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EP-3590958-B1 - TCR LIBRARIES

EP3590958B1EP 3590958 B1EP3590958 B1EP 3590958B1EP-3590958-B1

Inventors

  • JAKOBSEN, BENT KARSTEN
  • MOLLOY, PETER EAMON
  • VUIDEPOT, ANNELISE BRIGITTE
  • LIDDY, NATHANIEL ROSS

Dates

Publication Date
20260506
Application Date
20150313

Claims (16)

  1. A library of particles, the library displaying a plurality of different T cell receptors (TCRs), wherein: (a) the plurality of TCRs consists essentially of TCRs comprising an alpha chain comprising an alpha chain variable domain comprising a TRAV12-2 or a TRAV21 gene product and a beta chain comprising a beta chain variable domain comprising a TRBV6 gene product; (b) the alpha and beta chain variable domains of the TCRs are expressed from DNA sequences that have been obtained from human donors by producing cDNA from donor mRNA; and (c) the plurality of TCRs consists of TCRs comprising an alpha chain variable domain and a beta chain variable domain derived from naturally occurring sequences which have not been modified or mutated with reference to the donor mRNA that serves as the template for generating the cDNA from which the TCR chains are obtained.
  2. The library according to claim 1, wherein the TRBV6 gene product is a TRBV6-1, a TRBV6-2, a TRBV6-3, a TRBV6-5 or a TRBV6-6 gene product.
  3. The library according to claim 1 or claim 2, wherein the TCR alpha chain variable domain comprises a TRAV12-2 gene product, or a TRAV21 gene product.
  4. The library according to any one of claims 1 to 3, wherein the alpha chain variable domain and the beta chain variable domain are displayed as a single polypeptide chain.
  5. The library according to any one of claims 1 to 4 wherein the TCRs comprise a non-native disulphide bond or a native disulphide bond between a constant region of the alpha chain and a constant region of the beta chain.
  6. The library according to any one of claims 1 to 5 wherein the particles are phage particles, ribosomes, yeast cells or mammalian cells.
  7. A non-natural isolated T cell receptor (TCR) comprising a TCR alpha chain variable domain comprising a TRAV12-2 gene product or a TRAV21 gene product and a TCR beta chain variable domain comprising a TRBV6 gene product obtained from a library according to any one of claims 1 to 6.
  8. Use of a library according to any one of claims 1 to 6, to identify a TCR that specifically binds to a peptide antigen.
  9. A method of obtaining a T cell receptor that specifically binds a peptide antigen, comprising screening the library according to any one of claims 1 to 6 with the peptide antigen, the method comprising: a) panning the library using as a target the peptide antigen; b) repeating step a) one or more times; c) screening the phage clones identified in step a) or b); and d) identifying a TCR that specifically binds the peptide antigen.
  10. A nucleic acid encoding a TCR alpha chain variable domain and/or a beta chain variable domain of the TCR according to claim 7.
  11. A method of making a library of particles according to any one of claims 1 to 6, the library displaying a plurality of different TCRs, the method comprising: i) obtaining a plurality of nucleic acids that encode different TRAV12-2 or TRAV21 alpha chain variable domains; ii) obtaining a plurality of nucleic acids that encode different TRBV6 beta chain variable domains; iii) cloning the TRAV12-2 or TRAV21 alpha chain variable domain encoding nucleic acids into expression vectors; iv) cloning the TRBV6 beta chain variable domain encoding nucleic acids into the same or different vectors; and v) expressing the vectors in particles, thereby generating a library consisting essentially of TCRs comprising an alpha chain variable domain and a beta chain variable domain encoded by the nucleic acids.
  12. A method of making a library of particles according to any one of claims 1 to 6, the library displaying a plurality of different TCRs, the method comprising: i) obtaining a plurality of nucleic acids that encode different TRAV12-2 or TRAV21 alpha chain variable domains using primers that hybridise to nucleic acids encoding TRA12-2 or TRAV21 alpha chain variable domains; ii) obtaining a plurality of nucleic acids that encode different TRBV6 beta chain variable domains using primers that hybridise to nucleic acids encoding TRAV6 beta chain variable domains; iii) cloning the TRAV12-2 or TRAV21 alpha chain variable domain encoding nucleic acids into expression vectors; iv) cloning the TRBV6 beta chain variable domain encoding nucleic acids into the same or different vectors; and v) expressing the vectors in particles, thereby generating a library consisting essentially of TCRs comprising an alpha chain variable domain and a beta chain variable domain encoded by the nucleic acids to which said primers hybridise.
  13. The method of claim 11 or claim 12, wherein the nucleic acids of step (i) and step (ii) are obtained from a natural repertoire.
  14. A method according to any one of claims 11 to 13, wherein the TCR alpha chain variable domain and the TCR beta chain variable domain are expressed as a single chain polypeptide.
  15. A method of obtaining a T cell receptor that specifically binds a peptide antigen, comprising screening the library according to any one of claims 1 to 6 with the peptide antigen.
  16. A particle displaying on its surface a TCR according to claim 7, optionally wherein the particle is a phage particle, a ribosome, a yeast cell or a mammalian cell.

Description

Summary of the Invention The present invention relates to a library of particles, the library displaying a plurality of different T cell receptors (TCRs), wherein: (a) the plurality of TCRs consists essentially of TCRs comprising an alpha chain variable domain comprising a TRAV12-2 or a TRAV21 gene product and a beta chain variable domain comprising a TRBV6 gene product, wherein the alpha chain variable domain comprises a TRAV12-2 or a TRAV21 gene product and the beta chain variable domain comprises a TRBV6 gene product; (b) the alpha and beta chain variable domains of the TCRs are expressed from DNA sequences that have been obtained from human donors by producing cDNA from donor mRNA; and (c) the plurality of TCRs consists of TCRs comprising an alpha chain variable domain and a beta chain variable domain derived from naturally occurring sequences which have not been modified or mutated with reference to the donor mRNA that serves as the template for generating the cDNA from which the TCR chains are obtained. Background T cell receptors (TCRs) mediate the recognition of specific major histocompatibility complex (MHC)-restricted peptide antigens by T cells and are essential to the functioning of the cellular arm of the immune system. TCRs exist only in membrane bound form and for this reason TCRs are historically very difficult to isolate. Most TCRs are composed of two disulphide linked polypeptide chains, the alpha and beta chain. TCRs are described herein using the International Immunogenetics (IMGT) TCR nomenclature and links to the IMGT public database of TCR sequences. Native alpha-beta heterodimeric TCRs have an alpha chain and a beta chain. Broadly, each chain comprises variable, joining and constant regions, and the beta chain also usually contains a short diversity region between the variable and joining regions, but this diversity region is often considered as part of the joining region. Each variable region comprises three hypervariable CDRs (Complementarity Determining Regions) embedded in a framework sequence; CDR3 is believed to be the main mediator of antigen recognition. There are several types of alpha chain variable (Vα) regions and several types of beta chain variable (Vβ) regions distinguished by their framework, CDR1 and CDR2 sequences, and by a partly defined CDR3 sequence. The Vα types are referred to in IMGT nomenclature by a unique TRAV number. Thus "TRAV21" defines a TCR Vα region having unique framework and CDR1 and CDR2 sequences, and a CDR3 sequence which is partly defined by an amino acid sequence which is preserved from TCR to TCR but which also includes an amino acid sequence which varies from TCR to TCR. In the same way, "TRBV6-5" defines a TCR Vβ region having unique framework and CDR1 and CDR2 sequences, but with only a partly defined CDR3 sequence. For the purposes of this application, we are using the general assumption that there are 54 functional alpha variable genes and 67 functional beta variable genes within the alpha and beta loci respectively. However, this number may vary as research progresses and the number of alpha and beta variable genes may be considered to be different than at the present time due to definitions of functionality or duplications. Thus, for the sake of clarity, we consistently refer to the International Immunogenetics (IMGT) TCR nomenclature as found at the IMGT website www.imgt.org (as accessed 10 March 2013). The joining regions of the TCR are similarly defined by the unique IMGT TRAJ and TRBJ nomenclature, and the constant regions by the IMGT TRAC and TRBC nomenclature. The beta chain diversity region is referred to in IMGT nomenclature by the abbreviation TRBD, and, as mentioned, the concatenated TRBD/TRBJ regions are often considered together as the joining region. The gene pools that encode the TCR alpha and beta chains are located on different chromosomes and contain separate V, (D), J and C gene segments, which are brought together by rearrangement during T cell development. This leads to a very high diversity of T cell alpha and beta chains due to the large number of potential recombination events that occur between the 54 TCR alpha variable genes and 61 alpha J genes or between the 67 beta variable genes, two beta D genes and 13 beta J genes. The recombination process is not precise and introduces further diversity within the CDR3 region. Each alpha and beta variable gene may also comprise allelic variants, designated in IMGT nomenclature as TRAVxx*01 and *02, or TRBVx-x*01 and *02 respectively, thus further increasing the amount of variation. In the same way, some of the TRBJ sequences have two known variations. (Note that the absence of a"*" qualifier means that only one allele is known for the relevant sequence.). The natural repertoire of human TCRs resulting from recombination and thymic selection has been estimated to comprise approximately 106 unique beta chain sequences, determined from CDR3 diversity (Arstila, T. P., et al (