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.
OBJECTIVES: Folate receptor alpha (FolRα) is a cell-surface glycoprotein, highly expressed in ovarian and endometrial adenocarcinoma, and thus a promising target for cancer therapy using antibody drug conjugates (ADCs). Most ADCs currently in development are generated by random attachment of the cytotoxic payload to the antibody and result in a heterogeneous mixture, comprised of many different forms that are likely to vary in stability and activity, and therefore may be suboptimal therapeutic agents. We have employed an E. coli cell-free expression system (XpressCFTM) and site-specific conjugation technology, to generate STRO-002, a novel homogenous FolRα-targeting ADC. STRO-002 was optimized by selection of the antibody, drug-linker, conjugation site and drug-antibody ratio (DAR) that conferred the best pharmacological properties. We have conducted preclinical studies to evaluate the stability of STRO-002 and characterize the pharmacological properties of the cytotoxic metabolite SC209. In vitro cytotoxicity assays and in vivo efficacy studies were conducted to evaluate the activity of STRO-002 in multiple ovarian cancer cell lines and xenografts. IND enabling toxicology studies were conducted to determine the safety profiles for STRO-002 and its metabolite SC209 in cynomolgous monkeys and rats, respectively. RESULTS: Based on optimization studies, the anti-FolRα human IgG1 antibody (H01/SP8166) conjugated to a proprietary cleavable drug-linker (SC239) was selected for the lead ADC STRO-002. SC239 contains a tubulin-targeting 3-aminophenyl hemiasterlin warhead, SC209, which has potent cytotoxic activity. Based on most favorable anti-tumor activity, positions 180 and 404 on each heavy chain were selected for conjugation of SC239 to SP8166 to yield an ADC with DAR of ~ 4. The drug-linkage in STRO-002 is highly stable and the released warhead, SC209, is a very weak substrate for cellular drug-resistance efflux pumps and is cleared rapidly from plasma. STRO-002 has potent but highly specific cytotoxic activity (0.1-3 nM) on multiple FolRα-positive ovarian cancer cell lines in vitro and anti-tumor efficacy in ovarian xenograft models. STRO-002 exhibits dose-dependent tumor growth inhibition in Igrov-1 tumor xenografts at a single dose and complete regression is achieved in Igrov-1 and OVCAR-3 tumors with a single dose at 10 and 5 mg/kg, respectively. In addition, administration of STRO-002 in combination with carboplatin confers added benefit in efficacy in Igrov-1 tumors. Toxicology studies show favorable safety profiles for STRO-002 and SC209. The main toxicity finding in monkeys dosed up to 9 mg/kg consists of reversible hematopoietic/lymphoid tissue toxicity, which is considered antigen-independent and is consistent with the anti-proliferative effects of SC209 observed in single-dose toxicology studies in rats. No evidence of ocular toxicity due to SC209 were observed in either species. CONCLUSIONS: STRO-002 is a highly specific FolRα targeting ADC with minimal drug moiety release in circulation and the potential for an improved safety and activity profile, and a reduced risk of tumor drug resistance. Our data supports the advancement of STRO-002 to the clinic as a potential treatment of FolRα expressing malignancies such as ovarian cancer. Citation Format: Cristina Abrahams, Stellanie Krimm, Xiaofan Li, Sihong Zhou, Jeffrey Hanson, Mary Rose Masikat, Krishna Bajjuri, Tyler Heibeck, Dharti Kothari, Abigail Yu, Robert Henningsen, Cuong Tran, Gang Yin, James Zawada, Julie Hang, Maureen Bruhns, Willy Solis, Alexander Steiner, Adam Galan, Toni Kline, Ryan Stafford, Alice Yam, Venita I. De Almeida, Mark Lupher, Jr., Trevor Hallam. PRECLINICAL ACTIVITY AND SAFETY OF STRO-002, A NOVEL ADC TARGETING FOLATE RECEPTOR ALPHA FOR OVARIAN AND ENDOMETRIAL CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-090.
CD74, also known as HLA-DR-associated invariant chain, is a type II transmembrane glycoprotein highly expressed in many B-cell malignancies. The limited expression of CD74 in normal tissues suggests it may be a suitable ADC target for these tumor types. Accordingly, we engineered an anti-CD74 human IgG1 antibody (SP7219) using novel Fab-based ribosome display methods. The selected Fabs were readily reformatted and directly screened as IgGs using Sutro's unique high-throughput, cell-free protein synthesis platform, Xpress CFTM. We then developed novel, potent ADCs, SP7676 and SP7675 (STRO-001), comprised of our lead antibody (SP7219) conjugated to non-cleavable DBCO-maytansinoid linker-warheads with an average drug-antibody ratios (DAR) of 2. We used site-specific conjugation technology which results in a high degree of homogeneity characterized by the drug linker covalently binding to a single defined site. The sites for conjugation were selected based on highest cell killing activity and stablity in vitro and in vivo. Both ADCs demonstrate potent cell killing activity across multiple B-cell tumor lines in vitro, and anti-tumor activity in preclinical multiple myeloma xenograft models. In vitro cytotoxicity assays show nanomolar potency of STRO-001 in four MM cell lines: Mc/CAR (IC50 0.8 nM), MM.1S (IC50 10-11 nM), U266B1 (IC50 8.5 -9.3 nM), and ARP-1 (IC50 4.3-22 nM). CD74 cell surface expression is required for ADC anti-proliferative effect but the expression level does not seem to correlate with in vitro potency. SP7676 elicited a robust anti-tumor response in the ANBL-6 multiple myeloma xenograft model. Durable regressions were observed in all mice at ≥ 3 mg/kg, with equivalent efficacy (regression) at 3 mg/kg (every 3 days x5) and 10 mg/kg (every 3 days x5 or weekly x3). SP7676 also elicited a clear survival benefit in a disseminated multiple myeloma CAG xenograft model starting at 1mg/kg every 3 days x 5 doses. Similarly, both SP7676 and STRO-001 inhibited the formation of internal visceral tumors in the ARP-1 xenograft model after 3 weekly doses of 3 mg/kg. Evaluation of our lead candidate, STRO-001 in additional MM cell lines and primary patient samples is planned. The tolerability of STRO-001 in non-human primates is under evaluation. STRO-001 was administered to cynomolgous monkeys in an exploratory dose-escalating study up to 30 mg/kg x 2 doses on Day 1 and 15. STRO-001 reduces normal B-cell populations at ≥1 mg/kg after a single dose, providing pharmacodynamic evidence of B-cell targeting while other hematopoietic lineages are mostly affected only at the highest dose studied. Anticipated hematologic toxicities were readily reversible at 1, 3 and 10 mg/kg and target organs of interest were identified. Based on these encouraging data, STRO-001 is advancing to IND-enabling studies for the treatment of CD74 expressing multiple myeloma and other B-cell malignancies. Disclosures Abrahams: Sutro Biopharma: Employment. Li:Sutro Biopharma: Employment. Yu:Sutro Biopharma: Employment. Krimm:Sutro Biopharma: Employment. Kahana:Celgene: Employment. Narla:Celgene: Employment. Schwartz:Celgene: Employment. Boylan:Celgene: Employment. Hoffmann:Sutro Biopharma: Employment. Steiner:Sutro Biopharma: Employment. Zawada:Sutro Biopharma: Employment. Stephenson:Sutro Biopharma: Employment. Bruhns:Sutro Biopharma: Employment. DeAlmeida:Sutro Biopharma: Employment. Matheny:Sutro Biopharma: Employment. Bussell:Sutro Biopharma: Employment. Galan:Sutro Biopharma: Employment. Kline:Sutro Biopharma: Employment. Vasquez:Sutro Biopharma: Employment. Yam:Sutro Biopharma: Employment. Stafford:Sutro Biopharma: Employment. Heinsohn:Sutro Biopharma: Employment. Sato:Sutro Biopharma: Employment. Molina:Sutro Biopharma: Employment. Hallam:Sutro Biopharma: Employment. Lupher:Sutro Biopharma: Employment.
Introduction: Despite advances in cytotoxic and targeted therapies, recurrent disease remains the most significant obstacle to long-term survival in patients with childhood and adult leukemias. As part of our efforts to identify therapies that can be repurposed for immediate use in patients with leukemia, we interrogated a library of all available antibody-drug conjugates (ADCs) whose targets are expressed in leukemias (AML or ALL). A list of targets with available ADCs was merged with transcriptome data from over 3000 pediatric and adult leukemias (AML and ALL) to identify targets that are expressed in a large cohort of leukemias with immediate therapies for repurposing. The transcriptome data set included nearly 2000 pediatric AML cases sequenced as part of Target Pediatric AML (TpAML), 419 adult AML cases from TCGA LAML and the Beat AML program, as well as 853 ALL cases from COG and St. Jude trials. In this repurposing endeavor, CD74 emerged as the most expressed transcript in AML and ALL. CD74 encodes for a cell surface protein that associates with the class II major histocompatibility complex and is involved in the regulation of antigen presentation for immune response and B-cell differentiation. STRO-001 (Sutro Biopharma) is a CD74-directed, site-specific ADC developed for the treatment of multiple myeloma and lymphomas. Given broad expression of CD74 in leukemias, we evaluated the efficacy of STRO-001 in AML and ALL preclinical models. Methods: To characterize CD74 expression, RNA-seq data obtained from pediatric and adult AML and ALL patients was examined. Cell surface expression of CD74 was determined by flow cytometry using PE labeled anti-human CD74 antibody. The CD74-targeting ADC (STRO-001) was obtained from Sutro Biopharma.The preclinical efficacy of STRO-001 was evaluated against AML and ALL cell lines and patient samples expressing various levels of CD74 in vitro and in vivo. For in vivo studies, AML and ALL cell lines were transduced with GFP/Luciferase construct, and GFP+ cells were injected intravenously into NSG mice. Leukemia burden was measured by bioluminescence (IVIS) imaging weekly. Results: Transcriptomics analysis showed CD74 expression in a majority of adult and pediatric AML (>99% of cases) and at a much higher level compared CD33 and CD123 (targets currently developed for AML, Fig. 1A). CD74 is also broadly expressed in pediatric ALL, with a significant increase in expression observed compared to CD19 and CD22 (known targets in ALL, Fig. 1B). We confirmed that CD74 is expressed on the cell surface of AML blasts in primary patient samples (Fig. 1C) as well as AML and ALL cell lines (Fig. 1D). Given confirmation of cell surface expression of CD74, we investigated whether targeting CD74 can effectively eliminate leukemia cells. We evaluated the in vitro cytotoxicity of STRO-001 against K562 (a CML cell line that does not express CD74), AML cell lines (MV4;11 and NOMO-1), and ALL cell lines (REH1 and RS4;11) with varied CD74 expression. STRO-001 demonstrates target-specific cytotoxicity against CD74-expressing AML and ALL cell lines, but not K562 cells (Fig. 1E). STRO-001 exhibited high potency in CD74 expressing cells, with IC-50s of 41nM (MV4;11), 1.3nM (NOMO-1), 0.7nM (REH-1) and 3nM (RS4;11). In vivo studies in NSG mice transplanted with AML and ALL cell lines showed high potency. Treatment with STRO-001 at 3mg/kg once a week for 3 weeks effectively eradicated the leukemia in NOMO-1, REH-1, and RS4;11-bearing xenograft mice, while disease progression was observed in untreated control mice (Fig. 1F). We further evaluated the efficacy of STRO-001 in primary patient samples. Primary leukemia specimens from 3 patients with varied CD74 expression (Fig. 1G) were incubated with increasing concentrations of STRO-001 for 3 days. STRO-001 exhibited potent anti-leukemia activity against primary AML cells with IC-50s of 17.4nM, 12.8nM, and 4.07nM, respectively (Fig. 1H). Conclusion: Through transcriptomics profiling and validation of the cell surface expression by flow cytometry, we have identified CD74 as a viable therapeutic target for AML and ALL in children and adults. We further demonstrate that targeting CD74 with STRO-001 effectively eliminates leukemia cells both in vitro and in vivo, providing the preclinical data to compel evaluation of STRO-001 in clinical trials for childhood and adult leukemia. Figure 1 Figure 1. Disclosures Hylkema: Moderna: Current equity holder in publicly-traded company; Quest Diagnostics Inc: Current equity holder in publicly-traded company. Pardo: Hematologics, Inc.: Current Employment. Abrahams: Sutro Biopharma: Current Employment. Bedard: Sutro Biopharma: Current Employment. Molina: Sutro Biopharma: Current Employment. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: Equity Ownership. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.