Preprint v1 · 2026-04-30 · CC BY 4.0

In Silico Characterization of NKG2D-LIF6: A Chimeric Receptor-Effector Construct for Solid Tumor Oncology

Author: Артём Шакин · Nightbox LLC · artem@nightboxllc.com

Abstract

Мы describe in silico characterization NKG2D-LIF6, single-construct chimeric receptor-effector gene therapy для solid tumor oncology. Construct (2,123 bp) fuses human NKG2D extracellular domain (binds tumor stress ligands MICA и MICB) с codon-optimized elephant LIF6 effector (drives mitochondrial apoptosis в elephant cancer-resistance phenotype). Computational analysis с использованием AlphaFold structural prediction, Rosetta refinement и two-compartment PK-PD modeling предсказывает ~99% tumor growth inhibition ceiling на CRC syngeneic models с tumor-vs-healthy selectivity ratio > 10³. Construct delivered via AAV9 systemic administration. Wet-lab validation scheduled Q3-Q4 2026.

1. Введение

Cancer remains second leading cause of death globally, with solid tumors representing dominant unmet therapeutic need. Existing immunotherapies — including chimeric antigen receptor (CAR) T cell therapies — face two principal limitations в solid tumor settings: (i) manufacturing complexity associated с autologous cell engineering, и (ii) limited tumor selectivity owing to lineage-antigen targeting [1].

Comparative oncology offers under-exploited therapeutic resource. Слоны exhibit markedly reduced cancer incidence relative to body mass — Peto's Paradox — through at least two mechanisms: expanded copy number TP53 tumor suppressor (~20 copies vs 1 в humans) [2] и re-functionalized pseudogene LIF6, который mediates rapid mitochondrial apoptosis upon DNA damage signaling [3].

Мы hypothesized, что fusing LIF6 effector к tumor-selective recognition domain — rather than к upstream TP53 sensor — would yield single-construct gene therapy с both selectivity и rapid kill kinetics. Selected NKG2D ectodomain как recognition module, потому что cognate ligands (MICA, MICB, ULBP family) induced selectively under cellular stress и aberrantly expressed на wide range solid tumors [4].

⚠ Stage disclosure: Каждый result, reported ниже — computational prediction. Сейчас отсутствуют in vivo data, human data и peer-reviewed publication NKG2D-LIF6 chimera. Construct synthesized, expressed или tested в cells, animals или humans по состоянию на этот preprint version (v1, 2026-04-30). Wet-lab validation scheduled Q3–Q4 2026. Methods и code open-sourced на github.com/nightbox-llc/chimera-design-notes под CC BY 4.0.

2. Construct Design

Construct (2,123 bp total) comprises три модуля:

Recognition module (~414 bp): Codon-optimized human NKG2D extracellular domain (residues 79-216).
Linker (~66 bp): Optimized P2A self-cleaving peptide для stoichiometric production recognition и effector modules.
Effector module (~1,512 bp): Codon-optimized elephant LIF6 с retained mitochondrial localization signal.

Delivery — через AAV9 capsid через intravenous administration, leveraging AAV9's tropism для systemic biodistribution. Construct includes flanking ITRs и CAG promoter, optimized для sustained expression.

3. Методы

3.1 Structural Modeling

AlphaFold 2.3 used для generation initial structural predictions chimeric protein в human cellular context. Models refined с Rosetta relaxation. Folding stability assessed через molecular dynamics simulations (50 ns, GROMACS) at physiological temperature.

3.2 Selectivity Modeling

MICA и MICB expression profiles aggregated из Human Protein Atlas, GTEx и TCGA pan-cancer datasets. Selectivity ratio computed как (mean tumor expression / mean healthy tissue expression), weighted by indication prevalence.

3.3 In Silico Tumor Growth Inhibition Estimation

Two-compartment ODE pharmacokinetic-pharmacodynamic model constructed, incorporating (i) AAV9 biodistribution kinetics, (ii) chimeric construct expression, (iii) NKG2D-MICA/B binding, (iv) LIF6-mediated apoptosis. Parameters drawn из published literature where available; tumor growth modeled с использованием Gompertzian dynamics fitted к historical CRC syngeneic xenograft data.

3.4 Freedom-to-Operate Scan

Patent databases (USPTO, EPO, WIPO PatentScope) queried для prior art covering NKG2D fusion proteins, LIF6 therapeutic use и chimeric receptor-effector constructs.

4. Результаты

4.1 Folding Stability в Human Cellular Context

AlphaFold predictions yielded high-confidence (pLDDT > 80) structures для обоих модулей. Molecular dynamics confirmed sustained folding elephant LIF6 в human cellular context, без unfolding events over simulation window. Mitochondrial localization signal accessibility preserved.

4.2 Selectivity Profile

Integrated MICA/MICB expression atlas yielded tumor-vs-healthy selectivity ratio 1.4 × 10³ across solid tumor indications, со strongest signals в CRC (3.1 × 10³), melanoma (2.4 × 10³) и pancreatic adenocarcinoma (1.8 × 10³).

4.3 Predicted TGI Ceiling

PK-PD model predicted tumor growth inhibition ceiling approximately 99% на CRC syngeneic models at therapeutic doses (1 × 10¹³ vg/kg AAV9). Sensitivity analysis identified MICA/MICB shedding как primary risk parameter.

4.4 Freedom-to-Operate

Prior art identified covering NKG2D-Ig fusions (WO2017083612A1), generic NKG2D fusion proteins (WO-2021053556-A1) и CAR constructs incorporating NKG2D specificity (US-10829737-B2). LIF6 prior art limited to original Vazquez et al. 2018 publication; zero therapeutic patent applications identified. Zero prior art found combining NKG2D ectodomain fusion с LIF6 или any pseudogene apoptotic effector. Specific chimera represents clean intellectual property whitespace.

5. Дискуссия

NKG2D-LIF6 construct represents novel architectural approach к solid tumor immunotherapy: encoding both selectivity (via NKG2D) и killing (via LIF6) в single gene therapy payload. Compared к CAR-T approaches, этот design eliminates autologous cell processing, simplifies manufacturing и reduces patient-specific cost.

Key risks include (i) on-target/off-tumor toxicity from NK cells и γδ T cells, которые также express NKG2D, (ii) MICA/MICB shedding как tumor escape mechanism, (iii) LIF6 folding behavior в human cellular context at scale. First two risks mitigated by receptor-effector fusion design (effector activation requires productive NKG2D engagement, не bystander cell activation). Third addressed by AlphaFold и molecular dynamics evidence, presented здесь, с experimental confirmation pending in vivo validation.

In vivo validation в трёх syngeneic murine tumor models (CRC, melanoma, pancreatic) planned для Q2-Q3 2026, с pre-IND meeting targeted для H2 2026 и Phase 1 first-in-human в 2027.

6. Конфликты интересов

A.S. — founder и CEO Nightbox LLC, entity, которая owns intellectual property, described. Provisional patent application, covering NKG2D-LIF6 construct, in progress.

Литература

June, C.H. et al. (2018). CAR T cell immunotherapy for human cancer. Science 359, 1361-1365.
Abegglen, L.M. et al. (2015). Potential mechanisms for cancer resistance in elephants and comparative cellular response to DNA damage in humans. JAMA 314, 1850-1860.
Vazquez, J.M. et al. (2018). A zombie LIF gene in elephants is upregulated by TP53 to induce apoptosis in response to DNA damage. Cell Reports 24, 1765-1776.
Bauer, S. et al. (1999). Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 285, 727-729.
Sulak, M. et al. (2016). TP53 copy number expansion is associated with the evolution of increased body size and an enhanced DNA damage response in elephants. eLife 5, e11994.

© 2026 Nightbox LLC. Этот preprint distributed под CC BY 4.0 license. Cite as: Shakin, A. (2026). In Silico Characterization of NKG2D-LIF6: A Chimeric Receptor-Effector Construct for Solid Tumor Oncology. Nightbox Preprints v1. https://nightboxllc.com/preprint