastatic cancer. To improve the efficacy of adoptive T cell therapy targeting these tumor-specific mutations, we have proposed a new therapeutic strategy, which involves the genetic modification of autologous T cells with neoantigen-specific T cell receptors (TCRs) and the transfer of these modified T cells back to cancer patients. However, the current techniques to isolate neoantigen-specific TCRs are labor intensive, time consuming, and technically challenging, not suitable for clinical applications. To facilitate this process, a new approach was developed, which included the co-culture of TILs with tandem minigene (TMG)-transfected or peptide-pulsed autologous antigen-presenting cells (APCs) and the single-cell RNA sequencing (RNA-seq) analysis of T cells to identify paired TCR sequences associated with cells expressing high levels of interferon-γ (IFN-γ) and interleukin-2 (IL-2). Following this new approach, multiple TCRs were identified, synthesized, cloned into a retroviral vector, and then transduced into donor T cells. These transduced T cells were shown to specifically recognize the neoantigens presented by autologous APCs. In conclusion, this approach provides an efficient procedure to isolate neoantigen-specific TCRs for clinical applications, as well as for basic and translational research., (Published by Elsevier Inc.)

Purpose T cells genetically modified to express chimeric antigen receptors (CARs) targeting CD19 (CAR-19) have potent activity against acute lymphoblastic leukemia, but fewer results supporting treatment of lymphoma with CAR-19 T cells have been published. Patients with lymphoma that is chemotherapy refractory or relapsed after autologous stem-cell transplantation have a grim prognosis, and new treatments for these patients are clearly needed. Chemotherapy administered before adoptive T-cell transfer has been shown to enhance the antimalignancy activity of adoptively transferred T cells. Patients and Methods We treated 22 patients with advanced-stage lymphoma in a clinical trial of CAR-19 T cells preceded by low-dose chemotherapy. Nineteen patients had diffuse large B-cell lymphoma, two patients had follicular lymphoma, and one patient had mantle cell lymphoma. Patients received a single dose of CAR-19 T cells 2 days after a low-dose chemotherapy conditioning regimen of cyclophosphamide plus fludarabine. Results The overall remission rate was 73% with 55% complete remissions and 18% partial remissions. Eleven of 12 complete remissions are ongoing. Fifty-five percent of patients had grade 3 or 4 neurologic toxicities that completely resolved. The low-dose chemotherapy conditioning regimen depleted blood lymphocytes and increased serum interleukin-15 (IL-15). Patients who achieved a remission had a median peak blood CAR + cell level of 98/μL and those who did not achieve a remission had a median peak blood CAR + cell level of 15/μL ( P = .027). High serum IL-15 levels were associated with high peak blood CAR + cell levels ( P = .001) and remissions of lymphoma ( P < .001). Conclusion CAR-19 T cells preceded by low-dose chemotherapy induced remission of advanced-stage lymphoma, and high serum IL-15 levels were associated with the effectiveness of this treatment regimen. CAR-19 T cells will likely become an important treatment for patients with relapsed lymphoma.

Effective clinical cancer immunotherapies, such as administration of the cytokine IL-2, adoptive cell transfer (ACT) and the recent success of blockade of the checkpoint modulators CTLA-4 and PD-1, have been developed without clear identification of the immunogenic targets expressed by human cancers in vivo. Immunotherapy of patients with cancer through the use of ACT with autologous lymphocytes has provided an opportunity to directly investigate the antigen recognition of lymphocytes that mediate cancer regression in humans. High-throughput immunological testing of such lymphocytes in combination with improvements in deep sequencing of the autologous cancer have provided new insight into the molecular characterization and incidence of anti-tumor lymphocytes present in patients with cancer. Here we highlight evidence suggesting that T cells that target tumor neoantigens arising from cancer mutations are the main mediators of many effective cancer immunotherapies in humans.

The treatment of B-cell malignancies by adoptive cell transfer (ACT) of anti-CD19 chimeric antigen receptor T cells (CD19 CAR-T) has proven to be a highly successful therapeutic modality in several clinical trials. 1-6 The anti-CD19 CAR-T cell production method used to support initial trials relied on numerous manual, open process steps, human serum, and 10 days of cell culture to achieve a clinical dose. 7 This approach limited the ability to support large multicenter clinical trials, as well as scale up for commercial cell production. Therefore, studies were completed to streamline and optimize the original National Cancer Institute production process by removing human serum from the process in order to minimize the risk of viral contamination, moving process steps from an open system to functionally closed system operations in order to minimize the risk of microbial contamination, and standardizing additional process steps in order to maximize process consistency. This study reports a procedure for generating CD19 CAR-T cells in 6 days, using a functionally closed manufacturing process and defined, serum-free medium. This method is able to produce CD19 CAR-T cells that are phenotypically and functionally indistinguishable from cells produced for clinical trials by the previously described production process.

Purpose: Adoptive transfer of activated autologous tumor-infiltrating lymphocytes (TIL) can mediate complete, durable regressions in patients with metastatic melanoma. Responding patients generally do not have significant changes in noncutaneous RECIST targets before 30 to 60 days following TIL infusion, and complete responses are often not confirmed for 1 to 2 years. There is a critical need for a biomarker that can provide early information regarding the likelihood and duration of a response to enable rational decisions about altering therapy. We wished to evaluate the role of circulating tumor DNA (ctDNA) in separating responding from nonresponding patients., Experimental Design: We studied BRAF V600E ctDNA levels by a sensitive allele-specific PCR assay in 388 serum samples from 48 patients who received TIL immunotherapy at the NCI and correlated differences in the dynamic patterns of their ctDNA measurements with response outcomes., Results: A strong correlation was found between the presence or absence of an early serum peak of V600E ctDNA, and the likelihood of an objective response. Furthermore, patients that developed an early ctDNA peak and cleared their serum of V600E ctDNA were highly likely to achieve a complete response over the next 1 to 2 years. Patients that showed no peak of V600E ctDNA failed to achieve an objective response, with one exception., Conclusions: We show that the dynamic changes occurring in BRAF V600E ctDNA levels within the first month following T-cell transfer immunotherapy in metastatic melanoma can be used to rapidly identify responding from nonresponding patients, potentially allowing clinicians to make critical treatment-related decisions in a more timely manner. These data also suggest that the majority of tumor killing by TIL occurs very early after the initiation of therapy. Clin Cancer Res; 22(22); 5480-6. ©2016 AACR., (©2016 American Association for Cancer Research.)

Immunotherapy treatment of patients with metastatic cancer has assumed a prominent role in the clinic. Durable complete response rates of 20% to 25% are achieved in patients with metastatic melanoma following adoptive cell transfer of T cells derived from metastatic lesions, responses that appear in some patients to be mediated by T cells that predominantly recognize mutated antigens. Here, we provide a detailed analysis of the reactivity of T cells administered to a patient with metastatic melanoma who exhibited a complete response for over 3 years after treatment. Over 4,000 nonsynonymous somatic mutations were identified by whole-exome sequence analysis of the patient's autologous normal and tumor cell DNA. Autologous B cells transfected with 720 mutated minigenes corresponding to the most highly expressed tumor cell transcripts were then analyzed for their ability to stimulate the administered T cells. Autologous tumor-infiltrating lymphocytes recognized 10 distinct mutated gene products, but not the corresponding wild-type products, each of which was recognized in the context of one of three different MHC class I restriction elements expressed by the patient. Detailed clonal analysis revealed that 9 of the top 20 most prevalent clones present in the infused T cells, comprising approximately 24% of the total cells, recognized mutated antigens. Thus, we have identified and enriched mutation-reactive T cells and suggest that such analyses may lead to the development of more effective therapies for the treatment of patients with metastatic cancer. Cancer Immunol Res; 4(8); 669-78. ©2016 AACR., (©2016 American Association for Cancer Research.)

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 TCRβ (mTCRβ) expression. Rapid expansion of mTCRβ(+) 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.

Purpose: Progressive malignancy is the leading cause of death after allogeneic hematopoietic stem-cell transplantation (alloHSCT). After alloHSCT, B-cell malignancies often are treated with unmanipulated donor lymphocyte infusions (DLIs) from the transplant donor. DLIs frequently are not effective at eradicating malignancy and often cause graft-versus-host disease, a potentially lethal immune response against normal recipient tissues., Methods: We conducted a clinical trial of allogeneic T cells genetically engineered to express a chimeric antigen receptor (CAR) targeting the B-cell antigen CD19. Patients with B-cell malignancies that had progressed after alloHSCT received a single infusion of CAR T cells. No chemotherapy or other therapies were administered. The T cells were obtained from each recipient's alloHSCT donor., Results: Eight of 20 treated patients obtained remission, which included six complete remissions (CRs) and two partial remissions. The response rate was highest for acute lymphoblastic leukemia, with four of five patients obtaining minimal residual disease-negative CR. Responses also occurred in chronic lymphocytic leukemia and lymphoma. The longest ongoing CR was more than 30 months in a patient with chronic lymphocytic leukemia. New-onset acute graft-versus-host disease after CAR T-cell infusion developed in none of the patients. Toxicities included fever, tachycardia, and hypotension. Peak blood CAR T-cell levels were higher in patients who obtained remissions than in those who did not. Programmed cell death protein-1 expression was significantly elevated on CAR T cells after infusion. Presence of blood B cells before CAR T-cell infusion was associated with higher postinfusion CAR T-cell levels., Conclusion: Allogeneic anti-CD19 CAR T cells can effectively treat B-cell malignancies that progress after alloHSCT. The findings point toward a future when antigen-specific T-cell therapies will play a central role in alloHSCT., (© 2016 by American Society of Clinical Oncology.)

Adoptive transfer of receptor-engineered T cells has produced impressive results in treating patients with B cell leukemias and lymphomas. This success has captured public imagination and driven academic and industrial researchers to develop similar 'off-the-shelf' receptors targeting shared antigens on epithelial cancers, the leading cause of cancer-related deaths. However, the successful treatment of large numbers of people with solid cancers using this strategy is unlikely to be straightforward. Receptor-engineered T cells have the potential to cause lethal toxicity from on-target recognition of normal tissues, and there is a paucity of truly tumor-specific antigens shared across tumor types. Here we offer our perspective on how expanding the use of genetically redirected T cells to treat the majority of patients with solid cancers will require major technical, manufacturing and regulatory innovations centered around the development of autologous gene therapies targeting private somatic mutations.

Adoptive cell transfer (ACT) of purified naive, stem cell memory, and central memory T cell subsets results in superior persistence and antitumor immunity compared with ACT of populations containing more-differentiated effector memory and effector T cells. Despite a clear advantage of the less-differentiated populations, the majority of ACT trials utilize unfractionated T cell subsets. Here, we have challenged the notion that the mere presence of less-differentiated T cells in starting populations used to generate therapeutic T cells is sufficient to convey their desirable attributes. Using both mouse and human cells, we identified a T cell-T cell interaction whereby antigen-experienced subsets directly promote the phenotypic, functional, and metabolic differentiation of naive T cells. This process led to the loss of less-differentiated T cell subsets and resulted in impaired cellular persistence and tumor regression in mouse models following ACT. The T memory-induced conversion of naive T cells was mediated by a nonapoptotic Fas signal, resulting in Akt-driven cellular differentiation. Thus, induction of Fas signaling enhanced T cell differentiation and impaired antitumor immunity, while Fas signaling blockade preserved the antitumor efficacy of naive cells within mixed populations. These findings reveal that T cell subsets can synchronize their differentiation state in a process similar to quorum sensing in unicellular organisms and suggest that disruption of this quorum-like behavior among T cells has potential to enhance T cell-based immunotherapies.

Recent developments have demonstrated that immunotherapies are capable of achieving durable antitumor responses in patients with metastatic cancer. One modality that has been able to induce durable complete regressions in patients with melanoma has been adoptive cell therapy (ACT). This has slowly been expanded to other cancer types using new approaches such as genetically engineered T-cells and other methods of antigen targeting. It now appears that immune targeting of mutated "neoantigens" plays a major role in successful ACT, as well as in other immunotherapies such as checkpoint inhibitors. This realization presents not only new challenges to ACT but also new opportunities in that all tumors now may have potential antigens to attack that can be revealed by tumor genomic sequencing. There are a variety of exciting approaches to translate these new findings into clinical trials applying ACT to the majority of cancer types., (2016 Published by Elsevier Inc.)

Adoptively transferred tumor-infiltrating T lymphocytes (TILs) that mediate complete regression of metastatic melanoma have been shown to recognize mutated epitopes expressed by autologous tumors. Here, in an attempt to develop a strategy for facilitating the isolation, expansion, and study of mutated antigen-specific T cells, we performed whole-exome sequencing on matched tumor and normal DNA isolated from 8 patients with metastatic melanoma. Candidate mutated epitopes were identified using a peptide-MHC-binding algorithm, and these epitopes were synthesized and used to generate panels of MHC tetramers that were evaluated for binding to tumor digests and cultured TILs used for the treatment of patients. This strategy resulted in the identification of 9 mutated epitopes from 5 of the 8 patients tested. Cells reactive with 8 of the 9 epitopes could be isolated from autologous peripheral blood, where they were detected at frequencies that were estimated to range between 0.4% and 0.002%. To the best of our knowledge, this represents the first demonstration of the successful isolation of mutation-reactive T cells from patients' peripheral blood prior to immune therapy, potentially providing the basis for designing personalized immunotherapies to treat patients with advanced cancer.

Purpose: Although adoptive cell therapy can be highly effective for the treatment of patients with melanoma, the application of this approach to the treatment of other solid tumors has been limited. The observation that the cancer germline (CG) antigen NY-ESO-1 is expressed in 70% to 80% and in approximately 25% of patients with synovial cell sarcoma and melanoma, respectively, prompted us to perform this first-in-man clinical trial using the adoptive transfer of autologous peripheral blood mononuclear cells that were retrovirally transduced with an NY-ESO-1-reactive T-cell receptor (TCR) to heavily pretreated patients bearing these metastatic cancers., Experimental Design: HLA-*0201 patients with metastatic synovial cell sarcoma or melanoma refractory to standard treatments and whose cancers expressed NY-ESO-1 received autologous TCR-transduced T cells following a lymphodepleting preparative chemotherapy. Response rates using Response Evaluation Criteria in Solid Tumors (RECIST), as well as immunologic correlates of response, are presented in this report., Results: Eleven of 18 patients with NY-ESO-1(+) synovial cell sarcomas (61%) and 11 of 20 patients with NY-ESO-1(+) melanomas (55%) who received autologous T cells transduced with an NY-ESO-1-reactive TCR demonstrated objective clinical responses. The estimated overall 3- and 5-year survival rates for patients with synovial cell sarcoma were 38% and 14%, respectively, whereas the corresponding estimated survival rates for patients with melanoma were both 33%., Conclusions: The adoptive transfer of autologous T cells transduced with a retrovirus encoding a TCR against an HLA-A*0201 restricted NY-ESO-1 epitope can be an effective therapy for some patients bearing synovial cell sarcomas and melanomas that are refractory to other treatments., (©2014 American Association for Cancer Research.)

Purpose: T cells can be genetically modified to express an anti-CD19 chimeric antigen receptor (CAR). We assessed the safety and efficacy of administering autologous anti-CD19 CAR T cells to patients with advanced CD19(+) B-cell malignancies., Patients and Methods: We treated 15 patients with advanced B-cell malignancies. Nine patients had diffuse large B-cell lymphoma (DLBCL), two had indolent lymphomas, and four had chronic lymphocytic leukemia. Patients received a conditioning chemotherapy regimen of cyclophosphamide and fludarabine followed by a single infusion of anti-CD19 CAR T cells., Results: Of 15 patients, eight achieved complete remissions (CRs), four achieved partial remissions, one had stable lymphoma, and two were not evaluable for response. CRs were obtained by four of seven evaluable patients with chemotherapy-refractory DLBCL; three of these four CRs are ongoing, with durations ranging from 9 to 22 months. Acute toxicities including fever, hypotension, delirium, and other neurologic toxicities occurred in some patients after infusion of anti-CD19 CAR T cells; these toxicities resolved within 3 weeks after cell infusion. One patient died suddenly as a result of an unknown cause 16 days after cell infusion. CAR T cells were detected in the blood of patients at peak levels, ranging from nine to 777 CAR-positive T cells/μL., Conclusion: This is the first report to our knowledge of successful treatment of DLBCL with anti-CD19 CAR T cells. These results demonstrate the feasibility and effectiveness of treating chemotherapy-refractory B-cell malignancies with anti-CD19 CAR T cells. The numerous remissions obtained provide strong support for further development of this approach., (© 2014 by American Society of Clinical Oncology.)

Background: Chimeric antigen receptor (CAR) modified T cells targeting CD19 have shown activity in case series of patients with acute and chronic lymphocytic leukaemia and B-cell lymphomas, but feasibility, toxicity, and response rates of consecutively enrolled patients treated with a consistent regimen and assessed on an intention-to-treat basis have not been reported. We aimed to define feasibility, toxicity, maximum tolerated dose, response rate, and biological correlates of response in children and young adults with refractory B-cell malignancies treated with CD19-CAR T cells., Methods: This phase 1, dose-escalation trial consecutively enrolled children and young adults (aged 1-30 years) with relapsed or refractory acute lymphoblastic leukaemia or non-Hodgkin lymphoma. Autologous T cells were engineered via an 11-day manufacturing process to express a CD19-CAR incorporating an anti-CD19 single-chain variable fragment plus TCR zeta and CD28 signalling domains. All patients received fludarabine and cyclophosphamide before a single infusion of CD19-CAR T cells. Using a standard 3 + 3 design to establish the maximum tolerated dose, patients received either 1 × 10(6) CAR-transduced T cells per kg (dose 1), 3 × 10(6) CAR-transduced T cells per kg (dose 2), or the entire CAR T-cell product if sufficient numbers of cells to meet the assigned dose were not generated. After the dose-escalation phase, an expansion cohort was treated at the maximum tolerated dose. The trial is registered with ClinicalTrials.gov, number NCT01593696., Findings: Between July 2, 2012, and June 20, 2014, 21 patients (including eight who had previously undergone allogeneic haematopoietic stem-cell transplantation) were enrolled and infused with CD19-CAR T cells. 19 received the prescribed dose of CD19-CAR T cells, whereas the assigned dose concentration could not be generated for two patients (90% feasible). All patients enrolled were assessed for response. The maximum tolerated dose was defined as 1 × 10(6) CD19-CAR T cells per kg. All toxicities were fully reversible, with the most severe being grade 4 cytokine release syndrome that occurred in three (14%) of 21 patients (95% CI 3·0-36·3). The most common non-haematological grade 3 adverse events were fever (nine [43%] of 21 patients), hypokalaemia (nine [43%] of 21 patients), fever and neutropenia (eight [38%] of 21 patients), and cytokine release syndrome (three [14%) of 21 patients)., Interpretation: CD19-CAR T cell therapy is feasible, safe, and mediates potent anti-leukaemic activity in children and young adults with chemotherapy-resistant B-precursor acute lymphoblastic leukaemia. All toxicities were reversible and prolonged B-cell aplasia did not occur., Funding: National Institutes of Health Intramural funds and St Baldrick's Foundation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Purpose: Cancer immunotherapy with adoptive transfer of tumor-infiltrating lymphocytes (TIL) represents an effective treatment for patients with metastatic melanoma, with the objective regressions in up to 72% of patients in three clinical trials. However, the antigen targets recognized by these effective TILs remain largely unclear., Experimental Design: Melanoma patients 2359 and 2591 both experienced durable complete regressions of metastases ongoing beyond five years following adoptive TIL transfer. Two conventional screening approaches were carried out to identify the antigens recognized by these clinically effective TILs. In addition, a novel approach was developed in this study to identify mutated T-cell antigens by screening a tandem minigene library, which comprised nonsynonymous mutation sequences identified by whole-exome sequencing of autologous tumors., Results: Screening of an autologous melanoma cDNA library using a conventional approach led to the identification of previously undescribed nonmutated targets recognized by TIL 2359 or TIL 2591. In contrast, screening of tandem minigene libraries encoding tumor-specific mutations resulted in the identification of mutated kinesin family member 2C (KIF2C) antigen as a target of TIL 2359, and mutated DNA polymerase alpha subunit B (POLA2) antigen as a target of TIL 2591. Both KIF2C and POLA2 have been found to play important roles in cell proliferation., Conclusions: These findings suggest that the minigene screening approach can facilitate the antigen repertoire analysis of tumor reactive T cells, and lead to the development of new adoptive cell therapies with purified T cells that recognize candidate-mutated antigens derived from genes essential for the carcinogenesis., (©2014 American Association for Cancer Research.)

The ability of IL-2 to expand T cells with maintenance of functional activity has been translated into the first reproducible effective human cancer immunotherapies. The administration of IL-2 can lead to durable, complete, and apparently curative regressions in patients with metastatic melanoma and renal cancer. The growth of large numbers of tumor-infiltrating lymphocytes with in vitro anti-cancer activity in IL-2 has led to the development of cell transfer therapies that are highly effective in patients with melanoma. The genetic modification of T cells with genes encoding αβ TCRs or chimeric Ag receptors and the administration of these cells after expansion in IL-2 have extended effective cell transfer therapy to other cancer types.

Purpose: Metastasis heterogeneity presents a significant obstacle to the development of targeted cancer therapeutics. In this study, we sought to establish from a large series of human melanoma metastases whether there exists a determined pattern in tumor cellular heterogeneity that may guide the development of future targeted immunotherapies., Experimental Design: From a cohort of 1,514 patients with metastatic melanoma, biopsies were procured over a 17-year period from 3,086 metastatic tumors involving various anatomic sites. To allow specific tumor cell profiling, we used established immunohistochemical methods to perform semiquantitative assessment for a panel of prototypic melanocyte differentiation antigens (MDA), including gp100, MART-1, and tyrosinase. To gain insight into the endogenous host immune response against these tumors, we further characterized tumor cell expression of MHC I and MHC II and, also, the concomitant CD4(+) and CD8(+) T-cell infiltrate., Results: Tumor cell profiling for MDA expression demonstrated an anatomic site-specific pattern of antigen expression that was highest in brain, intermediate in soft tissues/lymph nodes, and lowest in visceral metastases. Hierarchical clustering analysis supported that melanoma metastases have a phylogenetically determined, rather than a stochastic, pattern of antigen expression that varies by anatomic site. Furthermore, tyrosinase expression was more frequently lost in metastatic sites outside of the brain and was uniquely correlated with both endogenous CD8(+) and CD4(+) T-cell infiltrates., Conclusion: Site-specific antigen heterogeneity represents a novel attribute for human melanoma metastases that should be considered in future therapy development and when assessing the responsiveness to antigen-specific immunotherapies., (©2014 American Association for Cancer Research.)

Two-dimensional (2D) kinetic analysis directly measures molecular interactions at cell-cell junctions, thereby incorporating inherent cellular effects. By comparison, three-dimensional (3D) analysis probes the intrinsic physical chemistry of interacting molecules isolated from the cell. To understand how T-cell tumor reactivity relates to 2D and 3D binding parameters and to directly compare them, we performed kinetic analyses of a panel of human T-cell receptors (TCRs) interacting with a melanoma self-antigen peptide (gp100209 -217 ) bound to peptide-major histocompatibility complex in the absence and presence of co-receptor CD8. We found that while 3D parameters are inadequate to predict T-cell function, 2D parameters (that do not correlate with their 3D counterparts) show a far broader dynamic range and significantly improved correlation with T-cell function. Thus, our data support the general notion that 2D parameters of TCR-peptide-major histocompatibility complex-CD8 interactions determine T-cell responsiveness and suggest a potential 2D-based strategy to screen TCRs for tumor immunotherapy., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

New treatments are needed for B-cell malignancies persisting after allogeneic hematopoietic stem cell transplantation (alloHSCT). We conducted a clinical trial of allogeneic T cells genetically modified to express a chimeric antigen receptor (CAR) targeting the B-cell antigen CD19. T cells for genetic modification were obtained from each patient's alloHSCT donor. All patients had malignancy that persisted after alloHSCT and standard donor lymphocyte infusions (DLIs). Patients did not receive chemotherapy prior to the CAR T-cell infusions and were not lymphocyte depleted at the time of the infusions. The 10 treated patients received a single infusion of allogeneic anti-CD19-CAR T cells. Three patients had regressions of their malignancies. One patient with chronic lymphocytic leukemia (CLL) obtained an ongoing complete remission after treatment with allogeneic anti-CD19-CAR T cells, another CLL patient had tumor lysis syndrome as his leukemia dramatically regressed, and a patient with mantle cell lymphoma obtained an ongoing partial remission. None of the 10 patients developed graft-versus-host disease (GVHD). Toxicities included transient hypotension and fever. We detected cells containing the anti-CD19-CAR gene in the blood of 8 of 10 patients. These results show for the first time that donor-derived allogeneic anti-CD19-CAR T cells can cause regression of B-cell malignancies resistant to standard DLIs without causing GVHD.

Adoptive cell transfer of tumor-infiltrating lymphocytes (TILs) can mediate cancer regression in patients with metastatic melanoma, but whether this approach can be applied to common epithelial malignancies remains unclear. In this study, we compared the phenotype and function of TILs derived from liver and lung metastases from patients with gastrointestinal (GI) cancers (n = 14) or melanoma (n = 42). Fewer CD3(+) T cells were found to infiltrate GI compared with melanoma metastases, but the proportions of CD8(+) cells, T cell differentiation stage, and expression of costimulatory molecules were similar for both tumor types. Clinical-scale expansion up to ~50 × 10(9) T cells on average was obtained for all patients with GI cancer and melanoma. From GI tumors, however, TIL outgrowth in high-dose IL-2 yielded 22 ± 1.4% CD3(+)CD8(+) cells compared with 63 ± 2.4% from melanoma (p < 0.001). IFN-γ ELISA demonstrated MHC class I-mediated reactivity of TIL against autologous tumor in 5 of 7 GI cancer patients tested (9% of 188 distinct TIL cultures) and in 9 of 10 melanoma patients (43% of 246 distinct TIL cultures). In these assays, MHC class I-mediated up-regulation of CD137 (4-1BB) expression on CD8(+) cells suggested that 0-3% of TILs expanded from GI cancer metastases were tumor-reactive. This study implies that the main challenge to the development of TIL adoptive cell transfer for metastatic GI cancers may not be the in vitro expansion of bulk TILs, but the ability to select and enrich for tumor-reactive T cells.

Despite significant progress in the development of adoptive cell-transfer therapies (ACTs) using gene-engineered T cells, little is known about the fate of cells following infusion. To address that, we performed a comparative analysis of gene expression between T-cell receptor-engineered lymphocytes persisting in the circulation 1 month after administration and the product that was infused. We observed that 156 genes related to immune function were differentially expressed, including underexpression of stimulators of lymphocyte function and overexpression of inhibitory genes in postinfusion cells. Of genes overexpressed postinfusion, the product of programmed cell death 1 (PDCD1), coinhibitory receptor PD-1, was expressed at a higher percentage in postinfusion lymphocytes than in the infusion product. This was associated with a higher sensitivity to inhibition of cytokine production by interaction with its ligand PD-L1. Coinhibitory receptor CD160 was also overexpressed in persisting cells, and its expression was associated with decreased reactivity, which surprisingly was found to be ligand-independent. These results contribute to a deeper understanding of the properties of transgenic lymphocytes used to treat human malignancies and may provide a rationale for the development of combination therapies as a method to improve ACT.

T cell receptors (TCRs) on T cells recognize peptide-major histocompatibility complex (pMHC) molecules on the surface of antigen presenting cells and this interaction determines the T cell immune response. Due to negative selection, naturally occurring TCRs bind self (tumor) peptides with low affinity and have a much higher affinity for foreign antigens. This complicates isolation of naturally occurring, high affinity TCRs that mediate more effective tumor rejection for therapeutic purposes. An attractive approach to resolve this issue is to engineer high affinity TCRs in vitro using phage, yeast or mammalian TCR display systems. A caveat of these systems is that they rely on a large library by random mutagenesis due to the lack of knowledge regarding the specific interactions between the TCR and pMHC. We have focused on the mammalian retroviral display system because it uniquely allows for direct comparison of TCR-pMHC-binding properties with T-cell activation outcomes. Through an alanine-scanning approach, we are able to quickly map the key amino acid residues directly involved in TCR-pMHC interactions thereby significantly reducing the library size. Using this method, we demonstrate that for a self-antigen-specific human TCR (R6C12) the key residues for pMHC binding are located in the CDR3β region. This information was used as a basis for designing an efficacious TCR CDR3α library that allowed for selection of TCRs with higher avidity than the wild-type as evaluated through binding and activation experiments. This is a direct approach to target specific TCR residues in TCR library design to efficiently engineer high avidity TCRs that may potentially be used to enhance adoptive immunotherapy treatments., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Most systemic cancer therapies target tumor cells directly, although there is increasing interest in targeting the tumor stroma that can comprise a substantial portion of the tumor mass. We report here a synergy between two T-cell therapies, one directed against the stromal tumor vasculature and the other directed against antigens expressed on the tumor cell. Simultaneous transfer of genetically engineered syngeneic T cells expressing a chimeric antigen receptor targeting the VEGF receptor-2 (VEGFR2; KDR) that is overexpressed on tumor vasculature and T-cells specific for the tumor antigens gp100 (PMEL), TRP-1 (TYRP1), or TRP-2 (DCT) synergistically eradicated established B16 melanoma tumors in mice and dramatically increased the tumor-free survival of mice compared with treatment with either cell type alone or T cells coexpressing these two targeting molecules. Host lymphodepletion before cell transfer was required to mediate the antitumor effect. The synergistic antitumor response was accompanied by a significant increase in the infiltration and expansion and/or persistence of the adoptively transferred tumor antigen-specific T cells in the tumor microenvironment and thus enhanced their antitumor potency. The data presented here emphasize the possible beneficial effects of combining antiangiogenic with tumor-specific immunotherapeutic approaches for the treatment of patients with cancer., (©2013 AACR.)

Purpose: Myeloid-derived suppressor cells (MDSC) have emerged as an immune-regulatory cell type that is expanded in tumor-bearing mice, but less is known about their immune-suppressive role in patients with cancer., Experimental Design: To study the importance of MDSC in patients with melanoma, we characterized the frequency, phenotype, and suppressive function of blood myeloid-derived cells and tumor-infiltrating myeloid cells in 26 freshly resected melanomas., Results: Blood and tumor-infiltrating myeloid cells (Lin(-) CD11b(+)) could be phenotypically and morphologically classified into monocytes/macrophages, neutrophils, eosinophils, and immature myeloid cells according to marker expression (CD14(+), CD14(-) CD15(hi), CD14(-) CD15(int), and CD14(-) CD15(-), respectively). In contrast to the expansion of MDSC reported in tumor-bearing mice, we found no differences in the frequency and phenotype of myeloid subsets in the blood of patients with melanoma compared with healthy donors. Myeloid cells represented 12% of the live cells in the melanoma cell suspensions, and were phenotypically diverse with high tumor-to-tumor variability. Interestingly, a positive association was found between the percentage of Tregs and granulocytic cells (Lin(-) CD11b(+) CD14(-)CD15(+)) infiltrating melanoma tumors. However, melanoma-infiltrating myeloid cells displayed impaired suppression of nonspecific T-cell proliferation compared with peripheral blood myeloid cells, in which monocytes and eosinophils were suppressive., Conclusions: Our findings provide a first characterization of the nature and suppressive function of the melanoma myeloid infiltrate and indicate that the suppressive function of MDSC in patients with melanoma seems far less than that based on murine tumor models.

No curative treatment exists for glioblastoma, with median survival times of less than 2 years from diagnosis. As an approach to develop immune-based therapies for glioblastoma, we sought to target antigens expressed in glioma stem cells (GSCs). GSCs have multiple properties that make them significantly more representative of glioma tumors than established glioma cell lines. Epidermal growth factor receptor variant III (EGFRvIII) is the result of a novel tumor-specific gene rearrangement that produces a unique protein expressed in approximately 30% of gliomas, and is an ideal target for immunotherapy. Using PCR primers spanning the EGFRvIII-specific deletion, we found that this tumor-specific gene is expressed in three of three GCS lines. Based on the sequence information of seven EGFRvIII-specific monoclonal antibodies (mAbs), we assembled chimeric antigen receptors (CARs) and evaluated the ability of CAR-engineered T cells to recognize EGFRvIII. Three of these anti-EGFRvIII CAR-engineered T cells produced the effector cytokine, interferon-γ, and lysed antigen-expressing target cells. We concentrated development on a CAR produced from human mAb 139, which specifically recognized GSC lines and glioma cell lines expressing mutant EGFRvIII, but not wild-type EGFR and did not recognize any normal human cell tested. Using the 139-based CAR, T cells from glioblastoma patients could be genetically engineered to recognize EGFRvIII-expressing tumors and could be expanded ex vivo to large numbers, and maintained their antitumor activity. Based on these observations, a γ-retroviral vector expressing this EGFRvIII CAR was produced for clinical application.

We conducted a clinical trial to assess adoptive transfer of T cells genetically modified to express an anti-CD19 chimeric Ag receptor (CAR). Our clinical protocol consisted of chemotherapy followed by an infusion of anti-CD19-CAR-transduced T cells and a course of IL-2. Six of the 8 patients treated on our protocol obtained remissions of their advanced, progressive B-cell malignancies. Four of the 8 patients treated on the protocol had long-term depletion of normal polyclonal CD19(+) B-lineage cells. Cells containing the anti-CD19 CAR gene were detected in the blood of all patients. Four of the 8 treated patients had prominent elevations in serum levels of the inflammatory cytokines IFNγ and TNF. The severity of acute toxicities experienced by the patients correlated with serum IFNγ and TNF levels. The infused anti-CD19-CAR-transduced T cells were a possible source of these inflammatory cytokines because we demonstrated peripheral blood T cells that produced TNF and IFNγ ex vivo in a CD19-specific manner after anti-CD19-CAR-transduced T-cell infusions. Anti-CD19-CAR-transduced T cells have great promise to improve the treatment of B-cell malignancies because of a potent ability to eradicate CD19(+) cells in vivo; however, reversible cytokine-associated toxicities occurred after CAR-transduced T-cell infusions.

Immunotherapy based on the adoptive transfer of naturally occurring or gene-engineered T cells can mediate tumour regression in patients with metastatic cancer. Here, we discuss progress in the use of adoptively transferred T cells, focusing on how they can mediate tumour cell eradication. Recent advances include more accurate targeting of antigens expressed by tumours and the associated vasculature, and the successful use of gene engineering to re-target T cells before their transfer into the patient. We also describe how new research has helped to identify the particular T cell subsets that can most effectively promote tumour eradication.

Purpose: We investigated the feasibility of delivering the proinflammatory cytokine interleukin (IL)-12 into tumor using T cells genetically engineered to express a chimeric antigen receptor (CAR) against the VEGF receptor-2 (VEGFR-2)., Experimental Design: Two different strains of mice bearing five different established subcutaneous tumors were treated with syngeneic T cells cotransduced with an anti-VEGFR-2 CAR and a constitutively expressed single-chain murine IL-12 or an inducible IL-12 gene after host lymphodepletion. Tumor regression, survival of mice, and persistence of the transferred cells were evaluated., Results: Adoptive transfer of syngeneic T cells cotransduced with an anti-VEGFR-2 CAR and a constitutively expressing single-chain IL-12 resulted in the regression of five different established tumors of different histologies without the need for IL-2 administration. T cells transduced with either anti-VEGFR-2 CAR or single-chain IL-12 alone did not alter the tumor growth indicating that both of them had to be expressed in the same cell to mediate tumor regression. Anti-VEGFR-2 CAR and IL-12-cotransduced T cells infiltrated the tumors, expanded, and persisted for prolonged periods. The antitumor effect did not require the presence of host T and B cells but was dependent on host IL-12R-expressing cells. The anti-VEGFR-2 CAR changed the immunosuppressive tumor environment by altering/reducing both the systemic and the intratumoral CD11b(+)Gr1(+) myeloid suppressor cell subsets that expressed VEGFR-2., Conclusions: These results suggest that targeted delivery of IL-12 into the tumor environment with T cells redirected against VEGFR-2 is a promising approach for treating patients with a variety of solid tumor types.

Administration of ex vivo cultured, naturally occurring tumor-infiltrating lymphocytes (TILs) has been shown to mediate durable regression of melanoma tumors. However, the generation of TILs is not possible in all patients and there has been limited success in generating TIL in other cancers. Advances in genetic engineering have overcome these limitations by introducing tumor-antigen-targeting receptors into human T lymphocytes. Physicians can now genetically engineer lymphocytes to express highly active T-cell receptors (TCRs) or chimeric antigen receptors (CARs) targeting a variety of tumor antigens expressed in cancer patients. In this review, we discuss the development of TCR and CAR gene transfer technology and the expansion of these therapies into different cancers with the recent demonstration of the clinical efficacy of these treatments., (Published by Elsevier Ltd.)

Cancer immunotherapy using the adoptive transfer of autologous tumor-infiltrating lymphocytes results in objective cancer regression in 49-72% of patients with metastatic melanoma. In a pilot trial combining cell transfer with a maximum lymphodepleting regimen, complete durable responses were seen in 40% of patients, with complete responses ongoing beyond 3 to 7 years. Current approaches to cell transfer therapy using autologous cells genetically engineered to express conventional or chimeric T-cell receptors have mediated cancer regression in patients with metastatic melanoma, synovial sarcoma, neuroblastoma and refractory lymphoma. Adoptive cell transfer immunotherapy is a rapidly developing new approach to the therapy of metastatic cancer in humans. This Review will emphasize the current available applications of cell transfer immunotherapy for patients with cancer.

In addition to the direct killing of tumor cells, radiation therapy can alter the balance of immune cells in vivo due to the differential radiosensitivity of different cell types. The addition of adjuvant radiation therapy before adoptive cell transfer therapy has been shown to enhance antitumor responses in both mouse models and clinical trials. This study examines the effects of in vitro irradiation on the phenotype and function of human antigen-presenting cells. The results indicated that irradiation upregulated CD70 expression on both B cells and mature dendritic cells (DCs). Expression of CD70 on mature DCs was enhanced in a dose-dependent manner, whereas under the same conditions, no significant upregulation of CD80, CD86, or CD40 was observed. The levels of expression of CD70 induced on mature DC by irradiation correlated highly with the ability of those cells to stimulate T-cell proliferation and interferon-γ production. Furthermore, significant reductions in T-cell proliferation and interferon-γ production were seen when CD70 expression on DCs was partially reduced using shRNA, as well as when DCs were incubated with a blocking anti-CD70 antibody. Radiation therapy may therefore enhance T-cell activation in vivo through the CD27 pathway by virtue of its ability to upregulate the expression of CD70 on antigen-presenting cells.

T cells that are genetically modified to express single-chain chimeric antigen receptors (CAR) have shown promise in early cancer immunotherapy clinical trials. Unfortunately, 2 recent deaths in cancer patients treated with CAR T cells have created some uncertainty on how to best mitigate patient risk, while continuing to advance this very promising therapeutic avenue. In order to address these concerns, the Recombinant DNA Advisory Committee (RAC) held a symposium, the objectives of which were to first review the reported treatment-associated toxicities and, second, to discuss methods for improving safety and efficacy. This report highlights the issues raised as part of this discussion, with a specific focus on protocols infusing CAR T cells. Because this was not a consensus conference, the opinions described should not be construed to represent those of any individual RAC member, the RAC as a body, conference participants, the National Institutes of Health, or the U.S. Food and Drug Administration.