Intratumoral T-cell Research Articles

EXTH-13. COMBINED ANTI-ANGIOGENIC TREATMENT TO INCREASE INTRATUMORAL PERSISTENCE OF CAR T-CELLS IN LUNG CANCER BRAIN METASTASES

Abstract BACKGROUND Metastatic lung cancer, especially when it spreads to the brain, shows a devastating prognosis. Immunotherapeutic approaches including CAR T-cells show promise in hematologic malignancies. However, the presence of immunosuppressive factors within the tumor microenvironment and physical barriers like elevated interstitial pressure due to dysfunctional tumor vessels are one of the main obstacles especially in solid tumors. Evaluation of the potential synergy between anti-angiogenetic drugs and CAR T-cells in this context are an ongoing investigation. METHODS Here, we set up an immunocompetent syngeneic orthotopic cerebral metastasis model in mice by combining a chronic cranial window with repetitive intracerebral two-photon laser scanning microscopy (TPLSM). This model allows in vivo characterization of fluorescent tumor cells and CAR T-cells on a single cell level over time. Intraparenchymal injection of red fluorescent EpCAM-transduced Lewis Lung carcinoma cells (EpCAM/tdtLL/2 cells) followed by injection of EpCAM-directed or undirected CAR T-cells into the adjacent brain tissue was performed. Additionally, aVEGF-A combined with aVEGF-R2 and the respective isotype control were injected intraperitoneally. RESULTS Compared to undirected CAR T-cells, mice receiving EpCAM-directed CAR T-cells showed higher intratumoral CAR T-cell densities after intraparenchymal injection. This finding was accompanied with reduced tumor growth and eventually translates into a survival benefit. However, intratumoral CAR T-cell numbers diminish over time indicating insufficient persistence inside of the tumor microenvironment. Additional anti-VEGF-A/VEGF-R2 treatment results in reduced tumor growth after EpCAM/GFPCAR T-cell and GFPT-cell treatment, respectively. Interestingly, in vivo imaging reveals higher intratumoral CAR T-cell numbers after anti-angiogenic treatment compared to isotype controls pointing towards enhanced CAR T-cell persistence resulting in a survival benefit. CONCLUSION Collectively, our findings indicate that locally injected CAR T-cells may safely induce relevant anti-tumor effects in brain metastases from lung cancer. Additional anti-angiogenic treatment provides a mechanism to enhance intratumoral CAR T-cell persistence and reduce tumor growth.

IMMU-14. TRANS-SPECIES ANALYSIS OF REPLICATION-REPAIR DEFICIENT (RRD) MEDULLOBLASTOMA AND RESPONSE TO IMMUNE CHECKPOINT INHIBITION: AN IRRDC REPORT

Abstract BACKGROUND Replication-repair deficiency (RRD) stemming from mismatch repair and polymerase proofreading gene defects (MMRD/PPD) lead to hypermutant childhood cancers, most frequently brain tumors. While medulloblastoma is reported, the clinical, genomic and immune landscape remains unknown. METHODS We analysed the genome, methylome, transcriptome (bulk, single-nuclei) and immune-microenvironment of the largest cohort of RRD-medulloblastoma patients enrolled by the International RRD Consortium and correlated these to clinical outcomes. RRD mice-models were used to preclinically assess response to immunotherapy. Results: RESULTS RRD-medulloblastoma (n=43) were enriched for anaplasia (61%) and localised disease (77%). Methylation/nano-string-based subgrouping failed to classify 40%, while the remaining clustered with the SHH-subgroup. Copy-number changes were notably infrequent (<20%). All tumors harboured hypermutation and microsatellite instability in contrast to non-RRD controls (p<0.0001). Pathogenic variants were frequent in POLE/POLD1 (80%), TP53 (48%) and SHH-pathway genes (PTCH1, SUFU, SMO: 56%). Interestingly, some tumors additionally had glioma-driver alterations (ATRX, NF1: ~50%), similar to RRD cerebellar glioblastoma, but distinct from both non-RRD medulloblastoma (n=791) and hemispheric glioblastoma (n=733) controls. Moreover, although bulk-transcriptome matched non-RRD SHH medulloblastoma, single-nuclei analyses suggested an earlier cell-of-origin potentially explaining the heterogenous phenotypes resulting from the driver mutations. Uniquely, immune analyses (gene expression and immunohistochemistry) demonstrated high intra-tumoral CD8 T-cell infiltration. Three-year progression-free survival was 60%. Outcome was worse for RRD-medulloblastoma harbouring TP53-mutation (p=0.04) and failing subgroup-classification (p=0.004). Nestin/Cre MSH2-/- POLE;p.S459F mice developing medulloblastoma recapitulated human disease and demonstrated response to anti-PD1 monotherapy. Recurrent/progressive human tumors including those harbouring TP53-mutations exhibited radiological responses to anti-PD1 monotherapy, which was associated with prolonged ongoing survival in comparison to those not treated with checkpoint-inhibitors (p=0.02). CONCLUSIONS Hypermutant RRD-medulloblastoma reveal heterogenous phenotypes due to unique spectrum of driver mutations and an early cell of origin. Their immune-hot microenvironment allows successful salvage treatment with checkpoint-inhibitors for those failing chemo-radiation, including the high-risk SHH/TP53-mutant and unclassified subgroups.

IMMU-44. EXTRINSIC ELECTRICAL MODULATION OF THE GLIOBLASTOMA TUMOR MICROENVIRONMENT

Abstract BACKGROUND Approximately 30 to 50% of patients with glioblastoma (GBM) present with glioma-related epilepsy (GRE). Despite morbidity associated with seizures, GRE constitutes an independent positive predictive predictor for survival in GBM. Our prior results showed that repeated seizure induction through extrinsic electrical modulation (EEM, a technique in which an electrical stimulus sufficient to produce a therapeutic seizure is applied) slows intracranial tumor growth and prolongs overall survival in immunocompetent CT-2A glioma-bearing mice. However, the overall survival advantage following EEM treatment was diminished in immunocompromised mice bearing human patient derived xenografts. We hypothesized that EEM induces immune-related tumor microenvironment (TME) changes that prolong survival in mice. METHODS C57BL/6J mice were orthotopically implanted with CT-2A xenografts and subjected to EEM of 2.0 millicoulombs or sham treatment. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed at 3 and 24 hours to determine mRNA level of inflammatory cytokines. Immunohistochemistry (IHC) and bulk RNA sequencing analysis were utilized to assess CD3+ T-cell infiltration and identify immune-related secreted genes, respectively. Flow cytometry at 3 and 96 hours was utilized to characterize tumor-infiltrating immune cell populations. Samples were subjected to Percoll gradient enrichment of immune cell populations and stained for extracellular (CD45, CD4, CD8, CD11b, F4/80, CD80, Ly6G, Ly6C, and CX3CR1) and intracellular (TNF-a, Il-1b, CCL2, and Arg1) antigens. RESULTS RT-qPCR showed an increase in pro-inflammatory cytokines TNF-α (P = 0.0087) and IL-1β (P = 0.000349). IHC analysis demonstrated an increase in intratumoral CD3+ T-cells in EEM-treated samples (P = 0.0171). From 1855 upregulated genes, 3 immune-related secreted genes (C1QTNF4, FAM19A4, and GREM2) were identified. Flow cytometric analysis showed a significant increase in CD11b+, CX3RC1+ microglia (P = 0.0306) and CD45+, CD11b+ tumor-associated myeloid cells (TAMCs) (P = 0.0192) at 3 hours post-EEM. CONCLUSIONS EEM treatment promotes an inflammatory TME with increased intratumoral microglia, TAMCs and T-cell infiltration. These inflammatory changes may prolong survival of CT-2A glioma-bearing mice.

PD-1 blockade does not improve efficacy of EpCAM-directed CAR T-cell in lung cancer brain metastasis

BackgroundLung cancer brain metastasis has a devastating prognosis, necessitating innovative treatment strategies. While chimeric antigen receptor (CAR) T-cell show promise in hematologic malignancies, their efficacy in solid tumors, including brain metastasis, is limited by the immunosuppressive tumor environment. The PD-L1/PD-1 pathway inhibits CAR T-cell activity in the tumor microenvironment, presenting a potential target to enhance therapeutic efficacy. This study aims to evaluate the impact of anti-PD-1 antibodies on CAR T-cell in treating lung cancer brain metastasis.MethodsWe utilized a murine immunocompetent, syngeneic orthotopic cerebral metastasis model for repetitive intracerebral two-photon laser scanning microscopy, enabling in vivo characterization of red fluorescent tumor cells and CAR T-cell at a single-cell level over time. Red fluorescent EpCAM-transduced Lewis lung carcinoma cells (EpCAM/tdtLL/2 cells) were implanted intracranially. Following the formation of brain metastasis, EpCAM-directed CAR T-cell were injected into adjacent brain tissue, and animals received either anti-PD-1 or an isotype control.ResultsCompared to controls receiving T-cell lacking a CAR, mice receiving EpCAM-directed CAR T-cell showed higher intratumoral CAR T-cell densities in the beginning after intraparenchymal injection. This finding was accompanied with reduced tumor growth and translated into a survival benefit. Additional anti-PD-1 treatment, however, did not affect intratumoral CAR T-cell persistence nor tumor growth and thereby did not provide an additional therapeutic effect.ConclusionCAR T-cell therapy for brain malignancies appears promising. However, additional anti-PD-1 treatment did not enhance intratumoral CAR T-cell persistence or effector function, highlighting the need for novel strategies to improve CAR T-cell therapy in solid tumors.

SAIL66, a next generation CLDN6-targeting T-cell engager, demonstrates potent antitumor efficacy through dual binding to CD3/CD137

BackgroundOvarian cancer remains a formidable challenge in oncology, necessitating innovative therapeutic approaches. Claudin-6 (CLDN6), a member of the tight junction molecule CLDN family, exhibits negligible expression in healthy tissues but...

T lymphocyte recruitment to melanoma brain tumors depends on distinct venous vessels

To improve immunotherapy for brain tumors, it is important to determine the principal intracranial site of Tcell recruitment from the bloodstream and their intracranial route to brain tumors. Using intravital microscopy in mouse models of intracranial melanoma, we discovered that circulating Tcells preferably adhered and extravasated at a distinct type of venous blood vessel in the tumor vicinity, peritumoral venous vessels (PVVs). Other vascular structures were excluded as alternative Tcell routes to intracranial melanomas. Anti-PD-1/CTLA-4 immune checkpoint inhibitors increased intracranial Tcell motility, facilitating migration from PVVs to the tumor and subsequently inhibiting intracranial tumor growth. The endothelial adhesion molecule ICAM-1 was particularly expressed on PVVs, and, in samples of human brain metastases, ICAM-1 positivity of PVV-like vessels correlated with intratumoral Tcell infiltration. These findings uncover a distinct mechanism by which the immune system can access and control brain tumors and potentially influence other brain pathologies.

Chemotherapy drives tertiary lymphoid structures that correlate with ICI-responsive TCF1+CD8+ T cells in metastatic ovarian cancer.

Patients with high-grade serous ovarian carcinoma (HGSOC) are virtually insensitive to immune checkpoint inhibitors (ICIs) employed as standalone therapeutics, at least in part reflecting microenvironmental immunosuppression. Thus, conventional chemotherapeutics and targeted anticancer agents that not only mediate cytotoxic effects but also promote the recruitment of immune effector cells to the HGSOC microenvironment stand out as promising combinatorial partners for ICIs in this oncological indication. We harnessed a variety of transcriptomic, spatial and functional assays to characterize the differential impact of neo-adjuvant paclitaxel-carboplatin on the immunological configuration of paired primary and metastatic HGSOC biopsies as compared to NACT-naïve HGSOC samples from 5 independent patient cohorts. We found neo-adjuvant chemotherapy (NACT)-driven endoplasmic reticulum stress and calreticulin exposure in metastatic HGSOC lesions culminates with the establishment of a dense immune infiltrate including follicular T cells (TFH cells), a prerequisite for mature tertiary lymphoid structure (TLS) formation. In this context, TLS maturation was associated with an increased intratumoral density of ICI-sensitive TCF1+PD-1+ CD8+ T cells over their ICI-insensitive TIM-3+PD-1+ counterparts. Consistent with this notion, chemotherapy coupled with a PD-1-targeting ICI provided a significant survival benefit over either therapeutic approach in syngeneic models of HGSOC bearing high (but not low) tumor mutational burden. Altogether, our findings suggest that NACT promotes TLS formation and maturation in HGSOC lesions, de facto preserving an intratumoral ICI-sensitive T-cell phenotype. These observations emphasize the role of rational design, especially relative to the administration schedule, for clinical trials testing chemotherapy plus ICIs in patients with HGSOC.

TNFR1 signaling promotes pancreatic tumor growth by limiting dendritic cell number and function

Pancreatic adenocarcinoma (PDAC) is one the most intractable cancers, in part due to its highly inflammatory microenvironment and paucity of infiltrating dendritic cells (DCs). Here, we find that genetic ablation or antibody blockade of tumor necrosis factor receptor 1 (TNFR1) enhanced intratumor Tcell activation and slowed PDAC growth. While anti-PD-1 checkpoint inhibition alone had little effect, it further enhanced intratumor Tcell activation in combination with anti-TNFR1. The major cellular alteration in the tumor microenvironment in the absence of TNFR1 signaling was a large increase in DC number and immunostimulatory phenotype. This may reflect a direct effect on DCs, because TNF induced TNFR1-dependent apoptosis of bone-marrow-derived DCs. The therapeutic response to anti-TNFR1 alone was superior to the combination of DC-activating agonistic anti-CD40 and Flt3 ligand (Flt3L). These observations suggest that targeting TNFR1, perhaps in concert with other strategies that promote DC generation and mobilization, may have therapeutic benefits.

SOX12 Facilitates Hepatocellular Carcinoma Progression and Metastasis through Promoting Regulatory T-Cells Infiltration and Immunosuppression.

Despite the success of immunotherapy in treating hepatocellular carcinoma (HCC), HCC remains a severe threat to health. Here, a crucial transcription factor, SOX12, is revealed that induces the immunosuppression of liver tumor microenvironment. Overexpressing SOX12 in HCC syngeneic models increases intratumoral regulatory T-cell (Treg) infiltration, decreases CD8+T-cell infiltration, and hastens HCC metastasis. Hepatocyte-specific SOX12 knockout attenuates DEN/CCl4-induced HCC progression and metastasis, whereas hepatocyte-specific SOX12 knock-in accelerates these effects. Mechanistically, SOX12 transcriptionally activates C-C motif chemokine ligand 22 (CCL22) expression to promote the recruitment and suppressive activity of Tregs. Moreover, SOX12 transcriptionally upregulates CD274 expression to suppress CD8+T-cell infiltration. Either knockdown of CCL22 or PD-L1 dampens SOX12-mediated HCC metastasis. Blocking of CC chemokine receptor 4 (CCR4), a receptor for CCL22, by inhibitor C-021 or Treg-specific knockout of CCR4 inhibits SOX12-mediated HCC metastasis. Transforming growth factor-β1 (TGF-β1)/TGFβR1-Smad2/3/4 is identified as a key upstream signaling for SOX12 overexpression in HCC cells. Combining C-021 or TGFβR1 inhibitor galunisertib with anti-PD-L1 exhibits an enhanced antitumor effect in two HCC models. Collectively, the findings demonstrate that SOX12 contributes to HCC immunosuppression through the CCL22/CCR4-Treg and PD-L1-CD8+T axes. Blocking of CCR4 or TGFβR1 improves the efficacy of anti-PD-L1 in SOX12-mediated HCC.

Intratumoral T-cell composition predicts epcoritamab-based treatment efficacy in B-cell non-Hodgkin lymphomas.

Leveraging endogenous tumor-resident T-cells for immunotherapy using bispecific antibodies (BsAb) targeting CD20 and CD3 has emerged as a promising therapeutic strategy for patients with B-cell non-Hodgkin lymphomas. However, features associated with treatment response or resistance are unknown. To this end, we analyzed data from patients treated with epcoritamab-containing regimens in the EPCORE NHL-2 trial (NCT04663347). We observed downregulation of CD20 expression on B-cells following treatment initiation both in progressing patients and in patients achieving durable complete responses (CR), suggesting that CD20 downregulation does not universally predict resistance to BsAb-based therapy. Single-cell immune profiling of tumor biopsies obtained following one cycle of therapy revealed substantial clonal expansion of cytotoxic CD4+ and CD8+ T-cells in patients achieving CR, and an expansion of follicular helper and regulatory CD4+ T-cells in patients whose disease progressed. These results identify distinct tumor-resident T-cell profiles associated with response or resistance to BsAb therapy.

Differences in tumor-associated T-cell receptor repertoires between early-onset and average-onset colorectal cancer.

The incidence of colorectal cancer (CRC) among individuals younger than age 50 (early-onset CRC [EOCRC]) has substantially increased, and yet the etiology and molecular mechanisms underlying this alarming rise remain unclear. We compared tumor-associated T-cell repertoires between EOCRC and average-onset CRC (AOCRC) to uncover potentially unique immune microenvironment-related features by age of onset. Our discovery cohort included 242 patients who underwent surgical resection at Cleveland Clinic from 2000 to 2020. EOCRC was defined as younger than age 50 years at diagnosis (N = 126) and AOCRC as 60 years of age or older (N = 116). T-cell receptor (TCR) abundance and clonality were measured by immunosequencing of tumors. Logistic regression models were used to evaluate the associations between TCR repertoire features and age of onset, adjusting for sex, race, tumor location, and stage. Findings were replicated in 152 EOCRC and 1984 AOCRC cases from the Molecular Epidemiology of Colorectal Cancer Study. EOCRC tumors had significantly higher TCR diversity compared with AOCRC tumors in the discovery cohort (odds ratio [OR] = 0.44, 95% confidence interval [CI] = 0.32 to 0.61, P < .0001). This association was also observed in the replication cohort (OR = 0.74, 95% CI = 0.62 to 0.89, P = .0013). No significant differences in TCR abundance were observed between EOCRC and AOCRC in either cohort. Higher TCR diversity, suggesting a more diverse intratumoral T-cell response, is more frequently observed in EOCRC than AOCRC. Further studies are warranted to investigate the role of T-cell diversity and the adaptive immune response more broadly in the etiology and outcomes of EOCRC.

Intermittent MEK Inhibition with GITR Costimulation Rescues T-cell Function for Increased Efficacy with CTLA-4 Blockade in Solid Tumor Models.

MEK inhibitors (MEKi) have shown limited success as a treatment for MAPK/ERK pathway-dependent cancers due to various resistance mechanisms tumor cells can employ. CH5126766 (CKI27) is an inhibitor that binds to MEK and prevents release of RAF, reducing the relief of negative feedback commonly observed with other MEKis. We observed that CKI27 increased MHC expression in tumor cells and improved T cell-mediated killing. Yet, CKI27 also decreased T-cell proliferation, activation, and cytolytic activity by inhibiting the MAPK/ERK pathway that is activated downstream of T-cell receptor signaling. Therefore, we aimed to balance the positive and negative immunomodulatory effects of MEKis for optimal combination with immunotherapy. Intermittent administration of CKI27 allowed T cells to partially recover and costimulation via GITR and OX-40 agonist antibodies completely alleviated inhibition of function. In Kras mutant lung and colon tumor mouse models, intermittent CKI27 and anti-GITR significantly decreased tumor growth and prolonged survival when further combined with CTLA-4 immune checkpoint blockade. Moreover, this triple combination increased CD8+ and CD4+ T-cell proliferation, activation, and effector/memory subsets in the tumor-draining lymph nodes and tumors and led to intratumoral regulatory T-cell destabilization. These data, collectively, will allow for more informed decisions when optimizing combination regimens by overcoming resistance, reducing toxicity, and generating long-term immune responses.

Intratumoral T-cell infiltration and response to nivolumab plus ipilimumab in patients with metastatic clear cell renal cell carcinoma from the CheckMate-214 trial.

4536 Background: We have previously shown that levels of antigen-experienced but not terminally exhausted CD8+ TILs (i.e. CD8+PD1+TIM3-LAG3- TILs) are associated with improved clinical outcomes in patients (pts) with metastatic clear cell RCC (mccRCC) treated with nivolumab monotherapy within three independent clinical trials (CheckMate-010, CheckMate-025, HCRN GU16-260). Here, we aimed to evaluate the performance of this biomarker in patients with mccRCC treated with nivolumab plus ipilimumab (Nivo+Ipi) versus sunitinib (Sun) as part of the CheckMate-214 (CM-214) clinical trial. Methods: Pre-treatment, formalin fixed paraffin embedded (FFPE) tumor samples from the CM-214 clinical trial were stained with a multiplex immunofluorescence (mIF) assay and the density of CD8+PD1+TIM3-LAG3- TILs (mIF biomarker) was quantified using Halo image analysis software. The correlation between the mIF biomarker and clinical outcomes, including progression-free survival (PFS), and objective response rate (ORR), was evaluated using Cox proportional hazards and logistic regression models. The significance level was set at 5% (2-sided). Results: Following quality control, mIF data were obtained for 255 intermediate-and poor-risk patients (Nivo+Ipi= 136, Sun= 119). A comparison between patients with mIF biomarker data available and those without, showed no imbalance with regards to ORR and PFS in the Nivo+Ipi group. However, in the Sun group, pts with mIF biomarker data showed a higher ORR compared to pts without available data (33% vs 24%). In Nivo+Ipi-treated pts, the mIF biomarker measured as continuous variable showed a trend for a positive association with ORR (OR = 1.23, p = 0.1162) but no association with PFS (HR 0.97, p = 0.688). In Sun-treated pts, the mIF biomarker measured as continuous variable showed a trend for a positive association with ORR (OR = 1.32, p = 0.0726) and a significant positive association with PFS (HR= 0.78, p = 0.004. However, given that Sun-treated pts with available mIF data were enriched for responders, results obtained in the Sun group should be interpreted with caution. Conclusions: In contrast to Nivo monotherapy, pre-treatment levels of CD8+ PD1+ TIM-3-LAG-3- TILs were not associated with improved clinical outcomes to Nivo+Ipi. These results suggest that the baseline levels of intratumoral T-cell inflammation do not represent a strong determinant of response to anti-CTLA4-based therapy in mccRCC, which is consistent with the knowledge that anti-CTLA4 therapy (Ipi) can recruit new T cells into the tumor. The association between CD8+ PD1+ TIM-3-LAG-3- TILs and outcomes on sunitinib needs further investigation.

Inhibition of P21-activated kinases 1 and 4 synergistically suppresses the growth of pancreatic cancer by stimulating anti-tumour immunity

BackgroundPancreatic ductal adenocarcinoma (PDA) is one of the most lethal types of cancer, and KRAS oncogene occurs in over 90% of cases. P21-activated kinases (PAK), containing six members (PAK1 to 6), function downstream of KRAS. PAK1 and PAK4 play important roles in carcinogenesis, but their combinational effect remains unknown. In this study, we have determined the effect of dual inhibition of PAK1 and PAK4 in PDA progression using knockout (KO) cancer cell lines.MethodsMurine wild-type (WT) and PAK1KO pancreatic cancer cell lines were isolated from PAK1+/+ and PAK1−/− KPC (LSL-KrasG12D/+; LSL-Trp53 R172H/+; Pdx-1-Cre) mice. KPC PAK4KO and KPC PAK1&4 KO cell lines were generated from KPC WT and KPC PAK1KO cell lines respectively using the CRISPR-CAS9 gene knockout technique. PAK WT and KO cell lines were used in mouse models of pancreatic tumours. Cells and tumour tissue were also used in flow cytometry and proteomic studies. A human PDA tissue microarray was stained by immunohistochemistry.ResultsDouble knock out of PAK1 and PAK4 caused complete regression of tumour in a syngeneic mouse model. PAK4KO inhibited tumour growth by stimulating a rapid increase of cytotoxic CD8+ T cell infiltration. PAK1KO synergistically with PAK4KO increased cytotoxic CD8+ T cell infiltration and stimulated a sustained infiltration of CD8+ T cells at a later phase to overcome the immune evasion in the PAK4KO tumour. The human PDA tissue microarray study showed the important role of PAK1 and PAK4 in intra-tumoral T-cell function.ConclusionOur results demonstrated that dual inhibition of PAK1 and PAK4 synergistically suppressed PDA progression by stimulating cytotoxic CD8 + T cell response.

PVRIG is Expressed on Stem-Like T Cells in Dendritic Cell-Rich Niches in Tumors and Its Blockade May Induce Immune Infiltration in Non-Inflamed Tumors.

Cancers that are poorly immune infiltrated pose a substantial challenge, with current immunotherapies yielding limited clinical success. Stem-like memory T cells (TSCM) have been identified as a subgroup of T cells that possess strong proliferative capacity and that can expand and differentiate following interactions with dendritic cells (DCs). In this study, we explored the pattern of expression of a recently discovered inhibitory receptor poliovirus receptor-related immunoglobulin domain protein (PVRIG) and its ligand, poliovirus receptor-related ligand 2 (PVRL2), in the human tumor microenvironment. Using spatial and single-cell RNA transcriptomics data across diverse cancer indications, we found that among the T-cell checkpoints, PVRIG is uniquely expressed on TSCM and PVRL2 is expressed on DCs in immune aggregate niches in tumors. PVRIG blockade could therefore enhance TSCM-DC interactions and efficiently drive T-cell infiltration to tumors. Consistent with these data, following PVRIG blockade in patients with poorly infiltrated tumors, we observed immune modulation including increased tumor T-cell infiltration, T-cell receptor (TCR) clonality, and intratumoral T-cell expansion, all of which were associated with clinical benefit. These data suggest PVRIG blockade as a promising strategy to induce potent antitumor T-cell responses, providing a novel approach to overcome resistance to immunotherapy in immune-excluded tumors.

IL-7-primed bystander CD8 tumor-infiltrating lymphocytes optimize the antitumor efficacy of T cell engager immunotherapy

Bispecific Tcell engagers (TCEs) show promising clinical efficacy in blood tumors, but their application to solid tumors remains challenging. Here, we show that Fc-fused IL-7 (rhIL-7-hyFc) changes the intratumoral CD8 Tcell landscape, enhancing the efficacy of TCE immunotherapy. rhIL-7-hyFc induces a dramatic increase in CD8 tumor-infiltrating lymphocytes (TILs) in various solid tumors, but the majority of these cells are PD-1-negative tumor non-responsive bystander Tcells. However, they are non-exhausted and central memory-phenotype CD8 Tcells with high Tcell receptor (TCR)-recall capacity that can be triggered by tumor antigen-specific TCEs to acquire tumoricidal activity. Single-cell transcriptome analysis reveals that rhIL-7-hyFc-induced bystander CD8 TILs transform into cycling transitional Tcells by TCE redirection with decreased memory markers and increased cytotoxic molecules. Notably, TCE treatment has no major effect on tumor-reactive CD8 TILs. Our results suggest that rhIL-7-hyFc treatment promotes the antitumor efficacy of TCE immunotherapy by increasing TCE-sensitive bystander CD8 TILs in solid tumors.

Glucose-driven histone lactylation promotes the immunosuppressive activity of monocyte-derived macrophages in glioblastoma

Immunosuppressive macrophages restrict anti-cancer immunity in glioblastoma (GBM). Here, we studied the contribution of microglia (MGs) and monocyte-derived macrophages (MDMs) to immunosuppression and mechanisms underlying their regulatory function. MDMs outnumbered MGs at late tumor stages and suppressed Tcell activity. Molecular and functional analysis identified a population of glycolytic MDM expressing GLUT1 with potent immunosuppressive activity. GBM-derived factors promoted high glycolysis, lactate, and interleukin-10 (IL-10) production in MDMs. Inhibition of glycolysis or lactate production in MDMs impaired IL-10 expression and Tcell suppression. Mechanistically, intracellular lactate-driven histone lactylation promoted IL-10 expression, which was required to suppress Tcell activity. GLUT1 expression on MDMs was induced downstream of tumor-derived factors that activated the PERK-ATF4 axis. PERK deletion in MDM abrogated histone lactylation, led to the accumulation of intratumoral Tcells and tumor growth delay, and, in combination with immunotherapy, blocked GBM progression. Thus, PERK-driven glucose metabolism promotes MDM immunosuppressive activity via histone lactylation.

Macrophage-coated tumor cluster aggravates hepatoma invasion and immunotherapy resistance via generating local immune deprivation

Immune checkpoint inhibitors (ICIs) represent a promising treatment for hepatocellular carcinoma (HCC) due to their capacity for abundant lymphocyte infiltration. However, some patients with HCC respond poorly toICI therapy due to the presence of various immunosuppressive factors in the tumor microenvironment. Our research reveals that a macrophage-coated tumor cluster (MCTC) signifies a unique spatial structural organization in HCC correlating with diminished recurrence-free survival and overall survival in a total of 572 HCC cases from 3 internal cohorts and 2 independent external validation cohorts. Mechanistically, tumor-derived macrophage-associated lectin Mac-2 binding protein (M2BP) induces MCTC formation andtraps immunocompetent cells at the edge of MCTCs to induce intratumoral cytotoxic Tcell exclusion and local immune deprivation. Blocking M2BP with a Mac-2 antagonist might provide an effective approach to prevent MCTC formation, enhance Tcell infiltration, and thereby improve the efficacy of ICI therapy in HCC.

Abstract 1421: The HDAC6-specific inhibitor AVS100 (SS208) blocks M2 polarization of tumor associated macrophages and potentiates immunotherapy in preclinical tumor models

Abstract Inhibition of HDAC6 was associated with an increased proinflammatory tumor microenvironment and an antitumoral response. Here, we show that a highly specific HDAC6 inhibitor AVS100 (SS208), blocks M2 polarization in murine and human macrophages while partially affecting M1 polarization. AVS100 effects were observed as blocked upregulation of M2-related gene signature under M2 polarizing conditions and blocked generation of CD206+ and Arg1+ macrophages. Oral administration of AVS100 had an antitumoral effect in SM1 melanoma and CT26 colon cancer models and increased the efficacy of anti-PD1 treatment, leading to complete remission in melanoma and increased response in colon cancer. Flow cytometry and scRNAseq analysis of tumor-infiltrating immune cells revealed an increase of proinflammatory/anti-inflammatory ratio in tumor-associated macrophages as well as an increase of intratumoral CD8 effector T-cells after AVS100 treatment. Interestingly, cured mice didn’t relapse and became resistant to a subsequent tumor challenge, suggesting acquired antitumoral T-cell immunity. T-cell repertoire analysis of effector/memory T-cells in cured mice revealed a higher number of immunodominant T-cell clones after AVS100 treatment, indicating increased T-cell expansion. Finally, AVS100 has demonstrated no mutagenicity and a strong safety profile in rats and dogs, leading to its recent U.S. FDA clearance of an Investigational New Drug (IND) application and planned initiation of Phase Ia/b clinical trials targeting locally advanced or metastatic solid tumors in the first half of 2024. Altogether, we have performed the preclinical characterization of a novel small molecule inhibitor targeting HDAC6 for solid cancers. AVS100 had an antitumoral effect as single agent and improved the efficacy of immune checkpoint inhibition by blocking the immunoregulatory tumor microenvironment and increasing T-cell immunity. Citation Format: Damian Kovalovsky, Satish Noonepalle, Manasa Suresh, Dileep Kumar, Michael Berrigan, Anelia Horvath, Allen Kim, David Quiceno-Torres, Karthik Musunuri, Alejandro Villagra. The HDAC6-specific inhibitor AVS100 (SS208) blocks M2 polarization of tumor associated macrophages and potentiates immunotherapy in preclinical tumor models [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 1421.

Abstract 2488: The penile cancer tumor microenvironment and immunotherapy response: Results from the PERICLES trial

Abstract Introduction: The phase II PERICLES trial investigated the efficacy of atezolizumab (anti-PD-L1) for advanced penile cancer, with or without radiotherapy. As previously published, durable responses (progression-free at 12 months) were observed in 4 out of 32 enrolled patients. Here, we present the final overall survival (OS) results and an analysis of potential biomarkers for immunotherapy response. Methods: Preplanned analysis projected that at 27/32 events, the study would have 80% power (2-sided α=0.05) to assess whether the OS for the entire cohort significantly differed from the hypothesized historical median for stage IV patients. Baseline tumor tissue samples were subjected to bulk-RNA sequencing (n=31), fluorescent (n=28) and chromogenic (n=30) multiplex immunohistochemistry. We quantified the densities of CD3+CD8+ cytotoxic T-cells, CD3+CD8−FoxP3− T-helper cells, CD3+CD8−FoxP3+ regulatory T-cells, CD68+ macrophages and CD20+ B-cells in both the tumor and stromal compartments and assessed spatial relationships. We also quantified CD8+PD1+ (potentially enriched for tumor reactivity) and CD8+PD1− T-cells. Differential expression and gene set enrichment analysis (GSEA) were performed using the hallmarks gene sets. Results: The median OS for the entire study cohort was 11.3 months (95% CI, 5.5-16.7), which is an improvement compared to the hypothesized historical median of 7.8 months for stage IV patients (one sample log-rank p=0.033). Biomarker analyses showed an increased density of stromal macrophages (p&amp;lt;0.001) and intratumoral cytotoxic T-cells (p=0.035) in durable responders compared to non-responders. Additionally, we observed an increased density of intratumoral CD8+PD1+ in durable responders (p&amp;lt;0.001). In contrast, intratumoral CD8+PD1− densities were comparable between durable and non-responders (p=0.057). Patients with a high density of intratumoral CD8+PD1+ also had an improved progression-free survival (PFS) (log-rank, p&amp;lt;0.001) and OS (log-rank, p=0.002). Analysis of the spatial relationships in the tumor microenvironment (TME) showed that durable responders had larger distances between cytotoxic T-cells and T-helper cells (p=0.034). GSEA revealed significant (FDR≤5%) up-regulation of IFN-α/γ responses in durable responders. In contrast, non-responders showed up-regulation of epithelial-mesenchymal transition, KRAS signaling and angiogenesis, suggesting potential barriers to an anti-tumor response. Conclusion: In the final clinical analysis, OS was improved with atezolizumab compared to the historical population of stage IV patients. CD8+PD1+ T-cells, a common proxy for tumor-reactive T-cells, and spatial relationships in the TME might serve as biomarkers of durable response to immunotherapy in advanced penile cancer. Citation Format: Tynisha S. Rafael, Alberto Gil-Jimenez, Hielke M. de Vries, Iris M. Seignette, Elise Bekers, Marta Lopez-Yurda, Dennis Peters, Erik Hooijberg, Annegien Broeks, Oscar R. Brouwer, Eva Schaake, Daniel J. Vis, Tanja D. de Gruijl, Lodewyk F.A. Wessels, Michiel S. van der Heijden. The penile cancer tumor microenvironment and immunotherapy response: Results from the PERICLES trial [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 2488.