METHOD OF DETECTING NEW IMMUNOGENIC T CELL EPITOPES AND ISOLATING NEW ANTIGEN-SPECIFIC T CELL RECEPTORS BY MEANS OF AN MHC CELL LIBRARY

• US 20180073013A1
• Filed: 03/15/2016
• Published: 03/15/2018
• Est. Priority Date: 03/16/2015
• Status: Active Grant

• Current Assignee: Helmholtz Zentrum Munchen Forschungszentrum Fur Gesundheit und Umwelt GmbH, Max-Delbruck-Centrum Fur Molekulare Medizin In Der Helmholtz-Gemeinschaft
• Original Assignee: Helmholtz Zentrum Munchen-Deutsches Forschungszentrum Fur Gesundheit Und, Max-Delbruck-Centrum Fur Molekulare Medizin In Der Helmholtz-Gemeinschaft
• Inventors: LORENZ, Felix, UCKERT, Wolfgang, ELLINGER, Christian, SCHENDEL, Dolores
• Granted Patent: US 11,001,830 B2
• Time in Patent Office: Days
• CPC Class Codes: A61P 31/20 for DNA viruses
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 2710/16122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2710/16134 Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 2710/20022 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2710/20034 Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

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

1. A nucleic acid encoding a T cell receptor (TCR) alpha chain construct (TRA) and/or TCR beta chain construct (TRB) of a TCR construct specific for an epitope in complex with a human MHC I, wherein the epitope is an epitope of human papillomavirus 16 oncoprotein E5,

wherein the nucleic acid is preferably obtainable from a method comprising

(a) stimulating T cells isolated from a donor with professional antigen presenting cells presenting epitopes of said defined antigen and sharing at least one MHC allele with the donor, 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 I alleles present in the donor, and wherein the cells of said library present epitopes of said defined antigen; and

(c) selecting T cells activated by said contact, 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 T cells.

2. The nucleic acid of claim 1, wherein the epitope is an epitope of human papillomavirus 16 oncoprotein E5 restricted to HLA-B*15:

01, which preferably comprises SEQ ID NO;

1.

3. The nucleic acid of claim 2, wherein the TRA comprises a CDR3 having at least 84% sequence identity to SEQ ID NO:

2, wherein the CDR3 preferably contains SEQ ID NO;

2, wherein the variable region of the TRA preferably has at least 80% sequence identity to SEQ ID NO;

3, and wherein the TRA preferably comprises SEQ ID NO;

4 and is encoded by SEQ ID NO;

5;

and/or wherein the TRB comprises a CDR3 having at least 84% sequence identity to SEQ ID NO;

6, wherein the CDR3 preferably contains SEQ ID NO;

6, wherein the variable region of TRB preferably has at least 80% sequence identity to SEQ ID NO;

7, and wherein the TRA preferably comprises SEQ ID NO;

8 and is encoded by SEQ ID NO;

9.

6. A recombinant host cell comprising a nucleic acid of claim 1 encoding the TRA and TRB of a TCR construct specific for an epitope of human papillomavirus 16 oncoprotein E5 or for an epitope of human cytomegalovirus protein pp65, and expressing said TCR, wherein the host cell preferably is a CD8+ T cell.

7. A protein encoded by the nucleic acid of claim 1, preferably, comprising TRA and TRB.

8. The protein of claim 7, wherein the TCR construct comprises TRA and TRB, for use in a pharmaceutical composition, preferably, for treatment of a patient infected with

a) human papillomavirus 16, wherein the TCR construct is specific for an epitope of human papillomavirus 16 oncoprotein E5 in complex with HLA-B*15:

01 and wherein the patient is HLA-B*15;

01-positive;

or

b) human cytomegalovirus, wherein the TCR construct is specific for an epitope of human cytomegalovirus protein pp65 in complex with HLA-B*07;

02 and wherein the patient is HLA-B*07;

02-positive.

9. A nucleic acid encoding a fragment of human papillomavirus 16 oncoprotein E5 having a length of up to 40 amino acids comprising an epitope, or a peptide fragment of human papillomavirus 16 oncoprotein E5 having a length of up to 40 amino acids comprising an epitope, wherein the epitope is capable of being recognized by the TCR construct of the recombinant host cell of claim 6,

wherein the epitope is preferably identifiable by a method comprising

(a) stimulating T cells isolated from a donor with professional antigen presenting cells presenting epitopes of said defined antigen and sharing at least one MHC allele with the donor, 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 I alleles present in the donor, and wherein the cells of said library present epitopes of said defined antigen; and

(c) selecting T cells activated by said contact, preferably, based on an activation marker expressed by said activated T cells; and

(d) identifying the epitope capable of activating said selected T cells.

10. The nucleic acid or peptide of claim 9, wherein the E5 epitope comprises SEQ ID NO:

1 and is selected from the group consisting of SEQ ID NO;

1, 45, 47, 49, 51, 52 and 57.

11. The nucleic acid or peptide of claim 9 for use in a pharmaceutical composition, preferably, for preventing infection with human papillomavirus 16, or for treatment of a patient infected with human papillomavirus 16.

17. The recombinant host cell of claim 6, wherein the TCR construct comprises TRA and TRB, for use in a pharmaceutical composition, preferably, for treatment of a patient infected with

a) human papillomavirus 16, wherein the TCR construct is specific for an epitope of human papillomavirus 16 oncoprotein E5 in complex with HLA-B*15:

01 and wherein the patient is HLA-B*15;

01-positive;

or

b) human cytomegalovirus, wherein the TCR construct is specific for an epitope of human cytomegalovirus protein pp65 in complex with HLA-B*07;

02 and wherein the patient is HLA-B*07;

02-positive.

18. The nucleic acid of claim 1, wherein the TCR construct comprises TRA and TRB, for use in a pharmaceutical composition, preferably, for treatment of a patient infected with

a) human papillomavirus 16, wherein the TCR construct is specific for an epitope of human papillomavirus 16 oncoprotein E5 in complex with HLA-B*15:

01 and wherein the patient is HLA-B*15;

01-positive;

or

b) human cytomegalovirus, wherein the TCR construct is specific for an epitope of human cytomegalovirus protein pp65 in complex with HLA-B*07;

02 and wherein the patient is HLA-B*07;

02-positive.

4. A nucleic acid encoding a TRA and/or TRB of a TCR construct specific for an epitope in complex with a human MEW I, wherein the TCR construct is specific for an epitope of human cytomegalovirus protein pp65 in complex with HLA-B*07:

02, which epitope consists of SEQ ID NO;

10, wherein the TRA comprises a CDR3 according to SEQ ID NO;

11 and/or wherein the TRB comprises a CDR3 according to SEQ ID NO;

15.

5. The nucleic acid of claim 4, wherein the variable region of the TRA has at least 80% sequence identity to SEQ ID NO:

12, and wherein the TRA preferably comprises SEQ ID NO;

13 and is encoded by SEQ ID NO;

14; and

/or wherein the variable region of the TRB has at least 80% sequence identity to SEQ ID NO;

16 and wherein the TRB preferably comprises SEQ ID NO;

17 and is encoded by SEQ ID NO;

18.

12. A method for preparing a nucleic acid encoding the TRA and TRB of a TCR construct specific for an 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 I or MHC II alleles present in the donor, and wherein the cells of said library present epitopes of said defined antigen; and

(c) selecting T cells activated by said contact, 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 T cells.

13. The method of claim 12, wherein the MHC is MHC I, wherein the library of cells preferably comprises MHC I-expressing K562 cells.

14. The method of claim 12, wherein the method further comprises optimizing the sequence, preferably, optimizing codon usage of the TRA and TRB and, optionally, combining human variable regions with murine constant regions or minimal murine constant regions.

15. 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 12, and expressing said nucleic acids encoding the TRA and TRB in a T cell.

16. 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 12 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.