Anna Pasetto

Landscape of immunogenic tumor antigens in successful immunotherapy of virally induced epithelial cancer

Targeting nonviral antigens in viral-driven cancer Adoptive cell transfer harnesses a patients own T cells to destroy cancer. The strategy can successfully treat epithelial tumors driven by human papillomavirus (HPV), but it remains unclear why only some patients respond. Stevanović et al. examined the antitumor T cell response associated with HPV+ cervical cancers that underwent complete regression. Unexpectedly, reactive T cells were not directed against virally associated antigens, but rather against cancer germline antigens or neoantigens not previously recognized by the immune system. These findings counter the widely held belief that T cell responses against viral antigens are responsible for therapeutic effects in HPV-driven cancers. Science, this issue p. 200 Reactive T cells directed against nonviral antigens lead to regression of human papilloma virus–positive cervical cancer. Immunotherapy has clinical activity in certain virally associated cancers. However, the tumor antigens targeted in successful treatments remain poorly defined. We used a personalized immunogenomic approach to elucidate the global landscape of antitumor T cell responses in complete regression of human papillomavirus–associated metastatic cervical cancer after tumor-infiltrating adoptive T cell therapy. Remarkably, immunodominant T cell reactivities were directed against mutated neoantigens or a cancer germline antigen, rather than canonical viral antigens. T cells targeting viral tumor antigens did not display preferential in vivo expansion. Both viral and nonviral tumor antigen–specific T cells resided predominantly in the programmed cell death 1 (PD-1)–expressing T cell compartment, which suggests that PD-1 blockade may unleash diverse antitumor T cell reactivities. These findings suggest a new paradigm of targeting nonviral antigens in immunotherapy of virally associated cancers.

Tumor- and neoantigen-reactive T-cell receptors can be identified based on their frequency in fresh tumor

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

Isolation of T cell receptors specifically reactive with mutated tumor associated antigens from tumor infiltrating lymphocytes based on CD137 expression.

Purpose: The adoptive transfer of lymphocytes genetically modified to express tumor reactive T-cell receptors (TCR) can mediate tumor regression. Some tumor-infiltrating lymphocytes (TIL) recognize somatic mutations expressed only in the patients tumors, and evidence suggests that clinically effective TILs target tumor-specific neoantigens. Here we attempted to isolate neoantigen-reactive TCRs as a prelude to the treatment of patients with autologous T cells genetically modified to express such TCRs. Experimental Design: Mutations expressed by tumors were identified using whole-exome and RNA sequencing. Tandem minigene (TMG) constructs encoding 12–24 mutated gene products were synthesized, each encoding the mutated amino acid flanked by 12 amino acids of the normal protein sequence. TILs were cultured with autologous dendritic cells (DC) transfected with in vitro transcribed (IVT) mRNAs encoding TMGs and were evaluated for IFNγ secretion and CD137 expression. Neoantigen-reactive T cells were enriched from TILs by sorting for CD137+ CD8+ T cells and expanded in vitro. Dominant TCR α and β chains were identified in the enriched populations using a combination of 5′ rapid amplification of cDNA ends, deep sequencing of genomic DNA, PairSeq analysis, and single-cell RT-PCR analysis. Human PBL retrovirally transduced to express the TCRs were evaluated for recognition of relevant neoantigens. Results: We identified 27 TCRs from 6 patients that recognized 14 neoantigens expressed by autologous tumor cells. Conclusions: This strategy provides the means to generate T cells expressing neoantigen-reactive TCRs for use in future adoptive cell transfer immunotherapy trials for patients with cancer. Clin Cancer Res; 23(10); 2491–505. ©2016 AACR.

TCR-Redirected Human T Cells Inhibit Hepatitis C Virus Replication: Hepatotoxic Potential Is Linked to Antigen Specificity and Functional Avidity

Virus-specific CTL with high levels of functional avidity have been associated with viral clearance in hepatitis C virus (HCV) infection and with enhanced protective immunity. In chronic HCV infection, lack of antiviral CTL is frequently observed. In this study, we aim to investigate novel HCV TCRs that differ in Ag specificity. This involved isolating new HCV-specific murine TCRs that recognize a conserved HLA-A2–restricted CTL epitope within the nonstructural protein (NS) 5A viral protein and comparing them with TCRs recognizing another conserved CTL target in the NS3 viral protein. This was done by expressing the TCRs in human T cells and analyzing the function of the resulting TCR-transduced T cells. Our result indicates that these TCRs are efficiently assembled in transduced human T cells. They recognize peptide-loaded targets and demonstrate polyfunctional features such as IL-2, IFN-γ, and TNF-α secretion. However, in contrast to NS3-specific TCRs, the NS5A TCR-transduced T cells consist of a smaller proportion of polyfunctional T cells and require more peptide ligands to trigger the effector functions, including degranulation. Despite the differences, NS5A TCRs show effective inhibition of HCV replication in human hepatoma cells with persistent HCV RNA replication. Moreover, cellular injury demonstrated by aspartate aminotransferase release and cell death is less significant in the hepatoma cells following coincubation with NS5A TCR-transduced T cells, which is a property consistent with noncytotoxic antiviral CTLs. Our results suggest that HCV TCR-transduced T cells may be promising for the treatment of patients with chronic HCV infections.

Generation of T-cell receptors targeting a genetically stable and immunodominant cytotoxic T-lymphocyte epitope within hepatitis C virus non-structural protein 3.

Hepatitis C virus (HCV) is a major cause of severe liver disease, and one major contributing factor is thought to involve a dysfunction of virus-specific T-cells. T-cell receptor (TCR) gene therapy with HCV-specific TCRs would increase the number of effector T-cells to promote virus clearance. We therefore took advantage of HLA-A2 transgenic mice to generate multiple TCR candidates against HCV using DNA vaccination followed by generation of stable T-cell–BW (T-BW) tumour hybrid cells. Using this approach, large numbers of non-structural protein 3 (NS3)-specific functional T-BW hybrids can be generated efficiently. These predominantly target the genetically stable HCV genotype 1 NS31073–1081 CTL epitope, frequently associated with clearance of HCV in humans. These T-BW hybrid clones recognized the NS31073 peptide with a high avidity. The hybridoma effectively recognized virus variants and targeted cells with low HLA-A2 expression, which has not been reported previously. Importantly, high-avidity murine TCRs effectively redirected human non-HCV-specific T-lymphocytes to recognize human hepatoma cells with HCV RNA replication driven by a subgenomic HCV replicon. Taken together, TCR candidates with a range of functional avidities, which can be used to study immune recognition of HCV-positive targets, have been generated. This has implications for TCR-related immunotherapy against HCV.

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

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

The Adjuvant Activity of Alphavirus Replicons Is Enhanced by Incorporating the Microbial Molecule Flagellin into the Replicon

Ligands of pattern recognition receptors (PRRs) including Toll-like receptors (TLRs) stimulate innate and adaptive immune responses and are considered as potent adjuvants. Combinations of ligands might act in synergy to induce stronger and broader immune responses compared to stand-alone ligands. Alphaviruses stimulate endosomal TLRs 3, 7 and 8 as well as the cytoplasmic PRR MDA-5, resulting in induction of a strong type I interferon (IFN) response. Bacterial flagellin stimulates TLR5 and when delivered intracellularly the cytosolic PRR NLRC4, leading to secretion of proinflammatory cytokines. Both alphaviruses and flagellin have independently been shown to act as adjuvants for antigen-specific antibody responses. Here, we hypothesized that alphavirus and flagellin would act in synergy when combined. We therefore cloned the Salmonella Typhimurium flagellin (FliC) gene into an alphavirus replicon and assessed its adjuvant activity on the antibody response against co-administered antigen. In mice immunized with recombinant alphavirus, antibody responses were greatly enhanced compared to soluble FliC or control alphavirus. Both IgG1 and IgG2a/c responses were increased, indicating an enhancement of both Th1 and Th2 type responses. The adjuvant activity of FliC-expressing alphavirus was diminished but not abolished in the absence of TLR5 or type I IFN signaling, suggesting the contribution of several signaling pathways and some synergistic and redundant activity of its components. Thus, we have created a recombinant adjuvant that stimulates multiple signaling pathways of innate immunity resulting in a strong and broad antibody response.

A Synthetic Codon-Optimized Hepatitis C Virus Nonstructural 5A DNA Vaccine Primes Polyfunctional CD8+ T Cell Responses in Wild-Type and NS5A-Transgenic Mice

The hepatitis C virus (HCV) nonstructural (NS) 5A protein has been shown to promote viral persistence by interfering with both innate and adaptive immunity. At the same time, the HCV NS5A protein has been suggested as a target for antiviral therapy. In this study, we performed a detailed characterization of HCV NS5A immunogenicity in wild-type (wt) and immune tolerant HCV NS5A-transgenic (Tg) C57BL/6J mice. We evaluated how efficiently HCV NS5A-based genetic vaccines could activate strong T cell responses. Truncated and full-length wt and synthetic codon-optimized NS5A genotype 1b genes were cloned into eukaryotic expression plasmids, and the immunogenicity was determined after i.m. immunization in combination with in vivo electroporation. The NS5A-based genetic vaccines primed high Ab levels, with IgG titers of >104 postimmunization. With respect to CD8+ T cell responses, the coNS5A gene primed more potent IFN-γ–producing and lytic cytotoxic T cells in wt mice compared with NS5A-Tg mice. In addition, high frequencies of NS5A-specific CD8+ T cells were found in wt mice after a single immunization. To test the functionality of the CTL responses, the ability to inhibit growth of NS5A-expressing tumor cells in vivo was analyzed after immunization. A single dose of coNS5A primed tumor-inhibiting responses in both wt and NS5A-Tg mice. Finally, immunization with the coNS5A gene primed polyfunctional NS5A-specific CD8+ T cell responses. Thus, the coNS5A gene is a promising therapeutic vaccine candidate for chronic HCV infections.

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

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

Abstract 4982: Regression of metastatic breast cancer after adoptive cell transfer of tumor infiltrating lymphocytes and checkpoint blockade

Background: Adoptive transfer of tumor infiltrating lymphocytes (TIL) can effect long-term durable regression in patients with metastatic melanoma but has not been widely tested in common epithelial cancers. Similar to metastatic melanoma, recent studies on epithelial GI cancers report that tumor regressions can be mediated by adoptively transferred TIL recognizing non-synonymous somatic mutations. Breast cancer metastatic tumors are infiltrated by TIL and reactivity against autologous somatic mutations can be identified. Methods: TIL were grown from a metastatic soft tissue tumor of a patient with hormone and chemotherapy-refractory ER+, HER2- breast cancer. DNA was extracted from tumor and matched normal peripheral blood samples for whole exome sequencing (WES) and RNAseq. Non-synonymous somatic mutations were identified and tested for potential recognition by autologous TIL using previously described tandem mini-gene and long peptide approaches. Recognition was assessed by IFN-γ release on ELISPOT and/or CD137 (4-1BB) upregulation with appropriate controls. Mutation reactive TIL were rapidly expanded in culture and transferred back to the patient. Adoptive cell transfer was preceded by a lymphodepleting preparative regimen and one dose of pembrolizumab. Cells were supported post-transfer with intravenous IL-2 administration, and pembrolizumab was administered for three additional doses. Deep sequencing of TCR rearrangement sequences was performed on the TIL and the pre- and post-infusion peripheral blood. Results: The resected subcutaneous tumor no longer expressed ER and was negative for PD-L1 staining. WES/RNASeq identified 96 non-synonymous mutations for testing. Multiple CD4+ clones specifically recognizing a mutation expressed in the solute carrier SLC3A2 (p.K94T) and a single CD8+ clone recognizing a mutation in the proteasome-associated protein KIAA0368 (p.S186F) were identified with no recognition of wild-type peptides. These eight T cell clones constituted 23% of the infusion bag TIL. Nine months after transfer of 82x109 cells, the patient has an ongoing partial response, with target lesions down 96% from baseline, including multiple hepatic metastases and disabling brachial plexus adenopathy. Seven of the eight T cell clones with known reactivity persisted in the peripheral blood comprising 2.4% of all CDR3 sequences at 7 months. Only two of these TCR clones were detectable, with frequencies totaling 0.005%, in the pre-treatment circulation. Conclusions: Tumor-infiltrating lymphocytes derived from a patient with metastatic breast cancer recognized tumor-specific non-synonymous somatic mutations, expanded in vitro and were used for adoptive cell transfer. TIL, co-administered with IL-2 and a short course of pembrolizumab, persisted in vivo and mediated regression of soft tissue, nodal and visceral metastases of breast cancer. Citation Format: Nikolaos Zacharakis, Katarzyna Trebska-McGowan, Robert Somerville, Yong-Chen Lu, Anna Pasetto, Mary Black, Harshini Chinnasamy, Hui Xu, Jared J. Gartner, Todd D. Prickett, Paul F. Robbins, Steven A. Rosenberg, Stephanie L. Goff, Steven A. Feldman. Regression of metastatic breast cancer after adoptive cell transfer of tumor infiltrating lymphocytes and checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4982. doi:10.1158/1538-7445.AM2017-4982