Antibody-dependent Cell Phagocytosis Research Articles

The FIPO23 trial: First-in-human phase I/II study of XON7 in advanced solids tumors.

TPS2673 Background: XON7 is a first-in-class glyco-humanized polyclonal antibody (GH-pAb) targeting selectively multiple tumor-associated antigens. XON7 induces tumor cell apoptosis and promotes the elimination of tumor cells by immune effector cells through CDC (Complement Dependent Cytotoxicity) and ADCP (Antibody-Dependent Cell Phagocytosis). XON7 demonstrated anti-tumor efficacy in mice xenograft models with human solid tumors such as colon, prostate, lung and triple negative breast cancers. Safety pharmacology and toxicology studies in non-human primates demonstrated an acceptable safety profile for XON7. Taken together, these preclinical data support the initiation of human trials in patients with advanced solid tumors. Methods: First-in-patient, multicenter, open-label, two phases (dose-escalation (ESC) followed by dose expansion (EXP)) study of XON7 single agent, in patients with relapsed refractory, locally advanced or metastatic solid tumors without standard available treatments ( NCT06154291). Key eligibility criteria include disease progression after ≤ 4 lines of therapy; age > 18 years; ECOG performance status ≤2; adequate organ functions; no known CNS involvement. All advanced or metastatic tumor types except glioblastoma, can be included during ESC. Primary endpoints: safety, tolerability and determination of MTD/RP2D. Secondary endpoints: pharmacokinetics (PK), pharmacodynamics, immunogenicity and preliminary anti-tumor activity (RECIST 1.1). AEs graded according to CTCAE 5.0. In the ESC part, XON7 is administered as 60-min intravenous infusion every 2 weeks according to an escalating schedule of doses from 1.5 to 20 mg/kg for up to 12, 28-day cycles. Dose escalation/de-escalation is based on the Bayesian Optimal Interval (BOIN) design, up to 45 pts are planned. At the recommended dose, up to 7 EXP cohorts (30 pts each) defined according to the primary tumor site are planned. Bayesian sequential monitoring will be used. Patient recruitment is planned or ongoing at 25 sites worldwide. Results: As of Feb 3rd, 2024, 3 pts are enrolled in the first dose cohort and have received XON7 at dose 1,5 mg/kg Q2W for the first 28-days cycle. Conclusions Accrual is ongoing in this first-in-human trial of XON7. Clinical trial information: NCT06154291 .

Development of ISB 1442, a CD38 and CD47 bispecific biparatopic antibody innate cell modulator for the treatment of multiple myeloma

Antibody engineering can tailor the design and activities of therapeutic antibodies for better efficiency or other advantageous clinical properties. Here we report the development of ISB 1442, a fully human bispecific antibody designed to re-establish synthetic immunity in CD38+ hematological malignancies. ISB 1442 consists of two anti-CD38 arms targeting two distinct epitopes that preferentially drive binding to tumor cells and enable avidity-induced blocking of proximal CD47 receptors on the same cell while preventing on-target off-tumor binding on healthy cells. The Fc portion of ISB 1442 is engineered to enhance complement dependent cytotoxicity, antibody dependent cell cytotoxicity and antibody dependent cell phagocytosis. ISB 1442 thus represents a CD47-BsAb combining biparatopic targeting of a tumor associated antigen with engineered enhancement of antibody effector function to overcome potential resistance mechanisms that hamper treatment of myeloma with monospecific anti-CD38 antibodies. ISB 1442 is currently in a Phase I clinical trial in relapsed refractory multiple myeloma.

Subtype recognition and identification of a prognosis model characterized by antibody-dependent cell phagocytosis-related genes in breast cancer.

Breast cancer (BC) is a heterogeneous tumor with a variety of etiology and clinical features. Antibody-dependent cell phagocytosis (ADCP) is the last step of immune checkpoint inhibition (ICI), and macrophages detect and recognize tumor cells, then destroy and engulf tumor cells. Despite the large number, negative regulators that inhibit phagocytic activity are still a key obstacle to the full efficacy of ICI. An ADCP-related risk score prognostic model for risk stratification as well as prognosis prediction was established in the Cancer Genome Atlas (TCGA) cohort. The predictive value of ADCP risk score in prognosis and immunotherapy was also further validated in the TCGA along with International Cancer Genome Consortium cohorts. To promote the clinical application of the risk score, a nomogram was established, with its effectiveness verified by different methods. In this study, the genes collected from previous studies were defined as ADCP-related genes. In BC patients, two ADCP-related subtypes were identified. The immune characteristics and prognostic stratification were significant different between them. We identified two subtypes associated with ADCP gene expression in breast cancer. They have significant differences in immune cells, molecular functions, HLA family genes, immune scores, stromal scores, and inflammatory gene expression, which have important guiding significance for the selection of clinical treatment methods. At the same time, we constructed a risk model based on ADCP, and the risk score can be used as a good indicator of prognosis, providing potential therapeutic advantages for chemotherapy and immunotherapy, thus helping the clinical decision-making of BC patients.

Strategies to Develop Anti-KIR Mab Lacutamab in Patients with Peripheral T-Cell Lymphoma: Preliminary Monotherapy Clinical Data and Pre-Clinical Combinability Data

Peripheral T-cell Lymphoma (PTCL) is a heterogeneous group of aggressive T-cell lymphomas associated with poor prognosis. Many patients with PTCL do not respond or relapse despite responding to first line systemic therapy. Despite the common use of chemotherapies, there is no universally accepted standard of care. This highlights an urgent and significant need for innovative treatment strategies that incorporate novel mechanisms of action, particularly for patients with relapsed or refractory (R/R) PTCL. One such novel target is KIR3DL2, a killer immunoglobulin-like receptor that is expressed across different subtypes of T-cell lymphomas including approximately 50% of PTCL patients. Lacutamab is a first-in-class monoclonal antibody designed to specifically deplete KIR3DL2-expressing cells through antibody-dependent cell-cytotoxicity (ADCC) and antibody-dependent cell-phagocytosis (ADCP). It is being developed as a novel treatment for patients with R/R PTCL as a single agent or in combination. In previous trials in patients with R/R cutaneous T-cell lymphoma (CTCL), lacutamab showed acceptable safety profile with no dose limiting toxicities and promising activity. Here we provide preclinical combination data supporting anti-tumor activity and rationale for the exploration of lacutamab in combination with approved and novel therapies in patients with PTCL and we present preliminary monotherapy data from an ongoing Phase 1b study in PTCL. To further develop novel-lacutamab combinations, the combinability of lacutamab with therapies used in R/R or frontline PTCL e.g., pralatrexate or CHOP, respectively, was tested. Pralatrexate is an antineoplastic folate analog approved for the treatment of R/R PTCL. In vitro, pralatrexate was shown to induce KIR3DL2 upregulation on several tumor cell lines with endogenous or forced KIR3DL2 expression and to enhance lacutamab-induced ADCC by NK cells. Importantly, the combination of lacutamab with pralatrexate in vivo delayed tumor growth (Figure 1A) and improved survival (Figure 1B) compared to each monotherapy. CHOP-based chemotherapy is a standard of care in the frontline treatment of PTCL subtypes. In a preclinical in vivo human tumor model, lacutamab in combination with CHOP was shown to reduce tumor growth compared with CHOP or lacutamab alone. An ongoing multi-center, open-label, Phase Ib trial (NCT05321147) evaluate the safety and efficacy of lacutamab monotherapy in patients with KIR3DL2-expressing R/R PTCL who have received at least one prior line of systemic therapy. Lacutamab 750 mg is administered as an intravenous infusion weekly x 5 weeks, every 2 weeks x 10 then every 4 weeks until disease progression or unacceptable toxicity. The primary objective was to assess the safety and tolerability of lacutamab in R/R PTCL. Secondary objectives were to assess clinical activity and to characterize the pharmacokinetics and immunogenicity of lacutamab. At the data cut-off, 10 patients were treated with lacutamab. Median age was 71.0 years (range: 61-77), median prior lines of therapies was 3 (range: 1-5), and median follow-up was 1.9 months (m) (range: 0.5-8.8 m). The majority (90%) of treatment-emergent adverse events (TEAEs) were of grade 1-2 severity, grade ≥3 related TEAEs were observed in 2 (20%) patients: 1 with serum sickness, and 1 with aspartate aminotransferase elevation. No serious TEAEs were observed and none of the patients discontinued study drug due to TEAE. Overall, preliminary Phase 1b data in patients with R/R PTCL confirm the acceptable safety profile of lacutamab monotherapy. The preclinical activity profile improves when assessed in combination. These data inform the future development of lacutamab to provide additional therapeutic options that may improve outcomes for PTCL patients, including those who relapse or are refractory to available therapies. A Phase 2 study evaluating the combination of lacutamab with GemOx is ongoing (NCT04984837) based on pre-clinical observations that GemOx improves lacutamab-induced ADCC by NK cells.

G protein-coupled receptor 183 mediates the sensitization of Burkitt lymphoma tumors to CD47 immune checkpoint blockade by anti-CD20/PI3Kδi dual therapy.

Immunotherapy-based regimens have considerably improved the survival rate of B-cell non-Hodgkin lymphoma (B-NHL) patients in the last decades; however, most disease subtypes remain almost incurable. TG-1801, a bispecific antibody that targets CD47 selectively on CD19+ B-cells, is under clinical evaluation in relapsed/refractory (R/R) B-NHL patients either as a single-agent or in combination with ublituximab, a new generation CD20 antibody. A set of eight B-NHL cell lines and primary samples were cultured in vitro in the presence of bone marrow-derived stromal cells, M2-polarized primary macrophages, and primary circulating PBMCs as a source of effector cells. Cell response to TG-1801 alone or combined with the U2 regimen associating ublituximab to the PI3Kδ inhibitor umbralisib, was analyzed by proliferation assay, western blot, transcriptomic analysis (qPCR array and RNA sequencing followed by gene set enrichment analysis) and/or quantification of antibody-dependent cell death (ADCC) and antibody-dependent cell phagocytosis (ADCP). CRISPR-Cas9 gene edition was used to selectively abrogate GPR183 gene expression in B-NHL cells. In vivo, drug efficacy was determined in immunodeficient (NSG mice) or immune-competent (chicken embryo chorioallantoic membrane (CAM)) B-NHL xenograft models. Using a panel of B-NHL co-cultures, we show that TG-1801, by disrupting the CD47-SIRPα axis, potentiates anti-CD20-mediated ADCC and ADCP. This led to a remarkable and durable antitumor effect of the triplet therapy composed by TG-1801 and U2 regimen, in vitro, as well as in mice and CAM xenograft models of B-NHL. Transcriptomic analysis also uncovered the upregulation of the G protein-coupled and inflammatory receptor, GPR183, as a crucial event associated with the efficacy of the triplet combination. Genetic depletion and pharmacological inhibition of GPR183 impaired ADCP initiation, cytoskeleton remodeling and cell migration in 2D and 3D spheroid B-NHL co-cultures, and disrupted macrophage-mediated control of tumor growth in B-NHL CAM xenografts. Altogether, our results support a crucial role for GPR183 in the recognition and elimination of malignant B cells upon concomitant targeting of CD20, CD47 and PI3Kδ, and warrant further clinical evaluation of this triplet regimen in B-NHL.

Abstract 3203: EVOcells Oncology: Tailored genetic engineering of iPSC-derived immune effector cells and combination with the right biologic therapeutics result in optimal killing of primary tumor cells from patients

Abstract Current autologous cell therapies, with blockbuster products on the market, have been leading for a decade to unprecedented clinical successes in patients with hematological malignancies. However, these patient-derived T-cell therapies are facing many challenges. The use of GMP iPSC lines to produce immune effector cells will reduce the complexity of the manufacturing process and will provide an unlimited source of starting material. The goal of the EVOcells Oncology platform is to offer a truly allogeneic cell therapy platform to treat a broad number of cancer patients with consistent quality and scalability of the final product. Besides, the versatility of our platform to produce different immune cell types combined to customized genetic engineering strategies will bring cell therapy to the level of personalized medicine. Our “off-the-shelf” cell therapy platform has already validated two pillars: iPSC-derived NK cells (iNK) and iPSC-derived Macrophages (iMACs). Through multiple genetic engineering strategies specific to each immune cell type, we are developing a comprehensive portfolio of cell therapy products to address specific tumor escape mechanism in liquid and solid tumors. Our initial effort aimed to develop these two innate immune cell types to propose efficacious cell therapies with an increased safety profile as they have low risk of graft-versus-host disease (GvHD) or CRS (Cytokine Release Syndrome). Thanks to the expression of a broad pattern of activatory receptors, iNK cells form Immunological Synapses with tumor cells leading in turn to efficient killing with and without addition of a CAR construct. Besides, we demonstrated the possibility to combine “naked” iNK cells with marketed therapeutic monoclonal antibodies (mAb) to further improve their efficacy. At the end of the differentiation process, iMACs are showing a M0 like phenotype with high plasticity allowing the in vitro differentiation of the cells towards either a M1 or a M2 polarization in response to the appropriate stimulations. iMACs produce key macrophages cytokines and are able to kill tumor cells via ADCP (Antibody-Dependent-Cell-Phagocytosis) mechanism when combined to a therapeutic mAb. Thanks to our collaboration with clinicians at the IUCT-Oncopole (Toulouse Cancer Hospital), we were able to identify appropriate cancer indications and further demonstrate in a translational fashion that both iNK and iMACs are able to kill primary resistant tumor cells which were isolated from patient’ samples. Taken together, these results are showing the versatility and the breadth of our EVOcells Oncology platform to produce a true arsenal of cell therapies and its potential for future clinical development. Citation Format: Michael Esquerré, Audrey Holtzinger, Nadja Wagner, Monika Braun, Mélanie Pichery, Stefanie Pfaender, Stephanie Sontag, Kathrin Haake, Michela Mirenda, Michael Paillasse, Davide Grandolfo, Chloé Beuraud, Mandy Richter, Philip Hublitz, Julien Bousquet, Marion Fabre, Mylène Gador, Daniel Sommermeyer, Tanja Schneider, Oriane Bombarde, Camille Esquerré, Loic Ysebaert, Fabien Despas, Matthias Austen, Andreas Scheel, Markus Dangl. EVOcells Oncology: Tailored genetic engineering of iPSC-derived immune effector cells and combination with the right biologic therapeutics result in optimal killing of primary tumor cells from patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3203.

Sub-Cutaneous Administration of Rituximab Triggers a Pro-Phagocytic Phenotype of Monocytic Compartment in Low-Tumor Burden Follicular Lymphoma Patients Included in the Flirt Clinical Trial, a Lysa Study

Introduction FLIRT (NCT02303119) is a phase III randomized clinical trial designed to evaluate whether Rituximab (R) induction by one IV infusion followed by 3 sub-cutaneous (SC) infusions and short maintenance using SC route (SC-arm) could improve PFS compared to 4-weekly IV R infusions without maintenance (IV-arm) in first line/low tumor burden (GELF criteria) Follicular Lymphoma (FL) patients (202 patients). This study met its primary endpoint and demonstrated that the SC-arm improves PFS of patients with low-tumor burden (#EHA 2022, #ASCO 2022). R is known to exert its anti-tumor activity through complement activation, antibody-dependent cell cytotoxicity, and antibody-dependent cell phagocytosis (ADCP). An ancillary study of the FLIRT trial planned to explore the differential impacts on immune response in the 2 arms and to identify predictive biomarkers. Methods Peripheral blood cells were analyzed in 39 patients from the SC-arm and 43 patients from the IV-arm before treatment (D1) and at M3, i.e. after 4 R infusions in both arms, by multicolor flow cytometry to evaluate circulating T, B, NK, dendritic, and monocyte subset counts and activation status. Results The 82 patients studied display the same clinical features than the whole cohort and the PFS was significantly longer in the SC-arm than in the IV-arm in this limited cohort (P=0.0059). When focusing only on the differences between the 2 arms, monocytes were differentially affected, with an upregulation at M3 of the Fcγ receptors CD32 and CD64 on the 3 main circulating monocyte subsets (CD14hiCD16lo classical, CD14loCD16hi non classical, and intermediate) exclusively in the SC-arm (P<0.05), whereas the SIRPα phagocytosis inhibitory receptor was significantly upregulated on these 3 monocyte subsets exclusively in the IV-arm (P<0.05). In addition, the percentage of CD25hiFoxp3pos Treg among CD4pos T cells was decreased only in the SC-arm (P<0.01), together with an increase of naïve (CD45RAposCCR7pos) and effector memory (CD45RAnegCCR7neg) CD4pos T cells (P<0.05), and naïve CD8pos T cells (P<0.05). Interestingly, CD32 and CD64 expression on M3 monocytes in the SC-arm was correlated with T-cell activation, as evaluated by the expression of HLA-DR on CD8pos T cells and PD-1 on CD4pos T cells (Spearman, P<0.05). Conversely, no modification of T cell subsets was found exclusively in the IV-arm. Additional studies are ongoing to establish whether this differential impact on monocyte phenotype translate to transcriptomic and functional differences. Conclusions Treatment of low-tumor burden FL with R only using SC route is associated with a specific pro-phagocytic activation profile of the monocytic compartment that is not found when R is administrated exclusively by the IV route that favors conversely an anti-phagocytic polarization. This SC-specific monocyte reprogramming is associated with a T-cell activation phenotype, opening the way for additional studies on the impact of R route of administration on ADCP and anti-tumor immune response activation.

Preclinical Evaluation of ISB 1442, a First-in-Class CD38 and CD47 Bispecific Antibody Innate Cell Modulator for the Treatment of AML and T-ALL

ISB 1442 is a bispecific antibody (BsAb) using Ichnos' proprietary Bispecific Engagement by Antibodies based on the TCR (BEAT®) platform. A fully human BsAb with anti-CD38 and CD47 binding arms, ISB 1442 was developed for the treatment of hematologic malignancies. 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 designed to block the interaction between CD47 and the signal-regulatory protein alpha (SIRPα) receptor present on phagocytes. 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 effects on red blood cells (RBC) compared to benchmark anti-CD47 monoclonal antibodies (mAb), such as 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). Preclinical activity of ISB 1442 for the treatment of relapsed/refractory multiple myeloma (rrMM), including increased killing potency relative to daratumumab and magrolimab benchmarks as well as more favorable off tumor/on target specificity, was reported previously (Sammicheli et al. ASH 2021). ISB 1442 is currently in a Phase 1 clinical trial for rrMM. Beyond MM, CD38 and CD47 are expressed in other hematologic cancers, including acute myeloid leukemia (AML) and T cell acute lymphoblastic leukemia (T-ALL). In AML, unleashing tumor cell phagocytosis using magrolimab shows clinical benefit in combination with demethylating agents and BCL-2 inhibitors. T-ALL is characterized with high CD38 expression, and daratumumab in combination with chemotherapy is under investigation (NCT03384654) for this indication. ISB 1442 has the potential to treat AML and T-ALL patients with its first-in-class molecular attributes of co-targeting CD38 and CD47 by a single antibody with enhanced Fc effector functions. ISB 1442 induced potent phagocytosis of both AML and T-ALL cell lines in vitro. The induction of phagocytosis was more prominent in cell lines expressing high level of CD38. ISB 1442 also induced higher phagocytosis of both AML and T-ALL tumor cells as compared to daratumumab and anti-CD47 (5F9) in cell lines expressing low level of CD38 and CD47. In addition to phagocytosis, ISB 1442 induced potent ADCC of both AML and T-ALL cell lines. To characterize the complex mechanisms of action of ISB 1442 in a single assay, a multiple mode of action of killing (MMoAK) in vitro assay was established where macrophages and PBMCs are incubated with AML or T-ALL tumor cells and human serum. With this approach, tumor cells can be targeted simultaneously by NK cells from PBMCs (ADCC Mechanism of Action (MOA)), macrophages (ADCP MOA), and complement from human serum (CDC MOA). ISB 1442 induced higher killing of AML and T-ALL cell lines in MMoAK assays as compared to daratumumab or anti-CD47 (5F9). ISB 1442 induced potent killing of primary tumor samples from both AML and T-ALL patients in a heterologous assay using macrophages from healthy donors as effector cells, suggesting that target cancer cells do not have intrinsic mechanisms protecting them from the immune cell-mediated MOA of ISB 1442 killing . In patients' samples having detectable levels of myeloid or NK cells in bone marrow aspirates, ISB 1442 induced killing of tumor cells in the autologous setting in a direct killing assay ex vivo. In summary, these data support the clinical development of 1442 in AML and T-ALL. Based on its unique design and multiple mechanisms of action, ISB 1442 is anticipated to have antitumor activity in AML and T-ALL patients in a single antibody relative to anti-CD38 or anti-CD47 monoclonal antibodies as well as their combination.

Establishing and validating an ADCP-related prognostic signature in pancreatic ductal adenocarcinoma.

With the progress of precision medicine treatment in pancreatic ductal adenocarcinoma (PDAC), individualized cancer-related examination and prediction is of great importance in this high malignant tumor, and antibody-dependent cell phagocytosis (ADCP) with changed pathways highly enrolled in the carcinogenesis of PDAC. High-throughput data of pancreatic ductal adenocarcinoma were downloaded and 160 differentially expressed ADCP-related genes (ARGs) were obtained. Secondly, GO and KEGG enrichment analyses show that ADCP is a pivotal biologic process in pancreatic carcinogenesis. Next, CALB2, NLGN2, NCAPG and SERTAD2 are identified through multivariate Cox regression. These 4 genes are confirmed with significant prognostic value in PDAC. Then, a risk score formula is constructed and tested in PDAC samples. Finally, the correlation between these 4 genes and M2 macrophage polarization was screened. Some pivotal differentially expressed ADCP-related genes and biologic processes, four pivotal subgroup was among identified in the protein-protein network, and hub genes was found in these sub group. Then, an ADCP-related formula was set: CALB2* 0.355526 + NLGN2* -0.86862 + NCAPG* 0.932348 + SERTAD2* 1.153568. Additionally, the significant correlation between M2 macrophage-infiltration and the expression of each genes in PDAC samples was identified. Finally, the somatic mutation landscape and sensitive chemotherapy drug between high risk group and low risk group was explored. This study provides a potential prognostic signature for predicting prognosis of PDAC patients and molecular insights of ADCP in PDAC, and the formula focusing on the prognosis of PDAC can be effective. These findings will contribute to the precision medicine of pancreatic ductal adenocarcinoma treatment.

P871: ISB 1442, A FIRST-IN-CLASS CD38 AND CD47 BISPECIFIC ANTIBODY INNATE CELL MODULATOR FOR THE TREATMENT OF CD38 POSITIVE HEMATOLOGICAL MALIGNANCIES

Background: ISB 1442 is a first in class 2 + 1 biparatopic bispecific antibody targeting CD38 x CD47 using the BEAT® 2.0 (Bispecific Engagement by Antibodies based on the TCR) platform. The CD38 binding arm consists of bi-paratopic Fabs that strongly bind to CD38 in two different epitopes on tumor cells and do not functionally compete with daratumumab. 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 high affinity anti-CD38 Fab arms preferentially drive binding to tumor cells and enables blocking of proximal CD47 receptors on the same cell via avidity-induced binding. 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). Aims: ISB 1442 was developed for the treatment of hematologic malignancies that express CD38, including multiple myeloma (MM), acute myeloid leukemia (AML), and T-acute lymphoblastic leukemia (T-ALL), with the intention of overcoming mechanisms of resistance to CD38-targeted therapies such as daratumumab and isatuximab, and minimizing hemagglutination/hemolysis on red blood cell (RBC) observed with anti-CD47 monoclonal antibodies (mAb) such as magrolimab. Methods: ISB 1442 was tested for its capacity in vitro to induce ADCP, ADCC and CDC relative to daratumumab and magrolimab across a broad range of MM, AML and T-ALL cell lines expressing different levels of CD38 and CD47. To assess the complex mechanisms of action of ISB 1442 in a single system, a multiple mode of action of killing (MMoAK) assay was established to allow for simultaneous killing by natural killer cells (ADCC), autologous macrophages (ADCP), and complement from human serum (CDC). In vivo, ISB 1442 was assessed in a therapeutic model of subcutaneously established Raji tumor xenograft in CB17/SCID mice compared to daratumumab and magrolimab. On-target specificity was evaluated in vitro in human and monkey whole blood assays. Results: In vitro, ISB 1442 exhibited higher killing potency compared to daratumumab across a range of CD38-expressing MM, AML and T-ALL tumor cells. Additionally, ISB 1442 showed in vitro tumor killing potency through phagocytosis comparable to magrolimab, acting mostly through ADCP. In the CDC, ADCC and MMoAK assays, ISB 1442 exhibited tumor cell killing that was twice as high as daratumumab in MM cell lines. In vivo, ISB 1442 induced higher tumor growth inhibition than daratumumab and comparable tumor regression to magrolimab. ISB 1442 did not cause any detectable hemolysis, RBC depletion or platelet aggregation and showed markedly lower hemagglutination relative to magrolimab, suggesting a more favorable on-target specificity profile in humans. In monkeys, ISB 1442 showed more pronounced binding on RBC than magrolimab due to higher expression of CD38, suggesting that monkey is a more sensitive species than human for toxicological evaluation of CD38-targeted therapies. Summary/Conclusion: In summary, we report a novel approach for the treatment for CD38 positive hematologic malignancies by co-targeting CD38 and CD47 using a first in class multispecific antibody. Based on its unique design and multiple mechanisms of action, ISB 1442 is anticipated to enhance antitumor activity in patients relative to anti-CD38 mAbs by overcoming primary and acquired tumor escape mechanisms of resistance.

Abstract 2903: ISB 1442, a first-in-class CD38 and CD47 bispecific antibody innate cell modulator for the treatment of CD38+ malignancies

Abstract ISB 1442 is a fully human bispecific antibody (BsAb) using BEAT (Bispecific Engagement by Antibodies based on the T-cell receptor) platform to target CD38 and CD47 as treatment for CD38+ malignancies, including multiple myeloma (MM). ISB 1442 is designed with a bi-paratopic anti-CD38 arm that strongly binds to CD38+ tumor cells and an anti-CD47 arm made of a single Fab designed to block interaction between CD47 and the signal-regulatory protein alpha (SIRPα) with low affinity. This approach is expected to induce minimal unintended effects on red blood cells (RBC) compared to anti-CD47 monoclonal antibody (mAb) magrolimab as it enables the CD47 binding only upon avidity induced CD38 crosslinking. 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). In vitro, ISB 1442 exhibited higher killing potency compared to daratumumab across a range of CD38-expressing tumor cells. Additionally, ISB 1442 showed in vitro tumor killing potency through phagocytosis comparable to magrolimab, acting mostly through ADCP. To assess the complex mechanisms of action of ISB 1442 in a single system, a multiple mode of action of killing (MMoAK) assay was established to allow for simultaneous killing by natural killer cells, autologous macrophages, as well as complement from human serum. In the MMoAK assay, ISB 1442 exhibited tumor cell killing that was twice as high as daratumumab. In vivo, in a therapeutic model of subcutaneously established Raji tumor xenograft in CB17/SCID mice, ISB 1442 induced higher tumor growth inhibition than daratumumab and comparable tumor regression to magrolimab. On-target specificity was evaluated in vitro in human and monkey whole blood assays. ISB 1442 did not cause any detectable hemolysis, RBC depletion or platelet aggregation and showed markedly lower RBC hemagglutination relative to magrolimab, suggesting a more favorable on-target specificity profile in humans. On the contrary, due to the higher expression of CD38 in monkey RBC compared to human, ISB 1442 showed more pronounced binding on RBC than magrolimab, suggesting that monkey is a more sensitive species than human for toxicological evaluation of CD38-targeted therapies. Finally, by integrating in vitro pharmacology data along with available clinical information on benchmark antibodies, a quantitative systems pharmacology model was developed to simulate potential efficacious dose range in MM. In summary, we report a novel approach for the treatment for CD38+ cancers by co-targeting CD38 and CD47. Based on its unique design and multiple mechanisms of action, ISB 1442 is anticipated to enhance antitumor activity by overcoming known primary and acquired tumor escape mechanisms of resistance relative to daratumumab. Citation Format: Stefano Sammicheli, Camille Grandclement, Elie Dheilly, Maria Panagopoulou, Evangelia Martini, Perrine Suere, Blandine Pouleau, Carole Estoppey, Julia Frei, Jeremy Loyau, Thierry Monney, Adam Drake, Alain Rubod, Marie Agnes Doucey, Vinu Menon, Venkatesha Udupa, Sunitha GN, Daniel Rasmussen, Jeppe Koch Olsen, Roberto Gionannini, Girish Gudi, Ankita Srivastava, Cyril Konto, Mario Perro. ISB 1442, a first-in-class CD38 and CD47 bispecific antibody innate cell modulator for the treatment of CD38+ malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2903.

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

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

The Ublituximab-Umbralisib (U2) Drug Regimen Potentiates the Activity of the Novel CD47-CD19 Bispecific Antibody, TG-1801, through the Activation of the G Protein-Coupled Receptor EBI2/GPR183

Introduction: TG-1801 is a novel, bispecific anti-CD47 and anti-CD19 fully human IgG1 antibody that targets CD47 selectively on CD19+ B-cells, sparing red blood cells or platelets and blocking the CD47-SIRPα macrophage checkpoint on mature B cells. TG-1801 is currently in clinical trial as a single agent or in combination with ublituximab, a glyco-engineered CD20 antibody, in B-cell non-Hodgkin lymphoma (B-NHL). The doublet therapy of ublituximab with the dual PI3Kδ/CK1e inhibitor umbralisib (“U2” regimen), provides a non-chemotherapy backbone regimen on which several novel multidrug combinations are being explored clinically. Here we explored in vitro and in vivo potential synergies between TG-1801 and ublituximab, umbralisib, and the U2 combination, in preclinical models of B-NHL.Methods: A panel of n=12 human B-cell lymphoma cell lines and primary samples were cultured in vitro in the presence of bone marrow-derived stromal cells (BMSCs), M2-polarized primary macrophages, and primary circulating PBMCs as a source of effector cells. Cell response to TG-1801 +/- U2 treatments was analyzed by proliferation assay, western blot, transcriptomic analysis (qPCR array and RNA sequencing followed by gene set enrichment analysis) and quantification of antibody-dependent cell death (ADCC) and antibody-dependent cell phagocytosis (ADCP). In vivo, drug efficacy was determined in a Raji xenograft model, by dosing tumor-bearing mice for 17 days with ublituximab (5mg/kg, qw) +/- umbralisib (150mg/kg, bid), the combination of both and/or TG-1801 (5mg/kg, qw).Results: Here we show on a panel of lymphoma cells lines that the number of receptors per cell and the ratio CD47/CD19 do not impact TG-1801-mediated ADCC or phagocytosis (ADCP). In addition, we show that TG-1801 potentiated ublituximab-mediated ADCC and ADCP and exhibited a similar additive effect when added to U2 combination. In vivo, ublituximab alone displayed a tumor growth inhibition (TGI) of 88%, with 3/8 mice harboring a barely palpable tumor, while the umbralisib alone treatment arm showed a TGI of 50%, with 2/8 mice lacking detectable tumors. TG-1801 exhibited a 76% TGI with 1/8 tumor free-mouse. Most importantly, the combination of TG-1801 with umbralisib alone, ublituximab alone, and U2 achieved TGI of 85%, 93% and 93% respectively, with more tumor-free mice 35 days after the last dose in these three groups. Interestingly, this superior anti-tumor effect of the different TG-1801 combinations was associated with a higher infiltration of mouse macrophages within the tumors as assessed by F4/80 IHC labeling. RNA-seq analysis of the Raji xenografts and of n=4 representative in vitro B-NHL co-cultures treated with TG-1801, U2 or the triple combination uncovered the upregulation of the G-protein coupled receptor EBI2/GRP183 as a common event associated with the synergistic antitumor effects of TG-1801 and U2 in vitro and in vivo. A critical role of EBI2 in the regulation of macrophage activity, B cell migration and in the promotion of a pro-inflammatory phenotype was demonstrated upon exposure of the co-cultures with the EBI2 small molecule inhibitor NIBR189, which impaired the U2/TG-1801-evoked ADCP, B-cell cytoskeleton remodeling and inflammatory cytokine production.Conclusion: The data presented here set the preclinical rationale and support a combination strategy of the novel CD47-CD19 bispecific antibody TG-1801 in B-NHL with other B-cell targeted mechanisms, including umbralisib and ublituximab. DisclosuresNormant: TG Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Miskin: TG Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Roue: TG Therapeutics, Inc.: 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.

Siplizumab, an Anti-CD2 Monoclonal Antibody, Induces a Unique Set of Immune Modulatory Effects Compared to Alemtuzumab and Rabbit Anti-Thymocyte Globulin In Vitro.

Antibodies are commonly used in organ transplant induction therapy and to treat autoimmune disorders. The effects of some biologics on the human immune system remain incompletely characterized and a deeper understanding of their mechanisms of action may provide useful insights for their clinical application. The goal of this study was to contrast the mechanistic properties of siplizumab with Alemtuzumab and rabbit Anti-Thymocyte Globulin (rATG). Mechanistic assay systems investigating antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell phagocytosis and complement-dependent cytotoxicity were used to characterize siplizumab. Further, functional effects of siplizumab, Alemtuzumab, and rATG were investigated in allogeneic mixed lymphocyte reaction. Changes in T cell activation, T cell proliferation and frequency of naïve T cells, memory T cells and regulatory T cells induced by siplizumab, Alemtuzumab and rATG in allogeneic mixed lymphocyte reaction were assessed via flow cytometry. Siplizumab depleted T cells, decreased T cell activation, inhibited T cell proliferation and enriched naïve and bona fide regulatory T cells. Neither Alemtuzumab nor rATG induced the same combination of functional effects. The results presented in this study should be used for further in vitro and in vivo investigations that guide the clinical use of immune modulatory biologics.

Antibody and antibody fragments for cancer immunotherapy

Antibody has become the most rapidly expanding class of pharmaceuticals for treating a wide variety of human diseases including cancers. Especially, with the fast development of cancer immunotherapy, antibody drugs have become the most promising therapeutic for curing cancers. Immune-mediated cell killing by antibodies including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) as well as regulation of T cell function through immune checkpoint blockade. Due to the absence of Fc fragment, antibody fragments including single-chain variable fragments (scFvs) and single-domain antibodies (sdAds) are mainly applied in chimeric antigen receptors (CAR) T cell therapy for redirecting T cells to tumors and T cell activation by immune checkpoint blockade. In this review, the cancer immunity is first discussed. Then the principal mechanisms of antibody-based immunotherapy will be reviewed. Next, the antibody and antibody fragments applied for cancer immunotherapy will be summarized. Bispecific and multispecific antibodies and a combination of cancer immunotherapy with other tumor treatments will also be mentioned. Finally, an outlook and perspective of antibody-based cancer immunotherapy will be given. This review would provide a comprehensive guidance for the researchers who are interested in and intended to involve in the antibodies- or antibody fragments-based tumor immunity.

Trispecific antibody against HIV-1 demonstrate anti-viral activity in vivo and have potent Fc-dependent effector functions

Abstract Broadly neutralizing antibodies (bNAbs) against HIV-1 have been suggested as a complementary immunotherapy to current combination small molecule anti-retroviral therapies (cART) for treatment of HIV-1 infection. Monoclonal bNAbs when used as a monotherapy demonstrate high susceptibility for escape in HIV-1 infected individuals, thus a trispecific bNAb targeting three independent HIV-1 envelope determinants was developed to achieve more robust suppression. Upon treatment of viremic simian-human immunodeficiency virus (SHIV)-infected macaques with the trispecific bNAb, plasma viral suppression of up to 1000-fold was observed. To elucidate a mechanism for the observed plasma viremia control, in vitro assays using the trispecific bNAb were conducted to determine Fc-mediated effector functions. First, we determined that the trispecific bNAb bound to HIV-1 infected cell lines with an equal or higher affinity than its parental bNAbs alone. We then conducted antibody-dependent cell cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP) assays and found that the trispecific bNAb had comparable or better activity compared to its parental bNAbs alone. Similarly, when we measured direct virolysis of HIV-1 virus using an antibody-dependent complement-mediated virus lysis (ADCML) assay, the trispecific bNAb had comparable or better activity compared to its parental bNAbs alone. These studies demonstrate that the trispecific bNAb shows anti-viral activity in vivo and has potent Fc-dependent effector functions that target both free virus and virally infected cells compared to monoclonal bNAbs alone. Thus, the trispecific bNAb is a promising single immunotherapeutic protein for treatment of HIV-1 infection.

Engineering of Fc Multimers as a Protein Therapy for Autoimmune Disease.

The success of Intravenous Immunoglobulin in treating autoimmune and inflammatory processes such as immune thrombocytopenia purpura and Kawasaki disease has led to renewed interest in developing recombinant molecules capable of recapitulating these therapeutic effects. The anti-inflammatory properties of IVIG are, in part, due to the Fc region of the IgG molecule, which interacts with activating or inhibitory Fcγ receptors (FcγRs), the neonatal Fc Receptor, non-canonical FcRs expressed by immune cells and complement proteins. In most cases, Fc interactions with these cognate receptors are dependent upon avidity—avidity which naturally occurs when polyclonal antibodies recognize unique antigens on a given target. The functional consequences of these avid interactions include antibody dependent cell-mediated cytotoxicity, antibody dependent cell phagocytosis, degranulation, direct killing, and/or complement activation—all of which are associated with long-term immunomodulatory effects. Many of these immunologic effects can be recapitulated using recombinant or non-recombinant approaches to induce Fc multimerization, affording the potential to develop a new class of therapeutics. In this review, we discuss the history of tolerance induction by immune complexes that has led to the therapeutic development of artificial Fc bearing immune aggregates and recombinant Fc multimers. The contribution of structure, aggregation and N-glycosylation to human IgG: FcγR interactions and the functional effect(s) of these interactions are reviewed. Understanding the mechanisms by which Fc multimers induce tolerance and attempts to engineer Fc multimers to target specific FcγRs and/or specific effector functions in autoimmune disorders is explored in detail.

Daratumumab displays in vitro and in vivo anti-tumor activity in models of B-cell non-Hodgkin lymphoma and improves responses to standard chemo-immunotherapy regimens.

CD38 is expressed in several types of non-Hodgkin lymphoma (NHL) and constitutes a promising target for antibody-based therapy. Daratumumab (Darzalex) is a first-in-class anti-CD38 antibody approved for the treatment of relapsed/refractory (R/R) multiple myeloma (MM). It has also demonstrated clinical activity in Waldenström macroglobulinaemia and amyloidosis. Here, we have evaluated the activity and mechanism of action of daratumumab in preclinical in vitro and in vivo models of mantle cell lymphoma (MCL), follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL), as monotherapy or in combination with standard chemo-immunotherapy. In vitro, daratumumab engages Fc-mediated cytotoxicity by antibody-dependent cell cytotoxicity and antibody-dependent cell phagocytosis in all lymphoma subtypes. In the presence of human serum, complement-dependent cell cytotoxicity was marginally engaged. We demonstrated by Selective Plane Illumination Microscopy that daratumumab fully penetrated a three-dimensional (3D) lymphoma organoid and decreased organoid volume. In vivo, daratumumab completely prevents tumor outgrowth in models of MCL and FL, and shows comparable activity to rituximab in a disseminated in vivo model of blastic MCL. Moreover, daratumumab improves overall survival (OS) in a mouse model of transformed CD20dim FL, where rituximab showed limited activity. Daratumumab potentiates the antitumor activity of CHOP and R-CHOP in MCL and FL xenografts. Furthermore, in a patient-derived DLBCL xenograft model, daratumumab anti-tumor activity was comparable to R-CHOP and the addition of daratumumab to either CHOP or R-CHOP led to full tumor regression. In summary, daratumumab constitutes a novel therapeutic opportunity in certain scenarios and these results warrant further clinical development.

Abstract 3955: Tumor macrophage-mediated antibody dependent cell phagocytosis (ADCP) is theprimary mechanism mediating HER2 mAb (Trastuzumab) anti-tumor responses which can be synergistically enhanced by CD47 innate immune checkpoint blockade

Abstract Approximately 20% of breast cancers (BC) are defined by HER2 overexpression and treated using HER2-specific monoclonal antibodies (mAb), such as Trastuzumab. Previous studies have demonstrated Trastuzumab can limit signaling, elicit antibody dependent cell cytotoxicity (ADCC), antibody dependent phagocytosis (ADCP), and adaptive immune responses to HER2, however its primary therapeutic mechanism of action (MOA) remains unclear. As HER2 mAb efficacy is subverted in advanced cancers, understanding and boosting their MOA is of critical clinical interest and scientific significance for HER2+ BC as well as for other targeted mAbs. Using a series of fully murine Trastuzumab mAbs of different isotypes, we found that the IgG2A (high A/I) isotype was essential for tumor growth suppression, suggesting an innate immune MOA. In contrast to previous reports, our studies revealed that NK cells, neutrophils, T-cell, or B-cell populations were unnecessary for this anti-tumor MOA. However, we found that a murine IgG2A Trastuzumab (mTras) altered the immune microenvironment in vivo through neutrophil suppression (~4 fold, p&amp;lt;.001) but stimulated a significant expansion (~7 fold, p&amp;lt;.001) and activation of resident tumor associated-macrophages (TAMs). Using DiD dye-labelled tumors, we confirmed that mTras elicited striking HER2-specific ADCP in ~50% of resident TAMs (versus ~16% in control), while in vitro studies confirmed that mTras-mediated macrophage anti-tumor efficacy was dependent upon ADCP, but not ADCC, through activation of FCGR4. Critically, we also found BC CD47 expression suppressed ADCP and resulted in suboptimal mTras anti-tumor efficacy. To explore this innate resistance pathway, we utilized CD47 KO BC lines and CD47 mAbs and found that both significantly enhanced mTras-mediated ADCP in vitro (&amp;gt;4 fold, p&amp;lt;.01) without altering ADCC activity. Moreover, we also found that this combination significantly enhanced TAM expansion and activation in vivo, which significantly enhanced human HER2+ BC anti-tumor efficacy (88% complete regression vs. 0% for single agent) and prolonged survival in multiple murine HER2+ BC models, including an endogenous treatment-resistant HER2+ BC mouse model. Thus, our study demonstrates the primary MOA of a clinically relevant HER2 mAb requires engagement with TAMs to stimulate their expansion and elicit ADCP, which could be significantly enhanced by CD47-SIRPa blockade. This suggests that the strategic use of CD47-SIRPa innate blockade may allow for significant enhancement of anti-tumor immunity in combination with other cancer antigen targeting mAb therapies (targeting EGFR, CD20, etc) as a more effective strategy to stimulate anti-tumor immune responses in advanced immunosuppressive cancers. Citation Format: Li-Chung Tsao, Jun-Ping Wei, Gang-jun Lei, Tao Wang, Xiao-Yi Yang, Cong-Xiao Liu, H. Kim Lyerly, Zachary C. Hartman. Tumor macrophage-mediated antibody dependent cell phagocytosis (ADCP) is theprimary mechanism mediating HER2 mAb (Trastuzumab) anti-tumor responses which can be synergistically enhanced by CD47 innate immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3955.