Targeted therapies that suppress B cell receptor (BCR) signaling have emerged as promising agents in autoimmune disease and B cell malignancies. Bruton's tyrosine kinase (Btk) plays a crucial role in B cell development and activation through the BCR signaling pathway and represents a new target for diseases characterized by inappropriate B cell activity. N-(3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide (CC-292) is a highly selective, covalent Btk inhibitor and a sensitive and quantitative assay that measures CC-292-Btk engagement has been developed. This translational pharmacodynamic assay has accompanied CC-292 through each step of drug discovery and development. These studies demonstrate the quantity of Btk bound by CC-292 correlates with the efficacy of CC-292 in vitro and in the collagen-induced arthritis model of autoimmune disease. Recently, CC-292 has entered human clinical trials with a trial design that has provided rapid insight into safety, pharmacokinetics, and pharmacodynamics. This first-in-human healthy volunteer trial has demonstrated that a single oral dose of 2 mg/kg CC-292 consistently engaged all circulating Btk protein and provides the basis for rational dose selection in future clinical trials. This targeted covalent drug design approach has enabled the discovery and early clinical development of CC-292 and has provided support for Btk as a valuable drug target for B-cell mediated disorders.
3485 Targeted therapies that suppress B cell receptor (BCR) signaling have emerged as promising agents in the treatment of several B cell malignancies. Bruton's tyrosine kinase (Btk) plays a key role in promoting B cell proliferation and survival through participation in the BCR signaling pathway and represents a promising new drug target. AVL-292 is a covalent, highly selective, orally active small molecule inhibitor of Btk currently being evaluated in a Phase 1b clinical trial in relapsed, refractory B cell malignancies including Chronic Lymphocytic Leukemia (CLL) and non-Hodgkin lymphomas. AVL-292 forms a covalent bond with Cys481 in Btk and potently inhibits Btk in biochemical (IC50 < 0.5nM) and cellular assays (EC50 1–10 nM) including anti-IgM stimulation of BCR signaling, B cell proliferation and activation. A quantitative pharmacodynamic assay to determine the level of AVL-292 bonded to Btk in vitro or in vivo was developed and this drug-target engagement by AVL-292 was shown to correlate directly with inhibition of Btk enzyme activity and substrate phosphorylation. To rationally determine the dose and dose frequency of AVL-292 most likely to benefit patients and to reduce the potential for sub-therapeutic dosing in initial oncology patient cohorts, AVL-292 was administered to healthy adult subjects in a double-blind, placebo controlled, single ascending dose study. This study assessed safety, pharmacokinetics, and quantitatively measured Btk protein levels and AVL-292-Btk engagement in freshly isolated peripheral B lymphocytes. In healthy human subjects, AVL-292 was found to be safe and well tolerated following oral administration at dose levels ranging from 0.5–7.0 mg/kg. AVL-292 plasma levels and pharmacodynamic measurement of Btk engagement was dose-proportional across cohorts. All subjects that received 1.0 mg/kg AVL-292 achieved >80% Btk engagement and mean peak plasma levels (Cmax 365 ng/mL) of AVL-292 were rapidly achieved (Tmax median 40 min). Subjects receiving 2.0 mg/kg AVL-292 had a mean peak plasma concentration of 542 ng/mL. All subjects demonstrated >84% Btk engagement at this dose, with 5 of 6 subjects achieving >98% drug-target engagement. Although AVL-292 plasma levels declined substantially by 8 hours, Btk engagement persisted throughout 24 hours, demonstrating that covalent inhibition of Btk with AVL-292 enables prolonged duration of activity without high levels of circulating drug. These results suggest that a once daily dosing schedule is sufficient for sustained Btk inhibition. Furthermore, the Btk protein level in circulating B lymphocytes from all study subjects was evaluated and the mean level was found to be 417.7 pg Btk/mg total protein. Interestingly, this finding in normal B cells correlates well with preclinical ex vivo analysis of Btk protein in primary CLL cells where comparable Btk protein levels were found. This suggests that the AVL-292 dose range and schedule identified for complete Btk engagement in this healthy volunteer trial is likely to inform appropriate dose selection in the subsequent phase 1b oncology study, allowing more rapid identification of a safe and clinically effective dose. Disclosures: Evans: Avila Therapeutics: Employment, Equity Ownership. Tester:Avila Therapeutics: Employment, Equity Ownership. Aslanian:Avila Therapeutics: Employment, Equity Ownership. Chaturvedi:Avila Therapeutics: Employment, Equity Ownership. Mazdiyasni:Avila Therapeutics: Employment, Equity Ownership. Sheets:Avila Therapeutics: Employment, Equity Ownership. Nacht:Avila Therapeutics: Employment, Equity Ownership. Stiede:Avila Therapeutics: Employment, Equity Ownership. Witowski:Avila Therapeutics: Employment, Equity Ownership. Lounsbury:Avila Therapeutics: Employment, Equity Ownership. Petter:Avila Therapeutics: Employment, Equity Ownership. Singh:Avila Therapeutics: Employment, Equity Ownership. Westlin:Avila Therapeutics: Employment, Equity Ownership.
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.
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