ALBERT

Description: Introduction Bone Marrow Transplant (BMT) is a potentially curative treatment for malignant and non-malignant blood disorders and has demonstrated impressive outcomes in autoimmune diseases. Prior to BMT, patients are prepared with high-dose chemotherapy alone or with total body irradiation, and both are associated with early and late morbidities, such as infertility, secondary malignancies and organ toxicity; and substantial risk of mortality. This greatly limits the use of BMT in malignant and non-malignant conditions. To address these issues, we are developing antibody drug conjugates (ADCs) targeting hematopoietic stem cells (HSCs) and immune cells to more safely condition patients for BMT. Results To enable simultaneous HSC and immune cell depletion for BMT we investigated targeting human CD45, a protein expressed exclusively on nearly all blood cells including HSCs. Antibody discovery campaigns identified several antibodies with sub-nanomolar affinities for human and non-human primate (NHP) CD45. We then created anti-CD45 ADCs with drug payloads including DNA-damaging, tubulin-targeting and RNA polymerase-inhibiting molecules. An ADC developed with alpha-amanitin (an RNA polymerase II inhibitor) enabled potent in vitro killing of primary human CD34+ HSCs and immune cells (40-120 picomolar IC50s). With this anti-CD45 amanitin ADC (CD45-AM), we explored depletion of HSCs and immune cells in vivo using humanized NSG mice. A single dose of 1 or 3 mg/kg CD45-AM enabled 〉95% depletion of human CD34+ cells in the bone marrow as assessed 7 or 14 days post-administration (Figure, n = 3/group, p values 〈 0.05); 〉95% depletion of human B-, T- and myeloid cells was observed in the periphery and bone marrow (Figure, p values 〈 0.05). Control non-targeting isotype matched-ADCs and anti-CD45 antibody not bearing a toxin had minimal effect on either HSC or immune cells. In hematopoietic malignancies, an anti-CD45 ADC would ideally reduce disease burden and enable BMT. In a model of acute lymphoblastic leukemia (REH cell line, n = 10 mice/group), and 3 patient-derived models of FLT3+NPM1+ acute myeloid leukemia (n = 4-5 mice/group per model), a single dose of 1 mg/kg CD45-AM more than doubled the median survival and several mice survived disease-free (p values 〈 0.001). Anti-CD45 antibodies have been investigated for BMT conditioning in patients as naked antibodies that rely on Fc-effector function to deplete lymphocytes (Biol Blood Marrow Transplant. 2003 9(4): 273-81); or as radioimmunotherapy (Blood. 2006 107(5): 2184-2191). These agents demonstrated infusion-related toxicities likely due to effector function elicited by the wild-type IgG backbone. To address this issue, we created anti-CD45 antibodies with reduced Fc-gamma receptor binding that prevented cytokine release in vitro and in humanized mice. As BMT will likely require fast clearing ADCs to avoid depleting the incoming graft, we also created fast-half-life CD45-AM variants with a t½ of 8-15 hours in mice. To determine the safety and pharmacokinetic properties of regular and fast half-life Fc-silent variants in an immune-competent large animal we tested these in cynomolgus monkeys. Single doses (3 mg/kg, iv, n = 3/group) of fast and regular half-life Fc-silent unconjugated anti-CD45 antibodies were both well tolerated in cynomolgus monkeys and displayed pharmacokinetic properties suitable for BMT. Conclusion These results demonstrate that targeting CD45 with an amanitin ADC results in potent in vitro and in vivo human HSC and immune cell depletion. This new CD45-AM ADC also significantly reduced disease burden in multiple leukemia models. Our results indicate Fc-silencing may avoid infusion-related toxicities observed with previous CD45 mAbs. An alpha-amanitin ADC targeted to CD45 may be appropriate for preparing patients for BMT since we hypothesize it may i) be non-genotoxic; ii) effectively deplete both HSC and immune cells; iii) avoid bystander toxicity, due to amanitin's poor cell permeability as a free toxin; and iv) kill cycling and non-cycling cells, the latter being necessary for effective HSC depletion. As our anti-CD45 ADCs are cross-reactive, we are currently investigating their HSC and immune cell depletion activity in vivo in NHPs to enable further preclinical development of these transplant conditioning agents. Disclosures Palchaudhuri: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties; Harvard University: Patents & Royalties. Pearse:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Proctor:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Aslanian:Magenta Therapeutics: Employment, Equity Ownership. McDonough:Magenta Therapeutics: Employment, Equity Ownership. Sarma:Magenta Therapeutics: Employment, Equity Ownership. Brooks:Magenta Therapeutics: Employment, Equity Ownership. Bhat:Magenta Therapeutics: Employment. Ladwig:Magenta Therapeutics: Employment, Equity Ownership. McShea:Magenta Therapeutics: Employment, Equity Ownership. Kallen:Magenta Therapeutics: Employment, Equity Ownership. Li:Magenta Therapeutics: Employment, Equity Ownership. Panwar:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Dushime:Magenta Therapeutics: Employment, Equity Ownership. Sawant:Magenta Therapeutics: Employment, Equity Ownership. Adams:Magenta Therapeutics: Employment, Equity Ownership. Falahee:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Lamothe:Magenta Therapeutics: Employment, Equity Ownership. Gabros:Magenta Therapeutics: Employment, Equity Ownership. Kien:Magenta Therapeutics: Employment, Equity Ownership. Gillard:Magenta Therapeutics: Employment, Equity Ownership. McDonagh:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Description: Background: Genotoxic conditioning prior to allogeneic and autologous bone marrow transplantation (BMT) limits the use of these potentially curative treatments due to risks of regimen-related morbidities and mortality, including risks of organ toxicity, infertility, and secondary malignancies. CD117, which is specifically expressed on hematopoietic stem cells (HSCs) and progenitors is rapidly internalized and is an ideal target for an antibody drug conjugate (ADC) based approach to conditioning. We have previously shown that a single dose of an anti-CD117 ADC depleted 〉95% of bone marrow HSCs in a humanized mouse model and reduced disease burden while extending survival in an AML tumor model (Hartigan et al., Blood 2017 130:1894). The aim of this translational study was to develop a potent anti-CD117 ADC highly effective in eliminating host HSCs with a short half-life and minimal adverse side effects in a non-human primate (NHP) model. Methods: Three different DNA-damaging cytotoxic payloads and amanitin (AM) were site specifically conjugated to an anti-CD117 antibody. The ADCs were titrated and evaluated for in vitro cytotoxicity using human bone marrow CD34+ cells. The ADCs were administered in ascending doses to humanized NSG mice. HSC depletion and immunophenotype of the human cells in the peripheral blood was determined by flow cytometry. For amanitin conjugates, NHP HSC depletion was evaluated in male cynomolgus monkeys in single ascending doses (3/group). HSC content in the bone marrow was monitored by flow cytometry and colony-forming unit (CFU) analysis on day 7 or 14 and 56 post dosing. Hematology and clinical chemistries were evaluated throughout the two-month study. Results: Of the toxins evaluated, only anti-CD117 conjugated with the RNA polymerase II inhibitor amanitin resulted in 〉90% depletion of human HSCs in humanized NSG mice at 0.3 mg/kg. The AM-conjugates also demonstrated a broad therapeutic window in this model (therapeutic index of 〉120). As a proof-of-concept for the depletion of HSCs in large animals, a single i.v. dose escalation study was performed with the cross-reactive anti-CD117-AM in NHP. On-target, dose-dependent decreases in phenotypic HSCs and CFUs were observed in the bone marrow at day 7 post anti-CD117-AM dosing with 〉95% HSC depletion observed with a single dose of 0.3 mg/kg (Fig. 1). In the periphery, a dose-dependent transient decrease in reticulocytes was observed at day 4 with a neutrophil and monocyte nadir at day 18. The depth and duration of the depletion was also dose-dependent. The anti-CD117-AM induced depletion was on target and amanitin dependent as the unconjugated antibody and isotype-AM had no effect. Notably, white blood cell and lymphocyte counts were stable through day 56, demonstrating that this strategy will spare the adaptive immune system. Thrombocytopenia occurred 4-8 days post infusion and was dose-dependent, transient and reversible. This also occurred with the isotype-AM, suggesting the effect was off-target. Because the half-life of the anti-CD117-AM was 5 days, a second dose escalation study with anti-CD117-AM engineered to have a short half-life (~18 h) was performed in NHPs. The short half-life anti-CD117-AM demonstrated similar potency on all cell parameters at 0.3 mg/kg and was well tolerated at the effective dose. As expected, the short half-life anti-CD117-AM was rapidly cleared with a half-life of 15-18 h. In both studies, a transient dose dependent elevation of liver enzymes was observed in groups treated with the highest doses of isotype-AM, anti-CD117-AM, and the short half-life anti-CD117-AM. Conclusions: Anti-CD117-AM exhibited potent elimination of NHP HSCs and progenitors in vivo. The potency of short half-life anti-CD117-AM was comparable, providing a model for target cell depletion and rapid clearance prior to BMT. Both ADCs were well tolerated at the efficacious doses. This strategy preserves the adaptive immune system with delayed onset of neutrophil nadir (18 days), potentially shortening the period of neutropenia. Targeted depletion of hematopoietic cell subtypes with limited off-target effects could provide a significant improvement in standard-of-care approaches to patient preparation prior to HSC transplant. Disclosures Pearse: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. McDonough:Magenta Therapeutics: Employment, Equity Ownership. Proctor:Magenta Therapeutics: Employment, Equity Ownership. Panwar:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Sarma:Magenta Therapeutics: Employment, Equity Ownership. McShea:Magenta Therapeutics: Employment, Equity Ownership. Kien:Magenta Therapeutics: Employment, Equity Ownership. Dushime:Magenta Therapeutics: Employment, Equity Ownership. Adams:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Brooks:Magenta Therapeutics: Employment, Equity Ownership. Palchaudhuri:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties; Harvard University: Patents & Royalties. Li:Magenta Therapeutics: Employment, Equity Ownership. Kallen:Magenta Therapeutics: Employment, Equity Ownership. Sawant:Magenta Therapeutics: Employment, Equity Ownership. McDonagh:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Description: Background. Allogeneic bone marrow transplant (BMT) is a potentially curative approach in patients with refractory or high risk hematologic malignancies, such as acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Prior to transplant, patients are prepared with non-specific, high dose chemotherapy alone or in combination with total body irradiation, which are associated with early and late morbidities, including organ toxicities, infertility, secondary malignancies, and substantial risk of mortality. As a result, many eligible patients do not consider transplant and of those transplanted, 2/3 can only tolerate reduced intensity conditioning, which is associated with increased relapse rates (Scott et al. Journal of Clinical Oncology 2017, 1154-1161). Thus, safer and more effective conditioning agents with improved disease control are urgently needed. To meet this need, we developed two novel antibody drug conjugates (ADCs) conjugated to amanitin (AM) targeting CD117 (C-KIT, Pearse 2018), which is expressed on hematopoietic stem and progenitor cells and AML and MDS cells in 〉60% of patients (Ludwig et al. Haematologica 1997, 617-621), and CD45 (Palchaudhuri 2018) which is expressed on all lympho-hematopoietic cells and nearly all hematologic malignancies except multiple myeloma. The aim of the project was to design an agent with the dual benefit of depleting primary human hematopoietic stem progenitor cells (HSPCs) while reducing disease burden in leukemia models. Methods. ADCs were tested in xenograft murine models inoculated with human AML cells from immortalized cell lines (Kasumi-1, a CD117 expressing leukemia cell line, and REH-Luc, a CD45 expressing AML cell line tagged with luciferase), and three patient-derived xenografts (PDX) developed from FLT-3+NPM1+ AML samples (J000106132 [prior treatment with Allogeneic BMT, Sorafenib, Hydroxyurea, and Decitabine], J000106565 [M4/5, prior treatment with Induction chemotherapy, Consolidation HiDAC, Allogeneic BMT], J000106134 [M4, no prior treatment reported]) with varying growth kinetics (median survival of vehicle treated groups was 43, 63, 82 days post inoculation) that express both CD117 and CD45 (Jackson Laboratories). Results. In the Kasumi model, a single injection of 0.3 mg/kg anti-CD117-AM administered on day 7 or 42 after AML inoculation resulted in a marked increase in survival (median 〉240 days) compared to vehicle treated controls (median 76 days) or unconjugated anti-CD117 antibody (median 86.5 days) (n=6-8 mice/group, p