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Alternative splicing (AS) is prevalent in cancer, generating an extensive but largely unexplored repertoire of novel immunotherapy targets. We describe I soform peptides from R NA splicing for I mmunotherapy target S creening (IRIS), a computational platform capable of discovering AS-derived tumor antigens (TAs) for T cell receptor (TCR) and chimeric antigen receptor T cell (CAR-T) therapies. IRIS leverages large-scale tumor and normal transcriptome data and incorporates multiple screening approaches to discover AS-derived TAs with tumor-associated or tumor-specific expression. In a proof-of-concept analysis integrating transcriptomics and immunopeptidomics data, we showed that hundreds of IRIS-predicted TCR targets are presented by human leukocyte antigen (HLA) molecules. We applied IRIS to RNA-seq data of neuroendocrine prostate cancer (NEPC). From 2,939 NEPC-associated AS events, IRIS predicted 1,651 epitopes from 808 events as potential TCR targets for two common HLA types (A*02:01 and A*03:01). A more stringent screening test prioritized 48 epitopes from 20 events with “neoantigen-like” NEPC-specific expression. Predicted epitopes are often encoded by microexons of ≤30 nucleotides. To validate the immunogenicity and T cell recognition of IRIS-predicted TCR epitopes, we performed in vitro T cell priming in combination with single-cell TCR sequencing. Seven TCRs transduced into human peripheral blood mononuclear cells (PBMCs) showed high activity against individual IRIS-predicted epitopes, providing strong evidence of isolated TCRs reactive to AS-derived peptides. One selected TCR showed efficient cytotoxicity against target cells expressing the target peptide. Our study illustrates the contribution of AS to the TA repertoire of cancer cells and demonstrates the utility of IRIS for discovering AS-derived TAs and expanding cancer immunotherapies.

Supplemental Table 3: Differential gene expression analysis between B16 KO and WT tumors in vivo at day 6. Showing genes with q < 0.05 and log2FC > 1 or < -1.

Supplementary Figure 1: Impact of PAK4 deletion on the expression of chemokines.

Supplementary Figure 10: PAK4 kinase inhibition modulates WNT signalling in vivo.

Supplementary Figure 8: PAK4 KO in human melanoma cells impairs B-catenin/WNT signalling.

Supplementary Figure 4: PAK4 deletion does not affect MHC-I and II surface expression.

Supplemental Table 6: List of extracellular matrix genes enriched in PAK4 KO anti-PD-1 treated samples.

Supplementary Figure 2: PAK4 deletion does not affect CCL21 secretion.

Supplementary Figure 3: No difference in signalling through different stimuli between PAK4 KO and WT cells.

Supplemental Table 4: Differential gene expression analysis between B16 PAK4 KO anti-PD-1 vs PAK4 KO isotype tumors in vivo at day 10. Showing genes with q < 0.05 and log2FC > 2 or < -2.

Supplemental Table 2: Differential gene expression analysis between B16 KO and WT cells in vitro. Showing genes with q < 0.05 and log2FC > 1 or < -1.

Supplementary Table 1: Panel of immune markers for dendritic cell characterization.

Supplementary Figure 6: Spatial colocalization of CD8 and CD31 positive cells.

Supplemental Table 5: Differential gene expression analysis between B16 PAK4 KO anti-PD-1 vs WT anti-PD-1 tumors in vivo at day 10. Showing genes with q < 0.05 and log2FC > 1 or < -1.

Supplementary Figure 9: PAK4 inhibitor, A0317859, also synergizes with PD-1 blockade in YUMM2.1 melanoma cells.

Supplementary Figure 7: PAK4 protein expression levels in the different cell lines.

Supplementary Figure 5: PAK4 deletion increases CXCL10 expression in vivo.

Supplementary Table S22: Segmented b-allele deficits

Supplementary Tables S16-S19, describing IHC results on NHS/HPFS data

Mechanism-based strategies to overcome resistance to PD-1 blockade therapy are urgently needed. We developed genetic acquired resistant models of JAK1, JAK2, and B2M loss-of-function mutations by gene knockout in human and murine cell lines. Human melanoma cell lines with JAK1/2 knockout became insensitive to IFN-induced antitumor effects, while B2M knockout was no longer recognized by antigen-specific T cells and hence was resistant to cytotoxicity. All of these mutations led to resistance to anti–PD-1 therapy in vivo. JAK1/2-knockout resistance could be overcome with the activation of innate and adaptive immunity by intratumoral Toll-like receptor 9 agonist administration together with anti–PD-1, mediated by natural killer (NK) and CD8 T cells. B2M-knockout resistance could be overcome by NK-cell and CD4 T-cell activation using the CD122 preferential IL2 agonist bempegaldesleukin. Therefore, mechanistically designed combination therapies can overcome genetic resistance to PD-1 blockade therapy.Significance:The activation of IFN signaling through pattern recognition receptors and the stimulation of NK cells overcome genetic mechanisms of resistance to PD-1 blockade therapy mediated through deficient IFN receptor and antigen presentation pathways. These approaches are being tested in the clinic to improve the antitumor activity of PD-1 blockade therapy.This article is highlighted in the In This Issue feature, p. 1079

Supplementary Table S21: Segmented copy-number ratios

Supplementary Table S2: Somatic mutations

Supplementary Tables S1, S3 through S15, S20, and S23

The supplementary data contains supplemental methods, figures, tables and the clinical trial protocol in which patients from this study were enrolled.

A promising arsenal of targeted and immunotherapy treatments for metastatic melanoma has emerged over the last decade. With these therapies, we now face new mechanisms of tumor-acquired resistance. We report here a patient whose metastatic melanoma underwent dedifferentiation as a resistance mechanism to adoptive T-cell transfer therapy (ACT) to the MART1 antigen, a phenomenon that had been observed only in mouse studies to date. After an initial period of tumor regression, the patient presented in relapse with tumors lacking melanocytic antigens (MART1, gp100) and expressing an inflammation-induced neural crest marker (NGFR). We demonstrate using human melanoma cell lines that this resistance phenotype can be induced in vitro by treatment with MART1 T cell receptor–expressing T cells or with TNFα, and that the phenotype is reversible with withdrawal of inflammatory stimuli. This supports the hypothesis that acquired resistance to cancer immunotherapy can be mediated by inflammation-induced cancer dedifferentiation.Significance: We report a patient whose metastatic melanoma underwent inflammation-induced dedifferentiation as a resistance mechanism to ACT to the MART1 antigen. Our results suggest that future melanoma ACT protocols may benefit from the simultaneous targeting of multiple tumor antigens, modulating the inflammatory response, and inhibition of inflammatory dedifferentiation-inducing signals. Cancer Discov; 8(8); 935–43. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 899

To understand the genetic drivers of immune recognition and evasion in colorectal cancer, we analyzed 1,211 colorectal cancer primary tumor samples, including 179 classified as microsatellite instability–high (MSI-high). This set includes The Cancer Genome Atlas colorectal cancer cohort of 592 samples, completed and analyzed here. MSI-high, a hypermutated, immunogenic subtype of colorectal cancer, had a high rate of significantly mutated genes in important immune-modulating pathways and in the antigen presentation machinery, including biallelic losses of B2M and HLA genes due to copy-number alterations and copy-neutral loss of heterozygosity. WNT/β-catenin signaling genes were significantly mutated in all colorectal cancer subtypes, and activated WNT/β-catenin signaling was correlated with the absence of T-cell infiltration. This large-scale genomic analysis of colorectal cancer demonstrates that MSI-high cases frequently undergo an immunoediting process that provides them with genetic events allowing immune escape despite high mutational load and frequent lymphocytic infiltration and, furthermore, that colorectal cancer tumors have genetic and methylation events associated with activated WNT signaling and T-cell exclusion.Significance: This multi-omic analysis of 1,211 colorectal cancer primary tumors reveals that it should be possible to better monitor resistance in the 15% of cases that respond to immune blockade therapy and also to use WNT signaling inhibitors to reverse immune exclusion in the 85% of cases that currently do not. Cancer Discov; 8(6); 730–49. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 663

Supplementary Figures

Supplemental Figure 1: Hypothetical model of peripheral blood TCR-transgenic cell repopulation. Supplemental Figure 2: GLP team organizational chart. Supplemental Figure 3. Bone marrow transplant (BMT) optimization studies in HLA-A2/Kb transgenic mice. Supplemental Figure 4. Body weight and hematology assessment at day 5 and 3 months after BMT. Supplemental Figure 5. Spleen and bone marrow cellularity at day 5 and 3 months after BMT. Supplemental Figure 6. Serum chemistry at 3 months after BMT. Supplemental Figure 7. Flow cytometry gating strategy for bone marrow and splenocytes phenotype characterization. Supplemental Figure 8. Survival and hematology 3 months after BMT with Lin- cells transduced with LV-empty, LV-NY-ESO-1 TCR or LV-NY-ESO-1 TCR/sr39TK. Supplemental Figure 9. Differentiation of NYESO TCR/sr39TK-engineered T cells in ATOs (artificial thymic organoids). Supplemental Table 1. Certificate of Analysis of the GMP-comparable LV-NY-ESO-1 TCR/sr39TK (RRL-MSCV-optNYESO-optsr39TK-WPRE, production volume: 20L). Supplemental Table 2. Certificate of Analysis of the Clinical Grade Lentivirus LV-NY-ESO- 1 TCR/sr39TK (RRL-MSCV-optNYESO-optsr39TK-WPRE, production volume 60L). Supplemental Table 3. Certificate of Analysis of the clinical grade RV-NY-ESO-1 TCR (MSGV1-A2aB-1G4A-LY3H10) (Production volume 18L)*. Supplemental Table 4. Certificate of Analysis of the GMP comparable RV-NY-ESO-1 TCR (MSGV1-A2ab-1G4A-Ly3H10, Production volume: 3L). Supplemental Table 5. Co-Administration of NY-ESO-1 TCR Genetically Modified T cells and Hematopoietic Stem Cells (HSCs) in HLA-A2.1/Kb mice. GLP studies cohort distribution. Supplemental Table 6. Cell manufacturing acceptance criteria. Supplemental Table 7. List of protocol-specific organs. Supplemental Table 8. Manufacturing validation runs. Supplemental Table 9. Comparison between fresh and cryopreserved product at 1, 30, 90 and 180# days.

Purpose:To improve persistence of adoptively transferred T-cell receptor (TCR)–engineered T cells and durable clinical responses, we designed a clinical trial to transplant genetically-modified hematopoietic stem cells (HSCs) together with adoptive cell transfer of T cells both engineered to express an NY-ESO-1 TCR. Here, we report the preclinical studies performed to enable an investigational new drug (IND) application.Experimental Design:HSCs transduced with a lentiviral vector expressing NY-ESO-1 TCR and the PET reporter/suicide gene HSV1-sr39TK and T cells transduced with a retroviral vector expressing NY-ESO-1 TCR were coadministered to myelodepleted HLA-A2/Kb mice within a formal Good Laboratory Practice (GLP)–compliant study to demonstrate safety, persistence, and HSC differentiation into all blood lineages. Non-GLP experiments included assessment of transgene immunogenicity and in vitro viral insertion safety studies. Furthermore, Good Manufacturing Practice (GMP)–compliant cell production qualification runs were performed to establish the manufacturing protocols for clinical use.Results:TCR genetically modified and ex vivo–cultured HSCs differentiated into all blood subsets in vivo after HSC transplantation, and coadministration of TCR-transduced T cells did not result in increased toxicity. The expression of NY-ESO-1 TCR and sr39TK transgenes did not have a detrimental effect on gene-modified HSC's differentiation to all blood cell lineages. There was no evidence of genotoxicity induced by the lentiviral vector. GMP batches of clinical-grade transgenic cells produced during qualification runs had adequate stability and functionality.Conclusions:Coadministration of HSCs and T cells expressing an NY-ESO-1 TCR is safe in preclinical models. The results presented in this article led to the FDA approval of IND 17471.

PAK4 inhibition can sensitize tumors to immune checkpoint blockade (ICB) therapy; however, the underlying mechanisms remain unclear. We report that PAK4 inhibition reverses immune cell exclusion by increasing the infiltration of CD8 T cells and CD103+ dendritic cells (DC), a specific type of DCs that excel at cross-presenting tumor antigens and constitute a source of CXCL10. Interestingly, in melanoma clinical datasets, PAK4 expression levels negatively correlate with the presence of CCL21, the ligand for CCR7 expressed in CD103+ DCs. Furthermore, we extensively characterized the transcriptome of PAK4 knockout (KO) tumors, in vitro and in vivo, and established the importance of PAK4 expression in the regulation of the extracellular matrix, which can facilitate immune cell infiltration. Comparison between PAK4 wild type and KO anti-PD-1 treated tumors revealed how PAK4 deletion sensitizes tumors to ICB from a transcriptomic perspective. In addition, we validated genetically and pharmacologically that inhibition of PAK4 kinase activity is sufficient to improve antitumor efficacy of anti-PD-1 blockade in multiple melanoma mouse models. Therefore, this study provides novel insights into the mechanism of action of PAK4 inhibition and provides the foundation for a new treatment strategy that aims to overcome resistance to PD-1 blockade by combining anti-PD-1 with a small-molecule PAK4 kinase inhibitor. Significance: Our findings provide new insights into PAK4 inhibition mechanism of action as well as the scientific foundation for specifically blocking PAK4 kinase activity using a novel and specific PAK4 kinase inhibitor to overcome resistance to PD-1 blockade.

First-generation adoptive T-cell transfer (ACT) administering tumor-infiltrating lymphocytes (TILs), and second-generation ACT using autologous T cells genetically modified to express tumor-specific T-cell receptors (TCRs) or chimeric antigen receptors (CARs) have both shown promise for the treatment of several cancers, including melanoma, leukemia and lymphoma. However, these treatments require labor-intensive manufacturing of the cell product for each patient, frequently utilize lentiviral or retroviral vectors to genetically modify the T cells, and have limited antitumor efficacy in solid tumors. Gene editing is revolutionizing the field of gene therapy, and ACT is at the forefront of this revolution. Gene-editing technologies can be used to re-engineer the phenotype of T cells to increase their antitumor potency, to generate off-the-shelf ACT products, and to replace endogenous TCRs with tumor-specific TCRs or CARs using homology-directed repair (HDR) donor templates. Adeno-associated viral vectors or linear DNA have been used as HDR donor templates. Of note, non-viral delivery substantially reduces the time required to generate clinical-grade reagents for manufacture of T-cell products—a critical step for the translation of personalized T-cell therapies. These technological advances in the field using gene editing open the door to the third generation of ACT therapies.

The success of T cell therapies for solid tumors has been limited by the scarcity of tumor-specific targets, the T cell exhaustion and immunosuppressive tumor microenvironment, and the limited persistence and T cell trafficking to the tumor, among others. To better understand how successful T cell responses induced by immune checkpoint blockade (ICB) eliminate metastatic tumors, we conducted a longitudinal landscape analysis of the neoepitope-specific T cells during treatment in patients with melanoma, with and without response to ICB. Using newly developed technologies to isolate neoepitope-specific T cells, we characterized the target of the T cell responses and their evolution over time in the peripheral blood and the tumor. Briefly, after computational prediction of patient-specific putative neoantigens, hundreds of capture reagents were made consisting of the patient HLA class I subtypes loaded with the corresponding predicted neoantigens; neoepitope-specific T cells were then isolated, and the TCR alpha and beta sequenced. Tumor reactivity of the neoantigen-specific TCRs was assessed upon co-culture of autologous melanoma cell lines from each patient with T cells gene-edited to replace their endogenous TCR with the isolated neoantigen-specific TCRs. In these studies, we showed that all patients presented neoantigen-specific T cell responses that were tumor-reactive and targeted a limited number of immunodominant epitopes. Interestingly, in patients with clinical benefit from ICB, these responses were polyclonal, with multiple TCR clonotypes specific for the same mutations, and recurrently detected over time. Our results show that even in patients without response to ICB, we could isolate neoantigen-specific TCRs that could potentially be used to engineer large numbers of T cells for adoptive T cell therapy. Another alternative for patients that do not respond to ICB is adoptive T cell therapy with T cells genetically modified to target shared antigens. We propose targeting the surface expression of TYRP1 for the treatment of cutaneous and rare subtypes of melanoma. TYRP1 is mainly expressed intracellularly in the melanosomes, with a small fraction of the total protein localized on the cell surface. Using a fine-tuning engineering approach, we developed a highly-sensitive CAR-T cell therapy that detects the low amounts of TYRP1 localized on the cell surface of tumor cells with high TYRP1 intracellular overexpression. The TYRP1 CAR-T cell therapy presents robust antitumor activity without toxicity in murine and patient-derived cutaneous, acral, and uveal melanoma models. Based on the efficacy and safety profile of this therapy, we are currently planning the phase I clinical trial. Citation Format: Cristina Puig Saus. Engineering a potent T-cell response against solid tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr SY19-02.

A logic to cell-cell recognition There has been exciting progress in the field of cancer immunotherapy, which harnesses a patient's own immune system to kill cancer cells. However, achieving precise recognition of cancer cells remains challenging. Cells engineered with synthetic Notch (synNotch) receptors bind to specific antigens, and binding induces the expression of defined genes. Williams et al. used synNotch modules as transcriptional connectors that daisy-chain together multiple receptors. They engineered T cells that can recognize up to three target antigens expressed on or inside cancer cells and integrated these inputs to achieve NOT, AND, and OR logic. The engineered cells achieved precise recognition of targeted cancer cells. Science , this issue p. 1099

The success of adoptive T cell therapies for solid cancers is challenged, in part, by the lack of targets expressed at sufficient levels by the tumor. Tyrosinase-related protein-1 (TYRP-1) is an enzyme that plays an essential role in melanin biosynthesis, which occurs in the pigmented cells of the skin and the eye. TYRP-1 is expressed in 70% of melanoma patients (Log2 FPKM ≥ 1) and presents a high overexpression (Log2FPKM ≥ 7) in 30% of all melanoma lesions included in The Cancer Genome Atlas (TCGA) dataset. TYRP-1 is localized intracellularly in the melanosomes. However, as a result of the fusion of the melanosomes with the plasma membrane, a small fraction of the cellular TYRP-1 is located on the cell surface. We have shown that between 1-50% (depending on the cell line) of the cellular TYRP-1 is expressed on the cell surface in melanoma cell lines with high levels of TYRP-1 but cannot be detected in cell lines with low or intermediate levels of expression, and we have designed a new chimeric antigen receptor (CAR) to target this fraction of the total TYRP-1. Upon co-culture with a panel of human melanoma cell lines with high expression of TYRP-1, the newly developed CAR-T cell therapy leads to tumor cell growth inhibition (94.4% ±0.9 in M207 melanoma cells, 94.4% ±0.9 in M230, 99.2% ±0.2 in M249, and 83.1% ±1.1 in M285 at 96h after co-culture using an effector to target cell ratio of 1:1) and cytokine release (between 13,325 - 5,558pg IFNgamma/mL was secreted 24h after co-culture depending on the target cell line). Additionally, it induces tumor growth control or tumor elimination after adoptive transfer of CAR-T cells in immunocompromised mice bearing human tumors established from the same cell lines (100% Complete responses (CR) in M207 and 90% CR in M249 tumor models at the end of the experiment). The epitope targeted by the TYRP-1 CAR-T cells is conserved between mouse and human. As a result, similar antitumor activity is obtained in a murine melanoma model in vitro (73.1% ± 1.2 tumor growth inhibition at 48h with a 1:1 effector to target ratio and 300,925pg IFNgamma/mL ± 19,657 secretion at 24h with a 5:1 effector to target ratio in the B16 murine melanoma model) and in vivo in immunocompetent mice (20% CR at the end of the experiment). Moreover, we have confirmed the specificity of the TYRP-1 CAR-T cells using TYRP-1-knocked out melanoma cell lines. And we have observed a lack of toxicity in the eye after adoptive T cell therapy of TYRP-1 CAR-T cells in immunocompetent mouse models. In summary, we have shown the relevance of the TYRP-1 expression on the cell surface as a widespread target for CAR-T cell therapy for melanoma patients and have designed a highly efficacious candidate that we now plan on translating to the clinic. Additionally, our results unravel a new source of potential CAR targets for solid tumors based on highly abundant intracellular membrane proteins translocated to the plasma membrane as a consequence of the cell secretory pathway. Citation Format: Cristina Puig Saus, Sameeha Jilani, Justin Saco, Clement Ng, Jane Coffman, Roxana Radu, Yvonne Chen, Antoni Ribas. CAR-T cell therapy for melanoma targeting surface expression of TYRP-1 [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 LB152.

Introduction: Loss-of-heterozygosity (LOH) events in chromosome 6p, comprising the human leukocyte antigen (HLA) genes, have been reported in about 10% of cutaneous melanoma (compared to 20-40% of squamous cell carcinomas), while copy number gains in this region have been observed in over 50% of melanoma. Recent studies focused in HLA allelic loss have been restricted to DNA-based approaches, and have not been validated orthogonally at the RNA or protein levels. Here, using clinical melanoma biopsies and patient-derived melanoma cell lines, we show that genetic alterations in HLA genes results in imbalanced allele expression, subsequently skewing antigen presentation by melanoma cells. Methods: Whole exome and RNA sequencing (WES, RNAseq) analyses were performed on 760 melanoma biopsies and 60 patient-derived melanoma cell lines. Patient-matched normal WES was used to perform HLA haplotyping across Class I and II HLA genes. Tumor WES was analyzed for copy number alterations in chromosome 6, identifying which alleles were lost or gained. Differential expression of HLA alleles was quantified in tumor RNAseq, correlating the allelic imbalance at the DNA and RNA levels. Melanoma cell lines heterozygous for HLA-A*02, A*03, and A*24 were analyzed by flow cytometry for surface-level HLA protein expression using allele-specific antibodies to quantify allelic densities and compare the imbalance of HLA-A alleles at the DNA, RNA, and protein levels. Results: Across 760 melanoma biopsies, copy number alterations in chromosome 6p were identified in 76% of tumors; 12% had LOH, and 54% had copy number gains that resulted in imbalanced copies of alleles. In paired tumor WES and RNAseq (N=682), genetic imbalance was correlated with imbalanced expression of HLA alleles in the classical Class I HLA genes (Spearman rho=0.64-0.7; p=2.2e-16); this association was strengthened in tumors with high tumor cellularity, and was not associated with the total expression of the HLA genes.These patterns were explored in a 60 patient-derived melanoma cell lines with matched tumor WES and RNAseq, confirming that alleles gained at the genetic level were also expressed at higher levels than alleles that were not gained or lost. In 10 cell lines heterozygous for either HLA-A*02, A*03, or A*24, allelic imbalance at the DNA and RNA level resulted in correlative imbalanced surface presentation of alleles at the protein level. Conclusions: Evaluation of paired tumor WES and RNAseq revealed orthogonal validation of HLA allelic imbalance, and analysis in cell lines suggested that these patterns were likely tumor intrinsic. Experimental validation of these findings at the protein level suggests that antigen presentation density can be modulated by chromosomal gains, and not just allelic loss, in HLA genes. This knowledge is important for the design of cancer vaccines or T cell therapies targeting neoantigens presented by HLA class I complexes. Citation Format: Katie M. Campbell, Justin Saco, Egmidio Medina, Meelad Amouzgar, Shannon M. Pfeiffer, Cynthia R. Gonzalez, Gabriela Steiner, Ameya Champhekar, Cristina Puig Saus, Jesse Zaretsky, Gabriel Abril Rodriguez, Agustin Vega-Crespo, Ignacio Baselga Carretero, Mito Tariveranmoshabad, Anusha Kalbasi, Christine Spencer, Zachary L. Skidmore, Malachi Griffith, Obi L. Griffith, Daniel K. Wells, Antoni Ribas. Infrequent chromosomal loss and recurrent gains lead to imbalanced expression of HLA genes in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3818.

Neoantigens are novel peptide sequences resulting from somatic mutations in tumors that upon loading onto major histocompatibility complex (MHC) molecules allow recognition by T cells. Accurate neoantigen identification is thus critical for designing cancer vaccines and predicting response to immunotherapies. Neoantigen identification and prioritization relies on correctly inferring whether the presenting peptide sequence can successfully induce an immune response. As the majority of somatic mutations are SNVs, changes between wildtype and mutant peptide are subtle and require cautious interpretation. An important, yet underappreciated, variable in neoantigen-prediction pipelines is the mutation position within the peptide relative to its anchor positions for the patient’s specific HLA alleles. While a subset of peptide positions are presented to the T-cell receptor for recognition, others are responsible for anchoring to the MHC, making these positional considerations critical for predicting T-cell responses. However, a systematic method for determining anchor locations for the wide range of HLA alleles present in the population and application of these to evaluate MT/WT peptide pairs arising in tumors has not been reported. As a result, many neoantigen studies have either failed to adequately consider this crucial factor or have used conventional assumptions to guide their neoantigen identification process. Here, we provide a computational workflow for predicting anchor locations for a wide range of HLA alleles, using a reference dataset generated from clinical and The Cancer Genome Atlas (TCGA) patient samples. We calculated high probability anchor positions for different peptide lengths for over 300 common HLA alleles. Analysis of these results showed clusters of different anchor trends among the HLA alleles analyzed. A subset of these HLA anchor results were orthogonally validated using protein crystallography structures. Analysis of 923 tumor samples showed that 7-41% of neoantigen candidates were potentially misclassified in the neoantigen selection process and can be rescued using allele-specific knowledge of anchor positions. These anchor predictions are currently undergoing experimental validation using both peptide-MHC stability assays as well as fluorescence-based competition binding assays. By incorporating our anchor prediction results into neoantigen prediction pipelines, such as pVACtools, we hope to formalize and streamline the identification process for relevant clinical studies. Citation Format: Huiming Xia, Joshua McMichael, Michelle Becker-Hapak, Onyinyechi C. Onyeador, Rico Buchli, Ethan McClain, Patrick Pence, Suangson Supabphol, Megan M. Richters, Anamika Basu, Cody A. Ramirez, Cristina Puig-Saus, Kelsy C. Cotto, Jasreet Hundal, Susanna Kiwala, S. Peter Goedegebuure, Tanner M. Johanns, Gavin P. Dunn, Antoni Ribas, Christopher A. Miller, William Gillanders, Todd A. Fehniger, Obi L. Griffith, Malachi Griffith. Computational prediction of MHC anchor locations guide neoantigen prediction and prioritization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5639.

Neoantigens are novel peptide sequences resulting from sources such as somatic mutations in tumors. Upon loading onto major histocompatibility complex (MHC) molecules, they can trigger recognition by T cells. Accurate neoantigen identification is thus critical for both designing cancer vaccines and predicting response to immunotherapies. Neoantigen identification and prioritization relies on correctly predicting whether the presenting peptide sequence can successfully induce an immune response. As the majority of somatic mutations are single nucleotide variants, changes between wildtype and mutated peptides are typically subtle and require cautious interpretation. A potentially underappreciated variable in neoantigen-prediction pipelines is the mutation position within the peptide relative to its anchor positions for the patient’s specific MHC molecules. While a subset of peptide positions are presented to the T-cell receptor for recognition, others are responsible for anchoring to the MHC, making these positional considerations critical for predicting T-cell responses. We computationally predicted high probability anchor positions for different peptide lengths for 328 common HLA alleles and identified unique anchoring patterns among them. Analysis of 923 tumor samples shows that 6-38% of neoantigen candidates are potentially misclassified and can be rescued using allelespecific knowledge of anchor positions. A subset of anchor results were orthogonally validated using protein crystallography structures. Representative anchor trends were experimentally validated using peptide-MHC stability assays and competition binding assays. By incorporating our anchor prediction results into neoantigen prediction pipelines, we hope to formalize, streamline and improve the identification process for relevant clinical studies.One Sentence SummaryNeoantigen prediction accuracy is significantly influenced by the mutation position within the neoantigen and its relative position to the patient’s allele-specific MHC anchor locations.

Melanoma dedifferentiation has been reported to be a state of cellular resistance to targeted therapies and immunotherapies as cancer cells revert to a more primitive cellular phenotype. Here, we show that, counterintuitively, the biopsies of patient tumors that responded to anti-programmed cell death 1 (anti-PD-1) therapy had decreased expression of melanocytic markers and increased neural crest markers, suggesting treatment-induced dedifferentiation. When modeling the effects in vitro, we documented that melanoma cell lines that were originally differentiated underwent a process of neural crest dedifferentiation when continuously exposed to IFN-γ, through global chromatin landscape changes that led to enrichment in specific hyperaccessible chromatin regions. The IFN-γ-induced dedifferentiation signature corresponded with improved outcomes in patients with melanoma, challenging the notion that neural crest dedifferentiation is entirely an adverse phenotype.

Author(s): Abril-Rodriguez, Gabriel; Torrejon, Davis Y; Liu, Wei; Zaretsky, Jesse M; Nowicki, Theodore S; Tsoi, Jennifer; Puig-Saus, Cristina; Baselga-Carretero, Ignacio; Medina, Egmidio; Quist, Michael J; Garcia, Alejandro J; Senapedis, William; Baloglu, Erkan; Kalbasi, Anusha; Cheung-Lau, Gardenia; Berent-Maoz, Beata; Comin-Anduix, Begona; Hu-Lieskovan, Siwen; Wang, Cun-Yu; Grasso, Catherine S; Ribas, Antoni

Lack of tumor infiltration by immune cells is the main mechanism of primary resistance to programmed cell death protein 1 (PD-1) blockade therapies for cancer. It has been postulated that cancer cell-intrinsic mechanisms may actively exclude T cells from tumors, suggesting that the finding of actionable molecules that could be inhibited to increase T cell infiltration may synergize with checkpoint inhibitor immunotherapy. Here, we show that p21-activated kinase 4 (PAK4) is enriched in non-responding tumor biopsies with low T cell and dendritic cell infiltration. In mouse models, genetic deletion of PAK4 increased T cell infiltration and reversed resistance to PD-1 blockade in a CD8 T cell-dependent manner. Furthermore, combination of anti-PD-1 with the PAK4 inhibitor KPT-9274 improved anti-tumor response compared with anti-PD-1 alone. Therefore, high PAK4 expression is correlated with low T cell and dendritic cell infiltration and a lack of response to PD-1 blockade, which could be reversed with PAK4 inhibition.

Mechanism-based strategies to overcome resistance to PD-1 blockade therapy are urgently needed. We developed genetic acquired resistant models of JAK1, JAK2, and B2M loss-of-function mutations by gene knockout in human and murine cell lines. Human melanoma cell lines with JAK1/2 knockout became insensitive to IFN-induced antitumor effects, while B2M knockout was no longer recognized by antigen-specific T cells and hence was resistant to cytotoxicity. All of these mutations led to resistance to anti¿PD-1 therapy in vivo. JAK1/2-knockout resistance could be overcome with the activation of innate and adaptive immunity by intratumoral Toll-like receptor 9 agonist administration together with anti¿PD-1, mediated by natural killer (NK) and CD8 T cells. B2M-knockout resistance could be overcome by NK-cell and CD4 T-cell activation using the CD122 preferential IL2 agonist bempegaldesleukin. Therefore, mechanistically designed combination therapies can overcome genetic resistance to PD-1 blockade therapy. Significance: The activation of IFN signaling through pattern recognition receptors and the stimulation of NK cells overcome genetic mechanisms of resistance to PD-1 blockade therapy mediated through deficient IFN receptor and antigen presentation pathways. These approaches are being tested in the clinic to improve the antitumor activity of PD-1 blockade therapy., This study was funded in part by the Parker Institute for Cancer Immunotherapy, NIH grants R35 CA197633 and P01 CA244118, the Ressler Family Fund, and support from Ken and Donna Schultz (all to A. Ribas). D.Y. Torrejon was supported by a Young Investigator Award from ASCO, a grant from the Spanish Society of Medical Oncology for Translational Research in Reference Centers, and the V Foundation-Gil Nickel Family Endowed Fellowship in Melanoma Research. It has been developed within the framework of a medical doctorate at the Autonomous University of Barcelona. G. Abril-Rodriguez was supported by the Isabel & Harvey Kibel Fellowship Award and the Alan Ghitis Fellowship Award for Melanoma Research. J. Tsoi and K.M. Campbell were supported by the NIH Ruth L. Kirschstein Institutional National Research Service Award #T32-CA009120 and the UCLA Tumor Immunology Training Grant (T32CA009120). G. Parisi was supported by the V Foundation-Gil Nickel Family Endowed Fellowship in Melanoma Research. J.M. Zaretsky was in the UCLA Medical Scientist Training Program supported by NIH training grant GM08042. S. Hu-Lieskovan was supported by a Conquer Cancer Foundation ASCO Career Development Award, a UCLA KL2 Translational Research Award, and a Melanoma Research Alliance Young Investigator Award. Flow and mass cytometry were performed in the UCLA Jonsson Comprehensive Cancer Center (JCCC) and Center for AIDS Research Flow Cytometry Core Facility that is supported by NIH awards P30 CA016042 and 5P30 AI028697, and by the JCCC, the UCLA AIDS Institute, and the David Geffen School of Medicine at UCLA. The purchase of the Helios mass cytometer that was used in this work was supported, in part, by funds provided by the James B. Pendleton Charitable Trust. We want to thank Dr. Cristiana Guiducci from Dynavax and Drs. Deborah Charych and Willem Overwijk from Nektar Therapeutics for helpful guidance in the performance of in vivo studies with SD-101 and bempegaldesleukin, respectively. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Background: Despite decades of experience with bone marrow transplantation, there has not been prior whole body visualization of the engraftment of hematopoietic stem cell (HSC) in bone marrow in humans. The herpes simplex virus type 1 sr39 thymidine kinase (sr39tk) gene is a positron emission tomography (PET) reporter/suicide gene that can be used to genetically label cells and track them in vivo based on the ability to retain the PET tracer [18F]FHBG, which is a penciclovir analogue. Methods: Three patients with metastatic sarcoma were treated with gene-modified autologous CD34+ peripheral blood stem cells (PBSCs) transduced with a lentiviral vector encoding both a transgenic T-cell receptor against the tumor antigen NY-ESO-1 and the sr39tk reporter/suicide gene after a reduced intensity conditioning regimen of busulfan and fludarabine. These gene-modified stem cells were co-administered with gene-modified autologous T-cells also encoding the NY-ESO-1 T-cell receptor. The primary endpoint was safety and feasibility, while an exploratory endpoint was standardized uptake value of [18F]FHBG PET between post-transplant day +25 and day +120 scans to study the biodistribution of the gene-modified PBSCs and progeny T cells. Results: Two patients (NYSCT-01 and NYSCT-03) demonstrated robust engraftment the gene-modified PBSCs in bone marrow niches by day +25-30, as demonstrated by whole body [18F]FHBG PET. PET signal was evident throughout the body in the bone marrow in the scalp, jaw, vertebrae, ribs, pelvis, femur and other bones with significant areas of bone marrow. The third patient (NYSCT-05), whose product had much lower transduction efficiency, did not display any engraftment signal at day +28. Patient NYSCT-01 demonstrated loss of [18F]FHBG signal at day +120, with no detectable T-cell progeny in peripheral circulation. Patient NYSCT-03 developed a post-transplant de novo multidrug-resistant cytomegalovirus (CMV) viremia at day +58 which ultimately required treatment with systemic ganciclovir, and passed away prior to her day +120 PET scan. Patient NYSCT-05 developed a post-transplant CMV reactivation viremia on day +45, which responded to oral valganciclovir treatment. Conclusions: PET imaging allowed visualization of the successful engraftment of sr39tk reporter gene-labeled HSCs in bone marrow niches. Further studies are needed to determine mechanisms of loss of engraftment due to immunogenicity of the sr39tk gene, or insufficient transduction efficiency. Citation Format: Theodore S. Nowicki, Beata Berent-Maoz, Cristina Puig-Saus, Paula Kaplan-Lefko, Ignacio Baselga Carretero, Ameya Champhekar, Giuseppe Carlucci, Mignonette Macabali, Ivan Perez Garcilazo, Agustin Vega-Crespo, Jonathan Rodriguez, Bartosz Chmielowski, Arun Singh, Martin Allen-Auerbach, Shaojun Zhu, Roger Slavik, Begonya Comin-Anduix, John Williams, Antoni Ribas. Whole body imaging of genetically labeled hematopoietic stem cells in human subjects [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 436.

Metabolic obstacles of the tumor microenvironment remain a challenge to T-cell-mediated cancer immunotherapies. To better understand the interplay of immune checkpoint signaling and immune metabolism, this study developed and used an optimized metabolite extraction protocol for non-adherent primary human T-cells, to broadly profile in vitro metabolic changes effected by PD-1 signaling by mass spectrometry-based metabolomics and isotopomer analysis. Inhibitory signaling reduced aerobic glycolysis and glutaminolysis. A general scarcity across the panel of metabolites measured supported widespread metabolic regulation by PD-1. Glucose carbon fate analysis supported tricarboxylic acid cycle reliance on pyruvate carboxylation, catabolic-state fluxes into acetyl-CoA and succinyl-CoA, and a block in de novo nucleoside phosphate synthesis that was accompanied by reduced mTORC1 signaling. Nonetheless, exogenous administration of nucleosides was not sufficient to ameliorate proliferation of T-cells in the context of multiple metabolic insufficiencies due to PD-L1 treatment. Carbon fate analysis did not support the use of primarily glucose-derived carbons to fuel fatty acid beta oxidation, in contrast to reports on T-memory cells. These findings add to our understanding of metabolic dysregulation by PD-1 signaling and inform the effort to rationally develop metabolic interventions coupled with immune-checkpoint blockade for increased treatment efficacy.

SUMMARY: We analyze the transcriptome of baseline and on-therapy tumor biopsies from 101 patients with advanced melanoma treated with nivolumab (anti-PD-1) alone or combined with ipilimumab (anti-CTLA-4). We find that T cell infiltration and interferon-γ (IFNγ) signaling signatures correspond most highly with clinical response to therapy, with a reciprocal decrease in cell cycle and WNT signaling pathways in responding biopsies. We model the interaction in 58 human cell lines, where IFNγ in vitro exposure leads to a conserved transcriptome response unless cells have IFNγ receptor alterations. This conserved IFNγ transcriptome response in melanoma cells serves to amplify the antitumor immune response. Therefore, the magnitude of the antitumor T cell response and the corresponding downstream IFNγ signaling are the main drivers of clinical response or resistance to immune checkpoint blockade therapy. ETOC BLURB: Analyzing the transcriptome of biopsies of patients during immune checkpoint blockade therapy, Grasso et al. show that the increase of T cell infiltration and the downstream IFNγ signaling drive clinical responses.

© 2016 Massachusetts Medical Society. BACKGROUND Approximately 75% of objective responses to anti-programmed death 1 (PD-1) therapy in patients with melanoma are durable, lasting for years, but delayed relapses have been noted long after initial objective tumor regression despite continuous therapy. Mechanisms of immune escape in this context are unknown. METHODS We analyzed biopsy samples from paired baseline and relapsing lesions in four patients with metastatic melanoma who had had an initial objective tumor regression in response to anti-PD-1 therapy (pembrolizumab) followed by disease progression months to years later. RESULTS Whole-exome sequencing detected clonal selection and outgrowth of the acquired resistant tumors and, in two of the four patients, revealed resistance-associated lossof- function mutations in the genes encoding interferon-receptor-associated Janus kinase 1 (JAK1) or Janus kinase 2 (JAK2), concurrent with deletion of the wild-type allele. A truncating mutation in the gene encoding the antigen-presenting protein beta-2-microglobulin (B2M) was identified in a third patient. JAK1 and JAK2 truncating mutations resulted in a lack of response to interferon gamma, including insensitivity to its antiproliferative effects on cancer cells. The B2M truncating mutation led to loss of surface expression of major histocompatibility complex class I. CONCLUSIONS In this study, acquired resistance to PD-1 blockade immunotherapy in patients with melanoma was associated with defects in the pathways involved in interferon-receptor signaling and in antigen presentation. (Funded by the National Institutes of Health and others.)

Melanoma is a cancer of melanocytes, which develop from neural crest cells during embryogenesis. One of the ways melanoma can resist targeted therapy or adoptive T cell transfer therapy (ACT) is by dedifferentiation, whereby the tumor becomes less melanocytic and more neural crest-like. ACT-induced dedifferentiation and therapy resistance was a result of tumor necrosis factor (TNF) from antitumor T cells (Landsberg, Nature 2012), which can serve as a positive control for inflammatory cytokine-induced melanoma plasticity. To study whether immune checkpoint blockade induces melanoma dedifferentiation, we analyzed RNA-seq data from paired biopsies of 68 patients before and during anti-PD-1 therapy. Surprisingly, the tumors of patients with complete or partial response dedifferentiate following therapy (paired p= 0.00015), but those of stable or progressive disease do not (paired p= 0.31 and 0.32, respectively). As the presence of interferon-gamma signatures in biopsies is best correlated with response to anti-PD-1 therapy, we hypothesized that interferon-gamma mediates dedifferentiation. We dedifferentiated four human melanoma cell lines by in vitro exposure to long-term (three to five weeks) interferon-gamma and compared it to three days of TNF as positive control. Although TNF and long-term interferon-gamma induced similar phenotypic changes based on flow cytometry, ATAC-seq showed that they each drive different global chromatin remodeling. Motif enrichment analyses revealed STAT6, TFAP2C, and SOX proteins are key transcription factors involved in interferon-gamma-induced dedifferentiation. Inferred protein activity analysis of the matching RNA-seq data showed that these regulators, as well as IRF3 and HMGA1, alter in activity selectively with dedifferentiation. In conclusion, dedifferentiation may be a marker of positive response to anti-PD-1 therapy, mediated by chronic exposure to T cells producing interferon-gamma. Citation Format: Yeon Joo Kim, Katherine M. Sheu, Jennifer Tsoi, Gabriel Abril-Rodriguez, Catherine Grasso, Alexander Hoffmann, Stephen T. Smale, Thomas G. Graeber, Cristina Puig-Saus, Antoni Ribas. Interferon-gamma-induced melanoma plasticity and response to PD-1 blockade therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3155.