DV-06 — Tumor Killing Models & Integrated Anti-Tumor Efficacy Report

PROJECT HELIX-HTT | Huntington’s-Adjacent Neuro-Oncology Vaccine Program

HELIX-VAX-01 / HELIX-VAX-02 Preclinical Efficacy Evaluation Package

DOCUMENT CONTROL HEADER

Program: PROJECT HELIX-HTT

Phase: II — Preclinical Vaccine Development

Module: 6 — Tumor Killing Models & Integrated Anti-Tumor Efficacy

Document Code: SCF-DV06-TKAR-NO-0001

Purpose: Establish whether HELIX-VAX-induced immune responses can infiltrate, recognize, and eliminate target tumor populations under clinically relevant microenvironment conditions.

1. EXECUTIVE SUMMARY

DV-06 is the first comprehensive efficacy module integrating:

✓ Vaccine Construct Engineering (DV-02)

✓ APC Activation (DV-04)

✓ T-Cell Activation & Cytotoxicity (DV-05)

✓ SCF Microenvironment Analysis

✓ Tumor Evolution Modeling

✓ Immune Escape Modeling

✓ Neuroimmune Safety

The central question becomes:

Can HELIX-VAX generate sufficient immune pressure to eliminate tumor cells faster than the tumor can evolve and escape?

2. SCF ANTI-TUMOR EXECUTION CASCADE

HELIX-VAX
      ↓
APC Activation
      ↓
CD8 / CD4 Priming
      ↓
Tumor Recognition
      ↓
Tumor Infiltration
      ↓
Tumor Cell Killing
      ↓
Clonal Collapse
      ↓
Tumor Elimination

3. MASTER EFFICACY ASSESSMENT PROGRAM

4. TK-A1 2D TUMOR CELL KILLING ASSAY

Objective

Measure direct cytotoxic killing.

Experimental Design

Tumor Cells
      +
HELIX-VAX Primed T Cells
      ↓
24h
48h
72h
96h

Primary Endpoints

Direct Cytotoxicity Score (DCS)

DCS =
Cell Death
+
Target Lysis
+
Apoptosis

5. TK-A2 3D ORGANOID ELIMINATION MODEL

Objective

Evaluate killing in a realistic tumor architecture.

Advantages

3D organoids preserve:

• Tumor heterogeneity
• Hypoxia gradients
• Immune exclusion zones
• Cellular complexity

Measurements

Organoid Elimination Score (OES)

6. TK-A3 IMMUNE INFILTRATION MODEL

Objective

Determine whether activated T cells enter tumors.

Biomarkers

Infiltration Index (II)

II =
CD8 Penetration
+
CD4 Penetration
+
Effector Density

7. TK-A4 CLONAL COLLAPSE ANALYSIS

Objective

Determine elimination of dominant tumor populations.

Clonal Categories

Clonal Collapse Score (CCS)

Measures:

• Founder clone elimination
• Subclone elimination
• Residual diversity

8. TK-A5 IMMUNE ESCAPE RESISTANCE MODEL

Objective

Determine whether tumors can evade vaccine pressure.

Escape Mechanisms

Escape Resistance Score (ERS)

ERS =
Antigen Stability
+
Presentation Stability
+
Clonal Coverage

-
Escape Events

9. TK-A6 MICROENVIRONMENT STRESS TESTING

Objective

Evaluate efficacy under hostile tumor conditions.

Stress Conditions

Hypoxia

Low oxygen

Oxidative Stress

High ROS

Immunosuppression

PD-L1

IDO1

TGFβ

Nutrient Restriction

Metabolic competition

MES Resilience Score (MRS)

Measures:

Ability to maintain efficacy despite adverse conditions.

10. RHENOVA OVERLAY

Hypoxia-Adjusted Killing Capacity

Determine:

Normal Killing
vs
Hypoxia Killing

Redox-Adjusted Killing Capacity

Determine:

Normal Killing
vs
High ROS Killing

RHENOVA Compatibility Index (RCI)

RCI =
Hypoxia Performance
+
Redox Performance

11. TK-A7 COMBINATION STRATEGY ANALYSIS

Objective

Evaluate SCF synergy strategies.

Strategy A

HELIX-VAX

+

Microenvironment Conditioning

Strategy B

HELIX-VAX

+

Checkpoint Modulation

Strategy C

HELIX-VAX

+

Redox Optimization

Strategy D

HELIX-VAX

+

Hypoxia Reduction

Synergy Score (SS)

SS =
Observed Effect

÷

Expected Additive Effect

Interpretation

12. TK-A8 INTEGRATED ANTI-TUMOR POTENCY

Composite Efficacy Score (CES)

CES =
Direct Killing
+
Organoid Killing
+
Infiltration
+
Clonal Collapse
+
Escape Resistance
+
Microenvironment Resilience

-
Safety Risks

13. NEUROIMMUNE SAFETY INTEGRATION

Mandatory Review

Monitor:

CNS Compatibility Score (CCS)

14. GO / NO-GO CRITERIA

GO

✓ CES ≥ 85

✓ OES ≥ 80

✓ CCS ≥ 75

✓ ERS ≥ 75

✓ CNS Safety Green

CONDITIONAL GO

✓ CES 70–84

✓ Correctable deficiencies

NO-GO

✗ CES < 70

✗ Severe immune escape

✗ Severe neuroimmune toxicity

✗ Failure of organoid killing

15. INTEGRATED EFFICACY DASHBOARD

Composite Program Advancement Score (CPAS)

CPAS =
DCAS
+
TCPS
+
CES

÷ 3

16. OUTPUT FILES

DV06-01

2D Tumor Killing Report

DV06-02

3D Organoid Elimination Report

DV06-03

Immune Infiltration Report

DV06-04

Clonal Collapse Analysis

DV06-05

Escape Resistance Assessment

DV06-06

Microenvironment Stress Testing Report

DV06-07

Combination Strategy Analysis

DV06-08

Integrated Anti-Tumor Efficacy Dashboard

17. STRATEGIC VALUE TO PROJECT HELIX-HTT

Successful completion of DV-06 demonstrates:

• Functional anti-tumor immunity
• Killing of heterogeneous tumor populations
• Resistance to immune escape
• Activity under hypoxic and immunosuppressive conditions
• Compatibility with SCF microenvironment conditioning strategies

This is the first module that provides a comprehensive efficacy bridge between vaccine immunogenicity and future IND-enabling development.

STATUS

DV-06 — Tumor Killing Models & Integrated Anti-Tumor Efficacy Report: COMPLETE

NEXT DELIVERABLE

DV-07 — Neuroimmune Safety & CNS Compatibility Report

This module will establish the maximum tolerated immune activation envelope, CNS autoimmunity risk profile, microglial activation thresholds, neuroinflammatory safety margins, BBB compatibility assessment, and SCF Neuroimmune Compatibility Index (SNCI) required before advancement into PK/PD and GLP toxicology studies.