Sprouting angiogenesis drives blood vessel growth in healthy and diseased tissues. Vegf and Dll4/Notch signalling cooperate in a negative feedback loop that specifies endothelial tip and stalk cells to ensure adequate vessel branching and function. Current concepts posit that endothelial cells default to the tip-cell phenotype when Notch is inactive. Here we identify instead that the stalk-cell phenotype needs to be actively repressed to allow tip-cell formation. We show this is a key endothelial function of neuropilin-1 (Nrp1), which suppresses the stalk-cell phenotype by limiting Smad2/3 activation through Alk1 and Alk5. Notch downregulates Nrp1, thus relieving the inhibition of Alk1 and Alk5, thereby driving stalk-cell behaviour. Conceptually, our work shows that the heterogeneity between neighbouring endothelial cells established by the lateral feedback loop of Dll4/Notch utilizes Nrp1 levels as the pivot, which in turn establishes differential responsiveness to TGF-β/BMP signalling.
Background: Wnt signaling causes phosphorylation of Dishevelled, but its functional significance is unclear. Results: Sites of Wnt-induced phosphorylation were mapped in Dvl2 and mutated to permit functional testing. Conclusion: Three CK1 phosphorylation sites in the C-terminal of Dvl2 account for the Wnt-induced mobility shift and modulate signaling. Significance: Wnt-induced phosphorylation of Dvl differentially regulates canonical and noncanonical Wnt signaling.
STRO-001 is a site-specific, predominantly single-species, fully human, aglycosylated anti-CD74 antibody-drug conjugate incorporating a non-cleavable linker-maytansinoid warhead with a drug-antibody ratio of 2 which was produced by a novel cell-free antibody synthesis platform. We examined the potential pharmacodynamics and anti-tumor effects of STRO-001 in multiple myeloma (MM). CD74 expression was assessed in MM cell lines and primary bone marrow (BM) MM biopsies. CD74 mRNA was detectable in CD138+ enriched plasma cells from 100% (892/892) of patients with newly diagnosed MM. Immunohistochemistry confirmed CD74 expression in 35/36 BM biopsies from patients with newly diagnosed and relapsed/refractory MM. Cytotoxicity assays demonstrated nanomolar STRO-001 potency in 4/6 MM cell lines. In ARP-1 and MM.1S tumor-bearing mice, repeat STRO-001 dosing provided significant antitumor activity with eradication of malignant hCD138+ BM plasma cells and prolonged survival. In a luciferase-expressing MM.1S xenograft model, dose-dependent STRO-001 efficacy was confirmed using bioluminescent imaging and BM tumor burden quantification. Consistent with the intended pharmacodynamic effect, STRO-001 induced dose-responsive, reversible B-cell and monocyte depletion in cynomolgus monkeys, up to a maximum tolerated 10 mg/kg, with no evidence of off-target toxicity. Collectively, these data suggest that STRO-001 is a promising therapeutic agent for the treatment of MM.
Folate receptor alpha (FolRα) is a glycosylphosphatidylinositol linked cell-surface glycoprotein that is widely expressed in serous and epithelial ovarian cancer, endometrial adenocarcinoma, non-small cell lung cancer and triple negative breast cancer. In contrast, FolRα expression is highly restricted on normal tissues, making it a highly promising target for cancer therapy using antibody drug conjugates (ADCs). We have designed a novel, FolRα-targeting ADC, STRO-002, with potent cytotoxic activity on FolRα expressing tumors in vitro and in vivo, including in cells with low expression levels (~0.2 million copies/cell) of FolRα. STRO-002 contains the anti-FolRa human IgG1 antibody (SP8166) conjugated to a proprietary cleavable drug-linker (SC239). SC239 contains a tubulin-targeting 3-aminophenyl hemiasterlin warhead, SC209, which has potent cytotoxic activity and is a weak substrate for efflux pumps. SP8166 was discovered and optimized using a Fab ribosome display selection and screening platform based on Sutro's Xpress CF+TM system. Four non-natural amino acid p-azidomethyl phenylalanine (pAMF) residues are incorporated into SP8166 at two defined sites on each heavy chain. These sites were selected based on optimal stability and activity in vitro and in vivo. The SC239 drug-linker is conjugated via a cleavable valine citrulline p-aminobenzyl carbamate linker functionalized with dibenzocyclooctyne (DBCO). The rapid and selective reaction of DBCO and pAMF results in a well-defined, homogeneous ADC with a drug-antibody ratio (DAR) of ~4. STRO-002 has potent cytotoxic activity (0.1-3 nM) on multiple FolRα-positive ovarian cancer cell lines in vitro and demonstrates strong anti-tumor response in KB, Igrov1 and OvCAR3 xenograft models in vivo. On Igrov1 xenografts, STRO-002 exhibits dose-dependent tumor growth inhibition starting at a single dose as low as 2.5 mg/kg. Evaluation of in vivo activity of STRO-002 in additional xenograft and PDX models, as well as in combination studies with chemotherapeutic agents is ongoing. Data from exploratory safety studies of STRO-002 in cynomolgus monkey and SC209 (active catabolite) in rats show a favorable safety profile. Our data suggests that STRO-002 is a promising clinical candidate for ovarian cancer, including tumors with low expression levels of FolRα, and IND enabling studies are currently being conducted. Citation Format: Xiaofan Li, Cristina Abrahams, Sihong Zhou, Stellanie Krimm, Robert Henningsen, Heather Stephenson, Jeffrey Hanson, Mary Rose Masikat, Krishna Bajjuri, Tyler Heibeck, Cuong Tran, Gang Yin, James Zawada, Ganapathy Sarma, Joy Chen, Maureen Bruhns, Willy Solis, Alexander Steiner, Adam Galan, Toni Kline, Ryan Stafford, Alice Yam, Venita I. De Almeida, Mark Lupher, Trevor Hallam. Discovery and activity of STRO-002, a novel ADC targeting folate receptor alpha for ovarian and endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1782.
CD74 is a type II transmembrane glycoprotein involved in the formation and transport of MHC class II protein. High expression of CD74 has been confirmed in follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) and other types of NHL with immunohistochemistry (IHC) using the LL1 antibody (Stein et al. Clin Cancer Res 2007). We employed site-specific conjugation technology to generate novel CD74-targeting ADCs, SP7676 and SP7675 (STRO-001) that exhibit a high degree of homogeneity characterized by the drug linker covalently binding to a single defined site. The human anti-CD74 IgG1 antibody (SP7219) used for ADCs SP7676 and STRO-001 was engineered using novel Fab-based ribosome display methods enabling selection from ~1012 different antibody variants. Hundreds of unique Fabs from this selection were converted to IgGs and expressed directly in Sutro's proprietary cell-free protein synthesis platform, Xpress CFTM, for extensive screening. The top antibody lead derived from this screen is further tested to identify the best sites for conjugation of linker-warheads. Sutro's SP7219 emerged as the top performing antibody and was conjugated to noncleavable DBCO-maytansinoid linker-warheads to form the ADCs SP7676 and STRO-001. Since conjugation sites were selected based on highest stability both in vitro and in vivo, these novel ADCs lose little drug moiety in circulation and have potential for improved PK, safety and activity profiles. In vitro cell proliferation/cytotoxicity assays show potent activity in 1) DLBCL (germinal center B-cell-like [GCB] and "double-hit") lines: SU-DHL-4, IC50 - 1nM; SU-DHL-6, IC50 - 0.4 nM; WSU-NHL, IC50 - 1.6 nM; Pfeiffer, IC50 - .09 nM; NUDUL-1, IC50 - 0.4 nM; HT, IC50 - 0.7 nM; OCI-LY-19, IC50 - 0.7 nM; WSU-DLBCL2, IC50 - 0.3 nM; 2) mantle cell lymphoma (MCL) cell lines: Mino, IC50 - 0.4-0.7 nM; JVM-2, IC50 - 1.7-2.9 nM; Jeko-1, IC50 - 0.4 - 0.6 nM; 3) Ph+ acute lymphoblastic leukemia (ALL): SUP-B15, IC50 - 3.9-4.6 nM; and 4) CLL (EBV-transformed): JVM-13, IC50 - 3.0-3.4 nM. SP7676 elicited strong anti-tumor response in the OCI-LY-10 lymphoma xenograft model with 100% of animals achieving complete regression of tumors at 3mg/kg every 3 days x 5 doses and 10 mg/kg weekly x 3 doses. In the WSU-DLCL2 "double-hit" lymphoma xenograft model, administration of SP7676 (with re-dosing at time of re-growth) produced tumor regressions at 10 mg/kg every 3 days x 5 (6/8 mice tumor free, remaining 2 with small tumors) and 10 mg/kg weekly x 3 (tumor regression in most animals, 4/8 tumor free). Additionally, STRO-001 exhibits dose-dependent tumor growth inhibition in SUDHL-6 xenografts starting at 2.5 mg/kg weekly x 3 doses. Exploratory testing of our lead candidate, STRO-001 in cynomologous monkeys showed dose-dependent B-cell depletion at 1 - 30 mg/kg doses on Day 1 and 15, confirming the intended pharmacodynamic effect. Our preliminary data demonstrate that SP7676 and STRO-001 generate potent cell killing activity across multiple B-cell lymphoma/leukemia cell lines in vitro, and anti-tumor activity in preclinical B-cell NHL xenografts. Evaluation of STRO-001 in other cell lines and xenograft models and in combination studies is ongoing. GLP toxicology and other IND-enabling studies are planned. Disclosures Li: Sutro Biopharma: Employment. Abrahams:Sutro Biopharma: Employment. Embry:Sutro Biopharma: Employment. Yu:Sutro Biopharma: Employment. Kahana:Celgene: Employment. Brown:Celgene: Employment. Narla:Celgene: Employment. Barnes:Celgene: Employment. Schwartz:Celgene: Employment. Boylan:Celgene: Employment. Zawada:Sutro Biopharma: Employment. Stephenson:Sutro Biopharma: Employment. Bruhns:Sutro Biopharma: Employment. Bussell:Sutro Biopharma: Employment. Steiner:Sutro Biopharma: Employment. Galan:Sutro Biopharma: Employment. Kline:Sutro Biopharma: Employment. Yam:Sutro Biopharma: Employment. Stafford:Sutro Biopharma: Employment. Hoffmann:Sutro Biopharma: Employment. Matheny:Sutro Biopharma: Employment. DeAlmeida:Sutro Biopharma: Employment. Vasquez:Sutro Biopharma: Employment. Heinsohn:Sutro Biopharma: Employment. Sato:Sutro Biopharma: Employment. Molina:Sutro Biopharma: Employment. Hallam:Sutro Biopharma: Employment. Lupher:Sutro Biopharma: Employment.
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