Innate Immune Checkpoint Research Articles

Enhancement of anticancer immunity by immunomodulation of apoptotic tumor cells using annexin A5 protein-labeled nanocarrier system

Chemotherapy promotes phosphatidylserine (PS) externalization in tumors undergoing apoptosis, forms an immunosuppressive tumor microenvironment (TME), and inhibits dendritic cell (DC) maturation and antigen presentation by binding PS receptors expressed in DCs, thereby limiting naive T cell education and activation. In this study, we demonstrate a selective nanocarrier system composed of annexin A5-labeled poly (lactide-co-glycolide) nanoparticles (PLGA_NPs) encapsulating tumor specific antigen or neoantigen, to target apoptotic tumor cells expressing PS as an innate immune checkpoint inhibitor (ICI) that induces active cancer immunotherapy. Moreover, PLGA_NPs enhanced tumor-specific antigen-based cytotoxic T cell immunity via the original function of DCs by converting the tumor antigen-rich environment. Therefore, chemotherapy combined with an immunomodulatory nanocarrier system demonstrated an enhanced anticancer immune response by increasing survival rates, immune-activating cells, and pro-inflammatory cytokines in the spleen and TME. In contrast, the tumor mass, immune-suppressive cells, and anti-inflammatory cytokines were decreased. Furthermore, the combination of a nanocarrier system with other ICIs against large tumors showed therapeutic efficacy by immunosuppression in the TME and further amplified the anticancer immunity of interferon gamma+ (IFN-γ) CD8+ (cluster of differentiation 8) T cells. Taken together, our Annexin A5-labeled PLGA-NPs can be applied in various combination therapeutic techniques for cancer immunotherapy.

Immune Checkpoint Inhibition in Acute Myeloid Leukemia and Myelodysplastic Syndromes.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of many solid tumors, with limited progress made in the area of myeloid malignancies. The low mutational burden of acute myeloid leukemia (AML) is one potential reason behind the lack of activity of T-cell harnessing ICIs, particularly CTLA-4 and PD-1 inhibitors. Innate immune checkpoints play a critical role in the immune escape of AML and myelodysplastic syndromes (MDS). The CD47 targeting agent, magrolimab, has shown promising activity when combined with azacitidine in early phase trials conducted in AML and higher-risk MDS, especially among patients harboring a TP53 mutation. Similarly, sabatolimab (an anti-TIM-3 monoclonal antibody) plus hypomethylating agents have shown durable responses in higher-risk MDS and AML in early clinical trials. Randomized trials are currently ongoing to confirm the efficacy of these agents. In this review, we will present the current progress and future directions of immune checkpoint inhibition in AML and MDS.

Targeting the CD47-SIRPα Innate Immune Checkpoint to Potentiate Antibody Therapy in Cancer by Neutrophils.

Simple SummaryImmunotherapy aims to engage various immune cells in the elimination of cancer cells. Neutrophils are the most abundant leukocytes in the circulation and have unique mechanisms by which they can kill cancer cells opsonized by antibodies. However, neutrophil effector functions are limited by the inhibitory receptor SIRPα, when it interacts with CD47. The CD47 protein is expressed on all cells in the body and acts as a ‘don’t eat me’ signal to prevent tissue damage. Cancer cells can express high levels of CD47 to circumvent tumor elimination. Thus, blocking the interaction between CD47 and SIRPα may enhance anti-tumor effects by neutrophils in the presence of tumor-targeting monoclonal antibodies. In this review, we discuss CD47-SIRPα as an innate immune checkpoint on neutrophils and explore the preliminary results of clinical trials using CD47-SIRPα blocking agents.In the past 25 years, a considerable number of therapeutic monoclonal antibodies (mAb) against a variety of tumor-associated antigens (TAA) have become available for the targeted treatment of hematologic and solid cancers. Such antibodies opsonize cancer cells and can trigger cytotoxic responses mediated by Fc-receptor expressing immune cells in the tumor microenvironment (TME). Although frequently ignored, neutrophils, which are abundantly present in the circulation and many cancers, have demonstrated to constitute bona fide effector cells for antibody-mediated tumor elimination in vivo. It has now also been established that neutrophils exert a unique mechanism of cytotoxicity towards antibody-opsonized tumor cells, known as trogoptosis, which involves Fc-receptor (FcR)-mediated trogocytosis of cancer cell plasma membrane leading to a lytic/necrotic type of cell death. However, neutrophils prominently express the myeloid inhibitory receptor SIRPα, which upon interaction with the ‘don’t eat me’ signal CD47 on cancer cells, limits cytotoxicity, forming a mechanism of resistance towards anti-cancer antibody therapeutics. In fact, tumor cells often overexpress CD47, thereby even more strongly restricting neutrophil-mediated tumor killing. Blocking the CD47-SIRPα interaction may therefore potentiate neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) towards cancer cells, and various inhibitors of the CD47-SIRPα axis are now in clinical studies. Here, we review the role of neutrophils in antibody therapy in cancer and their regulation by the CD47-SIRPα innate immune checkpoint. Moreover, initial results of CD47-SIRPα blockade in clinical trials are discussed.

PB1820: CD47-BLOCKER TTI-622 COMBINED WITH AZACITIDINE IN PATIENTS WITH TP53-MUTATED ACUTE MYELOID LEUKEMIA (AML) AND WITH AZACITIDINE + VENETOCLAX IN ELDERLY OR UNFIT PATIENTS WITH TP53-WILDTYPE AML

Background: CD47 is an innate immune checkpoint that binds signal regulatory protein alpha (SIRPα) and delivers a “don’t eat me” signal to suppress macrophage phagocytosis. TTI-622 is a fusion protein consisting of the CD47-binding domain of human SIRPα linked to the Fc region of human IgG4. It is designed to enhance phagocytosis and antitumor activity by preventing CD47 from delivering its inhibitory signal as well as generating a moderate pro-phagocytic signal via IgG4 Fc. Importantly, unlike many CD47-blocking agents, TTI-622 does not bind to human red blood cells. Increased expression of CD47 is an adverse prognostic factor in acute myeloid leukemia (AML) due to its association with reduced event-free and overall survival, and blockade of CD47 has shown pre-clinical and clinical activity in AML. In vitro, both azacitidine and venetoclax significantly enhanced the pro-phagocytic effect of TTI-622 on macrophages. Aims: To characterize the safety and preliminary efficacy profile of TTI-622 in combination with azacitidine in newly diagnosed TP53-mutated AML and in combination with azacitidine + venetoclax in elderly or unfit, newly diagnosed TP53-wildtype AML. Pharmacokinetics and immunogenicity of TTI-622 combined with azacitidine ± venetoclax will also be investigated. Primary endpoints include dose-limiting toxicities, incidence of adverse events (AEs) and complete response (CR). Minimal residual disease status will also be evaluated. Methods: As part of an ongoing dose-escalation and expansion trial of TTI-622 (NCT03530683), patients with AML meeting the below key entry criterion are enrolled. Cohort A includes patients with newly diagnosed TP53-mutated AML. Cohort B includes patients with newly diagnosed TP53-wildtype AML who are either elderly (375 years) or unfit. Unfit patients include those < 75 years with one or more of the following comorbidities: Eastern Cooperative Oncology Group (ECOG) performance status of 2, clinically significant heart or lung comorbidities, contraindication to anthracycline therapy, or other comorbidity the investigator regards as incompatible with intensive remission induction chemotherapy. All patients from both cohorts must have documented bone marrow or peripheral blast counts 3 20% and must have received no prior systemic therapy for AML nor hypomethylating agents for any antecedent hematologic disorders prior to transformation to AML. Patients must have no extramedullary disease or leukocytosis >25,000/mL. Renal function must be 330 mL/minute based on Cockcroft-Gault estimate. There are no restrictions based on neutrophil or platelet counts, or hemoglobin level. Patients with AML secondary to antecedent hematologic disorders or therapy induced will not comprise more than 50% of either Cohort. Up to 30 patients will be enrolled into each cohort. In 28-day cycles, patients in both Cohorts will receive TTI-622 8 mg/kg intravenous (IV) infusions weekly combined with azacitidine 75 mg/m2 administered either IV or subcutaneously for 7 consecutive days, repeated approximately every 4 weeks. Patients in Cohort B will also receive venetoclax 100 mg and 200 mg orally on Days 1 and 2, respectively, followed by 400 mg (or equivalent adjusted for azoles) daily thereafter. Safety monitoring includes clinical laboratories and assessments of AEs based on CTCAE v 4.03. Response assessments of both bone marrow and peripheral blood are performed at the end of Cycles 1, 2 and 4; and then every 3 cycles thereafter. Response assessment is per the ELN2017 criteria. Results: Results N/A (trial-in-progress) Summary/Conclusion: Conclusion N/A (trial-in-progress)

Abstract 607: Humanized mice model for efficacy and toxicity evaluation of drugs targeting CD47-SIRPα-PD1-PD-L1 axes

Abstract CD47 is widely expressed on the surface of cells. CD47 interacts with inhibitory receptor signaling protein alpha (SIRPα) to mediate a series of activities such as apoptosis, proliferation, and immune response. Tumor cells can escape the immune surveillance of macrophages through the CD47-SIRPa signaling pathway. Blocking CD47-SIRPα interactions has been shown to promote the destruction of cancer cells by phagocytes. Furthermore, there is growing evidence that targeting of the CD47-SIRPα axis may also promote antigen-presenting cell function. These identify the CD47-SIRPα axis as a promising innate immune checkpoint in cancer. We have generated humanized SIRPα and double humanized CD47/SIRPα mice model for drug efficacy and toxicity evaluation. The humanized mice with human extracellular domain and mouse cytoplasmic fragment of target protein showed similar immune cell population compared with wild type mice. The anti-CD47 antibody, Magrolimab (5F9) analog, displayed anti-tumor effect when administrated to humanized SIRPα mice bearing MC38-hCD47 (MC38 cells engineered to express human CD47) tumor cells. The tumor regression was also observed when treating humanized CD47/SIRPα mice bearing MC38-hCD47 with tested anti-CD47 antibody. Dual checkpoint blockade of CD47 and PD-L1 or PD1 have been reported as a synergistic therapy that activates both innate and adaptive immune response against tumors.We also generated humanized PD1/SIRPα and PD1/PDL1/SIRPα/CD47 mice model for efficacy and toxicity evaluation of drugs targeting for CD47/SIRPα-PD1/PD-L1 axes. Treatment with anti-CD47, anti-SIRPα and anti-PD-L1 alone showed tumor regression efficacy, and combination of anti-PD-L1 and anti-SIRPα or anti-CD47 showed improved anti-tumor effect compared with alone treatment. Those data suggested the humanized syngeneic mice model are useful for the efficacy evaluation of drugs targeting of axes.However, the administration of anti-CD47 blocking antibodies may cause haematotoxicity, including anaaemia and thrombocytopenia, because of the ubiquitous expression of CD47.In vitro Erythrocyte binding activity analysis showed that red blood cells(RBC) collected from humanized CD47/SIRPα mice could bind with Magrolimab analog, while another anti-CD47 antibody has low affinity with RBC. Furthermore, treatment with Magrolimab analog in CD47/SIRPα humanized mice caused haemagglutination and Hemolysis of human RBCs, which were not observed when treating with tested anti-CD47 antibody. Thus, the humanized CD47 and double humanized CD47/SIRPα mice are also suitable for CD47 antibody toxicity evaluation. In conclusion, humanized CD47/SIRPα and quadruple humanized PD1/PDL1/SIRPα/CD47 mice model provide a validated preclinical mouse model to interrogate new therapeutic agents targeting SIRPα-PD1 axes in immune oncology therapy. Citation Format: Juan Liang, Cunxiang Ju, Song Li, Weiwei Yu, Hongyan Sun, Santi Suryani Chen, Jing Zhao, Xiang Gao, Mark Moore. Humanized mice model for efficacy and toxicity evaluation of drugs targeting CD47-SIRPα-PD1-PD-L1 axes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 607.

Abstract 6281: Preclinical development of a bispecific antibody-trap selectively targeting CD47 and CD24 for cancer immunotherapy

Abstract Limited treatment beneficial rate and market saturation of the first generation of immune checkpoint inhibitors (Such as PD-(L)1 inhibitors) have turned the attention of pharmaceutical industry to the next generation of innate immune checkpoints such as CD47/SIRPα and CD24/Siglec-10. Various cancers of lung, breast, colon, prostate, ovarian, stomach, pancreas, myeloid and lymphoid tissues are known to highly express CD47 and/or CD24, which confer cancer cells means of immune-evasion via induction of “Don’t eat me” signals. Precisely and simultaneously neutralizing CD47 and CD24 signaling on tumors, when coupled with an IgG1 antibody’s potent phagocytosis and cytotoxicity activity, may serve as potent immunotherapy for multiple cancer types. We have developed a humanized CD24 neutralizing antibody which has been revealed to neutralize CD24-Siglec-10 interaction and completely eliminate established colon cancer in syngeneic mouse models via proposed mechanisms of antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and subsequent tumor-specific immunity as a result of macrophage activation. Using this mAb as backbone, we developed an CD24 mAb-CD47 trap by conjugating the SIRPα decoy receptor to the N-terminal of the antibody heavy chain. Initial in vitro studies indicated that this mAb-trap has simultaneous two-target binding as well as blocking activities. More promisingly, eight administrations of the mAb-trap at 3mg/kg dose in mice bearing triple-negative breast cancer resulted in a complete tumor growth inhibition. Furthermore, in a Herceptin-resistant xenograft tumor model, the mAb-trap is also efficacious and resulted in over 60% of tumor growth inhibition when administrated at 7.2mg/kg. Taken together, our data show that precise neutralization of CD47 and CD24 signaling on tumors using IgG1 antibody may serve as potent monotherapy for cancers. Citation Format: Wenzhi Tian, Song Li, Dianze Chen, Dandan Liu, Huiqin Guo, Chunmei Yang, Li Zhang, Wei Zhang, Xiaoping Tu, Liang Peng, Gui Zhao, Ruliang Zhang, Fan Zhang. Preclinical development of a bispecific antibody-trap selectively targeting CD47 and CD24 for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6281.

IMMU-21. Targeting the adenosinergic immune suppression pathway in high grade glioma synergizes with innate immune checkpoint blockade

Abstract Immune response in the tumor microenvironment is modulated by the conversion of eATP to AMP to adenosine via the ecto-enzymes CD39 and CD73. Overexpression of CD73 in tumors leads to an increase in extracellular adenosine concentration and decrease in eATP, resulting in immune suppression. In this study, CD73 knockouts were created in adult and pediatric glioma cell lines (DIPG17, T3691, BT245) and used to quantify the effects of CD73 knockout on macrophage phagocytosis in the presence of extracellular AMP. In cell line DIPG17, CD73 knockout significantly increased phagocytosis with and without external AMP compared to the WT. In the WT condition, addition of AMP significantly reduced phagocytosis, while this decrease was not significant in the CD73-KO. The addition of anti-CD47 antibody significantly increased phagocytosis with external AMP in the WT condition. Similar results were obtained with cell line T3691. However, CD73 knockout combined with anti-CD47 treatment and external AMP showed significantly greater phagocytosis than the WT condition. This suggests that a combination of anti-CD73 and anti-CD47 treatment may be more effective in the tumor microenvironment. Lastly, in cell line BT245, CD73 knockout significantly increased phagocytosis as seen in the other cell lines. However, no significant difference was observed between WT and CD73-KO with extracellular AMP or anti-CD47. In vivo studies were conducted in orthotopic xenograft mouse models with DIPG17 CD73 knockout cells. Combination treatment with anti-CD47 antibody significantly decreased tumor burden and prolonged survival in the CD73-KO tumors compared to anti-CD47 treated DIPG13-WT tumors. We conclude that the CD73 adenosinergic pathway and the CD47-SIRPα pathway may present a target for immunotherapy in pediatric and adult gliomas.

TTI-622-01: A phase 1a/1b dose-escalation and expansion trial of TTI-622 in patients with advanced hematologic malignancies, including multiple myeloma.

TPS8071 Background: CD47 is an innate immune checkpoint that binds signal regulatory protein alpha (SIRPα) and delivers a "don’t eat me" signal to suppress macrophage phagocytosis. TTI-622 is a fusion protein consisting of the CD47-binding domain of human SIRPα linked to the Fc region of human IgG4. It is designed to enhance phagocytosis and antitumor activity by preventing CD47 from delivering its inhibitory signal as well as generating a moderate pro-phagocytic signal via IgG4 Fc. CD47 is significantly increased in multiple myeloma (MM) bone marrow mononuclear cells and expression inversely correlates with survival in patients. Relapsed/Refractory (R/R) MM shows particularly high expression of CD47. Preclinical studies demonstrate that the addition of proteasome inhibitors to CD47 blockade significantly increases phagocytosis of MM cells in vitro and anti-myeloma activity in vivo. The ongoing phase 1a part of this study has been previously described. The phase 1b part of the study will determine the safety and efficacy of TTI-622 when given as monotherapy or in combination with selected approved anticancer treatments in patients with hematological malignancies where new treatments with novel mechanism of action are needed. Here we describe 5 RRMM cohorts within phase 1b of the study. Methods: TTI-622-01 is a multi-center Phase 1a/1b study. Phase 1a was designed to determine the MTD, pharmacokinetics (PK), pharmacodynamics, and preliminary antitumor activity of QW, Q2W, and Q3W single-agent TTI-622 in R/R lymphoma using a 3+3 dose escalation schema. Phase 1b, ongoing, will determine the safety and recommended dose of TTI-622 to be given as single agent and in combination with carfilzomib + dexamethasone (Kd) in RRMM and will evaluate the preliminary efficacy. Secondary objectives are to further characterize the safety, PK and immunogenicity of TTI-622 when combined with Kd. Patients will be enrolled in 5 separate cohorts: 3 cohorts will explore different doses and administration schedules of TTI-622 combined with the approved dose of Kd; 2 cohorts will explore different doses of TTI-622 monotherapy. Cohorts will be opened in a staggered manner. In each cohort 3 patients will be dosed and followed for 28 days (21 days in the monotherapy) before expanding enrolment to approximately an additional 27 patients. Key eligibility criteria include: relapse or progression following ≥3 prior lines of therapy (including a proteasome inhibitor, an immunomodulatory drug, and an anti-CD38 antibody), carfilzomib-refractory progressive and measurable disease per IMWG at study entry; age ≥18 years; ECOG performance status ≤2; adequate organ functions; no known CNS involvement; no prior anti-CD47 or anti-SIRPα therapy. Patient recruitment is planned or ongoing at 40 sites worldwide. Clinical trial information: NCT03530683.

TTI-622-01: A phase 1a/1b dose-escalation and expansion trial of TTI-622 in patients with advanced hematologic malignancies, including diffuse large B-cell lymphoma (DLBCL).

TPS7595 Background: CD47 is an innate immune checkpoint that binds signal regulatory protein alpha (SIRPα) and delivers a "don’t eat me" signal to suppress macrophage phagocytosis. Overexpression of CD47 on cancer cells serves as a mechanism of immune surveillance evasion, and is associated with poor prognosis in both hematologic and solid malignancies. TTI-622 is a fusion protein consisting of the CD47-binding domain of human SIRPα linked to the Fc region of human IgG4. It is designed to enhance phagocytosis and antitumor activity by preventing CD47 from delivering its inhibitory signal as well as generating a moderate pro-phagocytic signal via IgG4 Fc. Importantly, unlike many CD47-blocking agents, TTI-622 does not bind to human red blood cells. Preclinical studies demonstrate that TTI-622 induces macrophage-mediated phagocytosis of different malignant cell lines, including DLBCL cells, decreases tumor growth and improves survival in a DLBCL xenograft tumor model. Anti-CD47 antibody enhances rituximab stimulated macrophage-mediated phagocytosis of non-GCB DLBCL cell lines (Bouwstra et al, Cancer Immunol Res. 2019). The ongoing phase 1a part of this study has been previously described. Here we describe 2 cohorts within the phase 1b part of the study that are intended to determine the safety and preliminary efficacy of TTI-622 when given in combination with anti-CD20 targeting agent in patients with CD20+ relapsed/refractory (RR) DLBCL. Methods: TTI-622-01 is a multi-center Phase 1a/1b study. Phase 1a was designed to determine the MTD, pharmacokinetics (PK), pharmacodynamics, and preliminary antitumor activity of QW, Q2W, and Q3W single-agent TTI-622 in R/R lymphoma using a 3+3 dose escalation schema. Phase 1b, ongoing, will determine the safety, recommended dose and preliminary efficacy of TTI-622 in combination with select approved anticancer treatments for patients with hematological malignancies including, but not limited to anti-CD20 therapy in patients with CD20+ RR DLBCL. Secondary objectives are to further characterize safety, PK and immunogenicity of TTI-622 when combined with approved therapies. Patients will be enrolled in 2 cohorts exploring different doses of TTI-622 in combination with anti-CD20 therapy. Cohorts will open in a staggered manner. In each cohort 3 patients will be dosed and followed for 28 days before expanding enrolment to additional 27 patients per cohort. Key eligibility criteria include: age ≥18 years; relapsed and/or refractory disease after ≥1 prior line of therapy; not eligible for or have progressed after high dose chemotherapy (HDT)/auto-SCT; ≥1 site of measurable disease (Lugano 2014 classification); ECOG PS ≤2; adequate organ functions, no known CNS involvement; no prior anti-CD47 or anti-SIRPα therapy. Patient recruitment is planned or ongoing at 40 sites worldwide. Clinical trial information: NCT03530683.

CD24 Is a Potential Immunotherapeutic Target for Mantle Cell Lymphoma.

CD24 and its ligand Siglec-10 were described as an innate immune checkpoint in carcinoma. Here, we investigated this axis in B-cell lymphoma by assessing CD24 expression and evaluating pro-phagocytic effects of CD24 antibody treatment in comparison to hallmark immune checkpoint CD47. In mantle cell lymphoma (MCL) and follicular lymphoma patients, high mRNA expression of CD24 correlated with poor overall survival, whereas CD47 expression did not. Conversely, CD24 expression did not correlate with survival in diffuse large B-cell lymphoma (DLBCL), whereas CD47 did. CD24 was also highly expressed on MCL cell lines, where treatment with CD24 antibody clones SN3 or ML5 potently induced phagocytosis, with SN3 yielding >90% removal of MCL cells and triggering phagocytosis of primary patient-derived MCL cells by autologous macrophages. Treatment with CD24 mAb was superior to CD47 mAb in MCL and was comparable in magnitude to the effect observed in carcinoma lines. Reversely, CD24 mAb treatment was less effective than CD47 mAb treatment in DLBCL. Finally, phagocytic activity of clone SN3 appeared at least partly independent of antibody-dependent cellular phagocytosis (ADCP), suggesting CD24/Siglec-10 checkpoint activity, whereas clone ML5 solely induced ADCP. In conclusion, CD24 is an immunotherapeutic target of potential clinical relevance for MCL, but not DLBCL.

CD47xCD19 bispecific antibody triggers recruitment and activation of innate immune effector cells in a B-cell lymphoma xenograft model

BackgroundCD47/SIRPα axis is recognized as an innate immune checkpoint and emerging clinical data validate the interest of interrupting this pathway in cancer, particularly in hematological malignancies. In preclinical models, CD47/SIRPα blocking agents have been shown to mobilize phagocytic cells and trigger adaptive immune responses to eliminate tumors. Here, we describe the mechanisms afforded by a CD47xCD19 bispecific antibody (NI-1701) at controlling tumor growth in a mouse xenograft B-cell lymphoma model.MethodsThe contribution of immune effector cell subsets behind the antitumor activity of NI-1701 was investigated using flow cytometry, transcriptomic analysis, and in vivo immune-cell depletion experiments.ResultsWe showed that NI-1701 treatment transformed the tumor microenvironment (TME) into a more anti-tumorigenic state with increased NK cells, monocytes, dendritic cells (DC) and MHCIIhi tumor-associated macrophages (TAMs) and decreased granulocytic myeloid-derived suppressor cells. Notably, molecular analysis of isolated tumor-infiltrating leukocytes following NI-1701 administration revealed an upregulation of genes linked to immune activation, including IFNγ and IL-12b. Moreover, TAM-mediated phagocytosis of lymphoma tumor cells was enhanced in the TME in the presence of NI-1701, highlighting the role of macrophages in tumor control. In vivo cell depletion experiments demonstrated that both macrophages and NK cells contribute to the antitumor activity. In addition, NI-1701 enhanced dendritic cell-mediated phagocytosis of tumor cells in vitro, resulting in an increased cross-priming of tumor-specific CD8 T cells.ConclusionsThe study described the mechanisms afforded by the CD47xCD19 bispecific antibody, NI-1701, at controlling tumor growth in lymphoma mouse model. NI-1701 is currently being evaluated in a Phase I clinical trial for the treatment of refractory or relapsed B-cell lymphoma (NCT04806035).

CD47 Blockade Leads to Chemokine-Dependent Monocyte Infiltration and Loss of B Cells from the Splenic Marginal Zone.

CD47 is an important innate immune checkpoint through its interaction with its inhibitory receptor on macrophages, signal-regulatory protein α (SIRPα). Therapeutic blockade of CD47-SIRPα interactions is a promising immuno-oncology treatment that promotes clearance of cancer cells. However, CD47-SIRPα interactions also maintain homeostatic lymphocyte levels. In this study, we report that the mouse splenic marginal zone B cell population is dependent on intact CD47-SIRPα interactions and blockade of CD47 leads to the loss of these cells. This depletion is accompanied by elevated levels of monocyte-recruiting chemokines CCL2 and CCL7 and infiltration of CCR2+Ly6Chi monocytes into the mouse spleen. In the absence of CCR2 signaling, there is no infiltration and reduced marginal zone B cell depletion. These data suggest that CD47 blockade leads to clearance of splenic marginal zone B cells.

Activation of the Innate Immune Checkpoint CLEC5A on Myeloid Cells in the Absence of Danger Signals Modulates Macrophages' Function but Does Not Trigger the Adaptive T Cell Immune Response.

C-Type lectin receptor 5A (CLEC5A) is a spleen tyrosine kinase- (Syk-) coupled pattern recognition receptor expressed on myeloid cells and involved in the innate immune response to viral and bacterial infections. Activation of the CLEC5A receptor with pathogen-derived antigens leads to a secretion of proinflammatory mediators such as TNF-α and IL-6 that may provoke a systemic cytokine storm, and CLEC5A gene polymorphisms are associated with the severity of DV infection. In addition, the CLEC5A receptor was mentioned in the context of noninfectious disorders like chronic obstructive pulmonary disease (COPD) or arthritis. Altogether, CLEC5A may be considered as an innate immune checkpoint capable to amplify proinflammatory signals, and this way contributes to infection or to aseptic inflammation. In this study, we determined CLEC5A receptor expression on different macrophage subsets (in vitro and ex vivo) and the functional consequences of its activation in aseptic conditions. The CLEC5A surface expression appeared the highest on proinflammatory M1 macrophages while intermediate on tumor-associated phenotypes (M2c or TAM). In contrast, the CLEC5A expression on ex vivo-derived alveolar macrophages from healthy donors or macrophages from ovarian cancer patients was hardly detectable. Targeting CLEC5A on noninflammatory macrophages with an agonistic α-CLEC5A antibody triggered a release of proinflammatory cytokines, resembling a response to dengue virus, and led to phenotypic changes in myeloid cells that may suggest their reprogramming towards a proinflammatory phenotype, e.g., upregulation of CD80 and downregulation of CD163. Interestingly, the CLEC5A agonist upregulated immune-regulatory molecules like CD206, PD-L1, and cytokines like IL-10, macrophage-derived chemokine (MDC/CCL22), and thymus and activation chemokine (TARC/CCL17) which are associated with an anti-inflammatory or a protumorigenic macrophage phenotype. In the absence of concomitant pathogenic or endogenous danger signals, the CLEC5A receptor activation did not amplify an autologous T cell response, which may represent a protective innate mechanism to avoid an undesirable autoimmune adaptive response.

Innate immune checkpoint Siglec10 in cancers: mining of comprehensive omics data and validation in patient samples.

Sialic acid binding Ig-like lectin 10 (Siglec10) is a member of innate immune checkpoints that inhibits the activation of immune cells through the interaction with its ligand CD24 on tumor cells. Here, by analyzing public databases containing 64 517 patients of 33 cancer types, we found that the expression of Siglec10 was altered in 18 types of cancers and was associated with the clinical outcomes of 11 cancer types. In particular, Siglec10 was upregulated in patients with kidney renal clear cell carcinoma (KIRC) and was inversely associated with the prognosis of the patients. In 131 KIRC patients of our settings, Siglec10 was elevated in the tumor tissues of 83 (63.4%) patients compared with that in their counterpart normal kidney tissues. Moreover, higher level of Siglec10 was associated with advanced disease (stages III and IV) and worse prognosis. Silencing of CD24 in KIRC cells significantly increased the number of Siglec10-expressing macrophages phagocytosing KIRC cells. In addition, luciferase activity assays suggested that Siglec10 was a potential target of the transcription factors c-FOS and GATA1, which were identified by data mining. These results demonstrate that Siglec10 may have important oncogenic functions in KIRC, and represents a novel target for the development of immunotherapies.

Comprehensive Analysis of Innate Immunophenotyping Based on Immune Score Predicting Immune Alterations and Prognosis in Breast Cancer Patients.

Breast cancer is the most common cancer, with the highest mortality rate and the most diagnosed cancer type in women worldwide. To identify the effect innate immune checkpoint for breast cancer immunotherapy, the innate immune prognostic biomarkers were selected through the ICI score model and the risk model in breast cancer patients. Moreover, the reliability and accuracy of the ICI score model and the risk model were further examined through the analysis of breast cancer prognosis and immune cell infiltration. The pan cancer analysis further confirmed and selected CXCL9 as the key innate immune checkpoint for breast cancer immunotherapy and identified three small molecular drugs for target CXCL9 through molecular docking analysis. In summary, CXCL9 significantly correlated with the prognostic of breast cancer and immune cell infiltration and could be innate immune checkpoint for breast cancer immunotherapy.

Approaches of the Innate Immune System to Ameliorate Adaptive Immunotherapy for B-Cell Non-Hodgkin Lymphoma in Their Microenvironment.

Simple SummaryThe innate immune checkpoint blockade anti-CD47 antibodies combined with the anti-CD20 antibody rituximab can invigorate macrophage phagocytosis. Generally applied as vaccine adjuvants, TLR9 agonist CpG-oligodeoxynucleotides (CpG-ODNs) stimulate antigen presenting cells (APCs) and plasmacytoid dendritic cells (pDCs) to secrete interferon (IFN)-α/β, which activates natural killer (NK) cells. This innate immune activation and pDC maturation leads to potentiation of the adaptive immune response. In fact, stimulator of interferon gene agonists also induce APCs to secrete IFN-α/β. CpG-ODNs, combined with rituximab or irradiation, demonstrated clinical efficacy in indolent B-cell non-Hodgkin lymphoma patients. Furthermore, patients with mantle cell lymphoma presenting Ig-derived neoantigens and vaccinated with CpG-ODN priming T cells maintained minimal residual disease. Additionally, IFN-α–conditioned DC vaccination as well as 4-1BB and OX40 agonist antibodies activate NK cells as well as T cells. Collectively, these innate activators are promising agents for enhancing anti-B-cell-lymphoma adaptive immunity, such as chimeric antigen receptor-T-cell therapy.A dominant paradigm being developed in immunotherapy for hematologic malignancies is of adaptive immunotherapy that involves chimeric antigen receptor (CAR) T cells and bispecific T-cell engagers. CAR T-cell therapy has yielded results that surpass those of the existing salvage immunochemotherapy for patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) after first-line immunochemotherapy, while offering a therapeutic option for patients with follicular lymphoma (FL) and mantle cell lymphoma (MCL). However, the role of the innate immune system has been shown to prolong CAR T-cell persistence. Cluster of differentiation (CD) 47-blocking antibodies, which are a promising therapeutic armamentarium for DLBCL, are novel innate immune checkpoint inhibitors that allow macrophages to phagocytose tumor cells. Intratumoral Toll-like receptor 9 agonist CpG oligodeoxynucleotide plays a pivotal role in FL, and vaccination may be required in MCL. Additionally, local stimulator of interferon gene agonists, which induce a systemic anti-lymphoma CD8+ T-cell response, and the costimulatory molecule 4-1BB/CD137 or OX40/CD134 agonistic antibodies represent attractive agents for dendritic cell activations, which subsequently, facilitates initiation of productive T-cell priming and NK cells. This review describes the exploitation of approaches that trigger innate immune activation for adaptive immune cells to operate maximally in the tumor microenvironment of these lymphomas.

CD47 expression attenuates Ebola virus-induced immunopathology in mice

It has been shown that a very early cell-intrinsic response to infection is the upregulation of CD47 cell surface expression, a molecule known for delivering a “don't eat me signal” that inhibits macrophage-mediated phagocytosis and antigen presentation. Thus, blockade of CD47 signaling during lymphocytic choriomenigitis virus infections of mice has been shown to enhance the kinetics and potency of immune responses, thereby producing faster recovery. It seems counterintuitive that one of the earliest responses to infection would be immunoinhibitory, but it has been hypothesized that CD47 induction acts as an innate immune system checkpoint to prevent immune overactivation and immunopathogenic responses during certain infections. In the current study we examined the effect of CD47 blockade on lethal Ebola virus infection of mice. At 6 days post-infection, CD47 blockade was associated with significantly increased activation of B cells along with increases in recently cytolytic CD8+ T cells. However, the anti-CD47-treated mice exhibited increased weight loss, higher virus titers, and succumbed more rapidly. The anti-CD47-treated mice also had increased inflammatory cytokines in the plasma indicative of a “cytokine storm”. Thus, in the context of this rapid hemorrhagic disease, CD47 blockade indeed exacerbated immunopathology and disease severity.

EXTH-45. MICROGLIA-SPECIFIC DISRUPTION OF SIALIC ACID-SIGLEC-9/E INTERACTIONS: A NOVEL IMMUNOTHERAPY AGAINST GLIOBLASTOMA?

Abstract Recently, ‘don’t eat me’-signals like CD47 have emerged as novel innate immune checkpoints, enabling cancer cells to evade clearance by phagocytes such as monocyte-derived macrophages (MDM) or microglia (MG). Here, we aim at defining the role of inhibitory Siglec-9 in human and its mouse homologue Siglec-E in MG-centered immunotherapy against GBM. TCGA RNA-sequencing data revealed a significant correlation between high expression of immunoinhibitory SIGLEC9 and poor survival in GBM patients (log-rank p = 0.02). Siglec-E blockade increased murine MG mediated GBM cell in vitro phagocytosis (normalized phagocytosis of 1.00 in isotype vs. 1.76 in anti-Siglec-E antibody, p &amp;lt; 0.001). By employing a CT-2A orthotopic GBM mouse model with MG-specific spatio-temporal deletion of Siglece (Sall1 CreERT2 x Siglece flox ), we observed high MG-proliferation upon Siglec-E knockdown (Ki-67+ MG 14.8% in Cre- vs. 34.9% in Cre+, p &amp;lt; 0.0001) accompanied by an enhanced microglial GBM-cell uptake (5.6% in Cre- vs. 12.3% in Cre+, p &amp;lt; 0.001). This beneficial response was counteracted by an accentuated influx of pro-tumorigenic MDM in the Cre+ group (CD163high CD86low MDM of total MDM 47.1% in Cre- vs. 65.3% in Cre+, p = 0.002), which prevented an efficient adaptive anti-tumor immune response and survival benefit. Currently, we are investigating the cross-talk between GBM-associated MG and MDM upon Siglec-E knockdown by scRNAseq of the tumor-infiltrating immune compartment, including TCR-clonotype tracking. By genetically targeting sialic acids, the ligand for Siglec receptors, on CT-2A cells (GNE-KO), we observed a strong innate and adaptive immune response with less exhausted tumor-infiltrating CD8+ T-cells (14.8% in WT vs. 5.9% in GNE-KO, p = 0.003), which resulted in a prolonged survival (30d in WT vs. 41d post-tumor-injection in GNE-KO, p = 0.03). These data identify the sialic-acid-Siglec-E pathway as an anti-phagocytic signal in a pre-clinical GBM model, and demonstrate its therapeutic potential in GBM immunotherapy.

CD47-Blocker TTI-622 Shows Single-Agent Activity in Patients with Advanced Relapsed or Refractory Lymphoma: Update from the Ongoing First-in-Human Dose Escalation Study

CD47-Blocker TTI-622 Shows Single-Agent Activity in Patients with Advanced Relapsed or Refractory Lymphoma: Update from the Ongoing First-in-Human Dose Escalation Study

Updates from Ongoing, First-in-Human Phase 1 Dose Escalation and Expansion Study of TTI-621, a Novel Biologic Targeting CD47, in Patients with Relapsed or Refractory Hematologic Malignancies

Updates from Ongoing, First-in-Human Phase 1 Dose Escalation and Expansion Study of TTI-621, a Novel Biologic Targeting CD47, in Patients with Relapsed or Refractory Hematologic Malignancies