Checkpoint Blockers Research Articles

Targeting metabolism of breast cancer and its implications in T cell immunotherapy.

Breast cancer is a prominent health issue amongst women around the world. Immunotherapies including tumor targeted antibodies, adoptive T cell therapy, vaccines, and immune checkpoint blockers have rejuvenated the clinical management of breast cancer, but the prognosis of patients remains dismal. Metabolic reprogramming and immune escape are two important mechanisms supporting the progression of breast cancer. The deprivation uptake of nutrients (such as glucose, amino acid, and lipid) by breast cancer cells has a significant impact on tumor growth and microenvironment remodeling. In recent years, in-depth researches on the mechanism of metabolic reprogramming and immune escape have been extensively conducted, and targeting metabolic reprogramming has been proposed as a new therapeutic strategy for breast cancer. This article reviews the abnormal metabolism of breast cancer cells and its impact on the anti-tumor activity of T cells, and further explores the possibility of targeting metabolism as a therapeutic strategy for breast cancer.

Abstract CT181: First-in-human phase I of anti-VISTA monoclonal antibody W0180 with and without anti-PD-1 pembrolizumab in patients with locally advanced or metastatic solid tumors

Abstract Background: V-domain Immunoglobulin Suppressor of T cell Activation (VISTA) is an immune checkpoint regulator highly expressed on myeloid cells. VISTA has been shown to maintain tumor microenvironment (TME) in an immunosuppressive state. Its expression is reported as a resistance mechanism to immune checkpoint blockers, making it an attractive target for cancer treatment. Preclinical data showed that VISTA blockade mediates antitumor activity by activating the antitumor immune response and is enhanced in combination with anti-PD-1. W0180 is a humanized, Fc competent, IgG1 antibody targeting VISTA with high affinity. This study investigates W0180 with/without pembrolizumab (pembro) in patients (pts) with solid tumors. Methods: Pts initially received W0180 monotherapy in dose escalation (3.5-600 mg weekly (QW)). Following evaluation of the first 3 dose levels in monotherapy, dose escalation of W0180 (60 to 300 mg QW) in combination with pembro (200 mg Q3W) started in pts treated with prior anti-PD-(L)1. Objectives were to characterize safety and tolerability of W0180, determine RDE and PK/PD on blood and paired tumor biopsies. Results: By October 2023, 33 pts were treated in dose escalation, 24 pts with W0180 monotherapy and 9 pts with W0180 + pembro. Two dose-limiting toxicities were observed, Grade 2 subacute cerebral infarction and Grade 3 infusion related reaction (IRR). Overall, 33 pts experienced ≥1 treatment-emergent AE (TEAE). Most common TEAEs were IRR (32/33), fatigue (14/33), and anemia (11/33). Grade 3/4 TEAEs occurred in 23 pts, neutropenia (6/23) and anemia (6/23) were the most common. There was no treatment-related death. 6 (18%) pts discontinued due to AEs. Following the observation of Grade 3 arterial thromboembolism (n=1) and Grade 2 subacute cerebral infarction (n=1), eligibility criteria were amended to require adequate lipid profile, troponin and exclude pts with history of arterial thrombotic events and current smoking. W0180 PK was nonlinear with a rapid clearance at 3.5-600 mg doses. No objective response was observed but 5 pts had stable disease (SD) lasting for >16 weeks. One pt with appendix cancer treated at 600mg monotherapy had SD for >15 months. One pt with head and neck cancer treated at 300mg in combination had SD for 13 months. Biomarkers data suggested evidence of PD effect with a trend for dose response through sustained increase of proliferative CD8+ T-cells in blood at the highest W0180 exposures and increased density of tumor-infiltrating lymphocytes in 10 out of 16 pts. These changes were independent of tumor VISTA expression level and correlated to high PD-L1 expression (CPS>5) for 4 pts at baseline. Conclusions: Analysis is ongoing to assess the RDE of W0180 + pembro and collect additional evidence of proof of mechanism in pts with solid tumors. NCT04564417 Citation Format: Carlos Gomez-Roca, Stéphane Champiat, Philippe Cassier, David Jegou, Marie Primard, Aurélie Pétain, Geneviève Gueguen-Dorbes, Claire Fabre, Ignacio Melero, Aurélien Marabelle. First-in-human phase I of anti-VISTA monoclonal antibody W0180 with and without anti-PD-1 pembrolizumab in patients with locally advanced or metastatic solid 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 CT181.

Abstract LB057: Pre-clinical characterization of IBI343, a site-specifically conjugated anti-Claundin18.2 ADC, for treating solid tumors

Abstract Claudin18.2 is important for the formation and function of tight junctions between cells in epithelial tissues. Normally expressed only in gastric mucosa, Claudin18.2 is overexpressed in multiple types of solid tumors, including gastric, pancreatic, breast and colon cancers. Despite being a well-established tumor-associated antigen, the clinical value and feasibility of targeting Claudin18.2 by various modalities are yet-to-be fully demonstrated. IBI343 is a fully humanized anti-Claudin18.2 monoclonal antibody conjugated to Exatecan via site-specific glycol-conjugation with an average drug-to-antibody ratio (DAR) of 3.6. IBI343 showed robust antigen-specific in vitro cytotoxicity in cancer cell lines with varying levels of Claudin18.2 expression. In addition, due to the cleavable linker design and high hydrophilicity of the payload, IBI343 demonstrated potent bystander killing effects in vitro. The glycan-based conjugation technology leads to enhanced stability of the conjugated linker-payload and the entire ADC molecule, which results in superior pharmacokinetics. IBI343 showed potent in vivo anti-tumor efficacy with complete tumor regressions in multiple xenograft models. Furthermore, it demonstrated strong in vitro and in vivo synergistic effects with various anti-cancer agents, including immune checkpoint blockers and DNA damage repair pathway inhibitors. IBI343 displayed good safety profile in monkey GLP toxicology study (HNSTD = 30 mg/kg). These pre-clinical findings show that IBI343 is a promising anti-cancer agent with large therapeutic window, and support clinical exploration of IBI343 in patients with Claudin18.2-expressing tumors. A companion late-breaking abstract of clinical phase 1 study results (NCT05458219) will also be presented in AACR 2024. Citation Format: Shuaixiang Zhou, Xiong Yao, Jian Guan, Keke Fei, Jia Lu, Weiwei Wu, Yang Liu, Tianyu Zhu, Zhuangguang Liao, Shi Chen, Bingliang Chen, Kaijie He. Pre-clinical characterization of IBI343, a site-specifically conjugated anti-Claundin18.2 ADC, for treating solid 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 LB057.

Abstract LB310: Single-cell transcriptomics and TCR clonality reveal a role for immunotherapy in pediatric brain tumors

Abstract Central nervous system tumors are the most common solid tumors and the leading cause of cancer-related deaths in children. Outcomes remain dismal for a significant proportion of patients despite aggressive multimodal therapy and survivors suffer from treatment-related morbidity and neurological deficits. Despite their remarkable success in adult cancers, immunotherapies are not approved for pediatric brain tumors. Several factors like the relatively smaller patient population, a tumor location that makes them difficult to access through biopsies and poorly-studied tumor immune microenvironment (TME) have posed challenges in advancing immunotherapeutic options for these patients. Immune checkpoint blockers (ICB) tested in unstratified cohorts of children with brain tumors have not shown promising results; therefore, the role of immunotherapy remains unclear. Because the magnitude and nature of pre-existing T cell responses in the tumor has been shown to correlate with response to ICB, we asked if pediatric brain tumors show evidence of such favorable T cell responses. In the current study, we address this question using an unbiased approach to analyze the clonal relationship and transcriptional status of T cells present in tumor tissue of children with brain tumors using paired single-cell RNA-sequencing and TCR-sequencing. Our results demonstrate that a large fraction of T cells in the tumor tissue are clonally expanded with potential to recognize tumor antigens. Such clonally-expanded T cells display states linked to anti-tumor immunity, express higher levels of transcripts encoding for molecules linked to T cell activation, effector functions, immune cell recruitment, tissue-residency, immune checkpoints, and importantly, show significant enrichment of signatures linked to neoantigen-specific T cells and immunotherapy response. Notably, we identify several neoantigens in pediatric brain tumors, and show that neoantigen-specific T cell gene signatures are linked to better survival outcomes in high-grade glioma. We further show that PD1-expressing CD8+ T cells in pediatric brain tumors are indeed functional as evidenced by their capacity for cytotoxicity, cytokine production and proliferation. Among the patients in our cohort, we observe substantial heterogeneity in the degree of clonal expansion and expression of transcripts encoding immune checkpoints in tumor-infiltrating T cells. Overall, our findings suggest that accurate characterization of intra-tumoral T cell responses for features linked to immune check point therapy (ICB) response may enable selection of patients where immunotherapy is likely to be beneficial, an approach that requires prospective validation in future clinical trials. Citation Format: Anusha Preethi Ganesan, Aditi Upadhye, Kevin Meza Landeros, Ciro Ramírez-Suástegui1, Benjamin Schmiedel, Edwin Woo, Serena J. Chee, Denise Malicki, Nicole Coufal, David Gonda, Michael Levy, Jason Greenbaum, Gregory Seumois, John Crawford, William Roberts, Stephen Schoenberger, Hilde Cheroutre, Christian Ottensmeier, Pandurangan Vijayanand. Single-cell transcriptomics and TCR clonality reveal a role for immunotherapy in pediatric brain 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 LB310.

Retroelements in targeted antitumor therapy (review)

Background: Pathological expression of retroelements is determined in most malignant tumors, which reflects the pathogenesis of tumors, since activated retrotransposons cause genomic instability due to a cascade of mutual regulatory influence events between transposons, tumor suppressors and oncogenes. These relationships are reflected in the expression profiles of certain long non-coding RNAs and miRNAs in specific tumor tissues, since retroelements are key sources of non-coding RNAs. Since retroelements serve as drivers for the initiation and progression of carcinogenesis, it is promising to use drugs aimed at inhibiting their expression. The aim of the study: To determine the available methods of influencing retroelements in antitumor therapy, to evaluate the ways of their further improvement for the effective treatment of malignant neoplasms. Materials and methods: The databases Scopus, WoS, PubMed were used to analyze the role of retroelements in the development of tumors, methods of influencing them in the treatment of neoplasms. Results: Reverse transcriptase inhibitors have proven to be effective. Silencing of retrotransposons by acting on histone modifiers can lead to a weakening of the antitumor immune response; therefore, this approach requires RNA guides that direct the formation of heterochromatin in the area where strictly specific retroelements are located. A promising method is viral mimicry aimed at activating the interferon and T-cell response to artificially activated endogenous retroviruses. For this purpose, inhibitors of histone methyltransferases, DNA methyltransferases, and histone deacetylases are used. Their joint use is most effective, especially with PD-1 checkpoint blockers. The arrangement of retroelements in gene introns has become the basis for the development of spliceosomal targeted therapy, which also initiates an antitumor immune response to double-stranded RNA transcripts. We suggest the possibility of using antisense oligonucleotides targeting retroelements in anticancer therapy. Such approach is used to treat age-related macular degeneration, Fukuyama muscular dystrophy, amyotrophic lateral sclerosis. Antisense oligonucleotides targeting microRNAs, proto-oncogenes, and oncosuppressors are used in anticancer therapy and are closely related to retroelements, which could become promising targets for antisense oligonucleotides. Conclusion: In antitumor therapy, it is possible to use two diametrically opposed strategies for influencing retroelements: inhibition and activation. The most promising way is a combination of these methods with specific silencing of retrotransposons using guides (complementary RNAs) and stimulation of the expression of elements not involved in carcinogenesis, but activating the immune response.

Enzyme Core Spherical Nucleic Acid That Enables Enhanced Cuproptosis and Antitumor Immune Response through Alleviating Tumor Hypoxia.

Cuproptosis, a copper-dependent cell death process, has been confirmed to further activate the immune response and mediate the immune resistance. However, hypoxic tumor microenvironment hampers cuproptosis sensitivity and suppresses the body's antitumor immune response. Herein, we have successfully immobilized and functionalized catalase (CAT) with long single-stranded DNA containing polyvalent CpG sequences through rolling circle amplification (RCA) techniques, obtaining an enzyme-cored spherical nucleic acid nanoplatform (CAT-ecSNA-Cu) to deliver copper ions for cuproptosis. The presence of long-stranded DNA-protected CAT enhances mitochondrial respiration by catalyzing the conversion of H2O2 to O2, thereby sensitizing cuproptosis. Meanwhile, increased tumor oxygenation suppresses the expression of the hypoxia-inducible factor-1 (HIF-1) protein, resulting in the alleviation of the immunosuppressive tumor microenvironment. Of note, cuproptosis induces immunogenic cell death (ICD), which facilitates dendritic cell (DC) maturation and enhances antigen presentation through polyCpG-supported Toll-like receptor 9 (TLR9) activation. Furthermore, cuproptosis-induced PD-L1 upregulation in tumor cells complements checkpoint blockers (αPD-L1), enhancing antitumor immunity. The strategy of enhancing cuproptosis-mediated antitumor immune responses by alleviating hypoxia effectively promotes the activation and proliferation of effector T cells, ultimately leading to long-term immunity against cancer.

Long-Term Survival and Immune Response Dynamics in Melanoma Patients Undergoing TAPCells-Based Vaccination Therapy.

Cancer vaccines present a promising avenue for treating immune checkpoint blockers (ICBs)-refractory patients, fostering immune responses to modulate the tumor microenvironment. We revisit a phase I/II trial using Tumor Antigen-Presenting Cells (TAPCells) (NCT06152367), an autologous antigen-presenting cell vaccine loaded with heat-shocked allogeneic melanoma cell lysates. Initial findings showcased TAPCells inducing lysate-specific delayed-type hypersensitivity (DTH) reactions, correlating with prolonged survival. Here, we extend our analysis over 15 years, categorizing patients into short-term (<36 months) and long-term (≥36 months) survivors, exploring novel associations between clinical outcomes and demographic, genetic, and immunologic parameters. Notably, DTHpos patients exhibit a 53.1% three-year survival compared to 16.1% in DTHneg patients. Extended remissions are observed in long-term survivors, particularly DTHpos/M1cneg patients. Younger age, stage III disease, and moderate immune events also benefit short-term survivors. Immunomarkers like increased C-type lectin domain family 2 member D on CD4+ T cells and elevated interleukin-17A were detected in long-term survivors. In contrast, toll-like receptor-4 D229G polymorphism and reduced CD32 on B cells are associated with reduced survival. TAPCells achieved stable long remissions in 35.2% of patients, especially M1cneg/DTHpos cases. Conclusions: Our study underscores the potential of vaccine-induced immune responses in melanoma, emphasizing the identification of emerging biological markers and clinical parameters for predicting long-term remission.

Immune Checkpoint Inhibitors' challenges, mechanisms and management

Numerous studies have demonstrated that the ability of protecting malignant cells from immune destruction—particularly from T and B lymphocytes, macrophages, and natural killer cells—is linked to the aetiology of cancer. Malignant cells hold the capacity to activate specific immunological checkpoint pathways that have immunodepressant qualities, which is why immunosuppression is connected to the growth and spread of cancer. Immunotherapy has grown to be a crucial method of therapy for cancer. Immune checkpoint blocking increases anti-tumor immunity by preventing intrinsic down-regulated factors of immunity, like CTLA-4 and PD-1, or PD-L1. Unquestionably, ICIs have advanced the area of cancer immunotherapy. In spite of ICIs' success, various challenges to these drugs, such as resistance and immune side effects, have made treatment difficult. Therefore, more in-depth understanding of the mechanism of ICI treatment and its toxicity is needed. Ultimately, on the basis of these insights, biologists will eventually be able to refine ICIs to achieve longer lasting efficacy and improved security. Here, we review the mechanisms and clinical applications of ICIs, examine the various challenges facing ICIs, including the mechanisms of resistance to treatment and the manifestation of irAEs and explore more sound management measures on this basis.

Abstract 2717: Bifunctional fusion protein PDL1sFv/MICAe for cancer immunotherapy

Abstract Many cancer cells upregulate the expression of programmed death ligand 1 (PD-L1) to surpass T-cell mediated immune response. Immune checkpoint blockers targeting PD-L1 or its receptor PD1 have been shown to suppress the inhibitory signals and enhance T cell function. As a new immunotherapy, immune checkpoint blockers have been used to treat multiple cancers. However, most clinically available blockers only engage with one type of immune cell: the T cells. Natural killer (NK) cells, on the other hand, play a significant role in anti-cancer immune responses. It will represent an advancement in anti-cancer immunotherapy if NK cells are engaged with immune checkpoint blockers. In this study, we designed a unique bifunctional fusion gene, PDL1sFv/MICAe, consisting of the variable regions of an anti-PD-L1 antibody and the extracellular domain of stress protein MICA and hypothesized that fusion protein PDL1sFv/MICAe will simultaneously bind to PD-L1 on tumor cells to block the inhibitory signal and extend T cells activity, and to NKG2D to activate NK Cells. cDNA sequence of fusion gene PDL1sFv/MICAe was designed based on the variable region of an established clinically used antibody, Atezolizumab, and the extracellular domain of MICA with linkers in between. After a computational confirmation of its structure and binding affinities, the fusion gene was synthesized and cloned into an expressing vector, and the fusion protein was produced for evaluation. Western blotting and fluorescent microscopic analyses confirmed that the protein binds to PD-L1 of human tumor cells, including MCF7 and MDAMB231, and NKG2D of KHYG1 cells. Preliminary results also show that the fusion protein enhances NK cell cytotoxicity against tumor cells. Studies on confirming the fusion protein’s activities on other tumor cells, NK cell IFN-γ production, T cell function, etc., are underway. In summary, fusion protein PDL1sFv/MICAe represents an improved version of the immune checkpoint blockers and could treat cancer patients quickly and effectively. Citation Format: Junyi Li, Yanzhang Wei. Bifunctional fusion protein PDL1sFv/MICAe for cancer immunotherapy [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 2717.

Abstract 3719: Fasting-mimicking diet plus 2-DG drives antitumor immunity by targeting EDEM3-induced PD-L1 glycosylation

Abstract The PD-1/PD-L1 axis plays an essential role in immune escape and immunotherapy. Importantly, translated N-linked glycosylation of PD-L1 stabilizes protein, enters the cell membrane binding to PD-1, renders T cells anergic and leads to T-cell exhaustion and dysfunction. Targeting PD-L1 glycosylation as an attractive target by a rational combination of cancer immunotherapies. Cancer-associated fibroblasts (CAFs), a predominant cell component of the tumor microenvironment, exert crucial functions in promoting immune escape and immunotherapy resistance by increasing the tumor PD-L1 expression. However, whether and how CAFs upregulate the glycosylation of PD-L1 has never been reported. This study shows that CAFs can upregulate PD-L1 glycosylation and enhance PD-L1 cell surface expression via glycosyltransferase EDEM3, thus cancer immune evasion. Then, the study delves into the relationship between EDEM3 expression, prognosis and immunogenicity. We demonstrate that EDEM3 plays a role in tumor immunity during tumor progression, as evidenced by EDEM3 expression is only significantly correlated with poor prognosis in the CD8+ T cell-enriched population. In subcutaneous tumor models, we observed high cell-surface PD-L1 expression accompanied by decreased CD8+ T cell infiltration and increased M2 macrophage in immunocompetent mice transplanted with EDEM3 over-expressed CRC cells that promote resistance to PD-1/PD-L1 blockade cancer immunotherapy. By performing bioinformatic analysis, we found that the hexosamine biosynthesis pathway (HBP) is highly affected in EDEM3-upregulated CRC cancer. Subsequently, the production of the UDP-GlcNAc (the end product of HBP metabolic flow), a donor substrate for N-linked glycosylation, is significantly increased in EDEM3 over-expressed CRC cells by LC/MS. Meanwhile, glucose is confirmed to be the primary effector of PD-L1 glycosylation in CRC cells, and the glucose restriction attenuates EDEM3-modified PD-L1 glycosylation by reducing UDP-GlcNAc biosynthesis. Finally, we demonstrate that modulation of glucose metabolism through a fasting-mimicking diet (FMD) in combination with 2-deoxy glucose (2-DG) treatment enhances antitumor immunity, especially in tumors with high-EDEM3 expression that exhibit resistance to immunotherapy. We propose potential therapeutic targets for CRC that inhibit PD-L1 glycosylation through FMD+2-DG and could augment the efficacy of immune checkpoint blockers. Citation Format: Xiaoxia Liu, Shaoyong Peng, Xiaolin Wang, Huichuan Yu, Yanxin Luo. Fasting-mimicking diet plus 2-DG drives antitumor immunity by targeting EDEM3-induced PD-L1 glycosylation [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 3719.

Abstract 3208: A nano sized system for the treatment of gastrointestinal cancers

Abstract Despite the recent progress in cancer immunotherapies, most patients do not benefit from the currently available immune checkpoint blockers, and many develop severe immune-related adverse events. Therefore, it is urgent to develop alternative, effective immunotherapeutic approaches. An example of non-responsive cancers are gastrointestinal (GI) cancers, which account for 26% of the global cancer incidence and 35% of all cancer-related deaths. In many GI cancers, the failure of immunotherapies to yield clinical response is partially attributed to the inefficient antigen presentation by DC, which results in insufficient T cell stimulation despite their expression of tumor-associated antigens (TAA). To overcome these hurdles, we designed novel polymeric-based nanoparticles (NP) to deliver a combination of GI cancers antigens of CEACAM5 protein and toll-like receptor ligands to DC. We chose to encapsulate CEACAM5 antigens because it is overexpressed in GI cancers, such as gastric cancer, colorectal cancer, and pancreatic cancer. With this approach, we aim to modulate the tumor milieu-immune cell interactions to an anti-cancer phenotype in order to control tumor growth and improve overall survival. Our NP present an average diameter below 200 nm, narrow polydispersity index, slightly negative surface charge, spherical morphology, and high antigens and immune potentiators loading. Cy5-labeled NPs were found to be extensively internalized by DC, which triggered their activation in vivo, shown by the increasing expression of DC-related co-stimulatory molecules such as CD80, CD86, and CD40. Furthermore, we assessed the immunotherapeutic effect of our NP on mouse models and on a human 3D multi-cellular spheroid ex-vivo model, composing the patient’s tumor cells and peripheral blood mononuclear cells (PBMC). We found that the NP successfully induced a potent immune-mediated anti-tumor response against CEACAM5-positive GI tumors, sensitizing the tumor microenvironment to other therapies and decreasing tumor volume, ultimately leading to prolonged survival. Taken together, the developed NP induced a strong antigen-specific immune response and overall sensitized CEACAM5-expressing GI cancers to the clinically relevant treatments in vivo, that so far have shown limited clinical outcomes in addition to a specific T cell anti-tumor activity that was shown on our patient-derived ex vivo model. Citation Format: Daniella Vaskovich, Rita Acúrcio, Ron Kleiner, Barbara Carreira, Ana Matos, Helena Florindo, Ronit Satchi-Fainaro. A nano sized system for the treatment of gastrointestinal cancers [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 3208.

Abstract 1183: A first-in-class and SHP1 allosteric inhibitor, SB8091, shows a good efficacy and safety profile in preclinical models

Abstract Introduction: Src homology 2 domain-containing phosphatase 1 (SHP1) is a cytoplasmic protein tyrosine phosphatase (PTP) that is primarily expressed in hematopoietic cells and is commonly recognized as a negative regulator of multiple signaling pathways1. Previously, we showed that inhibition of the phosphatase activity of SHP1 by a novel SHP1 allosteric inhibitor, SB6299, results in the activation of innate and adaptive immune cells and enhanced anti-tumor immune responses2. Here, we report SB8091, an advanced SHP1 allosteric inhibitor with a promising initial safety profile and improved pharmacokinetic properties. Methods: SHP1 inhibitors were developed through a structure-based drug design approach, and optimized for cellular activity on target, in vitro ADME, and in vivo pharmacokinetic properties. Pharmacokinetic (PK) and preliminary toxicity studies were performed with mice. The lead series was assessed in in-vitro studies with mouse or human primary immune cells. In-vivo efficacy and pharmacodynamic data were generated using mouse syngeneic tumor models. Results: Structurally, SB8091 seemed to stabilize the autoinhibitory form of SHP1 as an allosteric inhibitor, in a nanomolar range and which can explain the outstanding selectivity compared to other PTP enzymes, including SHP2. In several human primary immune cell types, SB8091 significantly increased cytokine responses and demonstrated significantly enhanced activity in NK cells and macrophages against various cancer target cells. As a single agent, this compound exhibited inhibitory effects on tumor growth when administered orally, and the efficacy was further enhanced when co-administered with anti-PD1 antibodies in various syngeneic tumor models. Notably, this effect was associated with a pharmacodynamic response marked by increased levels of phosphorylated substrates regulated by SHP1. SB8091 exhibited favorable pharmacokinetic properties for oral administration, and SafetyScreen data showed a very low potential for off-target activity and drug-drug interactions. Additionally, during a 2-week repeat pilot toxicity study in mice, SB8091 demonstrated excellent tolerability even at high doses, indicating a high therapeutic index (TI &amp;gt; 100). Conclusion: Our advanced compound, SB8091, is a potent, highly selective, and orally bioavailable allosteric inhibitor of SHP1. It has demonstrated excellent efficacy as a monotherapy or in combination with immune checkpoint blockers, with favorable drug properties and safety profiles. Research to advance to the clinical stage is being conducted.

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.

Abstract 5068: Clonal Hematopoiesis of indeterminate potential (CHIP) in patients with advanced solid tumors treated with immune checkpoint blockers (ICB) as monotherapy: Analysis of the PREMIS study

Abstract Introduction: Clonal Hematopoiesis of indeterminate potential (CHIP) is a clonal expansion of mutations affecting genes involved in hematologic malignancies in patients without a hematological disease per se. It has emerged as a significant risk factor for cardiovascular events via skewing innate immune cells towards a pro-inflammatory state. Recently studies have shown its potential detrimental impact on overall survival in patients treated with chemotherapy. However, its impact on patients treated with immune checkpoint blockers in monotherapy remains unclear. Objective: Our aim is to evaluate CHIP prevalence in a prospective cohort of patients treated with ICB for advanced solid tumors and its impact on patient outcomes. Materials and methods: We performed a Next-Generation Sequencing (NGS) panel of 74-genes dedicated to hematological alterations on DNA extracted from whole blood collected before first administration of an ICB for an advanced solid tumor, within the PREMIS trial (NCT03984318). CHIP prevalence was assessed according to a variant allele frequency (VAF) threshold ≥ 2%. Results: We included 127 pts in the analysis; 88 pts (69%) were male with a median age of 70 years [range 37-100]. Most common tumor locations were skin (29%), genitourinary (25%) and lung (19%). ICB setting was 1°L in 40% of patients; 2°L in 43%; &amp;gt; 3°L in 17%, and the most used ICB was Nivolumab in 39% of patients. At least 1 CHIP mutation was found in 55 pts (43%). The most frequent mutations were found in DNMT3A (40%), TET2 (18%) and PPM1D (13%). Co-mutations were found in 20% of patients. Median PFS was 7.7 months (m) for CHIPm patients compared to 5.7 m in non-CHIPm patients (p=2.2) and median OS was 16.2 m in CHIPm patients compared to 14.9 m in non-CHIPm patients (p= .5). No pts developed hematological disease during the follow up. Conclusion: CHIP is commonly found in pts with solid tumors, with a prevalence in our cohort of 43% with no statistical significance neither in PFS nor in OS in the CHIP-mutated population. Citation Format: Julieta Elena Rodriguez, François Xavier Danlos, Alicia Larive, Aurelien Marabelle, Yohann Loriot, Caroline Robert, Laurence Albiges, Maxime Frelaut, Mihaela Aldea, Benjamin Besse, Michel Ducreux, Caroline Even, Jean Baptiste Micol, Santiago Ponce, Christophe Marzac, Nathalie Chaput, Christophe Massard, Capucine Baldini. Clonal Hematopoiesis of indeterminate potential (CHIP) in patients with advanced solid tumors treated with immune checkpoint blockers (ICB) as monotherapy: Analysis of the PREMIS study [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 5068.

Abstract 379: Landscape of HIF-1α expression across 24,186 solid tumors using comprehensive immune profiling

Abstract Background: Transcription factor Hypoxia Inducible Factor-1α (HIF-1α) is a key regulator of various cellular and systemic responses to hypoxia, with HIF-1α mediated pathways playing an important role in tumor progression and metastasis. Recent studies have highlighted the correlation between HIF-1α expression with tumor cell survival, angiogenesis, and invasion. In this study, we explore the landscape of HIF-1α expression across 35 solid tumor histologies, its co-expression with immune checkpoint and angiogenesis-related genes, as well as its association with outcomes from a retrospective cohort of patients with non-small cell lung cancer (NSCLC) treated with pembrolizumab. Methods: A discovery cohort of 24,186 solid tumors across 35 types were evaluated by comprehensive immune profiling assay. HIF-1α gene expression was normalized and ranked against a reference population of 735 samples yielding a percentile rank values [0-100], with rank classified as high (≥75), moderate (≥25 and &amp;lt;75) and low (&amp;lt;25). We investigated the distribution of HIF-1α expression, cellular proliferation signature, and co-expression with other immunotherapy targets using Spearman correlations (rs). Additionally, Kaplan-Meier analysis and Chi-squared tests were utilized for overall survival (OS), progression free survival (PFS) and objective response rate (ORR) differences in a retrospective cohort of 72 patients with NSCLC, treated with pembrolizumab. Results: Pan-cancer analysis confirmed a broad distribution of HIF-1α expression, with highest median expression levels observed in brain and central nervous system (CNS) (median=64), thyroid (median=49) and uterine (median=42) cancers. Significant associations (p&amp;lt;0.001) were observed between PD-L1 positivity (TPS≥1%, CPS≥1) and HIF-1α expression, with 60% of HIF-1α high cases (n=1,314/2,193) being PD-L1 positive, whereas 53% of HIF-1α low cases (n=4,834/9,135) were PD-L1 negative. Significant correlations (p&amp;lt;0.05) of ranked expression of HIF-1α with immune checkpoint blockage targets like PD-1 and CTLA-4, as well as angiogenesis-related genes like VEGFA and PTEN were observed. Within our pembrolizumab-treated retrospective cohort, there were no significant differences in OS and PFS between HIF-1α high and HIF-1α low tumors. However, the response to pembrolizumab was significantly higher in HIF-1α low tumors (ORR=58.82%, p&amp;lt;0.01) compared to HIF-1α high tumors. Conclusion: In clinically tested solid tumors, HIF-1α showed a dynamic expression range, with highest levels observed in brain, thyroid and uterine cancers. Significant co-expression of HIF-1α with various downstream genes, especially angiogenesis-related genes, enabled identification of gene signatures that characterize the hypoxic tumor microenvironment and may aid in the development of novel combination therapy strategies. Citation Format: Hardik I. Parikh, Sarabjot Pabla, Maria-Fernanda Senosain, Robert Seager, Erik Vanroey, Shuang Gao, Yamuna Pulivendula, Paul DePietro, Mary Nesline, Durgapras Dash, Jeffrey Conroy, Stephanie Hastings, Heidi Ko, Kyle Strickland, Rebecca Previs, Eric Severson, Taylor Jensen, Shakti Ramkissoon, Marcia Eisenberg, Brian Caveney. Landscape of HIF-1α expression across 24,186 solid tumors using comprehensive immune profiling [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 379.

Abstract 66: A new mechanism of tumor immune escape: TCF19 promotes the development of novel terminally exhausted T cells through E2F and MYC transcription family

Abstract Introduction: ICB (immune checkpoint blocking) has been a promising therapy in cancer treatment, but there are still some patients do not response to this therapy. And it is well known that the majority cells reactivated to ICB are TCF1+ precursor exhausted T cells. Thus, studying the formation and heterogeneity of exhausted T cells are very necessary for improving the efficacy of ICB therapy. In this research, we identified a new population as TCF19+PD1+TIM3+ exhausted CD8+ T cells, which blocked the responds to ICB therapy. Methods: We collected single cell sequencing data, including colorectal cancer, gastric cancer, melanoma, and lung cancer, and drew tumor immune cells map through cluster analysis. Then using bioinformatics tools CytoTRACE. T and CCAT. A to calculate the differentiation of exhausted T cell populations, which discovered a new subset of highly proliferative and poorly differentiated terminal exhausted T cells. Subsequently, the existence of this group of cells was verified through in vitro experiments, and their formation mechanism and correlation with immunotherapy were further studied. Results: We utilized CytoTRACE.T and CCAT.A to predict differentiation states of terminally exhausted T cells. Then GEO and KEGG pathway gene set enrichment analysis were used. After analyzing, we found out that there was a subset of cells in terminally exhausted T cells defined as TCF19+PD1+TIM3+ Tex cells, which presented low differentiated and high proliferative potential.Then we performed multiple immunofluorescence staining and flow cytometry to verified the existence of TCF19+PD1+TIM3+ Tex cells in vitro. Clarifing the generation of these cell subset through regulating the expression of E2F and MYC transcription factor families.By collecting pathological sections of patients with immunotherapy information, we found that the amount of TCF19+PD1+TIM3+ Tex cells was inversely proportional to the efficacy of ICB, through immunofluorescence staining and immunohistochemical analysis as well as organoid models cocultured with T cells. Conclusion: In this research, we found a new subpopulation of terminal Tex cells, which showed high proliferation and low differentiation. For cells, TCF19 mediated the generation of stemness terminal Tex cells may through regulating the expression of E2F and MYC. Finally, we discovered that the existence of TCF19+PD1+TIM3+ Tex cells can predict the efficacy of ICB, that the more TCF19+PD1+TIM3+ Tex cells contained, the less efficacy of ICB in patients.This finding may provide a new research direction for immunotherapy. Thus, more attention should be paid to this new cell subset. Citation Format: Fuhui Wang, Nan Dong, Changhua Zhang. A new mechanism of tumor immune escape: TCF19 promotes the development of novel terminally exhausted T cells through E2F and MYC transcription family [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 66.

Immune checkpoint inhibitor monotherapy is sufficient to promote microenvironmental normalization via the type Iinterferon pathway in PD-L1-expressing head and neck cancer.

Immune checkpoint blockers (ICBs) targeting programmed cell death protein 1 (PD-1) have been proven to be an effective first-line therapy against programmed cell death 1 ligand 1 (PD-L1; also known as CD274 molecule)-expressing head and neck squamous cell carcinoma (HNSCC) in recent KEYNOTE-048 trial. However, associated changes in the tumor microenvironment (TME) and underlying mechanisms remain elusive. Oral tumors in C57/BL6 mice were induced by administering 7,12-dimethylbenzanthracene into the buccal mucosa. Single-cell suspension was isolated from tumor tissue; proliferating cells were injected subcutaneously into the left flank of mice to establish Ajou oral cancer (AOC) cell lines. Subsequently, a syngeneic PD-L1-expressing HNSCC model was developed by injecting AOC cells into the buccal or tongue area. The model recapitulated human HNSCC molecular features and showed reliable in vivo tumorigenicity with significant PD-L1 expression. ICB monotherapy induced global changes in the TME, including vascular normalization. Furthermore, the antitumor effect of ICB monotherapy was superior to those of other therapeutic agents, including cisplatin and inhibitors of vascular endothelial growth factor receptor 2 (VEGFR2). The ICB-induced antitumorigenicity and TME normalization were alleviated by blocking the type I interferon pathway. In summary, ICB monotherapy is sufficient to induce TME normalization in the syngeneic model; the type I interferon pathway is indispensable in realizing the effects of ICBs. Furthermore, these results explain the underlying mechanism of the efficacy of ICB monotherapy against PD-L1-expressing HNSCC in the KEYNOTE-048 trial.

Xevinapant Combined with Pembrolizumab in Patients with Advanced, Pretreated, Colorectal and Pancreatic Cancer: Results of the Phase Ib/II CATRIPCA Trial.

Xevinapant is an orally available inhibitor of apoptosis proteins (IAP) inhibitor. Preclinical data suggest that IAP antagonism may synergize with immune checkpoint blockers by modulating the NFκB pathway in immune cells. Adult patients with non-high microsatellite instability advanced/metastatic pancreatic ductal adenocarcinoma (PDAC) or colorectal cancer were enrolled in this phase Ib/II study and received pembrolizumab 200 mg every 3 weeks intravenously, and ascending doses of oral xevinapant (100, 150, and 200 mg daily for 14 days on/7 days off). Dose escalation followed a 3+3 design with a 21-day dose-limiting toxicity (DLT) evaluation period. Following the determination of the recommended phase II dose (RP2D), 14 patients with PDAC and 14 patients with colorectal cancer were enrolled in expansion cohorts to assess preliminary efficacy. Forty-one patients (26 males) with a median age of 64 years were enrolled: 13 in the dose escalation and 28 in the two expansion cohorts. No DLT was observed during dose escalation. The RP2D was identified as xevinapant 200 mg/day + pembrolizumab 200 mg every 3 weeks. The most common adverse events (AE) were fatigue (37%), gastrointestinal AE (decreased appetite in 37%, nausea in 24%, stomatitis in 12%, and diarrhea and vomiting in 10% each), and cutaneous AE (pruritus, dry skin, and rash seen in 20%, 15%, and 15% of patients, respectively). The best overall response according to RECIST1.1 was partial response (confirmed) in 1 (3%), stable disease in 4 (10%), and progressive disease in 35 (88%). Xevinapant combined with pembrolizumab was well tolerated with no unexpected AEs. However, antitumor activity was low.

Computational analysis of PD-L1 dimerization mechanism induced by small molecules and potential dynamical properties

The design of small molecule inhibitors that target the programmed death ligand-1 (PD-L1) is a forefront issue in immune checkpoint blocking therapy. Small-molecule inhibitors have been shown to exert therapeutic effects by inducing dimerization of the PD-L1 protein, however, the specific mechanisms underlying this dimerization process remain largely unexplored. Furthermore, there is a notable lack of comparative studies examining the binding modes of structurally diverse inhibitors. In view of the research gaps, this work employed molecular dynamics simulations to meticulously examine the interactions between two distinct types of inhibitors and PD-L1 in both monomeric and dimeric forms, and predicted the dimerization mechanism. The results revealed that inhibitors initially bind to a PD-L1 monomer, subsequently attracting another monomer to form a dimer. Notably, symmetric inhibitors observed superior binding efficiency compared to other inhibitors. Key residues, including Ile54, Tyr56, Met115 and Tyr123 played a leading role in binding. Structurally, symmetric inhibitors were capable of thoroughly engaging the binding pocket, promoting a more symmetrical formation of PD-L1 dimers. Furthermore, symmetric inhibitors formed more extensive hydrophobic interactions with protein residues. The insights garnered from this research are expected to significantly contribute to the rational design and optimization of small molecule inhibitors targeting PD-L1.

Magnetic natural lipid nanoparticles for oral treatment of colorectal cancer through potentiated antitumor immunity and microbiota metabolite regulation

The therapeutic efficacy of oral nanotherapeutics against colorectal cancer (CRC) is restricted by inadequate drug accumulation, immunosuppressive microenvironment, and intestinal microbiota imbalance. To overcome these challenges, we elaborately constructed 6-gingerol (Gin)-loaded magnetic mesoporous silicon nanoparticles and functionalized their surface with mulberry leaf-extracted lipids (MLLs) and Pluronic F127 (P127). In vitro experiments revealed that P127 functionalization and alternating magnetic fields (AMFs) promoted internalization of the obtained P127-MLL@Gins by colorectal tumor cells and induced their apoptosis/ferroptosis through Gin/ferrous ion-induced oxidative stress and magneto-thermal effect. After oral administration, P127-MLL@Gins safely passed to the colorectal lumen, infiltrated the mucus barrier, and penetrated into the deep tumors under the influence of AMFs. Subsequently, the P127-MLL@Gin (+ AMF) treatment activated antitumor immunity and suppressed tumor growth. We also found that this therapeutic modality significantly increased the abundance of beneficial bacteria (e.g., Bacillus and unclassified-c-Bacilli), reduced the proportions of harmful bacteria (e.g., Bacteroides and Alloprevotella), and increased lipid oxidation metabolites. Strikingly, checkpoint blockers synergistically improved the therapeutic outcomes of P127-MLL@Gins (+ AMF) against orthotopic and distant colorectal tumors and significantly prolonged mouse life spans. Overall, this oral therapeutic platform is a promising modality for synergistic treatment of CRC.