# US 12,215,313 B2 · Method of detecting new immunogenic T cell epitopes and isolating new antigen-specific T cell receptors by means of an MHC cell library

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- Number: US 12,215,313 B2
- Document ID: US12215313B2
- Status: Granted
- Kind: B2
- Application number: 17/313,761
- Filed: 2021-05-06
- Published: 2021-08-26
- Granted: 2025-02-04
- Earliest claimed: 2015-03-16
- Assignees: Max-DelbrüCk-Centrum FüR Molekulare Medizin In Der Helmholtz-Gemeinschaft; Zentrum MüNchen-Deutsches Forschungszentrum FüR Gesundheit Und Umwelt (GmbH)
- Applicants: Max-DelbrüCk-Centrum FüR Molekulare Medizin In Der Helmholtz-Gemeinschaft; Helmholtz Zentrum MüNchen-Deutsches Forschungszentrum FüR Gesundheit Und Umwelt (GmbH)
- Inventors: Felix Lorenz; Wolfgang Uckert; Christian Ellinger; Dolores Schendel
- Web page: https://patentgrep.com/patent/US12215313B2

## CPC

- A61P 31/20: for DNA viruses
- C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display}
- A61P 37/04: Immunostimulants
- C07K 14/005: from viruses
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C12N 2710/16122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2710/16134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2710/20022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2710/20034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The present invention relates to the field of immunotherapy, in particular, to adoptive T cell therapy, T cell receptor (TCR) gene therapy and vaccination. The invention provides a method for preparing a nucleic acid encoding the TCR alpha chain construct (TRA) and TCR beta chain construct (TRB) of a TCR construct specific for an epitope from an antigen presented on major histocompatibility complex (MHC), comprising contacting T cells isolated from a donor with a library of artificial antigen presenting cells (APC) comprising cells expressing all MHC I or MHC II alleles present in the donor, preferably, in K562 cells. The TCR construct can be expressed in a T cell, which is useful for adoptive T cell therapy, e.g., of cancer, viral infections or autoimmune diseases. The invention further provides a method for identifying the epitope recognized by said TCR. Immunogenic epitopes recognized by said TCRs can be used to develop vaccine formulations to induce antigen-specific T cell immunity in patients. The invention further provides pairs of two TCR constructs and respective immunogenic epitopes obtained by the method of the invention, wherein the epitopes are from human papillomavirus (HPV) 16 (also designated alphapapillomavirus 9) oncoprotein E5 and human cytomegalovirus (CMV) protein pp65.

## Claims

### Claim 1

A method for preparing a nucleic acid encoding the TRA and TRB of a TCR construct specific for an immunodominant epitope from a defined antigen presented on a MHC, comprising
(a) stimulating T cells isolated from a donor with professional antigen presenting cells presenting epitopes of said defined antigen, to enrich antigen-specific T cells; and
(b) contacting said T cells with a library of cells, wherein each cell expresses a single MHC allele, wherein the library comprises cells expressing all MHC II alleles present in the donor, and wherein the cells of said library present epitopes of said defined antigen; wherein T cells expressing TCR specific for said epitopes become activated T cells; and
(c) selecting T cells activated by said contact in step b, preferably, based on an activation marker expressed by said activated T cells; and
(d) isolating the nucleic acids encoding the TCR alpha and TCR beta chains of the TCR of said activated T cells selected in step c.

### Claim 2 (depends on 1)

The method of claim 1, wherein the library of cells comprises MHC II-expressing K562 cells.

### Claim 3 (depends on 1)

The method of claim 1, wherein the method further comprises optimizing the sequence of the nucleic acid of step d.

### Claim 4 (depends on 1)

A method for preparing a T cell expressing a TCR construct specific for an epitope from a defined antigen presented on a MHC, comprising carrying out the method of claim 1, and expressing said nucleic acids encoding the TRA and TRB in a T cell.

### Claim 5 (depends on 1)

A method for identifying an epitope capable of being presented by a MHC in a defined antigen, comprising carrying out steps (a)-(d) of claim 1 and identifying the epitope capable of activating T cells transfected with nucleic acids encoding the isolated TRA and TRB constituting the TCR construct, wherein the epitope is optionally prepared in peptide or nucleic acid form.

### Claim 6 (depends on 3)

The method of claim 3, further comprising optimizing codon usage of the TRA and TRB.

### Claim 7 (depends on 3)

The method of claim 3, further comprising combining human variable regions with murine constant regions or minimal murine constant regions.

### Claim 8 (depends on 1)

The method of claim 1, wherein the professional antigen presenting cells express CD74 and HLA-DM.