Peptide-MHC Complex Production via Plasmid Segmentation
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Solution Overview
Problem
Current methods for producing peptide-MHC multimers are laborious, yield low amounts of properly folded complexes, and are not suitable for rapid, high-throughput identification of tumor-specific TCRs for personalized immunotherapy.
Innovation Solution
The development of compositions and processes for producing peptide-MHC multimers, including a polynucleotide molecule with specific sequences for antigenic peptides, universal target sequences, Beta 2 Microglobulin, and Major Histocompatibility Complex alleles, which enables rapid and efficient production of properly folded peptide-MHC complexes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If solid-phase synthesis and E. coli expression are used for peptide-MHC production, then peptide-MHC complexes can be produced, but the process is laborious and yields low amounts of properly folded complexes
Solution Approach 1:
The invention divides the peptide-MHC production process into separate functional components: a plasmid encoding the MHC molecule and a separate peptide synthesis step. This segmentation allows each component to be optimized independently, with the plasmid providing controlled expression of properly folded MHC molecules and the peptide being added separately, thereby improving both manufacturing precision and productivity.
Solution Approach 2:
The invention performs preliminary action by pre-expressing the MHC molecule in plasmid form before adding the peptide. This ensures that the MHC is already properly folded and ready to bind the peptide, eliminating the need for complex refolding procedures and inclusion body processing, thus improving both the quality and efficiency of peptide-MHC complex production.
2Reliability
If commercial peptide synthesis is used, then peptide ligands can be produced, but the turnaround time is too long for personalized immunotherapy
Solution Approach 1:
The invention performs preliminary action by pre-synthesizing and storing plasmids encoding various MHC molecules before they are needed for personalized immunotherapy. When a patient's neoantigens are identified, the corresponding plasmid is already available, allowing rapid production of peptide-MHC multimers without waiting for de novo peptide synthesis, thus dramatically reducing turnaround time while maintaining reliability.
Solution Approach 2:
The invention introduces dynamics by creating a flexible system where plasmids encoding different MHC molecules can be rapidly selected and adapted to match the specific neoantigens identified in each patient. This dynamic approach allows the system to respond quickly to individual patient needs, enabling personalized immunotherapy on clinically relevant timescales.
3Adaptability or versatility
If photocleavable peptide exchange is used, then parallel production of MHC molecules with different peptide ligands is enabled, but exchange efficiency is poor and many predicted ligands cannot be used
Solution Approach 1:
The invention extracts the peptide binding step from the MHC production process. Instead of attempting to exchange peptides on already-formed MHC molecules (which has poor efficiency), the invention separates the MHC expression (in plasmid form) from the peptide binding step, allowing any peptide that can bind MHC to be used without being constrained by the limitations of photocleavable exchange chemistry, thus improving manufacturing precision while maintaining adaptability.
Data Source
AI summary
Disclosed herein are antigenic peptide-MHC molecules, termed comPACTs, and methods of producing such molecules. Also disclosed herein are methods of producing libraries of comPACT polynucleotides and polypeptides, and their exemplary use in capturing cancer neoepitope-reactive T cells.