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Factor VIII (FVIII) replacement products enable comprehensive care in hemophilia A. Treatment goals in severe hemophilia A are expanding beyond low annualized bleed rates to include long-term outcomes associated with high sustained FVIII levels. Endogenous von Willebrand factor (VWF) stabilizes and protects FVIII from degradation and clearance, but it also subjects FVIII to a half-life ceiling of ∼15 to 19 hours. Increasing recombinant FVIII (rFVIII) half-life further is ultimately dependent upon uncoupling rFVIII from endogenous VWF. We have developed a new class of FVIII replacement, rFVIIIFc-VWF-XTEN (BIVV001), that is physically decoupled from endogenous VWF and has enhanced pharmacokinetic properties compared with all previous FVIII products. BIVV001 was bioengineered as a unique fusion protein consisting of a VWF-DʹD3 domain fused to rFVIII via immunoglobulin-G1 Fc domains and 2 XTEN polypeptides (Amunix Pharmaceuticals, Inc, Mountain View, CA). Plasma FVIII half-life after BIVV001 administration in mice and monkeys was 25 to 31 hours and 33 to 34 hours, respectively, representing a three- to fourfold increase in FVIII half-life. Our results showed that multifaceted protein engineering, far beyond a few amino acid substitutions, could significantly improve rFVIII pharmacokinetic properties while maintaining hemostatic function. BIVV001 is the first rFVIII with the potential to significantly change the treatment paradigm for severe hemophilia A by providing optimal protection against all bleed types, with less frequent doses. The protein engineering methods described herein can also be applied to other complex proteins.

In a phase 1b study of acalabrutinib (a covalent Bruton tyrosine kinase (BTK) inhibitor) in combination with vistusertib (a dual mTORC1/2 inhibitor) in patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL), multiple ascending doses of the combination as intermittent or continuous schedules of vistusertib were evaluated. The overall response rate was 12% (3/25). The pharmacodynamic (PD) profile for acalabrutinib showed that BTK occupancy in all patients was >95%. In contrast, PD analysis for vistusertib showed variable inhibition of phosphorylated 4EBP1 (p4EBP1) without modulation of AKT phosphorylation (pAKT). The pharmacokinetic (PK)/PD relationship of vistusertib was direct for TORC1 inhibition (p4EBP1) but did not correlate with TORC2 inhibition (pAKT). Cell-of-origin subtyping or next-generation sequencing did not identify a subset of DLBCL patients with clinical benefit; however, circulating tumor DNA dynamics correlated with radiographic response. These data suggest that vistusertib does not modulate targets sufficiently to add to the clinical activity of acalabrutinib monotherapy. Clinicaltrials.gov identifier: NCT03205046.

Aggressive B-cell lymphomas are genetically and clinically heterogeneous. Standard clinical trial designs do not efficiently evaluate the safety and efficacy of multiple drug combinations within the context of underlying molecular biology. The rapid identification of oncogenic driver pathways and development of multiple targeted drugs in lymphomas create many potential combinations. To address this need, we developed a Phase 1 master protocol termed PRISM (NCT03527147) to evaluate multiple targeted therapies alone or in combination for the treatment of relapsed/refractory (R/R) aggressive B-cell lymphoma. Each study arm is conducted in a predefined disease subset with the aim of addressing clinical and translational questions within an overarching protocol. All study arms are open label and not randomized. Enrolment of subjects into a given study arm is based on meeting inclusion/exclusion criteria and available slots. Pertinent inclusion criteria for the master protocol are: (a) a diagnosis of R/R non-Hodgkin lymphoma based on established World Health Organization criteria; (b) ≥1 prior line of therapy for the treatment of current histology, no known curative treatment options available, or the subject is ineligible for potential curative options; (c) the presence of radiographically measurable lymphadenopathy or extranodal lymphoid malignancy and; (d) an ECOG performance status ≤2. Exclusion criteria for the master protocol include: (a) a history of prior malignancy, severe or uncontrolled disease or conditions; (b) use of anti-lymphoma therapy within 14 days of the first dose of study drug and; (c) a requirement for ongoing immunosuppressive therapy. Treatment-specific inclusion/exclusion criteria are also provided (see www.clinicaltrials.gov). As PRISM has multiple study arms, subjects can be simultaneously screened for multiple arms. In each arm, a safety review for dose-limiting toxicity (DLT) is performed after 6 subjects have completed the protocol-defined DLT window. Further enrolment will only proceed in that arm if ≤1 subject experiences a DLT (Figure 1). The sample size for each respective arm is determined based on prior clinical/experimental data on anticipated/clinically meaningful activity of each drug combination. This determines a minimally acceptable response and a desirable response. For each arm, a futility analysis occurs after approximately 10 sequentially enrolled subjects. An arm is considered futile if there is 80% chance for the response rate to be above the minimally acceptable response. The study endpoints include safety, ORR, duration of response, progression-free survival, overall survival, and standard pharmacokinetic parameters. Exploratory analyses include in depth translational studies employing peripheral blood and tumor tissue collected at screening and during treatment. These investigations aim to discover predictive biomarkers, identify the molecular correlates of response based on known genetic subtypes, investigate pharmacodynamic and pathway changes and define the depth of response using assays for measurable residual disease (MRD). Exploratory translational endpoints may inform additional biomarker selection strategies for future arms of the PRISM study. All study arms within PRISM to date have combined acalabrutinib, a highly selective BTK inhibitor, with additional targeted agents in subjects with R/R diffuse large B-cell lymphoma. The mechanism of action of the drugs combined with acalabrutinib are as follows: 1. AZD9150 is a 16-nucleotide antisense oligonucleotide designed to target and down-regulate expression of human STAT3 mRNA; administered intravenously. 2. AZD6738 is an inhibitor of ATR; administered orally. 3. Hu5F9-G4 is an anti-CD47 antibody and rituximab is an anti-CD20 antibody; both administered intravenously. 4. AZD5153 is a BRD4 inhibitor; administered orally. In summary, PRISM is a unique platform protocol designed to efficiently evaluate targeted agents in R/R aggressive B-cell lymphoma with an emphasis on comprehensive translational and molecular investigations. Disclosures Izumi: AstraZeneca: Equity Ownership; Acerta Pharma: Employment, Equity Ownership, Patents & Royalties: Acalabrutinib patents. Hamdy:AstraZeneca: Equity Ownership; Acerta Pharma: Employment, Equity Ownership, Patents & Royalties: Acalabrutinib patents. Arkenau:Acerta Pharma: Research Funding. de Vos:Portola Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy; Verastem: Consultancy. Reagan:Kite, A Gilead Company: Consultancy; Curis: Consultancy; Seattle Genetics: Research Funding. Zinzani:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy; Verastem: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Eusapharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Portola: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Servier: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sandoz: Membership on an entity's Board of Directors or advisory committees; Immune Design: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Therapeutics: Honoraria, Speakers Bureau. Davies:BioInvent: Research Funding; ADCT Therapeutics: Honoraria, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Research Funding; Karyopharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Research Funding; Pfizer: Honoraria, Research Funding; Acerta Pharma: Honoraria, Research Funding; MorphoSys AG: Honoraria, Membership on an entity's Board of Directors or advisory committees. Pagel:AstraZeneca: Consultancy; Pharmacyclics, Inc.: Consultancy. Vose:Legend Pharmaceuticals: Honoraria; Acerta Pharma: Honoraria, Other: Grants, Research Funding; Bristol-Meyers Squibb Company: Research Funding; Celgene Corporation: Research Funding; Incyte Corporation: Research Funding; Kite Pharma: Honoraria, Other: Grants, Research Funding; Novartis: Research Funding; Seattle Genetics: Research Funding; AbbVie: Consultancy, Honoraria; Epizyme: Consultancy, Honoraria. Bitman:Acerta Pharma: Employment; AstraZeneca: Equity Ownership. Brock:Acerta Pharma: Employment; AstraZeneca: Equity Ownership. Clark:AstraZeneca: Employment, Equity Ownership. Frigault:Acerta Pharma: Employment; AstraZeneca: Employment, Equity Ownership. Ware:Acerta Pharma: Employment; Astrazeneca: Employment, Equity Ownership. Yang:Acerta Pharma: Employment; AstraZeneca: Equity Ownership. Staudt:Nanostring: Patents & Royalties. Flinn:TG Therapeutics, Trillum Therapeutics, Abbvie, ArQule, BeiGene, Curis, FORMA Therapeutics, Forty Seven, Merck, Pfizer, Takeda, Teva, Verastem, Gilead Sciences, Astra Zeneca (AZ), Juno Therapeutics, UnumTherapeutics, MorphoSys, AG: Research Funding; Acerta Pharma, Agios, Calithera Biosciences, Celgene, Constellation Pharmaceuticals, Genentech, Gilead Sciences, Incyte, Infinity Pharmaceuticals, Janssen, Karyopharm Therapeutics, Kite Pharma, Novartis, Pharmacyclics, Portola Pharmaceuticals: Research Funding; AbbVie, Seattle Genetics, TG Therapeutics, Verastem: Consultancy; TG Therapeutics, Trillum Therapeutics, Abbvie, ArQule, BeiGene, Curis, FORMA Therapeutics, Forty Seven, Merck, Pfizer, Takeda, Teva, Verastem, Gilead Sciences, Astra Zeneca (AZ), Juno Therapeutics, UnumTherapeutics, MorphoSys, AG: Research Funding; F. Hoffmann-La Roche Ltd: Research Funding. OffLabel Disclosure: acalabrutinib in DLBCL

Depolymerized holothurian glycosaminoglycan (DHG) is a fucosylated chrondroitin sulfate that possesses antithrombin-independent antithrombotic properties in rodent thrombosis and dog hemodialysis models. DHG demonstrates significantly less bleeding in template or tail transection assays than therapeutically equivalent doses of heparins. Several potential in vitro mechanisms have been described for DHG, including acceleration of thrombin inhibition by heparin cofactor II (HCII), inhibition of factor VIII activation by thrombin, and inhibition of factor X activation by the intrinsic tenase complex (factor IXa-factor VIIIa). The relevant mechanism(s) for inhibition of tissue factor (TF) induced plasma thrombin generation by DHG were examined in HCII or mock-immunodepleted, and factor-deficient human plasmas, using selected recombinant factor IX(a) with mutations in the heparin-binding exosite. Plasma thrombin generation was detected by fluorogenic substrate cleavage in the presence of corn trypin inhibitor to block contact activation, and compared to a standard curve generated with α2-macroglobulin-thrombin complex. The dose-dependent decrease in velocity index, a parameter reflecting the rate of thrombin generation between lag phase and peak thrombin concentration, was used to compare DHG potency. When triggered by 0.2 pM TF, the EC50 for inhibition of thrombin generation by DHG was 0.16 ± 0.01 μM in both HCII-depleted and mock-depleted plasma, suggesting that DHG acts independently of HCII. When triggered by excess (4 pM) TF, plasma thrombin generation was independent of factors VIII and IX. Under these conditions, the EC50 for DHG inhibition of thrombin generation was increased 13-fold in mock-depleted plasma (2.02 ± 0.09 μM) and 28-fold in HCII-depleted plasma (4.31 ± 0.23 μM). These results suggest that components of the intrinsic tenase complex contribute to inhibition of plasma thrombin generation by DHG, and HCII contributes only at high tissue factor concentrations. In the presence of 0.2 pM TF, Western blotting under nonreducing conditions showed preservation of the prothrombin/meizothrombin band and delayed/reduced thrombin generation in the presence of 0.5 μM DHG, confirming that the inhibition involves reduced prothrombin activation rather than accelerated thrombin inhibition. When triggered by 0.2 pM TF in factor VIII-deficient plasma supplemented with 700 pM factor VIII or thrombin-activated factor VIIIa, the EC50 for inhibition by DHG was 0.41 ± 0.02 μM and 0.44 ± 0.05 μM, respectively. Similarly, the EC50 for DHG inhibition of thrombin generation in factor IX deficient plasma supplemented with 0.2 pM TF and 100% plasma-derived factor IX (90 nM), or 100 pM plasma-derived factor IXa alone, was 0.36 ± 0.01 μM and 0.34 ± 0.02 μM, respectively. Thus, activation of factors VIII and IX do not contribute significantly to the inhibition mechanism for DHG. The contribution of intrinsic tenase activity to DHG inhibition of plasma thrombin generation was assessed using recombinant factor IX(a) mutants with moderate (R170A) or marked (R233A) reductions in heparin affinity. Factor IX deficient plasma was supplemented with 0.2 pM TF and 100% recombinant factor IX, or 100 pM factor IXa, with increasing concentrations of DHG. Similar to plasma-derived factor IX(a), DHG demonstrated an EC50 of 0.38 ± 0.01 μM for inhibition of thrombin generation in the presence of factor IX(a) wild type (WT) zymogen or protease. In the presence of factor IX(a) R170A, the EC50 for DHG was 0.86 ± 0.06 μM and 1.02 ± 0.02 μM, respectively, a 2–3 fold increase relative to WT (P ≤ 0.01). For factor IX(a) R233A, the EC50 for DHG was 3.55 ± 0.47 μM for zymogen and 2.98 ± 0.64 μM for protease, an 8–9 fold increase relative to WT (P ≤ 0.01). Thus, mutations in the factor IXa heparin-binding exosite induced resistance to DHG inhibition of thrombin generation as follows: factor IX(a) R233A> R170A> WT. These findings are consistent with the common mechanism for intrinsic tenase inhibition demonstrated for heparin and DHG in purified systems, and establish the factor IXa heparin-binding exosite as the relevant molecular target for inhibition of plasma thrombin generation by DHG. This antithrombin-independent mechanism likely mediates the antithrombotic efficacy of DHG and related glycosaminoglycans, and may represent a novel therapeutic target with lower bleeding risk.