Systems And Methods For Generating Class 1 Major Histocompatibility Complex Multimer Screening Reagents Using Chaperone Mediated Peptide Exchange

Tech ID: 32985 / UC Case 2018-408-0

Background

MHC Class I multimers are key reagents that are used in the identification of antigen specific T cells + an antigenic peptide. The most useful form of such a reagent involves a Class I MHC molecule that is provided ready to be loaded with an antigenic peptide of interest. However, such molecules are inherently unstable. 

Potential solutions to the instability have major drawbacks. Some MHC Class I molecules are provided with a conditional ligand that can be cleaved by exposure to UV light. These constructs are prone to aggregation and precipitation, must be stored and worked with in the dark, and they can have relatively poor peptide exchange efficiency. Other peptide receptive MHC class I molecules are engineered to have disulfide links holding the peptide to the MHC-I binding groove. In addition to altering natural peptide-MHC-I binding, such molecules are diffficult to express in bacterial vectors.

UC Santa Cruz researchers and the Office of Innovation Transfer have collaborated to produce a robust patent portfolio around a new method of producing peptide receptive class I MHC multimers that produce MHC-I molecules at a high yield with highly efficient peptide exchange. 

A description of the original system (2018-408) is below. Related inventions include: 

2019-975 - a system that uses peptide receptive MHC-I made using TAPBPR to purify a antigenic peptides from a peptide library. The peptides bind to immobilized peptide receptive MHC-I conjugated to a column, then are later eluted. 

2020-284 - specific placeholder peptides and methods for creating peptide receptive HLA-A*68:02 and HLA*A24:02

2020-297 -  an invention resulting from the surprising result that TAPBPR acts catalytically on MHC-I/placeholder peptide complexes to form peptide receptive MHC-I. A 1:10,000 ration of TAPBPR:MHC-I placeholder peptide produces peptide receptive MHC-I as efficiently as a 1:1 ratio. 

2020-251 - co-expression of TAPBPR with MHC-I in mammalian cells. This has the following advantages: (a) placeholder peptides are not needed, (b) peptide receptive MHC-I can be purified directly from the culture, (c) the MHC-I molecules are glycosylated as they would be in a mammalian immune system. 

 

Technology Description

This system involves expression of MHC class I heavy chains and light chains in bacteria. The heavy chains and light chains are refolded in the presence of  placeholder peptide and the chaperone TAPBPR. The placeholder peptide is a peptide with a Tm value of the particular MHC class I epitope that is below 50 degrees Celsius. 

The system has advantages over other systems in that it provides greater MHC-I protein expression in E. coli cells but also provides more efficient peptide exchange. Existing systems can only do one. Result is 5-10x yield in total peptide-MHC compared to other systems

 


 Photosensitive Peptide Disulfide Mutant TAPBPR
Yield/Liter of refolding  4.0 +/- 0.5 mg 0.5 +/- 0.2 mg 5.0 +/- 1.5 mg
 Peptide exchange efficiency 30-40% 80-90% 80-90%
 Total pMHC yield 1.6 mg    0.4 mg 4 mg 

 Any MHC can be adapted to this technique. Specific placeholder peptides are provided for HLA-A*02, H-2D, and H-2L. 

 DNA barcoded MHC-I molecules can readily be prepared to better pair TCR sequence with antigen specificity: 

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Applications

Peptide-receptive MHC-I multimer reagents 

MHC-I reagents 

Identifying antigenic peptides

Identifying and purifying T cell populations  

 

 

Advantages

Peptide receptive MHC-I is provided - user can select the peptide

More efficient MHC-I expression than other methods

More efficient peptide exchange than other methods

More biologically accurate (no engineered disulfide bonds)

Easier to make (no need to work in the dark) 

Intellectual Property Information

Country Type Number Dated Case
United States Of America Published Application 20230059548 02/23/2023 2019-975
European Patent Office Published Application EP 4 136 098 02/22/2023 2020-284
European Patent Office Published Application 4085069 11/09/2022 2019-975
European Patent Office Published Application 4085068 11/09/2022 2020-297
European Patent Office Published Application 402841.2 07/20/2022 2020-251
United States Of America Published Application 20210371499 12/20/2021 2020-284
United States Of America Published Application 20210155670 05/27/2021 2020-251
United States Of America Published Application 20210079461 03/18/2021 2018-408
 

Additional Patent Pending

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Keywords

MHC-I, Class I MHC, Antigenic peptide, Peptide receptive MHC, MHC multimers, MHC tetramers, MHC multimer reagents, MHC tetramer reagents, MHCI, Major Histocompatibility Complex, Class I, HLA-A*24, HLA-A*68, HLA-A*2, H-2D, H-2L, chaperone, TAPBPR

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