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Fig. S1. Establishment of SB-3123 murine melanoma model. Fig. S2. Schematic of primer design for BRAF c.1799T > A (V600E) mutation analysis in SB-3123p murine melanoma cells. Fig. S3. SB-3123p melanoma is highly sensitive to vemurafenib. Fig. S4. BRAFV600E specific effect of vemurafenib upon SB-3123p established melanomas. Fig. S5. Establishment of SB-3123 murine melanoma cells expressing mouse gp100. Fig. S6. Concurrent vemurafenib therapy is required for sustained tumor growth control. Fig. S7. SB-3123 proliferative capacity is reduced when cultured under combinatorial conditions of vemurafenib and IFN-γ/TNF-α cytokines. Fig. S8. Isobologram analysis from our 8x8 screen for the synergistic (black circle) drug combination of vemurafenib + IFN-γ/TNF-α. Fig. S9. IFN-γ and TNF-α combined with vemurafenib induce growth arrest of SB-3123 melanoma cells. Fig. S10. Melanoma proliferative capacity is reduced when cultured under combinatorial conditions of vemurafenib and IFN-γ/TNF-α cytokines in a BRAFV600E dependent manner. Fig. S11. Human melanoma proliferative capacity is reduced when cultured under combinatorial conditions of vemurafenib and IFN-γ/TNF-α cytokines in a BRAFV600E dependent manner. Fig. S12. Gene set enrichment analysis of vemurafenib + IFN-γ/TNF-α dependent gene expression signature enriched in genes induced by IFN-γ/TNF-α treatment of SB3123. Supplementary Table 1 - All Genes, One Way Anova p FDR < 0.05 - Gene Cluster 1. Supplementary Table 2 - All Genes, One Way Anova p FDR < 0.05 - Gene Cluster 2. Supplementary Table 3 - All Genes, One Way Anova p FDR < 0.05 - Gene Cluster 3. Supplementary Table 4 - All Genes, One Way Anova p FDR < 0.05 - Gene Cluster 4. Supplementary Table 5 - All Genes, One Way Anova p FDR < 0.05 - Gene Cluster 5. Table S6. Gene set enrichment analysis of vemurafenib + IFN-γ/TNF-α dependent gene expression signature normalized to the vemurafenib only dependent gene expression signature in SB3123. Table S7. Gene set enrichment analysis of vemurafenib + IFN-γ/TNF-α dependent gene expression signature normalized to the IFN- γ/TNF-α only dependent gene expression signature in SB3123. Table S8. Gene set enrichment analysis of vemurafenib dependent gene expression signature normalized to control treated SB3123. Supplementary Table 9 - IFNy/TNFB induced genes vs Control Treatment, fold change > 2.0, p-value < 0.01. Supplementary Table 10 - Vemurafenib induced genes vs control treatment, fold change > 2.0, p-value < 0.01. Supplementary Table 11 - Vem + IFNg/TNFa vs IFNg/TNFa differentially expressed genes, FC > 1.5.

Supplementary Materials and Methods

A CONSORT diagram of patients on trials not previously reported

Purpose: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 and Methods: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.Results: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%.Conclusions: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

Table S2 describes treatment characteristics by clinical response to treatment

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.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.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.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.This study was funded by Intima Bioscience, U.S. and in part through the Intramural program CCR at the National Cancer Institute.

Purpose: 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 and Methods: 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. Results: 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%. Conclusions: 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

TPS2678 Background: T cell receptor (TCR)-T cell therapy is an emerging cancer treatment strategy. Thus far, demonstration of clinical activity has been limited to a subset of solid tumors including melanoma, synovial cell sarcoma and HPV-associated cancer. It is estimated that metastatic epithelial cancers are responsible for approximately 90% of cancer deaths in the United States. The estimated 600,000 cancer deaths each year is driven largely by lung adeno- and squamous cell carcinoma and invasive breast cancer, which account for approximately 30% of all cancer-related deaths. Kita-Kyushu Lung Cancer Antigen 1 (KK-LC-1) is a cancer germline antigen with expression restricted to germ cells in adults and certain epithelial cancers including lung, breast, gastric and cervical. We identified a KK-LC-1 TCR from the tumor-infiltrating lymphocytes (TIL) of a patient with cervical cancer who had a complete tumor response to TIL therapy. The KK-LC-1 TCR was the most dominate clonotype in the infused TIL product and persisted in the peripheral blood following infusion, suggesting that it may have contributed to cancer regression in this patient. Methods: We are conducting a phase I cell dose escalation trial to test the safety and efficacy of KK-LC-1 TCR-T cell therapy in patients with metastatic KK-LC-1 positive epithelial cancer. Patients receive a lymphocyte depleting conditioning regimen followed by a one-time infusion of genetically engineered T cells expressing the KK-LC-1 TCR (KK-LC-1 TCR-T cells) and high-dose systemic aldesleukin. KK-LC-1 positivity is determined by RNAscope assay measuring the percentage of cancer cells expressing CT83 (gene encoding KK-LC-1) with a percentage of 25 or greater considered positive. Main inclusion criteria include HLA-A*01:01 allele, prior first-line therapy, ECOG of 0 or 1 and adequate organ and hematologic function. Main exclusion criteria include active major medical illness of the cardiovascular, respiratory or immune system, primary or secondary immunodeficiency and autoimmune disease. Participants will be entered in sequential dose levels and receive escalating doses of cells beginning at 1x108 and ending with 6x1010. Dose-limiting toxicities will be assessed during the first 30 days of cell infusion. The primary objective is to determine the maximally tolerated dose of KK-LC-1 TCR-T cells. Exploratory objectives include assessing the safety and efficacy of KK-LC-1 TCR-T cells and to conduct immunologic studies to understand and improve the administered treatment. Clinical trial information: NCT05035407.

While neoantigen-specific tumor infiltrating lymphocytes (TIL) can be derived from in antigen-expressing tumors, their adoptive transfer fails to consistently elicit durable tumor regression. There has been much focus on the role of activation/exhaustion markers such as PD1, CD39 and TOX in TIL senescence. We found these markers were inversely expressed to Cytokine-Induced SH2 protein (CISH), a negative regulator of TCR signaling and tumor immunity in mice. To evaluate the physiological role of CISH in human TIL we developed a high-efficiency CRIPSR-based method to knock out CISH in fully mature TIL. CISH KO resulted in increased T cell receptor (TCR) avidity, tumor cytolysis and neoantigen recognition. CISH expression in the tumor resections correlated with TIL inactivity against p53 hotspot mutations and CISH KO in TIL unmasked reactivity against these universal neoantigens. While CISH KO resulted in T cell hyperactivation and expansion it did not alter maturation, perhaps by preferential PLCγ-1 and not AKT inhibition. Lastly, CISH KO in T cells increased PD1 expression and the adoptive transfer of Cish KO T cells synergistically combines with PD1 antibody blockade resulting in durable tumor regression and survival in a preclinical animal model. These data offer new insights into the regulation of neoantigen recognition, expression of activation/exhaustion markers, and functional/maturation signals in tumor-specific T cells.

While neoantigen-specific tumor infiltrating lymphocytes (TIL) can be derived from in antigen-expressing tumors, their adoptive transfer fails to consistently elicit durable tumor regression. There has been much focus on the role of activation/exhaustion markers such as PD1, CD39 and TOX in TIL senescence. We found these markers were inversely expressed to Cytokine-Induced SH2 protein (CISH), a negative regulator of TCR signaling and tumor immunity in mice. To evaluate the physiological role of CISH in human TIL we developed a high-efficiency CRIPSR-based method to knock out CISH in fully mature TIL. CISH KO resulted in increased T cell receptor (TCR) avidity, tumor cytolysis and neoantigen recognition. CISH expression in the tumor resections correlated with TIL inactivity against p53 hotspot mutations and CISH KO in TIL unmasked reactivity against these universal neoantigens. While CISH KO resulted in T cell hyperactivation and expansion it did not alter maturation, perhaps by preferential PLCγ-1 and not AKT inhibition. Lastly, CISH KO in T cells increased PD1 expression and the adoptive transfer of Cish KO T cells synergistically combines with PD1 antibody blockade resulting in durable tumor regression and survival in a preclinical animal model. These data offer new insights into the regulation of neoantigen recognition, expression of activation/exhaustion markers, and functional/maturation signals in tumor-specific T cells.

The correlations between tumor mutational load and higher response rates to immune-checkpoint inhibitor (ICI) therapies revealed the importance of targeting tumor neoantigens in cancer immunotherapy. Although ICI therapy has been revolutionary, many patients still do not respond to these treatments. One reason for this may be the absence of an adequate anti-tumor T-cell repertoire. A potential solution is the adoptive transfer of T-cells targeting neoantigens. Despite having some common shared neoantigens such as KRAS and p53, the majority of patients have highly individualized neoantigens and personalized T-cell responses. One method for identifying neoantigen-reactive T-cells from a patient's tumor-infiltrating lymphocytes (TIL) involves: (1) identifying non-synonymous mutations via whole-exome sequencing (WES), (2) using autologous dendritic cells (DC) to display these mutations in the form of electroporated minigenes (as RNA) or pulsed synthetic peptides (to create a tumor cell surrogate) and (3) co-culturing the TIL with these DCs to detect neoantigen-reactive T-cells through the upregulation of T-cell activation markers (e.g., 4-1BB). Each of these steps is labor-intensive, costly, and time-consuming. To address this, we sought to identify neoantigen-reactive TCRs directly from fresh tumors by developing a signature based on the cell surface protein and transcriptomic phenotype of cells proven to be mutation reactive. Knowing the V(D)J sequences for T-cells known to be neoantigen reactive (by the above screening), we performed 10x Genomics single cell RNA-Seq with CITE-Seq analysis on TIL isolated from frozen samples of those fresh tumors. Using the known CDR3 sequences, we compared neoantigen-reactive T-cells (six CD8 reactivities and two CD4 reactivities) versus other T-cells. This analysis led to identifying a set of cell surface proteins and genes that are specifically over- or under-expressed (including but not limited to CD62L-, CD45RA-, IL7Rlow, CD39+, CD27+, CD74+, TIGIT+, CXCL13+, LAYN+, HMOX1+, BATF+) on neoantigen-reactive T-cells. We tested whether CD39+ and CXCL13+ signature in combination with the TCR clonotype frequency could prospectively identify other neoantigen-reactive TCRs. This proved to be the case and applied not only to CD8 TIL but strongly to CD4 reactivities. In all cases, we could identify neoantigen-reactive TCRs, and in one case, 8 out of 10 candidate receptors identified by phenotype signature proved to be neoantigen-reactive. Not only were they neoantigen specific, but new, previously unidentified neoantigens were found when their specificities were analyzed. This method can open the path to treating patients with custom-made TCRs in a timely manner and expand the repertoire of tumor-specific neoantigens and the diversity of the T-cell response discoverable in human tumors. Citation Format: Kenichi Hanada, Chihao Zhao, Raul Gil-Hoyos, Jared Gartner, Christopher Chow-Parmer, Frank Lowery, Sri Krishna, Samuel Chatmon, Prickett D. Todd, Scott Kivitz, Maria Parkhurst, Michelle Langhan, Thomas Shelton, Zulmarie Franco, Robert Somerville, John Wunderlich, David Danforth, Zachary Rae, Kelly Michael, Nathan Wong, Paul Robbins, Steven Rosenberg, James Yang. A signature for tumor neoantigen-reactive T-cells in fresh human lung cancers allows rapid cloning of their receptors [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 1509.

Palmer et al. find that Cish, a member of the SOCS family, is induced by TCR stimulation in CD8+ T cells and inhibits their functional avidity against tumor. The authors uncover a novel mechanism of suppression for a SOCS member., Improving the functional avidity of effector T cells is critical in overcoming inhibitory factors within the tumor microenvironment and eliciting tumor regression. We have found that Cish, a member of the suppressor of cytokine signaling (SOCS) family, is induced by TCR stimulation in CD8+ T cells and inhibits their functional avidity against tumors. Genetic deletion of Cish in CD8+ T cells enhances their expansion, functional avidity, and cytokine polyfunctionality, resulting in pronounced and durable regression of established tumors. Although Cish is commonly thought to block STAT5 activation, we found that the primary molecular basis of Cish suppression is through inhibition of TCR signaling. Cish physically interacts with the TCR intermediate PLC-γ1, targeting it for proteasomal degradation after TCR stimulation. These findings establish a novel targetable interaction that regulates the functional avidity of tumor-specific CD8+ T cells and can be manipulated to improve adoptive cancer immunotherapy.

Lymphodepleting regimens are used before adoptive immunotherapy to augment the antitumor efficacy of transferred T cells by removing endogenous homeostatic "cytokine sinks." These conditioning modalities, however, are often associated with severe toxicities. We found that microRNA-155 (miR-155) enabled tumor-specific CD8(+) T cells to mediate profound antitumor responses in lymphoreplete hosts that were not potentiated by immune-ablation. miR-155 enhanced T-cell responsiveness to limited amounts of homeostatic γc cytokines, resulting in delayed cellular contraction and sustained cytokine production. miR-155 restrained the expression of the inositol 5-phosphatase Ship1, an inhibitor of the serine-threonine protein kinase Akt, and multiple negative regulators of signal transducer and activator of transcription 5 (Stat5), including suppressor of cytokine signaling 1 (Socs1) and the protein tyrosine phosphatase Ptpn2. Expression of constitutively active Stat5a recapitulated the survival advantages conferred by miR-155, whereas constitutive Akt activation promoted sustained effector functions. Our results indicate that overexpression of miR-155 in tumor-specific T cells can be used to increase the effectiveness of adoptive immunotherapies in a cell-intrinsic manner without the need for life-threatening, lymphodepleting maneuvers.

Background Variant Creutzfeldt-Jakob disease (vCJD) is a fatal neurodegenerative infection that can be transmitted by blood and blood products from donors in the latent phase of the disease. Currently, there is no validated antemortem vCJD blood screening test. Several blood tests are under development. Any useful test must be validated with disease-relevant blood reference panels. Study Design and Methods To generate blood reference materials, we infected four cynomolgus macaques with macaque-adapted vCJD brain homogenates. Blood was collected throughout the preclinical and clinical phases of infection. In parallel, equivalent blood was collected from one uninfected macaque. For each blood collection, an aliquot was stored as whole blood and the remainder was separated into components. Aliquots of plasma from terminally ill macaques were assayed for the presence of PrPTSE with the protein misfolding cyclic amplification (PMCA) method. Infectivity of the macaque brain homogenate used to infect macaques was titrated in C57BL/6 and RIII J/S inbred wild-type mice. Results We sampled blood 19 times from the inoculated monkeys at various stages of the disease over a period of 29 months, generating liters of vCJD-infected macaque blood. vCJD was confirmed in all inoculated macaques. After PMCA, PrPTSE was detected in plasma from infected monkeys, but not from uninfected animals. Both mouse models were more sensitive to infection with macaque-adapted vCJD agent than to primary human vCJD agent. Conclusion The macaque vCJD blood panels generated in this study provide a unique resource to support vCJD assay development and to characterize vCJD infectivity in blood.

Both targeted inhibition of oncogenic driver mutations and immune-based therapies show efficacy in treatment of patients with metastatic cancer, but responses can be either short lived or incompletely effective. Oncogene inhibition can augment the efficacy of immune-based therapy, but mechanisms by which these two interventions might cooperate are incompletely resolved. Using a novel transplantable BRAFV600E-mutant murine melanoma model (SB-3123), we explored potential mechanisms of synergy between the selective BRAFV600E inhibitor vemurafenib and adoptive cell transfer (ACT)–based immunotherapy. We found that vemurafenib cooperated with ACT to delay melanoma progression without significantly affecting tumor infiltration or effector function of endogenous or adoptively transferred CD8+ T cells, as previously observed. Instead, we found that the T-cell cytokines IFNγ and TNFα synergized with vemurafenib to induce cell-cycle arrest of tumor cells in vitro. This combinatorial effect was recapitulated in human melanoma–derived cell lines and was restricted to cancers bearing a BRAFV600E mutation. Molecular profiling of treated SB-3123 indicated that the provision of vemurafenib promoted the sensitization of SB-3123 to the antiproliferative effects of T-cell effector cytokines. The unexpected finding that immune cytokines synergize with oncogene inhibitors to induce growth arrest has major implications for understanding cancer biology at the intersection of oncogenic and immune signaling and provides a basis for design of combinatorial therapeutic approaches for patients with metastatic cancer. Cancer Immunol Res; 3(1); 37–47. ©2014 AACR. See related commentary by Riddell, p. 23

Variant Creutzfeldt-Jakob disease (vCJD) is a fatal neurodegenerative infection that can be transmitted by blood and blood products from donors in the latent phase of the disease. Currently, there is no validated antemortem vCJD blood screening test. Several blood tests are under development. Any useful test must be validated with disease-relevant blood reference panels.To generate blood reference materials, we infected four cynomolgus macaques with macaque-adapted vCJD brain homogenates. Blood was collected throughout the preclinical and clinical phases of infection. In parallel, equivalent blood was collected from one uninfected macaque. For each blood collection, an aliquot was stored as whole blood and the remainder was separated into components. Aliquots of plasma from terminally ill macaques were assayed for the presence of PrP(TSE) with the protein misfolding cyclic amplification (PMCA) method. Infectivity of the macaque brain homogenate used to infect macaques was titrated in C57BL/6 and RIII J/S inbred wild-type mice.We sampled blood 19 times from the inoculated monkeys at various stages of the disease over a period of 29 months, generating liters of vCJD-infected macaque blood. vCJD was confirmed in all inoculated macaques. After PMCA, PrP(TSE) was detected in plasma from infected monkeys, but not from uninfected animals. Both mouse models were more sensitive to infection with macaque-adapted vCJD agent than to primary human vCJD agent.The macaque vCJD blood panels generated in this study provide a unique resource to support vCJD assay development and to characterize vCJD infectivity in blood.