Methods and compositions for producing a cell expressing a t cell receptor

Patent metadata

Field	Value
Publication number	US-2016340403-A1
Application number	US-201515111639-A
Country	US
Kind code	A1
Filing date	Jan 28, 2015
Priority date	Jan 29, 2014
Publication date	Nov 24, 2016
Grant date	—

Abstract

Provided is a method for determining a TCR polypeptide chain that can form a TCR specific for a peptide of interest. Also provided are methods and compositions for producing a cell expressing a T cell receptor (TCR) specific for a peptide of interest, methods and compositions for producing a TCR chain nucleic acid and/or pair of TCR chain polypeptides and/or nucleic acids encoding a TCR, a cell population comprising the cell harboring the nucleic acids encoding a TCR obtained by said method, and a method for treating a disorder comprising administering to the subject said cell population.

First claim

1 . A method for obtaining a high affinity T cell receptor TCR, a recombinant cell expressing a TCR specific for a peptide of interest and/or a nucleic acid encoding a TCR polypeptide chain that can form a TCR specific for a peptide of interest comprising:
a. transducing a cell population comprising cells able to express a TCR and/or differentiate into cells able to express a TCR, with a bait nucleic acid encoding a bait TCR polypeptide chain, wherein the bait TCR polypeptide chain can constitute a parent TCR with a counterchain TCR polypeptide chain that specifically binds said peptide of interest; b. culturing the transduced cell population under conditions that permit the bait TCR polypeptide chain to be expressed; and c. one or more of:
i) obtaining a recombinant cell expressing a TCR comprising the bait TCR polypeptide chain and a prey TCR polypeptide chain that selectively binds said peptide of interest from the transduced cell population obtained in step (b);
ii) isolating a prey nucleic acid encoding the prey TCR polypeptide chain from the cell obtained in step (c)(i); and/or
iii) pairing the isolated prey nucleic acid isolated in step (c)(ii) with the bait nucleic acid which when expressed provide a high affinity TCR.

2 . The method of claim 1 , wherein the step of obtaining the recombinant cell expressing a TCR comprising the bait TCR polypeptide chain and the prey TCR polypeptide chain that selectively binds said peptide of interest from the transduced cell population obtained in step (b) comprises isolating one or more cells of the cell population that express the transduced bait TCR polypeptide chain and which bind the peptide of interest.

3 . The method of claim 1 , wherein the prey nucleic acid is isolated by cloning the prey nucleic acid.

4 . The method of claim 1 , wherein the prey TCR polypeptide chain comprises a CDR3 region comprising at least one amino acid modification relative to the CDR3 region of a control TCR polypeptide CDR3 region, optionally the CDR3 region of the cognate polypeptide chain in the parent TCR.

5 . The method of claim 1 , wherein the method further comprises: i) introducing the isolated prey nucleic acid and the bait nucleic acid into a cell able to express a TCR or differentiate into a cell able to express a TCR under conditions that permit the bait TCR polypeptide chain to be expressed; ii) measuring the avidity and/or affinity of the TCR comprising the prey TCR polypeptide chain and the bait TCR polypeptide chain; and iii) isolating a prey nucleic acid clone encoding the prey TCR polypeptide chain, wherein the bait TCR polypeptide chain and the prey TCR polypeptide chain constitute a TCR having increased avidity and/or affinity for the peptide of interest compared to the control TCR, optionally the parent TCR.

6 . The method of claim 1 for obtaining a nucleic acid encoding a TCR polypeptide chain which in combination with a counterchain TCR polypeptide constitutes a TCR specific for a peptide of interest, the method comprising the steps of:
a. transducing a cell population comprising cells able to express a TCR or differentiate into cells able to express a TCR, with a bait nucleic acid which encodes a bait TCR polypeptide chain,
optionally selected from TCRalpha and TCRbeta polypeptide chains, which in combination with a counterchain TCR polypeptide chain constitutes a TCR, wherein the bait TCR polypeptide chain was previously isolated from a T cell recognizing said peptide of interest;
b. culturing the transduced cell population under conditions that permit the bait TCR polypeptide chain to be expressed;
c. obtaining a cell expressing the TCR specific for a peptide of interest from the transduced cell population obtained in step (b); and
d. isolating a prey nucleic acid encoding a prey TCR polypeptide chain which constitutes a TCR with the polypeptide encoded by the bait nucleic acid transduced, into said cell population from the cell obtained in step (c).

7 . The method of claim 1 , wherein the step of obtaining the recombinant cell expressing the TCR comprises the step of culturing the transduced cell population with an antigen presenting cell presenting the peptide of interest.

8 . The method of claim 1 , wherein the step of obtaining the recombinant cell expressing the TCR specific for a peptide of interest from the transduced cell population comprises using cell sorting, optionally fluorescence-activated cell sorting (FACS).

9 . The method of claim 1 for obtaining a pair of nucleic acids encoding a TCR specific for a peptide of interest, which comprises the steps of:
a. transducing a cell population with a bait nucleic acid which encodes a bait TCR polypeptide chain selected from the group of bait polypeptide chains constituting a TCR, wherein the bait TCR polypeptide chain was expressed and previously isolated from a T cell recognizing the peptide of interest, wherein the cell population comprises cells able to express a TCR or differentiate into cells able to express a TCR;
b. culturing the transduced cell population under conditions that permit the bait TCR polypeptide chain to be expressed;
c. obtaining a recombinant cell expressing the TCR specific for a peptide of interest from the transduced cell population obtained in step (b);
d. isolating a prey nucleic acid encoding a polypeptide which constitutes a TCR with the polypeptide encoded by the bait nucleic acid transduced into said cell population from the cell obtained in step (c); and
e. pairing the bait nucleic acid transduced into said cell population in step (a) with the prey nucleic acid isolated in step (d).

10 . The method of claim 1 , wherein the obtaining the recombinant cell step (c) comprises the step of culturing the transduced cell population with an antigen presenting cell presenting the peptide of interest.

11 . The method of claim 1 , wherein the cell population is a population of peripheral blood mononuclear cells (PBMCs) optionally PBMCs activated with a CD3 ligand.

12 . The method of claim 1 , wherein the bait nucleic acid transduced into said cell population in step (a) encodes a TCR alpha chain or a TCR beta chain.

13 . The method of claim 1 , wherein the bait nucleic acid transduced into said cell population in step (a) encodes a TCR chain which predominantly contributes to peptide recognition by a TCR.

14 . The method of claim 1 , wherein the transduction is repeated a second, third, fourth, fifth and/or sixth time.

15 . The method of claim 1 , wherein the isolated prey nucleic acid encoding the prey TCR polypeptide chain is transduced into a cell able to express a TCR or differentiate into a cell able to express a TCR, optionally wherein the isolated prey nucleic acid is transduced in combination with a nucleic acid encoding a TCR polypeptide chain that in combination with the prey TCR polypeptide chain constitutes a TCR, optionally the bait TCR nucleic acid, to produce a transduced cell population comprising recombinant cells expressing a TCR specific for a peptide of interest.

16 . The method of claim 1 , wherein the cell population or cell is also transduced with an antisense molecule for suppressing expression of an endogenous TCR chain.

17 . The method of claim 1 wherein one or more of the nucleic acids being transduced, optionally the bait nucleic acid, is codon optimized.

18 . An isolated and/or recombinantly engineered TCR polypeptide chain and/or a TCR comprising said TCR polypeptide chain, said TCR polypeptide chain comprising a sequence selected from SEQ ID NO: 4, 6, 8, 10, 12, 14, 52,

Assignees

• Univ Health Network

Inventors

• Hirano Naoto
• Nakatsugawa Munehide
• Ochi Toshiki

Classifications

• A61P31/16

  for influenza or rhinoviruses · CPC title

• A61P31/06

  for tuberculosis · CPC title

• A61P31/10

  Antimycotics · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• A61P31/18

  for HIV · CPC title