IF a trispecific NK cell engager (TriKE) constructed in a single-chain diabody-IL-15 scaffold format — incorporating (i) a nanobody domain against IL-1 receptor accessory protein (IL-1RAP), a surface co-receptor transcriptionally derepressed on DNMT3A- and TET2-mutant hematopoietic stem and progenitor cells (HSPCs) due to epigenetic dysregulation; (ii) a single-chain variable fragment (scFv) against NKp46 (NCR1), an NK activating receptor not subject to competitive displacement by circulating IgG; and (iii) a transpresented IL-15/IL-15Rα sushi-domain moiety for NK persistence — is administered systemically (intravenous, 3×/week, starting dose 0.1 mg/kg, escalating to 1 mg/kg) to aged (20–22 month) female C57BL/6J mice bearing experimentally validated CHIP-like clonal expansions (via competitive transplantation of Dnmt3a R882H/+ or Tet2−/− Lin−Sca-1+c-Kit+ [LSK] cells at ≥30% chimerism),

THEN a ≥50% reduction in the variant allele frequency (VAF) of mutant clones in peripheral blood and bone marrow, measured by droplet digital PCR (ddPCR) at 8 and 16 weeks post-treatment, accompanied by a ≥2-fold expansion of CD56+CD3− NK cells in the bone marrow niche and a reduction in downstream inflammatory cytokines (IL-6, IL-1β) characteristic of CHIP-driven inflammaging, will be observed,

BECAUSE the following causal chain operates:

1. DNMT3A and TET2 mutations cause epigenetic derepression of IL-1RAP at the transcriptional level. Both DNMT3A loss-of-function and TET2 loss-of-function alter CpG methylation patterns at promoters of surface-expressed co-receptors; IL-1RAP (also known as IL1RAP/C3orf13) is silenced in normal adult HSCs via polycomb and DNA methylation but is aberrantly re-expressed in AML stem cells — a state that CHIP clones partially recapitulate as a pre-malignant intermediate. DNMT3A and TET2 mutations are established drivers of this epigenetic de-silencing trajectory. [SPECULATIVE link: the magnitude of IL-1RAP upregulation in CHIP vs. frank AML is unknown and constitutes a critical evidence gap — see below.] (Literature Task Output — Surface-Expressed Markers section)

2. IL-1RAP surface density on CHIP clones exceeds that on normal HSCs, creating a differential targeting window. Even if expression is lower than frank AML, the TriKE format with its avidity-enhanced bivalent IL-1RAP nanobody arm can exploit sub-stoichiometric surface density differences that monovalent antibody formats cannot, as demonstrated by NK engager valency studies. (Literature Task Output — BiKE/TriKE Formats section)

3. The anti-NKp46 arm engages NK cells via an activating receptor not subject to competitive displacement by polyclonal IgG (unlike CD16a/FcγRIIIa, which is saturated by circulating immunoglobulins in vivo, a major limitation of CD16a-based BiKEs in aged hosts with high serum IgG). NKp46 engagement initiates cytotoxic granule release and ADCC-independent killing, bypassing this saturation problem. (Literature Task Output — BiKE/TriKE ...

SENS category: GlycoSENS