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US-12624349-B2 - Methods of high-throughput identification of T cell epitopes by capturing cytokines on the surface of antigen-presenting cells

US12624349B2US 12624349 B2US12624349 B2US 12624349B2US-12624349-B2

Abstract

The present disclosure relates to methods that combine identification of T cell-secreted cytokines by modified antigen-presenting cells (APCs), cell sorting, and next-generation sequencing to identify class I- and class II-restricted epitopes starting from massively-complex peptide-encoding oligonucleotide pools. APCs are modified to express anti-cytokine antibodies, a library of DNA-encoded peptides, and multiple HLA class I or II molecules. Upon co-culture with T cells, these modified APCs form HLA/epitope/TCR complexes, enabling the production of T cell activation-dependent cytokines with the DNA that encodes the presented peptide. After co-culture, the APCs are sorted, and the peptide-encoding DNA is sequenced to determine the identity of the immunogenic peptides. Thus, the disclosure allows pooled screening of thousands of encoded peptides to enable epitope discovery for orphan T cell receptors.

Inventors

  • Matthew Meyerson
  • Mark N. Lee

Assignees

  • DANA-FARBER CANCER INSTITUTE, INC.

Dates

Publication Date
20260512
Application Date
20200923

Claims (20)

  1. 1 . A method of identifying epitopes that activate T cells, comprising: providing a plurality of antigen presenting cells (APCs), wherein each APC expresses a) a nucleic acid encoding a candidate epitope or a nucleic acid encoding a peptide that may be processed into a candidate epitope, b) a nucleic acid encoding an HLA molecule, and c) a nucleic acid encoding an anti-cytokine antibody; mixing the plurality of APCs with a plurality of T cells, wherein each T cell expresses a T cell receptor (TCR) on its surface, wherein binding of the TCR on the T cells to the candidate epitope activates the T cells, wherein the activated T cells secrete a cytokine that binds to the anti-cytokine antibody; and identifying APCs bound to the cytokine; and sequencing the nucleic acid encoding the candidate epitope contained in the APCs to which the cytokine is bound.
  2. 2 . The method of claim 1 , wherein the APCs are professional APCs.
  3. 3 . The method of claim 2 , wherein the professional APCs are dendritic cells, macrophages, monocytes or B cells.
  4. 4 . The method of claim 1 , wherein the APCs are non-professional APCs.
  5. 5 . The method of claim 1 , wherein the APCs are human APCs.
  6. 6 . The method of claim 1 , wherein the candidate epitope is an infectious disease-associated candidate epitope, an autoimmune disease-associated candidate epitope, or a tumor-associated candidate epitope, or wherein the APCs are non-professional APCs of an immortalized cell line; or wherein the HLA molecule is a class I HLA molecule.
  7. 7 . The method of claim 1 , wherein the HLA molecule is a class II HLA molecule.
  8. 8 . The method of claim 1 , wherein the anti-cytokine antibody is an anti-IL-2 antibody, or wherein the anti-cytokine antibody is an anti-INF-γ antibody.
  9. 9 . The method of claim 1 , wherein the T cells comprise CD8 + T cells.
  10. 10 . The method of claim 1 , wherein the T cells comprise CD4 + T cells.
  11. 11 . The method of claim 1 , wherein the identifying comprises contacting the APCs after contact with the plurality of T cells with a detectable label that binds the cytokine; and detecting the label.
  12. 12 . The method of claim 11 , wherein the detectable label comprises a fluorescently-labeled, secondary anti-cytokine antibody.
  13. 13 . The method of claim 1 , further comprising sorting labeled APCs from non-labeled APCs that do not bear a T cell-activating epitope.
  14. 14 . The method of claim 13 , wherein the sorting is conducted by magnetic or flow cytometry; or wherein the sequencing is conducted using next generation sequencing.
  15. 15 . The method of claim 1 , wherein the plurality of APCs comprises a library of APCs that expresses a library of the candidate epitopes.
  16. 16 . The method of claim 1 , wherein the candidate epitope is 8-24 amino acids in length, or wherein the candidate epitope is 8-15 amino acids in length, or wherein the candidate epitope is 8-12 amino acids in length.
  17. 17 . A modified APC, wherein the modified APC expresses a) an exogenous nucleic acid encoding a candidate epitope or an exogenous nucleic acid encoding a peptide that may be processed into a candidate epitope, b) an exogenous nucleic acid encoding an HLA molecule, and c) an exogenous nucleic acid encoding an anti-cytokine antibody.
  18. 18 . The modified APC of claim 17 , which is a professional APC.
  19. 19 . The modified APC of claim 18 , wherein the professional APC is a dendritic cell, macrophage, monocyte or a B cell.
  20. 20 . The modified APC of claim 17 , wherein the APC is a non-professional APC.

Description

RELATED APPLICATIONS This application is a National Stage application, filed under 35 U.S.C. § 371, of International Application No. PCT/US2020/052164, filed Sep. 23, 2020, which claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/904,473, filed on Sep. 23, 2019, each of which is incorporated herein by reference in its entirety. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 2, 2020, is named 52095-639001WO_SL.txt and is 239.79 kilobytes in size. GOVERNMENT LICENSE RIGHTS This invention was made with government support under grant number R35 CA197568 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION T cell activation cascade is critical for the initiation of the immune response and within the human system revolves around three stages. The first stage involves binding between a T cell receptor (TCR) with programmed specificity to a particular foreign molecule (antigen), specifically an epitope of the antigen presented on an antigen presenting cell (APC). The second stage involves binding of a T cell to the APC to initiate T cell proliferation. The third stage involves secretion of cytokines from activated T cells that send signals to different types of immune responder cells. Activation of T cells enables cytotoxic CD8 T cells to develop cell-mediated immune system mechanisms. It also promotes the engagement of accessory immune cells such as macrophages. Further, the activation cascade increases antibody responses through the T-helper cells (CD4) and the enhancement of antibody production by B cells. (T-Cell Activation, Susan Pross, xPharm: The Comprehensive Pharmacology Reference, 2007). Improper or defective T cell activation cause a variety of autoimmune diseases (e.g., type 1 diabetes mellitus in infancy, hypothyroidism, and Addison's disease) which attack a subject's own immune cells, as well as uncontrolled cell proliferation associated with cellular checkpoint blockades in cancer immunotherapies. T cell receptors (TCRs) on the surface of T cells are used by the immune system to identify foreign molecules in order to trigger an immune response. TCRs recognize small peptides, called epitopes that are bound to human leukocyte (HLA) antigen proteins. The number of possible candidate epitopes is large. Variation in human leukocyte antigen (HLA) and T cell receptor (TCR) genes is associated with risk of infection and autoimmunity (International HIV Controllers Study et al., 2010 Science, 330:1551-7; Gutierrez-Arcelus et al., 2016 Nat. Rev. Genet., 17:160-174; Miyadera et al., 2015 J. Hum. Genet., 60:697-702), and can influence patient survival to checkpoint blockade immunotherapy (Zaretsky et al., 2016 N. Engl. J. Med., 375:819-829; Chowell et al., 2018 Science, 359:582-587). Identification of the specific complexes between HLA molecules epitopes, and TCRs—resulting in T cell stimulation—provides fundamental information about disease pathogenesis (Miyadera et al., 2015 J. Hum. Genet., 60:697-702; Latorre et al., 2018 Nature, 562:63-68; Tran et al., 2016 N. Engl. J. Med., 375:2255-2262; Zacharakis et al., 2018 Nat. Med., 24:724-730). Productive interactions between a T cell (e.g., cytotoxic T cell) and an antigen, such as an antigen presented by an APC, are rare. They may often occur among fewer than one out of one million target cells. An antigen recognized by a given T cell is typically present at exceedingly low frequencies, e.g., 1 in 100,000 antigens or less. Further, not every target cell displaying a given antigen will encounter its cognate T cell, especially given the specificities of mixed T cell populations. Other factors of a technological nature present barriers to identifying which HLA, epitopes, and TCRs that productively lead to T cell activation. These barriers, in part, emerge from the significant inter- and intra-individual variation in HLA (Dendrou et al., 2018 Nat. Rev. Immunol., 18:325-339 (2018)) and TCR genes (Robins et al., 2009 Blood., 114:4099-107; Robins et al., 2010 Sci. Transl. Med., 2, 47ra64; Emerson et al., 2017 Nat. Genet., 49:659-665), as well as the vast potential space of candidate peptide epitopes (Lundegaard et al., 2010 Immunome Res., 6:S3623-629). Accordingly, efforts to identify T cell receptor interactions with epitopes thereof have focused on individual or small numbers of pairs of interactions based on direct measurement of T cell responses. Functional assays, notably enzyme-linked immunospot (ELISpot) assays (Czerkinsky et al., 1988 J. Immunol. Methods., 110:29-36) and HLA multimer assays (Altman et al., 1996 Science., 274:94-6; Newell et al., 2013 Nat. Biotechnol., 31:623-629; Bentzen et al., 2016 Nat. Biotechnol., 34:1037-1045), have been in widespread use to detect HLA-epitope-TC