Over the past decade, Immuno-Oncology has largely focused on blocking inhibitory surface receptors like PD-1 to enhance T cell anti-tumor activity. However, intracellular immune checkpoints such as CISH, which function independently of tumor-expressed ligands, offer powerful and previously untapped therapeutic potential. As a downstream regulator of TCR signaling, CISH controls T cell activation, expansion, and neoantigen reactivity. Though historically considered undruggable, recent advances in CRISPR engineering have enabled functional interrogation of these targets. We demonstrate that CISH deletion enhances T cell activation and anti-cancer functions more effectively than other emerging intracellular checkpoints. In CAR-T cells, CISH inactivation significantly increased sensitivity to tumor antigen, enabling robust recognition and killing even at low antigen levels, conditions that often lead to treatment failure with conventional T cell therapies, mirroring antigen escape scenarios seen in solid tumors. Our findings further validate CISH as a potent and druggable intracellular checkpoint capable of boosting anti-tumor T cell responses across diverse cancer types, independent of PD-L1 status. The underlying mechanisms of CISH inhibition may help explain the positive outcomes reported in recent clinical studies of this approach in solid tumor immunotherapy.

Patients with relapsed/refractory (r/r) HPV-associated epithelial cancers have a poor prognosis. Engineered T cells expressing a T cell receptor (TCR) specific for HPV16 E7 can induce tumor regression. We conducted a Phase 1 trial of KITE-439, an investigational autologous T-cell product expressing TCR specific for HPV16 E7 in patients with r/r HPV16+ epithelial cancers. CD4+ and CD8+ T cells selected from leukapheresed peripheral blood mononuclear cells were stimulated with anti-CD3/anti-CD28 antibodies followed by retroviral transduction and expansion in the presence of interleukin-7/15 and an AKT inhibitor. Patients received lymphodepleting chemotherapy (cyclophosphamide 30 mg/kg/day for 2 days and fludarabine 25 mg/m2/day for 5 days) followed by a single infusion of KITE-439 with daily IL-2 (2.5×105 IU/kg, up to 7 doses). The primary objectives were safety, tolerability, and efficacy; the primary endpoint was dose-limiting toxicities (DLTs). Eight HLA-A*02:01+ patients received KITE-439 (1×106–1×108 cells/kg). No DLTs occurred during the trial. In all patients, KITE-439 cells were detected in peripheral blood within 7 days post-infusion. Three patients experienced Grade 1–2 cytokine release syndrome related to KITE-439 (resolved within 1–5 days) and 4 had KITE-439–related Grade 1–2 neurologic events (resolved within a day). One patient achieved a partial response (from Day 35 to Month 3) and 7 had a best response of stable disease. These results suggest that KITE-439 has an acceptable safety profile for the treatment of patients with HPV-associated epithelial cancers. Further studies are needed to determine optimal manufacturing conditions and T-cell characteristics required for enhanced antitumor activity.

T cell receptor (TCR) restriction by highly polymorphic major histocompatibility complex (MHC) proteins is a foundation of cellular immunity. Although the effects of MHC polymorphisms on peptide binding and selection are well established, how micropolymorphisms within MHC supertypes impact immune recognition is poorly understood. Here, we identified a mechanism through which the micropolymorphisms in two closely related HLA-A3 superfamily members govern TCR specificity. We previously showed that TCRs specific for a public neoantigen arising from a PIK3CA oncogenic hotspot mutation restricted by HLA-A*03:01 were unable to recognize the same epitope in the context of HLA-A*03:02 despite equivalent processing and presentation by both alleles. We found here that the two micropolymorphisms distinguishing A*03:02 from A*03:01 prevent TCR binding not by altering peptide binding or static structures, but by altering the conformational ensemble of the neoantigen, preventing it from adopting a binding-permissive state. The effect is rooted in how the two polymorphic sites interact with other covarying, evolutionarily coupled polymorphisms, reflecting a cross-groove network of interactions that controls the conformational adaptability of the peptide/HLA complex. We suggest polymorphism-dependent adaptability reflects an evolved feature of class I MHC proteins, further diversifying epitopes and contributing to how TCRs and other immunoreceptors differentiate between antigens. Beyond this mechanistic insight, our findings emphasize the need for high-resolution HLA typing in efforts across immunology, including antigen-specific immunotherapy.

Immune elimination of chronic infection or cancer requires cytotoxic CD8+ T cells that adopt and maintain an effector phenotype. Cytotoxic T cell function is a bioenergetically demanding process and T cells subjected to chronic antigen exposure have compromised effector function despite high rates of glycolysis. Here we report the ability of the short-chain α-hydroxy acid, D-α-hydroxybutyrate, to act as a signaling molecule that increases mitochondrial ATP production and drives the conversion of proliferating T cells into cytotoxic effector cells. DAHB signaling switches ATP production from glycolysis to oxidative phosphorylation supported by fatty acid oxidation, even in glucose-replete media. This conversion suppresses both AMPK phosphorylation and the integrated stress response (ISR) in activated T cells while significantly elevating the level of the phosphagen, phosphocreatine (PCr). Both the PCr bioenergetic reserve and oxidative phosphorylation were required for T cell effector differentiation. DAHB-induction of CD8-effector gene transcription was coupled to bioenergetics by enhanced ATP-dependent remodeling of chromatin accessibility at effector gene loci. DAHB enhanced CD8+ T cell antitumor activity both in vitro and in vivo, and DAHB treatment of transferred T cells led to persistent in vivo antitumor effects. Together, these findings link cellular bioenergetics to the regulation of chromatin accessibility and gene expression required to support effector function.

T cells exposed to persistent antigen in the context of chronic viral infections or cancer lose self-renewal and cytotoxic capacity. Several transcriptional, epigenetic, and metabolic drivers of this process have been identified. However, the post-transcriptional regulatory mechanisms influencing the proteome of dysfunctional T cells are not well understood. Here we present a time-resolved molecular landscape of human T cells during the development of chronic antigen-driven dysfunction. Persistent T cell receptor stimulation significantly remodeled the proteome, including changes in canonical T cell exhaustion-associated proteins and proteins related to mitochondrial function, redox homeostasis, nucleotide metabolism, and cell-cycle progression. Dysfunctional T cells displayed activation of stress response pathways that were recapitulated in vivo; targeting these pathways altered the cytotoxic capacity of T cells during persistent tumor exposure. Our comprehensive proteomic resource reveals unique post-transcriptional changes in dysfunctional T cells and lays the groundwork for novel cysteine-directed therapeutics to enhance cancer immunotherapy.

Abstract T cell-engaging therapies have achieved limited success in microsatellite stable (MSS) metastatic colorectal cancer (mCRC), in part because of a paucity of truly cancer-specific targets. Systematic interrogation of the immunopeptidome, the repertoire of peptides presented by human leukocyte antigen (HLA) class I molecules, can expand the pool of druggable antigens beyond conventional surface receptors. Nonetheless, both neoantigenic driver mutations and recently described oncofetal peptides are rare or absent in most CRCs. Our recent work shows that mCRC cells adopt a highly stereotyped fetal-like phenotype, characterized by activation of a developmental WNT signaling program that is further enriched in metastasis-initiating cells and conserved across diverse patients. We hypothesized that this transcriptional reprogramming generates a cancer-specific, developmentally fixed HLA-I ligandome that can be exploited for T cell therapy. Using an integrated platform for systematic collection and multimodal profiling of matched normal colon, primary tumor, and metastases from patients undergoing CRC surgery, we established ex vivo patient-derived organoids (PDOs) that faithfully capture patient-specific CRC cell states and provide an effectively inexhaustible source of tumor cells for immunopeptidomic discovery and functional validation. In HLA-A2+ PDOs, representing the most common HLA allele, immunopeptidomic analyses identified recurrent presentation of peptides derived from fetal WNT program genes, in particular NKD1, that are prevalent across mCRC PDOs from HLA-A2+ patients and undetectable in healthy tissues. These peptides were immunogenic in vitro, eliciting robust reactivity from healthy donor T cells and supporting their suitability as therapeutic targets. Moreover, naive CD8+ T cells engineered with NKD1-specific T cell receptors (TCRs) showed strong cytokine activation and potent, antigen-dependent cytotoxicity against mCRC PDOs without detectable off-target activity, consistent with a favorable therapeutic index. Together, these data nominate NKD1 as a conserved antigen in mCRC and support the clinical advancement of an NKD1-directed TCR-T cell product as a first-in-class precision therapy for patients with MSS mCRC. Citation Format: Jaeyop Lee, Swara Patel, Iñaki Etxeberria, Elizabeth Benitez, Jura Pintar, Andres Rettig, Christopher Cowley, Stefanie Gerstberger, Kathleen Luckett, Asha Saxena, Zita Aretz, Tatyana Korontsvit, Zhuoning Li, Kevin Soares, Emmanouil Pappou, T. Peter Kingham, William Jarnagin, Philip B. Paty, Martin R. Weiser, Michael D’Angelica, Julio Garcia-Aguilar, Jinru Shia, Mara Monetti, Christopher A. Klebanoff, Karuna Ganesh, David A. Scheinberg, . Immunopeptidomic discovery of fetal WNT-associated antigen NKD1 enables HLA-A2+ restricted TCR-T therapy for MSS mCRC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3714.

The oncogenic impact of somatic driver alterations is shaped by tissue context. Classifying alterations by cancer type and evaluating their context-specific properties requires large cohorts of genomically profiled and clinically annotated tumors. Here, we define cancer type-specific patterns of driver alterations, including 164 newly identified hotspots, in 54,331 tumors from 48,179 patients spanning 448 histological cancer subtypes. One-third of all drivers arose in non-canonical contexts and exhibited distinct features, including increased subclonality, later emergence, and divergent biological properties. Within cancer types, gene fusions and other distinct patterns of co-occurring drivers are indicative of earlier age of disease onset. We also identify ancestry-specific differences in human leukocyte antigen (HLA)-restricted driver neoantigens affecting T cell receptor therapy eligibility, and demonstrate cancer-type-specific patterns of intrinsic resistance via somatic HLA loss. Our findings highlight that functional roles of driver alterations depend on the cancer types and clinical contexts in which they arise.

BACKGROUND:Over the past decade, immunotherapeutic strategies-mainly targeting the PD-1-PD-L1 immune checkpoint axis-have altered cancer treatment for many solid tumours, but few patients with gastrointestinal forms of cancer have benefited to date. There remains an urgent need to extend immunotherapy efficacy to more patients while addressing resistance to current immune checkpoint inhibitors. The aim of this study was to determine the safety and anti-tumour activity of knockout of CISH, which encodes cytokine-inducible SH2-containing protein, a novel intracellular immune checkpoint target and a founding member of the SOCS family of E3-ligases, using tumour infiltrating lymphocyte (TILs) genetically edited with CRISPR-Cas9 in patients with metastatic gastrointestinal epithelial cancers. METHODS:For this first-in-human, single-centre, phase 1 trial, patients aged 18-70 years with a diagnosis of metastatic gastrointestinal epithelial cancer with progressive disease following at least one first line standard therapy, measurable disease with at least one lesion identified as resectable for TIL generation and at least one other lesion meeting RECIST criteria as measurable to serve as an indicator of disease response, and an ECOG performance status of 0 or 1 were screened and enrolled if meeting these and all other eligibility criteria. TILs procured from tumour biopsies were expanded on the basis of neoantigen reactivity, subjected to CRISPR-Cas9-mediated CISH knockout, and infused intravenously into 12 patients after non-myeloablative lymphocyte depleting chemotherapy (cyclophosphamide 60 mg/kg per dose on study days -6 and -5, and fludarabine 25 mg/m2 per dose on days -7 to -3) followed by high-dose IL-2 (aldesleukin; 720 000 IU/kg per dose). The primary endpoint was safety of administration of neoantigen-reactive TILs with knockout of the CISH gene, and a key secondary endpoint was anti-tumour activity measured as objective radiographic response and progression-free and overall survival. This study is registered with ClinicalTrials.gov, NCT04426669, and is complete. FINDINGS:Between May 12, 2020, and Sept 16, 2022, 22 participants were enrolled in the trial (one patient was enrolled twice owing to lack of TIL outgrowth on the first attempt); ten patients were female, and 11 were male (self-defined). One patient was Asian, the remainder were White (self-defined). We successfully manufactured CISH knockout TIL products for 19 (86%) of the patients, of whom 12 (63%) received autologous CISH knockout TIL infusion. The median follow-up time for the study was 129 days (IQR 15-283). All 12 (100%) patients had treatment-related severe adverse events. The most common grade 3-4 adverse events included haematological events (12 patients [100%]) attributable to the preparative lymphodepleting chemotherapy regimen or expected effects of IL-2, fatigue (four patients [33%]), and anorexia (three patients [25%]). Deaths of any cause for patients on study were attributed to the underlying disease under study (metastatic gastrointestinal cancer) and related complications (10 patients) or infection (grade 5 septicaemia in one patient). There were no severe (≥grade 3) cytokine release or neurotoxicity events. Six (50%) of 12 patients had stable disease by day 28, and four (33%) had stable disease ongoing at 56 days. One young adult patient with microsatellite-instability-high colorectal cancer refractory to anti-PD1/CTLA-4 therapies had a complete and ongoing response (>21 months). INTERPRETATION:These results support the safety and potential antitumour activity of inhibiting the immune checkpoint CISH through the administration of neoantigen-reactive CISH-knockout TILs, with implications for patients with advanced metastatic cancers refractory to checkpoint inhibitor immunotherapies, and provide the first evidence that a novel intracellular checkpoint can be targeted with therapeutic effect. FUNDING:Intima Bioscience.

Chimeric antigen receptor (CAR) T cells can mediate durable complete responses in individuals with certain hematologic malignancies, but antigen downregulation is a common mechanism of resistance. Although the native T cell receptor can respond to very low levels of antigen, engineered CARs cannot, likely due to inefficient recruitment of downstream proximal signaling molecules. We developed a platform that endows CAR T cells with the ability to kill antigen-low cancer cells consisting of a membrane-tethered version of the cytosolic signaling adaptor molecule SLP-76 (MT-SLP-76). MT-SLP-76 can be expressed alongside any CAR to lower its activation threshold, overcoming antigen-low escape in multiple xenograft models. Mechanistically, MT-SLP-76 amplifies CAR signaling through recruitment of ITK and PLCγ1. MT-SLP-76 was designed based on biologic principles to render CAR T cell therapies less susceptible to antigen downregulation and is poised for clinical development to overcome this common mechanism of resistance. Majzner and colleagues show that engineering T cells with a membrane-tethered version of the signaling adaptor molecule SLP-76 alongside a chimeric antigen receptor (CAR) enhances CAR T cell activity against low-antigen-density tumors.

Abstract Neoantigens (NeoAgs) are a critical class of human cancer rejection antigens. The vast majority arise from random passenger mutations unique to a single individual, which limits the systematic study of the molecular basis of NeoAg immunogenicity in patients. In contrast, “public” NeoAg are clonally conserved epitopes derived from recurrently mutated driver genes that are shared among patients. In this study, we developed of a unique research platform to perform a comprehensive structural, biophysical, genetic, and immunogenic analysis of a family of shared NeoAgs derived from mutant NRAS Q61, the second most prevalent RAS mutation. We combined a mass-spectrometry screen, x-ray crystallography, dextramer-based T cell detection, single cell TCRa/b-sequencing, and functional immune validation assays to determine the immunogenicity of NRAS(Q61) epitopes. From our mass-spectrometry screen, we discovered that neoepitopes derived from the three most common NRAS(Q61) hotspot substitutions (R, K, and L) are naturally processed and presented in HLA-A*01, thereby generating a family of public NeoAgs. By resolving the x-ray structures of the mutant pep/HLA complexes and their wild-type counterpart, we established that each hotspot substitution “untethers” a TCR contact residue from the side wall of HLA, increasing solvent exposure of the mutated residue and facilitating immune recognition. We subsequently performed immune monitoring of peripheral blood and tumor samples from n=47 HLA-A*01+ patients with an NRAS(Q61) mutated cancer, the largest biorepository from patients who express an identical NeoAg ever assembled. Detectable NRAS public NeoAg-specific T cell responses were observed across all hotspot substitutions; however, the proportion of patients who developed a response correlated with the relative hydrophobicity of the mutant sidechains (L>R>K). In addition, we found that exposure to immune checkpoint blockade, but not tumor mutational burden, was significantly correlated with the detection and clonal dynamics of CD8+ T cells reactive to NRAS NeoAgs. Using single-cell sequencing, we retrieved, functionally validated, and characterized a panel of n=30 TCRs from patient samples that specifically confer recognition to cancer cells expressing NRAS Q61 public NeoAgs. Each of these TCRs display high functional-avidity and function in a CD8 co-receptor independent manner. Moreover, a subset of TCRs demonstrate therapeutic “cross-protection” in vitro and in vivo towards multiple NRAS Q61 mutated variants, allowing a single receptor to provide therapeutic coverage for >90% of NRAS mutations. Importantly, cross-protective TCRs were not more likely to be cross-reactive to normal human proteins compared with TCRs that recognize a single NRAS hotspot substitution. Together, these findings reveal that NRAS mutations give rise to immunogenic public NeoAgs that can be studied across patients. Further, our data helps establish generalizable principles regarding NeoAg immunogenicity while also providing a therapeutically actionable target for a TCR-based approach. Citation Format: Inaki Etxeberria, Gihan Perera, Olga Lyudovyk, Smita Chandran, Lauren Banks, Michael Gormally, Michael Postow, Taha Merghoub, Jedd Wolchok, Benjamin Greenbaum, Brian Baker, Christopher Klebanoff. Immunogenic landscape and therapeutic targeting of mutant NRAS ”public” neoantigens [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr PR011.

Mutations in RNA splicing factors are prevalent across cancers and generate recurrently mis-spliced mRNA isoforms. Here, we identified a series of bona fide neoantigens translated from highly stereotyped splicing alterations promoted by neomorphic, leukemia-associated somatic splicing machinery mutations. We utilized feature-barcoded peptide-major histocompatibility complex (MHC) dextramers to isolate neoantigen-reactive T cell receptors (TCRs) from healthy donors, patients with active myeloid malignancy, and following curative allogeneic stem cell transplant. Neoantigen-reactive CD8+ T cells were present in the blood of patients with active cancer and had a distinct phenotype from virus-reactive T cells with evidence of impaired cytotoxic function. T cells engineered with TCRs recognizing SRSF2 mutant-induced neoantigens arising from mis-splicing events in CLK3 and RHOT2 resulted in specific recognition and cytotoxicity of SRSF2-mutant leukemia. These data identify recurrent RNA mis-splicing events as sources of actionable public neoantigens in myeloid leukemias and provide proof of concept for genetically redirecting T cells to recognize these targets.

The inherent antigen cross-reactivity of the T cell receptor (TCR) is balanced by high specificity. Surprisingly, TCR specificity often manifests in ways not easily interpreted from static structures. Here we show that TCR discrimination between an HLA-A*03:01 (HLA-A3)-restricted public neoantigen and its wild-type (WT) counterpart emerges from distinct motions within the HLA-A3 peptide binding groove that vary with the identity of the peptide’s first primary anchor. These motions create a dynamic gate that, in the presence of the WT peptide, impedes a large conformational change required for TCR binding. The neoantigen is insusceptible to this limiting dynamic, and, with the gate open, upon TCR binding the central tryptophan can transit underneath the peptide backbone to the opposing side of the HLA-A3 peptide binding groove. Our findings thus reveal a novel mechanism driving TCR specificity for a cancer neoantigen that is rooted in the dynamic and allosteric nature of peptide/MHC-I binding grooves, with implications for resolving long-standing and often confounding questions about T cell specificity. While static structures can provide insight into T cell receptor (TCR) antigen specificity, this often fails and auxiliary information is needed. Here the authors show, by focusing on an HLA-A3-bound neoantigen and its WT counterpart, the allosteric formation of a peptide-dependent dynamic gate that permits selective TCR recognition of a neoantigen.

T cell-based immunotherapies hold promise in treating cancer by leveraging the immune system's recognition of cancer-specific antigens1. However, their efficacy is limited in tumours with few somatic mutations and substantial intratumoural heterogeneity2-4. Here we introduce a previously uncharacterized class of tumour-wide public neoantigens originating from RNA splicing aberrations in diverse cancer types. We identified T cell receptor clones capable of recognizing and targeting neoantigens derived from aberrant splicing in GNAS and RPL22. In cases with multi-site biopsies, we detected the tumour-wide expression of the GNAS neojunction in glioma, mesothelioma, prostate cancer and liver cancer. These neoantigens are endogenously generated and presented by tumour cells under physiologic conditions and are sufficient to trigger cancer cell eradication by neoantigen-specific CD8+ T cells. Moreover, our study highlights a role for dysregulated splicing factor expression in specific cancer types, leading to recurrent patterns of neojunction upregulation. These findings establish a molecular basis for T cell-based immunotherapies addressing the challenges of intratumoural heterogeneity.

The T-cell receptor (TCR) initiates T-lymphocyte activation, but the mechanism of TCR activation remains uncertain. Here, we present cryogenic electron microscopy structures for the unliganded and human leukocyte antigen (HLA)-bound human TCR–CD3 complex in nanodiscs that provide a native-like lipid environment. Distinct from the open and extended conformation seen in detergent, the unliganded TCR–CD3 in nanodiscs adopts two related closed and compacted conformations that represent its physiologic resting state in vivo. By contrast, the HLA-bound complex adopts the open and extended conformation, and conformation-locking disulfide mutants show that ectodomain opening is necessary for maximal ligand-dependent T-cell activation. These structures also reveal conformation-dependent protein–lipid and glycan–glycan interactions within the TCR. Together, these results establish allosteric conformational change during TCR activation, reveal avenues for immunotherapeutic engineering, and highlight the importance of native-like lipid environments for membrane protein structure determination. The T-cell receptor (TCR) activation mechanism has remained uncertain. Here, the authors present molecular structures for the apo and ligand-bound human TCR–CD3 complex in lipid nanodiscs, revealing large conformational changes during activation.

2519 Background: Tumor heterogeneity and low mutational burden limits the availability of effective immunotherapy targets. Aberrant RNA-splicing (neojunctions) represents an underexplored yet promising source of neoantigens. To address this, we developed a neoantigen discovery platform (SNIPP) that characterizes a novel class of clonally-expressed, splicing-derived neoantigens. Furthermore, we validated the immunogenicity of these neoantigens by identifying specific TCRs that drive CD8+ T-cell-mediated tumor killing. Methods: SNIPP identified public neojunctions by analyzing TCGA RNA-seq data, selecting neojunctions with a positive sample rate (PSR) > 10% and filtering out those found in GTEx normal tissue RNA-seq data (PSR < 1%) across 12 cancer types. To characterize intratumorally conserved neojunctions, we performed maximally-distanced multi-site biopsies ( n = 535) within glioma patients ( n = 56) and generated RNA-seq data for each intratumoral site. Two independent algorithms were utilized to predict peptide processing likelihood and HLA-binding affinity of splicing-derived neoantigen candidates. Neoantigen-specific TCR sequences were identified via in vitro sensitization of PBMCs and subsequent 10x V(D)J scRNA-seq. These TCRs were transduced into CD8+ T-cells, which were tested downstream for immunogenicity and cytotoxicity against glioma cell lines. Results: Our pipeline identified 789 public neojunctions, including 32 neojunctions concurrently detected in transcriptomic and proteomic glioma datasets and confidently predicted to be presented by HLA-A*02:01. IVS and subsequent 10x V(D)J scRNA-seq identified TCR clonotypes reactive against neojunctions in RPL22 ( n = 7) and GNAS ( n = 1), with the latter exhibiting high intratumoral conservation (detected in > 90% of spatially-mapped biopsies across 17/56 patients (26.78%)). TCR-transduced CD8+ T-cells recognized and were immunogenically activated and demonstrated cytotoxicity against endogenously processed and presented neoantigens in GBM and melanoma lines. Additionally, IDH1-mutant oligodendrogliomas exhibited significantly higher neojunction expression compared to IDH1-mutant astrocytomas and IDH1wt subtypes. Differential gene expression analysis (DESeq2) revealed reduced expression of splicing factors in oligodendrogliomas, attributed to their specific co-deletion of chromosomes 1p and 19q. CRISPRi-mediated knockdown of these splicing factors (e.g. SF3A3, SNRPD2) in IDH1wt glioma cells resulted in significantly increased expression of corresponding neojunctions. Conclusions: Our study highlights a novel class of neoantigens derived from tumor-wide aberrant RNA splicing. The SNIPP platform effectively identifies public intratumorally-conserved neojunctions with strong therapeutic potential. Furthermore, elevated neojunction expression in oligodendroglioma underscores the mechanistic link between dysregulated splicing factor expression and RNA splicing abnormalities.

Abstract A major roadblock preventing durable responses to CD8+ T cell-based immunotherapies in melanoma and other solid cancers is loss or downregulation of human leukocyte antigen class I (HLA-I) on tumor cells. We hypothesized this immune escape mechanism might be overcome by tumor-reactive cytotoxic CD4+ T cells which can directly engage tumor cells that express HLA-II. Unlike HLA-I, the molecular mechanisms governing response and resistance to HLA-II antigen presentation by tumor cells have previously not been studied in a systematic fashion. We performed a genome-scale CRISPR knockout (KO) screen to interrogate the molecular mechanisms of direct CD4+ T cell engagement of tumor cells. Using the HLA-II+/HLA-I+ melanoma cell line A375, we identified genes that either confer sensitivity or resistance to primary human CD4+ T cells expressing an HLA-II restricted TCR. CD4+ T cell killing was preserved following KO of genes that confer intrinsic resistance to CD8+ T cells, including B2M, TAP1/2, and TAPBP. This confirms that HLA-II restricted TCRs can serve as an orthogonal strategy to overcome CD8+ T cell resistance. We further discovered that KO of the RNA-binding protein PCBP2 led to enhanced depletion (i.e. sensitization) under CD4+ T cell selection. We established the generalizability of this finding through focused CRISPR KO screens in n=3 unique HLA-II+ cell lines using distinct guide RNA sequences and a different HLA-II restricted TCR. Mechanistically, we found that deletion of PCBP2 sensitized tumor cells to apoptosis induced by CD4+ T cell-derived cytokines, including TNF and IFNγ. PCBP2 encodes multiple protein isoforms that differ in the linker region separating two of PCBP2’s three RNA binding KH domains. In genetic complementation experiments, we discovered that these isoforms can be functionally grouped by inclusion (i.e. PCBP2 long) and exclusion (i.e. PCBP2 short) of coding exon 8. Only re-expression of short PCBP2 isoforms reverted the cytokine-sensitized phenotype of PCBP2 KO tumor cells. Reflecting this observation, specific deletion of PCBP2 long isoforms using CRISPR/Cas9 KO of PCBP2 exon 8 did not affect cytokine sensitivity. We further found that disabling the RNA binding capacity of the PCBP2 short isoforms’ third KH domain, but not others, blocked protection from cytokine-induced cell death. In vivo, we discovered that adoptive transfer of CD4+ T cells targeting an HLA-II restricted tumor antigen resulted in superior control of PCBP2 KO tumors compared to control tumors. In conclusion, we establish that CD4+ T cells can be employed to overcome acquired resistance to CD8+ T cell killing and describe a previously unknown isoform-specific role of the RNA-binding protein PCBP2 in regulating antitumor immunity. These findings establish a novel, mechanism-based immunotherapeutic approach to augment the antitumor efficacy of HLA-II restricted CD4+ T cells. Citation Format: Korbinian N Kropp, Inaki Etxeberria, Smita S Chandran, Fei Yi, Rebecca Nadler, Michael V Gormally, Jahan Rahman, Salima Benbarche, Jeetayu Biswas, Marco V Ruzzo, Ralph Garippa, Omar Abdel-Wahab, Christopher A Klebanoff. Deletion of the RNA-binding protein PCBP2 sensitizes tumor cells to CD4+ T cell killing [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B051.

T-cell receptors (TCRs) recognize antigens derived from fragments of somatically expressed proteins that are degraded by the proteasome and presented by specific human leukocyte antigen (HLA) molecules. Recent therapeutic advances using the TCR as a tumor-targeting moiety have focused attention on HLA loss of heterozygosity (LOH) as a potential resistance mechanism. Allele-specific LOH, rather than allele-agnostic, is particularly pertinent, but rarely evaluated. Using a real-world dataset comprising 78,418 cases, we demonstrate that allele-specific LOH occurs at a relatively low frequency (<10%) across all patients with cancer. We observed a modest increase in allele-specific HLA LOH in cancers harboring associated neoantigen driver mutations (eg, KRAS or TP53 mutations), but the overall frequency remained consistently low. Furthermore, using an orthogonal dataset, we integrated clinical outcomes with HLA LOH and identified distinct impacts on overall survival in colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) cohorts. For instance, A*02:01-specific LOH was linked to worse survival in CRC (HR 0.5355, 95% CI 0.2991 to 0.9589, p=0.0094) but showed a trend toward improved survival in NSCLC (HR 1.249, 95% CI 0.7778 to 2.005). These findings underscore the relevance of allele-specific HLA LOH assessments and reveal nuanced differences in its clinical implications, which should be accounted for in the optimization of TCR-based immunotherapies.

Chimeric antigen receptor (CAR)-engineered lymphocytes treat B cell malignancies; however, limited persistence can restrain the full therapeutic potential of this approach. FAS ligand (FAS-L)/FAS interactions govern lymphocyte homeostasis. Knowledge of which cells express FAS-L in patients with cancer and whether these sources compromise CAR persistence remains incomplete. Here, we constructed a single-cell atlas of diverse cancers to identify cellular subsets expressing FASLG, the gene encoding FAS-L. We discovered that FASLG expression is limited primarily to endogenous T cells, natural killer (NK) cells and CAR-T cells, while tumor and stromal cell expression is minimal. To establish whether CAR-T and CAR-NK cell survival is FAS-L regulated, we performed competitive fitness assays using FAS-dominant negative receptor (ΔFAS)-modified lymphocytes. Following transfer, ΔFAS-expressing CAR-T/CAR-NK cells became enriched, a phenomenon that mechanistically was reverted through FASLG knockout. By contrast, FASLG was dispensable for CAR-mediated tumor killing. In multiple models in female mice, ΔFAS coexpression enhanced antitumor efficacy. Together, these findings reveal that CAR-engineered lymphocyte persistence is governed by a FAS-L/FAS autoregulatory circuit. Klebanoff and colleagues report that survival and persistence of CAR-T and CAR-NK cells are regulated by a FAS ligand–FAS autoregulatory circuit, showing that disabling FAS signaling enhances their antitumor efficacy in preclinical models.