Adoptive Transfer Of Tumor-infiltrating Lymphocytes Research Articles

Abstract LB073: Adoptive transfer of one CD4TCR and one CD8TCR targeting autochthonous neoantigens are essential and sufficient for eradication of naturally heterogeneous solid tumors

Abstract Despite advances in checkpoint blockade therapy or adoptive transfer of tumor-infiltrating lymphocytes (TILs), achieving cure remains challenging, but success of immunotherapy seems to depend on recognition of tumor-specific antigens (neoantigens). We aimed to determine how many neoantigens need to be recognized by how many different TCRs for eradication of solid tumors. Unmanipulated, naturally expressed (autochthonous) neoantigens were targeted with adoptively transferred TCR-engineered autologous T cells (TCR-therapy). Investigated were effects of TCR-engineered CD8+ T cells, TCR-engineered CD4+ T cells or a combination of both. The targeted tumors were established for at least three weeks and derived from primary autochthonous cancer cell cultures, resembling natural solid tumors and their heterogeneity as found in humans. CD8TCR-therapy was only effective against homogenous tumors and relapse was common in heterogeneous tumors, even when targeting multiple different autochthonous neoantigens. By contrast, a combination of CD8TCR-therapy with CD4TCR-therapy, each targeting an independent neoantigen, eradicated large and established solid tumors of natural heterogeneity. CD4TCR-therapy targeted a mutant neoantigen on tumor stroma while direct cancer cell recognition by CD8TCR-therapy was essential for cure. Thus, two cancer-specific TCRs can be essential and sufficient to eradicate heterogeneous solid tumors expressing unmanipulated, autochthonous neoantigens. Citation Format: Steven Wolf, Vasiliki Anastasopoulou, Kimberley Drousch, Markus Diehl, Boris Engels, Poh Yin Yew, Kazuma Kiyotani, Yusuke Nakamura, Karin Schreiber, Hans Schreiber, Matthias Leisegang. Adoptive transfer of one CD4TCR and one CD8TCR targeting autochthonous neoantigens are essential and sufficient for eradication of naturally heterogeneous solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB073.

Abstract 15: Massively parallel base-editing screens to map variant effects on anti-tumor hallmarks of primary human T cells and improve cell-based cancer immunotherapies

Abstract Base editing enables precise generation of single nucleotide variants, but large-scale screening in primary human T cells has been limited by low editing efficiency. Here, we developed a high-throughput approach for highly efficient, massively parallel adenine and cytosine base-editor screening in primary human T cells. We performed multiple large-scale pooled screens editing 108 genes with central functions in T cells and whole-gene tiling mutagenesis of selected genes. All screens were functionally read out to capture the effect of variants on hallmarks of T cell anti-tumor immunity, including activation, proliferation, long-term persistence, and cytokine production. We discovered a broad landscape of gain-of-function and loss-of-function mutations and characterized their impact across multiple axes of T cell functions. Among others, we identified gain-of-function mutations in PIK3CD and its regulatory subunit PIK3R1, LCK, AKT1, and loss-of-function mutants of CTLA-4, driving increased T cell polyfunctionality. We validated several of these mutations by probing downstream signaling and functional outputs. To demonstrate the feasibility of using such screen results for improving cell-based cancer immunotherapies, we used human T cells with engineered T cell receptors recognizing specific epitopes in melanoma models. We base-edited these T cells with mutations identified in our screens and demonstrate that these mutations indeed significantly enhance cytotoxic cytokine production and tumor-lytic capacity with minimal off-target activity. Lastly, we also provide proof-of-feasibility for high-efficiency combinatorial base-editing in T cells. This study presents a framework for large-scale base editing screens with multi-dimensional functional readouts in cells that are typically difficult to molecularly engineer in a precise manner. This work also has important therapeutic implications: T cells are the substrate for clinically active cell-based therapies, such as adoptive transfer of tumor-infiltrating lymphocytes (TILs) or chimeric-receptor antigen T (CAR-T) cells. Our screen-informed base editing to achieve an improved T cell product against melanoma exemplifies the therapeutic power of generating synthetic protein variants that improve a wide range of existing and future T cell-based cancer immunotherapies. Citation Format: Zachary Hudson Walsh, Parin Shah, Neeha Kothapalli, Gergo Nikolenyi, Shivem Shah, Giuseppe Leuzzi, Neil Vasan, Mohammed AlQuarishi, Alberto Ciccia, Johannes Melms, Benjamin Izar. Massively parallel base-editing screens to map variant effects on anti-tumor hallmarks of primary human T cells and improve cell-based cancer immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 15.

Engineered Tcell therapy for viral and non-viral epithelial cancers.

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

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

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) fails to consistently elicit tumor rejection. Manipulation of intrinsic factors that inhibit Tcell 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 Tcell functional avidity in mice. Here, we investigate the mechanistic role of CISH in regulating human Tcell effector function in solid tumors and demonstrate that CRISPR/Cas9 disruption of CISH enhances TIL neoantigen recognition and response to checkpoint blockade. 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 Tcells and TIL. Tumor-specific Tcells with disrupted Cish function were adoptively transferred into tumor-bearing mice and evaluated for efficacy with or without checkpoint blockade. CISH expression was associated with Tcell dysfunction. CISH deletion using CRISPR/Cas9 resulted in hyper-activation and improved functional avidity against tumor-derived neoantigens without perturbing Tcell maturation. Cish knockout resulted in increased susceptibility to checkpoint blockade invivo. CISH negatively regulates human Tcell effector function, and its genetic disruption offers a novel avenue to improve the therapeutic efficacy of adoptive TIL therapy. This study was funded by Intima Bioscience, U.S. and in part through the Intramural program CCR at the National Cancer Institute.

TIL expansion with high dose IL-2 or low dose IL-2 with anti-CD3/anti-CD28 stimulation provides different quality of TIL-expanded T cell clones

Tumor infiltrating lymphocytes (TILs) are cells that are present inside the tumor environment, of which include T cells, B cells and natural killer (NK) cells. At present, TILs are used for immunotherapy in various cancers. Knowledge on adoptive transfer of TILs in ovarian cancer is still limited, especially regarding TIL expansion methods. Therefore, the aim of our study was to compare the quality of T cell clones between two expansion methods for ovarian cancer TILs. We show that TILs stimulated with the mitogenic stimulation method (low dose IL-2 with anti-human CD3/CD28) and the standard stimulation method (high dose IL-2 only) both increased total number of T cells. TCR repertoire analyses revealed different TCR repertoire patterns between TIL-expanded T cells that were stimulated with the standard stimulation method (high dose IL-2 only) and the mitogenic stimulation method (low dose IL-2 with anti-human CD3/CD28). Regardless, when TILs were expanded using the standard stimulation method (high dose IL-2 only), the predominant T cell receptor beta variable (TRBV) chains that were used in both TIL-expanded clones of the CD4+ and CD8+ subpopulations were similar. In addition, there were also TIL-expanded CD4+ and CD8+ T cell clones that were dominant in only one or the other subpopulations. These results reveal the bias in TIL quality after being stimulated with different protocols. Further studies are required to understand the selection of TIL expansion, in order for a more efficacy adoptive transfer treatment.

Adoptive Transfer of Tumor-Infiltrating Lymphocytes in Metastatic Melanoma

Adoptive Transfer of Tumor-Infiltrating Lymphocytes in Metastatic Melanoma

Impact of Prior Treatment on the Efficacy of Adoptive Transfer of Tumor-Infiltrating Lymphocytes in Patients with Metastatic Melanoma.

Adoptive cell transfer (ACT) of autologous tumor-infiltrating lymphocytes (TIL) can mediate durable responses in patients with metastatic melanoma. This retrospective analysis provides long-term follow-up and describes the effect of prior therapy on outcomes after ACT-TIL. Patients with metastatic melanoma underwent surgical resection of a tumor for generation of TILs and were treated with a lymphodepleting preparative regimen followed by adoptive transfer of TILs and intravenous IL2. Clinical characteristics of enrolled patients and treatment characteristics of TIL infusion products over two decades of ACT were analyzed to identify predictors of objective response. Adoptive transfer of TILs mediated an objective response rate of 56% (108/192) and median melanoma-specific survival of 28.5 months in patients naïve to anti-programmed cell death-1 (PD-1) therapy compared with 24% (8/34) and 11.6 months in patients refractory to anti-PD-1 (aPD-1). Among patients with BRAF V600E/K-mutated disease, prior treatment with targeted molecular therapy was also associated with a decreased response rate (21% vs. 60%) and decreased survival (9.3 vs. 50.7 months) when compared with those patients naïve to targeted therapy. With a median potential follow-up of 89 months, 46 of 48 complete responders in the aPD-1-naïve cohort have ongoing responses after a single treatment and 10-year melanoma-specific survival of 96%. Patients previously treated with PD-1 or MAPK inhibition are significantly less likely to develop durable objective responses to ACT-TIL. While ACT-TIL is currently being investigated for treatment-refractory patients, it should also be considered as an initial treatment option for eligible patients with metastatic melanoma. See related commentary by Sznol, p. 5156.

Abstract 127: Single cell mapping of tumor infiltrating lymphocytes enables neoantigen-reactive T cell identification in metastatic human cancer

Abstract The adoptive transfer of tumor-infiltrating lymphocytes (TILs) expanded in vitro can mediate regressions of established tumors, yet only a minority of intratumoral T cells are reactive to cancer antigens while the majority represent bystander cells. This complexity underpins the need to phenotypically characterize T cells infiltrating the tumor microenvironment. In this study, we performed single-cell RNA and T cell receptor (TCR) sequencing (scRNA/TCR-seq) on over 45,000 T cells isolated from ten archival metastatic tumor samples whose primary histologies included colon, rectal, breast, and melanoma. By transcriptomic clustering of the T cells and mapping TCR identity data onto these blueprints, we observed a diversity of T cell phenotypes spanning from memory-like to dysfunctional effector-like, with clonal expansion occurring more widely in the effector-like cells. Through sequencing of peripheral blood lymphocytes (PBL) obtained at the time of tumor resection, we found T cell clusters that were either tumor enriched or distinctly PBL enriched, potentially representing recent tumor immigrants. Prior knowledge of 30 CD4 and CD8 tumor neoantigen-reactive TCRs (NeoTCRs) from eight patients allowed us to project these NeoTCRs onto the TIL transcriptomic map where we observed 380 total T cells bearing these NeoTCRs. The majority (>85%) of NeoTCRs were expressed by T cells within 2 clusters, one CD4+ and one CD8+, marked by expression of CXCL13, ENTPD1 (CD39), TOX, and PDCD1 (PD-1), indicating a dysfunctional state. We reasoned that other dominant T cell clonotypes within the NeoTCR clusters could contain additional novel NeoTCR clonotypes. We expressed these predicted NeoTCRs in donor PBL and screened them in vitro with antigen presenting cells (APCs) pulsed with peptides or tandem minigenes encoded by patient-specific tumor mutations, yielding 24 new NeoTCR clones (251 cells), which included neoantigen reactivities undetected in the same patients' fragment TIL screens. Using a NeoTCR signature score derived from the archival samples, three prospective patients' TIL were analyzed by scRNA/TCRseq and clusters scored according to their NeoTCR signature. TCRs reconstructed from these clusters and tested against patients' tumor-specific reagents yielded novel CD4 and CD8 NeoTCRs. Altogether, this study enabled successful enrichment and detection of tumor-specific NeoTCRs in the sequenced TIL of 12/12 patients for whom reactivity was either previously assessed or prospectively identified. Deconvolution of NeoTCRs from bystander TCRs within the tumor-immune microenvironment represents an important step in the development of personalized immunotherapy, as prospective NeoTCR isolation based on transcriptional phenotype of TIL will allow for rapid therapeutic development in the form of T cells expressing these tumor-specific TCRs. Citation Format: Frank J. Lowery, Sri Krishna, Rami Yossef, Praveen D. Chatani, Neilesh B. Parikh, Yong-Chen Lu, Nikolaos Zacharakis, Noam Levin, Ken-ichi Hanada, James C. Yang, Paul F. Robbins, Maria R. Parkhurst, Steven A. Rosenberg. Single cell mapping of tumor infiltrating lymphocytes enables neoantigen-reactive T cell identification in metastatic human cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 127.

Naturally Occurring Genetic Alterations in Proximal TCR Signaling and Implications for Cancer Immunotherapy.

T cell-based immunotherapies including genetically engineered T cells, adoptive transfer of tumor-infiltrating lymphocytes, and immune checkpoint blockade highlight the impressive anti-tumor effects of T cells. These successes have provided new hope to many cancer patients with otherwise poor prognoses. However, only a fraction of patients demonstrates durable responses to these forms of therapies and many develop significant immune-mediated toxicity. These heterogeneous clinical responses suggest that underlying nuances in T cell genetics, phenotypes, and activation states likely modulate the therapeutic impact of these approaches. To better characterize known genetic variations that may impact T cell function, we 1) review the function of early T cell receptor-specific signaling mediators, 2) offer a synopsis of known mutations and genetic alterations within the associated molecules, 3) discuss the link between these mutations and human disease and 4) review therapeutic strategies under development or in clinical testing that target each of these molecules for enhancing anti-tumor T cell activity. Finally, we discuss novel engineering approaches that could be designed based on our understanding of the function of these molecules in health and disease.

Randomized phase II trial of lymphodepletion plus adoptive cell transfer of tumor-infiltrating lymphocytes, with or without dendritic cell vaccination, in patients with metastatic melanoma

BackgroundThe adoptive transfer of tumor-infiltrating lymphocytes (TIL) has demonstrated robust efficacy in metastatic melanoma patients. Tumor antigen–loaded dendritic cells (DCs) are believed to optimally activate antigen-specific T lymphocytes. We hypothesized...

Cell therapies in ovarian cancer.

Epithelial ovarian cancer (EOC) is the most important cause of gynecological cancer-related mortality. Despite improvements in medical therapies, particularly with the incorporation of drugs targeting homologous recombination deficiency, EOC survival rates remain low. Adoptive cell therapy (ACT) is a personalized form of immunotherapy in which autologous lymphocytes are expanded, manipulated ex vivo, and re-infused into patients to mediate cancer rejection. This highly promising novel approach with curative potential encompasses multiple strategies, including the adoptive transfer of tumor-infiltrating lymphocytes, natural killer cells, or engineered immune components such as chimeric antigen receptor (CAR) constructs and engineered T-cell receptors. Technical advances in genomics and immuno-engineering have made possible neoantigen-based ACT strategies, as well as CAR-T cells with increased cell persistence and intratumoral trafficking, which have the potential to broaden the opportunity for patients with EOC. Furthermore, dendritic cell-based immunotherapies have been tested in patients with EOC with modest but encouraging results, while the combination of DC-based vaccination as a priming modality for other cancer therapies has shown encouraging results. In this manuscript, we provide a clinically oriented historical overview of various forms of cell therapies for the treatment of EOC, with an emphasis on T-cell therapy.

Adoptive Cell Therapy in Breast Cancer: A Current Perspective of Next-Generation Medicine.

Immunotherapy has become a cornerstone in the treatment of cancer and changed the way clinicians and researchers approach tumor vulnerabilities. Durable responses are commonly observed with immune checkpoint inhibitors in highly immunogenic tumors, while the infusion of T cells genetically engineered to express chimeric antigen receptors (CARs) has shown impressive efficacy in certain types of blood cancer. Nevertheless, harnessing our own immunity has not proved successful for most breast cancer patients. In the era of genomic medicine, cellular immunotherapies may provide a more personalized and dynamic tool against tumors displaying heterogeneous mutational landscapes and antigenic pools. This approach encompasses multiple strategies including the adoptive transfer of tumor-infiltrating lymphocytes, dendritic cells, natural killer cells, and engineered immune components such as CAR constructs and engineered T cell receptors. Although far from permeating the clinical setting, technical advances have been overwhelming in recent years, with continuous improvement in traditional challenges such as toxicity, adoptive cell persistence, and intratumoral trafficking. Also, there is an avid search for neoantigens that can be targeted by these strategies, either alone or in combination. In this work, we aim to provide a clinically-oriented overview of preclinical and clinical data regarding the use of cellular immunotherapies in breast cancer.

Tumor-infiltrating lymphocytes from human prostate tumors reveal anti-tumor reactivity and potential for adoptive cell therapy

ABSTRACTAdvanced prostate cancer remains incurable and is the second leading cause of mortality in men. Immunotherapy based on the adoptive transfer of tumor-infiltrating lymphocytes (TIL) has demonstrated promising clinical results in patients with metastatic melanoma and lately also in other solid tumors. However, the ability to obtain TIL from patients with prostate cancer, considered poorly immunogenic, remains unknown. In this study, we investigate the feasibility of isolating and expanding TIL from primary prostate tumors. We collected tumor specimens from eight patients with diagnosed prostate adenocarcinoma undergoing radical prostatectomy and were able to successfully expand multiple autologous TIL cultures from all patients. Twenty-eight prostate-TIL cultures were further expanded using a standard rapid expansion procedure under Good Manufacturing Practice conditions. TIL cultures were phenotypically characterized for T cell subset composition, differentiation status and co-inhibitory/stimulatory markers such as PD-1, TIM-3, LAG-3, and CD28 and were found to have in general similarity to TIL obtained from patients with melanoma and lung carcinoma previously treated at our center. All analyzed TIL cultures were functional as determined by the capability to produce high level of IFNγ upon stimuli. Most importantly, co-culture assays of prostate-TIL with autologous tumors demonstrated anti-tumor reactivity.In conclusion, these findings demonstrate that functional and anti-tumor reactive TIL can be obtained, despite the immunosuppressive microenvironment of the cancer, thus this study supports the development of TIL therapy for prostate cancer patients.

Tissue Harvesting for Adoptive Tumor Infiltrating Lymphocyte Therapy in Metastatic Melanoma

Adoptive transfer of tumor-infiltrating lymphocytes (TILs) combined with non-myeloablative chemotherapy (NMA) has been shown to prolong survival in patients with metastatic disease. Tissue harvesting was performed form a variety of sites. TILs were isolated, expanded and infused with bolus high-dose IL-2. Between 2008 and 2018, 242 lesions were resected for TILs harvesting from a range of sites form 196 patients without mortality and with minimal morbidity. Of those harvested, 75 were unable to complete therapy because of clinical deterioration during the wait period. Of 121 evaluable treated patients, there was no effect of metastatic site biopsied on the mean fold TIL expansion. Those receiving prior ipilimumab had a higher TIL fold expansion but a lower TIL fold expansion than those exposed to anti-PD1 therapy. Harvesting may be safely performed with successful TIL expansion from most sites. Prior check point inhibitory immunotherapy may potentially influence TIL fold expansion.

Adoptive transfer of tumor-infiltrating lymphocytes in patients with sarcomas, ovarian, and pancreatic cancers.

TPS2650 Background: Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TIL) has a long history of efficacy in metastatic melanoma, and is being increasingly considered across other solid tumors. Preclinical data generated at MD Anderson Cancer Center has demonstrated the ability to grow TIL from a variety of tumor types including various types of sarcomas, ovarian and pancreas cancers. We are testing the efficacy of TIL across multiple tumor types using two different manufacturing protocols. Methods: We are conducting two ongoing investigator initiated basket TIL therapy trials. The first (NCT03449108) includes cohorts with poorly differentiated soft tissue and bone sarcomas, osteosarcoma, and platinum resistant ovarian cancer. The TIL product used in this trial is an investigational cell product (LN-145, Iovance Biotherapeutics, Inc.). The second trial (NCT03610490) includes cohorts of osteosarcoma, platinum resistant ovarian cancer, and pancreatic cancer (who have progressed on, or received maximal benefit from, front-line therapy). For this trial, TIL are manufactured at MD Anderson Cancer Center using a protocol that includes the use of urelumab (an agonistic anti-CD137 antibody) combined with T cell receptor activation during TIL expansion. In both trials eligible subjects undergo tumor harvest using a surgical excisional biopsy of the tumor for TIL manufacturing, receive a modified cyclophosphamide and fludarabaine lymphodepletion regimen and up to six doses of IL-2 (600,000 IU/kg) following TIL infusion. No intervening therapy is allowed between tumor harvest and initiation of lymphodepletion. The primary endpoint for each cohort is ORR as assessed by investigators using RECIST 1.1 criteria. The Simon’s two stage design is used to monitor the efficacy of each cohort independently. In the first stage, 10 patients will be treated per cohort. If there is no confirmed response in these 10 evaluable patients, the cohort will be terminated. If the cohort moves forward to Stage II, an additional 8 patients will be treated leading to a total of 18 patients. Three or more responders out of 18 treated patients for the cohort will be considered clinically relevant to justify further investigation. Enrollment is ongoing in all cohorts in both trials. An accrual update will be provided at the annual meeting. Clinical trial information: NCT03449108, NCT03610490.

BRAF Inhibition: Bridge or Boost to T-cell Therapy?

The concept of treatment-refractory disease has evolved as checkpoint modulation has changed the therapeutic landscape for patients with metastatic melanoma. Developing meaningful salvage strategies will involve the exploration of combination therapies and new immunotherapeutics, including adoptive transfer of tumor-infiltrating lymphocytes or other T-cell-based therapy.See related article by Atay et al., p. 2783.

Combined Expression of Genetic Adjuvants Via mRNA Electroporation Exerts Multiple Immunostimulatory Effects on Antitumor T Cells.

Adoptive transfer of tumor-infiltrating lymphocytes (TILs) or gene-modified T cells expressing antitumor TCRs or chimeric antigen receptors often yields a high rate of clinical response in several types of cancer. New approaches for enhancing the functional properties of antitumor T cells could improve the clinical outcome of these treatments. To this end, we created 3 classes of genes, each designed to operate autonomously upon expression in T cells. We recently reported on the enhancing effects of constitutively active toll-like receptor 4 (caTLR4), membrane (mem) interleukin-2, memIL-12, and memIL-15, and self-oligomerizing, constitutively active CD40 (caCD40). Here, we evaluated their combined effects on peripheral blood CD8 T cells and different antimelanoma TIL cultures following mRNA electroporation. Expression in CD8 T cells induced transient production of interferon-γ and prolonged and robust upregulation of CD25, CD69, 4-1BB, and OX40. The adjuvants enhanced cytolytic activity of TILs and production of interferon-γ and TNF-α in the presence of autologous, but not mismatched, melanoma for at least 3 days after electroporation. Expression of the 3 adjuvants in young TILs from different patients markedly increased the expression of CD25, OX40, 4-1BB, CD127, and CD28 and exhibited cooperative and, at times, synergistic effects. Furthermore, predefined mixtures of mRNA encoding these adjuvants markedly enhanced the specific antitumor response of selected TILs and killing of autologous melanoma cells by young TILs. Our findings suggest that combinations of these new genetic adjuvants can substantially improve the functional properties of antitumor T cells, offering a new tool of unique versatility in adoptive cell therapy.

Tumor-infiltrating BRAFV600E-specific CD4+ T cells correlated with complete clinical response in melanoma.

T cells specific for neoantigens encoded by mutated genes in cancers are increasingly recognized as mediators of tumor destruction after immune checkpoint inhibitor therapy or adoptive cell transfer. Unfortunately, most neoantigens result from random mutations and are patient specific, and some cancers contain few mutations to serve as potential antigens. We describe a patient with stage IV acral melanoma who achieved a complete response following adoptive transfer of tumor-infiltrating lymphocytes (TILs). Tumor exome sequencing surprisingly revealed fewer than 30 nonsynonymous somatic mutations, including oncogenic BRAFV600E. Analysis of the specificity of TILs identified rare CD4+ T cells specific for BRAFV600E and diverse CD8+ T cells reactive to nonmutated self-antigens. These specificities increased in blood after TIL transfer and persisted long-term, suggesting they contributed to the effective antitumor immune response. Gene transfer of the BRAFV600E-specific T cell receptor (TCR) conferred recognition of class II MHC-positive cells expressing the BRAF mutation. Therapy with TCR-engineered BRAFV600E-specific CD4+ T cells may have direct antitumor effects and augment CD8+ T cell responses to self- and/or mutated tumor antigens in patients with BRAF-mutated cancers.

Abstract B12: Immune checkpoint blockade to improve T cell infiltration and function in colorectal cancer

Abstract Adoptive transfer of tumor infiltrating lymphocytes (TIL) has emerged as a promising approach to induce effective anti-tumor immunity and tumor regression in various types of cancers. Current studies suggest that the presence of TIL play a role in patient outcomes and can predict tumor stage, node positivity, and in some reports, disease free and overall survival in patients with colorectal cancer. TIL may be inactivated in patients due to factors in the local tumor microenvironment, such as regulatory T cells, myeloid derived suppressor cells and the expression of a number of co-inhibitory immune checkpoints including PD-L1, BTLA, PD-1, CTLA-4, Lag-3 and Tim-3. We first examined the feasibility of expanding TIL from the tumors of three colorectal patients. Tumors were minced into fragments and cultured in media supplemented with 6000 IU IL2 / mL. TIL growth was observed in tumor fragments from each of the three patients with a total of 12 out of 41 fragments demonstrating TIL expansion. TIL grown out of these fragments were predominantly CD3+ with 32% CD4+T cells and 46% CD8+ T cells and 18.5% NK cells. Seven of the 12 fragments tested were tumor specific as demonstrated by IFN-gamma production in response to autologous tumor. CD8+ T cells within the patient TIL expressed PD-1, BTLA, Lag-3 and Tim-3 and a co-stimulatory molecule, 41BB. In addition, we found high levels of PD-L1 expression on tumors of colorectal patients and human colorectal cell lines. Since we observed high levels of immune checkpoints expression in colorectal patients, we wanted to develop a preclinical colorectal model to investigate the effect of immune checkpoint blockade on tumor growth and TIL function. A previous study from our lab has shown that TIL from our preclinical MC38 colorectal murine model express high levels of co-inhibitory checkpoints and that blockade of PD-L1 leads to enhanced infiltration and anti-tumor specificity of TIL. However, given the dynamic nature of immune responses to tumors and expression of multiple immune checkpoints, it may be necessary to block additional checkpoints to improve clinical efficacy. In this study, we examined whether blockade of the immune checkpoint receptor BTLA had an effect on TIL infiltration and tumor growth. Mice bearing MC-38 tumors treated with anti-BTLA antibody demonstrated a delay in tumor growth (150mm2, p<0.01) on day 28 post tumor injection compared to isotype control (300mm2). We observed an increase in CD8+ T cell infiltration (30%, p<0.001) in the tumors of anti-BTLA antibody treated mice compared to mice treated with isotype control (13%). TIL isolated from the anti-BTLA antibody treated mice demonstrated increased IFN-gamma and granzyme B secretion compared to isotype controls (p<0.001). Dual immune checkpoint blockade with anti-PD-L1 and anti-BTLA antibodies had a synergistic effect in delaying tumor growth. We also measured a significant increase in CD8+CD107a+IFN-gamma+ secreting T cells (37%) compared to TIL from mice treated with anti-PD-L1 (27%) or anti-BTLA antibody alone (25%) (p<0.002). These findings suggest that targeting immune checkpoint molecules can improve T cell infiltration and anti-tumor immunity in a preclinical model of colorectal cancer. In addition, we demonstrate that tumor-specific TIL can be expanded from the tumors of colorectal patients. This study presents a rationale for the potential synergy between blockade of multiple immune checkpoints and adoptive transfer of TIL for the treatment of colorectal cancer. Citation Format: Krithika N. Kodumudi, James Charles, Carrie Luu, John Mullinax, Julie Li, MacLean Hall, Amy Weber, Amod A. Sarnaik, Shari Pilon-Thomas. Immune checkpoint blockade to improve T cell infiltration and function in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B12.

Abstract B063: Oncolytic adenoviruses armed with tumor necrosis factor alpha and interleukin-2 enable successful adoptive T cell therapy of solid tumors

Abstract Adoptive T-cell based therapy can promote dramatic tumor regressions in both pre-clinical tumor models and in patients with CD19+ hematological tumors. However, in the case of human solid tumors, anti-tumor effects are limited by T-cell tolerance and the immunosuppressive tumor microenvironment. Human data from cancer patients treated with oncolytic adenovirus indicated that lymphocytes traffic to tissues following virotherapy. Therefore, we hypothesized to enable successful T-cell therapy by arming viruses with immunostimulatory cytokines which can counteract tumor immunosuppression locally. Previously, we have identified interleukin-2 (IL-2) and Tumor Necrosis Factor alpha (TNFa) as the most promising factors to stimulate the graft used in adoptive T-cell therapy. Also, we have established that our arming approach results in long lasting, high level cytokine expression locally but low levels systemically in vivo and in patients, which is important for safety versus efficacy. One attractive aspect of this approach is the ease of combinations with standard therapies including checkpoint inhibiting antibodies (ongoing experiments). Non-replicating adenovirus vectors coding for murine TNFa and IL-2 were used together with intraperitoneally administered OT-I TCR transgenic T-cells in the murine B16.OVA melanoma model. The best antitumor efficacy was obtained when virally coded IL-2 and TNFa were both used with T-cell transfer. Moreover, IL-2/TNFa virus combination treatment was able to attract (111)In-labelled OT-I cells into B16.OVA tumors in SPECT/CT imaging experiments. In addition to mouse studies, we developed oncolytic adenoviruses expressing human TNFa and/or human IL-2 that were used in combination with intratumoral adoptive transfer of tumor-infiltrating lymphocytes (TIL) in immunocompetent and adenovirus-permissive Syrian hamsters. These viruses improved the efficacy of TIL therapy and protected animals from tumor rechallenge. Furthermore, we have shown that adenovirally delivered IL-2 is superior to systemically administered IL-2 with regard to antitumor efficacy and safety, in both mice and hamsters. TILT Biotherapeutics is in the process of confirming the results in clinical trials. Citation Format: Mikko Siurala, Riikka Havunen, João Santos, Siri Tähtinen, Dipongkor Saha, Markus Vähä-Koskela, Michael Behr, Dirk M. Nettelbeck, Anja Ehrhardt, Suvi Parviainen, Akseli Hemminki. Oncolytic adenoviruses armed with tumor necrosis factor alpha and interleukin-2 enable successful adoptive T cell therapy of solid tumors [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B063.