Search

CD47 expressed on cells functions as a "don't eat me" signal by binding SIRPa on macrophages and blocking phagocytosis. Cancer cells including AML evade phagocytosis by overexpressing CD47, and high CD47 expression in AML has been associated with inferior outcomes. Here we studied the pre-clinical activity of the 5F9 (magrolimab), an antibody that blocks the CD47, combined with BCL-2 inhibitor venetoclax with azacitidine (VEN/AZA) in AML both in vitro and in vivo. We first tested the combined efficacy of VEN/AZA and 5F9 in vitro in phagocytosis assay, utilizing monocytes isolated from seven healthy donors by CD16 positive selection and inducing macrophage differentiation with recombinant human M-CSF (rhM-CSF, 500ng/ml) for 1 week. Three paired isogenic AML cell lines MOLM-13, VEN-resistant (VEN-RE) MOLM-13, and p53-mutant MOLM-13 harboring R248W mutation, were pre-treated with DMSO or combination of 10nM VEN and 1uM AZA for 24hrs. Leukemia cells were labeled with Calcein and co-cultured with macrophages for 1h at a ratio of 2:1 (T:M), followed by flow cytometry read-out, quantifying Calcein+ macrophages that engulfed leukemia cells. The active phagocytosis was calculated as the percentage of Calcein+CD206+ cells from total CD206+ cells (Figure 1A, experimental schema). The 5F9/VEN-AZA combination significantly increased phagocytosis not only in parental but also in VEN-RE and p53-mutant MOLM-13 cells when compared with 5F9 or VEN/AZA alone treatments (Figure 1B). The treatment of VEN or AZA induced AnnexinV in MOLM-13 cells which may function as a pro-phagocytic signal and contribute to the enhanced phagocytosis (Figure 1C). Further proteomics studies to identify additional "eat me" signals following VEN/AZA treatment are ongoing and will be presented. Next, we examined the therapeutic efficacy of 5F9 alone or combined with VEN/AZA in the AML patient-derived xenografts (PDX) models. NSG mice were engrafted with VEN-RE AML PDX #344 (no canonical AML mutations detected by targeted sequencing) and #574 (FLT3-ITD, DNMT3A, NPM1-mutated). After confirmed bone marrow (BM) engraftment by flow cytometry, mice were randomized to receive vehicle, 5F9 (10mg/kg/d, ip. twice a week, 3 weeks), VEN (50mg/kg/g, po. qd, 14 days) / AZA (2.5mg/kg/d, ip, qd, 7 days), or their combination. Circulating leukemia burden was monitored by hCD45 flow cytometry in peripheral blood (PB) (Figure 1D), and mice survival was followed. 5F9 reduced leukemia burden, delayed leukemia progression and significantly extended mice' survival in both models, with the median survival of 276.5 days of 5F9 group compared to 197 days of vehicle in #344, and 81.5 vs 52 days in #574 (Figure 1E ). On the contrary, VEN/AZA therapy had no or minimal impact on tumor burden or survival. Notably, the combination of 5F9 and VEN/AZA resulted in elimination of circulating blasts and significant extension of survival in both models, median of 385 days in #344 and 124.5 days in #574. In summary, the VEN/AZA combined with 5F9 increased phagocytosis in isogenic MOLM-13 cells in vitro regardless of VEN resistance or p53 status and prolonged the survival in vivo in VEN/AZA refractory AML PDX models. The mechanisms of increased phagocytosis by combination treatment and the induction of "eat-me signals" by VEN/AZA is currently under investigation and will be presented. This triplet combination is currently being tested in a clinical trial (NCT04435691, Daver et al., ASH 2021) with high preliminary efficacy. Figure 1 Figure 1. Disclosures Chao: Gilead Sciences, Inc.: Current Employment; TigaTx: Membership on an entity's Board of Directors or advisory committees; Stanford University: Patents & Royalties; Hepatx Inc: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Iconovir Bio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Leukemia and Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; Stanford University Medical School: Membership on an entity's Board of Directors or advisory committees; Foresite capital: Consultancy; Bioverge: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Chimera Bioengineering: Current equity holder in publicly-traded company. Maute: Gilead Sciences: Ended employment in the past 24 months. Andreeff: Syndax: Consultancy; Karyopharm: Research Funding; ONO Pharmaceuticals: Research Funding; Senti-Bio: Consultancy; AstraZeneca: Research Funding; Daiichi-Sankyo: Consultancy, Research Funding; Breast Cancer Research Foundation: Research Funding; Medicxi: Consultancy; Aptose: Consultancy; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Oxford Biomedica UK: Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Glycomimetics: Consultancy; Amgen: Research Funding. Daver: Hanmi: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Novimmune: Research Funding; Trillium: Consultancy, Research Funding; Glycomimetics: Research Funding; ImmunoGen: Consultancy, Research Funding; Sevier: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; FATE Therapeutics: Research Funding; Trovagene: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding. Konopleva: Rafael Pharmaceuticals: Other: grant support, Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Ascentage: Other: grant support, Research Funding; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; KisoJi: Research Funding; Stemline Therapeutics: Research Funding; Ablynx: Other: grant support, Research Funding; AstraZeneca: Other: grant support, Research Funding; Cellectis: Other: grant support; Sanofi: Other: grant support, Research Funding; Calithera: Other: grant support, Research Funding; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; Agios: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding.

Intro: Relapsed/refractory diffuse large B cell lymphoma (r/r DLBCL) is associated with poor outcomes, and remains an area of unmet clinical need. Emerging data indicate that a key macrophage immune checkpoint termed CD47 can be effectively targeted in r/r DLBCL. CD47 is a potent "don't eat me" signal and impairs tumor cell phagocytosis by engaging the inhibitory macrophage receptor, SIRPα. Importantly, early clinical studies demonstrate that blocking CD47-SIRPα interactions with an anti-CD47 antibody (magrolimab) yields promising activity in r/r DLBCL patients, particularly when combined with rituximab, which further potentiates lymphoma cell phagocytosis via its Fc domain. Moving forward, it will be crucial to identify combinatorial strategies that improve the efficacy of anti-CD47 therapy in r/r DLBCL. Tumor-associated macrophages (TAMs) contribute to an immunosuppressive environment in cancer through secretion of cytokines, metabolites, and expression of immune checkpoints. Sustained PI3K-γ signaling is critical to maintain immune suppressive TAM functions, and PI3K-γ inhibition polarizes TAMs into a pro-inflammatory state that culminates in potent anti-tumor T cell responses, sensitizing cancers to PD-1 blockade therapy. We hypothesized that activating TAMs through PI3K-δ/γ inhibition would also augment their inherent phagocytic capacity and synergize with phagocytosis-targeting treatments such as CD47 blockade. Thus, we investigated the impact of the dual PI3K-δ/γ inhibitor, duvelisib, on expression of key phagocytic receptors and pathways in human macrophages, as well as its effect on enhancing lymphoma cell phagocytosis with the anti-CD47 antibody, magrolimab. Results: RNA sequencing of duvelisib-treated human monocyte-derived macrophages revealed that PI3K-δ/γ inhibition induced broad transcriptional changes suggestive of an activated macrophage state (Fig 1A), including upregulation of interferon response genes (IRF9) and costimulatory molecules (CD86), while decreasing expression of immune suppressive cytokines (IL10) and inhibitory ligands (PDL1). Moreover, duvelisib upregulated transcription of canonical pro-phagocytic receptors (LRP1, FCGR2A,CD44), lysosomal degradation components (LYZ, CTSS) and phosphatidylserine-bridging genes (MFG-E8, GAS6), while inhibiting metabolic pathways, most notably mTOR, each of which has been associated with increased and more efficient phagocytosis. Given these findings, we evaluated the effect of dual PI3K-δ/γ inhibition on macrophage phagocytosis of DLBCL cells in the context of CD47 blockade in vitro. Across a broad panel of human DLBCL cell lines and multiple monocyte donors, pre-treatment of macrophages with duvelisib significantly increased lymphoma cell phagocytosis by magrolimab (Fig 1B-C). This effect reflected a direct increase in the phagocytic capacity of treated macrophages, as lymphoma cells were not exposed to duvelisib. In separate experiments, we found that duvelisib rarely induced full apoptosis of DLBCL cells, but did promote the expression of "eat me" signals, such as calreticulin and phosphatidylserine on many of the cell lines examined (Fig 1D). Collectively, these results suggest that PI3K-δ/γ inhibition enhances the inherent phagocytic capacity of macrophages, while also sensitizing lymphoma cells for engulfment. Similarly, potent effects were not observed with PI3K-δ- or PI3K-γ-specific inhibitors, suggesting that combined PI3K-δ/γ inhibition is critical to mediating these beneficial effects. Finally, to determine whether duvelisib impacted the efficacy of magrolimab in vivo, we treated human DLBCL xenografts and observed that while duvelisib had minimal impact on lymphoma growth, the combination of duvelisib and magrolimab induced complete tumor rejection in a high proportion of mice, leading to increased survival compared to mice treated with magrolimab alone (Fig 1E). Conclusion: Collectively, our findings suggest that targeting the PI3K-δ/γ axis favorably induces key macrophage phagocytic pathways, while simultaneously upregulating important "eat me" signals on lymphoma cells. Together, these alterations lower the phagocytic threshold of TAMs and dramatically increase the efficacy of anti-CD47 therapy in DLBCL xenograft models. This work paves the way for early-phase studies of duvelisib and magrolimab in r/r DLBCL and other lymphomas. Disclosures Godfrey: Verastem: Research Funding; Merck: Research Funding; Gilead: Research Funding. Coma:Verastem Oncology, Inc: Current Employment, Current equity holder in publicly-traded company. Maute:Gilead Sciences, Inc.: Current Employment. Chao:Gilead Sciences: Current Employment. Pachter:Verastem Therapeutics: Current Employment. Kline:Kite/Gilead: Speakers Bureau; Merck: Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees.

PURPOSE To evaluate the safety, pharmacokinetics, and pharmacodynamics of Hu5F9-G4 (5F9), a humanized IgG4 antibody that targets CD47 to enable phagocytosis. PATIENTS AND METHODS Adult patients with solid tumors were treated in four cohorts: part A, to determine a priming dose; part B, to determine a weekly maintenance dose; part C, to study a loading dose in week 2; and a tumor biopsy cohort. RESULTS Sixty-two patients were treated: 11 in part A, 14 in B, 22 in C, and 15 in the biopsy cohort. Part A used doses that ranged from 0.1 to 3 mg/kg. On the basis of tolerability and receptor occupancy studies that showed 100% CD47 saturation on RBCs, 1 mg/kg was selected as the priming dose. In subsequent groups, patients were treated with maintenance doses that ranged from 3 to 45 mg/kg, and most toxicities were mild to moderate. These included transient anemia (57% of patients), hemagglutination on peripheral blood smear (36%), fatigue (64%), headaches (50%), fever (45%), chills (45%), hyperbilirubinemia (34%), lymphopenia (34%), infusion-related reactions (34%), and arthralgias (18%). No maximum tolerated dose was reached with maintenance doses up to 45 mg/kg. At doses of 10 mg/kg or more, the CD47 antigen sink was saturated by 5F9, and a 5F9 half-life of approximately 13 days was observed. Strong antibody staining of tumor tissue was observed in a patient at 30 mg/kg. Two patients with ovarian/fallopian tube cancers had partial remissions for 5.2 and 9.2 months. CONCLUSION 5F9 is well tolerated using a priming dose at 1 mg/kg on day 1 followed by maintenance doses of up to 45 mg/kg weekly.

7507 Background: Magrolimab (Hu5F9-G4) is an antibody blocking CD47, a macrophage immune checkpoint and don’t eat me signal on cancers. It induces tumor phagocytosis and eliminates leukemia stem cells. Azacitidine (AZA) synergizes with magrolimab by inducing eat me signals on leukemic cells, enhancing phagocytosis. We report Ph1b data including a potential MDS registration cohort. Methods: Magrolimab+AZA was given to untreated intermediate to very high risk IPSS-R MDS and intensive chemo unfit AML patients. A magrolimab priming/intrapatient dose escalation regimen (1-30 mg/kg QW, Q2W Cycle 3+) was used. AZA was dosed 75mg/m2 days 1-7. Efficacy was assessed by IWG 2006 (MDS) and ELN 2017 (AML) criteria. Results: 68 patients (39 MDS, 29 AML) with a median age of 72 were treated with magrolimab+AZA. 19% were intermediate cytogenetic risk with 68% poor risk (13% unknown). 27% were TP53 mutant. The combo was well-tolerated with safety similar to AZA alone. Common treatment-related AEs were anemia (38%), fatigue (21%), neutropenia (19%), thrombocytopenia (18%) and infusion reaction (16%). Treatment-related febrile neutropenia was 1.5%. Only 1 patient (1.5%) discontinued due to an AE. In RBC transfusion dependent patients, 58% of MDS and 64% of AML patients became transfusion independent. 30/33 (91%) efficacy evaluable MDS patients had an objective response (42% CR, 24% marrow CR (4/8 also with HI), 3% PR, 21% HI alone, 9% SD). MDS patient responses deepened on study, with a 56% CR rate in patients with ≥ 6 mo follow-up. In AML, 16/25 (64%) responded (40% CR, 16% CRi, 4% PR, 4% MFLS, 32% SD, 4% PD). In 12 TP53 mutant AML patients, 75% had a CR+CRi (42% CR, 33% CRi, 17% SD, 8% PD). Cytogenetic CR was seen in 35% and 50% of responding MDS and AML patients. 22% of MDS and 50% of AML patients with CR/CRi/marrow CR were MRD negative by flow cytometry. Median duration of response is not reached in either MDS or AML, including TP53 mutant AML, with a median follow-up of 5.8, 8.8 and 9.4 mos, respectively (range: 1.9 – 16.8 mos). 91% of MDS and 100% of AML responding patients are in response at 6 mos. The 6 mo overall survival estimate is 100% in MDS and 91% in TP53 mutant AML patients. Conclusions: Magrolimab is a macrophage targeting immunotherapy that with AZA is well tolerated with durable efficacy in MDS, AML, particularly TP53 mutant, a poor prognostic group. A potential registration single arm MDS cohort is ongoing (NCT03248479). ENHANCE, a randomized Ph3 MDS trial is planned. Additional patients/analyses will be reported. Funded by Forty Seven and CIRM. Clinical trial information: NCT03248479 .

e15098 Background: Magrolimab (Hu5F9-G4) is a monoclonal IgG4 antibody that binds to CD47 receptor and blocks its interaction with SIRPA, thereby enabling tumor cell phagocytosis and activation of an anti-tumor T-cell response. Magrolimab has shown encouraging efficacy and tolerable safety profiles in treatment-naïve myelodysplastic syndromes/acute myeloid leukemia (MDS/AML) patients on an every week (QW) maintenance dosing schedule. Reduced dosing frequency (e.g. every other week – Q2W) is desired to enhance patient convenience and quality of life. Methods: Interim pharmacokinetic (PK) and CD47 receptor occupancy (RO) data from study 5F9005 (Clinical Trial: NCT03248479) were used for this analysis. A previously developed population PK (PopPK) model of magrolimab was verified with observed PK data from study 5F9005. The PK model was linked to peripheral blood and bone marrow blast CD47 RO. The model-predicted RO was validated with observed RO data. Simulations of PK and RO were conducted to justify the proposed reduced dosing frequency of Q2W starting in Cycle 3 onward. Simulations were compared with preliminary observed RO data from the ongoing 5F9005 study after switching to Q2W dosing. Results: The results showed that observed magrolimab PK in study 5F9005 was consistent with model predictions. Model predicted RO was aligned with observed RO data. PK and RO simulations based on the model indicated > 99% RO in peripheral blood and ~90% RO in bone marrow are expected after switching to Q2W dosing regimen from Cycle 3 onward. Preliminary observed RO data confirmed these predictions. Conclusions: Magrolimab PK in AML/MDS is consistent with other patient populations including solid tumors and lymphomas. Model-based simulations and preliminary RO data support Q2W dosing of magrolimab from Cycle 3 onward as optimal RO is expected with the proposed dosing regimen. Clinical trial information: NCT03248479 .

BACKGROUND: The Hu5F9-G4 (hereafter, 5F9) antibody is a macrophage immune checkpoint inhibitor blocking CD47 that induces tumor-cell phagocytosis. 5F9 synergizes with rituximab to eliminate B-cell non-Hodgkin’s lymphoma cells by enhancing macrophage-mediated antibody-dependent cellular phagocytosis. This combination was evaluated clinically. METHODS: We conducted a phase 1b study involving patients with relapsed or refractory non-Hodgkin’s lymphoma. Patients may have had diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma. 5F9 (at a priming dose of 1 mg per kilogram of body weight, administered intravenously, with weekly maintenance doses of 10 to 30 mg per kilogram) was given with rituximab to determine safety and efficacy and to suggest a phase 2 dose. RESULTS: A total of 22 patients (15 with DLBCL and 7 with follicular lymphoma) were enrolled. Patients had received a median of 4 (range, 2 to 10) previous therapies, and 95% of the patients had disease that was refractory to rituximab. Adverse events were predominantly of grade 1 or 2. The most common adverse events were anemia and infusion-related reactions. Anemia (an expected on-target effect) was mitigated by the strategy of 5F9 prime and maintenance dosing. Dose-limiting side effects were rare. A selected phase 2 dose of 30 mg of 5F9 per kilogram led to an approximate 100% CD47-receptor occupancy on circulating white and red cells. A total of 50% of the patients had an objective (i.e., complete or partial) response, with 36% having a complete response. The rates of objective response and complete response were 40% and 33%, respectively, among patients with DLBCL and 71% and 43%, respectively, among those with follicular lymphoma. At a median follow-up of 6.2 months among patients with DLBCL and 8.1 months among those with follicular lymphoma, 91% of the responses were ongoing. CONCLUSIONS: The macrophage checkpoint inhibitor 5F9 combined with rituximab showed promising activity in patients with aggressive and indolent lymphoma. No clinically significant safety events were observed in this initial study. (Funded by Forty Seven and the Leukemia and Lymphoma Society; ClinicalTrials.gov number, NCT02953509.)

114 Background: Magrolimab (M, Hu5F9-G4) is an antibody targeting CD47, a macrophage “don’t eat me” signal that demonstrates preclinical synergy with cetuximab (C) in refractory KRAS wild type (KRASwt) and KRAS mutant (KRASm) colorectal (CRC) tumors. Methods: Phase (Ph) 1 doses of M+C were escalated in solid tumor patients (pts) and Ph 2 efficacy was explored in previously treated KRASwt and KRASm CRC patients. Day 1 priming with 1 mg/kg of M was used to mitigate on-target anemia followed by maintenance doses ranging from 10 to 45 mg/kg in combination with C. Ph 2 pts were treated with 30 or 45 mg/kg of M and 400/250 mg/m2 of C. Results: In 78 treated pts (32 Ph 1 and 46 Ph 2), the median age was 59 years (range 19-82), and median prior therapies was 5 (range 1-14). No maximum tolerated dose was reached. Treatment-related adverse events (TRAEs) of any Grade (G) included dermatitis acneiform 36%, dry skin 33%, fatigue 32%, infusion reactions 31%, headache 30%, diarrhea 23%, nausea 23%, chills 23%, and anemia 22%. There were no fatal TRAEs and 3/78 (4%) discontinued M treatment due to any adverse events. In the combined Ph 1+2 study, 2 of 30 evaluable KRASwt CRC pts had confirmed PRs for 7.0 and 12.5 months (mo), for a 6.7% objective response rate (ORR). Both had prior C treatment. The median progression-free survival (mPFS) and median overall survival (mOS) was 3.6 mo (95%CI 1.8-5.4) and 10.1 mo (95%CI: 6.9-14.4), respectively. In 40 evaluable KRASm pts, there were no responses but 45% had stable disease (SD) and the mPFS and mOS were 1.9 mo (95%CI: 1.8-3.5) and 10.4 mo (95%CI: 5.7-16.4), respectively. In 28 KRASm pts who were TAS102/regorafenib naïve, preliminary mOS was 12.4 mo (95%CI: 5.9-not reached) which is longer than that reported for historical controls. Tumor biopsies showed treatment-related increases in macrophage immune cell infiltrates in SD pts, and baseline T cell infiltration was associated with longer OS. Pharmacokinetic profiles will be presented. Conclusions: M+C is a novel, well-tolerated combination immunotherapeutic treatment regimen. Responses were observed in two previously treated CRC pts and survival is encouraging in KRASm pts. Funded by Forty Seven and California Institute for Regenerative Medicine. Clinical trial information: NCT02953782.

Background Hematopoietic stem cell (HSC) transplantation (HSCT) is a well-established procedure that, with or without gene therapy, is curative for numerous severe life-threatening diseases including genetic blood disorders and blood cancers. While advances have been made, there are still substantial concerns since these chemo- and radiation therapy based procedures cause long-term toxicities such as infertility and secondary malignancies or even result in high mortality. We have previously established in a series of preclinical studies a novel chemo- and radiation-free non-toxic monoclonal antibody (Ab) -based conditioning regimen for autologous and allogeneic HSCT (Czechowicz et al., Akanksha et al. and George et al.). This cKIT-CD47 Ab-based regimen selectively depletes host HSCs for HSCT while sparing off-target toxicities caused by chemotherapy/radiation. By significantly decreasing morbidity/mortality associated with traditional conditioning regimens, antibody-mediated conditioning could expand the patient population eligible to receive HSCT for a variety of disorders. We developed a novel cKIT Ab (FSI-174), with an active Fc, and in combination with our CD47 magrolimab (previously 5F9, blocks the don't eat me pathway) could be utilized to translate the promising preclinical findings into clinical studies for safe and less toxic bone marrow conditioning for HSCT. Here we present the functional characterization of FSI-174 as single Ab and in combination with magrolimab in vitro and in non-human primate (NHP) studies. Methods We tested if FSI-174 could block stem cell factor signaling and we explored if FSI-174 alone or in combination with magrolimab could promote phagocytosis of cKIT positive cells (Kasumi-1). In addition, we determined if FSI-174 could cause mast cell degranulation. Subsequently, we explored the potential of FSI-174 alone (Phase A) or in combination with magrolimab (Phase B) to deplete HSCs in NHPs (rhesus macaques)in vivo. In Phase A, single doses of FSI-174 (0.3, 1, or 3 mg/kg) were administered alone. In Phase B, FSI-174 (0.3 or 3 mg/kg) was administered in combination with magrolimab (5mg/kg priming and 20 mg/kg maintenance dose). Bone marrow aspirates and core biopsies and peripheral blood were sampled before the study start and throughout the study. Frequency of bone marrow HSCs and cKIT receptor occupancy (RO) was determined by flow cytometry. In addition, the PK profile of FSI-174 was determined. Results In-vitro analysis demonstrated that FSI-174 decreases proliferation of HSPCs and enhances phagocytosis of cKIT positive cells, and the addition of magrolimab synergistically enhances the phagocytosis. Strikingly, FSI-174 did not cause mast cell degranulation in vitro. In the NHPs, complete (100%) cKIT receptor occupancy was achieved at all FSI-174 dose levels and was maintained for 1 to 9 days correlating with increasing doses and pharmacokinetics. The FSI-174 Cmax was found to be proportional to dose and mean Cmax increased from 6.25 ug/mL to 49.2 ug/mL. In Phase A, FSI-174 alone did not decrease the frequency of bone marrow HSCs compared to PBS control and had no effect on the peripheral blood cell counts. However, in Phase B, when FSI-174 was combined with magrolimab it significantly decreased the frequency of bone marrow HSCs with the nadir at day 9 and no recovery over 85 days compared to PBS control. Notably, there were no changes in peripheral blood cell counts over the course of the studies with no cytopenias in combination treatment. Conclusions We have developed a novel cKIT Ab (FSI-174) that meets the desired profile of stem cell factor block, promotion of phagocytosis, but without promoting mast cell degranulation. Furthermore, in the NHPs studies we have confirmed our chemo- and radiation-free cKIT-CD47 Ab -based conditioning approach with FSI-174 and magrolimab. As anticipated by our previous preclinical studies, monotherapy with FSI-174 does not deplete bone marrow HSCs in NHPs. Notably, no cytopenias are observed with either monotherapy or combination therapy. These data demonstrate the specificity, efficacy and safety of FSI-174/ magrolimab combination have great potential for conditioning regimen for HSCT in a chemotherapy and radiation free manner. Given the favorable safety profile of magrolimab across several clinical studies, these results are paving the way to the first-in-human trials for this novel conditioning for HSCT. Disclosures Marjon: Forty Seven Inc: Employment, Equity Ownership. Chen:Forty Seven Inc.: Consultancy, Equity Ownership. Duan:Forty Seven Inc.: Employment, Equity Ownership. Choi:Forty Seven inc: Employment, Equity Ownership. Sompalli:Forty Seven Inc: Employment, Equity Ownership. Feng:Forty Seven Inc: Employment, Equity Ownership. Mata:Forty Seven inc: Employment, Equity Ownership. Chen:Forty Seven Inc: Employment, Equity Ownership. Kean:HiFiBio: Consultancy; BlueBirdBio: Research Funding; Gilead: Research Funding; Regeneron: Research Funding; EMDSerono: Consultancy; FortySeven: Consultancy; Magenta: Research Funding; Bristol Meyers Squibb: Patents & Royalties, Research Funding; Kymab: Consultancy; Jazz: Research Funding. Chao:Forty Seven Inc: Employment, Equity Ownership. Chao:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties. Takimoto:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties. Agoram:Forty Seven Inc.: Employment, Equity Ownership. Majeti:FortySeven: Consultancy, Equity Ownership, Other: Board of Director; BioMarin: Consultancy. Weissman:Forty Seven Inc.: Consultancy, Equity Ownership, Patents & Royalties. Liu:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Volkmer:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties.

Introduction Magrolimab (Hu5F9-G4, 5F9) is a first-in-class IgG4 antibody targeting CD47, a macrophage immune checkpoint and "don't eat me" signal expressed on cancer cells. Blockade of CD47 leads to phagocytosis of tumor cells. Magrolimab synergizes with rituximab to eliminate CD20-positive lymphoma by enhancing antibody-dependent cellular phagocytosis. Magrolimab +rituximab demonstrated encouraging safety and efficacy in a Phase (Ph)1b dose escalation cohort in patients with relapsed/refractory (r/r) DLBCL and FL that were rituximab-refractory (Advani et al., NEJM 2018). CD24 is an additional "don't eat me" signal, and has been proposed as a potential target for immunotherapy (Barkal et al. Nature 2019). Here we describe immunohistochemical analysis of CD47 and CD24 in primary patient biopsies from an ongoing follow-up Phase 2 trial of Non-Hodgkin's lymphoma (including DLBCL and indolent lymphoma) patients treated with magrolimab+rituximab. Results By immunohistochemistry, 54 out of 54 patients assessed were positive for expression of CD47 at screening. High levels of CD47 expression were maintained during treatment with clinically-efficacious doses of magrolimab. Therapeutic response did not correlate with CD47 H-score expression levels. At screening, patients presented with highly variable levels of CD24 expression, with 40 of 54 samples showing positive staining in >30% of cells. We observed no significant correlation between CD24 expression by H-score and response to therapy (complete response + partial response) in either DLBCL or indolent lymphoma. Conclusions CD47 shows consistently high expression in primary biopsies from Non-Hodgkin's lymphoma patients and remains persistently high during treatment. In our Phase 2 trial, therapeutic response did not correlate with CD47 expression levels, suggesting that the degree of target expression is not a primary driver of magrolimab efficacy in Non-Hodgkin's lymphoma. Although we observe wide variation in CD24 expression within this cohort, its levels are not predictive of outcome for this disease indication. We are evaluating the expression of additional biomarkers to identify those Non-Hodgkin's lymphoma patients most likely to benefit from magrolimab+rituximab combination therapy. Disclosures Maute: Forty Seven Inc.: Employment, Equity Ownership, Patents & Royalties. Chen:Forty Seven Inc.: Consultancy, Equity Ownership. Marjon:Forty Seven Inc.: Employment, Equity Ownership. Duan:Forty Seven Inc.: Employment, Equity Ownership. Choi:Forty Seven Inc.: Employment, Equity Ownership. Chao:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties. Takimoto:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties. Agoram:Forty Seven Inc.: Employment, Equity Ownership. Volkmer:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties.

7009 Background: Hu5F9-G4 (5F9) is an antibody targeting CD47, a macrophage immune checkpoint and “don’t eat me” signal on cancers. CD47 blockade induces tumor phagocytosis and eliminates leukemia stem cells (LSC) in AML models. Azacitidine (AZA) synergizes with 5F9 by inducing “eat me” signals on AML, enhancing phagocytosis. This trial explored the safety/efficacy of 5F9 alone or with AZA in AML/MDS patients (pts). Methods: This Phase 1b treated: r/r AML/MDS pts with 5F9; and untreated AML (induction chemo ineligible) and higher risk MDS pts with 5F9+AZA. A 5F9 priming/intrapatient dose escalation regimen (1-30 mg/kg weekly) was used to mitigate on target anemia. Results: 10 (6 AML, 4 MDS) r/r pts received 5F9 (median 2 prior therapies (range 1-6). 24 untreated pts (15 AML, 9 MDS) received 5F9+AZA. In total, median age was 73, 62% of AML pts were intermediate or poor cytogenetic risk (38% unknown), all MDS pts were intermediate or high risk by IPSS-R. 5F9 alone or with AZA was well-tolerated with no MTD reached. 5F9 did not potentiate AZA toxicities. Treatment-related AEs ( > 10% of pts) for 5F9+AZA were anemia (25%), thrombocytopenia (20%), and infusion reactions (15%). In 25 efficacy evaluable pts, 8/15 (53%) untreated AML/MDS pts had a CR/CRi to 5F9+AZA (5/10 (50%) in AML, 3/5 (60%) in MDS). 1/10 (10%) r/r AML/MDS pts had a response (MLFS) to 5F9 alone. LSC frequency was reduced/eliminated in most 5F9+aza responders; 50% of responders were MRD negative by flow cytometry. 4/10 (40%) AML pts became RBC transfusion independent and 4/5 (80%) MDS pts had hematologic improvement. Time to response was more rapid (median 1.9 mos) than expected for AZA alone. As of Jan 2019, no responder has relapsed (median follow-up of 3.4 mos (range 1.1 – 6.8 mos). 2 pts had successful allogeneic transplant. Conclusions: 5F9+AZA is a novel immunotherapy blocking a key macrophage checkpoint. It has been well tolerated with robust activity in AML/MDS pts with rapid CRs and MRD negativity. Adding 5F9 to cytotoxic agents may be a promising treatment strategy. An expansion cohort is ongoing. Funded by Forty Seven and the California Institute for Regenerative Medicine. Clinical trial information: NCT03248479.

CD47 exerts different effects on disease in distinct cell types and locations and during different stages of experimental autoimmune encephalomyelitis., Comparison of transcriptomic and proteomic data from pathologically similar multiple sclerosis (MS) lesions reveals down-regulation of CD47 at the messenger RNA level and low abundance at the protein level. Immunohistochemical studies demonstrate that CD47 is expressed in normal myelin and in foamy macrophages and reactive astrocytes within active MS lesions. We demonstrate that CD47−/− mice are refractory to experimental autoimmune encephalomyelitis (EAE), primarily as the result of failure of immune cell activation after immunization with myelin antigen. In contrast, blocking with a monoclonal antibody against CD47 in mice at the peak of paralysis worsens EAE severity and enhances immune activation in the peripheral immune system. In vitro assays demonstrate that blocking CD47 also promotes phagocytosis of myelin and that this effect is dependent on signal regulatory protein α (SIRP-α). Immune regulation and phagocytosis are mechanisms for CD47 signaling in autoimmune neuroinflammation. Depending on the cell type, location, and disease stage, CD47 has Janus-like roles, with opposing effects on EAE pathogenesis.

CD47, a “don't eat me” signal for phagocytic cells, is expressed on the surface of all human solid tumor cells. Analysis of patient tumor and matched adjacent normal (nontumor) tissue revealed that CD47 is overexpressed on cancer cells. CD47 mRNA expression levels correlated with a decreased probability of survival for multiple types of cancer. CD47 is a ligand for SIRPα, a protein expressed on macrophages and dendritic cells. In vitro, blockade of CD47 signaling using targeted monoclonal antibodies enabled macrophage phagocytosis of tumor cells that were otherwise protected. Administration of anti-CD47 antibodies inhibited tumor growth in orthotopic immunodeficient mouse xenotransplantation models established with patient tumor cells and increased the survival of the mice over time. Anti-CD47 antibody therapy initiated on larger tumors inhibited tumor growth and prevented or treated metastasis, but initiation of the therapy on smaller tumors was potentially curative. The safety and efficacy of targeting CD47 was further tested and validated in immune competent hosts using an orthotopic mouse breast cancer model. These results suggest all human solid tumor cells require CD47 expression to suppress phagocytic innate immune surveillance and elimination. These data, taken together with similar findings with other human neoplasms, show that CD47 is a commonly expressed molecule on all cancers, its function to block phagocytosis is known, and blockade of its function leads to tumor cell phagocytosis and elimination. CD47 is therefore a validated target for cancer therapies.

Non-Hodgkin lymphoma (NHL) presents as both localized and disseminated disease with spread to secondary sites carrying a worse prognosis. Although pathways driving NHL dissemination have been identified, there are few therapies capable of inhibiting them. Here, we report a novel role for the immunomodulatory protein CD47 in NHL dissemination, and we demonstrate that therapeutic targeting of CD47 can prevent such spread. We developed 2 in vivo lymphoma metastasis models using Raji cells, a human NHL cell line, and primary cells from a lymphoma patient. CD47 expression was required for Raji cell dissemination to the liver in mouse xenotransplants. Targeting of CD47 with a blocking antibody inhibited Raji cell dissemination to major organs, including the central nervous system, and inhibited hematogenous dissemination of primary lymphoma cells. We hypothesized that anti-CD47 antibody-mediated elimination of circulating tumor cells occurred through phagocytosis, a previously described mechanism for blocking anti-CD47 antibodies. As predicted, inhibition of dissemination by anti-CD47 antibodies was dependent on blockade of phagocyte SIRPα and required macrophage effector cells. These results demonstrate that CD47 is required for NHL dissemination, which can be therapeutically targeted with a blocking anti-CD47 antibody. Ultimately, these findings are potentially applicable to the dissemination and metastasis of other solid tumors.

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and constitutes 15% of adult leukemias. Although overall prognosis for pediatric ALL is favorable, high-risk pediatric patients and most adult patients have significantly worse outcomes. Multiagent chemotherapy is standard of care for both pediatric and adult ALL, but is associated with systemic toxicity and long-term side effects and is relatively ineffective against certain ALL subtypes. Recent efforts have focused on the development of targeted therapies for ALL including monoclonal antibodies. Here, we report the identification of CD47, a protein that inhibits phagocytosis, as an antibody target in standard and high-risk ALL. CD47 was found to be more highly expressed on a subset of human ALL patient samples compared with normal cell counterparts and to be an independent predictor of survival and disease refractoriness in several ALL patient cohorts. In addition, a blocking monoclonal antibody against CD47 enabled phagocytosis of ALL cells by macrophages in vitro and inhibited tumor engraftment in vivo. Significantly, anti-CD47 antibody eliminated ALL in the peripheral blood, bone marrow, spleen, and liver of mice engrafted with primary human ALL. These data provide preclinical support for the development of an anti-CD47 antibody therapy for treatment of human ALL. Cancer Res; 71(4); 1374–84. ©2010 AACR.

CD47 is an anti-phagocytic (i.e. "don't eat me") signal and macrophage checkpoint that cancer cells utilize to evade innate immunity and establish disease. 5F9 is a humanized IgG4 monoclonal antibody (mAb) that binds to human CD47 and blocks its interaction with SIRPα, its cognate inhibitory receptor.5F9 is undergoing investigation in several clinical trials and preliminary analyses have revealed encouraging therapeutic potential. We previously established that administration of 5F9 clears a subset of aged RBCs, resulting in a compensatory reticulocytosis. This occurs transiently, predominantly after the first dose, and does not worsen with subsequent doses. These findings, aligned with the observations of Oldenborg et al., which established that removal of aged RBCs, is dependent on the balance between the accumulating pro-phagocytic signals and loss of inhibitory anti-phagocytic signals. We therefore hypothesized that an initial lower 5F9 "priming dose", sufficient to trigger clearance of aged RBCs, would yield an overall younger pool of RBCs that are less vulnerable to subsequent higher 5F9 therapeutic "maintenance doses". Indeed, this priming and maintenance dosing strategy has substantially mitigated the on-target anemia and has allowed for 5F9 treatment in multiple clinical trials and indications. To further probe this observed selective RBC clearance, we employed a receptor occupancy assay to quantitate the CD47 expression on the cell surface of RBCs, leukocytes (WBCs), and acute myeloid leukemia (AML) blasts. We discovered that the priming dose of 5F9 not only triggered clearance of a subset of RBCs, but also resulted in a near complete loss of CD47 - a phenomenon we term CD47 pruning. This effect was RBC-specific, as WBCs and AML bone marrow (BM) blasts did not exhibit this same CD47 pruning (Fig 1). To explore the functional consequence of these unexpected clinical findings, we investigated whether we could recapitulate them in a preclinical mouse model. Similar to the clinical setting, anti-mouse CD47 blocking Ab treatments exhibited a similar pruning effect and transient anemia. Using this model, we asked whether, in the context of anti-CD47 treatment, these pruned RBCs were protected compared to unpruned RBCs. We transfused fluorescently labeled RBCs from CD47 Ab-untreated and CD47-treated mice into animals receiving continuous anti-CD47 treatment. We observed that CD47-pruned RBCs exhibited a significantly longer half-life compared to unpruned RBCs, suggesting that CD47 pruning is protective for RBCs. To distinguish whether this protection is cell-intrinsic or cell-extrinsic, we transfused fluorescently labeled RBCs into CD47 Ab-untreated and -treated mice and observed that CD47-pruned RBCs are rapidly cleared in untreated recipients, suggesting that this treatment-induced protection of RBCs is cell extrinsic and a host-based alteration. Through depletion of distinct immune subsets by chemical depletion, antibody depletion, splenectomy, and partial hepatectomies, we demonstrated that host immune effector cells (e.g. red-pulp macrophages, neutrophils, T cells etc.) were not necessary for this pruning phenomenon. Critically, we also found that this CD47 pruning and resulting tolerance was Fc-independent. In order to understand whether these findings were generalizable to other anti-human CD47 blocking agents, we conducted similar studies in double humanized huCD47 and huSIRPa micewith additional agents and observed this same CD47 pruning and tolerance phenomenon. Thereby suggesting this represents a class-effect of this emerging potential therapy class. Notably, these clinical and pre-clinical findings have been RBC-specific and have not precluded anti-tumor efficacy. The loss of CD47 on RBCs after the priming dose also suggests that the potential risk of CD47 Ab-mediated RBC agglutination following subsequent maintenance dosing is substantially reduced. Unlike prior RBC antigen modulation reports, to our knowledge, these findings represent a novel RBC antigen depletion phenomenon that is independent of the known RBC regulatory compartments, including the spleen, liver, major immune effectors cells, complement, and is critically Fc-independent. These findings provide fundamental insight into the mechanism underlying how anti-CD47 Abs are tolerated without impairing therapeutic efficacy. Disclosures Chen: Forty Seven Inc: Consultancy, Equity Ownership. McKenna:Forty Seven Inc.: Equity Ownership. Choi:Forty Seven Inc: Employment, Equity Ownership. Duan:Forty Seven Inc: Employment, Equity Ownership. Sompalli:Forty Seven Inc: Employment, Equity Ownership. Schrier:Forty Seven Inc.: Consultancy. Weissman:Forty Seven, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Elrod:Forty Seven Inc.: Employment, Equity Ownership. Chao:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Takimoto:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Liu:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Volkmer:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties.

Tumor immunosurveillance is a well-established mechanism for regulation of tumor growth. In this regard, most studies have focused on the role of T- and NK-cells as the critical immune effector cells. However, macrophages play a major role in the recognition and clearance of foreign, aged, and damaged cells. Macrophage phagocytosis is negatively regulated via the receptor SIRPalpha upon binding to CD47, a ubiquitously expressed protein. We recently showed that CD47 is up-regulated in myeloid leukemia and migrating hematopoietic progenitors, and that the level of protein expression correlates with the ability to evade phagocytosis. These results implicate macrophages in the immunosurveillance of hematopoietic cells and leukemias. The ability of macrophages to phagocytose tumor cells might be exploited therapeutically by blocking the CD47-SIRPalpha interaction.

7504Background: Targeted non-cytotoxic therapies are needed in relapsed/refractory (r/r) NHL. Hu5F9-G4 (5F9) is a first-in-class humanized antibody targeting CD47, a protective “don’t eat me” signa...

2525Background: Hu5F9-G4 is a humanized monoclonal antibody targeting CD47, a “don’t eat me” signal on cancer cells, that stimulates tumor cell phagocytosis and activates an anti-tumor T-cell respo...

CD47 is a widely expressed cell surface protein that functions as a regulator of 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, SIRP-alpha, which in turn delivers an inhibitory signal for phagocytosis. We previously found increased expression of CD47 on primary human acute myeloid leukemia (AML) stem cells, and demonstrated that blocking monoclonal antibodies directed against CD47 enabled the phagocytosis and elimination of AML, non-Hodgkin's lymphoma (NHL), and many solid tumors in xenograft models. Here, we report the development of a humanized anti-CD47 antibody with potent efficacy and favorable toxicokinetic properties as a candidate therapeutic. A novel monoclonal anti-human CD47 antibody, 5F9, was generated, and antibody humanization was carried out by grafting its complementarity determining regions (CDRs) onto a human IgG4 format. The resulting humanized 5F9 antibody (Hu5F9-G4) bound monomeric human CD47 with an 8 nM affinity. Hu5F9-G4 induced potent macrophage-mediated phagocytosis of primary human AML cells in vitro and completely eradicated human AML in vivo, leading to long-term disease-free survival of patient-derived xenografts. Moreover, Hu5F9-G4 synergized with rituximab to eliminate NHL engraftment and cure xenografted mice. Finally, toxicokinetic studies in non-human primates showed that Hu5F9-G4 could be safely administered intravenously at doses able to achieve potentially therapeutic serum levels. Thus, Hu5F9-G4 is actively being developed for and has been entered into clinical trials in patients with AML and solid tumors (ClinicalTrials.gov identifier: NCT02216409).

Multiple lines of investigation have demonstrated that the immune system plays an important role in preventing tumor initiation and controlling tumor growth. Accordingly, many cancers have evolved diverse mechanisms to evade such monitoring. While multiple immune cell types mediate tumor surveillance, recent evidence demonstrates that macrophages, and other phagocytic cells, play a key role in regulating tumor growth through phagocytic clearance. In this review we highlight the role of tumor immune evasion through the inhibition of phagocytosis, specifically through the CD47-signal-regulatory protein-α pathway, and discuss how targeting this pathway might lead to more effective cancer immunotherapies.

Under normal physiologic conditions, cellular homeostasis is partly regulated by balancing pro- and anti-phagocytic signals. CD47 is highly expressed on several human cancers including acute myeloid leukemia, non-Hodgkin lymphoma, and bladder cancer, allowing cancer cells to evade phagocytosis by the innate immune system. Blockade of CD47 with a monoclonal antibody enables phagocytosis of cancer cells and leads to in vivo tumor elimination, but leaves most normal cells unaffected. In order for target cells to be phagocytosed upon blockade of an anti-phagocytic signal, we postulate that the cells must also display a potent pro-phagocytic signal. Here we identify calreticulin as a pro-phagocytic signal highly expressed on the surface of several human cancers including acute myeloid and lymphoblastic leukemias, chronic myeloid leukemia, non-Hodgkin lymphoma (NHL), bladder cancer, glioblastoma, and ovarian cancer, but minimally expressed on most normal cells. Increased CD47 expression correlated with high calreticulin levels on cancer cells, and was necessary for protection from calreticulin-mediated phagocytosis. Phagocytosis induced by anti-CD47 antibody required the interaction of target cell calreticulin with its receptor low density lipoprotein-receptor related protein (LRP) on phagocytic cells, as blockade of the calreticulin/LRP interaction prevented anti-CD47 antibody mediated phagocytosis. Lastly, increased calreticulin expression was an adverse prognostic factor in diverse tumors including neuroblastoma, bladder cancer, and NHL. These findings identify calreticulin as the dominant pro-phagocytic signal on several human cancers, provide an explanation for the selective targeting of tumor cells by anti-CD47 antibody, and highlight the balance between pro- and anti-phagocytic signals in the immune evasion of cancer.

Major clinical issues in bladder cancer include the identification of prediction markers and novel therapeutic targets for invasive bladder cancer. In the current study, we describe the isolation and characterization of a tumor-initiating cell (T-IC) subpopulation in primary human bladder cancer, based on the expression of markers similar to that of normal bladder basal cells (Lineage-CD44 + CK5 + CK20 − ). The bladder T-IC subpopulation was defined functionally by its enriched ability to induce xenograft tumors in vivo that recapitulated the heterogeneity of the original tumor. Further, molecular analysis of more than 300 bladder cancer specimens revealed heterogeneity among activated oncogenic pathways in T-IC (e.g., 80% Gli1, 45% Stat3, 10% Bmi-1, and 5% β-catenin). Despite this molecular heterogeneity, we identified a unique bladder T-IC gene signature by gene chip analysis. This T-IC gene signature, which effectively distinguishes muscle-invasive bladder cancer with worse clinical prognosis from non-muscle-invasive (superficial) cancer, has significant clinical value. It also can predict the progression of a subset of recurring non-muscle-invasive cancers. Finally, we found that CD47, a protein that provides an inhibitory signal for macrophage phagocytosis, is highly expressed in bladder T-ICs compared with the rest of the tumor. Blockade of CD47 by a mAb resulted in macrophage engulfment of bladder cancer cells in vitro. In summary, we have identified a T-IC subpopulation with potential prognostic and therapeutic value for invasive bladder cancer.

SummaryMacrophages clear pathogens and damaged or aged cells from the blood stream via phagocytosis. Cell-surface CD47 interacts with its receptor on macrophages, SIRPα, to inhibit phagocytosis of normal, healthy cells. We find that mobilizing cytokines and inflammatory stimuli cause CD47 to be transiently upregulated on mouse hematopoietic stem cells (HSCs) and progenitors just prior to and during their migratory phase, and that the level of CD47 on these cells determines the probability that they are engulfed in vivo. CD47 is also constitutively upregulated on mouse and human myeloid leukemias, and overexpression of CD47 on a myeloid leukemia line increases its pathogenicity by allowing it to evade phagocytosis. We conclude that CD47 upregulation is an important mechanism that provides protection to normal HSCs during inflammation-mediated mobilization, and that leukemic progenitors co-opt this ability in order to evade macrophage killing.

Zhao et al[1][1] raise several issues from our prior work[2][2],[3][3] characterizing the mechanism of targeting CD47 in human hematopoietic malignancies. We reported that a blocking anti-CD47 antibody can eliminate multiple human tumor types and that a major mechanism is through phagocytosis via

Monoclonal antibodies are standard therapeutics for several cancers including the anti-CD20 antibody rituximab for B cell non-Hodgkin lymphoma (NHL). Rituximab and other antibodies are not curative, and must be combined with cytotoxic chemotherapy for clinical benefit. Here we identify CD47 as an attractive monoclonal antibody target in NHL. A major function of CD47 is to inhibit phagocytosis, through binding its receptor, SIRP-alpha, on phagocytes. We hypothesize that NHL cells over-express CD47 to evade immune phagocytosis and that blockade of CD47 signaling by a monoclonal antibody can eliminate NHL cells by enabling phagocytic engulfment. Here we investigate the therapeutic potential of an anti-CD47 antibody alone and in combination with rituximab for the treatment of NHL. We predict that the combination of anti-CD47 antibody and rituximab through utilization of two independent antibody mechanisms will result in synergistic phagocytosis and elimination of NHL cells. We found that CD47 protein is highly expressed on primary human NHL cells compared to normal B cell counterparts. Higher CD47 gene expression predicted an inferior outcome in several cohorts of NHL patients, providing independent prognostic value. Blocking anti-CD47 antibodies preferentially enabled phagocytosis of NHL cells but not normal cell counterparts in vitro while the combination of anti-CD47 antibody and rituximab resulted in synergistic phagocytosis of NHL cells. In vivo, anti-CD47 antibody treatment of human NHL-engrafted mice reduced lymphoma burden and prolonged survival compared to IgG control treatment. Furthermore, anti-CD47 antibody treatment in combination with rituximab led to elimination of lymphoma and cure of NHL-engrafted mice compared to either antibody treatment as a single agent. The above in vivo results were observed in both a disseminated and localized human NHL cell line mouse model as well as in primary human lymphoma xenotransplants. These antibodies synergized through a novel mechanism combining Fc receptor (FcR)-dependent and FcR-independent stimulation of phagocytosis that might be applicable to many other cancers. Together, these data provide the rationale for utilizing an anti-CD47 antibody either alone or in combination with rituximab in treating human NHL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2440.

Abstract 2716 Poster Board II-692 The anti-CD20 antibody, rituximab, is standard therapy for many CD20 positive B-cell lymphomas, significantly improving long-term survival in combination with chemotherapy. However, rituximab alone is not curative in many non-Hodgkin lymphoma (NHL) patients with observation of primary and acquired resistance, arguing for a need for additional targeted therapies. We identified the cell surface protein CD47 as a potential therapeutic antibody target in human NHL. A major function of CD47 is to inhibit phagocytosis through binding its receptor SIRPα, on phagocytes. We hypothesize that NHL cells over-express CD47 to evade immune phagocytosis and that blockade of CD47 signaling with a monoclonal antibody can eliminate NHL cells by enabling phagocytic engulfment. We have previously shown that an anti-CD47 antibody enables phagocytosis of acute myeloid leukemia (AML) stem cells and eliminates AML in vivo. Here we investigate the therapeutic potential of an anti-CD47 antibody alone and in combination with rituximab for the treatment of NHL. We predict that the combination of anti-CD47 antibody and rituximab will result in synergistic elimination of NHL cells by both blocking an inhibitory signal and delivering a positive signal for phagocytosis. We found that CD47 protein is highly expressed on primary human B-cell NHL cells compared to normal peripheral blood B-cells. Higher CD47 gene expression independently predicted a worse clinical outcome in several cohorts of NHL patients including diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, and chronic lymphocytic leukemia. Blocking anti-CD47 antibodies preferentially enabled phagocytosis of NHL cells but not normal cell counterparts in vitro. Using isobologram analyses, the combination of anti-CD47 antibody and rituximab resulted in synergistic phagocytosis of NHL cells at higher levels compared to either anti-CD47 antibody or rituximab alone. In addition, anti-CD47 antibody and rituximab mediated their therapeutic effects through Fc-receptor-independent and Fc-receptor-dependent effector mechanisms, respectively. In vivo, anti-CD47 antibody treatment of NHL-engrafted mice reduced lymphoma burden and prolonged survival compared to IgG control. Furthermore, combination treatment with anti-CD47 antibody and rituximab led to elimination of lymphoma and cure of NHL-engrafted mice. These in vivo results were observed in both a disseminated and localized human NHL cell line mouse model as well as in primary human DLBCL mouse xenotransplants. Together, these data provide the rationale for utilizing an anti-CD47 antibody either alone or in combination with rituximab in treating human NHL. Disclosures: Weissman: U.S. Patent Application 11/528,890 entitled “Methods for Diagnosing and Evaluating Treatment of Blood Disorders.”: Patents & Royalties; Stem Cells Inc.: Cofounder and director; Cellerant Inc.: cofounder; Amgen: Equity Ownership.

18 Background: Magrolimab (M, Hu5F9-G4) is an antibody targeting CD47, a “don’t eat me” signal for macrophages that enhances ovarian cancer cell phagocytosis in preclinical models in combination with the PD-L1 inhibitor avelumab. CD47 blockade can also enhance cross-priming of T cells. Methods: In Part 1 (P1) M+A doses were escalated in ST patients (pts) while Part 2 (P2) enrolled platinum-resistant or refractory OC pts. All received a 1 mg/kg Day 1 priming dose of M to mitigate on-target anemia due to macrophage-mediated extravascular hemolysis followed by 30 or 45 mg/kg maintenance doses in P1 or 45 mg/kg in P2, in combination with 800 mg of A Q2 wks. Results: In the 34 total pts (13 in P1 and 21 in P2), median age was 66 years (range 47-88), and median # of prior therapies was 5 (range 1-10). In P1, no dose limiting toxicities occurred. In P1+P2, no Grade (G) 4 or 5 treatment-related adverse events (TRAEs) occurred. TRAEs of any G in > 20% pts included headache 62%, fatigue 47%, infusion related reaction 44%, pyrexia 38%, chills 35%, nausea 35%, anemia 24%, and vomiting 21%. In P1, a patient (pt) with metastatic papillary adenocarcinoma of the finger on 45 mg/kg of M had a confirmed partial response (PR) lasting for 4 months before progressing. In P2, 1 pt had an unconfirmed PR but progressed per RECIST v1.1 on the next scan achieving a best response of stable disease (SD). In the 18 OC P1+P2 pts with at least one response evaluation, 56% had SD and 44% had progression. Analysis of tumor biopsies for immune cells infiltration and CD47, PD-L1, and other marker expression is ongoing. In the 13 OC tumors analyzed to date, only 2 were PD-L1 positive. One PD-L1+ tumor had PD-L1 expression only on infiltrating immune cells. The other PD-L1+ had tumor cell expression and was the only OC pt with tumor shrinkage. This supports the potential importance of PD-L1 expression for this combination. Pharmacokinetic profiles will be presented. Conclusions: M+A is a novel, well-tolerated combination treatment regimen with 1 observed PR in a ST pt and a 56% SD rate in OC pts. Tumor shrinkage was noted in the only OC pt with tumor cell expression of PD-L1 which warrants further evaluation in PD-L1+ OC pts. Clinical trial information: NCT03558139.