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The YPEL family genes are highly conserved across a diverse range of eukaryotic organisms and thus potentially involved in essential cellular processes. Ypel4, one of five YPEL family gene orthologs in mouse and human, is highly and specifically expressed in late terminal erythroid differentiation (TED). In this study, we investigated the role of Ypel4 in murine erythropoiesis, providing for the first time an in-depth description of a Ypel4-null phenotype in vivo. We demonstrated that the Ypel4-null mice displayed a secondary polycythemia with macro- and reticulocytosis. While lack of Ypel4 did not affect steady-state TED in the bone marrow or spleen, the anemia-recovering capacity of Ypel4-null cells was diminished. Furthermore, Ypel4-null red blood cells (RBC) were cleared from the circulation at an increased rate, demonstrating an intrinsic defect of RBCs. Scanning electron micrographs revealed an ovalocytic morphology of Ypel4-null RBCs and functional testing confirmed reduced deformability. Even though Band 3 protein levels were shown to be reduced in Ypel4-null RBC membranes, we could not find support for a physical interaction between YPEL4 and the Band 3 protein. In conclusion, our findings provide crucial insights into the role of Ypel4 in preserving normal red cell membrane integrity.

2559 Background: Stimulating CD40 may support anti-cancer immune responses; however, on-target toxicity limits systemic dosing. NG-350A is a tumor-selective and blood stable adenoviral T-SIGn vector expressing a potent fully human IgG agonistic anti-CD40 antibody (mAb). NG-350A was designed to selectively deliver anti-CD40 to multiple tumor sites via IV delivery, driving immunological tumor re-engineering while avoiding systemic toxicity. We report results from a first-in-human trial after completion of enrollment. Methods: FORTITUDE (NCT03852511) is a phase 1 study of NG-350A ± pembro in patients (pts) with metastatic/advanced epithelial tumors. NG-350A monotherapy (mTx) was dose-escalated in separate intratumoral (IT; increasing numbers of doses) or IV (one cycle; three increasing dose levels) cohorts. IV NG-350A + pembro (200 mg Q3W for ≤8 cycles) was then assessed. Results: As of Jan 2022, 28 heavily pre-treated pts had received NG-350A, either as IT mTx (n=9; two dose levels), IV mTx (n=16; IV dose levels 1, 3 & 4) or IV + pembro (n=3; IV dose level 2). The MTD of NG-350A ± pembro was not reached, with no DLTs at the highest IT and IV dose levels. The safety profile of NG-350A was consistent with acute reactions to viral particles and asymptomatic aPTT prolongations (Table). No systemic CD40 transgene protein was detected at any dose level and the only SAE to occur in >1 pt was pneumonia. No objective responses were observed; however, 3/6 patients treated with NG-350A mTx at IV dose level 4 achieved stable disease (dose not yet tested with pembro). Dose-dependent specific increases in serum IL-12, IFNγ and IL-17a were detected in pts treated with IV NG-350A mTx from ̃Wk 2. Increases were sustained at ≥5x baseline levels 7 wks after dosing in the majority of evaluable pts treated at higher IV dose levels. These responses did not occur with IT dosing (or in prior studies with an unarmed vector); further follow-up is ongoing for NG-350A + pembro. IV NG-350A also led to the expansion of T cell clones in blood; most of these were newly detected. Conclusions: NG-350A ± pembro was well-tolerated, with no evidence of CD40-related toxicity. NG-350A IV mTx led to specific and sustained cytokine responses consistent with the MoA of the encoded anti-CD40 Ab. Peak cytokine elevations were typically higher than reported with systemic anti-CD40 Abs, suggesting NG-350A can drive local immunological tumor changes while avoiding systemic toxicity. A further trial (FORTIFY, NCT05165433) will continue dose-escalation of NG-350A + pembro to identify a dose level for efficacy assessments. Clinical trial information: NCT03852511. [Table: see text]

2572 Background: Tumor-selective viruses, particularly those dosed systemically to deliver transgenes, are potentially powerful cancer therapies. However, acute cytokine reactions to viral particle (vp) infusion may affect vector tolerability [Small 2006], thereby limiting the maximum tolerated dose (MTD) and subsequent transgene delivery. T-SIGn vectors (e.g. NG-641 and NG-350A) are transgene-armed variants of the epithelial tumor-selective adenovirus enadenotucirev (EnAd). Acute serum cytokine increases post-dosing have been seen at the MTD of EnAd (“flat” dosing of 3 x 1012 vp on Days [D] 1, 3 and 5) [Machiels 2019]. Following supportive preclinical data [McElwaine-Johnn 2019], we explored if a LHH dosing regimen, in which a lower dose is given on D1 prior to two higher doses on D3 and 5, may improve vector safety/tolerability thereby allowing higher cumulative doses to be given. Methods: Data were pooled from three Phase 1 dose-escalation studies in advanced/metastatic epithelial cancer: SPICE (EnAd + pembrolizumab/nivolumab; NCT02636036), FORTITUDE (NG-350A ± pembrolizumab; NCT03852511) and STAR (NG-641; NCT04053283). Serum cytokines were measured using a 17-analyte Luminex assay. IL-6/MCP-1 data for D1, 3 and 5 (pre- and 6-10 hrs post-dose) were analyzed to examine acute cytokine changes. TNFα/IFNγ were examined due to their association with cytokine release syndrome (CRS). Samples analyzed from SPICE/FORTITUDE were taken before PD-1 inhibitor administration. Results: 84 patients (SPICE n=51; FORTITUDE n=18; STAR n=15) were included in these analyses; 79 had cytokine data. AEs and Gr≥3 AEs within 1 wk of first dose, and DLTs at any time, were less frequent with a LHH vs flat dosing regimen (Tbl). Importantly, a LHH dose of 1-6-6 (1 x 1012 vp on D1; 6 x 1012 vp on D3 and 5; greater than the previous flat MTD) was tolerated. Acute increases in TNFα/IFNγ were limited and no severe CRS was seen. Increases in IL-6/MCP-1 with 1 x 1011 or 1 x 1012 vp flat dosing were negligible, whereas acute increases in IL-6/MCP-1 were seen after the first dose of 3 x 1012 vp when given as a flat dose (negligible increases on D3/5). Notably, cytokine responses with 1-3-3 dosing (1 x 1012 vp on D1; 3 x 1012 vp on D3 and 5), including after the first 3 x 1012 vp dose on D3, were negligible. Cytokine responses after the first dose of 6 x 1012 vp in the 1-6-6 regimen were similar to those seen with the first dose of the flat 3 x 1012 vp regimen. Conclusions: LHH dosing appears to induce a desensitization mechanism allowing higher cumulative doses of T-SIGn vectors to be given without the associated acute reactions to viral infusions. This finding may have implications for optimizing safety-efficacy profiles of viral vectors in cancer. Clinical trial information: NCT02636036, NCT03852511 and NCT04053283. [Table: see text]