DEVELOPMENT OF IPVAC MUT
The primary objective of the MUTAVAC project is to develop mutated versions of IPVAC (iPVAC Mut) to increase antigen diversity and enhance immune responses against multiple cancer associated targets. IPVAC is the first vaccine candidate developed by IPSirius and is based on engineered induced pluripotent stem cell (iPSC) lines shown to effectively target cancer stem cells (CSCs), resulting in a significant reduction of tumor burden and metastatic spread in vivo.
IPSirius has generated multiple iPSC lines from healthy donors as well as patients with hematological malignancies, oncogenic somatic mutations, or hereditary cancer predispositions using the Yamanaka reprogramming approach. Within MUTAVAC, highly mutated iPSC lines (iPVAC Mut) were generated from multiple donors through controlled exposure to the mutagen N ethyl N nitrosourea (ENU), following a proprietary IPSirius protocol. All iPSC lines were produced under research grade conditions, with mutagenesis optimized to induce multiple mutations while preserving cell viability.
KEY OUTCOMES – MUTAGENESIS AND GENOMIC CHARACTERIZATION
Three main outcomes were achieved. First, an ENU based in vitro mutagenesis process compatible with future clinical development was standardized. Six distinct iPSC lines were exposed to ENU at varying doses for 60 days and expanded for genomic analysis.
Second, cancer like mutational signatures were characterized by whole exome sequencing of parental iPSC lines (IPVAC) and ENU treated lines (iPVAC Mut). Between 150 and 300 mutations per line were identified, including pathogenic somatic variants and a tumor mutational burden of approximately 11.9 mutations/Mb, comparable to primary human cancers.
These data demonstrate that iPVAC Mut lines:
-Were successfully generated across all donor derived iPSCs
-Recapitulate genomic and transcriptomic features of primary cancers
-Harbor hundreds of novel cancer associated mutations
-Maintain mutational stability over multiple culture passages
DEVELOPMENT AND VALIDATION OF A HUMAN IMMUNE POTENCY BIOASSAY
A human immune potency bioassay was developed to assess T cell reactivity against the mutational diversity of iPVAC Mut candidates. A standardized Mixed Lymphocyte Reaction (MLR) assay was established using PBMCs from HLA A2 healthy donors provided by the French Blood Establishment.
The assay relies on co culture of antigen loaded dendritic cells derived from CD14⁺ monocytes with naïve CD8⁺ T cells, with immune activation quantified by IFN γ ELISPOT. Robust isolation, dendritic cell differentiation, and expansion of functional CD8⁺ T cells were demonstrated.
The MLR platform was validated using the highly immunogenic MART 1 peptide in PBMCs from ten HLA A2 donors, resulting in strong IFN γ secretion, priming of up to 22% MART 1 specific CD8⁺ T cells, and increased CD137 expression, confirming effective T cell activation.
IMMUNOGENICITY AND MECHANISM OF ACTION
The immunogenicity of IPVAC and iPVAC Mut was assessed using dendritic cells loaded with iPSC derived lysates. CD8⁺ T cells primed with these antigens exhibited cytotoxic activity against iPSCs and multiple cancer cell lines (SK MEL 5, MDA MB 231, SW 620). Notably, iPVAC Mut induced stronger cytotoxic responses than IPVAC, accompanied by increased expression of activation and effector markers (CD107a, CD137, IFN γ, TNF α).
Mechanistic insights were obtained through single cell RNA sequencing of CD8⁺ T cells, revealing over 70,000 distinct TCR α/β clonotypes and a 22% increase in clonotype diversity in iPVAC Mut relative to IPVAC. This was associated with enhanced neoantigen diversity, increased NKG7 expression, and elevated granzyme A production.
PORTFOLIO ACTIVITY PLAN
To address genetic instability risks inherent to iPSC based therapies, IPSirius implemented Optical Genome Mapping (OGM). Genome integrity analysis of 50 cGMP iPSC samples across multiple stages revealed structural variants undetected by conventional karyotyping.
These results confirm OGM as a robust tool for baseline genomic profiling and quality control of iPSC master cell lines. While regulatory acceptance criteria for ATMPs are still evolving, relevant parameters include the absence of large (>1 Mb) oncogenic rearrangements, tumor associated translocations, and unexpected aneuploidies, consistent with EMA and FDA expectations.
