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TO THE EDITOR: Successful engraftment of hematopoietic stem cells (HSCs) involves overcoming nonimmunologic barriers that hinder access to the HSC niches in the bone marrow (BM).[1][1],[2][2] Cytotoxic chemotherapy and/or radiation are currently used to reduce these barriers in clinical

Successful hematopoietic cell transplantation (HCT) relies on conditioning of recipients to deplete hematopoietic stem cells (HSC) from marrow niches to allow healthy HSC to engraft. Currently, only genotoxic chemotherapy and/or radiation are used to achieve such niche clearance. We have developed a targeted non-genotoxic approach to deplete HSC using a monoclonal antibody, JSP191, that binds human CD117 (c-Kit), a receptor tyrosine kinase expressed on HSC and progenitor cells (HSPC). We have opened a phase 1 dose escalation trial using JSP191 as the sole conditioning agent in patients with SCID (ClinicalTrials.gov: NCT02963064). HCT is the only definitive cure for SCID, a uniformly lethal genetic immune disorder. Because infants with SCID lack functional T cells which can mediate transplant rejection and because of toxicity concerns, SCID infants may receive HCT without conditioning. This approach is associated with presumed engraftment of donor lymphoid progenitors but not HSC, resulting in incomplete and poorly sustained immune reconstitution, evidenced by naïve T cell production that wane over time and absent functional B cells requiring life-long immunoglobulin (Ig) replacement. Second donor HSC transplants ("boosts") without conditioning have not led to HSC engraftment and thus immune defects persist. We previously reported the initial safety, tolerability, and proof-of-concept of JSP191 as a safe and effective conditioning agent for SCID patients in whom an initial HCT failed to engraft HSC. Here, we present data supporting a recommended phase 2 dose (RP2D) of JSP191 that achieves donor HSC engraftment predictive of clinically meaningful lymphocyte reconstitution. Study endpoints include 1) donor chimerism of stringently flow-sorted CD15 + blood granulocytes ≥3% (18-24 wks post-HCT) as a measure of HSC engraftment, 2) lymphocyte reconstitution of donor-derived naïve CD4 +CD45RA + T cells >85/μL (36-104 wks post HCT), and 3) improved clinical outcomes, including reduced Ig dependence. Twelve patients with prior HCT (3 to 38 years old) of different genotypes (Table 1) were dosed in the initial dose escalation (0.1, 0.3 and 1.0 mg/kg JSP191) and at the RP2D 0.6 mg/kg. At study entry all patients lacked donor HSC engraftment evidenced by 0% donor CD15 + granulocyte chimerism. JSP191 was infused as a single IV dose and serum pharmacokinetics (PK) measured in real-time. CD34 +-selected cells obtained from the original donors were infused when the estimated JSP191 serum level fell below 500 ng/mL. JSP191 clearance displayed saturable non-linear kinetics, which was prolonged at the highest dose cohort of 1.0 mg/kg dose. JSP191 and HSPC infusions were uniformly well tolerated. Pre-and post-infusion marrow analyses in 7 evaluable patients showed a dose-dependent decline in CD117 +HSPC following JSP191 treatment (range 30-89%). Table 1a shows that 6 of 9 non-IL2RG SCID patients who are >24 wks post-HCT reached the predefined endpoint of HSC engraftment with >3% donor CD15 + blood granulocytes. This level of HSC chimerism correlated with the subsequent development of naïve donor derived T lymphocytes. Importantly, beyond HSC engraftment and naïve donor T lymphocyte production, patients who achieved HSC donor chimerism have demonstrated clinical improvement including resolution of chronic diarrhea, healthy weight gain, reduced Ig dependence, and antibody responses to vaccines. Three non-IL2RG patients did not achieve the chimerism threshold (one at 0.3 mg/kg and two at 1.0 mg/kg), potentially due to insufficient dose or prolonged JSP191 clearance and time to HSC infusion, respectively. Hence, an intermediate dose of 0.6 mg/kg which displays a shorter, more favorable half-life and higher HSC engraftment compared to the other dose cohorts was selected as the RP2D. Two of 3 IL2RG patients did not reach the donor chimerism threshold (Table 1b) reflecting the fact that re-transplanted IL2RG patients can have higher immune-mediated resistance, that may necessitate additional immune ablation. Conclusion: This study is the first to show the long-term benefit of HSC engraftment following targeted single-agent JSP191 conditioning. These results suggest JSP191 conditioning has the potential to provide benefit for HCT in a wide spectrum of disorders without the deleterious risks of genotoxic agents. This study is actively enrolling previously transplanted and newly diagnosed SCID patients. Figure 1 Figure 1. Disclosures Dvorak: Jazz Pharma: Consultancy; Alexion, Inc: Consultancy; Omeros Corp.: Consultancy. Prockop: Memorial Sloan Kettering Cancer Center: Other: S Prockop receives support for the conduct of sponsored clinical trials through MSK from Atara Biotherapeutics, Jasper and AlloVir. , Patents & Royalties: S Prockop is a co-inventor on intellectual property (IP) licensed to Atara. S Prockop has waived rights to this IP to MSK and has no personal financial interests in Atara. MSK has financial interests in Atara and IP interests relevant to this abstract. ; Atara Biotherapeutics: Other: support for the conduct of sponsored trials and Inventor; Jasper: Other: support for the conduct of sponsored trials; AlloVir: Other: support for the conduct of sponsored trials; Neovii: Consultancy; ADMA Biologics: Consultancy; MSK: Other: Inventor. Kwon: Jasper Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Long-Boyle: Jasper Therapeutics: Consultancy. Arulprakasam: Jasper Therapeutics: Current Employment. Harada: Jasper Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Prohaska: Jasper Therapeutics: Consultancy, Current holder of stock options in a privately-held company. Pang: Jasper Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Heller: NextCure: Consultancy, Current equity holder in publicly-traded company; Jasper Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Weissman: FortySeven: Patents & Royalties. Cowan: Homology Medicines, Inc: Consultancy, Current equity holder in publicly-traded company; Bluebird Bio, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Leadiant, Inc: Consultancy; Ensoma Inc.: Consultancy; Rocket Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees. Logan: Amgen, Pfizer, AbbVie: Consultancy; Pharmacyclics, Astellas, Jazz, Kite, Kadmon, Autolus, Amphivena: Research Funding. O'Reilly: Atara Biotherapeutics: Consultancy, Patents & Royalties: for EBV-specific T-Cell Bank, Research Funding. Parkman: Jasper Biotech: Consultancy. Shizuru: Jasper Therapeutics: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; FortySeven (Gilead): Patents & Royalties: Inventor listed on a patent, licensed by FortySeven.

Osteoarthritis (OA) is the leading cause of joint failure, yet the underlying mechanisms remain elusive, and no approved therapies that slow progression exist. Dysregulated integrin function was previously implicated in OA pathogenesis. However, the roles of integrin α(V)β(3) and the integrin-associated receptor CD47 in OA remain largely unknown. Here, transcriptomic and proteomic analyses of human and murine osteoarthritic tissues revealed dysregulated expression of α(V)β(3), CD47, and their ligands. Using genetically deficient mice and pharmacologic inhibitors, we showed that α(V)β(3), CD47, and the downstream signaling molecules Fyn and FAK are crucial to OA pathogenesis. MicroPET/CT imaging of a mouse model showed elevated ligand-binding capacities of integrin α(V)β(3) and CD47 in osteoarthritic joints. Further, our in vitro studies demonstrated that chondrocyte breakdown products, derived from articular cartilage of individuals with OA, induced α(V)β(3)/CD47-dependent expression of inflammatory and degradative mediators, and revealed the downstream signaling network. Our findings identify a central role for dysregulated α(V)β(3) and CD47 signaling in OA pathogenesis and suggest that activation of α(V)β(3) and CD47 signaling in many articular cell types contributes to inflammation and joint destruction in OA. Thus, the data presented here provide a rationale for targeting α(V)β(3), CD47, and their signaling pathways as a disease-modifying therapy.

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.

Successful hematopoietic cell transplantation (HCT) requires vacating recipient hematopoietic stem cell (HSC) niches to permit transplanted HSC to engraft. Currently, DNA damaging radiation or chemotherapy are used to eliminate recipient HSC and achieve niche clearance. We have pursued a non-genotoxic approach to target and deplete HSC using a humanized monoclonal antibody, AMG 191, that binds human CD117 (c-Kit), a receptor tyrosine kinase expressed on the surface of HSC and progenitor cells (HSPC). We have shown that AMG 191 suppresses human hematopoiesis in vitro, depletes human HSC in mice xenografted with human cells, and safely depletes HSC of non-human primates. We have initiated a Phase I dose escalation trial to test AMG 191 as the sole conditioning agent to achieve donor CD34-enriched HSPC engraftment in patients undergoing HCT for severe combined immunodeficiency (SCID) (ClinicalTrials.gov: NCT02963064). SCID is a severe genetic immune disorder curable only by HCT. Because of toxicity concerns, infants with SCID often receive donor hematopoietic grafts without conditioning, resulting in a lack of donor HSC engraftment. Instead, mature T lymphocytes and possibly lymphoid progenitors engraft but support only donor T cell development. This approach is associated with incomplete and poorly sustained immune reconstitution, and many patients have either no donor B cells and/or poor B cell function requiring life-long immunoglobulin replacement therapy. Second unconditioned donor HSC "boosts" can be performed, but they do not result in HSC engraftment and immune defects may persist. The primary endpoint of our study is to assess the safety and tolerability of AMG 191 as a conditioning agent in SCID patients. Secondary endpoints include AMG 191 pharmacokinetics (PK), host HSC depletion, and the determination of the dose of AMG 191 that achieves adequate donor HSC engraftment, defined as >5% donor blood granulocyte chimerism in peripheral blood at 24 weeks. Seven patients have been treated to date who are >12 weeks post-HCT (Table 1): three in each of the first two dose cohorts (0.1 and 0.3 mg/kg AMG 191), and one patient in the third cohort (1.0 mg/kg). An eighth patient has been treated at the 1.0 mg/kg dose and is three weeks post-HCT. Patients have a mixture of SCID genotypes. All patients treated to date had prior HCT with lack of donor HSC engraftment as evidenced by 0% donor sorted granulocyte chimerism at study entry. AMG 191 administration and infusion of original donor CD34+-selected cells were uniformly well tolerated. Pre- and post-infusion marrow analyses in five evaluable patients demonstrated dose-dependent decline in CD117+HSPC following AMG 191 treatment. Table 1 shows that four of six patients, who are >24 weeks post-HCT, reached the predefined endpoint of >5% granulocyte chimerism at 24 weeks, demonstrating donor HSC engraftment. The two patients who did not show donor engraftment at 24 weeks had detectable, low level (36 weeks show the production of recent thymic emigrants and/or de novo production of naïve T and/or B cells. In addition to improved lymphocyte values, patients have demonstrated clinical improvement including resolution of chronic diarrhea, significant weight gain, and reduced IVIG requirements. Conclusion: This study is the first demonstration of HSC engraftment following monoclonal antibody-based conditioning of patients without chemo(radio)therapy. Specifically, this first-in-human HCT trial shows that an anti-CD117 antibody safely clears HSC niches and facilitates donor HSPC engraftment in patients with SCID. Clinical benefit has been observed with minimal to no toxicity. Four of six evaluable patients have sustained evidence of donor myeloid engraftment along with T and B lymphopoiesis, indicative of engraftment of multipotent HSC. These results suggest that antibody conditioning for HCT may be preferable to traditional chemo(radio)therapy conditioning, especially in patients with non-malignant diseases and/or increased risk of toxicities due to such agents, such as certain forms of SCID, Fanconi anemia and sickle cell disease. Anti-CD117 antibody conditioning may also be applicable to gene therapy with genetically corrected autologous HSC. The AMG 191 study is actively enrolling previously transplanted SCID patients and newly diagnosed SCID patients. Disclosures Dvorak: Alexion Inc: Consultancy; Jazz Pharmaceuticals: Consultancy. Prohaska:Forty Seven Inc: Equity Ownership, Patents & Royalties. Weissman:Forty Seven Inc.: Consultancy, Equity Ownership, Patents & Royalties. Cowan:Rocket Pharma: Consultancy; Homology Medicine: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; California Institute Of Regenerative Medicine: Research Funding; UpToDate: Honoraria; Leadiant: Consultancy; NIH NIAD: Research Funding. Logan:Kadmon: Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kite: Research Funding; TeneoBio: Consultancy; Novartis: Consultancy; Astellas: Research Funding; Abbvie: Consultancy; Incyte: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Research Funding; Kiadis: Consultancy; Jazz: Research Funding; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees. Weinberg:U.S. Patent Office: Patents & Royalties: patent pending - submitted for aldehyde dehydrogenase 2 (ALDH2) activators to expand hematopoietic stem cells. Shizuru:Forty Seven Inc: Equity Ownership, Patents & Royalties.

Successful hematopoietic cell transplantation (HCT) requires vacating recipient hematopoietic stem cell (HSC) niches to permit donor HSC engraftment to provide life-long hematopoietic and immune function. Currently HCT relies on DNA damaging radiation or chemotherapy to achieve HSC niche clearance. We have pursued a non-toxic approach to target and deplete HSC using humanized monoclonal antibody, AMG 191, that binds human CD117 (c-Kit). We opened a Phase 1 dose escalation trial using AMG 191 as the sole conditioning agent to achieve donor HSC engraftment in patients undergoing HCT for severe combined immunodeficiency (SCID). The primary endpoint is to assess the safety of administering AMG 191. Secondary endpoints include AMG 191 pharmacokinetics (PK), host HSC depletion, and determination of the dose of AMG 191 that achieves adequate donor HSC engraftment, defined as >5% donor granulocyte chimerism at 24 weeks. We have completed the first dose cohort of patients receiving 0.1 mg/kg AMG 191 and treated the first two patients in the second cohort (0.3 mg/kg). All five patients tolerated the AMG 191 infusion and the subsequent infusion of their CD34-selectd donor cells without clinical problems. Here we report efficacy in the first two patients, who have reached the 24-week timepoint. Both patients had T-B-NK+ SCID with mutations in the DCLRE1C (Artemis) gene. Both previously received unconditioned HCT as infants, failed to develop donor B cells and remained dependent on exogenous immunoglobulin. CD34-selected mobilized peripheral blood cells from the original donors were infused when the AMG 191 serum level was Conclusion: These data are proof of concept that a humanized monoclonal antibody targeting CD117 can safely clear human HSC niches and facilitate donor HSC engraftment. This study is ongoing and open for enrollment.

Cancer immunotherapies hold much promise, but their potential in veterinary settings has not yet been fully appreciated. Canine lymphomas are among the most common tumors of dogs and bear remarkable similarity to human disease. In this study, we examined the combination of CD47 blockade with anti-CD20 passive immunotherapy for canine lymphoma. The CD47/SIRPα axis is an immune checkpoint that regulates macrophage activation. In humans, CD47 is expressed on cancer cells and enables evasion from phagocytosis. CD47-blocking therapies are now under investigation in clinical trials for a variety of human cancers. We found the canine CD47/SIRPα axis to be conserved biochemically and functionally. We identified high-affinity SIRPα variants that antagonize canine CD47 and stimulate phagocytosis of canine cancer cells in vitro. When tested as Fc fusion proteins, these therapeutic agents exhibited single-agent efficacy in a mouse xenograft model of canine lymphoma. As robust synergy between CD47 blockade and tumor-specific antibodies has been demonstrated for human cancer, we evaluated the combination of CD47 blockade with 1E4-cIgGB, a canine-specific antibody to CD20. 1E4-cIgGB could elicit a therapeutic response against canine lymphoma in vivo as a single agent. However, augmented responses were observed when combined with CD47-blocking therapies, resulting in synergy in vitro and in vivo and eliciting cures in 100% of mice bearing canine lymphoma. Our findings support further testing of CD47-blocking therapies alone and in combination with CD20 antibodies in the veterinary setting. Cancer Immunol Res; 4(12); 1072–87. ©2016 AACR.

Tobacco use is associated with an increase in the white blood cell (WBC) count. This association has been attributed to bronchopulmonary inflammation and/or infection. It is not known if nicotine itself may play a role. The objective of this study was to determine whether nicotine itself could affect the WBC count, and to determine whether this was due to a direct effect on hematopoietic stem cells (HSC). C57Bl6J mice received nicotine orally, and measurements of the WBC count, bone marrow and spleen cellularity, and HSC count were made. To determine the functionality of HSCs, irradiated animals received bone marrow transplants from vehicle or nicotine-treated mice. Nicotine increased leukocytes in the peripheral blood, bone marrow and spleen. The peripheral red cell and platelet count were unaffected. Nicotine increased the frequency of HSC in the bone marrow. Isolated long-term HSCs from nicotine-treated mice transplanted into irradiated mice regenerated all hematopoietic cell lineages, demonstrating the functional competence of those HSCs. HSCs expressed nicotinic acetylcholine receptors (nAChRs), as documented by FITC-conjugated alpha-bungarotoxin binding. Nicotine increased soluble Kit ligand, consistent with stem cell activation. In conclusion, the data suggest a new mechanism for the increased WBC associated with tobacco use. The effect of nicotine to activate hematopoiesis may contribute to tobacco-related diseases.

Murine peritoneal exudate cells (PEC), predominantly macrophages, were insonated in vitro with burst-mode ultrasound and assayed for their ability to phagocytose and kill Staphylococcus aureus . PEC were exposed at 37°C in rotating tubes to 1-MHz, burst-mode (10 ms on, 10 ms off) ultrasound at 3.7 ± 0.2 W/cm 2 I SPTA (7.4 ± 0.4 W/cm 2 I SPBA ) for 150 s. Bactericidal activity was assayed at 1, 2, and 3 h after exposure and subsequent 37°C incubation with the bacteria for 20 min. In these experiments, which comprised 17 treated and 7 sham-treated control samples, there was no significant difference in results between treated and control samples ( p > 0.29).