Signal Regulatory Protein Alpha Research Articles

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.

Soluble SIRP-Alpha Promotes Murine Acute Lung Injury Through Suppressing Macrophage Phagocytosis.

Soluble signal regulatory protein-alpha (SIRP-alpha) is elevated in bronchoalveolar lavage (BAL) of mice with lipopolysaccharides (LPS)-induced acute lung injury (ALI). To define the role of soluble SIRP-alpha in the pathogenesis of ALI, we established murine ALI in wild-type (WT) and SIRP-alpha knock-out (KO) mice by intratracheal administration of LPS. The results indicated that lack of SIRP-alpha significantly reduced the pathogenesis of ALI, in association with attenuated lung inflammation, infiltration of neutrophils and expression of pro-inflammatory cytokines in mice. In addition, lack of SIRP-alpha reduced the expression of pro-inflammatory cytokines in LPS-treated bone marrow-derived macrophages (BMDMs) from KO mice, accompanied with improved macrophage phagocytosis. Blockade of soluble SIRP-alpha activity in ALI BAL by anti-SIRP-alpha antibody (aSIRP) effectively reduced the expression of TNF-alpha and IL-6 mRNA transcripts and proteins, improved macrophage phagocytosis in vitro. In addition, lack of SIRP-alpha reduced activation of Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) and improved activation of signal transducer and activator of transcription-3 (STAT3) and STAT6. Suppression of SHP-1 activity by tyrosine phosphatase inhibitor 1 (TPI-1) increased activation of STAT3 and STAT6, and improved macrophage phagocytosis, that was effectively reversed by STAT3 and STAT6 inhibitors. Thereby, SIRP-alpha suppressed macrophage phagocytosis through activation of SHP-1, subsequently inhibiting downstream STAT3 and STAT6 signaling. Lack of SIRP-alpha attenuated murine ALI possibly through increasing phagocytosis, and improving STAT3 and STAT6 signaling in macrophages. SIRP-alpha would be promising biomarker and molecular target in the treatment of murine ALI and patients with acute respiratory distress syndrome (ARDS).

Endothelial SIRPα signaling controls VE-cadherin endocytosis for thymic homing of progenitor cells.

Thymic homing of hematopoietic progenitor cells (HPCs) is tightly regulated for proper T cell development. Previously we have identified a subset of specialized thymic portal endothelial cells (TPECs), which is important for thymic HPC homing. However, the underlying molecular mechanism still remains unknown. Here, we found that signal regulatory protein alpha (SIRPα) is preferentially expressed on TPECs. Disruption of CD47-SIRPα signaling in mice resulted in reduced number of thymic early T cell progenitors (ETPs), impaired thymic HPC homing, and altered early development of thymocytes. Mechanistically, Sirpa-deficient ECs and Cd47-deficient bone marrow progenitor cells or T lymphocytes demonstrated impaired transendothelial migration (TEM). Specifically, SIRPα intracellular ITIM motif-initiated downstream signaling in ECs was found to be required for TEM in an SHP2- and Src-dependent manner. Furthermore, CD47 signaling from migrating cells and SIRPα intracellular signaling were found to be required for VE-cadherin endocytosis in ECs. Thus, our study reveals a novel role of endothelial SIRPα signaling for thymic HPC homing for T cell development.

Expression of CD47 and SIRPα Macrophage Immune-Checkpoint Pathway in Non-Small-Cell Lung Cancer

Simple SummaryCancer cells escape macrophage phagocytosis by exploiting the CD47/SIRPα axis. We found that extensive membranous CD47 expression by cancer cells characterized 29/98 cases. SIRPα and CD68 were expressed by tumor-associated macrophages (Μφ, TAMs). A high CD68Mφ-score was linked with improved overall survival. High expression, however, of SIRPα by CD68+ TAMs was linked with CD47 expression by cancer cells, low TIL-score, and poor prognosis. A direct association of CD47 expression by cancer cells and the % FOXP3+ TILs was also noted. The CD47/SIRPα axis is a sound target for adjuvant immunotherapy policies, aiming to improve the cure rates in operable NSCLC.Background: Cancer cells escape macrophage phagocytosis by expressing the CD47 integrin-associated protein that binds to the SIRPα ligand (signal regulatory protein alpha) expressed by macrophages. Immunotherapy targeting this pathway is under clinical development. Methods: We investigated the expression of CD47/SIRPα molecules in a series of 98 NSCLCs, in parallel with the infiltration of tumor stroma by CD68+ macrophages, tumor-infiltrating lymphocytes (TILs), and PD-L1/PD-1 molecules. Results: Extensive membranous CD47 expression by cancer cells characterized 29/98 cases. SIRPα and CD68 were expressed, to a varying extent, by tumor-associated macrophages (Μφ, TAMs). A high CD68Mφ-score in inner tumor areas was linked with improved overall survival (p = 0.005); and this was independent of the stage (p = 0.02, hazard ratio 0.4). In contrast, high SIRPα expression by CD68+ TAMs (SIRPα/CD68-ratio) was linked with CD47 expression by cancer cells, low TIL-score, and poor prognosis (p = 0.02). A direct association of CD47 expression by cancer cells and the % FOXP3+ TILs (p = 0.01, r = 0.25) was also noted. Conclusions: TAMs play an important role in the prognosis of operable NSCLC. As SIRPα+ macrophages adversely affect prognosis, it is suggested that the CD47/SIRPα axis is a sound target for adjuvant immunotherapy policies, aiming to improve the cure rates in operable NSCLC.

Checkpoint CD47 expression in classical Hodgkin lymphoma.

SummaryThe glycoprotein CD47 regulates antiphagocytic activity via signal regulatory protein alpha (SIRPa). This study investigated CD47 expression on Hodgkin and Reed–Sternberg (HRS) cells in the classical Hodgkin lymphoma (cHL) tumour microenvironment and its correlation with prognosis, programmed‐death (PD) immune markers, and SIRPa+ leukocytes. We conducted immunohistochemistry with CD47 and SIRPa antibodies on diagnostic biopsies (tissue microarrays) from cHL patients from two cohorts (n = 178). In cohort I (n = 136) patients with high expression of CD47 on HRS cells (n = 48) had a significantly inferior event‐free survival [hazard ratio (HR) = 5.57; 95% confidence interval (CI), 2.78–11.20; p < 0.001] and overall survival (OS) (HR = 8.54; 95% CI, 3.19–22.90; p < 0.001) compared with patients with low expression (n = 88). The survival results remained statistically significant in multivariable Cox regression adjusted for known prognostic factors. In cohort II (n = 42) high HRS cell CD47 expression also carried shorter event‐free survival (EFS) (HR = 5.96; 95% CI, 1.20–29.59; p = 0.029) and OS (HR = 5.61; 95% CI, 0.58–54.15; p = 0.136), although it did not retain statistical significance in the multivariable analysis. Further, high CD47 expression did not correlate with SIRPa+ leukocytes or PD‐1, PD‐L1 and PD‐L2 expression. This study provides a deeper understanding of the role of CD47 in cHL during an era of emerging CD47 therapies.

Anomalous Levels of CD47/Signal Regulatory Protein Alpha in the Hippocampus Lead to Excess Microglial Engulfment in Mouse Model of Perioperative Neurocognitive Disorders.

BackgroundPerioperative neurocognitive disorders (PNDs) are common complications of surgical patients, which can lead to prolonged hospitalization, increased complications, and decreased independence and quality of life. However, the underlying molecular mechanisms of PND remain largely obscure. Microglia activation and synapse loss were observed in PND. Cluster of differentiation 47 (CD47), which can bind to its receptor signal regulatory protein alpha (SIRPα) and generate “do not eat me” signal, protects synapses from excessive pruning. Therefore, we aimed to evaluate the potential role of CD47–SIRPα signaling in PND.MethodsThe tibial fracture surgery was performed in aged C57BL/6 mice for PND model establishment. The expression of CD47 and SIRPα in the hippocampus was assessed. Synaptic plasticity, dendritic spine density, microglial engulfment, and hippocampal-dependent memory function were evaluated after model establishment and intervention with SIRPα overexpression.ResultsCD47 and SIRPα expression in the hippocampus were both decreased after the surgery. SIRPα overexpression showed reduced engulfment within host microglia, but a total effect of excessive synapse engulfment decreased dendritic spine density and post-synaptic density protein 95 (PSD95) expression. SIRPα overexpression could not improve the synaptic dysfunction and cognitive impairment in PND. In addition, SIRPα overexpression led to increased CD47 and Iba1 expression.ConclusionAnesthesia and surgery affect CD47–SIRPα signaling. SIRPα overexpression could not ameliorate the cognitive impairment in PND mice. One reason may be that the increased Iba1 expression leads to a total effect of excessive synapse engulfment, which results in decreased dendritic spine density and PSD95 expression.

High SIRPA Expression Predicts Poor Prognosis and Correlates with Immune Infiltrates in Patients with Esophageal Carcinoma.

Background Signal regulatory protein alpha (SIRPA) is an inhibitory receptor expressed in macrophages and a potential therapeutic target in cancers. This study aims to investigate the functional role of SIRPA in esophageal carcinoma (ESCA). Methods Based on the Oncomine and The Cancer Genome Atlas (TCGA) database, SIRPA expression and clinical value were determined. Gene set enrichment analysis (GSEA) was performed to predict the mechanism underlying the oncogene role of SIRPA. Spearman's correlation analysis was used to analyze the effects of SIRPA on the molecular relationship and immune landscape. Results SIRPA was highly expressed across Oncomine and TCGA databases and correlated with poor overall survival and disease-specific survival. There was an expression difference among clinical characteristics. Functional annotation showed that cancer-related biological function and pathways were enriched in the high SIRPA expression group. Besides, SIRPA strongly and extensively affected the immune infiltrates. Conclusion SIRPA might be an oncogene and a target of immunotherapy in ESCA.

3127 – ENHANCING RESOLUTION PATHWAYS LIMITS INFLAMMATION AND MITIGATES BONE MARROW FAILURE IN MURINE SEVERE APLASTIC ANEMIA

Severe aplastic anemia (SAA) is an acquired bone marrow failure (BMF) disease typically treated with immunosuppressive therapies or transplantation. Disease is driven by uncontrolled inflammation, including elevated interferon gamma (IFNγ), and the loss of hematopoietic stem and progenitor cells (HSC/HSPCs). In a mouse model of SAA, we observed robust cell death and progressive accumulation of necrotic cells in the BM. In health, inflammation resolves via efficient clearance of dead and damaged cells initiating reparative programs and limiting inflammatory signals. We hypothesized that delayed/impaired resolution hinged on defective efferocytosis and contributed to the pathophysiology of SAA. Indeed, efferocytosis was impaired in the BM of mice with SAA. To test whether failed clearance of apoptotic cells was due to reduced expression of, or cleavage of, efferocytic receptors we analyzed their expression of by flow cytometry. Unexpectedly, BM monocytes and macrophages in SAA exhibited a significant increase in Mer tyrosine kinase (MerTK) expression. Mechanistically, high MerTK expression required cell autonomous IFNγ signaling, and occurred in conjunction with increased expression of CD47 and signal regulatory protein alpha (SIRPα), an ITIM-containing inhibitory receptor that prevents phagocytosis via CD47 engagement. To test whether the CD47-SIRPα axis contributed to SAA pathogenesis, we treated mice with a blocking antibody to SIRPα. SAA mice treated with anti-SIPRα antibodies had increased HSCs and platelets, as compared to controls, along with reduced inflammatory cytokines and reduced necrotic cells in the BM. Mice expressing mutant Mertk, lacking the cleavage site, also exhibited reduced SAA pathogenesis. Together, our findings support a novel role for impaired efferocytosis and delayed inflammation resolution in the pathophysiology of SAA. Severe aplastic anemia (SAA) is an acquired bone marrow failure (BMF) disease typically treated with immunosuppressive therapies or transplantation. Disease is driven by uncontrolled inflammation, including elevated interferon gamma (IFNγ), and the loss of hematopoietic stem and progenitor cells (HSC/HSPCs). In a mouse model of SAA, we observed robust cell death and progressive accumulation of necrotic cells in the BM. In health, inflammation resolves via efficient clearance of dead and damaged cells initiating reparative programs and limiting inflammatory signals. We hypothesized that delayed/impaired resolution hinged on defective efferocytosis and contributed to the pathophysiology of SAA. Indeed, efferocytosis was impaired in the BM of mice with SAA. To test whether failed clearance of apoptotic cells was due to reduced expression of, or cleavage of, efferocytic receptors we analyzed their expression of by flow cytometry. Unexpectedly, BM monocytes and macrophages in SAA exhibited a significant increase in Mer tyrosine kinase (MerTK) expression. Mechanistically, high MerTK expression required cell autonomous IFNγ signaling, and occurred in conjunction with increased expression of CD47 and signal regulatory protein alpha (SIRPα), an ITIM-containing inhibitory receptor that prevents phagocytosis via CD47 engagement. To test whether the CD47-SIRPα axis contributed to SAA pathogenesis, we treated mice with a blocking antibody to SIRPα. SAA mice treated with anti-SIPRα antibodies had increased HSCs and platelets, as compared to controls, along with reduced inflammatory cytokines and reduced necrotic cells in the BM. Mice expressing mutant Mertk, lacking the cleavage site, also exhibited reduced SAA pathogenesis. Together, our findings support a novel role for impaired efferocytosis and delayed inflammation resolution in the pathophysiology of SAA.

Involvement of Protein tyrosine phosphatase delta (PTPd) in the cortical pyramidal dendritic growth of through the regulation of Signal Regulatory Protein alpha (SIRPa) phosphorylation.

Involvement of Protein tyrosine phosphatase delta (PTPd) in the cortical pyramidal dendritic growth of through the regulation of Signal Regulatory Protein alpha (SIRPa) phosphorylation.

Harnessing Macrophages through the Blockage of CD47: Implications for Acute Myeloid Leukemia.

Simple SummaryThe immune system is the first line of protection against infected and tumor cells. Macrophages are specialized immune cells that recognize these abnormal cells and eliminate them by a mechanism called phagocytosis. All normal cells express a protein called CD47 or “don’t eat me signal” to prevent their elimination through phagocytosis. Cancer cells, including leukemic cells, express higher levels of CD47 as a mechanism of protection against macrophage phagocytosis. CD47 blockade leads to an increase in phagocytosis of leukemic cells and better control of the disease. In this review, we explore CD47 function in normal conditions, its role in acute myeloid leukemia progression, and possible ways to block CD47 to enhance elimination of the leukemic cells improving the therapeutic options for patients with acute myeloid leukemia.CD47 is a surface membrane protein expressed by all normal tissues. It is the so-called “don’t eat me signal” because it protects the cells against phagocytosis. The CD47 interacts with the signal regulatory protein alpha (SIRPα) on the surface of macrophages, leading to downstream inhibitory signaling that dampens phagocytic capacity. Since macrophages exert immune surveillance against cancers, cancer cells overexpress CD47 to defend themselves against phagocytosis. Acute myeloid leukemia (AML) is a cancer of hematopoietic stem/progenitor cells (HSPC), and similar to other types of cancers, leukemic blasts show enhanced levels of CD47. In patients with AML, CD47 has been associated with a higher disease burden and poor overall survival. Blockage of CD47-SIRPα signaling leads to improved phagocytosis of AML cells and better overall survival in xenograft models. However, the introduction of a pro-phagocytic signal is needed to induce greater phagocytic capacity. These pro-phagocytic signals can be either Fc receptor stimulants (such as monoclonal antibodies) or natural pro-phagocytic molecules (such as calreticulin). Based on these pre-clinical findings, various clinical trials investigating the blockade of CD47-SIRPα interaction have been designed as monotherapy and in combination with other anti-leukemic agents. In this review, we will discuss CD47 biology, highlight its implications for AML pathophysiology, and explore the potential clinical translation of disrupting CD47-SIRPα to treat patients with AML.

Imaging of Glioblastoma Tumor-Associated Myeloid Cells Using Nanobodies Targeting Signal Regulatory Protein Alpha

Glioblastoma (GBM) is the most common malignant primary brain tumor. Glioblastomas contain a large non-cancerous stromal compartment including various populations of tumor-associated macrophages and other myeloid cells, of which the presence was documented to correlate with malignancy and reduced survival. Via single-cell RNA sequencing of human GBM samples, only very low expression of PD-1, PD-L1 or PD-L2 could be detected, whereas the tumor micro-environment featured a marked expression of signal regulatory protein alpha (SIRPα), an inhibitory receptor present on myeloid cells, as well as its widely distributed counter-receptor CD47. CITE-Seq revealed that both SIRPα RNA and protein are prominently expressed on various populations of myeloid cells in GBM tumors, including both microglia- and monocyte-derived tumor-associated macrophages (TAMs). Similar findings were obtained in the mouse orthotopic GL261 GBM model, indicating that SIRPα is a potential target on GBM TAMs in mouse and human. A set of nanobodies, single-domain antibody fragments derived from camelid heavy chain-only antibodies, was generated against recombinant SIRPα and characterized in terms of affinity for the recombinant antigen and binding specificity on cells. Three selected nanobodies binding to mouse SIRPα were radiolabeled with 99mTc, injected in GL261 tumor-bearing mice and their biodistribution was evaluated using SPECT/CT imaging and radioactivity detection in dissected organs. Among these, Nb15 showed clear accumulation in peripheral organs such as spleen and liver, as well as a clear tumor uptake in comparison to a control non-targeting nanobody. A bivalent construct of Nb15 exhibited an increased accumulation in highly vascularized organs that express the target, such as spleen and liver, as compared to the monovalent format. However, penetration into the GL261 brain tumor fell back to levels detected with a non-targeting control nanobody. These results highlight the tumor penetration advantages of the small monovalent nanobody format and provide a qualitative proof-of-concept for using SIRPα-targeting nanobodies to noninvasively image myeloid cells in intracranial GBM tumors with high signal-to-noise ratios, even without blood-brain barrier permeabilization.

A versatile platform for the tumor-targeted delivery of immune checkpoint-blocking immunoglobin G

Immunotherapies based on immune checkpoint-blocking antibodies have been considered the most attractive cancer treatments in recent years. However, the systemic administration of immune checkpoint-blocking antibodies is limited by low response rates and high risk of inducing immune-related adverse events (irAEs), which might be overcome by the tumor-targeted delivery of these antibodies. To achieve tumor-targeted delivery, immune checkpoint-blocking antibodies are usually modified with tumor-homing ligands through difficult genetic fusion or chemical conjugation. As most immune checkpoint-blocking antibodies are immunoglobin G (IgG) antibodies, we hypothesize that these IgG antibodies might be noncovalently modified with a tumor-homing ligand fused to an IgG-binding domain (IgBD). To test this hypothesis, the tumor-homing ZPDGFRβ affibody, which targets platelet-derived growth factor receptor β (PDGFRβ), was fused to the Fab-selective IgBD in a trimeric format. After mixing ZPDGFRβ fused to the IgBD with immune checkpoint-blocking IgG against programmed death-ligand 1 (αPD-L1), a novel homogenous complex was formed, indicating that αPD-L1 had been successfully modified with ZPDGFRβ fused to the IgBD. ZPDGFRβ-modified αPD-L1 bound to both PDGFRβ and PD-L1, thus leading to greater tumor uptake and antitumor effects in mice bearing PDGFRβ+PD-L1+ tumor grafts. In addition, due to the broad spectrum of IgBD for IgG, immune checkpoint-blocking IgG antibodies against cytotoxic T-lymphocyte-associated protein 4 (αCTLA-4) and signal regulatory protein alpha (αSIRPα) were also modified with ZPDGFRβ fused to the IgBD. These results demonstrated that a tumor-homing ligand fused to the IgBD might be developed as a versatile platform for the modification of immune checkpoint-blocking IgG antibodies to achieve tumor-targeted delivery.

ISB 1442, a First-in-Class CD38 and CD47 Bispecific Antibody Innate Cell Modulator for the Treatment of Relapsed Refractory Multiple Myeloma

ISB 1442 is a bispecific antibody (BsAb) using Ichnos' proprietary Bispecific Engagement by Antibodies based on the T-cell receptor (BEAT ®) platform. A fully human BsAb with anti-CD38 and CD47 binding arms, ISB 1442 was developed for the treatment of relapsed/refractory multiple myeloma (rrMM). The CD38 binding arm consists of two bi-paratopic Fabs that strongly bind to CD38 through avidity-induced interactions. The anti-CD47 arm comprises a single Fab arm designed to block the interaction between CD47 and the signal-regulatory protein alpha (SIRPα) receptor present on phagocytes (including macrophages, monocytes and dendritic cells). With this design, the CD38 Fab arm preferentially drives binding to tumor cells and enables blocking of proximal CD47 receptors on the same cell via avidity-induced binding. Hence, ISB 1442 is anticipated to induce minimal unintended effects on red blood cell (RBC) hemagglutination compared to benchmark anti-CD47 monoclonal antibodies (mAb) magrolimab. The Fc portion of ISB 1442 is engineered to enhance antibody dependent cell phagocytosis (ADCP), antibody dependent cell cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). ISB 1442 is the first-in-class with these unique molecular attributes, which are designed to overcome mechanisms of resistance to daratumumab in rrMM patients.In vitro, ISB 1442 exhibited a higher killing potency compared to benchmark daratumumab across a broad range of CD38-expressing tumor cells. Specifically, ISB 1442 showed a high potency to kill CD38 high tumor cells through CDC and a superior potency to kill CD38 low expressing tumor cells through ADCC and ADCP compared to daratumumab. Additionally, ISB 1442 showed in vitro tumor killing potency through phagocytosis comparable to magrolimab, an anti-CD47 mAb (IgG4) acting mostly through ADCP. Consistent with its molecular design, which includes a functional Fc, ISB 1442 induced more potent killing of CD38 high and CD38 low tumor cells by CDC and ADCC compared to magrolimab. To characterize the complex mechanisms of action of ISB 1442 in a single system more fully, a multiple mode of action of killing (MMoAK) in vitro assay was established where autologous macrophages and PBMCs from healthy donors were incubated with CD38 low expressing tumor cells and human serum. With this approach, tumor cells can be targeted simultaneously by NK cells from PBMCs, autologous macrophages, and complement from human serum. In MMoAK, ISB 1442 exhibited prominent tumor cell killing that was twice as high as daratumumab. The presence of soluble CD38 or RBCs, the main source of antigen sinks for CD38 and CD47, did not affect the killing potency of ISB 1442 across in vitro assays using CD38 high or CD38 low expressing tumor cells. These data suggest that the molecular design of ISB 1442 mitigates the potential risk of CD47 and CD38 antigen sink and the related side effects.On-target specificity was evaluated in vitro by measuring binding to human RBCs, induction of hemagglutination and RBC depletion, hemolysis and platelet aggregation. ISB 1442 did not cause any detectable hemolysis, RBC depletion or platelet aggregation in vitro and showed a marked reduction in human RBC hemagglutination relative to magrolimab, suggesting a more favorable on-target specificity profile.Finally, the potency of ISB 1442 was assessed in vivo in a therapeutic model of subcutaneously established Raji tumor xenograft in CB17/SCID mice which have functional complement, macrophages and NK cells of murine origin. ISB 1442 induced higher tumor growth inhibition relative to daratumumab and comparable tumor regression compared to magrolimab.In summary, we report a novel approach for the treatment for rrMM by co-targeting CD38 and CD47 using a 2+1 biparatopic bispecific antibody. Based on its unique design and multiple mechanisms of action, ISB 1442 is anticipated to enhance antitumor activity in rrMM patients relative to anti-CD38 mAbs by overcoming primary and acquired tumor escape mechanisms of resistance. DisclosuresNo relevant conflicts of interest to declare.

Upregulation of CD47 Expression in De Novo Diffuse Large B-Cell Lymphoma Is More Frequent in Activated B-Cell Type

CD47 is a marker of self that provides a “don't-eat-me-signal” through activation of signal-regulatory protein alpha (SIRPa), a cell surface receptor expressed on monocytes/macrophages and granulocytes. This interaction negatively regulates effector functions such as, phagocytosis, migration, and superoxide production. Upregulation of CD47 expression in cancer, including diffuse large B-cell lymphoma (DLBCL), has emerged as a mechanism to escape innate immune surveillance. Using conventional immunohistochemical detection, we assessed CD47 expression in DLBCL and interrogated its association with clinicopathologic features.Patients with de novo DLBCL were identified from two large institutions and were uniformly treated with R-CHOP and had sufficient material for study. Immunohistochemical stains (IHC) were performed on FFPE tissue (Hans algorithm, BCL2, MYC, and CD47) and scored semi-quantitatively from no reactivity (0) to strong (2 and 3; Figure 1). Mutational analysis using a 334 gene target sequencing panel, gene expression profiling using Lymph2Cx to determine the cell of origin (COO), and FISH analysis for MYC, BCL2, and BCL6 translocations, were performed. The Lymphgen tool (Wright et al, 2020) was also used to determine the DLBCL group. Fisher's exact test and Kaplan-Meier survival analysis for overall survival (OS) were performed and P <0.05 was considered significant.CD47 expression was assessed by IHC in a cohort of 152 cases of de novo DLBCL, including 107 cases of germinal center B-cell (GCB) type (70%), 37 cases of activated B-cell (ABC) type (24%), and 8 cases of intermediate type (5%). A total of 17 cases (11%) showed strong and diffuse CD47 expression with IHC scores of 2 or above (CD47hi). CD47hi cases were significantly more frequent in ABC DLBCL (24%, 9/37) than GCB DLBCL (6%, 6/107; P=0.003). The remaining 2 CD47hi cases were in the intermediate DLBCL group (25%, 2/8). ABC DLBCL with CD47hi showed more frequent mutations with TET2 (33% vs 7%; P=0.08) and ZFP36L1 (22% vs 0%; P=0.05) compared to cases with low expression of CD47 with IHC scores of less than 2 (CD47low). ABC DLBCL with CD47low showed more frequent mutations of NOTCH2 (18% vs 0%; P=0.31) and MYD88 (29% vs 11%; P=0.4) compared to CD47hi. GCB DLBCL with CD47hi showed frequent mutations of TP53 (67% vs 21%; P=0.026) and CCND1 (33% vs 0%; P=0.003) compared to CD47low. None of the 13 cases with double- or triple-hit for MYC, BCL2 and/or BCL6 showed CD47 expression. The Lymphgen tool showed that cases of DLBCL with CD47hi were mostly in the ‘other’ group (50%), with other groups represented such as ST2 (21%), EZB (14%), MCD and BN2 (1 case each). There was no difference in overall survival (OS) between CD47hi and CD47low DLBCL (5-year OS, 75% vs 72%; P=0.57), or with the GCB or ABC subtypes.Strong expression with CD47 is more frequent in ABC DLBCL and is seen in a subset of GCB DLBCL with mutations in TP53 and/or CCND1. The level of CD47 expression does not appear to predict OS in patients with DLBCL treated with R-CHOP. This study demonstrates that conventional immunohistochemical methods can readily identify DLBCL with high CD47 expression, and these patients may benefit from the use of anti-CD47 therapy. [Display omitted] DisclosuresHerrera: Gilead Sciences: Research Funding; Takeda: Consultancy; Tubulis: Consultancy; Karyopharm: Consultancy; Bristol Myers Squibb: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; ADC Therapeutics: Consultancy, Research Funding; Seagen: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Merck: Consultancy, Research Funding; Kite, a Gilead Company: Research Funding.

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

Expression of CD47 in the pancreatic β-cells of people with recent-onset type 1 diabetes varies according to disease endotype.

We are studying the dialogue between β-cells and the immune system in type 1 diabetes and have identified a cell surface receptor, signal regulatory protein-alpha (SIRPα) as an important component in the regulation of β-cell survival. SIRPα interacts with another protein, CD47, to mediate signalling. In the present work, we have studied the expression and role of CD47 in human islet cells in type 1 diabetes. Clonal EndoC-βH1 cells were employed for functional studies. Cells were exposed to pro-inflammatory cytokines and their viability monitored by flow cytometry after staining with propidium iodide. Targeted knockdown of CD47 or SIRPα was achieved with small interference RNA molecules and the expression of relevant proteins studied by Western blotting or immunocytochemistry. Human pancreas sections were selected from the Exeter Archival Diabetes Biobank and used to examine the expression of CD47 by immunofluorescence labelling. Image analysis was employed to quantify expression. CD47 is abundantly expressed in both α and β cells in human pancreas. In type 1 diabetes, the levels of CD47 are increased in α cells across all age groups, whereas the expression in β-cells varies according to disease endotype. Knockdown of either CD47 or SIRPα in EndoC-βH1 cells resulted in a loss of viability. We conclude that the CD47 plays a previously unrecognised role in the regulation of β-cell viability. This system is dysregulated in type 1 diabetes suggesting that it may be targeted therapeutically to slow disease progression.

Targeting the cluster of differentiation 47/signal-regulatory protein alpha axis in myeloid malignancies.

The antitumor activity of macrophages is regulated by a balance of prophagocytic and antiphagocytic signals. Cluster of differentiation 47 (CD47), the dominant macrophage immune checkpoint ('do not eat me' signal), interacts with its receptor signal-regulatory protein alpha (SIRPα) to suppress phagocytic activities. This axis plays a pivotal role in immune evasion in myeloid malignancies as well as multiple cancers providing strong rationale for therapeutic exploitation. Preclinical studies have revealed overexpression of CD47 on leukemic stem cells and myeloblasts from patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), which contributes to immune surveillance evasion and is associated with poor outcomes. Blockade of CD47 with different approaches has demonstrated proof-of-concept antitumor activities mainly through phagocytic clearance. Early phase clinical trials combining the anti-CD47 mAb magrolimab with the hypomethylating agent azacitidine have showed synergistic activities, deep and durable responses, as well as a tolerable safety profile in these patients, including those with TP53 mutations. Targeting CD47/SIRPα axis, in combination with other therapeutic agents, represents a promising treatment approach for patients with myeloid malignancies, particularly the challenging TP53-mutated subgroup.

Progress of CD47 immune checkpoint blockade agents in anticancer therapy: a hematotoxic perspective.

CD47, a transmembrane protein, acts as a "do not eat me" signal that is overexpressed in many tumor cell types, thereby forming a signaling axis with its ligand signal regulatory protein alpha (SIRPα) and enabling the tumor cells to escape from macrophage-mediated phagocytosis. Several clinical trials with CD47 targeting agents are underway and have achieved impressive results preliminarily. However, hematotoxicity (particularly anemia) has emerged as the most common side effect that cannot be neglected. In the development of CD47 targeting agents, various methods have been used to mitigate this toxicity. In this review, we summarized five strategies used to alleviate CD47 blockade-induced hematotoxicity, as follows: change in the mode of administration; dual targeting bispecific antibodies of CD47; CD47 antibodies/SIRPα fusion proteins with negligible red blood cell binding; anti-SIRPα antibodies; and glutaminyl-peptide cyclotransferase like inhibitors. With these strategies, the development of CD47 targeting agents can be improved.