Checkpoint For Cancer Immunotherapy Research Articles

Complement factor H: a novel innate immune checkpoint in cancer immunotherapy.

The elimination of cancer cells critically depends on the immune system. However, cancers have evolved a variety of defense mechanisms to evade immune monitoring, leading to tumor progression. Complement factor H (CFH), predominately known for its function in inhibiting the alternative pathway of the complement system, has recently been identified as an important innate immunological checkpoint in cancer. CFH-mediated immunosuppression enhances tumor cells' ability to avoid immune recognition and produce an immunosuppressive tumor microenvironment. This review explores the molecular underpinnings, interactions with immune cells, clinical consequences, and therapeutic possibilities of CFH as an innate immune checkpoint in cancer control. The difficulties and opportunities of using CFH as a target in cancer immunotherapy are also explored.

Targeting the CD24-Siglec10 Axis: A Potential Strategy for Cancer Immunotherapy

Abstract CD24, also known as heat-stable protein, is a highly glycosylated glycosylphosphatidylinositol junction membrane protein. CD24 specifically binds sialic-acid-binding Ig-like lectin 10 (Siglec10) on macrophages and serves as a “don’t eat me” signal, thus blocking the phagocytosis of tumor cells by macrophages and triggering tumor immune escape. Blocking the CD24-Siglec10 axis to reprogram the tumor immune microenvironment is a current research hotspot in cancer immunotherapy. Targeting the CD24-Siglec10 axis has received widespread attention, because of the high expression of CD24 on a variety of tumor cells and absence of blood toxicity. Targeting the CD24-Siglec10 axis as a cancer immunotherapy has shown favorable results and progress in preclinical studies. In this review, we summarize the discovery and functions of the CD24-Siglec10 axis, and review the roles and effects of this axis as a novel immune checkpoint in cancer immunotherapy. We also highlight recent advances in nanoparticle-mediated treatments targeting the CD24-Siglec10 axis for enhancing cancer immunotherapy.

Diversity of immune checkpoints in cancer immunotherapy.

Finding effective treatments for cancer remains a challenge. Recent studies have found that the mechanisms of tumor evasion are becoming increasingly diverse, including abnormal expression of immune checkpoint molecules on different immune cells, in particular T cells, natural killer cells, macrophages and others. In this review, we discuss the checkpoint molecules with enhanced expression on these lymphocytes and their consequences on immune effector functions. Dissecting the diverse roles of immune checkpoints in different immune cells is crucial for a full understanding of immunotherapy using checkpoint inhibitors.

The role of ubiquitin pathway-mediated regulation of immune checkpoints in cancer immunotherapy.

With the continuous cognition of the relationship between tumor cells and tumor immune microenvironment, immunotherapy based on the immune checkpoint blockade has achieved great breakthroughs, led to improved clinical outcomes, and prolonged survival for cancer patients in recent years. Nevertheless, the de novo or acquired resistance to immunotherapy has greatly counteracted the efficacy, leading to a 20%-40% overall response rate. Thus, further in-depth understanding of the regulation of the tumor microenvironment and antitumor immunity is urgently warranted. Ubiquitination-mediated protein degradation plays vital roles in protein stabilization, activation, and dynamics as well as in cellular homeostasis modulation. The dysregulated ubiquitination and deubiquitination are closely related to the changes in physiological and pathological processes, which subsequently result in a variety of diseases including cancer. In this review, the authors first summarize the current knowledge about the involvement of the ubiquitin-proteasome system in tumor development with the ubiquitin conjugation-regulated stability of p53, phosphatase and tensin homolog, and Myc protein as examples, then dissect the potential implications of ubiquitination-mediated immune checkpoints degradation in tumor microenvironment and immune responses, and finally discuss the effects of therapeutically targeting the ubiquitin-proteasome pathway on immunotherapy, with the goal of providing deep insights into the exploitation of more precise and effective combinational therapy against cancer.

V-Set and immunoglobulin domain containing (VSIG) proteins as emerging immune checkpoint targets for cancer immunotherapy.

The development of immune checkpoint inhibitors is becoming a promising approach to fight cancers. Antibodies targeting immune checkpoint proteins such as CTLA-4 and PD-1 can reinvigorate endogenous antitumor T-cell responses and bring durable advantages to several malignancies. However, only a small subset of patients benefit from these checkpoint inhibitors. Identification of new immune checkpoints with the aim of combination blockade of multiple immune inhibitory pathways is becoming necessary to improve efficiency. Recently, several B7 family-related proteins, TIGIT, VSIG4, and VSIG3, which belong to the VSIG family, have attracted substantial attention as coinhibitory receptors during T-cell activation. By interacting with their corresponding ligands, these VSIG proteins inhibit T-cell responses and maintain an immune suppressive microenvironment in tumors. These results indicated that VSIG family members are becoming putative immune checkpoints in cancer immunotherapy. In this review, we summarized the function of each VSIG protein in regulating immune responses and in tumor progression, thus providing an overview of our current understanding of VSIG family members.

Abstract LBA019: The immune checkpoint protein BTN1A1 suppresses T cell activation through interactions with Gal9 and PD-1

Abstract Butyrophilin (BTN) proteins are members of the B7 immunoglobulin superfamily and exhibit well-characterized immunomodulatory functions in mammals. We have recently identified BTN1A1 as an immune checkpoint protein prominently upregulated in response to acute inflammatory insults. Further in vitro and in vivo assays have validated BTN1A1 as an immune checkpoint target, particularly for patients refractory to anti-PD-1/PD-L1 antibody treatment. We have also developed a humanized antibody targeting human BTN1A1, hSTC810, which is expected to enter into Phase I clinical trials in the first quarter of 2022. In this study, a cell microarray from Retrogenix (Whaley Bridge, UK) was used to identify binding partner(s) for the extracellular domain of human BTN1A1. Through this screening approach, we found that BTN1A1 binds to galectin-1 (Gal1), galectin-9 (Gal9), and neuropilin 2 (NRP2). These three putative binding partners could specifically bind to wild-type BTN1A1 but not to this protein's unglycosylated (2NQ) form. Of these three targets, immunoprecipitation and Biacore binding assays revealed that Gal9 exhibited the greatest affinity for human BTN1A1, followed by Gal1, with respective KD values of 22.7 nM and 1.88 μM - an 83-fold difference. Gal9 binding to human BTN1A1 was dependent on BTN1A1 glycosylation status and required the carbohydrate recognition domain (CRD) of Gal9. As Gal9 is a known PD-1-binding protein, the KD of Gal9 for PD-1 was additionally assessed and found to be 19.7 nM. These results thus predicted the potential formation of BTN1A1/Gal9/PD-1 complexes. Consistent with these predictions, immunoprecipitation assays performed using cells expressing Myc-tagged versions of these three proteins demonstrated the formation of BTN1A1/Gal9, PD-1/Gal9, and BTN1A1/Gal9/PD-1 complexes. CRISPR-mediated BTN1A1 knockout in Jurkat T cells induced both PD-1 expression and T cell activation. BTN1A1 also suppressed T cell receptor (TCR) signaling in Jurkat cells, and the addition of exogenous recombinant Gal9 protein further blunted such BTN1A1-mediated TCR-signaling downregulation. Such downregulation was not observed in PD-1 knockout Jurkat cells. Together with the observation that BTN1A1 does not bind to PD-1 directly, the data suggest that BTN1A1 suppresses T cell activation by interacting with PD-1 through Gal9. As high Gal9 expression levels are correlated with poor prognosis in multiple cancers, our results highlight this BTN1A1-Gal9-PD-1 axis as a novel therapeutic target for immunotherapeutic drug development. (1) Chung EM, Bong YS, Kim YS, Park A, You YO, Sharma A, Lin SH, Lee YJ, Jung H, Yoo SS. BTN1A1: a novel immune checkpoint for cancer immunotherapy beyond the PD-1/PD-L1 axis. Cancer Res 2021;81(13_Suppl): Abstract nr 1643. Citation Format: Ezra M Chung, Young-Seung Kim, Chunai Wu, Andrew H Park, Hyunjin Jung, Stephen S Yoo. The immune checkpoint protein BTN1A1 suppresses T cell activation through interactions with Gal9 and PD-1 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA019.

832 Deep learning reveals predictive sequence concepts within immune repertoires to immunotherapy

832 Deep learning reveals predictive sequence concepts within immune repertoires to immunotherapy

Combinatorial Approaches with Checkpoint Inhibitors to Enhance Anti-Tumor Immunity as Advantages of Targeting the Tumor Immune Microenvironment over Blocking Immune Checkpoint in Cancer Immunotherapy

Aging is a condition in which the cell cycle is essentially irreversible and is caused by a variety of stressors such as obesity, radiation and chemotherapy. Aging cells that accumulate in the body during this period communicate with surrounding tissues through the production of proinflammatory proteins, called the SASP, and play a number of physiological and pathological roles. In the elderly, inflammatory agents of SASP increase various age-related diseases, including cancer; therefore, clarification of the SASP monitoring mechanism is essential for the development of new prevention and treatment strategies against age-related cancer. A group of Cancer Research Institute (CRI) of California South University researchers have hypothesized that the abnormal chromatin architecture observed in aging cells is related to SASP, and have begun analyzing chromatin interaction at the genome level and gene expression using next-generation sequencing techniques. They showed that the region containing the pericentromeric repetitive sequences called Human Satellite II (hSATII), which is genetically inactive in normal cells, has a significant state in aging cells; In addition, non-coding RNA expression (hSATII RNA) was significantly regulated during cellular aging. Further analysis showed that hSATII RNA regulated the expression of SASP-like inflammatory genes by disrupting chromatin interactions in some regions of the SASP gene through dysfunction of the CCCTC-binding factor (CTCF), which is important for maintaining genomic integrity. Small extracellular vehicles (EVs) secreted by cancer cells and stromal cells are dynamically involved in the development and progression of non-cellular tumors in the tumor microenvironment, and surprisingly more than the amount of hSATII RNA in small EVs caused by aging cells. Thus, our data suggest that hSATII RNA derived from aging stromal cells is transported to surrounding cells via small EVs and acts as an SASP-like inflammatory agent in the tumor microenvironment. In addition, the researchers found that hSATII RNA could be detected in cancer cells in surgical specimens of patients with primary colon cancer. Significantly, the population of hSATII-positive RNA cells was higher among cancer-associated fibroblasts than in fibroblasts in normal stromal tissues. These findings demonstrate the new role of hSATII RNA, which supports non-cellular tumor growth by secreting inflammatory agents similar to SASPs and small EVs. Understanding this molecular mechanism could facilitate new preventive and therapeutic development and provide solutions for future age-related injuries. Keywords: Cancer; Cells; Tissues; Tumors; Prevention; Prognosis; Diagnosis; Imaging; Screening; Treatment; Management

Trends in the Research Into Immune Checkpoint Blockade by Anti-PD1/PDL1 Antibodies in Cancer Immunotherapy: A Bibliometric Study.

The programmed death receptor 1 (PD1) and its ligand programmed death receptor ligand 1 (PDL1) are the most widely used immune checkpoints in cancer immunotherapy. The related literature shows the explosive growth trends due to the promising outcomes of tumor regression. The present study aimed to provide a comprehensive bibliometric analysis of the literature on anti-PD1/PDL1 from three perspectives including molecular mechanisms, randomized clinical trials (RCT), and meta-analysis, thus producing a knowledge map reflecting the status of the research, its historical evolution, and developmental trends in related research from 2000 to 2020. We included 11,971, 191, and 335 documents from the Web of Science Core Collection database, respectively, and adopted various bibliometric methods and techniques thereto. The study revealed the major research themes and emergent hotspots based on literature and citation data and outlined the top contributors in terms of journals and countries. The co-occurrence overlay of keywords and terms pertaining to the PD1/PDL1 molecule reflected the progress from the discovery of the PD1/PDL1 molecule to the clinical application of anti-PD1/PDL1. Immune-related adverse events (irAEs) formed a unique cluster in the term co-occurrence analysis of meta-analysis. The historical direct citation network of RCT indicated the development and transformation of cancers and therapy strategies. irAEs and the strategies of combination therapy might become a future focus of research in this cognate area. In summary, the bibliometric study provides a general overview of the landscape on anti-PD1/PDL1 research, allowing researchers to identify the potential opportunities and challenges therein.

Abstract 1643: BTN1A1: a novel immune checkpoint for cancer immunotherapy beyond the PD-1/PD-L1 axis

Abstract Cancer immunotherapy is an effective treatment against individuals with late-stage cancer forms. The PD-1/PD-LI axis is a main therapeutic target used in clinical settings, but only 15-20% of cancer patients are responsive. Thus, there is an urgent unmet need to identify other immuno-therapeutic targets to overcome this limitation. Butyrophilin (BTN) belongs to the B7 family, peripheral membrane proteins containing an immunoglobulin domain found in PD-L1 and CTLA4. Among the BTN family, BTN2A2 and BTN3A1 are shown as alternative co-inhibitory or co-stimulatory cancer immune checkpoint(s) in the effector T cell compartment. However, the immune-modulatory roles of BTN1A1 are largely unknown, especially in vivo. Here, we report the immune-modulatory role of BTN1A1 in T cell proliferation and activation in vitro and in vivo. We have found that BTN1A1 inhibits the proliferation of T cells that are activated by anti-CD3 and anti-CD28 antibodies in vitro. The overexpression of BTN1A1 in PC3 cells also inhibits T cell-mediated cancer cell killing. BTN1A1-overexpressing B16-Ova melanoma cancer cell lines also accelerate tumor growth compared to B16-Ova wild type in a syngeneic mouse model. The effect of BTN1A1 was fully attenuated in an immune-compromised SCID mouse. BTN1A1-deficient MC38 colorectal cancer cell lines also grew significantly slower than wild-type MC38 cell lines when they were inoculated subcutaneously into a syngeneic mouse model. This result strongly supports BTN1A1's role in immune evasion. BTN1A1 is expressed on immune cells. These cells include macrophages, B cells or activated CD8 T cells. We found that BTN1A1 is expressed in human tumors, with an expression that was mutually exclusive to PD-L1. We have developed the monoclonal antibody (STC810) against the human BTN1A1. STC810 has anti-tumor activity in a human immune environment in vitro and ex vivo. Since STC810 has no cross-reactivity with mouse BTN1A1 proteins, we generated mouse surrogate antibodies to study the effects of blocking BTN1A1 in vivo in mice tumor models. The anti-BTN1A1 surrogate antibody not only exhibits a single agent but has a synergistic effect in combination with the anti-PD-L1 antibody. Overall, using mouse models, genetic ablation, and antibody tests ex vivo we demonstrate that BTN1A1 is a bona fide immune checkpoint inhibitor. Late-stage preclinical studies of humanized STC810 are underway. The Phase I clinical trial is set to start in late 2021. Citation Format: Ezra M. Chung, Yong-Sik Bong, Young-Seung Kim, Andrew Park, Young-Ok You, Amrish Sharma, Steven H. Lin, Young-Joon Lee, Hyunjin Jung, Stephen S. Yoo. BTN1A1: a novel immune checkpoint for cancer immunotherapy beyond the PD-1/PD-L1 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1643.

Abstract 1636: CLEC-1 is a novel myeloid immune checkpoint for cancer immunotherapy limiting tumor cells phagocytosis and tumor antigen cross-presentation

Abstract Myeloid cells represent one of the most abundant immune cell types in solid tumors that impede myeloid phagocytosis by triggering “don't eat me” and “don't find me” signals. Recent literature demonstrates that C-type lectin receptors (CLRs) are powerful pattern recognition receptors shaping immune cell-mediated tissue damage by positively or negatively regulating myeloid cell functions and hence tumor elimination or evasion. We previously reported that the orphan CLR CLEC-1 expressed by dendritic cells (DCs) and macrophages (MPs) is enhanced by TGFβ and tempers downstream T cells responses. Furthermore, we observed that CLEC-1 is highly expressed by myeloid cells purified from the human tumor microenvironment, in particular, tumor-associated MPs. We evaluated whether CLEC-1 could also be a receptor for DAMPs and influences phagocytosis. We found that CLEC-1 fusion protein binds specifically to secondary necrotic healthy or tumor cells induced by chemotherapy, radiation (UV, X-ray), or culture stress conditions suggesting that ligands of CLEC-1 are generated upon stress and programmed cell death. Importantly, further to promising results in KO CLEC-1 mice, we observed in vivo that CLEC-1 deficient mice, in contrast to wild-type littermates, eradicate MC38 colorectal tumors in combination with cytotoxic and immunogenic chemotherapy. Importantly, disruption of CLEC-1 signaling by Fc-CLEC-1 fusion protein also promotes tumor eradication. We then generated and identified different anti-human CLEC-1 antagonist monoclonal antibodies (mAbs) with the capacity to block CLEC-1/CLEC-1L interaction. We developed innovative antagonist CLEC-1 mAbs which, in contrast to non-antagonist CLEC-1 control mAb, increase the phagocytosis of CLEC-1L-positive human tumor cells by human TGFβ-polarized DCs or MPs. Indeed, TGFβ-polarized DCs phagocytose more efficiently a NSCLC cell line (A549) as well as Rituximab (anti-CD20 mAb)-opsonized Burkitt lymphoma cells (Raji) when CLEC-1 is antagonized by Abs. Similarly, macrophages significantly more efficiently engulfed human tumors in the presence of CLEC-1 antagonist Abs, in particular when tumor cells were opsonized such as Rituximab-opsonized Raji cells, Cetuximab opsonized colon carcinoma cells (DLD-1; EGFR+) or Trastuzumab opsonized mammary carcinoma cells (SK-BR-3; Her2+). Importantly, we observed both in vitro and in vivo that DCs from Clec1a deficient mice cross-present more efficiently dead cell-associated antigens to CD8+ T cells (OT-1). We generated hCLEC-1 knock-in mice and in vivo preclinical evaluation of CLEC-1 blocking mAbs is ongoing. Altogether, these data illustrate that CLEC-1 broadly notably inhibits tumor-cell phagocytosis and synergized with tumor-targeted cytotoxic monoclonal antibodies in both solid and hematological tumors, and hampers DC antigen cross-presentation. Citation Format: Vanessa Gauttier, Sabrina Pengam, Marion Drouin, Javier Saenz, Bérangère Evrard, Kevin Biteau, Caroline Mary, Géraldine Teppaz, Ariane Desselle, Virginie Thépénier, Emmanuelle Wilhelm, Elise Chiffoleau, Nicolas Poirier. CLEC-1 is a novel myeloid immune checkpoint for cancer immunotherapy limiting tumor cells phagocytosis and tumor antigen cross-presentation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1636.

Methylation of the NT5E Gene Is Associated with Poor Prognostic Factors in Breast Cancer.

Cluster of differentiation (CD) 73, which is encoded by the NT5E gene, regulates production of immunosuppressive adenosine and is an emerging checkpoint in cancer immunotherapy. Despite the significance of CD73 in immuno-oncology, the roles of the NT5E gene methylation in breast cancer have not been well-defined yet. Therefore, we aimed to investigate the prognostic significance of the NT5E gene methylation in breast cancer. The DNA methylation status of the NT5E gene was analyzed using pyrosequencing in breast cancer tissues. In addition, the levels of inflammatory markers and lymphocyte infiltration were evaluated. The mean methylation level of the NT5E gene was significantly higher in breast cancer than in normal breast tissues. In the analysis of relevance with clinicopathologic characteristics, the mean methylation levels of the NT5E gene were significantly higher in patients with large tumor size, high histologic grade, negative estrogen receptor expression, negative Bcl-2 expression, and premenopausal women. There was no difference in disease-free survival according to the methylation status of the NT5E gene. We found that the NT5E gene methylation was related to breast cancer development and associated with poor prognostic factors in breast cancer. Our results suggest that the NT5E gene methylation has potential as an epigenetic biomarker in breast cancer.

Licochalcone A inhibits interferon-gamma-induced programmed death-ligand 1 in lung cancer cells

BackgroundProgrammed death-ligand 1 (PD-L1), which can be induced by interferon-gamma (IFN-γ) in the tumor microenvironment, is a critical immune checkpoint in cancer immunotherapy. Natural products which reduce IFN-γ-induced PD-L1 might be exert immunotherapy effect. Licochalcone A (LCA), a natural compound derived from the root of Glycyrrhiza inflata Batalin. (Fabaceae), was found to interfere IFN-γ-induced PD-L1. PurposeThe aim of this study is to further clarify the effect and the mechanism of LCA on inhibiting IFN-γ-induced PD-L1 in lung cancer cells. MethodsThe expression levels of PD-L1 were evaluated by flow cytometry, western blot and qRT-PCR. Click-iT protein synthesis assay and luciferase assay were used to identify the effect of LCA on protein synthesis. Jurkat T cell proliferation and apoptosis in the co-culture system were detected by flow cytometry. Flow cytometry was also applied to evaluate reactive oxygen species (ROS) generation. ResultsLCA downregulated IFN-γ-induced PD-L1 protein expression and membrane localization in human lung cancer cells, regardless of inhibiting PD-L1 mRNA level or promoting its protein degradation. LCA decreased apoptosis and proliferative inhibition of Jurkat T cells caused by IFN-γ-induced PD-L1-expressing in A549 cells in the co-culture system. Strikingly, LCA was verified as a protein synthesis inhibitor, which reduced both cap-dependent and -independent translation. LCA inhibited PD-L1 translation, likely due to inhibition of 4EBP1 phosphorylation (Ser 65) and activation of PERK-eIF2α pathway. Furthermore, LCA induced ROS generation in a time-dependent manner in lung cancer cells. N-acetyl-L-cysteine (NAC) not only revered ROS generation triggered by LCA but also restored IFN-γ-induced expression of PD-L1. Both the inhibition of 4EBP1 phosphorylation (Ser 65) and activation of PERK-eIF2α axis triggered by LCA was restored by co-treatment with NAC. ConclusionLCA abrogated IFN-γ-induced PD-L1 expression via ROS generation to abolish the protein translation, indicating that LCA has the potential to be applied in cancer immunotherapy.

A Prospective Study of the Relevance of the HLA-G Immune Checkpoint in Cancer Immunotherapy

A Prospective Study of the Relevance of the HLA-G Immune Checkpoint in Cancer Immunotherapy

Efficient development and expression of scFv recombinant proteins against PD-L1 surface domain and potency in cancer therapy.

PD-L1 is a 40kDa trans-membrane protein of B7 family and an important T cell regulator. Binding of PD-L1 and PD-1 inhibits proliferation and activation of T cell results cell exhaustion. This phenomenon can be reversed by blocking PD-L1/PD-1 interactions with single chain variables fragment (scFv) fusion proteins and by direct inhibition of tumor cells with drug conjugates. The human phage-displayed scFv library was utilized to generate scFv against the PD-L1 antigen by affinity bio-panning. The positive clones were selected by continuous transfection of bacterial cells and sequence analysis. The binding affinity and specificity of the scFv and antibody fragments were determined by using surface plasmon resonance biosensor, western blot analysis, and immunofluorescence assay. After three rounds of panning selection, about 30% of clones have a binding affinity with targeted PD-L1 antigen. Eight positive clones with accurate sequences were isolated and analyzed for binding affinity with PD-L1 antigen. Three of those with accurate sequences and binding affinity were selected for the recombinant formation and soluble expression by Escherichia coli host machinery. The highly positive recombinant clones with the exact orientation of FR and CDR domains were developed and can be used as a drug carrier tools in ADC formation or direct inhibition of immune checkpoint in cancer immunotherapy. The conjugate achieved its initial potency and need efficient improvement to enhance direct tumor suppression and bio-therapeutics strategies enrichment.

Checkpoint ahead - be prepared to stop!

Two studies published in Cell have characterized the inhibitory immune receptor NKG2A expressed on cytotoxic lymphocytes, such as natural killer cells and CD8+ T cells, as a novel checkpoint for cancer immunotherapy.

MS28.02 Combination IO+IO

MS28.02 Combination IO+IO

New checkpoints in cancer immunotherapy.

Immune responses must be fine-tuned to allow effective clearance of invading pathogens, while maintain tolerance to self-antigens. T cells are the major effector cells for fighting and killing tumor cells. Immune checkpoints play a pivotal role in T cell activation, and determine the functional outcome of T cell receptor (TCR) signaling. The blockade of immune checkpoints CTLA-4 and PD-1 has already been one of the most successful cancer immunotherapies. In this review, we will focus on three novel inhibitory B7 family checkpoint molecules, B7-H3, B7S1 and VISTA. The aim of this article is to summarize their expressions in tumors as well as their roles in controlling and suppressing T cell immune responses and anti-tumor immunity. These pathways may be explored in future cancer immunotherapy.

Abstract 3218: Disruption of the CD39 immune checkpoint pathway increases the efficacy of various anticancer therapies in syngeneic mouse models

Abstract The CD39-CD73-adenosine pathway is an emerging regulator of the immune antitumor response. CD39 is expressed within tumors and the tumor microenvironment by several cell population including immune and cancer cells. In tumor tissues, the pathway leads to the accumulation of immunosuppressive adenosine together with decreased levels of immunoactivating peritumoral ATP. We reported previously that CD39 blockade increased T cell and NK cell-mediated cytotoxic activity in vitro and disclose, during this meeting, the development of the first human-CD39-blocking humanized antibody (S. Augier et al., Preclinical development of a humanized blocking antibody targeting the CD39 immune checkpoint for cancer immunotherapy). Here we demonstrated that this pathway is involved in tumor-induced resistance to various cancer therapies in syngeneic mouse melanoma, colon cancer and fibrosarcoma models. We used therapy-resistant mouse models or inefficacious treatment regimens in the CD39 knockout mice to assess the capacity of CD39 to affect the response to chemotherapies, tumor associated antigen (TAA)-targeting antibodies and immunomodulators such as anti-PD1 antibodies. We achieved increased response rates, increased response duration and some complete and long lasting tumor regressions in the CD39 deficient context. These preclinical proof-of-concept studies highlight the role of the CD39 immune checkpoint pathway in limiting the efficacy of various anticancer therapies in syngeneic mouse models and thereby support the potential clinical value of the humanized CD39-neutralizing antibody under development. Citation Format: Marion Lapierre, Cécile Dejou, Carine Paturel, Henri-Alexandre Michaud, Laurent Gros, Armand Bensussan, Jean-François Eliaou, Jeremy Bastid, Nathalie Bonnefoy. Disruption of the CD39 immune checkpoint pathway increases the efficacy of various anticancer therapies in syngeneic mouse models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3218.

Abstract 3222: Preclinical development of a humanized blocking antibody targeting the CD39 immune checkpoint for cancer immunotherapy

Abstract CD39 (ENTPD1) is a cell membrane ectonucleotidase that hydrolyzes extracellular immunoactivating ATP and ADP into AMP, which can be further hydrolyzed by ectonucleotidase CD73 into immunosuppressive adenosine. Within the tumor microenvironment, adenosine accumulation causes immune suppression and dysregulation of immune cell infiltrates resulting in tumor spreading. The role of CD39 expression on both Tregs and on tumor cells in promoting immunosuppression has been demonstrated in several reports. Blockade of CD39 may promote anti-tumor immunity by directly accumulating immunostimulating ATP and indirectly by reducing adenosine accumulation. Here, we describe the discovery and preclinical development of an anti-huCD39 blocking antibody for cancer immunotherapy. Parental anti-huCD39 mouse monoclonal antibody was humanized. The humanized mAb specifically binds huCD39 protein, but not CD39-like proteins. Nanomolar affinities for human CD39 were measured in SPR studies on recombinant CD39 protein and in flow cytometry titration studies on CD39 expressing transfectants and tumor cell lines. The humanized mAb blocked human CD39 ATPase activity in vitro in the nanomolar range, as demonstrated using transfected cells, CD39-expressing tumor cell lines, as well as human PBMC and ex-vivo isolated fresh tumor samples. The humanized mAb cross-reacted on cynomolgus CD39 and blocked ATPase activity on cynomolgus PBMC with similar efficacy as on human PBMC. Finally, treatment with blocking anti-CD39 mAb inhibited tumor growth in vivo in mouse tumor models. Taken together, these data support the clinical development of anti-CD39 neutralizing mAb for cancer immunotherapy. Citation Format: Severine Augier, Ivan Perrot, Cecile Dejou, Rachel Joly, Stephane Delahaye, Helene Rispaud Blanc, Caroline Denis, Laurent Gauthier, Armand Bensussan, Jean-francois Eliaou, Yannis Morel, Nathalie Bonnefoy, Jeremy Bastid, Carine Paturel. Preclinical development of a humanized blocking antibody targeting the CD39 immune checkpoint for cancer immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3222.