Human CD4 Research Articles

Abstract LB079: Cell-specific deletion of Bhlhe40 reveals distinct mechanisms underlying anti-PD-1 and anti-CTLA-4

Abstract T cell-dependent anti-tumor activity unleashed by immunotherapies such as immune checkpoint therapy (ICT) can generate durable clinical responses. However, not all patients benefit, and epigenetic and transcriptional features required by T cells for effective ICT remain to be fully established. We previously documented that the transcription factor Bhlhe40 is upregulated by ICT in tumor specific CD4 and CD8 T cells in preclinical tumor models. We validated the relevance of this by ablating Bhlhe40 in both CD4 and CD8-T cells which rendered αPD-1 or αCTLA-4 ICT ineffective in tumor models normally responsive to ICT. Our findings extended beyond preclinical models as we recently found that human tumor-specific T cells recognizing mutant neoantigens display upregulation of Bhlhe40 and CRISPR-mediated Bhlhe40 deletion in human CD8 T cells recognizing the MART-1 tumor-antigen abrogates IFNG expression. Since we previously used CD4-Cre Bhlhe40f/f (B40ΔT) mice where Bhlhe40 was deleted in both CD4 and CD8 T cells, we could not definitively delineate how Bhlhe40 was regulating CD4 versus CD8 T cells under ICT. We thus used a model where Bhlhe40 is deleted specifically in CD8 T cells (B40ΔCD8). We found that like B40ΔT mice, B40ΔCD8 mice bearing the 1956 sarcoma were insensitive to αPD-1 ICT. While B40ΔT mice were also insensitive to αCTLA-4 ICT, interestingly, B40ΔCD8 mice displayed nearly normal tumor rejection upon αCTLA-4 ICT. Thus, Bhlhe40 is required in CD8 T cells for αPD-1 response, whereas is dispensable in CD8 T cells for αCTLA-4 efficacy. Single-cell protein and transcriptomic analysis of intratumoral immune-cells from ICT-treated control B40f/f, B40ΔCD8 and B40ΔT mice revealed overlapping and distinct changes to CD4 and CD8 T cells depending on which cells had Bhlhe40 deleted and which treatment was employed. With αPD-1 or αCTLA-4, defects in expression of IFN-γ, glycolytic enzyme transcripts and cytotoxic molecules, along with increased IL-10 production in CD4 and CD8 T cells from B40ΔT mice and in B40−/− CD8 T cells in B40ΔCD8 mice was observed. Whereas αCTLA-4-treated B40ΔT mice failed to reject tumors, addition of αIL-10R led to modest tumor growth inhibition, while with αPD-1 treatment, IL-10R blockade had little effect on tumor growth. We also noted that in control B40f/f mice, αPD-1 predominately altered CD8 T cells, whereas αCTLA-4 modified CD8 and CD4 T cells, including induction of an ICOS+ IFNγ+ CD4 T cell population expressing Bhlhe40. This population was largely preserved in B40ΔCD8 mice undergoing αCTLA-4 ICT. Analysis of intratumoral macrophages revealed profound defects in ICT-induced macrophage remodeling from CX3CR1+CD206+ to iNOS+ macrophages in B40ΔT mice lacking Bhlhe40 in CD4 and CD8 T cells. Although αCTLA-4-treated B40ΔCD8 mice reject their tumors, we observed a modestly higher frequency of CX3CR1+CD206+ macrophages and lower frequency of iNOS+ macrophages as compared to αCTLA-4-treated B40f/f mice. The highest percentage of CX3CR1+CD206+ macrophages and the fewest iNOS+ macrophages was observed in αPD-1-treated B40ΔCD8 mice that completely fail to reject tumors. These results reveal distinct requirements of Bhlhe40 in CD4 and CD8 T cells that depend on which ICT is used and further highlight non-redundant mechanisms of effective αPD-1 and αCTLA-4 Citation Format: Akata Saha, Alexander S. Shavkunov, Shailbala Singh, Avery J. Salmon, Sunita Keshari, Qi Miao, Brian T. Edelson, Cassian Yee, Ken Chen, Matthew Gubin. Cell-specific deletion of Bhlhe40 reveals distinct mechanisms underlying anti-PD-1 and anti-CTLA-4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB079.

Abstract LB118: HM16390, a novel long-acting IL-2 analog with fine-tuned binding affinities to IL-2 receptor subunits for favorable safety profile, exhibits potent tumor killing effect in the various tumor syngeneic models

Abstract Although recombinant human IL-2 (rhIL-2, aldesleukin) produced approximately a 15% objective response rate in patients with renal cell carcinoma (RCC) and metastatic melanoma, its IL-2Rα (CD25)-biased binding and short half-life (t1/2 in human: 13-85 min) require a high dose and frequent dosing interval, leading to systemic toxicity such as vascular leak syndrome (VLS) and cytokine release syndrome (CRS). To overcome this limitation, various research groups are developing engineered IL-2 muteins with abolished CD25 binding affinity. While such IL-2 muteins may reduce CD25-mediated toxicity, there is a risk of a lopsided immune response. Furthermore, concomitantly reduced affinity to IL-2Rβ (CD122) may cause insufficient anti-tumor activity. We are developing HM16390, a long-acting IL-2 analog with a significantly increased binding affinity to CD122 for potent tumor killing effect. Simultaneously, optimal binding property to CD25 was introduced for a suppression of uncontrolled systemic immune response via peripheral Treg expansion. From these novel sequence modifications, HM16390 exhibited approximately 14-fold potent activity in human NK and CD8+ T cells compared to rhIL-2 and comparable activity with rhIL-2 in human Tregs (EC50 of STAT5 phosphorylation) without undesired release of cytokines associated with CRS. In B16F10 mouse model, HM16390 has a longer-lasting PK property (t1/2: 14.2~26.0 hr) after single subcutaneous (SC) administration. CD8+ T cells were significantly increased in peripheral blood along with a transient increase of peripheral Tregs, suggesting that HM16390 not only has a significant effect on expansion of CD8+ T cells but can simultaneously modulate exaggerated peripheral immune response. It is also noteworthy that, unlike in the peripheral, Tregs were drastically reduced in the tumor immune microenvironment, and CD8+ T cells were significantly increased. Next, in orthotopic model of mouse RCC (RENCA-luc cell implantation), across treatment groups, 40%(n=4/10) to 80%(n=8/10) of animals achieved complete tumor regression in dose-dependent manner after once weekly SC injection of HM16390 for 4 weeks. Furthermore, tumor nodules were not observed in lung and kidney after intravenous (IV) rechallenge of RENCA cells in the mice cured by HM16390 for at least 4 months after drug discontinuation. This indicates a long-term immunological memory response of generated memory cells by treatment of HM16390. In conclusion, HM16390 demonstrates potent and durable anti-tumor activity in various murine tumor models via its enhanced CD122 binding affinity, and incorporated optimal CD25 binding affinity may alleviate systemic toxicity by immunomodulatory role of peripheral Tregs. Citation Format: Sol-Bi Shin, Jinyoung Kim, Jaehyuk Choi, Seongju Jeong, Yunjae Kim, Jooyun Byun, Sungmin Bae, Daejin Kim, In Young Choi. HM16390, a novel long-acting IL-2 analog with fine-tuned binding affinities to IL-2 receptor subunits for favorable safety profile, exhibits potent tumor killing effect in the various tumor syngeneic models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB118.

Abstract LB067: Tumor-reactive CD8 TIL with an exhausted phenotype can be expanded and regress human tumors

Abstract Tumor-reactive human CD8 T cells are found predominantly in the tumor microenvironment with an exhausted phenotype (extremely high levels of CD39, PD-1, CTLA-4, and TIM-3). The tumor-reactive cells can be enriched from patients’ tumors by sorting CD8+ TIL that express both CD39 and CD103. Our unique expansion method allowed these phenotypically exhausted cells to grow from thousands into billions. After expansion the CD39 and CD103 Double Positive (DP) CD8 TIL can recognize/kill autologous tumor cells in vitro. We tested whether the CD8 DP TIL could recognize/kill autologous tumor in vivo using a xenograft model with immune-compromised mice that constitutively secrete human IL-2 (NOG-hIL-2). The CD8 DP TIL were able to persist long-term, traffic to the tumor site, recognize autologous tumor, and facilitate tumor regression. Human IL-2 was necessary for their long-term survival in the periphery and for tumor regression. These preclinical data were the basis for a human clinical trial design for the adoptive transfer of CD8 DP TIL (product termed, AGX148). We recently opened a Phase 1 clinical trial using the CD39/103 DP CD8 TIL in a first-in-human protocol for adults with solid tumors (NCT05902520). The trial design has three cohorts based on giving IL-2 for 2 weeks, 3 weeks, or 4 weeks after adoptive transfer with six patients/cohort (3 receiving DP TIL alone and 3 receiving DP TIL combined with PD-1 siRNA knockdown with the INTASYLTM compound PH-762). The first 3 patients have been treated and received 27, 40, and 8 billion DP TIL, respectively. All 3 patients had failed standard therapy prior to their enrollment, including checkpoint blockade. No serious adverse events were observed in the first three patients. Two of three patients had partial responses 29 days after treatment and the third patient had stable disease for >56 days. Blood draws and tumor biopsies were obtained prior to and after treatment and we are currently assessing the CD8 T cell repertoire (TCRseq) and phenotype from these patient samples. In conclusion, this is the first study showing that CD8 T cells with an exhausted phenotype isolated from human tumors can be expanded to billions and induce tumor regression in vivo. Citation Format: Andrew Weinberg, Brendan Curti, Ryan Montler, Colin Thalhofer, Elaine Ballinger, Jessica Cross, Eric Tran, Nelson Sanjuan, Matthew Taylor, Christopher Fountain, Roxanne Payne, James Cardia, Nicholas Morris. Tumor-reactive CD8 TIL with an exhausted phenotype can be expanded and regress human tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB067.

Abstract LB426: Evidence for a role of GPR35 in IDO1-mediated tumor immune escape by regulating Hippo-YAP pathway

Abstract IDO1 is an immune checkpoint regulator and mediator of tumor immune evasion (1). The cellular and molecular mechanisms of IDO1-mediated immune suppression are not fully understood. We describe here that GPR35, the putative receptor for kynurenic acid (KYNA), a metabolite of IDO1-mediated tryptophan catabolism, couples to Hippo-YAP pathway. GPR35 activation, either by overexpression or by treatment with tryptophan metabolites resulted in Hippo inactivation and YAP-directed transcription via TEAD family of transcription factors. Recently, YAP is found to be essential for tumor immune escape. Yap1 deficiency in T-lineage-specific knockout mice, or by dosing a YAP inhibitor, impairs Treg formation and function, leading to tumor growth inhibition (2). YAP also plays an inhibitory role in CD8 T cell function, especially in activated CTL usually found in the TME [3,4]. Through medicinal chemistry design and a structure-vs-activity relationship study effort, we discovered a GPR35-selective inhibitor, here termed TMER1i. TMER1i potently blocks GPR35 in Hippo suppression and YAP activation. We showed that GPR35 is expressed in the T-cell lineage of the immune cells including naïve CD4 T cells, Treg and CD8 T cells. Treatment of purified human naïve CD4 T cells with anti-CD3/anti-CD28 under an IDO1+ TME-like culture condition in vitro induced a robust T cell activation and differentiation toward Treg phenotype, as shown previously using mouse naïve CD4 T cells by others (5). Importantly, TMER1i efficiently blocked the Treg differentiation under these conditions. When dosed in human-PBMC reconstituted NCG mice bearing HT29 tumors, TMER1i dose-dependently increased the infiltration of human CD8 T cells within tumors in a statistically significant manner, and a trending lower Treg cell infiltration, accompanied by a tumor growth inhibition. qPCR analysis of the HT29 tumors grown in the PBMC-NCG mice indicated that IDO1 was highly expressed in the tumors. These results indicated that GPR35 selective inhibitor TMER1i enhanced an anti-tumor immune activity in an IDO1+ tumor microenvironment. Taken together, these studies provided evidence for a role of GPR35 in mediating IDO1 immune suppression by regulating Hippo-YAP pathway in Treg and CTL cells in the immune system. Thus, GPR35 inhibitors have a potential to be novel immuno-therapeutic agents effective in treating patients with IDO1-positive tumors. Reference1.Munn, DH, and Mellor, AL. (2007) Indoleamine 2,3-dioxygenase and tumor-induced tolerance. J. Clin. Invest. 117:1147-11542.Ni, X. et al. Yap is essential for Treg-mediated suppression of antitumor Immunity. Cancer Discovery 2018; 8:1026-10433.Lebid, A. et al. (2020) YAP attenuates CD8 T cell-mediated anti-tumor response. Frontiers in Immunology 11:1-84.Stampouloglou, E. et al. (2020) YAP suppresses T-cell function and infiltration in the tumor microenvironment. PLoS Biol 18(1): e30005915.Fallarino, F. et al. (2006) The combined effects of tryptophan starvation and tryptophan catabolites down-regulate T cell receptor æ-chain and induce a regulatory phenotype in naïve T cells. J Immunol 176:6752-6761 Citation Format: Hongmei He, Nam Hee Kim, Sangdon Han, Zhi Liang Chu. Evidence for a role of GPR35 in IDO1-mediated tumor immune escape by regulating Hippo-YAP pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB426.

Extracellular CIRP co-stimulated T cells through IL6R/STAT3 in pediatric IgA vasculitis

Immunoglobulin A vasculitis (IgAV) is the most common vasculitis of childhood. Disordered immune responses play important roles in its pathogenesis, but the comprehensive immune profile of the disease and the underlying mechanisms are still largely unknown. Here we found a potential disease biomarker cold inducible RNA binding protein (CIRP) in our pediatric IgAV cohort. Serum CIRP level in these patients were elevated and positively correlated with the increased early memory (CD45RA+CD62L+CD95+) T cells revealed using multicolor flow cytometry. Immune phenotyping of the patients showed they had more activated T cells with higher IL6Ra expression. T cell culture experiment showed CIRP further activated both human CD4+ and CD8+ T cells as indicated by increased perforin secretion and phosphorylation of STAT3. Blockade of IL6Rα attenuated CIRP-induced T cell toxicity in vitro. RNA-sequencing data further supported CIRP stimulation promoted human T cell activation and migration, fueled inflammation through the JAK-STAT signaling pathway. Therefore, IL6Ra-mediated T cell activation by extracellular CIRP may contribute to pathogenesis of IgAV in children, both CIRP and IL6Ra could be new therapeutic targets for IgAV.

DR30318, a novel tri-specific T cell engager for Claudin 18.2 positive cancers immunotherapy

BackgroundClaudin 18.2 (CLDN18.2) is a highly anticipated target for solid tumor therapy, especially in advanced gastric carcinoma and pancreatic carcinoma. The T cell engager targeting CLDN18.2 represents a compelling strategy for enhancing anti-cancer efficacy.MethodsBased on the in-house screened anti-CLDN18.2 VHH, we have developed a novel tri-specific T cell engager targeting CLDN18.2 for gastric and pancreatic cancer immunotherapy. This tri-specific antibody was designed with binding to CLDN18.2, human serum albumin (HSA) and CD3 on T cells.ResultsThe DR30318 demonstrated binding affinity to CLDN18.2, HSA and CD3, and exhibited T cell-dependent cellular cytotoxicity (TDCC) activity in vitro. Pharmacokinetic analysis revealed a half-life of 22.2–28.6 h in rodents and 41.8 h in cynomolgus monkeys, respectively. The administration of DR30318 resulted in a slight increase in the levels of IL-6 and C-reactive protein (CRP) in cynomolgus monkeys. Furthermore, after incubation with human PBMCs and CLDN18.2 expressing cells, DR30318 induced TDCC activity and the production of interleukin-6 (IL-6) and interferon-gamma (IFN-γ). Notably, DR30318 demonstrated significant tumor suppression effects on gastric cancer xenograft models NUGC4/hCLDN18.2 and pancreatic cancer xenograft model BxPC3/hCLDN18.2 without affecting the body weight of mice.

Abstract 7532: NB005.1, a fully humanized anti-KLRG1 blocking mAb enhances effector function of human CD8 T-cells

Abstract Introduction: Immune checkpoint blockers (ICBs) have revolutionized treatment, enhancing survival and benefiting many patients. However, many patients fail to respond to the treatment, necessitating research for new targets and improved approaches. The Killer cell Lectin-like Receptor G1 (KLRG1), expressed on a subset of NK and T-cells harbors an immune receptor tyrosine-based inhibitory motif (ITIM) within its cytoplasmic domain, indicating its inhibitory function. On CD8+ T-cells, KLRG1 is expressed on highly differentiated antigen-specific effector memory T-cells (TEMs) and CD45RA+ effector memory T cells (TEMRA). These cells are known to exhibit strong cytotoxicity potential and display developmental plasticity following KLRG1 blockade. And 16-48% of CD8+ T-cells that infiltrate the tumor microenvironment (TME) are KLRG1 positive. We hypothesize that the blockade of KLRG1 should enhance the effector function of KLRG1+ CD8 T-cells in the TME and provide therapeutic benefit as a standalone or in combination with existing ICBs. Methods: Monoclonal antibodies (mAbs) targeting the human KLRG1 extracellular domain (ECD) were generated through mouse immunization and hybridoma technology. Purified mAbs from positive clones were screened using CHO-K1 and Jurkat cells overexpressing human or cynomolgus KLRG1 and human primary CD8 T-cells (n=3) to determine EC50 values. Epitope binning was performed, and high-affinity clones from each bin with cyno cross-reactivity were humanized to IgG1-Fc LALA variant. The Abs were evaluated for ligand blocking and enhancement of CD8 T-cell effector function through multiple functional assays. The assay measured enhancement of IFNγ or TNFα by KLRG1+ human CD8+ T-cells co-cultured with CHO-K1 expressing OKT3 and E-cadherin as artificial antigen-presenting cells (aAPCs) or PBMCs stimulated in with anti-CD3 and anti-CD28 Abs in presence of soluble E-cadherin. A human IgG1-LALA antibody (Ab) served as a negative control. PD-1 and KLRG1 synergy was evaluated in hPBMC assays. Results: NB005.1 demonstrated single digit nanomolar (nM) binding potency on human primary CD8 T-cells and Jurkat cells expressing human KLRG1 as identified by its EC50 value. Blocking of KLRG1 with NB005.1 on purified human CD8 T-cells in co-culture assay with aAPC expressing E-cad and OKT3 led to CD8 T-cell activation and increase in IFNγ production confirming inhibitory function of KLRG1. Similar result was obtained when the Ab was tested on hPBMCs stimulated with anti-CD3 and anti-CD28 Abs in presence of soluble E-cadherin. Conclusion: NB005.1, a fully humanized mAb, binds to human KLRG1 in the single digit nM range and effectively blocks the interaction with its ligand. This blockade enhances T-cell effector function as measured by IFNγ production, supporting the advancement of NB005.1 for preclinical testing, either as a standalone therapeutic or in combination with existing ICBs. Citation Format: Bishnu P. Nayak, Meiguang Xiong, Yajuan Xue, Binbin Wang, Shahrzad Lighvani, Shilpi Sharma, Dong Wang, Haojai Wang, Baiyang Wang, Yu Zhang, Xin Dong. NB005.1, a fully humanized anti-KLRG1 blocking mAb enhances effector function of human CD8 T-cells [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 7532.

JAK/STAT signaling pathway affects CCR5 expression in human CD4+ T cells.

CCR5 serves as R5-tropic HIV co-receptor. Knocking out CCR5 in HIV patients, which has occurred <10 times, is believed important for cure. JAK/STAT inhibitors tofacitinib and ruxolitinib inhibit CCR5 expression in HIV+ viremic patients. We investigated the association of JAK/STAT signaling pathway with CCR5/CCR2 expression in human primary CD4+ T cells and confirmed its importance. Six of nine JAK/STAT inhibitors that reduced CCR5/CCR2 expression were identified. Inhibitor-treated CD4+ T cells were relatively resistant, specifically to R5-tropic HIV infection. Furthermore, single JAK2, STAT3, STAT5A, and STAT5B knockout and different combinations of JAK/STAT knockout significantly reduced CCR2/CCR5 expression of both RNA and protein levels, indicating that CCR5/CCR2 expression was positively regulated by JAK-STAT pathway in CD4+ T cells. Serum and glucocorticoid-regulated kinase 1 (SGK1) knockout affected CCR2/CCR5 gene expression, suggesting that SGK1 is involved in CCR2/CCR5 regulation. If cell surface CCR5 levels can be specifically and markedly down-regulated without adverse effects, that may have a major impact on the HIV cure agenda.

Abstract 4042: Cbl inhibition increases the response of cytotoxic T cells to sub-optimal stimulation and drives enhanced anti-tumor responses alone or in combination with PD-1 blockade

Abstract INTRODUCTION: Herein, we describe a series of experiments to define the unique biology of dual Cbl-b/c-Cbl (Cbl) inhibition in amplifying T cell activation and anti-tumor responses. Through their E3 ubiquitin ligase activity, Cbl-b and c-Cbl have been shown to negatively regulate multiple signaling proteins downstream of the T cell receptor (TCR), suggesting that inhibition may yield enhanced anti-tumor T cell activity. Indeed, using a dual Cbl-b/c-Cbl small molecule inhibitor, we demonstrate that Cbl blockade enhances T cell activity under standard and sub-optimal stimulation conditions. Moreover, Cbl inhibition increases mouse syngeneic tumor control alone or in combination with α-PD-1, as described below. METHODS: Human CD8+ T cells were isolated from healthy human blood and activated using α-CD3 or α-CD3/CD28 stimulation. Mouse OT-I splenocytes were stimulated with SIINFEKL or lower affinity ovalbumin peptides and cytokine production was assessed. For tumor studies, mice were inoculated with syngeneic cancer cells and were treated with Cbl inhibitor (30 mg/kg, QD) +/- α-PD-1 (10 mg/kg, Q5D) starting at a tumor volume of 75 mm3. RESULTS: Human CD8+ T cells, stimulated in the presence of a Cbl inhibitor, demonstrated a concentration-dependent increase in activation hallmarks including cytokine secretion (IL-2, IFN-γ and granzyme B), proliferation, and the presence of highly polyfunctional cells. Furthermore, using serially stimulated CD8+ T cells with reduced overall activity, Cbl inhibition showed a marked enhancement of IFN-γ production and cytotoxicity compared to controls upon re-stimulation. We then used an antigen-specific T cell assay where OT-I splenocytes were stimulated by peptides with optimal or reduced TCR affinities, Cbl inhibition produced significantly higher IFN-γ levels with all peptides relative to controls demonstrating that Cbl inhibition can increase the response of T cells to sub-optimal stimulation. Using mouse CT26 and MC38 tumor models, Cbl inhibition was able to significantly enhance tumor control in combination with α-PD-1 and as a monotherapy in CT26 tumors. Notably, a greater number of tumor antigen-specific cytotoxic T cells were found in Cbl inhibitor-treated tumors. Higher expression levels of the co-stimulatory molecule CD226 and the TCR in these anti-tumor cells were also observed, demonstrating the unique biology of Cbl inhibition in enhancing anti-tumor responses. Finally, Cbl inhibition had no effect on the growth or survival of cancer cells in vitro consistent with the notion that enhanced tumor control was driven by immune activity. CONCLUSIONS: Altogether, these data demonstrate that pharmacologic Cbl inhibition robustly enhances T cell activity in vitro and in vivo and provide a mechanistic rationale for targeting Cbl-b/c-Cbl to amplify anti-tumor immune responses. Citation Format: Livia Yamashiro, Sachie Marubayashi, Rameshwari Rayaji, Anudari Battsooj, Karim Mrouj, Hema Singh, Yue Tong Lee, Ada Chen, Lunda Shen, Artur Mailyan, Kai Yu, Masha Podunavac, Julie Yu, Dongdong Liu, Clayton Hardman, Johnny Yin, Srikanth Gangam, Ken Lawson, Manmohan Leleti, Matthew Walters, Daniel DiRenzo. Cbl inhibition increases the response of cytotoxic T cells to sub-optimal stimulation and drives enhanced anti-tumor responses alone or in combination with PD-1 blockade [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 4042.

Abstract 1689: CRISPR functional genetic screening for immune enhancers of inhibitor receptors in human primary CD8 T cells

Abstract The restrained efficacy of T cell therapies in cancer treatment is associated with T cell exhaustion (TEX), an impaired effector function state that is marked by distinct epigenetic landscape. These unique epigenetic features promoted the progression of TEX, hindered reinvigoration of TEX state and controlled the abnormal expression of inhibitor receptors (IRs), which could serve as target sites in cancer treatment. Thus, deciphering the regulatory elements in primary human T cells will deeply reveal the mechanism controlling TEX and provide novel therapeutic strategies to promote clinical response. However, how enhancers can be effectively applied in anti-cancer treatment is still poorly understood because high throughput identifying the functional enhancers of inhibitor receptors at scale is technically limited in human T cells. Here, we first developed a pooled screening platform that employs a compact CRISPRi system and enabled efficient one-step delivery of pooled screening libraries to primary human T cells. Leveraging this platform, we screened large genomic regions (~300kb) at saturation around key TEX genes, two IRs—PDCD1, HAVCR2 and one transcription factor (TF)—TBX21 to interrogate functional enhancers in human primary CD8 T cells. We identified two enhancers for PDCD1, three enhancers for HAVCR2 and three enhancers for TBX21, all of which are located over 12kb away from each gene’s transcription start sites. Then, to parse the essential sequences that fulfill enhancer function, we employed Cas9-based in-situ saturation mutagenesis screening to the identified enhancers of PDCD1 and HAVCR2. We found disrupt essential sequences impaired the entire enhancer function, quantitatively phenocopied the effect of targeting the entire enhancer with CRISPRi. Transcription factor motifs analyses of these sequences revealed potential regulation of ZFX for controlling PDCD1 expression and STAT for HAVCR2 expression. Finally, we explored the functional consequences of applying enhancers to anti-cancer treatment. Indeed, targeting the identified enhancers of PDCD1 and HAVCR2 in CAR T cells lead to enhanced cancer cell killing efficiency in vitro. Together, our study provides a platform and paradigm to interrogate functional enhancers in human T cells, illuminated the great potential of manipulating enhancers in T cell therapies. Citation Format: Ping Wang, Josephine R. Giles, Hua Huang, Junwei Shi, E.John Wherry. CRISPR functional genetic screening for immune enhancers of inhibitor receptors in human primary CD8 T cells [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 1689.

Abstract 1906: UNO-A001 (A001), a clinical candidate CD38 x CD3 bispecific with a novel structure, potently kills multiple myeloma cells in vitro and is highly effective in vivo

Abstract CD38 is a validated target for the treatment of multiple myeloma (MM) as evidenced by the important clinical activity of CD38 targeting monoclonal antibodies. Bispecific T Cell Engagers (TCEs) kill cancer cells by distinct mechanisms as compared to standard format antibody therapeutics and several TCEs have demonstrated impressive clinical activity, and have been approved for clinical use, in hematologic malignancies. Here we describe a novel bispecific leveraging the potent killing mechanism of TCEs to target CD38 bearing cancer cells. UNO-A001 is a TCE binding the cell surface receptor CD38 on target human cancer cells and CD3 on T cells. The molecule has a differentiated format, binding CD38 with a high affinity humanized camelid VHH moiety and CD3 on T cells through a unique antibody Fab arm. The affinities of each arm were optimized so that the CD38 arm bound with high affinity and the CD3 arm with much lower relative binding affinity. This configuration was designed to engage CD38 bearing target cells with multiple TCE molecules at lower doses and concentrations while minimizing T cell binding in the periphery, and in the absence of cell surface CD38. A001 potently and specifically killed human CD38 bearing multiple myeloma cell lines in T cell killing assays in vitro with EC50s in the 2.7-6.6 pM range. This potency, despite the designed lower affinity to CD3, may be due to the unique structure of A001 enabling a productive immunologic synapse between T cells and the target cancer cells, and an optimal TAA/CD3 binding affinity ratio. A001 was also highly effective in inhibiting tumor growth in H929 xenograft models. In a flank model of H929 tumor growth A001 dose dependently inhibited tumor growth with complete growth inhibition of established tumors at the highest dose level tested. In a systemic disease model with NCI-H929-luc cells A001 was also highly effective and improved survival relative to control treated animals. In preparation for clinical studies a robust and high yield production cell line was created and characterized. It achieved high expression levels of quality monodisperse UNO-A001 product that was stable in all relevant storage conditions. A001 has similar affinities for human and cynomolgus macaque CD38 and CD3. Dose ranging studies in cynomolgus macaques demonstrated tolerability consistent with clinically approved TCEs, and the expected dose dependent pharmacodynamic effects on CD38 expressing myeloid and lymphoid cell populations. These data support planned clinical testing of A001 in multiple myeloma and other CD38 bearing hematologic malignancies, including mantle cell lymphoma, T cell lymphoma, and acute myelogenous leukemia. Citation Format: Mark Anderson, Yan Ling, Mark L. Chiu, Peter Cheung. UNO-A001 (A001), a clinical candidate CD38 x CD3 bispecific with a novel structure, potently kills multiple myeloma cells in vitro and is highly effective in vivo [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 1906.

Abstract 4586: ITF3756 increases NKG2D expression and cytotoxic activity of activated human CD8 T cells

Abstract Effector cytolytic CD8 T cells play a major role in the recognition and killing of cancer cells. These cells are short living due to the loss of their self-renewal capacity and rapid exhaustion. Enduring antitumor immune response relies on the capacity of regenerating effector cells upon antigen re-encounter by memory cells. Focusing on molecules that could modulate the differentiation of T cells towards a memory subset with a prolonged anti-tumor activity is strategic for an efficient anti-tumor immunotherapy. Thus, we developed a selective HDAC6 inhibitor currently in Phase I clinical trial, ITF3756, which in vitro drives the differentiation of human CD8 T cells towards a central memory phenotype with reduced expression of exhaustion markers and higher expression of IFNγ and Granzyme b upon tumor cells coculture. This suggests that ITF3756-treated CD8 T cells could have a greater tumor killing activity. Moreover, if this augmented cytotoxic activity is directed not only towards tumor cells expressing MHC I but also towards MHC I negative cells, the anti-tumor efficacy could be improved. We thus selected the MHC I negative K562 leukemic cell line as target in an in vitro model of CD8 T cell killing assay. CD8 T cells were stimulated with anti CD3CD28 beads with or without ITF3756, and then co-cultivated with K562 cells for 4h. ITF3756 treatment improved significantly CD8 mediated K562 killing over this short period of time. However, since the effectiveness of memory cells is based on their capacity of maintaining a long-lasting immune response, we monitored the cytolytic activity over a period of 2 weeks. After 14 days of co-culture, ITF3756 treated CD8 T cells showed a higher cytotoxic capacity compared to control cells. These results indicate that ITF3756-treated CD8 T cells can kill a target tumor cell in a more efficient way independently of MHC I restricted tumor recognition, both after short and long term in vitro co-culture. Since it has been described that CD8 T cells can also kill MHC I-negative tumor cells through the NKG2D-NKG2DL axis, we assessed if ITF3756 could modulate the expression of NKG2D on CD8 T cells. Indeed, ITF3756-treated CD8 T cells had a significantly higher expression of NKG2D compared to untreated cells. In conclusion, we show that ITF3756 promotes the differentiation of cells that possess better effector activity when co-cultured with tumor cells for both short and long periods of time. ITF3756 increased the expression of NKG2D and this could be a driver of the increased cytolytic activity. One mechanism of tumor immune escape is the downregulation of MHC I. Our results suggest that ITF3756 could be used to improve the efficacy of T cell therapy by enhancing the cytolytic activity even in the absence of MHC I. Citation Format: Chiara Ripamonti, Valeria Spadotto, Christian Steinkuhler, Gianluca Fossati. ITF3756 increases NKG2D expression and cytotoxic activity of activated human CD8 T cells [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 4586.

Abstract 1365: INB03: a new immune checkpoint inhibitor that reprograms macrophage polarization, boosts ADCP and reverts T-cell exhaustion markers

Abstract Trastuzumab resistance is an important clinical issue in HER2+ breast cancer (BC), and few actionable targets are available. We demonstrated that soluble TNF (sTNF) upregulates mucin 4 (MUC4) expression, which shields trastuzumab epitope on HER2 hindering its therapeutic effect, and that sTNF blockade with INB03 decreases MUC4 expression which, together with trastuzumab, triggers an effective antitumor immune response based on antitumoral macrophage-NK cell collaboration. Because immune checkpoint molecules (ICP) and T cell exhaustion foster tumor immune escape, we addressed whether INB03 could modulate macrophage polarization, T cell exhaustion, and ICP expression in both populations to boost an antitumor immune response. For in vitro assays, human monocytes and T cells were isolated from buffy coats and enriched with specific RosetteSep. Macrophages (Mϕ) were differentiated for 6 days with M-CSF (10 ng/ml) to M0, and polarized for 48h to the M1-like subtype with LPS (50 ng/ml)+IFNγ (20 ng/ml), or to the M2-like subtype with IL-4 (20 ng/ml)+IL-10 (20 ng/ml). Then, INB03 was added (10 μg/ml) for 96h with corresponding cytokines to already-polarized Mϕ to test its ability to revert polarization. Also, Mϕ antibody-dependent cellular phagocytosis (ADCP) against human HER2+ breast cancer cell line JIMT-1 was assessed. T cells were activated with CD3/CD28 beads and cultured for 7 or 14 days. INB03 was added at those times for 48h to test its ability to modulate ICP and exhaustion markers. For in vivo assays, tumor-free mice or bearing the HER2+ C4HD tumor were treated with vehicle or INB03 (10 mg/kg, i.p.) for 21 days. Polarization, exhaustion and ICP markers were studied in Mϕ and T cells from spleen and tumor. Adding INB03 to already-polarized human Mϕ enhanced polarization towards M1-like (CD86+CD206−). Polarized Mϕ exposed to INB03 showed decreased PD1 and PDL-1 expression. Also, the expression of ADCP-inhibitory markers B7H4 and SIRPα was diminished when INB03 was present. These Mϕ exhibited increased ADCP against JIMT-1 cells treated with INB03, which showed downregulation of the inhibitory signal CD47. Murine Mϕ from tumor-bearing mice treated with INB03 showed increased polarization to the M1-like phenotype. Human CD8+ T cells showed decreased expression of PD1, PD-L1, LAG3, TIM3 and TIGIT. However, only TIM3 and PD1 were downregulated in murine CD8+ T cells of tumor-bearing mice treated with INB03. In all, sTNF blockade skews Mϕ to the M1-like phenotype and reprograms already-polarized pro-tumoral M2-like Mϕ to antitumoral ones. INB03 can tailor the tumor microenvironment by promoting Mϕ ADCP against tumor cells and by acting as an ICP inhibitor for CD8+ T cells, possibly relieving their exhaustion. HER2+MUC4+ BC patients could benefit from the administration of INB03 to boost targeted therapy efficacy and overcome tumor-induced macrophage and T-cell immune escape. Citation Format: Sofia Bruni, Maria F. Mercogliano, Roxana Schillaci. INB03: a new immune checkpoint inhibitor that reprograms macrophage polarization, boosts ADCP and reverts T-cell exhaustion markers [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 1365.

Abstract 4013: Increasing mitochondrial respiration by silencing MCJ/DnaJC15 enhances CD8 CAR-T cell therapy efficacy

Abstract Chimeric antigen receptor (CAR)-T cell therapy is a promising treatment against leukemia and lymphoma. However, poor initial treatment response and post-therapy relapse have found to be common and remained to be resolved. Modulating T cell immunometabolism is considered a novel therapeutic approach to enhance CAR-T cell therapy. We have identified MCJ (DnaJC15) as an endogenous negative regulator of Complex I of the electron transport chain in mitochondria. MCJ is abundantly expressed in mouse CD8 cells relative to other immune cells. We propose that MCJ can be a therapeutic target to enhance mitochondrial respiration in CD8 CAR-T cells. We will show that loss of MCJ in mouse CD8 cells leads to superior anti-tumor response against melanoma. In addition, we will show that MCJ-deficient murine CD8 CAR-T cells have enhanced killing activity against leukemia both in vitro and in vivo. We have recently revealed that MCJ is also expressed in human CD8 cells and the levels of MCJ are heterogenous among human population. Similar to murine CD8 cells, MCJ knockdown in human CD8 cells enhances mitochondrial membrane potential and effector functions. We have designed a new CAR construct to silence MCJ in human CD8 CAR-T cells. MCJ-deficient human CD8 cells show superior cytotoxicity against leukemia with increased mitochondrial metabolism. Together, these data demonstrate that MCJ is a promising target to improve CAR-T cell therapy through modulating the T cell metabolism. We are currently investigating the effect of MCJ-deficient CD8 CAR-T cells against solid malignancy. Citation Format: Meng-Han Wu, Emily L. Giddings, Felipe Valenca-Pereira, Catherine Pham-Danis, Francesca Cendali, Michael C. Yarnell, Amanda J. Novak, Jorge Henao-Mejia, Richard A. Flavell, Angelo D'Alessandro, Mark Eric Kohler, Mercedes Rincon. Increasing mitochondrial respiration by silencing MCJ/DnaJC15 enhances CD8 CAR-T cell therapy efficacy [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 4013.

Abstract 4995: Impact of radiotherapy on extracellular vesicle mediated anti-tumor immunity in oligometastatic prostate cancer

Abstract Metastasis-ablative radiotherapy is a new therapeutic approach that prolongs survival in patients with advanced cancers. Tumour irradiation can elicit an immunogenic cell death resulting in the expansion of tumour-reactive T cells and eradication of both irradiated and distant non-irradiated metastases, referred to as the “abscopal effect”. This abscopal response, nevertheless, is observed in a minority of patients suggesting uncharacterized mechanisms that prevent robust systemic anti-tumor immune response. Tumour-derived extracellular vesicles (TdEVs) have recently emerged as mediators of antitumor immunity but their immunomodulatory role in response to radiotherapy remains largely unknown. Recent clinical studies by our lab have demonstrated that circulating levels of tumour-derived extracellular vesicles (tdEVs) are elevated in response to radiotherapy and high tdEV concentrations are associated with reduced levels of peripheral cytotoxic CD8+ T cells and higher risk of disease recurrence in advanced prostate cancer. Based on our findings, we hypothesize that radiotherapy induces the release of tumor-derived EVs that suppress anti-tumor immunity. Prostate cancer cells (PC3 and DU145) were irradiated with clinically used doses of ionizing radiation (20Gy x 1 or 10Gy x 3 fractions) and tdEVs were co-cultured with human CD8+ T cells to measure their proliferation and cytotoxic activity. Increased levels of tdEVs were released by tumour cells treated with radiotherapy. A higher frequency of impaired T cell cytotoxic activity and a reduction in CD8 T cell proliferation was found in the 10Gy x 3 radiation regimen. Proteomic analysis and western blot showed an increased expression of the immune checkpoint molecule B7-H3 on the tumour cell surface and tdEVs in response to radiotherapy. While coculture of CD8 T cells with B7-H3 depleted tdEVs led to recovery of T-cell cytotoxic ability, overexpression of the B7-H3 in tdEVs inhibited cytotoxic activity. This study provides a rationale for future studies in which we will test the immune-mediated effects of radiotherapy-induced tdEVs in immunocompetent mouse models and investigate B7-H3 blockade as a therapeutic approach to boost abscopal response in the treatment of advanced prostate cancer. Citation Format: Chisom J. Uzendu, Yohan Kim, Roxane R. Lavoie, Haidong Dong, Sean Park, Fabrice Lucien-Matteoni. Impact of radiotherapy on extracellular vesicle mediated anti-tumor immunity in oligometastatic prostate cancer [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 4995.

Abstract 6704: Development and characterization of a tri-specific selective CD8 T cell engager (CD8xCD3xCD19) for treatment of B cell lymphoma

Abstract The recent development of CD3 bi-specific antibodies (BsAbs) targeting CD20, BCMA, GPRC5D and CD19 to treat B cell malignancies have resulted in significant clinical benefit to patients. However, long-term remission rates and survival remain to be demonstrated. Current CD3 BsAbs preferentially bind to and activate CD4 T cells. However, CD8 cytotoxic T cells also play the key role in anti-tumor response and T cell exhaustion in cancer immunity. To potentially enhance anti-tumor efficacy by augmenting CD8 T-cell response, we developed a novel tri-specific T cell engager (TriTE) IM-8319, which simultaneously binds human CD3, CD8 and CD19 epitopes with KD at 14.2 nM, 3.3 nM and 0.3 nM, respectively. The binding affinity to human CD8 T cells and CD4 T cells was measured with EC50 at 0.02 nM and 1.2 nM, respectively. In human PBMC-killing assays, IM-8319 was found to preferentially activate CD8 T cells with EC50 of 6.0 pM, compared to an EC50 of 94 pM for activating CD4 T cells. Unique cytokine release responses were seen with IM-8319 treatment. Compared to bi-specific TCE controls without CD8 binding domain, IM-8319 in human PBMC assays increased INFγ and TNFα in CD8 T cells and blunted IL-2 release in CD4 T cells. We observed robust cytotoxic killing of CD19+ B cells with IM-8319. Compared the bi-specific TCE controls lacking CD8 binding domain, in vitro CD19+ B-cell depletion was increased 6-fold with IM-8319. In addition, we observed notable structure activity relationships, with &amp;gt;100-fold increases in TriTE cytotoxicity with a weak CD3 binding domain. T-cell fratricide has hindered the development of antibodies containing 2+ T-cell epitopes. We observed correctable, dose-dependent CD8 T cell depletion at high concentrations of CD8xCD3xCD19 TriTEs in vitro and xenograft mouse models. This unwanted biological effect was not a surprise when the TriTE binds to CD8 T cells in trans. However, by structure engineering IM-8319 to allow for cis binding of CD3 and CD8, we effectively eliminated CD8 T cell fratricide at therapeutic doses. In a Raji-inoculated murine xenograft that was reconstituted with human PBMC, IM-8319 significantly inhibited tumor growth without obvious CD8 T cell depletion. In summary, we report a novel TriTE (IM-8319) that selectively binds to and activates CD8 T cells. IM-8319 results in robust on-target cytotoxicty with specific activation of CD8 T cells, suggesting potential to reduce systemic toxicities such as cytokine release syndrome (CRS) while augmenting direct antitumor efficacy. Further preclinical evaluation of IM-8319 is warranted in patients with B-cell leukemias and lymphomas. Citation Format: Zheng Yang, Wuzhong Shen, Guiyun Tu, Shenjie Xue, Ying Tan, Bingbin Xie, Jason Xu, Hanyang Chen, Shui Liu, Xiaoqiang Yan. Development and characterization of a tri-specific selective CD8 T cell engager (CD8xCD3xCD19) for treatment of B cell lymphoma [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 6704.

Abstract 5252: Expansion of human T cells and NK cells for chimeric antigen receptor based therapies using human platelet lysate

Abstract T cells and NK cells expressing chimeric antigen receptors (CAR) have demonstrated potent clinical efficacy in patients with hematological malignancies. One of the issues to overcome in the treatment of solid tumors using CARs is the lack of a process that generates large amounts of CAR-T cells, reduces cell exhaustion and differentiation, and improves long term survival after infusion into patients. Previously, Torres Chavez et al. demonstrated in a series of in vitro and in vivo experiments, performed in both hematologic and solid tumor models, the profound qualitative impact on CAR-T cell performance after using human platelet lysate (hPL) as a cell growth supplement. T cells expanded with their hPL showed a good proliferative capacity and enhanced long-term in vivo persistence compared to Fetal Bovine Serum (FBS) or human AB Serum (ABS), resulting in superior anti-tumor effects. Additionally, this group demonstrated that the transduction of T cells to generate the CAR-T cells was significantly improved by the use of hPL. Mill Creek’s human platelet lysate (hPL) is produced using expired human platelets obtained from accredited blood banks in the United States. These platelets were originally intended for use in patient transfusion. The safety of platelets used in transfusion is managed by the U.S. Food &amp; Drug Administration (FDA), as well as the Association for the Advancement of Blood &amp; Biotherapies (AABB). These organizations set standards, including testing for transmissible diseases. The United States record for blood safety is well established, with extremely low rates of disease transmission, making the platelet units used for hPL manufacture low risk. The Covid-19 pandemic has increased awareness of emerging infectious diseases, even though transmission of Covid-19 via blood transfusion has not been documented. For that reason, we developed a gamma irradiated version of our products, which offers an additional safety measure in the clinic. Our data from experiments performed using human CD3+ and purified NK cells from donor peripheral blood suggests that our human platelet lysates offer an improved cell phenotype and expansion capacities versus serum derived products, but also versus other platelet lysates on the market. PLTGold® and PLTGold®-GI efficiently promote cell expansion, with higher cell yields and lower cell exhaustion rate. Additionally, our hPLs are suitable for suspension culture, potentially facilitating the large-scale expansion of allogeneic CAR cells. The use of our platelet lysates has the potential of significantly improving CAR-T and CAR-NK manufacture, but also the outcome of the therapy itself by improving the quality and potency of the end product. Citation Format: Vanesa Alonso-Camino, William Mirsch. Expansion of human T cells and NK cells for chimeric antigen receptor based therapies using human platelet lysate [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 5252.

Abstract 7495: Validation of microfluidic isolation of circulating pancreatic tumor cells from blood

Abstract Background. Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis and is the fourth leading cause of cancer-related deaths in the US. PDAC is diagnosed in the late stages, which limits treatment options and yields poor clinical outcomes. Recurrence is a significant issue for patients undergoing surgery for PDAC. Circulating tumor cells (CTCs) are thought to be a potential mediator of recurrence, facilitating the formation of secondary tumor sites after surgery. Thus, there is a need to isolate CTCs to improve understanding of disease recurrence, to ultimately improve outcomes. Herein, we aim to validate our inertial microfluidic (iMF) system for isolating pancreatic CTCs from blood. We also benchmark our iMF system to a commercially available immunomagnetic negative selection system, EasySep™, and quantify target cell recovery and cell enrichment. Methods. Our iMF system employs the differential cell sizes of CTCs from blood cells and inertial migration of such cells in a straight microchannel (150 µm × 50 µm × 24 mm) to enrich CTCs from blood. The iMF system sample flow rate used is 100 µL/min, with a 200 µL/min PBS buffer flow rate. The EasySep™ Direct Human CTC Enrichment kit targets CD2, CD14, CD16, CD19, CD45, CD61, CD66b, and Glycophorin to remove blood cells from the sample using magnetic particles, thereby separating them from CTCs. We spiked low cell concentrations of 50, 100, and 500 Hoechst-stained pancreatic carcinoma cells, PANC1, in 1 mL of healthy blood then captured using either EasySep™ or iMF. Cell enumeration was performed after centrifugation or Cytospin. Results. PANC1 cells and WBCs have average sizes of 18 µm and 8.4 µm, respectively. We characterized our iMF device to capture cells ≥ 12 µm, which yields capture of most PANC1 cells. Indeed, our iMF system achieved 82.8±19.2%, 72.5±16.3%, and 59.3±3.8% recovery for 50, 100, and 500 cell-spikes in lysed blood, respectively. For the same 50, 100, and 500 cell-spikes, the total iMF CTC yields after Cytospin were 46.3±11.8, 44.3±9.1, and 32.7±2.1%, while the total CTC yields from EasySep™ were 4.6±3, 5.3±2.1, 8.3±1%, respectively. When we processed whole blood directly, EasySep™ had lower cell recovery, 3.8±0.2, 4.6±1, and 3.3±0.4%, respectively. Our system also demonstrated 8 × higher target cell enrichment compared to EasySep™. Blood lysis before iMF separation did not drastically affect the recovery of target cells. Conclusions. We determined that our system has improved performance over the commercial isolation system, EasySep™, thereby validating the effectiveness of microfluidic isolation for PDAC. In future studies, we can then use our iMF system in CTC isolation from routine blood draws from PDAC patients undergoing surgical resection. We may then isolate real patient samples and study relevant phenotypes of PDAC CTCs which may therefore be used for identifying certain mechanisms of PDAC progression. Citation Format: Celine Macaraniag, Ifra Khan, Jian Zhou, Pier Cristoforo Giulianotti, Alain Borgeat, Gina Votta-Velis, Ian Papautsky. Validation of microfluidic isolation of circulating pancreatic tumor cells from blood [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 7495.

Abstract 4094: Immune modulatory cancer vaccines against IDO1 and PD-L1 trigger distinct pathways and cooperatively reduce tumor growth in preclinical models

Abstract While immune checkpoint inhibitors have shown clinical efficacy in many cancers, drug and immune resistance remain challenging. Indoleamine 2,3-dehydrogenase (IDO1) and Programmed Death Ligand 1 (PD-L1) both contribute to immunosuppression, leading to immune escape and cancer progression. In this context, vaccination to promote T-cell immunity against IDO1+ and PD-L1+ cells is an attractive strategy that demonstrated encouraging clinical results in melanoma (Kjeldsen et al. Nat Med 2021). Our study aims to further evaluate the efficacy and mode of action of combined IDO1 and PD-L1 peptide vaccines. Mice injected with tumor cells and treated with IDO1 or PD-L1 peptides were monitored for tumor growth. Vaccine activity was determined by IFNγ Elispot assay. Gene expression analysis was performed on tumors using Nanostring nCounter PanCancer IO360 Panel. IDO1 and PD-L1 expression were also examined via multiplex immunofluorescence in a human tumor tissue microarray (TMA) panel. Human IDO1 and PD-L1 specific CD4+ T cell clones were isolated and expanded from melanoma patients treated with IDO1/IO102 or PD-L1/IO103 peptide vaccines in combination with aPD1 therapy. Immunofluorescence analysis of murine tumor sections showed expression of IDO1 and PD-L1 by different cell populations. Vaccination against each target led to expansion of specific T cells in splenocytes and reduced tumor growth in MC38 and CT26 models. Combining IDO1 and PD-L1 treatments further enhanced the anti-tumor response. Gene expression analysis of tumors revealed distinct signatures following monotherapy and combination treatments. While PD-L1 vaccination predominantly enhanced the cytotoxic effector T cell function in the tumor microenvironment (TME) and concomitantly increased gene signatures associated with enhanced activation of T effector cells, IDO1 vaccine primarily exerted its effect via reduction in the immune suppression loop. TMA analysis of IDO1+ and PD-L1+ cells in human tumors also revealed that they represent largely distinct cell populations. Lastly, IDO1 and PDL1-specific CD4+ T cell clones from melanoma patients treated with IO102-IO103 vaccine could selectively target cells differentially expressing IDO1 or PDL1. Our data collectively show that cells expressing IDO1 and PD-L1 represent distinct populations in the TME thus targetable by the IDO1-PD-L1 vaccination approach. The vaccines targeting IDO1 and PD-L1 cooperatively reduce tumor outgrowth, and each contributes to the anti-tumor effect through distinct molecular programs regulating immunosuppression in the TME and T cell activation respectively. These data are supported by ex vivo functional assays using target specific T cells from vaccinated patients. Altogether, our data support the use of a dual antigen approach to reduce the immunosuppression and enhance anti-tumor effect. Citation Format: Marion Chapellier, Evelina Martinenaite, Preeyam Patel, Lea Svendsen, Shamaila Munir Ahmad, Hannah Jorinde Glöckner, Marco Carretta, Erika Sutanto-Ward, James DuHadaway, Souvik Dey, Shih-Chun Shen, Dema Ghaban, Marcos Iglesias, Megan Biller, Inés Lecoq, Alexander J. Muller, Mads Hald Andersen, Ayako Wakatsuki Pedersen. Immune modulatory cancer vaccines against IDO1 and PD-L1 trigger distinct pathways and cooperatively reduce tumor growth in preclinical 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 4094.

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.