anti-CD18 Monoclonal Antibody Research Articles

Critical Involvement of CD44 in T Helper Type 2 Cell-Mediated Eosinophilic Airway Inflammation in a Mouse Model of Acute Asthma.

Interactions between CD44 and hyaluronan (HA) are crucial for recruiting leukocytes to inflamed tissues. This review summarizes findings from our studies of the roles of CD44-HA interactions in leukocyte trafficking, with a particular focus on airway T helper type 2 (Th2) cells in mouse models of acute asthma. In a mite allergen-induced model of acute asthma, intraperitoneal injection of anti-CD44 monoclonal antibodies blocked lymphocytes and eosinophils from accumulating in the lung, and suppressed both the antigen-induced increase in Th2 cytokines in the bronchoalveolar lavage fluid (BALF) and airway hyperresponsiveness (AHR). CD44 deficiency was associated with decreased mite allergen-induced Th2 cell-mediated airway inflammation and AHR in sensitized mice. Asthmatic responses to antigen-sensitized splenic CD4+ T cells transferred from CD44-deficient mice were weaker than in wild-type mice. Administration of anti-CD44 monoclonal antibodies preferentially suppressed the airway accumulation of antigen-specific Th2 cells induced by antigen challenge, without affecting Th1 and Th17 cells. Increased HA-binding ability of CD44 and expression of Neu1 sialidase were observed on antigen-specific Th2 cells compared with antigen-specific Th1 and Th17 cells. Finally, in a mouse model of acute asthma, neuraminidase 1-deficient SM/J mice exhibited a lower Th2 cytokine concentration and a lower absolute Th2 cell number in the BALF, as well as an attenuated AHR. Our findings indicate that CD44 critically contributes to the antigen challenge-induced airway accumulation of antigen-specific Th2 cells, without affecting Th1 and Th17 cells, in mice. Furthermore, neuraminidase 1 activity is necessary for the interaction between HA and CD44, and Th2 cell-mediated airway inflammation.

CD24/Siglec-10 “Don't Eat Me” Signal Blockade Is a Potential Immunotherapeutic Target in Mantle-Cell Lymphoma

IntroductionMantle-cell lymphoma (MCL) is a B-cell non-Hodgkin Lymphoma (NHL) characterized by heterogenous behavior, ranging from indolent phenotype to highly aggressive and drug resistant one with dismal prognosis. Drug resistance may be generated by Tumor Microenvironment (TME), owing that Tumor-Associated Macrophages (TAM) are pathologically functional in providing survival signals to MCL cells (Pham, Front Oncol. 2018).Recently, “Don't Eat Me” signal (DEMs) blockade with anti-CD47 monoclonal Antibody (moAb) showed promising activity in pretreated NHL, through increase of phagocytosis by TAM (Advani, NEJM. 2019).CD24 was also demonstrated to be involved in DEMs and, in a preclinical model of solid cancer, blocking the CD24/Siglec-10 interaction provided an improvement of M2-like TAM-mediated phagocytosis in vitro and an increase of survival in vivo (Barkal, Nature. 2019).CD24 can be expressed in some phases of B-cell differentiation and MCL derives from a B-cell precursor with upregulated CD24. To date, there are no functional studies showing an improvement of phagocytosis through CD24/Siglec-10 pathway inhibition in hematologic malignancies and MCL.Here, we present our in vitro results of CD24/Siglec-10 DEMs blockade in MCL subset.MethodsA panel of MCL cell lines (Jeko-1, Granta-519, Mino) has been analyzed for CD24 surface expression by flow cytometry (FC) (clone SN3). Consequently, we performed co-culture experiments with MCL cell lines and macrophages from healthy donors. Briefly, Peripheral Blood Mononucleated Cells (PBMC) were collected from healthy volunteers through density gradient centrifugation technique. CD14+ monocytes were isolated through CD14 Microbeads isolation kit and cultured in plates with 50 ng/ml human GM-CSF for 7-9 days. In order to create M2-like Siglec-10+ TAM, 50 ng/ml human IL-10 and 50 ng/ml human TGF-β 1 were added on days 3-4 of differentiation until use on days 7-9. Siglec-10 expression on TAM was checked by FC (clone 5G6).M2-like macrophages were then collected and co-cultured with CFSE-labelled MCL target cells for 1-2 hours in a serum-free medium. Anti-CD24 moAb (clone SN3) or the appropriate IgG 1 isotype control were added at a concentration of 10 μg/ml. Phagocytosis was then stopped on ice and CD11b-PE staining (anti-CD11b moAb, clone REA713) was performed to identify human macrophages by FC.Phagocytosis was measured as the number of CD11b+/CFSE+ macrophages, quantified as a percentage of the total CD11b+ macrophages. Each phagocytosis reaction was performed in technical triplicate and phagocytosis was normalized to the highest technical replicate per donor in order to consider raw phagocytic level among donor-derived macrophages.ResultsMCL cell lines express surface CD24 by FC, with higher levels in Mino cell line (Figure 1A). Differentiated M2-like macrophages showed an upregulation of Siglec-10 expression after immunosuppressive stimuli, which is fundamental owing that Siglec-10 is the ligand of CD24 (Figure 1B).As pertains to the phagocytic assay, we documented an improvement of phagocytosis when M2-like macrophages and MCL cell lines were co-cultured together with anti-CD24 moAb (Figure 2 and Figure 3A). Furthermore, it is worth mentioning that phagocytosis seemed to be much higher in MCL cell lines with higher surface levels of CD24 (e.g., Mino), presenting increased number of CD11b+/CFSE+ M2-like TAM by FC (Figure 3B).ConclusionsMCL was found to be sensitive to CD24/Siglec-10 DEMs blockade when co-cultured with M2-like macrophages in vitro. We can argue that most of the observed increase of phagocytosis after the addition of anti-CD24 moAb may be secondary to loss of CD24 signalling rather than Fc-mediated opsonization, as already documented in previous analysis about solid cancer (Barkal, Nature. 2019). We can therefore hypothesize that the blockade of this DEMs pathway can improve phagocytosis in a non-opsonization manner in NHL as well.Furthermore, CD24 surface density seemed to be positively correlated to the intensity of phagocytic activity, suggesting that MCL subtypes expressing higher CD24 levels are much more dependent on this DEMs pathway than others with low CD24 density.Overall, CD24 turned out to be a potential immunotherapeutic target in MCL, aiming at improving innate immune system through DEMs blockade. In vivo studies are needed to confirm the activity we documented in vitro in this NHL subset. [Display omitted] DisclosuresGambacorti-Passerini: Bristol-Myers Squibb: Consultancy; Pfizer: Honoraria, Research Funding.

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.

IPSC-Derived Natural Killer Cells and Macrophages Synergistically Kill Acute Myeloid Leukemia Blasts

Despite many advances, the treatment of acute myeloid leukemia (AML) remains challenging, and few patients are cured by therapies other than allogeneic hematopoietic cell transplant. Treatment with natural killer (NK) cells from allogeneic donors is a promising therapy that can achieve remissions in 30-50% of AML patients. To generate an improved cell-based therapy for AML, our group has produced NK cells from induced pluripotent stem cells (iPSCs). iPSC-derived NK cells effectively kill AML cells, but may benefit from additional modifications or combination with other therapies to durably cure AML. Based on studies that demonstrate that targeting the CD47 pathway on macrophages and NK cells improves anti-tumor activity and is an effective treatment for patients with AML, we investigated the combination of iPSC-derived NK cells with iPSC-derived macrophages with and without CD47 blockade for the treatment of AML.AML clinical trials combining anti-CD47 monoclonal antibodies (mAb) with chemotherapy have demonstrated an anti-tumor effect primarily thought to be mediated though a macrophage immune-checkpoint blockade mechanism. To determine if addition of iPSC-derived macrophages can improve the cytotoxicity of NK cells against AML blasts, we co-cultured iPSC-NK cells and MOLM13 or MV-4-11 AML cells with iPSC-macrophages in a standard cytotoxicity assay. Similar results were found for cytotoxicity tests against both AML cell lines (Figure panels A and B). While macrophages alone did not kill AML blasts, the addition of iPSC-macrophages to iPSC-NK cells significantly improved killing of the AML cells by 50% (p<0.01). Addition of an anti-CD47 mAb (B6H12) further increased killing of these AML cells by the iPSC-NK cell + iPSC-macrophage combination treatment by an additional 23% (p<0.01). Intriguingly, the addition of just the anti-CD47 mAb to the iPSC-NK cells also significantly increased anti-AML activity, although this increased killing was consistently lower than what was seen with addition of iPSC-macrophages combined with anti-CD47 and iPSC-NK cells. Addition of the CD47 mAb to iPSC-macrophages without NK cells did not result in increased anti-AML cytotoxicity. We also demonstrated that blockade of SIRPα (the receptor on NK cells for CD47) significantly increased NK cell killing of AML blasts by 16% (p<0.05). Furthermore, the combination of iPSC-NK cells + iPSC-macrophages + SIRPα mAb led to a 37% increase in cytotoxicity compared to iPSC-NK cells + iPSC-macrophages alone (p<0.01).To confirm that addition of anti-CD47 or anti-SIRPα antibodies increased NK cell activation via loss of the inhibitory CD47-SIRPα interaction between NK cells and AML blasts and not by another mechanism, we tested the effect of adding both CD47 mAb and anti-SIRPα antibodies. Compared to addition of either anti-CD47 or anti-SIRPα mAb alone, the combination induced no additional increase in anti-AML activity by the NK cells. These results suggest that the CD47-SIRPα interaction between AML and NK cells is an important inhibitory immune-checkpoint on NK cells. To control for the effect of blocking SIRPα on macrophages, we tested the addition of the SIRPα mAb to iPSC-macrophages. This combination did not improve on the lack of cytotoxicity exhibited by macrophages alone. We also evaluated if ADCC mediated by the anti-CD47 mAb binding AML blasts could account for the increase in cytotoxicity and found that blockade of Fc-receptors on NK cells does not diminish the increase in cytotoxicity seen with addition of the mAb, excluding a role for ADCC.To investigate if these findings with AML could be extended to other hematologic malignancies, we tested the combination of iPSC-NK cells, iPSC-macrophages and CD47 mAb against the RPMI-8226 multiple myeloma cell line (Figure C). Here, we again demonstrate that CD47 blockade combined with iPSC-macrophages leads to increased NK cell-mediated anti-myeloma activity. In vivo studies testing the combination of iPSC-NK cells, iPSC-macrophages and CD47 mAb against human AML and myeloma cells in mouse xenograft-models are ongoing. Together our results indicate that blocking the CD47-SIRPα interaction between NK cells and tumor cells consistently mediates improved anti-tumor activity. Furthermore, iPSC-derived NK cells and macrophages provide an important, standardized, “off-the-shelf” cell therapy approach that can be translated into novel clinical therapies. [Display omitted] DisclosuresKaufman: Shoreline Biosciences: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Qihan Biotech: Consultancy, Current holder of stock options in a privately-held company; VisiCELL Medical: Consultancy, Current holder of stock options in a privately-held company.

ADG153, an Anti-CD47 Monoclonal Antibody Prodrug, Has Strong In Vivo Anti-Tumor Activity, Minimal RBC-Related and Antigen Sink Liabilities, and Extended Half Life in Comparison with Benchmark Clinical Antibodies of the Same IgG Subclass

Binding of the CD47 membrane protein, overexpressed on many tumor types, to the SIRPα inhibitory receptor on myeloid cells results in the inhibition of the activation of macrophages and other phagocytes against tumors. Therapies targeting the CD47/SIRPα axis have shown success in various preclinical models and are now in clinical trials for both solid and hematologic malignancies. Although anti-CD47 therapies have demonstrated promising clinical activities, the expression of CD47 on many different normal human cell types, including red blood cells (RBCs), serves as a large antigen sink for anti-CD47 antibodies. Blocking CD47 on RBCs, such as by magrolimab (Hu5F9), has led to transient anemia, requiring step-up dosing in the clinic.To circumvent these challenges, we developed ADG153, a fully human anti-CD47 SAFEbody masked by conditionally activable peptides. In normal tissues, the SAFEbody masking moiety can function to block ADG153 from binding to CD47; however, in an activable condition such as the tumor microenvironment where protease activity has been reported to be elevated, the masked antibody can be activated, enabling the activated ADG153 antibody to bind to and inhibit CD47 function on tumor cells.For head-to-head comparisons, in vitro studies were performed to compare the activity of Hu5F9 and unmasked ADG153 parental antibody of the IgG4 isotype. Both antibodies (1) blocked human SIRPα from binding to human CD47, (2) had similar potencies for binding to human CD47 protein, CD47-positive tumor cell lines, and human RBCs, and (3) induced macrophage phagocytosis. In contrast, the masked ADG153 SAFEbody demonstrated significantly reduced activities (>450-fold) in the same in vitro assays, showing strong masking efficiencies. Unlike Hu5F9, both the ADG153 parental and SAFEbody molecules did not cause in vitro human RBC hemagglutination.Although the ADG153 SAFEbody had significantly reduced binding to CD47 in vitro as expected, it demonstrated strong anti-tumor activity in in vivo mouse tumor models. In both the disseminated and subcutaneous CD47-positive Raji tumor models, the ADG153 SAFEbody of the IgG4 isotype showed similar anti-tumor activities to Hu5F9. However, in exploratory toxicology studies in cynomolgus monkeys, the ADG153 SAFEbody showed significantly less decreases than Hu5F9 in RBCs, hemoglobin, and hematocrit. Hu5F9 at 10 mg/kg caused ~49% maximum decrease in RBCs, while ADG153 SAFEbody at 60 mg/kg showed ~23% maximum decrease in RBCs (Panel A). Pharmacokinetic (PK) studies of single intravenous dose ADG153 SAFEbody compared to Hu5F9 in monkeys demonstrated ~8-fold longer apparent half-life and ~9-fold higher Area Under the Curve at 10 mg/kg (Panel B). Collectively, ADG153 SAFEbody is a differentiated anti-CD47 antibody that has strong in vivo anti-tumor activity with reduced RBC-related and antigen sink liabilities and favorable PK properties. This preclinical profile with an enhanced therapeutic index provides a strong rationale for advancing ADG153 SAFEbody into clinical development. [Display omitted] DisclosuresCai: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Nguyen: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company; Sparcbio, LLC: Ended employment in the past 24 months. Zheng: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company; Janssen Pharmaceuticals: Ended employment in the past 24 months. Shi: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Liu: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Li: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Du: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company. Luo: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Xu: Adagene Inc.: Current Employment, Current equity holder in publicly-traded company; Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months.

448 Lemzoparlimab (TJ011133), an anti-CD47 antibody, with/without dexamethasone plus anti-myeloma regimens for relapsed/refractory multiple myeloma: a phase 1b dose escalation and expansion study

BackgroundDespite therapeutic advancements for multiple myeloma (MM), most patients with MM develop relapsed/refractory (R/R) disease, which is associated with high mortality and highlights an unmet need for novel treatments.A key characteristic of MM cells is the overexpression of CD47, which downregulates phagocytosis, thereby allowing malignant plasma cells to evade destruction by the immune system. Blocking CD47 with lemzoparlimab, an anti-CD47 monoclonal antibody, may enhance macrophage-mediated anti-tumor activity.This study will characterize the safety, dose-limiting toxicity, and recommended dosing of lemzoparlimab (TJ011133) with or without dexamethasone and combined with other anti-myeloma regimens in patients with R/R MM.MethodsThis phase 1b, open-label, dose-escalation and expansion study (NCT04895410) will enroll adults with R/R MM, an Eastern Cooperative Oncology Group Performance Status ≤2, prior treatment history (varies based on treatment arm; Figure 1), and measurable disease (serum monoclonal paraprotein [M-protein] ≥0.5 g/dL, urine M-protein ≥200 mg/24 hours, or serum-free light chain ≥100 mg/L).Patients will be enrolled in 2 phases (escalation and expansion), divided into 4 arms to receive treatment in 28-day cycles (figure 1): (A) lemzoparlimab ± dexamethasone; (B) lemzoparlimab + dexamethasone + pomalidomide (4 mg orally daily [Days 1–21]); (C) lemzoparlimab + dexamethasone + carfilzomib (56 mg/m2 intravenously, 6 doses/cycle [first 2 doses in Cycle 1: 20 mg/m2]); and (D) lemzoparlimab + dexamethasone + daratumumab (1800 mg subcutaneously [Cycle 1, start on Day 2; Cycles 1–2, once weekly; Cycles 3–6, every 2 weeks; Cycles ≥7, Day 1]). Dexamethasone will be administered orally twice weekly at 20 mg for Arm C, and orally or intravenously weekly at 40 mg for other arms. Dose escalation will follow Bayesian optimal interval design. Once maximum tolerated dose/recommended phase 2 dose (RP2D) is determined, patients will be enrolled in the expansion. Treatment discontinuation criteria are: unacceptable toxicity, disease progression, consent withdrawal, or investigator’s discretion.The primary endpoint is determination of the RP2D. Secondary endpoints include safety and efficacy measures. Time-to-event endpoints will be analyzed using the Kaplan–Meier method.AcknowledgementsAbbVie is funding this study and participating in the study design, research, analysis, data collection, interpretation of data, reviewing, and approval of the publication. All authors had access to relevant data and participated in the drafting, review, and approval of this publication. No honoraria or payments were made for authorship.Medical writing support was provided by Marta Rossi, PhD, of Fishawack Communications Ltd, and funded by AbbVie.Trial RegistrationClinicalTrials.gov: NCT04895410Ethics ApprovalThe protocol, informed consent form(s), recruitment materials, and all patient materials will be submitted to the Independent Ethics Committee/Institutional Review Board for review and approval.Abstract 448 Figure 1Study schematicThe dose escalation phase will follow BOIN; it will start with lemzoparlimab (TJ011133) monotherapy at DL1 in Arm A. If DL1 is not tolerated as monotherapy or as combination therapy, DL −1 will be evaluated. Investigation of combination arms will start after evaluation of monotherapy.Arms A, B, C and D will include patients with R/R MM after progressing on defined therapies and number of prior lines of therapy.BOIN, Bayesian optimal interval; DL, dose level; MM, multiple myeloma; R/R, relapsed/refractory.

Epitope Mapping of the Anti-CD44 Monoclonal Antibody (C44Mab-46) Using Alanine-Scanning Mutagenesis and Surface Plasmon Resonance.

CD44 is a type I transmembrane protein expressed in various kinds of normal cancer cells, including pancreatic, breast, and oral cancers. CD44 is associated with cancer progression, metastases, and treatment resistance. CD44 consists of 20 exons, and various isoforms exist due to alternative splicing of the central 10 exons. Some splicing variants show cancer-specific expression patterns and are related to prognosis of patients with cancer. Therefore, CD44 targeting therapy has been attracting attention. In a previous study, we established an anti-CD44 monoclonal antibody, C44Mab-46 (IgG1, kappa), useful for flow cytometry, Western blotting, and immunohistochemistry by immunizing mice with CD44v3-10 ectodomain. This study investigated the binding epitope of C44Mab-46 using enzyme-linked immunosorbent assay (ELISA) and the surface plasmon resonance (SPR) with the synthesized peptide. ELISA results using deletion mutants showed that C44Mab-46 reacted with the amino acids (aa) of 161-180 aa of CD44. Further examination of the C44Mab-46 epitope using ELISA with point mutants in 161-180 aa of CD44 demonstrates that the C44Mab-46 epitope comprised Thr174, Asp177, and Val178. The SPR with point mutants in 161-180 aa of CD44 demonstrated that the C44Mab-46 epitope comprises Thr174, Asp175, Asp176, Asp177, and Val178. Together, the C44Mab-46 epitope was determined to be located in exon 5 of CD44.

Epitope Mapping of the Anti-CD44 Monoclonal Antibody (C44Mab-46) Using the REMAP Method.

CD44 functions as a major hyaluronan receptor on most cell types, with roles in cell adhesion, migration, proliferation, differentiation, and survival. The CD44 gene comprises 20 exons, with alternative splicing producing many different isoforms. CD44 variant isoforms exhibit tissue-specific expression patterns and have been studied as therapeutic targets for several cancers; therefore, anti-CD44 monoclonal antibodies (mAbs) are useful for investigating CD44 expression in various cancers. Previously, we established an anti-CD44 mAb, C44Mab-46 (IgG1, κ), by immunizing mice with the CD44v3-10 ectodomain. Although C44Mab-46 recognized all CD44 isoforms, the binding epitope of C44Mab-46 has not been determined. In this study, we first checked the reactivity of C44Mab-46 to several CD44v3-10 deletion mutants such as dN79, dN124, dN147, and dN224. We found the N-terminus of the C44Mab-46-binding epitope between residues 147 and 224 of CD44v3-10. We next investigated this epitope using a novel mapping system: RIEDL insertion for epitope mapping (REMAP) method. We constructed 31 CD44 standard (CD44s) mutants where the RIEDL tag was inserted into the expected epitope region in CD44s. We observed that the C44Mab-46 epitope constituted five amino acids: 174-TDDDV-178 of CD44s. Thus, the REMAP method could be used to determine mAb binding epitopes for membrane proteins.

Targeting CD47 As a Novel Therapeutic Strategy in Multiple Myeloma

Introduction: CD47 is an immune checkpoint coordinating the innate and adaptive immune systems which allows cancer cells to escape the innate immune response by sending inhibitory signals to macrophages and regulates the adaptive immune anti-tumor response. Hence, CD47 has been identified as a novel therapeutic target in cancers, including multiple myeloma (MM) which is localized mainly in the bone marrow (BM). MM cells rely heavily on the interactions with tumor microenvironment (TME) and immune system. Macrophages are enriched in the BM of MM patients compared to healthy controls, and were demonstrated to support MM growth, in opposition to their intrinsic phagocytic properties. We study the new class of immune checkpoint inhibitor such as anti-CD47 monoclonal antibody (mAb) which enhance MM killing. We tested two anti-CD47 mAbs provided; Vx1000R (blocking only) - blocking CD47/SIRPα binding and Vx1004R (dual function) - blocking CD47/SIRPα binding and inducing cell death activity.The objectives of this study were: (1) to test the expression of CD47 (Vx1000R and Vx1004R binding) to MM cells, stromal cells (MSP-1 and HS5) and peripheral blood mononuclear cell (PBMC) sub-populations; (2) to study anti-CD47 mAbs binding in hypoxic conditions; (3) to investigate the effect of anti-CD47 mAbs on MM cell apoptosis in 2D and the 3D tissue engineered BM (3DTEBM; a scaffold incorporating BM plasma from MM patients, MM cells, and accessory cells) to reproduce the TME; (4) to test the cytotoxicity of Vx1004R on PBMC sub-populations in 3D culture; and (5) to examine the effect of Vx1004R on MM cell phagocytosis ex vivo by macrophages.Methods: We performed meta-analysis of CD47 mRNA expression in dataset available online in healthy, MGUS, smoldering MM and MM subjects. We tested the binding of Vx1000R and Vx1004R mAbs on MM.1S, H929, RPMI8226 and U266 cells and stromal cells (MSP-1 and HS5) in normoxia (21% O2) and hypoxia (1% O2), and in the 3DTEBM at 24h and 72h. We also tested Vx1000R binding to PBMC sub-populations (from 12 MM patients) including CD3 (T cells), CD14 (monocytes/macrophages), CD16 (NK cells, eosinophils, and neutrophils), CD19 (B cells), CD123 (pDC and basophils) and CD138 (MM cells).Next, we examined the cytotoxic activity of CD47 mAbs on MM cells alone and in co-culture with the BM negative fraction (CD138-depleted) and stromal cells (MSP-1) in 2D cultures by performing MTT survival assay, and in 3DTEBM cultures by flow cytometry. The cytotoxicity of Vx1004R was studied on PBMCs incorporated into a similar 3D model and treated with Vx1004R; proliferation and apoptosis of different sub-populations of PBMC were assessed via flow cytometry. Lastly, the effect of Vx1004R on MM cell phagocytosis by macrophages (CD14+; green) was tested after 2h of co-culture with MM cells (pre-labeled with DiD+; red) in the presence or absence of Vx1004R. Phagocytosis was determined as double positive (green+ red+) cells by flow cytometry.Results: The meta-analysis results demonstrated that CD47 mRNA expression increases with tumor progression (compared to early stage disease and healthy subjects). We demonstrated that MM.1S, H929, U266 and RPMI8226 cells and stromal cells (MSP-1 and HS5) express high levels of CD47, which does not alter in hypoxia, and that MM cells increase CD47 expression in 3DTEBM at 72h. CD47 (Vx1000R) is expressed in different sub-populations of PBMCs with the highest expression in MM cells. In addition, we have shown that anti-CD47 dual function mAb inhibits proliferation of MM cells, especially when co-cultured with negative fraction and MSP-1 stroma in 3DTEBM cultures. Moreover, we showed that anti-CD47 mAb induces phagocytosis of MM cells ex vivo by macrophages.Conclusions: CD47 is a promising target candidate for MM treatment, since CD47 mRNA increases with MM progression, and CD47 protein increases in MM cells in the presence of TME in the 3DTEBM, suggesting involvement in pathophysiology of MM. Moreover, anti-CD47 mAb enhances killing of MM cells when co-cultured with TME; however, it does not affect apoptosis of PBMC sub-populations in the 3DTEBM. Finally, anti-CD47 mAb induces phagocytosis of MM cells ex vivo by macrophages. These results suggest that anti-CD47 mAbs are applicable for MM treatment. Further studies are warranted to examine the effect of anti-CD47 mAbs as a novel strategy to inhibit the progression and dissemination of MM in vivo, especially when combined with chemotherapy. DisclosuresVij:Takeda: Honoraria, Research Funding; Jazz: Honoraria; Abbvie: Honoraria; Celgene: Honoraria; Amgen: Honoraria, Research Funding; Janssen: Honoraria; Bristol-Meyers-Squibb: Honoraria; Konypharma: Honoraria. Capoccia:Tioma: Employment. Manning:Tioma: Employment. Azab:Abbvie: Research Funding; Verastem: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner; Selexys: Research Funding; Karyopharm: Research Funding; Cell Works: Research Funding; Cleave Bioscience: Research Funding; Cellatrix LLC: Other: Founder and owner; Glycomimetics: Research Funding; Tioma: Research Funding.

Humanized Anti-CD26 Monoclonal Antibody Has Anti-Myeloma Efficacy for the Treatment of High-Risk Multiple Myeloma

Background: Despite remarkable progress in treatment options of multiple myeloma (MM) over the last decade with proteasome inhibitors and immunomodulatory drugs (IMiDs), most of patients with high-risk multiple myeloma (MM) relapse and ultimately become refractory. Recently, novel monoclonal antibody directly targeting CS1(elotuzumab) or CD38 (daratuzumab) induced durable responses in patients with multidrug-refractory MM. CD26, a 110-kDa transmembrane glycoprotein which is expressed on several tumor cells including malignant lymphoma, has been implicated in tumorigenesis, whereas its role in plasma cell malignancies has not been characterized yet. Our recent work has revealed that CD26 is intensely expressed on activated osteoclasts (OCs) in MM. In addition, in vitro studies showed that although CD26 expression is only slightly detected on MM cell lines cultured alone, it is intensely and uniformly expressed on MM cell lines co-cultured with OCs. In this study, we clarify the direct on-tumor and immune mechanism of action with huCD26mAb, a humanized monoclonal IgG1 antibody specifically targeting CD26, alone or in combination with approved agents in high-risk MM.Methods and Results: Immunostaining on bone marrow biopsy specimens showed that CD26 is expressed on plasma cells in several MM patients, but not in those of healthy individuals. So, we first analyzed the effect of huCD26mAb on survival of MM cell lines (KMS18, KMS26, KMS27, KMS28, KMS34, U266). HuCD26mAb had no direct effect on proliferation in any of tested CD26- MM cell lines cultured alone, but it inhibited growth of CD26+ MM cell lines co-cultured with OCs at higher concentrations (>10μg/ml), as measured by the MTT assay. Next, we examined the ability of huCD26mAb to lyse CD26 positive MM cell lines by antibody-dependent cellular cytotoxicity (ADCC) using the calcein-AM release assay. HuCD26mAb, but not isotype control IgG1, triggered ADCC against CD26+ KMS18, KMS26, KMS28, KMS34 and U266 with t(4;14) by natural killer (NK) effector cells in a dose-dependent manner with lytic activity starting at 0.0001μg/ml and maximum lysis at 10μg/ml and in an effector to target (E/T) ratio-dependent manner. Moreover, huCD26mAb dose-dependently induced lysis of lenalidomide-resistant MM.1R via ADCC. We further asked whether pretreatment with conventional or novel agents is able to induce superior ADCC activity by huCD26mAb. CD26+ KMS18, KMS26 and KMS28 were pretreated with bortezomib (3nM), lenamlidomide (0.5μM) or dexamethasone (25nM) overnight. Indeed, subsequent MM cell lysis triggered by huCD26mAb (10μg/ml) was significantly augumented. Treatment with lenalidomide (0.5μM) followed by huCD26mAb (10μg/ml) also markedly reduced the viable MM.1R in the presence of NK effector cells. The synergistic ADCC efficacy of huCD26mAb in combination with lenalidomide resulted in 2.0-fold increase in MM.1R lysis. In addition, pretreatment of NK effector cells with lenalidomide also potentiated ADCC against MM.1R mediated by huCD26mAb in a dose-dependent fashion. Complement dependent cytotoxicity (CDC) of CD26+ MM cells by huCD26mAb was also tested using human serum as a source of complement. However, in the presence of human serum, the incubation with huCD26mAb or isotype IgG1 exerted no effect on MM cell lysis. These results indicate that huCD26mAb (≦10μg/ml) kills CD26+ MM cells by inducing ADCC but not CDC, regardless of sensitivity or resistance to conventional or novel agents. Finally, to further determine whether huCD26mAb alone or combination with lenalidomide affects side population (SP) fractions of drug-resistant MM cells, we first examined CD26 expression in SP and main population (MP) fractions by flow cytometry. Both CD26+ RPMI8226 and KMS11 exhibited SP fractions which equally expressed CD26 at high levels as MP fractions. So, we examined ADCC activity by huCD26mAb (10μg/ml) against these SP fractions, mixed with NK effector cells. Interestingly, although lenalidomide alone did not decrease the ratio of SP fractions, huCD26mAb substantially reduced their ratio and its further reduction was observed with both in combination against CD26+ RPMI8226 and KMS11.Conclusions: HuCD26mAb induced significant anti-MM efficacy chiefly through ADCC. CD26 may be a novel valuable target for antibody therapy and huCD26mAb, alone or in combination, may be a promising immunotherapeutic strategy for the treatment of high-risk MM. DisclosuresMorimoto:Y's Therapeutics: Consultancy, Research Funding. Yamada:Y's therapeutics: Consultancy, Research Funding.

Combined strategies for effective cancer immunotherapy with a novel anti-CD47 monoclonal antibody.

CD47 is a widely expressed cell-surface protein that regulates phagocytosis mediated by cells of the innate immune system, such as macrophages and dendritic cells. CD47 serves as the ligand for a receptor on these innate immune cells, signal regulatory protein (SIRP)-α, which in turn inhibits phagocytosis. Several targeted CD47 therapeutic antibodies have been investigated clinically; however, how to improve its therapeutic efficacy remains unclear. Herein, we developed a CD47 blocking antibody, named IBI188, that could specifically block the CD47-SIRP-α axis, which transduces the "don't eat me" signal to macrophages. In vitro phagocytosis assays demonstrated the pro-phagocytosis ability of IBI188. Furthermore, several in vivo models were chosen to evaluate the anti-tumor efficacy of IBI188. IBI188 treatment upregulated cell movement- and inflammation-related genes in macrophages. Synergism was observed when combined with an anti-CD20 therapeutic antibody, whose function depends on antibody-dependent cellular cytotoxicity/phagocytosis (ADCC/ADCP). CD47 expression was evaluated following azacytidine (AZA) treatment, a standard-of-care for patients with multiple myeloma; enhanced anti-tumor efficacy was observed in the combination group in AML xenograft models. Notably, IBI188 treatment increased vascular endothelial growth factor-A (VEGF-A) levels in a solid tumor model, and combined treatment with an anti-VEGF-A antibody and IBI188 resulted in an enhanced anti-tumor effect. These data indicate that IBI188 is a therapeutic anti-CD47 antibody with anti-tumor potency, which can be enhanced when used in combination with standard-of-care drugs for cancer treatment.

MO244CD47 BLOCKADE AMELIORATES AUTOIMMUNE VASCULITIS VIA THE EFFEROCYTOSIS OF NEUTROPHIL EXTRACELLULAR TRAPS*

Abstract Background and Aims Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is characterized by systemic necrotizing vasculitis in small vessels. The necrotic lesions consist of ANCA-mediated neutrophil extracellular traps (NETs) which represent a form of lytic cell death. The persistent NETs serve as autoantigens against ANCAs and cause organ damage in a vicious cycle. Considering dead cells are essentially cleared by phagocytic cells as a process of efferocytosis, why the NETs persist in tissue remains unclear. During efferocytosis, macrophages engulf apoptotic cells to prevent the leakage of intracellular components including toxic enzyme into the surrounding cells and these processes are regulated by the expression of CD47 as a “don’t eat me” signal. In this study, we hypothesized that ANCA-mediated NETs in AAV escape from efferocytosis via the up-regulation of CD47 and the persistent NETs amplify the disease. Method Human data: Human kidney biopsy specimens from patients with AAV and minor glomerular abnormality (MGA, as a case control) were subjected to immunohistochemistry (IHC) staining for CD47. In vitro: The expression of CD47 on neutrophils was evaluated by flow cytometry (FCM). Human neutrophils from healthy donor were treated with ANCA-IgGs from MPO-AAV patients or control IgGs. For the efferocytosis assay, macrophages were co-incubated with unstimulated, apoptotic, and ANCA-IgGs treated neutrophils in the presence of anti-CD47 monoclonal antibody (mAb) or a control antibody. The neutrophils were labeled with CFMDA cell tracker (fluorescent probe) and the efferocytosis was evaluated as neutrophil engulfed (CFMDA positive) macrophages using fluorescent microscopy. In vivo: Spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj) mice (8-week-old age) were treated with intraperitoneal injection of anti-CD47 mAb or a control antibody every 5 days for two weeks. The severity of glomerulonephritis was assessed by the levels of serum creatinine, haematuria, mRNA expression of pro-inflammatory genes, and histopathological findings. To address the immune response against the CD47 blockade, the titre of MPO-ANCA and the number of splenic cell subset was assessed by ELISA and FCM analysis, respectively. Results Human data: The IHC analysis of human renal specimens revealed that the positive area of CD47 of AAV was greater than that of MGA. In particular, the CD47-overexpressed cells were seen in glomeruli with necrotic crescent formation. In vitro: Mean fluorescence intensity (MFI) of CD47 in ANCA-IgGs treated neutrophils (NETs) was significantly higher than that in control IgGs treated neutrophils (ANCA-IgG; 442±21.4 a.u. vs control IgG; 402±10 a.u., p&amp;lt;0.05). In efferocytosis assay, apoptotic neutrophils were engulfed by macrophages (efferocytosis rate/ apoptotic neutrophil; 20.5±3.8%, live neutrophils; 0.9±0.5%). The efferocytosis rate of ANCA-induced NETs significantly decreased compared to apoptotic neutrophil, but anti-CD47 mAb improved the efferocytosis of ANCA-NETs (efferocytosis rate/ anti-CD47 mAb; 19.1±4.2%, control antibody; 7.7±2.2%, p&amp;lt;0.05). In vivo: the renal histopathological severity score, serum creatinine level of AAV mice treated with anti-CD47 mAb decreased compared to that of AAV mice treated with a control antibody (anti-CD47 mAb; 0.96±0.30 vs control antibody; 0.61±0.32 mg/dL). Although there was no significant difference in the number of splenic cells between anti-CD47 and control antibody treated mice, CD47 blockade therapy significantly reduced serum MPO-ANCA titre (28.5±10.4 vs 45.2±14.5 μg/mL) and renal mRNA expression (IFNα, IFNγ, MCP-1 and perforin) of AAV mice. Conclusion ANCA-mediated NETs might escape from efferocytosis through up-regulation of CD47 and provoke necrotizing vasculitis. CD47 blockade could be a potential novel therapeutic strategy for AAV.

Safety of AK117, an anti-CD47 monoclonal antibody, in patients with advanced or metastatic solid tumors in a phase I study.

2630 Background: AK117 is a novel humanized IgG4 monoclonal antibody (mAb) targeting CD47, a macrophage immune checkpoint that allows tumor cells to evade immune destruction by phagocytic cells. CD47 is a target expressed in many cancers. However, the initial dose of anti-CD47 therapy may be limited by severe anemia due to ubiquitous CD47 expression on senescent red blood cells (RBCs). Here, we present encouraging preliminary AK117 safety and receptor occupancy (RO) data from an ongoing dose-escalation study in patients (pts) with advanced or metastatic solid tumors. Methods: This is a first-in-human, phase 1a/1b, multicenter, open label, single arm, dose escalation and dose expansion study of AK117 administered intravenously to adult pts with resistant/refractory advanced or metastatic solid tumors or lymphomas. In the dose escalation phase (phase 1a), an accelerated titration followed by a 3+3+3 design was used to assess the safety and tolerability of AK117 monotherapy (dose range 0.3 mg/kg to 45 mg/kg); and determine the maximum tolerated dose (MTD). AK117 was administered QW on a 28-day treatment cycle and dose limiting toxicity (DLT) observation period. Tumor assessments per RECIST v1.1 were performed once every 8 weeks (2 cycles). Results: As of 15 Feb 2021, 15 pts were enrolled in phase 1a with DLT evaluation of the 30 mg/kg cohort currently in progress. There were no DLTs up to 20 mg/kg QW AK117, inclusive. Five treatment-related adverse events (TRAEs) occurred in 4 subjects as shown in the table below. All pts with TRAEs continue to receive AK117, except the pt on 1 mg/kg AK117 who discontinued due to disease progression. G2 anemia and G1 thrombocytopenia occurred after Cycle 1 in a pt (liposarcoma, 10 mg/kg cohort), who had a medical history of anemia (hemoglobin 119 g/L at screening). No hematological TRAEs were seen in other pts, including those who received 20 mg/kg AK117 QW. There were no infusion-related reactions (IRRs) or grade ≥ 3 TRAEs. Target engagement in the periphery was confirmed by measuring CD47 RO of AK117 on RBCs and T lymphocytes. 100% RO on RBCs and T lymphocytes was achieved after the first dose and continued to Day 8 (prior to second dose) in the 10 mg/kg and 20 mg/kg cohorts. Conclusions: AK117 is safe and well-tolerated up to 20 mg/kg QW, inclusive, with no IRRs or severe TRAEs observed. There were no hematological TRAEs, except in a pt with baseline G1 anemia receiving 10 mg/kg AK117. Unlike other anti-CD47 therapies, AK117 does not require a lower ‘priming’ dose to prevent anemia. Safety evaluation of the 30 mg/kg dose level is in progress. Full and durable RO in the periphery was seen at 10 mg/kg and above. Further evaluation of AK117 in combination with AK104, an anti-PD-1/CTLA-4 bispecific antibody, shall commence imminently. Clinical trial information: NCT04349969. [Table: see text]

Preliminary results of a first-in-human phase I dtudy of IMM01, SIRPα Fc protein in patients with relapsed or refractory lymphoma.

2550 Background: IMM01 is a recombinant human signal regulatory protein α (SIRPα) IgG 1 fusion protein that exerts dual-mechanism antitumor activity via engagement of activating tumor cell phagocytosis and stimulating T-cell anti-tumor responses by binding CD47 on tumor cell membrane. IMM01 displays promising preclinical characteristics regarding its receptor occupancy/tumor exposure/efficacy relationship. Unlike anti-CD47 monoclonal antibodies, IMM01 shows unique property of weak human erythrocyte conjugation so as avoiding severe hemolysis. Methods: Monotherapy of IMM01 was conducted in 14 enrolled subjects with relapsed or refractory lymphoma who had failed standard therapies. Dose escalation was performed in routine design of accelerated single-patient followed by standard 3+3 to establish the preliminary data of safety as well as determination of a recommended expansion dosage. Each cycle contains 4 dosing weekly followed by a week rest. The tumor responses were evaluated based on Lugano Classification 2014. IMM01 pharmacokinetics (PK) and pharmacodynamics (PD) analyses were performed. Results: As of February 08, 2021, a total of 14 patients (median age 49 y; median prior therapy 3) were enrolled in 6 escalated dose cohorts (0.003 mg/kg, 0.01 mg/kg, 0.05 mg/kg, 0.15 mg/kg, 0.5 mg/kg and 1.0 mg/kg). The common tumor types were follicular lymphoma, Hodgkin lymphoma, diffuse large B-cell lymphoma. No DLTs were observed up to 1.0 mg/kg. One SAE (grade 2 increased amylase and grade 3 increased lipase) was reported, which induced by disease progression on pancreas. The most common treatment related adverse events were thrombocytopenia (54%), neutrophil count decreased (36%), Pyrexia (36%) and Anaemia (27%). There were grade 1 or 2 except for one patient experienced a grade 3 platelet count decreased (lower baseline at 70×109/L). Transient platelet count decrease after 2 hours and return to baseline at 24 to 48 hours post first infusion. In 12 evaluated patients, one patient with FL had a CR and maintained a 26-week response at the dose of 0.01 mg/kg. One patient with HL who had failed PD-1 inhibitor was confirmed PR at 27 weeks and continues the therapy, and one patient with MZL maintained SD for 12 weeks at the dose of 0.15 mg/kg. One patient with HL failed PD-1 inhibitor and one patient with FL maintained a shrunk SD for 12 weeks at the dose of 0.5 mg/kg. One patient with AITL was evaluated as a shrunk SD after 5 doses treatment at the dose of 1.0 mg/kg. Terminal half-life of IMM01 range from 53.8 hours to 73.3 hours. The AUC and Cmax of IMM01 show nonlinear increases in the dose range of 0.05 mg/kg to 0.5 mg/kg. Conclusions: Preliminary data from the present phase 1 study of IMM01, a SIRPα IgG 1 fusion protein, demonstrate that IMM01 has an excellent preliminary safety, tolerability and promising anti-tumor activity up to doses of 1.0 mg/kg. Clinical trial information: ChiCTR1900024904.

Phase 2 Study of YS110, a Recombinant Humanized Anti-CD26 Monoclonal Antibody, in Japanese Patients With Advanced Malignant Pleural Mesothelioma

IntroductionYS110, a humanized monoclonal antibody with a high affinity to CD26, exhibited promising antitumor activity and was generally well-tolerated in the phase 1 part of a phase 1 and 2 Japanese trial in patients with malignant pleural mesothelioma (MPM). Here we report the results of the phase 2 part of the study. MethodsThe patients included were aged 20 years and older, had histologically confirmed MPM, were refractory to or intolerant of existing antineoplastic agents, and were not candidates for standard therapy. YS110 6 mg/kg, determined in the phase 1 dose-determination part, was given in 6-weekly cycles (5 × once-weekly infusions, followed by a 1-wk rest). ResultsThe study included 31 patients (median age = 68 y, 90.3% men); 64.5% had stage IV MPM, 90.3% had greater than or equal to 20% CD26 expression in tumor tissue, and 38.7% (12 patients) had previously received nivolumab. The 6-month disease control rate was 3.2%. The best overall response was partial response in one patient and stable disease in 14 patients. The median progression-free survival was 2.8 months (both in patients who had and had not previously received nivolumab—groups A and B, respectively). Respective progression-free survival rates at 6 months were 9.1% and 31.6% in groups A and B. The median overall survival was 9.7 months. A total of 30 patients (96.8%) had at least one adverse event. Common treatment-related adverse events were infusion-related reaction (16.1%), hiccups (9.7%), and interstitial lung disease (9.7%). There were no treatment-related deaths. ConclusionsThe 6-month disease control rate did not exceed the predefined threshold, but YS110 revealed modest efficacy in response rate as salvage therapy in difficult-to-treat patients with MPM. YS110 was generally well tolerated.

New monoclonal antibodies that recognize an unglycosylated, conserved, extracellular region of CD44 in vitro and in vivo, and can block tumorigenesis.

CD44 is a transmembrane glycoprotein that binds to hyaluronic acid, plays roles in a number of cellular processes and is expressed in a variety of cell types. It is up-regulated in stem cells and cancer. Anti-CD44 monoclonal antibodies affect cell motility and aggregation, and repress tumorigenesis and metastasis. Here we describe four new anti-CD44 monoclonal antibodies originating from B cells of a mouse injected with a plasmid expressing CD44 isoform 12. The four monoclonal antibodies bind to the terminal, extracellular, conserved domain of CD44 isoforms. Based on differences in western blot patterns of cancer cell lysates, the four anti-CD44 mAbs separated into three distinct categories that include P4G9, P3D2, and P3A7, and P3G4. Spot assay analysis with peptides generated in Escherichia coli support the conclusion that the monoclonal antibodies recognize unglycosylated sequences in the N-terminal conserved region between amino acid 21–220, and analyses with a peptide generated in human embryonic kidney 293 cells, demonstrate that these monoclonal antibodies bind to these peptides only after deglycosylation. Western blots with lysates from three cancer cell lines demonstrate that several CD44 isoforms are unglycosylated in the anti-CD44 target regions. The potential utility of the monoclonal antibodies in blocking tumorigenesis was tested by co-injection of cells of the breast cancer-derived tumorigenic cell line MDA-MB-231 with the anti-CD44 monoclonal antibody P3D2 into the mammary fat pads of mice. All five control mice injected with MDA-MB-231 cells plus anti-IgG formed palpable tumors, while only one of the six test mice injected with MDA-MB-231 cells plus P3D2 formed a tiny tumor, while the remaining five were tumor-free, indicating that the four anti-CD44 mAbs may be useful therapeutically.

A Study of Microvascular Density in Carcinoma of Breast with CD34 Immunostaining

Introduction: Tumor agiogenesis is generally classified in to two types, namely, sprouting and intussusceptive angiogenesis. The formation of new blood vessels from the already existing is termed as sprouting angiogenesis. Many growth factors including endothelial growth factor (VEGF) contribute the formation of new vessels at the tumor sites. On other hand, the tumor able to split the existing blood vessels, this type is termed as intussuscepted angiogenesis and discovered in human colon adenocarcinoma xenograft. In intussuscepted angiogenesis, an existing blood vessel splits into two new blood vessels by formation of trans vascular pillars.&#x0D; Objective: The present study aimed to analyse the nicrovascular density on breast cancer patients using CD 34 immunostaining method.&#x0D; Results: This mis-regulation may lead to the development of cancers. Histological grading with Grade II were more with 19 (63.3%) cases followed by Grade I with 4 (13.3%) and Grade III with 3 (10%) cases. There was increase in mean vascular density in Grade II when compared with Grade I and Grade III. However no significant correlation was observed statistically with a P value of 0.176. Using different antibodies such as CD34, CD31, Factor VIII and CD105 to microvessels differentiation was highlighted.&#x0D; Conclusion: The results showed that the anti-CD34 monoclonal antibody is more sensitive than the anti- CD31 antibody in calculation of MVD in breast cancer as mentioned in previous studies.

Serum soluble CD26/DPP4 titer variation is a potential prognostic biomarker in cancer therapy with a humanized anti-CD26 antibody

BackgroundThe phase I trial of the humanized anti-CD26 monoclonal antibody YS110 for CD26-expressing tumors was conducted recently. The present study identifies a potential prognostic biomarker for CD26-targeted therapy based on the phase I data.MethodsBox and Whisker plot analysis, Scatter plot analysis, Peason product moment correlation/Spearman’s rank-difference correlation, Bar graph analysis, and Receiver Operating Characteristics (ROC) were used to examine the correlation between sCD26 titer variation with YS110 administration and tumor volume change, RECIST criteria evaluation and progression free survival (PFS). Mechanism for serum sCD26 titer variation was confirmed by in vitro experimentation.ResultsSerum sCD26/DPP4 titer was reduced following YS110 administration and gradually recovered until the next infusion. Serum sCD26/DPP4 titer before the next infusion was sustained at lower levels in Stable Disease (SD) cases compared to Progressive Disease cases. ROC analysis defined the cut-off level of serum sCD26/DPP4 titer variation at day 29 pre/post for the clinical outcome of SD as tumor response or PFS. In vitro experimentation confirmed that YS110 addition reduced sCD26 production from CD26-expressing tumor and non-tumor cells.ConclusionsOur study indicates that serum sCD26/DPP4 titer variation in the early phase of YS110 treatment is a predictive biomarker for evaluating therapeutic efficacy.

CD26/Dipeptidyl Peptidase IV and Its Multiple Biological Functions.

CD26/Dipeptidyl peptidase IV (DPPIV) is a cell surface glycoprotein with numerous roles including glucose metabolism, immunomodulation, and tumorigenesis. CD26/DPPIV is well recognized in diabetes, with DPPIV inhibitors being a class of oral hypoglycemic drugs called gliptins that are commonly used to treat type two diabetes mellitus. Recent work also indicated a potential role for CD26 in infectious diseases, including COVID-19, and immune-mediated disorders such as rheumatoid arthritis, inflammatory bowel disease, and graft-versus-host disease.In cancer, CD26/DPPIV expression has been characterized in numerous tumors such as hematologic malignancies, malignant pleural mesothelioma (MPM), renal cell carcinoma (RCC), hepatocellular carcinoma (HCC), gastrointestinal stromal tumor (GIST), and prostate, lung, colorectal, and ovarian (PLCO) cancer. Hence, CD26 has been frequently studied as a tumor biomarker and therapeutic target. CD26/DPPIV-targeted therapies have been evaluated in various cancers, including the use of anti-CD26 monoclonal antibodies as anticancer treatment in selected neoplasms.This review highlights our current understanding of the role of CD26 in cancer, diabetes, immune-mediated diseases, and infectious diseases. Enhanced understanding of CD26 biology and function may lead to novel therapeutic approaches in multiple human diseases.

Abstract PS17-06: Humanized anti-CD47 monoclonal antibody magrolimab (Hu5F9-G4) plus trastuzumab potentiates antibody-dependent cellular phagocytosis (ADCP), and cooperate to inhibit human HER2+ breast cancer (BC) xenografts growth in vivo

Abstract Background: Cancer cells overexpress CD47 to evade phagocytic programmed cell removal (PrCR) by macrophages of the innate immune system. Blocking CD47 using magrolimab (Hu5F9-G4), an anti-CD47 humanized monoclonal antibody, works via preventing CD47 signaling via macrophage Sirpα to allow PrCR, as well as using an Fc-receptor mechanism to encourage macrophage-mediated phagocytosis. We have previously shown that magrolimab in combination with tumor-targeting antibodies (e.g. rituximab) further enhances magrolimab’s anti-cancer effects in preclinical models and in the clinic [Advani R, et al. N Engl J Med 2018; 379:1711-1721]. We therefore hypothesized that magrolimab combined with anti-HER2 monoclonal antibody would synergize to promote ADCP in vitro, and HER2+ breast xenograft growth inhibition in vivo. Methods: To test this hypothesis, 1 x 105 CFSE-labeled HER2+ BT474 and SKBR3 cells were plated in 96-well ultra-low attachment plates in serum-free media. Isotype control antibody, trastuzumab, magrolimab, and trastuzumab + magrolimab combination (10µg/ml) were added, and allowed to incubate at 37°C for 30 mins. 5 x 104 human macrophages were then added each well, and were co-cultured for 2 hours. Human macrophages were stained with anti-CD11b, and phagocytosis was determined as the percentage of cells that were CD11b+ and CSFE+ using a BD LSR Fortessa Analyzer. For in vivo tumor xenograft growth kinetics, GFP+/Luciferase+ BT474 cells (1X105) were implanted with 25% Matrigel into the mammary fat pads of 4-8 week-old NOD scid gamma (NSG) female mice. Twenty-five days after engraftment, trastuzumab (100ug) was administered via intraperitoneal (IP) injection weekly, magrolimab 250ug IP was administered every other day, and PBS control was administered IP at 100uL once weekly, and tumor growth was monitored for 17 weeks by bioluminescence (IVIS) after D-luciferin injection, and quantified using Image 4.0. Results: SKBR3 and BT474 parental and Trastuzumab-resistant lines demonstrated significantly increased susceptibility to ADCP when opsonized with combination treatment of trastuzumab + magrolimab (15.34, 95%CI= 12.2 to 16.6) compared to trastuzumab (9.66, 95%CI=1.861 to 7.7579, p = 0.034), or magrolimab alone (10.23, 95%CI=1.254 to 7.037, p = 0.0083). Treatment with magrolimab maintained tumor burden within the starting range but further progressed upon treatment cessation (2.09x109, CI95% = 2801531434 to 15343840962, p = 0.0006). Trastuzumab treatment resulted in lower starting range tumor burden, but also demonstrated progression once treatment was stopped (1.72x106, CI95% = -695208489 to -86402608, p = 0.0052). However, in the combinatorial treatment arm, tumors were significantly below the starting range and did not show signs of tumor progression within the 10-week non-treatment period (8.51x105, CI95%= -311406356 to -66914472, p = &amp;lt; 0.0001). Conclusion: We conclude magrolimab plus trastuzumab cooperate to inhibit HER2+ xenograft growth in vivo, and that treatment effect persists even after treatment is stopped. Our ex vivo data suggests one mechanism to explain the observed tumor growth inhibition is increased susceptibility to ADCP when HER2+ tumors are opsonized by the combination of trastuzumab + magrolimab. Future clinical translation of this combination is warranted. Citation Format: Rosalynd Upton, Dongdong Feng, Allison M Banuelos, Tanuka Biswas, Stephen Willingham, Kevin S Kao, Kelly McKenna, Benyamin Rosenthal, Michal C Tal, Jens-Peter Volkmer, Mark D Pegram, Irving L Weissman. Humanized anti-CD47 monoclonal antibody magrolimab (Hu5F9-G4) plus trastuzumab potentiates antibody-dependent cellular phagocytosis (ADCP), and cooperate to inhibit human HER2+ breast cancer (BC) xenografts growth in vivo [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-06.