PROJECT HELIX-HTT | Huntington’s-Adjacent Neuro-Oncology Vaccine Program — T-Cell Activation & Cytotoxicity Report

DV-05 — T-Cell Activation & Cytotoxicity Report

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

HELIX-VAX-01 / HELIX-VAX-02 Preclinical Immunogenicity Assessment

DOCUMENT CONTROL HEADER

Program: PROJECT HELIX-HTT

Phase: II — Preclinical Vaccine Development

Module: 5 — T-Cell Activation & Cytotoxicity Program

Document Code: SCF-DV05-TCAR-NO-0001

Purpose: Determine whether HELIX-VAX induces functional antigen-specific T-cell responses capable of recognizing and eliminating target tumor cells while maintaining neuroimmune safety.

1. EXECUTIVE SUMMARY

The ultimate goal of a therapeutic cancer vaccine is not merely antigen presentation—it is the generation of effective tumor-killing immunity.

DV-05 evaluates whether HELIX-VAX successfully converts:

Vaccine Antigen
      ↓
Dendritic Cell Activation
      ↓
CD4 + CD8 T-Cell Priming
      ↓
Memory Formation
      ↓
Tumor Recognition
      ↓
Tumor Cell Killing

This module determines:

✓ Magnitude of T-cell activation

✓ Specificity of immune response

✓ Durability of immune memory

✓ Cytotoxic killing capacity

✓ Neuroimmune safety

✓ Risk of exhaustion or tolerance

2. SCF T-CELL ACTIVATION ARCHITECTURE

Functional Activation Cascade

Antigen Presentation
        ↓
TCR Recognition
        ↓
T-Cell Activation
        ↓
Clonal Expansion
        ↓
Effector Differentiation
        ↓
Tumor Killing
        ↓
Memory Formation

3. MASTER ASSAY PROGRAM

Assay	Objective	Output
TC-A1	CD8 Activation	CD8 Activation Score
TC-A2	CD4 Activation	CD4 Helper Score
TC-A3	Cytokine Function	Functional Cytokine Score
TC-A4	Tetramer Specificity	Antigen Specificity Score
TC-A5	Cytotoxicity Assay	Tumor Killing Score
TC-A6	Memory Formation	Memory Durability Score
TC-A7	Exhaustion Analysis	Exhaustion Risk Score
TC-A8	Neuroimmune Safety	CNS Compatibility Score

4. TC-A1 CD8 CYTOTOXIC T-CELL ACTIVATION ASSAY

Objective

Measure expansion of antigen-specific cytotoxic T cells.

Biomarkers

Marker	Interpretation
CD8	Cytotoxic population
IFNγ	Effector activation
Granzyme B	Killing machinery
Perforin	Cytolytic activity
Ki67	Proliferation

CD8 Activation Score (CD8-AS)

CD8-AS =
Expansion
+
IFNγ
+
Granzyme B
+
Perforin
+
Ki67

Classification

Score	Interpretation
>90	Exceptional
75–90	Strong
60–74	Moderate
<60	Weak

5. TC-A2 CD4 HELPER T-CELL ACTIVATION ASSAY

Objective

Assess helper-cell support for durable immunity.

Biomarkers

Marker	Function
CD4	Helper T cells
IL-2	Expansion support
IFNγ	Th1 polarization
TNFα	Effector support
CD40L	APC interaction

CD4 Helper Score (CD4-HS)

Measures:

Helper activation
APC support
Memory development potential

6. TC-A3 CYTOKINE FUNCTIONALITY ASSAY

Objective

Evaluate functional immune programming.

Desired Cytokines

Cytokine	Role
IFNγ	Anti-tumor immunity
IL-2	Expansion
TNFα	Effector support
CXCL9	Recruitment
CXCL10	Recruitment

Undesired Cytokines

Cytokine	Concern
IL-10	Immune suppression
Excess IL-6	Neurotoxicity risk
Excess TNFα	Neuroinflammation risk

Functional Cytokine Score (FCS)

FCS =
IFNγ
+
IL2
+
TNFα
+
CXCL9
+
CXCL10

-
IL10
-
Excess IL6

7. TC-A4 ANTIGEN-SPECIFIC TETRAMER ASSAY

Objective

Confirm antigen-specific recognition.

Measurement

Determine:

Frequency of antigen-specific T cells
Recognition breadth
Antigen hierarchy

Antigen Specificity Score (ASS)

Score	Interpretation
>85	Strong specificity
70–85	Functional
55–69	Limited
<55	Weak

8. TC-A5 CYTOTOXICITY ASSAY

Objective

Measure actual tumor-cell killing.

Model Systems

2D Co-Culture

Tumor cells + activated T cells

3D Tumor Organoids

Immune infiltration model

Patient-Derived Systems

Highest translational value

Endpoints

Endpoint	Interpretation
Tumor Cell Death	Direct killing
Caspase Activation	Apoptosis
Target Lysis	Cytotoxicity
Tumor Burden Reduction	Functional efficacy

Tumor Killing Score (TKS)

TKS =
Tumor Cell Death
+
Target Lysis
+
Apoptosis
+
Tumor Reduction

9. TC-A6 MEMORY FORMATION ASSAY

Objective

Determine durability of immunity.

Memory Subsets

Subset	Function
TCM	Central memory
TEM	Effector memory
TRM	Tissue-resident memory
TSCM	Stem-like memory

Memory Durability Score (MDS)

Measures:

Persistence
Recall potential
Long-term protection

10. TC-A7 T-CELL EXHAUSTION ANALYSIS

Objective

Detect dysfunctional immune states.

Exhaustion Markers

Marker	Meaning
PD-1	Exhaustion
LAG3	Dysfunction
TIGIT	Dysfunction
TIM3	Dysfunction
CTLA4	Suppression

Exhaustion Risk Score (ERS)

Score	Interpretation
Low	Favorable
Moderate	Monitor
High	Combination therapy needed
Severe	Redesign required

11. TC-A8 NEUROIMMUNE SAFETY ASSESSMENT

Objective

Prevent CNS-directed autoimmunity.

Safety Biomarkers

Marker	Concern
IL-1β	Neuroinflammation
TNFα	Microglial activation
IL-6	CNS toxicity
IFNγ excess	Neuroimmune imbalance

Neuroimmune Compatibility Score (NCS)

Status	Meaning
Green	Acceptable
Yellow	Monitor
Red	Redesign

12. APC CARGO INTEGRATION

Outputs from DV-04 directly feed DV-05.

APC Cargo Layer	T-cell Consequence
Antigen Cargo	Specificity
HLA Cargo	Recognition
Co-Stimulatory Cargo	Expansion
Cytokine Cargo	Functional differentiation
Migration Cargo	Priming efficiency

13. RHENOVA MICROENVIRONMENT OVERLAY

Assess impact of:

Factor	Risk
Hypoxia	Reduced killing
Oxidative stress	T-cell dysfunction
Immune suppression	Reduced efficacy
Neuroinflammation	Safety risk

RHENOVA T-Cell Suitability Index (TSI)

TSI =
Activation
+
Killing
+
Memory

-
Hypoxia
-
Redox Burden
-
Suppression

14. COMPOSITE T-CELL POTENCY SCORE

TCPS

TCPS =
CD8 Activation
+
CD4 Activation
+
Functional Cytokines
+
Specificity
+
Tumor Killing
+
Memory

-
Exhaustion

-
Neuroimmune Risk

15. GO / NO-GO CRITERIA

GO

✓ TCPS ≥ 85

✓ TKS ≥ 80

✓ MDS ≥ 75

✓ NCS = Green

✓ ERS = Low

CONDITIONAL GO

✓ TCPS 70–84

✓ Correctable deficiencies

NO-GO

✗ TCPS < 70

✗ Severe exhaustion

✗ Neuroimmune safety failure

✗ Poor tumor killing

16. OUTPUT FILES

DV05-01

CD8 Activation Report

DV05-02

CD4 Helper Report

DV05-03

Functional Cytokine Report

DV05-04

Tetramer Specificity Report

DV05-05

Tumor Killing Report

DV05-06

Memory Formation Report

DV05-07

Exhaustion Analysis Report

DV05-08

Integrated T-Cell Potency Dashboard

17. SCF CLINICAL TRANSLATION VALUE

Successful completion of DV-05 demonstrates:

Vaccine-generated adaptive immunity
Antigen-specific tumor recognition
Durable immune memory
Resistance to immune exhaustion
Favorable neuroimmune safety

This represents the primary proof-of-concept that HELIX-VAX can generate functional anti-tumor immunity.

STATUS

DV-05 — T-Cell Activation & Cytotoxicity Report: COMPLETE

NEXT DELIVERABLE

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

This module will integrate APC activation, T-cell activation, tumor organoid killing, immune infiltration, antigen escape resistance, and SCF microenvironment compatibility into a comprehensive anti-tumor efficacy package.