Fusion Protein Targeting Mesothelin via scFv Linkers
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Solution Overview
Problem
Current cancer immunotherapy strategies face challenges in effectively targeting tumor cells due to the downregulation of MHC class I molecules, which are essential for recognizing and killing tumor cells by cytotoxic T-lymphocytes, and existing fusion proteins are limited by stability issues and large size, hindering their ability to penetrate tumors and recruit CD8+ T-cells efficiently.
Innovation Solution
A recombinant fusion protein is developed, comprising a single-chain MHC class I molecule covalently linked with a tumor-specific scFv fragment via peptide linkers, specifically targeting mesothelin-expressing tumor cells, allowing for efficient recruitment of CD8+ T-cells regardless of the tumor cells' MHC expression level.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If chemical conjugation is used to couple MHC peptide complexes to tumor-specific antibodies, then tumor cell targeting is achieved, but production homogeneity and tumor penetration capability are limited due to large molecular size
Solution Approach 1:
The patent divides the antibody into smaller functional fragments (scFv, Fab, or single-domain antibodies) while maintaining tumor-specific binding capability. This segmentation reduces the overall molecular size of the conjugate, improving tumor penetration while preserving targeting effectiveness through the retained antigen-binding function of the antibody fragment.
Solution Approach 2:
The patent extracts and removes the Fc region and other non-essential portions of the full antibody, retaining only the antigen-binding variable regions. This extraction eliminates the large molecular components that hinder tumor penetration while preserving the essential tumor-targeting function through the retained variable domains.
2Reliability
If MHC class I molecules are used for tumor cell recognition, then CD8+ T-cell mediated killing is enabled, but tumor evasion through downregulation of MHC class I molecules reduces effectiveness
Solution Approach 1:
The patent modifies the MHC class I molecule by replacing or modifying the alpha-3 domain with alternative structures (such as IgG Fc fragments or other stabilizing domains). This local modification maintains the peptide-binding groove functionality for T-cell recognition while altering the extracellular region to improve stability and reduce dependence on endogenous MHC expression levels on tumor cells.
Solution Approach 2:
The patent introduces alternative molecular structures (such as IgG Fc fragments or engineered stabilizing domains) as intermediaries to replace the native MHC alpha-3 domain. These intermediary structures provide structural stability and enable alternative recognition pathways that can function even when endogenous MHC class I expression is downregulated on tumor cells.
3Reliability
If fusion proteins are designed to target tumor-specific antigens and recruit CD8+ T-cells, then immunotherapy effectiveness is improved, but stability issues and large size hinder tumor penetration
Solution Approach 1:
The patent systematically varies key parameters of the fusion protein including: (1) antibody fragment type (scFv, Fab, single-domain); (2) MHC class I domain configuration (full-length, alpha-1/alpha-2 only, or modified alpha-3); (3) linker peptide sequences and lengths; (4) peptide-MHC complex stoichiometry. These parameter changes optimize the balance between stability, size, and immunogenicity to achieve effective tumor penetration and T-cell recruitment.
Data Source
Figure 1A~1B
Figure 2
Figure 3A~3B
AI summary
This invention provides fusion proteins comprising consecutive amino acids which beginning at the amino terminus of the protein correspond to consecutive amino acids present in (i) a cytomegalovirus human MHC-restricted peptide, (ii) a first peptide linker, (iii) a human ß-2 microglobulin, (iv) a second peptide linker, (v) a HLA-A2 chain of a human MHC class I molecule, (vi) a third peptide linker, (vii) a variable region from a heavy chain of a scFv fragment of an antibody, and (viii) a variable region from a light chain of such scFv fragment, wherein the consecutive amino acids which correspond to (vii) and (viii) are bound together directly by a peptide bond or by consecutive amino acids which correspond to a fourth peptide linker, wherein the antibody from which the scFv fragment is derived specifically binds to mesothelin. This invention provides nucleic acid constructs encoding same, processes for producing same, compositions, and uses thereof.