To mL eedom, Mark Foster,a nd Curt W. Bradshaw [a] Oligonucleotides are important therapeutic approaches, as evidenced by recent clinicals uccesses with antisense oligonucleotides (ASOs)a nd double-stranded short interfering RNAs (siRNAs). Phosphorothioate (PS) modifications are as tandard feature in the current generation of oligonucleotide therapeutics, but generatei someric mixtures, leadingt o2 n isomers. All currently marketed therapeutic oligonucleotides (ASOs and siRNAs)a re complex isomeric mixtures. Recent chemical methodologiesf or stereopure PS insertions have resulted in preliminary rules for ASOs, with multiple stereopure ASOs moving into clinicald evelopment. Although siRNAs have comparatively fewer PSs, the field has yet to embrace the idea of stereopure siRNAs.H erein, it has been investigated whether the individual isomersc ontributee qually to the in vivo activity of ar epresentative siRNA. The results of as ystematic evaluation of stereopure PS incorporation into antithrombin-3 (AT3) siRNA are reported and demonstrate that individualP Sisomersd ramatically affect in vivo activity.Astandard siRNA design with six PS insertions was investigated and it was found that only about 10 %o ft he 64 possible isomersw ere as efficacious as the stereorandomc ontrol.B ased on this data, it can be concluded that G1R stereochemistry is critical, G2R is important,G 21S is preferable, and G22 and P1/P2 tolerate both isomers. Surprisingly,t he disproportionate loss of efficacy for most isomers does not translate into significant gain for the productive isomers, and thus, warrants further mechanistic studies.Since the discovery by Fire and Mello in 1998 that doublestranded short interfering RNA (siRNA) can cleave mRNA and inhibit protein translation, [1] this new class of therapeutics has moved towards clinical utility.T he advent of double-stranded siRNAs as am odality to harnessanatural catalytic pathway, called RNA interference (RNAi), excited scientific and business communities alike because of its implications in therapeutics, particularly for targets difficult to drug with small-molecules and proteins. [2] Interesti ns iRNAs has had periodic boom and bust cycles following the recognition of and solutions for technical challenges. One of the significant challenges is delivery of these large, charged siRNA molecules across the cell membrane.B oth lipid nanoparticle and ligand-based approaches are clinically validated, with ar ecent approvalo ft he first RNAi therapeutic, Patisiran, al ipid complex from Alnylam, and nu-merousG alNAc-targeteds iRNAs in clinical trials. [3] Naturally occurring siRNA molecules have two complementary strands (19b ase pairs), with an additional two unpaired bases at the 3'-ends, so-called 21/21. Aw ide variety of variations weree xploredi ne arly optimization, includings ignificant modifications in size (19-to 27-mers) and shape (Dicer-substrate siRNA, asymmetric and blunt-ended designs). [4] In addition, siRNA components, such as sugars,n ucleobases, and the phosphate backbon...
CVX-045 is produced by covalently attaching a thrombospondin 1 (TSP-1) mimetic comprising a peptidic sequence and a linker to the Fab binding site of a proprietary scaffold antibody. CVX-045 possesses the potency of the TSP-1-derived peptide, along with the advantageous pharmacokinetics of an antibody. Antitumor activity of CVX-045 was evaluated in human xenograft models alone and in combination with standard chemotherapies and targeted molecules. In A549 and A431 xenograft models, CVX-045 demonstrated significant (P < .05) antiangiogenic activity, reducing tumor microvessel density and increasing the levels of necrosis within treated tumors. In an HT-29 xenograft model, CVX-045 in combination with 5-fluorouracil significantly (P < .01) decreased tumor growth rate compared with vehicle, CVX-045, or 5-fluorouracil alone. Cotreatment of CVX-045 plus CPT-11 delayed progression of tumor growth from day 28 to 60. In contrast CVX-045 alone treatment did not delay the progression of tumor growth, and CPT-11 alone delayed progression of tumor growth to day 39. Cotreatment of CVX-045 with sunitinib extended the time to reach tumor load from day 26 to 40. In summary, CVX-045 exhibits significant antiangiogenic activity in several tumor models and enhances antitumor activity in combination with chemotherapy or targeted therapies. These data suggest future avenues for effective combination therapy in treating solid tumors. CVX-045 has recently completed a phase 1 trial in solid tumors where it has been well tolerated.
Background T-ALL/LBL represent a class of devastating hematologic cancers with high rates of relapse and mortality in both children and adults. Despite intensive multi-agent chemotherapy regimens, fewer than 50% of adults and 85% of children with T-ALL survive beyond five years. For those who relapse after initial therapy, salvage regimens induce remissions in only ~20-30% of cases, and survival is dismal. T-ALL/LBL is a genetically diverse group, but with universal overexpression of CD7, making this a suitable target for immunotherapy. Despite the success of CAR-T cells in B-cell malignancies, CAR-T cell development in T-cell malignancies has proven challenging due to fratricide and high risk of contamination of the genetically modified CAR-T product with the patient's malignant T cells. WU-CART-007 is a CD7-directed, genetically modified, allogeneic, 'off the shelf', fratricide-resistant chimeric antigen receptor (CAR) T-cell product for the treatment of CD7+ve hematologic malignancies. Methods This multicenter, open-label, dose-escalation, Phase 1/2 study (NCT#04984356) of WU-CART-007 in patients ≥ 12 years old, with relapsed or refractory T-ALL/LBL is designed to characterize the safety, tolerability, dose-limiting toxicities (DLTs), and maximum tolerated dose (MTD)/maximum administered dose (MAD; if no MTD defined) (Phase 1), and to investigate the preliminary anti-tumor activity, as measured by objective response rate (ORR) and duration of response (DOR) (Phase 2). Phase 1 is comprised of a dose escalation segment and will proceed according to a standard 3+3 design testing up to 4 dose levels from 1 to 9 x 10 8 cells. Adolescent patients, ages 12-17, will be eligible for enrollment in Phase 1 Dose Escalation beginning at Dose level 3 and 4, and during Phase 2 Cohort Expansion. Upon reaching the MTD and/or RP2D, the Phase 2 portion comprised of the cohort expansion segment will be launched. A Simon's optimal two-stage design will be implemented to enroll patients (an interim analysis for futility in the first stage and the final analysis in the second stage) for Phase 2 dose expansion cohort to confirm safety and explore preliminary efficacy. All patients will receive a single infusion of WU-CART-007 cells on day 1 following a lymphodepleting conditioning therapy consisting of fludarabine and cyclophosphamide on days -5 to -3. Patients will be hospitalized for a minimum of 7 days following WU-CART-007 administration. Response will be assessed on Cycle 1 Day 28 (± 1 days), and at Months 3, 6, 12, and 24, by bone marrow aspirate and biopsy and PET/CT if indicated. Response will be defined as per modified NCCN Guidelines Version 2.2020. Disclosures Ghobadi: Atara: Consultancy; Amgen: Consultancy, Research Funding; Wugen: Consultancy; Celgene: Consultancy; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding. Locke: Janssen: Consultancy, Other: Scientific Advisory Role; Kite, a Gilead Company: Consultancy, Other: Scientific Advisory Role, Research Funding; Iovance Biotherapeutics: Consultancy, Other: Scientific Advisory Role; Legend Biotech: Consultancy, Other; Novartis: Consultancy, Other, Research Funding; Takeda: Consultancy, Other; Wugen: Consultancy, Other; Cowen: Consultancy; Umoja: Consultancy, Other; Bluebird Bio: Consultancy, Other: Scientific Advisory Role; Calibr: Consultancy, Other: Scientific Advisory Role; BMS/Celgene: Consultancy, Other: Scientific Advisory Role; GammaDelta Therapeutics: Consultancy, Other: Scientific Advisory Role; Cellular Biomedicine Group: Consultancy, Other: Scientific Advisory Role; Amgen: Consultancy, Other: Scientific Advisory Role; Allogene Therapeutics: Consultancy, Other: Scientific Advisory Role, Research Funding; EcoR1: Consultancy; Emerging Therapy Solutions: Consultancy; Gerson Lehrman Group: Consultancy; Moffitt Cancer Center: Patents & Royalties: field of cellular immunotherapy. Maude: Wugen: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding. Davidson-Moncada: Wugen: Current Employment. Cooper: Wugen: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Patents & Royalties; NeoImmune Tech: Patents & Royalties; RiverVest: Consultancy.
BackgroundAllogeneic Natural Killer (NK) cells are emerging as a safe and effective modality for the treatment of cancer, overcoming several limitations associated with adoptive T cell therapies. Cytokine induced memory-NK cells offer several advantages over conventional NK cells, including enhanced functional persistence, efficacy, and metabolic fitness. Additionally, unlike iPSC and cord blood derived NK cells, they do not require engineering to enable functionality. Here we describe the use of WU-PRIME, a GMP-grade fusion protein complex to generate memory NK cells, and WU-EXPAND, a feeder cell free expansion system to expand memory-NK cells and create WU-NK-101. Further cryopreservation enables the large-scale, off-the-shelf manufacture of memory NK for cancer immunotherapy, with high anti-tumor activity.MethodsNK cells derived from healthy donor leukopheresate were either activated with WU-PRIME and then expanded with WU-EXPAND to form WU-NK-101 or immediately expanded with WU- EXPAND as controls and then cryopreserved. We compared NK cell expansion as well as post- thaw NK cell functionality as assessed by cytokine secretion and short-term and long-term anti- tumor functionality, long-term persistence in NSG mice, as well as anti-tumor activity in vivo.ResultsNK cells activated with WU-PRIME followed by WU-EXPAND (WU-NK-101), expand robustly in large-scale reactions, over 250-fold in 14 days. The cells maintain durable expression of CD25 after expansion, as well as several other hallmarks of the memory-NK phenotype as assessed by mass cytometry. As compared to cells expanded with WU-EXPAND only, WU-NK-101 cells have improved in vitro activity against K562 cells, as well as AML cell lines (TF-1, THP-1, and HL-60). Notably, this functionality is maintained long-term upon repeated challenge. In vivo, WU-NK-101 cells, compared to expanded NK cells have improved in vivo persistence (figure 1; 50,290 v. 9,623, p<0.0001). In vivo anti-tumor activity was also assessed in leukemia models, where Memory NK cells demonstrate superior anti-tumor activity compared to expanded NK cells.Abstract 188 Figure 1NK cell persistence in tumor-bearing mice. 10e6 cryopreserved NK cells were injected into K562 tumor-bearing mice, and supported with 50,000IU human IL-2 every other day. After 9 days, blood was harvested by cheek bleed and assessed for NK cells (hCD45+, CD56+, CD3) in the blood by flow cytometry.ConclusionsThe data demonstrate that WU-NK-101 generated using a feeder cell-free expansion system has a memory phenotype and improved in vitro and in vivo anti-tumor activity compared to conventional NK cells. This activation and expansion platform will enable the development and clinical translation of multiple allogeneic NK cell therapies.
170 Background: CRC is the 4th leading cause of global cancer-related deaths, and novel therapeutic strategies for advanced CRC are urgently needed. Adoptive cell therapy (ACT) is effective in treating hematological malignancies; however, ACT in solid tumors is hindered by target antigen identification, restricted migration into tumors, and survival in the tumor microenvironment (TME) due to immunosuppressive signals and scarcity of nutrients. NK cells are central to anti-tumor immunity and can directly eliminate tumor cells without prior sensitization. Through cytokine reprogramming, NK cells can also gain memory-like features that augment their anti-tumor potential. WU-NK-101 is a cytokine-reprogrammed, expanded, cryopreserved, off-the-shelf NK cell product derived from peripheral blood mononuclear cells, with no additional engineering. Methods: WU-NK-101 ± Ctx was evaluated in vitro in 2D cytotoxicity assays in complete (N) and TME-aligned medias. WU-NK-101 cytotoxicity was further assessed against primary CRC surgical samples in native-TME-aligned 3D assays. Proteomic analysis was performed using tandem-mass spectrometry. In vivo efficacy of WU-NK-101 ± Ctx was evaluated in NSG mice bearing LoVo xenograft CRC tumors. Cell trafficking/penetration to TME was measured by tracking labeled WU-NK-101 ± trastuzumab in NSG mice bearing subcutaneous SKOV-3 xenografts. Results: Compared to conventional NK cells (cNK), WU-NK-101 had a unique phenotype consistent with rapid activation and proliferation (higher expression of activating receptors, Ki67, and GZMB), and showed enhanced in vitro cytotoxicity. WU-NK-101 also exhibited potent cytotoxicity against LoVo CRC tumors in vivo, which was further enhanced in combination with Ctx. Antibody combination improved WU-NK-101 penetration, and persistence in TME. WU-NK-101’s metabolic profile was consistent with aerobic (Warburg) glycolysis, potentially facilitating effector functions in the TME. WU-NK-101 also showed enhanced metabolic fitness and flexibility, as proteins in several metabolic pathways were upregulated in TME vs N media. Consistent with this, WU-NK-101 had increased cell-surface expression of nutrient transporters. While cNK and T cells cytotoxicity was significantly suppressed in TME-aligned media, WU-NK-101’s function was not impacted. Conclusions: We show that WU-NK-101 exerted potent activity against CRC, and in combination with Ctx showed improved intra-tumor infiltration/persistence and anti-tumor activity. Also, WU-NK-101 cells had enhanced metabolic fitness/flexibility and decreased susceptibility to immunosuppression, overcoming limitations encountered by ACT for solid tumors. A Phase 1b clinical trial is in development, which may reshape ACT in CRC and other EGFR-expressing tumors. [Table: see text]
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