ALBERT

Description: Abstract 637 The hematopoietic stem cell niche is known to govern the function of hematopoietic and leukemic stem cells. One constituent of the niche is the extracellular matrix comprised of several elements including a group of molecules called heparan sulfate proteoglycans (HSPGs). These molecules are present in the microenvironment of all tissue stem cells and are critical in the dynamic interactions between stem cells and niche elements. Heparan sulfate proteoglycan synthesis depends upon the glycosyltransferase, Ext1, a known tumor suppressor with a skeletal phenotype (exostoses) when mutated in affected humans. In order to investigate the role of HSPGs in the hematopoietic stem cell niche we conditionally deleted the Ext1 gene in the recently described Mx1+ osteolineage stem/progenitor cell population (Park et al., 2012). Our studies show that Ext1 and its biological product, HSPGs, play a major role in the maintenance of hematopoietic stem cells (HSCs) by modulating the bone marrow microenvironment. Ext1 deletion in osteolineage cells results in a marked loss of hematopoietic stem and progenitor cells (HSPCs) from the bone marrow and an increase in circulating colony forming units (CFU-Cs) and HSCs (designated as lineage -, c-Kit+, Sca1+, CD 150 + and CD48-) both in the spleen and peripheral blood, suggesting a key role of heparan sulfate proteoglycans in the retention of hematopoietic stem/progenitor cells (HSPCs) in the bone marrow. Importantly, histomorphometry analysis showed no defect in bone remodeling upon Ext1 deletion. In order to investigate whether the above phenotype could be therapeutically exploited in the context of bone marrow stem/progenitor cell mobilization, we administered the heparan sulfate proteoglycan inhibitors, heparin and protamine sulfate to mice in combination with conventional mobilization regimes such as G-CSF. Pharmacological inhibition of HSPG induced a significant improvement of hematopoietic stem/progenitor cell mobilization over G-CSF treatment alone, an effect that was completely abrogated in the absence of endogenous HSPGs. Furthermore, by combining HSPG inhibition with G-CSF, we could mobilize a population of HSPCs with superior in-vivo self-renewing ability. Mechanistically, we observed that Ext1 deletion in stromal cells leads to a constitutive activation of the AKT pathway through the PDK1-mediated phosphorylation of AKT-Thr-308, which in turn phosphorylates and inactivate FOXO1 transcription factor. Expression profiling of genes transcriptionally regulated by FOXO1 revealed that VCAM1, a key molecule driving HSCs adhesion to their niche, was highly down regulated in Ext1 deficient cells. VCAM1 protein levels were also decreased accompanied by a profound reduction in HSC adhesion to Ext1 deficient cells in vitro. Taken together our results suggest that Ext1/HSPGs control hematopoietic stem/progenitor cells retention in the bone marrow through an AKT/FOXO1/VCAM1 dependent pathway and that targeting heparan sulfate proteoglycans is a strategy for mobilizing stem cells with superior self renewing capability to the blood. We are grateful to Lexicon Genetics and the MMRRC for kindly providing the Ext1 mouse model. Disclosures: Wagers: BD Biosciences: Consultancy; iPierian, Inc.: Consultancy; MPM Capital: Consultancy; Novartis: Honoraria. Scadden:Fate Therapeutics: Consultancy, Equity Ownership; Genzyme: Consultancy.

Description: Key Points Mx1 + stromal cells and/or their descendants provide functional niches for HSPCs and regulate their localization. Targeting Ext1 or HSPG can mobilize more potent reconstituting cells and enable engraftment without cytotoxic conditioning.

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: Conditioning chemotherapy is used to deplete hematopoietic stem cells in the recipient's marrow prior to a bone marrow transplant to facilitate engraftment of donor cells. While effective, some major issues with chemotherapy remain which includes off-target genotoxic effects increasing the risk of secondary malignancies. These complications are compounded in disease settings that arise from a deficit in DNA repair pathways where cytotoxic treatments can result in oncogenic transformation, such as Fanconi anemia (FA). FA is an inherited bone marrow failure disorder resulting from an intrinsic defect in DNA repair affecting approximately 130,000 children each year. Currently mutations in 22 genes have been implicated in the pathogenesis of FA. While novel gene-therapy based protocols are showing early promise for this patient populations, the standard treatment for the hematologic complications for FA is a bone marrow transplant. However, secondarily to an underlying sensitivity to DNA damage, these patients are unable to receive standard myeloablative conditioning, which can reduce the efficiency of reconstitution. Avoiding alkylating agents could improve outcomes and success rates in these patients. Furthermore, this methodology could be translated to the allogeneic bone marrow transplantation setting, decreasing the toxicity of this treatment modality. Our approach for characterizing an alternative conditioning regimen for FA patients utilizes immunotoxin conjugates specifically targeting hematopoietic stem cell populations. These non-genotoxic antibody-based drug conjugates utilize saporin (SAP), a ribosomal toxin, to eliminate cells in a targeted manner while leaving the remainder of the marrow compartment intact. Antibodies targeting either CD45 or CD117 were used in a mouse model of FA where expression of their FANCA gene, one of the most common mutations in humans, has been knocked out. Mice conditioned with either of the drugs received various doses of whole marrow from healthy heterozygous littermates. On the day of transplant, mice conditioned with either immunotoxin demonstrated significantly reduced LSK stem cell populations in the marrow similar to cyclophosphamide (Cy) controls (Figure 1A). Peripheral engraftment of donor cells was monitored for 6 months, after which mice were sacrificed for complete analysis of engraftment in the bone marrow compartment. No significant difference was observed in engraftment between Cy and immunotoxin conditioned mice (Figure 1B), and all treatment groups exhibited robust multilineage reconstitution in a cell dose dependent manner. Additionally, Cy treated mice demonstrated greater and sustained weight loss and lower gastrointestinal losses compared to immunotoxin treated mice. Ongoing clonal analysis studies of engrafted cells has demonstrated polyclonal reconstitution, indicating a large number of donor stem cells are actively contributing to hematopoiesis. In this study, we demonstrate that non-genotoxic conditioning approaches both facilitate multilineage engraftment of donor marrow and significantly deplete host hematopoietic stem cell populations. These are crucial since persistence of host hematopoiesis could eventually result in clonal evolution and leukemogenesis in post-transplant FA patients. Achieving both of these conditions through targeted elimination with immunotoxin conjugates represents a major advancement in bone marrow transplantation for FA. We now are initiating studies using immunotoxin-based conditioning for the transplantation of gene-modified syngeneic stem cells and allogeneic cells. We think these studies will inform future clinical trials and provide the groundwork for the next-generation of therapy for FA patients. Disclosures Hartigan: Magenta Therapeutics: Employment. Palchaudhuri:Harvard University: Patents & Royalties; Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Kiem:Homology Medicine: Consultancy; Magenta: Consultancy; Rocket Pharmaceuticals: Consultancy.