Human Tumor-infiltrating Lymphocytes Research Articles

Rapid parallel reconstruction and specificity screening of hundreds of T cell receptors.

The ability to screen the reactivity of T cell receptors (TCRs) is essential to understanding how antigen-specific T cells drive productive or dysfunctional immune responses during infections, cancer and autoimmune diseases. Methods to profile large numbers of TCRs are critical for characterizing immune responses sustained by diverse T cell clones. Here we provide a medium-throughput approach to reconstruct dozens to hundreds of TCRs in parallel, which can be simultaneously screened against primary human tissues and broad curated panels of antigenic targets. Using Gibson assembly and miniaturized lentiviral transduction, individual TCRs are rapidly cloned and expressed in T cells; before screening, TCR cell lines undergo combinatorial labeling with dilutions of three fluorescent dyes, which allows retrieval of the identity of individual T cell effectors when they are organized and tested in pools using flow cytometry. Upon incubation with target cells, we measure the upregulation of CD137 on T cells as a readout of TCR activation. This approach is scalable and simultaneously captures the reactivity of pooled TCR cell lines, whose activation can be deconvoluted in real time, thus providing a path for screening the reactivity of dozens of TCRs against broad panels of synthetic antigens or against cellular targets, such as human tumor cells. We applied this pipeline to systematically deconvolute the antitumoral and antiviral reactivity and antigenic specificity of TCRs from human tumor-infiltrating lymphocytes. This protocol takes ~2 months, from experimental design to data analysis, and requires standard expertise in cloning, cell culture and flow cytometry.

Soluble Tim-3 serves as a tumor prognostic marker and therapeutic target for CD8+ T cell exhaustion and anti-PD-1 resistance

Resistance to PD-1 blockade in onco-immunotherapy greatly limits its clinical application. Tcell immunoglobulin and mucin domain containing-3 (Tim-3), a promising immune checkpoint target, is cleaved by ADAM10/17 to produce its soluble form (sTim-3) in humans, potentially becoming involved in anti-PD-1 resistance. Herein, serum sTim-3 upregulation was observed in non-small cell lung cancer (NSCLC) and various digestive tumors. Notably, serum sTim-3 is further upregulated in non-responding patients undergoing anti-PD-1 therapy for NSCLC and anti-PD-1-resistant cholangiocarcinoma patients. Furthermore, sTim-3 overexpression facilitates tumor progression and confers anti-PD-1 resistance in multiple tumor mouse models. Mechanistically, sTim-3 induces terminal Tcell exhaustion and attenuates CD8+ Tcell response to PD-1 blockade through carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1). Moreover, the ADAM10 inhibitor GI254023X, which blocks sTim-3 production, reduces tumor progression in Tim-3 humanized mice and reverses anti-PD-1 resistance in human tumor-infiltrating lymphocytes (TILs). Overall, human sTim-3 holds great predictive and therapeutic potential in onco-immunotherapy.

Abstract 734: Inhibition of equilibrative nucleoside transporter 1 relieves intracellular adenosine-mediated immune suppression

Abstract The profound benefit of immune checkpoint blockade for cancer therapy is restricted to limited subsets of patients with specific cancers. Many factors contribute to primary and acquired resistance to immune checkpoint blockade, including local accumulation of immunosuppressive metabolites such as the nucleoside adenosine and downstream adenosine receptor signaling. Pharmacological inhibition of adenosine generation and signaling are active areas of clinical investigation. Here we report a novel mechanism whereby adenosine suppresses anti-cancer immune responses by intracellular accumulation. We show that human T cells readily take up adenosine via equilibrative nucleoside transporter 1 (ENT1), suppressing proliferation and effector function by inhibition of de novo pyrimidine nucleotide synthesis in T cells. ENT1 expression increases upon T cell activation, suggesting uptake of extracellular adenosine is a mechanism to limit T cell responses in high adenosine environments. Quantitative mass spectrometry imaging reveals adenosine concentrations in human tumors up to the range of 100 µM - levels significantly greater than previously demonstrated and consistent with suppression of T cell responses. Deletion of ENT1 leads to potent control of tumor growth in syngeneic mouse models including KPC, a poorly immunogenic model of pancreatic ductal adenocarcinoma, and is associated with increased CD8+ T cell frequency, proliferation and cytokine production within tumors. Furthermore, ENT1 expression was observed by flow cytometry on human tumor-infiltrating CD8+ T cells. We have discovered and characterized EOS301984, a potent ENT1 antagonist that is currently being evaluated in patients with advanced solid tumors. EOS301984 blocks intracellular adenosine transport and restores pyrimidine levels in T cells activated in the presence of adenosine, resulting in enhanced tumor cell killing by memory T cells and increased ex vivo expansion of functional human tumor-infiltrating lymphocytes in adenosine-rich environments. Finally, in a humanized mouse model of triple negative breast cancer resistant to anti-PD-1 blockade (MDA-MB-231), combination of EOS301984 with nivolumab synergistically led to control of tumor growth. Thus, ENT1 inhibition represents a novel approach to augment anti-cancer immune responses through restoring pyrimidine nucleotide synthesis in T cells suppressed by adenosine. Citation Format: Theodore J. Sanders, Christopher S. Nabel, Margreet Brouwer, Annelise L. Hermant, Lucas Chaible, Jean-Philippe Deglasse, Angelique Pabois, Wilfried Cathou, Aurore Smets, Michael Deligny, Joao Marchante, Quentin Dubray, Marie-Claire Lettelier, Chiara Martinoli, Reece Marillier, Olivier De Henau, Yvonne McGrath, Matthew G. Vander Heiden, Erica Houthuys. Inhibition of equilibrative nucleoside transporter 1 relieves intracellular adenosine-mediated immune suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 734.

Integrated single-cell profiling dissects cell-state-specific enhancer landscapes of human tumor-infiltrating CD8+ T cells

Despite extensive studies on the chromatin landscape of exhausted Tcells, the transcriptional wiring underlying the heterogeneous functional and dysfunctional states of human tumor-infiltrating lymphocytes (TILs) is incompletely understood. Here, we identify gene-regulatory landscapes in a wide breadth of functional and dysfunctional CD8+ TIL states covering four cancer entities using single-cell chromatin profiling. We map enhancer-promoter interactions in human TILs by integrating single-cell chromatin accessibility with single-cell RNA-seq data from tumor-entity-matching samples and prioritize cell-state-specific genes by super-enhancer analysis. Besides revealing entity-specific chromatin remodeling in exhausted TILs, our analyses identify a common chromatin trajectory to TIL dysfunction and determine key enhancers, transcriptional regulators, and deregulated genes involved in this process. Finally, we validate enhancer regulation at immunotherapeutically relevant loci by targeting non-coding regulatory elements with potent CRISPR activators and repressors. In summary, our study provides a framework for understanding and manipulating cell-state-specific gene-regulatory cues from human tumor-infiltrating lymphocytes.

GITR Ligation Improves Anti-PD1-Mediated Restoration of Human MMR-Proficient Colorectal Carcinoma Tumor-Derived T Cells.

In contrast to mismatch repair deficient colorectal carcinoma (CRC), MMR proficient (pMMR) CRC does not respond to immune checkpoint blockade. We studied immune checkpoint stimulation via glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) on exvivo functionality of human tumor-infiltrating lymphocytes (TIL) isolated from pMMR primary CRC and liver metastases (CRLM). Using lymphocytes from resected tumor, adjacent tissues, and peripheral blood mononuclear cells (PBMC) of 132 pMMR primary CRC or CRLM patients, we determined GITR expression and the invitro T-cell agonistic activity of recombinant GITR ligation. Here, we show that GITR was overexpressed on TIL when compared with other stimulatory immune checkpoints (4-1BB, OX40). Its expression was enhanced in TIL compared with PBMC and adjacent tissues. Among CD4+ TIL, GITR expression was primarily expressed by CD45RA- FoxP3hi activated regulatory T cells. Within CD8+ TIL, GITR was predominantly expressed on functionally exhausted and putative tumor-reactive CD103+ CD39+ TIL. Strikingly, recombinant GITRL reinvigorated exvivo TIL responses by significantly enhancing CD4+ and CD8+ TIL numbers. Dual treatment with GITRL and nivolumab (anti-PD1) enhanced CD8+ TIL expansion compared with GITRL monotherapy. Moreover, GITRL/anti-PD1 dual therapy further improved anti-PD1-mediated reinvigoration of interferon gamma secretion by exhausted CD8 TIL from primary CRC. GITR is overexpressed on CD4+ and CD8+ TIL from pMMR CRC and CRLM. Agonistic targeting of GITR enhances exvivo human TIL functionality and may therefore be a promising approach for novel monotherapy or combined immunotherapies in primary pMRR CRC and CRLM.

Divergent clonal differentiation trajectories of T cell exhaustion.

Chronic antigen exposure during viral infection or cancer promotes an exhausted T cell (Tex) state with reduced effector function. However, whether all antigen-specific T cell clones follow the same Tex differentiation trajectory remains unclear. Here, we generate a single-cell multiomic atlas of T cell exhaustion in murine chronic viral infection that redefines Tex phenotypic diversity, including two late-stage Tex subsets with either a terminal exhaustion (Texterm) or a killer cell lectin-like receptor-expressing cytotoxic (TexKLR) phenotype. We use paired single-cell RNA and T cell receptor sequencing to uncover clonal differentiation trajectories of Texterm-biased, TexKLR-biased or divergent clones that acquire both phenotypes. We show that high T cell receptor signaling avidity correlates with Texterm, whereas low avidity correlates with effector-like TexKLR fate. Finally, we identify similar clonal differentiation trajectories in human tumor-infiltrating lymphocytes. These findings reveal clonal heterogeneity in the T cell response to chronic antigen that influences Tex fates and persistence.

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

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) fails to consistently elicit tumor rejection. Manipulation of intrinsic factors that inhibit Tcell effector function and neoantigen recognition may therefore improve TIL therapy outcomes. We previously identified the cytokine-induced SH2 protein (CISH) as a key regulator of Tcell functional avidity in mice. Here, we investigate the mechanistic role of CISH in regulating human Tcell effector function in solid tumors and demonstrate that CRISPR/Cas9 disruption of CISH enhances TIL neoantigen recognition and response to checkpoint blockade. Single-cell gene expression profiling was used to identify a negative correlation between high CISH expression and TIL activation in patient-derived TIL. A GMP-compliant CRISPR/Cas9 gene editing process was developed to assess the impact of CISH disruption on the molecular and functional phenotype of human peripheral blood Tcells and TIL. Tumor-specific Tcells with disrupted Cish function were adoptively transferred into tumor-bearing mice and evaluated for efficacy with or without checkpoint blockade. CISH expression was associated with Tcell dysfunction. CISH deletion using CRISPR/Cas9 resulted in hyper-activation and improved functional avidity against tumor-derived neoantigens without perturbing Tcell maturation. Cish knockout resulted in increased susceptibility to checkpoint blockade invivo. CISH negatively regulates human Tcell effector function, and its genetic disruption offers a novel avenue to improve the therapeutic efficacy of adoptive TIL therapy. This study was funded by Intima Bioscience, U.S. and in part through the Intramural program CCR at the National Cancer Institute.

Abstract 3104: Inverse correlation of circulating CD8+ T cells with tumor burden from immune checkpoint treatment in humanized orthotopic model of renal cell carcinoma

Abstract Background: Renal cell carcinoma (RCC) is a highly heterogeneous and metastatic disease with a widely varying prognosis. RCC is the first solid tumor treated with immune checkpoint blockade and approved by the FDA for showing improvement of clinical outcomes. Immune checkpoints physiologically regulate immune response via appropriate signals to T cells and maintain homeostasis, while pathologically they can be co-opted by cancer cells to evade the immune system. Here we developed a humanized (Hu)-mice model with adaptive transferred human immune cells to test the role of ICB [anti-PD-1/PD-L1, or anti-CTLA-4 antibody (Ab)] in an orthotopic human RCC tumor-bearing xenograft mouse model and analyzed the correlation of innate immune cells with tumor burden. Methods: Luciferase-tagged RCC cell line 786-O were injected intra-kidney into immune-deficient Rag2 mice. After tumor formation, mice were humanized by intraperitoneal injection of donor PBMCs (20×106/per mouse). Treatment groups included control, anti-PD-1/PD-L1 Abs or anti-CTLA-4 Ab (all Abs 200 µg/mouse, iv) weekly for 4 weeks. Tumor growth was measured by weekly bioluminescent imaging (BLI) and tumor weight at necropsy. The presence of human immune cells and tumor infiltrating lymphocytes was confirmed by flow cytometry and immunohistochemistry staining. The significance of the differences between groups was determined by student t test. The Pearson correlation coefficient was obtained by analysis of correlation between different study parameters. Results: BLI level showed a steady xenograft tumor progression for nine weeks. Anti-PD-1/PD-L1 Abs treatment trended toward reducing tumor BLI level while anti-CTLA-4 Ab significantly decreased tumor BLI level (p<0.01). Furthermore, anti-PD-1/PD-L1 Abs reduced lung metastasis with a significant decrease of BLI level compared to control group (p<0.05). Meanwhile, humanization was evidenced by robust, elevated levels of human CD45+ lymphocytes in the peripheral blood compared to non-reconstituted Rag2 mice. Infiltrated human CD3+CD8+ cells were found in mouse blood and spleen. Granzyme B+ cells, CD31+ cells, and pan-cytokeratin+ cells were found in tumors. Interestingly, we observed inverse correlation between circulating human CD8+ T cells and mouse RCC tumor burden. Conclusion: We successfully generated a humanized RCC mouse model which allows the RCC xenograft to grow steadily within the human immune system reconstituted Rag2 mice. Circulating CD8+ T cells are inversely correlated to tumor growth in this model. The PD-1/PD-L1 or CTLA-4-targeted immunotherapies showed distinct therapeutic effects in our humanized orthotopic xenograft model, which may lead to personalized treatment targeting different immune checkpoints to reinvigorate anti-tumor responses in RCC patients. Citation Format: Xin Zhang, Amita Bhattarai, Ravan Moret, Grace Maresh, Alicia Nicole Ray, David Woods, Rachel Graham, Maria Latis, Stephen Bardot, Li Li. Inverse correlation of circulating CD8+ T cells with tumor burden from immune checkpoint treatment in humanized orthotopic model of renal cell carcinoma [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 3104.

Targeting of Nrf2 improves antitumoral responses by human NK cells, TIL and CAR T cells during oxidative stress

BackgroundAdoptive cell therapy using cytotoxic lymphocytes is an efficient immunotherapy against solid and hematological cancers. However, elevated levels of reactive oxygen species (ROS) in the hostile tumor microenvironment can impair...

Anti-TIGIT antibodies promote immune activation relevant to targeting stem-like and tumor-specific T cells in combination with anti-PD-1

Abstract TIGIT is an inhibitory receptor expressed primarily on NK and T cell subsets, and binding to its cognate receptor ligand, CD155, results in multiple mechanisms of immunosuppression. Blocking the TIGIT-CD155 interaction in the context of cancer promotes anti-tumor immunity. We characterized cellular subsets that TIGIT blockade may impact and the pharmacology of two anti-TIGIT antibodies - representing functional (AB308) and non-functional (AB154) Fc domain classes - undergoing clinical evaluation. Surrogate antibodies were leveraged to interrogate TIGIT biology in mouse syngeneic tumor models. Human tumor-infiltrating lymphocytes from a variety of cancer types expressed appreciable levels of TIGIT on relevant immune populations, including Tregs and CD8+ T cells with tumor reactive or pre-dysfunctional stem-like phenotypes. Both antibodies potently bound TIGIT and blocked the TIGIT-CD155 interaction as well as displayed the predicted phenotypes in terms of Fcγ receptor (FcγR) engagement. In line with FcγRIII binding, AB308 demonstrated a capacity to induce ADCC against TIGIT-expressing target cells. Combination of anti-TIGIT antibodies with other therapeutic approaches that promote T cell activation resulted in enhanced immune responses. In mice, while combining Fc-silent or Fc-enabled anti-mouse TIGIT antibody with anti-PD-1 resulted in greater tumor growth inhibition than with anti-PD-1 alone, the activity of Fc-enabled anti-TIGIT was associated with intratumoral Treg depletion. These data provide a rationale for combination with immune-activating agents and support ongoing clinical evaluation of AB154 and AB308 with biomarker strategies focused on understanding the role of Fc functionality.

An activation to memory differentiation trajectory of tumor-infiltrating lymphocytes informs metastatic melanoma outcomes.

There is a need for better classification and understanding of tumor-infiltrating lymphocytes (TILs). Here, weapplied advanced functional genomics to interrogate 9,000 human tumors and multiple single-cell sequencing sets using benchmarked Tcell states, comprehensive Tcell differentiation trajectories, human and mouse vaccine responses, and other human TILs. Compared with other Tcell states, enrichment of T memory/resident memory programs was observed across solid tumors. Trajectory analysis of single-cell melanoma CD8+ TILs also identified a high fraction of memory/resident memory-scoring TILs in anti-PD-1 responders, which expanded post therapy. In contrast, TILs scoring highly for early Tcell activation, but not exhaustion, associated with non-response. Late/persistent, but not early activation signatures, prognosticate melanoma survival, and co-express with dendritic cell and IFN-γ response programs. These data identify an activation-like state associated to poor response and suggest successful memory conversion, above resuscitation of exhaustion, is an under-appreciated aspect of successful anti-tumoral immunity.

Multimodal single-cell analysis of human TILs across multiple tumor types reveals heterogeneity and potential opportunities for personalized immunotherapy

Abstract Immune checkpoint blockade (ICB) efficacy varies among tumor types likely due to differences in tumor infiltrating lymphocyte (TIL) composition and function within the tumor microenvironment (TME). To help understand these differences, we conducted multimodal single-cell analysis of TILs including single-cell RNA sequencing (scRNA-seq), CITE-seq (oligo-tagged antibodies), and scTCR-seq (10× Genomics) in (non-small cell lung cancer: NSCLC; head and neck squamous cell carcinoma: HNSCC; renal cell carcinoma: RCC; and breast cancer: BrCa; n=48). We found that regulatory T cell (Treg) frequency was higher in HNSCC, whereas exhausted T cells (Tex) were higher in NSCLC and RCC. In contrast to other tumor types, Tex in RCC lacked the expression of CD103, a hallmark of tissue-resident T cells. On the other hand, expression of PD-1, TIM-3, and LAG-3 were more prominent in Tex in RCC. Interestingly, Tex in HNSCC showed higher expression of TIGIT than other tumor types. Previous work has demonstrated an increased presence of CD4+CD8+ double-positive T cells (DPT) in RCC, which was associated with better overall survival. Therefore, we used CITE-seq to identify DPT and then compared the composition of DPT among different tumor types. DPT were CD39+, a marker for tumor-reactive T cells, and the vast majority were transcriptionally categorized as CD8+ T cells in RCC, whereas DPT in other tumor types are mixture of CD4+ or CD8+ T cell subsets. We also found overlap of TCR profiles between DPT and CD8+ T cell subsets (Tex, ZNF683-CD8, and GZMK-CD8) in RCC. Together, multimodal single-cell analysis of TILs highlighted heterogeneity among tumor types that may provide insight into novel strategies to treat cancer. Supported by a research grant from GlaxoSmithKline and the Providence Portland Medical Foundation. Supported by a research grant from GlaxoSmithKline and the Providence Portland Medical Foundation.

Immunological profiling of tumor-infiltrating CD8+ T lymphocytes in non-small cell lung cancer, head and neck squamous cell carcinoma, breast cancer, and renal cell cancer

Abstract To explore whether the variation in clinical response to immune checkpoint blockade (ICB) reflects intrinsic characteristics of tumor-infiltrating lymphocytes (TIL), TILs from multiple tumor types were analyzed by multiparameter flow cytometry. Recent work identified CD39+CD103+ double positive (DP) CD8 TIL as tumor-reactive, therefore we assessed this phenotype in primary NSCLC (n=28), BCa (n=23), HNSCC (n=23), and RCC (n=23) specimens. TILs from NSCLC and HNSCC had significantly higher frequencies of DP T cells than BCa and RCC (NSCLC: median=34.2, IQR=15.0–53.8%; HNSCC: median=28.6%, IQR=13.5–44.5%), while TILs from BCa and RCC had low frequencies of DP T cells (BCa: median=2.4%, IQR=1.1–3.9%; RCC: median=4.2%, IQR=1.5–16.6%). Additionally, DP cells in NSCLC and HNSCC were co-expressed immune checkpoint markers PD-1, TIM-3, and LAG-3 at a higher frequency than RCC or BCa (P<0.05). DP CD8 T cells in NSCLC and HNSCC also exhibited increased effector potential (granzyme B+) as compared to BCa and RCC, suggesting functional differences. Expression of the transcription factor Eomesodermin, associated with T cell exhaustion, was higher in effector CD8 T cells from RCC (median=84.5%, IQR=44.9–97.4%) as compared to NSCLC (median=28.7%, IQR=20.7–61.4%), BCa (median=30.1%, IQR=0–50%), and HNSCC (median=40.3%, IQR=20.1–61.2%). Additionally, 4-1BB, an indicator of TCR engagement, trended higher in RCC (mean=8.6%, IQR=1.4–46.7%) as compared to BCa (median=1.9%, IQR=0.4–8.6%). These data highlight the heterogeneity of human TILs isolated from distinct tumor types and provide insight into the basal expression of actionable therapeutic targets. Supported by GlaxoSmithKline and the Providence Portland Medical Foundation

B cells imprint adoptively transferred CD8+ T cells with enhanced tumor immunity

BackgroundAdoptive T cell transfer (ACT) therapy improves outcomes in patients with advanced malignancies, yet many individuals relapse due to the infusion of T cells with poor function or persistence. Toll-like...

Isolation of TCR genes with tumor-killing activity from tumor-infiltrating and circulating lymphocytes in a tumor rejection cynomolgus macaque model

To develop effective adoptive cell transfer therapy using T cell receptor (TCR)-engineered T cells, it is critical to isolate tumor-reactive TCRs that have potent anti-tumor activity. In humans, tumor-infiltrating lymphocytes (TILs) have been reported to contain CD8+PD-1+ T cells that express tumor-reactive TCRs. Characterization of tumor reactivity of TILs from non-human primate tumors could improve anti-tumor activity of TCR-engineered T cells in preclinical research. In this study, we sought to isolate TCR genes from CD8+PD-1+ T cells among TILs in a cynomolgus macaque model of tumor transplantation in which the tumors were infiltrated with CD8+ T cells and were eventually rejected. We analyzed the repertoire of TCRα and β pairs obtained from single CD8+PD-1+ T cells in TILs and circulating lymphocytes and identified multiple TCR pairs with high frequency, suggesting that T cells expressing these recurrent TCRs were clonally expanded in response to tumor cells. We further showed that the recurrent TCRs exhibited cytotoxic activity to tumor cells in vitro and potent anti-tumor activity in mice transplanted with tumor cells. These results imply that this tumor transplantation macaque model recapitulates key features of human TILs and can serve as a platform toward preclinical studies of non-human primate tumor models.

An Engineered IL15 Cytokine Mutein Fused to an Anti-PD1 Improves Intratumoral T-cell Function and Antitumor Immunity.

The use of cytokines for immunotherapy shows clinical efficacy but is frequently accompanied by severe adverse events caused by excessive and systemic immune activation. Here, we set out to address these challenges by engineering a fusion protein of a single, potency-reduced, IL15 mutein and a PD1-specific antibody (anti-PD1-IL15m). This immunocytokine was designed to deliver PD1-mediated, avidity-driven IL2/15 receptor stimulation to PD1+ tumor-infiltrating lymphocytes (TIL) while minimally affecting circulating peripheral natural killer (NK) cells and T cells. Treatment of tumor-bearing mice with a mouse cross-reactive fusion, anti-mPD1-IL15m, demonstrated potent antitumor efficacy without exacerbating body weight loss in B16 and MC38 syngeneic tumor models. Moreover, anti-mPD1-IL15m was more efficacious than an IL15 superagonist, an anti-mPD-1, or the combination thereof in the B16 melanoma model. Mechanistically, anti-PD1-IL15m preferentially targeted CD8+ TILs and single-cell RNA-sequencing analyses revealed that anti-mPD1-IL15m treatment induced the expansion of an exhausted CD8+ TIL cluster with high proliferative capacity and effector-like signatures. Antitumor efficacy of anti-mPD1-IL15m was dependent on CD8+ T cells, as depletion of CD8+ cells resulted in the loss of antitumor activity, whereas depletion of NK cells had little impact on efficacy. The impact of anti-hPD1-IL15m on primary human TILs from patients with cancer was also evaluated. Anti-hPD1-IL15m robustly enhanced the proliferation, activation, and cytotoxicity of CD8+ and CD4+ TILs from human primary cancers in vitro, whereas tumor-derived regulatory T cells were largely unaffected. Taken together, our findings showed that anti-PD1-IL15m exhibits a high translational promise with improved efficacy and safety of IL15 for cancer immunotherapy via targeting PD1+ TILs.See related Spotlight by Felices and Miller, p. 1110.

Alternative Splicing of the Inhibitory Immune Checkpoint Receptor SLAMF6 Generates a Dominant Positive Form, Boosting T-cell Effector Functions.

SLAMF6 is a homotypic receptor of the Ig-superfamily associated with progenitor-exhausted T cells. Here we show that in humans, SLAMF6 has three splice isoforms involving its V-domain. Although the canonical receptor inhibited T-cell activation through SAP recruitment, the short isoform SLAMF6Δ17-65 had a strong agonistic effect. The costimulatory action depended on protein phosphatase SHP1 and led to a cytotoxic molecular profile mediated by the expression of TBX21 and RUNX3. Patients treated with immune checkpoint blockade showed a shift toward SLAMF6Δ17-65 in peripheral blood T cells. We developed splice-switching antisense oligonucleotides (ASO) designed to target the relevant SLAMF6 splice junction. Our ASOs enhanced SLAMF6Δ17-65 expression in human tumor-infiltrating lymphocytes and improved their capacity to inhibit human melanoma in mice. The yin-yang relationship of SLAMF6 splice isoforms may represent a balancing mechanism that could be exploited to improve cancer immunotherapy.

Inhibition of vascular adhesion protein-1 enhances the anti-tumor effects of immune checkpoint inhibitors.

Modulation of the immunosuppressive tumor microenvironment (TME) is essential for enhancing the anti‐tumor effects of immune checkpoint inhibitors (ICIs). Adhesion molecules and enzymes such as vascular adhesion protein‐1 (VAP‐1), which are expressed in some cancers and tumor vascular endothelial cells, may be involved in the generation of an immunosuppressive TME. In this study, the role of VAP‐1 in TME was investigated in 2 murine colon cancer models and human cancer cells. Intraperitoneal administration of the VAP‐1‐specific inhibitor U‐V296 inhibited murine tumor growth by enhancing IFN‐γ‐producing tumor antigen‐specific CD8+ T cells. U‐V296 exhibited significant synergistic anti‐tumor effects with ICIs. In the TME of mice treated with U‐V296, the expression of genes associated with M2‐like macrophages, Th2 cells (Il4, Retnla, and Irf4), angiogenesis (Pecam1), and fibrosis (Acta2, Loxl2) were significantly decreased, and the Th1/Th2 balance was increased. H2O2, an enzymatic product of VAP‐1, which promoted the production of IL‐4 by mouse Th2 and inhibited IFN‐γ by mouse Th1 and human tumor‐infiltrating lymphocytes, was decreased in tumors and CD31+ tumor vascular endothelial cells in the TMEs of mice treated with VAP‐1 inhibitor. TCGA database analysis showed that VAP‐1 expression was a negative prognostic factor in human cancers, exhibiting a significant positive correlation with IL‐4, IL4R, and IL‐13 expression and a negative correlation with IFN‐γ expression. These results indicated that VAP‐1 is involved in the immunosuppressive TMEs through H2O2‐associated Th2/M2 conditions and may be an attractive target for the development of combination cancer immunotherapy with ICIs.

FcγRIIB engagement drives agonistic activity of Fc-engineered αOX40 antibody to stimulate human tumor-infiltrating T cells

BackgroundOX40 (CD134) is a costimulatory molecule of the tumor necrosis factor receptor superfamily that is currently being investigated as a target for cancer immunotherapy. However, despite promising results in murine...

Abstract 2175: KSQ-001: A CRISPR/Cas9-engineered tumor infiltrating lymphocyte (eTILTM) therapy for solid tumors

Abstract Adoptive cell therapy (ACT) with ex vivo expanded tumor infiltrating lymphocytes (TIL) offers a potentially transformative treatment for treatment refractory solid tumors. However, the immunosuppressive tumor microenvironment (TME) limits the effectiveness of TIL therapy. To systematically identify targets that have the potential to improve T-cell function in the TME, we conducted two genome-wide CRISPR/Cas9 functional screens of immune cells using our proprietary CRISPRomics® platform. The first screen employed primary mouse T cells and assessed in vivo T cell infiltration into tumors by measuring sgRNA guide enrichment. Notably, this screen identified clinically active targets, such as PD-1, and identified multiple targets, including Cell Therapy-1 (CT-1), that demonstrated anti-tumor T-cell function superior to PD-1. The potential of inactivating CT-1 for ACT was subsequently validated in both PD-1 sensitive and insensitive syngeneic tumor models in the absence of any additional conditioning regimens. CT-1 edited T cells established durable anti-tumor memory characterized by a T central memory state. A second genome-wide screen employed human TIL and assessed the impact of gene inactivation on human TIL expansion under standard manufacturing conditions. This screen also identified CT-1 as a top target. We therefore prioritized the development of KSQ-001, an engineered TIL (eTILTM) therapy, created via CRISPR/Cas9-mediated editing of CT-1. Potent and selective sgRNAs targeting CT-1 were identified and characterized, and manufacturing methods to engineer human TIL with high efficiency have been developed. Together, these data demonstrate that our CRISPRomics® platform enables comprehensive target identification and validation of compelling new targets for the development of robust eTILTM therapies, and support the clinical evaluation of KSQ-001 as a next generation adoptive cell therapy in a variety of treatment-refractory solid tumors. Citation Format: Mike Schlabach, Nick Colletti, Anja Hohmann, Christopher Wrocklage, Hugh Gannon, Alejandra Falla, Noah Tubo, Robert A. LaMothe, Caroline Bullock, Pete T. Mitchell, Nafeeza Hafeez, Suphinya Sathithloetsakun, Paula E. Grasberger, Isabelle Le Mercier, Tracy E. Garrett, Sean Keegan, Sol Shenker, Charlotte Franco, Frank Stegmeier, Gregory V. Kryukov, Louise Cadzow, Micah Benson. KSQ-001: A CRISPR/Cas9-engineered tumor infiltrating lymphocyte (eTILTM) therapy for solid tumors [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 2175.