ALBERT

Description: Mucopolysaccharidosis Type VII (MPS VII) is one of many lysosomal storage diseases and is caused by a deficiency of beta-glucuronidase (GUS). Progressive accumulation of undegraded glycosaminoglycan (GAG) intermediates occurs in the lysosome diminishing intellect, mobility, organ function, and life span. Previously, the efficacy of enzyme replacement therapy (ERT), bone marrow transplantation, and gene therapy have been tested in MPS VII mice. GUS supplied to serum by ERT or released into extracellular spaces from transplanted (Tx) normal or gene transduced cells is taken up by receptor mediated endocytosis and transported to the lysosome. All these treatments reduce lysosomal storage in visceral organs, but poorly deliver sufficient enzyme to the central nervous system (CNS). We hypothesized that in utero intrathecal transplantation of primary neuronal stem cells (NSC) would remedy the GUS deficiency of MPS VII CNS. We isolated NSC from the cortical ventricular zone (CVZ) of E14 GUS+ eGFP+ fetuses. CVZ NSC incubated with EGF and bFGF formed neurospheres in 3–5 days. E14 MPS VII GUS- recipients were Tx intrathecally with NSC and examined for engraftment by histological staining for GUS and eGFP expressing cells. Successful engraftment occurred in 13 of 23 (56.5%) MPS VII recipients. Three animals Tx with 5–25,000 (K) had GUS+ cells in the rostral migratory stream (RMS). Two of seven animals Tx with 50-80K had donor cells in the RMS and five had donor cells in olfactory bulb, cerebrum (cortical layers, corpus callosum and striatum), midbrain, hippocampus (including dentate gyrus), and the cerebellum (Purkinje layers, peduncle, and lobular regions). This demonstrates “complete” dispersal. One mouse Tx with 90K had donor cells lining the lateral ventricle. Another Tx with 160K had donor cells lining ventricular spaces and throughout the cerebrum away from the RMS. An animal Tx with 180K NSC had “complete” donor engraftment like those above. Histological staining for GUS+ donor cells in mice with “complete” engraftment showed levels of staining greater than untreated heterozygous controls, both in numbers of cells positive and in intensity of staining. Biochemistry of tissue sections from these animals averaged 59.5 ±9.2% (mean ±SE) of normal (+/+) activity. The oldest animal detected with “complete” engraftment thus far was 7 months old at sacrifice, demonstrating long-term durability of engraftment. Using anti-eGFP, beta-tubulin III (neurons), and GFAP (astrocytes) antibodies, we confirmed the “complete” engraftment was due to widespread dispersal and integration of donor derived cells and not to diffusion of GUS enzyme from a few engrafted cells. The lineage markers confirmed normal differentiation of donor CVZ NSC. Further evaluations are underway to measure cognitive function in treated animals by the Repeated Acquisition and Performance Chamber (RAPC). Comparison of untreated MPS VII adult mice to normal controls by the RAPC indicates significant deficiencies in learning and memory in untreated MPS VII mice and confirms our ability to measure functional changes in treated animals. In summary, GUS+ eGFP+ fetal NSC primary isolates engraft brain of MPS VII fetal recipients and restore GUS activity ≥ phenotypically normal MPS VII heterozygotes.

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: Introduction Targeted antibody drug conjugates (ADCs) to mouse CD45 or mouse CD117 have recently been shown to effectively prepare immunocompetent mice for whole bone marrow transplants (Palchaudhuri et al. Nature Biotech 2016 34:738-745; and Czechowicz et al. Blood 2016 128:493). This new targeted approach to conditioning using ADCs has the potential to expand the utility of transplantation if it can be successfully translated to humans. The anti-CD45 or anti-CD117 antibodies used previously were coupled to saporin (SAP), a ribosome-inhibiting protein, which once internalized elicits cytotoxicity in a cell cycle-independent manner. Both anti-CD45-saporin (CD45-SAP) and anti-CD117-saporin (CD117-SAP) ADCs have been shown to effectively deplete bone marrow hematopoietic stem cells (HSCs) as single dosed agents, creating vacancies that enable efficient autologous HSC engraftment (〉95% long-term donor chimerism). Results To further investigate and develop the utility of these tool ADCs in murine transplant models, we explored CD45-SAP (1.9 mg/kg, iv) and CD117-SAP (1 mg/kg, iv) in an allogeneic minor mismatch transplant model (Balb/c donor into DBA/2 recipients). The ADCs were used alone or in combination with an additional immune depleting agent, clone 30F11 (25 mg/kg, IP), a naked anti-CD45 antibody that mimics ATG by relying on effector function to enable potent peripheral B- and T -cell depletion. In addition to the lymphodepleting antibody, we included post-transplant Cytoxan (200 mg/kg, IP) to prevent GvHD. To compare the CD45-SAP and CD117-SAP to conventional conditioning methods, we investigated sub-lethal total body irradiation (TBI, 2Gy) or pre-transplant Cytoxan (200 mg/kg, IP) in combination with the immunosuppressants. Conditioned mice were transplanted with 2x107 whole bone marrow cells, and chimerism assessed over 12 weeks. CD45-SAP or CD117-SAP in combination with immunosuppressants (30F11 and post-transplant Cytoxan) enabled 〉85% peripheral donor chimerism at 12 weeks post-transplantation. Multilineage reconstitution was observed in the T-, B- and myeloid cell compartments with 〉80%, 〉90% and 〉90% donor chimerism respectively in both CD45-SAP and CD117-SAP groups. In contrast, 2Gy TBI in combination with immunosuppressants (30F11 and post-transplant Cytoxan) resulted in only 5% donor engraftment. Multi-dosing with 30F11 (QDx3) plus 2Gy TBI and post-transplant Cytoxan increased the peripheral donor chimerism to 40%. Pre-transplant Cytoxan plus 30F11 (QDx3) and post-transplant Cytoxan yielded 20% donor chimerism. For all groups, stem cell chimerism in the bone marrow matched the peripheral chimerism. Conclusion These results indicate anti-CD45 or anti-CD117 ADCs may be used in combination with immunosuppression to enable highly efficient allogeneic transplants in a minor mismatch model (〉85% donor chimerism). CD45-SAP and CD117-SAP were more effective at conditioning versus 2Gy TBI or pre-transplant Cytoxan. Future experiments will investigate anti-CD45 and anti-CD117 ADCs in additional allogeneic models. Disclosures Palchaudhuri: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties; Harvard University: Patents & Royalties. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Proctor:Magenta Therapeutics: Employment, Equity Ownership. Adams:Magenta Therapeutics: Employment, Equity Ownership. Pearse:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Sarma:Magenta Therapeutics: Employment, Equity Ownership. Aslanian:Magenta Therapeutics: Employment, Equity Ownership. Gillard:Magenta Therapeutics: Employment, Equity Ownership. Lamothe:Magenta Therapeutics: Employment, Equity Ownership. Burenkova:Magenta Therapeutics: Employment, Equity Ownership. Brooks:Magenta Therapeutics: Employment, Equity Ownership. Gabros: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: Resetting the immune system through autologous hematopoietic stem cell transplant (autoHSCT) is a highly effective treatment in selected patients with autoimmune diseases. AutoHSCT can induce long-term remission (up to 15 years) with 70-80% progression free survival in patients with relapsed refractory and secondary progressive multiple sclerosis (Muraro 2017) that is superior to standard of care agents in a randomized study (Burt 2019). Likewise, use of autoHSCT in scleroderma patients achieved superior outcomes in two randomized studies (Tyndall 2014, Sullivan 2018). These impressive results are achieved by a combination of the eradication of autoreactive immune effector cells and re-establishment of a self-tolerant immune system, i.e., immune system reset. However, only a small fraction of eligible patients undergo autoHSCT, in part due to toxicity associated with current conditioning protocols. To address these issues, we are developing antibody drug conjugates (ADCs) that selectively target CD45 to eradicate autoimmune cells and enable autoHSCT as a potential one-time curative treatment for patients with autoimmune disease. To model this approach in mice, we generated an anti-mouse CD45 ADC that was evaluated for the ability to condition recipients in a murine congenic transplant model following a single myeloablative dose. This ADC was further evaluated for its ability to eliminate pathogenic host-reactive cells and enable immune reset in recipients in multiple murine models of autoimmune disease, including MOG-induced experimental autoimmune encephalitis (EAE), proteoglycan-induced arthritis (PGIA), and sclerodermatous graft-vs-host disease (scGVHD). A single-dose of tool anti-mouse CD45-ADC at 3 mg/kg achieved full myeloablation in recipient mice (〉99% depletion of LT-HSCs (Lin-Kit+Sca-1+CD150+CD48-). Transplanted mice achieved full engraftment with congenic BMT (〉90% chimerism at 16 weeks). In EAE, conditioning with a non-myeloablative dose of the CD45-ADC followed by congenic transplant prior to disease onset significantly delayed disease onset and reduced disease severity (onset at 21 days, peak disease score 2.1 for with 1 mg/kg CD45-ADC; onset at 42 days, peak disease score 0.75 for 3 mg/kg CD45-ADC + BMT; onset at 11 days with peak disease score 3.1 for vehicle-treated)[Figure 1]. In active EAE, treatment with 3 mg/kg of CD45-ADC on day 10 or 13 followed by congenic transplant halted progression of disease activity (no increase from disease score at time of treatment; peak disease scores of 0.75 and 2.3, respectively). The effect observed with CD45-ADC treatment at day 13 with congenic transplant was comparable to that achieved by treatment with a clinically validated standard of care, FTY-720 (approved S1P1 antagonist equivalent to Gilenya) at day 13 which also halted disease at a peak score of 2.3. Disease control in this study compared favorably to a prior study where mice were treated with 9 Gy TBI + congenic BMT at day 9. These data show that CD45-ADC conditioning followed by congenic transplant is effective at immune reset and shows comparable efficacy to clinically validated therapies. Evaluation of this ADC in additional autoimmune models of arthritis and scleroderma are ongoing and will be presented. To translate these encouraging pre-clinical data, we generated novel anti-human CD45 ADCs that cross react with nonhuman primates (NHP) and evaluated these for the ability to deplete hematopoietic and immune cells in vitro and in vivo in humanized NSG (hNSG) mice. The CD45-ADC showed efficient killing of human BM CD34+ (EC50 2.44 x 10-9 M) and peripheral CD3+ cells from normal donor (EC50 7.6 x10-10 M) and MS patients (EC50 1.5 x 10-10 M). In vivo in hNSG, single doses of the CD45-ADCs were well-tolerated and led to substantial (〉95%) depletion of human cells. In NHPs, single doses of CD45-ADCs were well tolerated and achieved 〉90% peripheral lymphocyte depletion and 〉80% depletion of HSCs. Dose escalation studies are continuing and will be reported. These results suggest that targeted immune depletion with a single treatment of CD45-ADC may be sufficient for auto-HSCT and allow immune reset and re-establishment of immune tolerance. Targeted CD45-ADCs may represent a safer and better tolerated approach for conditioning patients prior to immune reset through autoHSCT and may significantly reduce the side effects associated with current conditioning. Disclosures Gillard: Magenta Therapeutics: Employment, Equity Ownership. Proctor:Magenta Therapeutics: Employment, Equity Ownership. Brooks:Magenta Therapeutics: Employment, Equity Ownership. Lamothe:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Mikse:Magenta Therapeutics: Employment, Equity Ownership. McDonough:Magenta Therapeutics: Employment, Equity Ownership. Palchaudhuri:Magenta Therapeutics: Employment, Equity Ownership. Bhat:Magenta Therapeutics: Employment, Equity Ownership. Sarma:Magenta Therapeutics: Employment, Equity Ownership. Bhattarai:Magenta Therapeutics: Employment, Equity Ownership. Sawant:Magenta Therapeutics: Employment, Equity Ownership. Pearse:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Mcdonough: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: 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 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.

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

Description: The subventricular zone (SVZ) of lateral ventricles in brain contains neuronal stem cells (NSC) that form neurospheres when cultured with EGF and/or bFGF. Progeny from transplanted NSC migrate throughout the brain and replace multiple differentiated cells. Our goal is to develop this phenomenon into a cellular/gene therapeutic approach for treatment of neurological disease. Progeny of normal or gene transduced NSC can replace defective host cells or act as enzyme delivery vehicles. One model for testing this approach is the Mucopolysaccharidosis Type VII (MPS VII) mouse that is deficient in β-glucuronidase (GUS) expression, causing lysosomal storage disease (Sly Syndrome). Undegraded substrates accumulate in brain, causing cognitive dysfunction. A second model is deficient in palmitoyl-protein thioesterase 1 (PPT1), causing neurodegeneration in humans and mice known as infantile neuronal ceroid lipofuscinosis, or Battens Disease. By 24–30wks of age, mice develop motor abnormalities and seizures. Obstacles to NSC therapy include suitable source of donor cells and immunological rejection. Since mesenchymal stem cells (MSC) derived from bone marrow and cord blood have neuronal differentiation capacity, we believe these tissues can generate NSC. If so, self bone marrow or cord blood derived NSC could be transduced with a lentivirus that restores enzyme expression, solving both obstacles. We cultured 15d fetal liver (the murine equivalent of cord blood) from eGFP transgenic mice in neurosphere medium containing EGF and bFGF +/− Noggin/Fc. Noggin is an antagonist for bone morphogenic protein, is expressed in ependymal cells of the SVZ, upregulates neurogenesis, and inhibits bone formation. Growth factors were added days 1–5, and alternating days thereafter. Half the media was replaced every 4d. At 14d, the +Noggin culture had 2–3x the confluency of the -Noggin culture and contained spindle-shaped cells resembling MSC. After another 14d with EGF and bFGF alone, the +Noggin cells formed neurospheres. Neurospheres reformed after trituration, indicating self-renewal capacity. The neurospheres were Nestin+, a marker for NSC, and upon differentiation with serum, β-NGF, BDNF, and NT-3, stained positive for neurons (MAP2) and astrocytes (GFAP). Next, we transplanted 250,000 normal eGFP fetal liver derived NSC (FL NSC) into the lateral ventricles of PPT1−/ − neonatal recipients. Brains 18–54 days post transplant revealed cells in the SVZ of the lateral, dorsolateral, and third ventricles. Donor cells migrated away from the ventricles, into the hippocampal fimbria, under the corpus callosum, and into the rostral migratory stream. Later time points revealed increased migration away from ventricles. We also tested transduction of NSC from MPS VII fetal brain using a lentiviral SIN vector driving human GUS from a PGK promoter. Transduction efficiency was 88–90%. Transplantation of 250,000 transduced NSC into neonatal MPS VII recipients revealed a similar pattern of transplantation as described above for FL NSC. Donor GUS+ cells were detected 4mo post transplant, indicating long-term gene expression and donor cell survival. In conclusion, NSC are efficiently transduced with lentivirus, can be cultured from hematopoietic tissue, and engraft long-term following neonatal injection. These results demonstrate a method to circumvent both donor cell availability and immune barriers to transplantation, providing hope for patients with devastating neurological disorders.