Autologous hematopoietic stem cell transplantation (Auto-HSCT) with gene-modification techniques represents a potential cure for multiple genetic blood diseases. Despite its broad curative potential, auto-gene modified HSCT is currently limited due to morbidity/mortality from cytotoxic chemotherapy-based conditioning, including risks of secondary malignancies, organ toxicity, and infertility. To overcome these limitations, we have developed antibody drug conjugates (ADC) targeting CD117 (C-KIT) to specifically deplete the hematopoietic stem and progenitor cells (HSPC) prior to auto-gene modified HSCT. We have previously shown that the anti-CD117 ADC is highly effective at killing human CD117+ cells in vitro and in vivo (Pearse et al., Blood 2018 132:3314). To validate CD117 as an appropriate antigen for targeted ADC-mediated depletion prior to HSCT, we developed an optimized non-human primate (NHP) tool anti-CD117 ADC and evaluated it in an auto-gene modified HSCT in the rhesus macaque model. The tool CD117-ADC is potent on primary human and NHP CD34+ cells in vitro with EC50 of 0.2 and 0.09 pM respectively (Figure 1A). Humanized NSG mice treated with the tool CD117-ADC had full depletion of human HSPCs in the bone marrow 21 days after a single administration of the ADC, while maintaining the peripheral immune cells. We next tested the efficacy and safety of the tool CD117-ADC in NHPs. A single administration of the tool CD117-ADC was fully myeloablative (>99% HSPC depletion) and comparable to HSPC depletion observed following busulfan conditioning (6 mg/kg, once daily for 4 consecutive days). There was no effect on the peripheral and bone marrow lymphocytes and the ADC was well tolerated. To facilitate the use in HSCT, the tool CD117-ADC was engineered to have a fast clearance and in this study the half-life was <10 hours. Based on these encouraging results, we explored whether the tool CD117-ADC could enable engraftment of autologous gene modified hematopoietic stem cells in the rhesus macaque model. A single rhesus macaque was mobilized with granulocyte-colony stimulating factor (G-CSF, 20 mcg/kg/day x 5) and plerixafor (1 mg/kg on day 5 of G-CSF) prior to apheresis. The isolated CD34+ cells were transduced with a lentivirus encoding the β-globin gene and cryopreserved. The tool CD117-ADC was dosed on day -6 and the cryopreserved gene modified cells were thawed and infused (3.3 x 106 CD34+ cells/kg) on day 0. A bone marrow aspirate analyzed on the day of infusion (day 0) demonstrated >99% depletion of the HSPCs and preserved of the bone marrow lymphocytes (Figure 1B). The primate engrafted neutrophils and platelets on day 8 and 10 respectively, and the peripheral lymphocytes were maintained throughout the transplant (Figure 1C). The gene marking in the granulocytes was detectable at day 9, and additional follow up and data from additional animals will be presented. In summary, we have developed a tool CD117 ADC that shows potent activity on NHP CD34+ cells. This optimized CD117-ADC is fully myeloablative with a single dose in NHPs, has a favorable safety profile, spares the immune system and is cleared rapidly as designed. In a rhesus model of autologous gene modified HSCT, a single dose of the ADC enables engraftment of auto-gene modified HSC. These proof of concept studies validate the use of CD117-ADC for targeted HSPC depletion prior to transplant and support its use as a new conditioning agent for autologous gene modified HSCT. This targeted approach for safer conditioning could improve the risk benefit profile for patients undergoing stem cell transplant and enable more patients to benefit from these potentially curative therapies. Disclosures Pearse: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. McDonough:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Proctor:Magenta Therapeutics: Employment, Equity Ownership. Panwar:Magenta Therapeutics: Employment, Equity Ownership. Sarma:Magenta Therapeutics: Employment, Equity Ownership. Kien:Magenta Therapeutics: Employment, Equity Ownership. Latimer:Magenta Therapeutics: Employment, Equity Ownership. Dushime:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Brooks:Magenta Therapeutics: Employment, Equity Ownership. Palchaudhuri:Magenta Therapeutics: Employment, Equity Ownership. Li:Magenta Therapeutics: Employment, Equity Ownership. Sawant:Magenta Therapeutics: Employment, Equity Ownership. McDonagh:Magenta Therapeutics: Employment. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.
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.
Antibody-drug conjugates (ADCs) are promising biotherapeutic agents for the treatment of cancer. The careful monitoring of critical quality attributes is important for ADCs' development, manufacturing and production. In this work, the effect of the presence of a trisulfide bond in the monoclonal antibody (mAb) conjugated to DM4 cytotoxic payload through a disulfide-bond linker sulfo-SPDB (sSPDB) was investigated. Three lots of antibody containing variable levels of trisulfide bonds were used. The identity and levels of trisulfide bonds were determined by liquid chromatography/ mass spectrometry (MS)/MS analysis. The antibodies were conjugated to sSPDB-DM4 to generate ADCs. Further analysis indicated that the drug-to-antibody ratio (DAR) value, a critical quality attribute, slightly increased for the conjugates made from antibody containing higher levels of trisulfide bond. Also, higher fragmentation levels were observed in the conjugates with more trisulfide bond. Detailed characterization by MS revealed that a small amount of DM4 payload was directly attached to inter-chain cysteine residues by disulfide or trisulfide bonds. Overall, our investigation indicated that the trisulfide bond present in the mAb could react with DM4 during the conjugation process. Therefore, the presence of trisulfide bonds in the antibody moiety should be carefully monitored and well controlled during the development of a maytansinoid ADC.
Hematopoietic stem cell transplant (HSCT) can be a highly effective, and often curative, treatment for patients with AML. At present, myeloablative conditioning (MAC) regimens are associated with severe acute and long-term toxicities. A subset of transplant-eligible AML patients undergoes reduced-intensity conditioning (RIC) regimens; while reducing toxicities, RIC regimens have higher incidence of post-transplant relapse and graft failure vs MAC. Thus, the need for safe and effective targeted conditioning agents for a broader use in transplant in both malignant as well as non-malignant settings is critical. We developed MGTA-117, an anti-human CD117 (c-kit)-targeted antibody (Ab) engineered for a short-half life (t1/2) conjugated to amanitin, an RNA polymerase II inhibitor, to enable ADC clearance prior to HSCT. MGTA-117 has a t1/2 of 91 hours in humanized NSG (hNSG) mice compared to the parental wild type (WT) Ab with a t1/2 of 217 hours in human FCRN mice. Via optimization of the linker-toxin moiety, the maximum tolerated dose (MTD) in C57BL/6 mice was improved 17-fold compared to previous generations of the ADC. The antibody component specifically binds CD117, which is expressed on hematopoietic stem and progenitor cells (HSPC) and in ~80% of patient cells with AML and MDS (Gao et al. PLOS One. 2015). When conjugated to amanitin, this ADC robustly depletes both CD117+ HSPCs and leukemic blasts. This targeted and optimized approach not only broadens the therapeutic window across preclinical models, but also provides the dual benefit of effective conditioning for HSCT and reduction of target-expressing tumor cells. Previously we have shown MGTA-117 elicits potent cytotoxicity on both primary human CD34+ cells and a CD117+ AML cell line in vitro. In hNSG mice, a single dose (0.1 mg/kg) of MGTA-117 selectively depleted ≥95% human HSPCs (Figures 1A and B). To demonstrate anti-leukemic activity of MGTA-117, we studied it in multiple human leukemic xenograft murine models that mimic untreated and refractory AML, including both cell line derived (Kasumi-1, CD117-expressing leukemia cell line, in NSG mice) and patient derived (AML PDX 1: J000106134, treatment naïve AML PDX and AML PDX 2: J000106132, heavily pretreated relapsed refractory AML PDX in NSG-SGM3 mice) xenograft models. MGTA-117 was well-tolerated in all three AML xenograft models both as a single dose (1-10 mg/kg) and as a multi-dose (3 mg/kg QODx2) regimen. In the Kasumi-1 model (Figure 1C), MGTA-117, both as a single dose (3 & 10 mg/kg) and as a multi-dose (3 mg/kg QODx2), resulted in a 2.4-2.7-fold increase in median survival compared to vehicle (PBS), isotype-ADC, or standard of care (SOC) cytarabine (ARA-C, 30 mg/kg QDx5). Furthermore, a single dose of MGTA-117 significantly decreased peripheral tumor burden leading to delayed tumor growth resulting in a 2-3.3 fold and 1.3-1.8 fold increase in median survival in the treatment naïve AML PDX 1 and highly pretreated AML PDX 2 models (respectively) compared to vehicle, isotype-ADC and SOC. ARA-C treatment demonstrated a modest improvement in survival in both the AML PDX 1 (1.3 fold), AML PDX 2 (1.3 fold) models compared to control. These data demonstrate that MGTA-117 has potential to be a potent targeted conditioning and anti-leukemia agent. Together with prior reports demonstrating MGTA-117 as an effective conditioning agent in an animal model, this targeted ADC approach has the potential to improve HSCT outcomes in AML by reducing leukemic burden peri-transplant and may decrease the toxicities associated with current conditioning regimens. Disclosures Lanieri: Magenta Therapeutics: Current Employment. Lamothe:Magenta Therapeutics: Current Employment. Miske:Magenta Therapeutics: Current Employment. McDonough:Magenta Therapeutics: Current Employment. Sarma:Magenta Therapeutics: Ended employment in the past 24 months. Bhattarai:Magenta Therapeutics: Current Employment. Latimer:Magenta Therapeutics: Current Employment. Dushime:Magenta Therapeutics: Current Employment. Jain:Magenta Therapeutics: Current Employment. Palchaudhuri:Magenta Therapeutics: Current Employment. Knihtila:Magenta Therapeutics: Current Employment. Pearse:Magenta Therapeutics: Ended employment in the past 24 months. Proctor:Magenta Therapeutics: Current Employment. Boitano:Magenta Therapeutics: Ended employment in the past 24 months, Patents & Royalties. Cooke:Magenta Therapeutics: Ended employment in the past 24 months, Patents & Royalties. Davis:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company.
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