# Optimizing rWTC-MBTA Vaccine Formulations, Dosing Regimens, and Cryopreservation Techniques to Enhance Anti-Metastatic Immunotherapy

## TL;DR

This study improves a cancer vaccine by optimizing its formulation, dosing, and freezing methods to enhance its effectiveness against metastatic tumors.

## Contribution

The study introduces optimized vaccine formulations and dosing regimens for practical, scalable anti-metastatic immunotherapy.

## Key findings

- Simplified vaccine formulations with multiple TLR ligands or Resiquimod showed best efficacy in TNBC models.
- Booster dosing schedules (3-1-1-1 or 3-3-3-3) effectively reduced metastatic burden.
- Gradual freezing with DMSO preserved vaccine efficacy by maintaining cell structure.

## Abstract

Metastatic cancer poses significant clinical challenges, necessitating effective immunotherapies with minimal systemic toxicity. Building on prior research demonstrating the rWTC-MBTA vaccine’s ability to inhibit tumor metastasis and growth, this study focuses on its clinical translation by optimizing vaccine composition, dosing regimens, and freezing techniques. The vaccine formula components included three TLR ligands (LTA, Poly I:C, and Resiquimod) and an anti-CD40 antibody, which were tested in melanoma and triple-negative breast cancer (TNBC) models. The formulations were categorized as rWTC-MBT (Mannan-BAM with LTA, Poly I:C, Resiquimod), rWTC-MBL (LTA), rWTC-MBP (Mannan-BAM with Poly I:C), and rWTC-MBR (Resiquimod). In the melanoma models, all the formulations exhibited efficacy that was comparable to that of the full vaccine, while in the “colder” TNBC models, the formulations with multiple TLR ligands or Resiquimod alone performed the best. Vaccine-induced activation of dendritic cell (DC) subsets, including conventional DCs (cDCs), myeloid DCs (mDCs), and plasmacytoid DCs (pDCs), was accompanied by significant CD80+CD86+ population induction, suggesting robust innate immune stimulation. An initial three-dose schedule followed by booster doses (3-1-1-1 or 3-3-3-3) reduced the metastatic burden effectively. Gradual freezing (DMSO-based preservation) maintained vaccine efficacy, underscoring the importance of intact cell structure. These findings highlight the potential of simplified formulations, optimized dosing, and freezing techniques in developing practical, scalable immunotherapies for metastatic cancers.