Method for identifying MHC-presented peptide epitopes for T cells

• US 20040110253A1
• Filed: 08/13/2003
• Published: 06/10/2004
• Est. Priority Date: 08/13/2002
• Status: Abandoned Application

• Current Assignee: National Jewish Medical and Research Center
• Original Assignee: National Jewish Medical and Research Center
• Inventors: Crawford, Frances G., Marrack, Philippa, Kappler, John W.
• Application Number:
  US10/640,881
• Publication Number:
  US 20040110253A1
• Time in Patent Office: Days
• US Class Current: 435/69.1
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: G01N 33/56977 HLA or MHC typing
• CPC Class Codes: G01N 33/6878 in eptitope analysis

Abstract

Described are three basic components: (1) methods for the display of functional MHC molecules with covalently attached antigenic peptides on the surface of baculovirus and baculovirus infected insect cells; (2) methods for the identification and physical isolation of baculovirus or baculovirus infected insect cells bearing a displayed MHC/peptide combination that is recognized by a particular T cell antigen receptor; and (3) methods for producing libraries of baculovirus or baculovirus infected insect cells displaying a particular MHC molecule and many different potential antigenic peptides.

Claims

1. A recombinant baculovirus expression vector for expression of functional MHC-peptide molecules, comprising a baculovirus genome comprising:

a) a first nucleic acid sequence inserted into a first baculovirus structural gene at a position under control of a promoter for the first baculovirus structural gene, wherein the first nucleic acid sequence encodes at least a portion of the extracellular domains of the α

chain of a major histocompatibility complex (MHC) Class I molecule or at least a portion of the extracellular domains of the α

chain of a MHC Class II molecule;

b) a second nucleic acid sequence inserted into a second baculovirus structural gene at a position under control of a promoter for the second baculovirus structural gene, wherein the second nucleic acid sequence encodes at least a portion of the extracellular domains of;

i) a β

2-microglobulin (β

2m) chain of a MHC Class I molecule if the first nucleic acid sequence encodes at least a portion of the extracellular domains of the α

chain of a MHC Class I molecule;

or

ii) a β

chain of a MHC Class II molecule if the first nucleic acid sequence encodes at least a portion of the extracellular domains of the α

chain of a MHC Class II molecule;

c) a third nucleic acid sequence encoding an MHC-binding peptide;

d) a fourth nucleic acid sequence encoding a peptide linker, wherein the third nucleic acid sequence encoding the MHC-binding peptide is connected to the 5′

end of the first or second nucleic acid sequence by the fourth nucleic acid sequence; and

e) a fifth nucleic acid sequence encoding at least a transmembrane region of a membrane protein, wherein the first or the second nucleic acid sequence is inserted into the baculovirus genome in frame with the fifth nucleic acid sequence, the fifth nucleic acid sequence being located after the 3′

end of the first or second nucleic acid sequence;

wherein the portion of the extracellular domains of the α

chain of the MHC Class I molecule and the portion of the extracellular domains of the β

2m chain of the MHC Class I molecule, or the portion of the extracellular domains of the α

chain of the MHC Class II molecule and the portion of the extracellular domains of the β

chain of the MHC Class II molecule, form a peptide binding groove of an MHC molecule, and wherein the MHC-binding peptide comprises a sequence of amino acids that binds to the peptide binding groove.

2. The recombinant baculovirus expression vector of claim 1, wherein the first nucleic acid sequence encodes at least a portion of the extracellular domains of the α

chain of a MHC Class I molecule, and wherein the second nucleic acid sequence encodes at least a portion of the extracellular domains of a β

2m chain of a MHC Class I molecule.

3. The recombinant baculovirus expression vector of claim 2, wherein the third nucleic acid sequence encoding the MHC-binding peptide is connected to the 5′

end of the second nucleic acid sequence encoding at least a portion of the extracellular domains of a β

2m chain of a MHC Class I molecule by the fourth nucleic acid sequence encoding a peptide linker.

4. The recombinant baculovirus expression vector of claim 1, wherein the first nucleic acid sequence encodes at least a portion of the extracellular domains of the α

chain of a MHC Class II molecule, and wherein the second nucleic acid sequence encodes at least a portion of the extracellular domains of a β

chain of a MHC Class II molecule.

5. The recombinant baculovirus expression vector of claim 4, wherein the third nucleic acid sequence encoding the MHC-binding peptide is connected to the 5′

end of the second nucleic acid sequence encoding at least a portion of the extracellular domains of a β

chain of a MHC Class II molecule by the fourth nucleic acid sequence encoding a peptide linker.

6. The recombinant baculovirus expression vector of claim 1, wherein the fifth nucleic acid sequence encodes at least the transmembrane portion of a membrane protein selected from the group consisting of:

baculovirus envelope protein gp64, MHC Class I, MHC Class II, and p26.

7. The recombinant baculovirus expression vector of claim 1, wherein the fifth nucleic acid sequence encodes at least the transmembrane portion of baculovirus envelope protein gp64.

8. The recombinant baculovirus expression vector of claim 1, wherein the fifth nucleic acid sequence encodes a full-length gp64.

9. The recombinant baculovirus expression vector of claim 1, wherein the fifth nucleic acid sequence encodes only the transmembrane portion and cytoplasmic tail of gp64.

10. The recombinant baculovirus expression vector of claim 1, wherein the first nucleic acid sequence further comprises, 3′

of the nucleic acid sequence encoding the extracellular domains of the α

chain of an MHC molecule, a nucleic acid sequence encoding a basic leucine zipper dimerization helix.

11. The recombinant baculovirus expression vector of claim 1, wherein the second nucleic acid sequence further comprises, 3′

of the nucleic acid sequence encoding the extracellular domains of the β

chain of a Class II MHC molecule or the Class I β

2m molecule, a nucleic acid sequence encoding an acidic leucine zipper dimerization helix.

12. The recombinant baculovirus expression vector of claim 1, wherein the peptide linker encoded by the fourth nucleic acid molecule comprises at least about 8 amino acid residues, wherein the linker facilitates the binding of the MHC-binding peptide to the peptide binding groove of the MHC molecule.

13. The recombinant baculovirus expression vector of claim 1, wherein the MHC-binding peptide is from a library of candidate antigenic peptides, wherein the each of the peptides in the library comprises conserved amino acids in a specific sequence sufficient to enable the peptide to bind to the peptide binding groove of the MHC molecule that is encoded by the vector.

14. The recombinant baculovirus expression vector of claim 1, wherein the MHC-binding peptide is from a library of candidate antigenic peptides, wherein each of the peptides in the library comprises between about 4 and 5 conserved amino acids in a specific sequence sufficient to enable the peptide to bind to the peptide binding groove of the MHC molecule that is encoded by the vector.

15. The recombinant baculovirus expression vector of claim 1, wherein the MHC-binding peptide is from a library of candidate antigenic peptides representing from between about 10³ and about 10⁹ different candidate antigenic peptides.

16. A recombinant baculovirus comprising the recombinant baculovirus expression vector of claim 1, wherein the recombinant baculovirus expresses and displays on its surface a functional MHC-peptide molecule encoded by the vector.

17. A population of cells infected with the recombinant baculovirus of claim 16, wherein the cells display the functional MHC-peptide molecules expressed by the baculovirus on their surfaces.

18. A recombinant insect cell that displays on its surface a functional MHC-peptide molecule, wherein the recombinant insect cell:

a) has been transfected with recombinant nucleic acid molecules that encode at least the extracellular domains of an MHC molecule, the recombinant nucleic acid molecules comprising;

i) a first nucleic acid sequence operatively linked to an expression control sequence, wherein the first nucleic acid sequence encodes at least a portion of the extracellular domains of the α

chain of a major histocompatibility complex (MHC) Class I molecule or at least a portion of the extracellular domains of the α

chain of a MHC Class II molecule; and

ii) a second nucleic acid sequence operatively linked to an expression control sequence under control of a baculovirus promoter and enhancer, wherein the second nucleic acid sequence encodes at least a portion of the extracellular domains of;

(1) a β

2-microglobulin (β

2m) chain of a MHC Class I molecule if the first nucleic acid sequence encodes at least a portion of the extracellular domains of the α

chain of a MHC Class I molecule;

or

(2) a β

chain of a MHC Class II molecule if the first nucleic acid sequence encodes at least a portion of the extracellular domains of the α

chain of a MHC Class II molecule;

wherein the portion of the extracellular domains of the α

chain of the MHC Class I molecule and the portion of the extracellular domains of the β

2m chain of the MHC Class I molecule, or the portion of the extracellular domains of the α

chain of the MHC Class II molecule and the portion of the extracellular domains of the β

chain of the MHC Class II molecule, form a peptide binding groove of an MHC molecule; and

b) has been infected with a recombinant baculovirus comprising a third nucleic acid sequence under control of a baculovirus promoter and comprising a signal sequence, wherein the third nucleic acid sequence encodes an MHC-binding peptide, wherein the MHC-binding peptide comprises a sequence of amino acids that binds to the peptide binding groove of the MHC Class I molecule or the MHC Class II molecule.

19. A method for production of libraries of functional MHC-peptide molecules displayed on the surface of baculovirus and baculovirus-infected cells, comprising:

a) producing a population of recombinant baculoviruses by introducing into the genome of the baculoviruses;

i) a first nucleic acid sequence encoding at least a portion of the extracellular domains of the α

chain of a major histocompatibility complex (MHC) Class I molecule or at least a portion of the extracellular domains of the α

chain of a MHC Class II molecule, wherein the first nucleic acid sequence is introduced into the baculovirus genome at a position under control of a promoter for a first baculovirus structural gene;

ii) a second nucleic acid sequence encoding at least a portion of the extracellular domains of;

(1) a β

2-microglobulin (β

2m) chain of a MHC Class I molecule if the first nucleic acid sequence encodes at least a portion of the extracellular domains of the α

chain of a MHC Class I molecule;

or

(2) a β

chain of a MHC Class II molecule if the first nucleic acid sequence encodes at least a portion of the extracellular domains of the α

chain of a MHC Class II molecule;

wherein the second nucleic acid sequence is introduced into the baculovirus genome at a position under control of a promoter for a second baculovirus structural gene; and

wherein the portion of the extracellular domains of the α

chain of the MHC Class II molecule and the portion of the extracellular domains of the β

chain of the Class II MHC molecule, or the portion of the extracellular domains of the α

chain of the Class I MHC molecule and the portion of the extracellular domains of the β

2m chain of the Class I MHC molecule, respectively, form a peptide binding groove;

iii) a third nucleic acid sequence encoding a candidate antigenic peptide, wherein the candidate antigenic peptide is randomly produced from a possible library of candidate antigenic peptides so that each baculovirus in the population may express a different candidate antigenic peptide, wherein each of the peptides in the library comprises;

(1) conserved amino acid residues at specific positions in the sequence sufficient to enable the peptide to bind to the MHC molecule; and

(2) randomly generated amino acid residues in the remaining positions in the sequence;

wherein the third nucleic acid sequence is introduced into the baculovirus genome before the 5′

end of the first or second nucleic acid sequence;

iv) a fourth nucleic acid sequence encoding a peptide linker, wherein the third nucleic acid sequence encoding a candidate antigenic peptide is connected to the first or second nucleic acid sequence by the fourth nucleic acid sequence;

v) a fifth nucleic acid sequence encoding at least the transmembrane portion of a membrane protein, the membrane protein-encoding sequence being in frame with and located after the 3′

end of the first or second nucleic acid sequence; and

b) expressing the nucleic acid sequences of (i)-(v) on the surface of each of the baculoviruses in the population, wherein expression of the nucleic acid sequences of (i)-(v) results in the production of at least a portion of an MHC molecule which is covalently linked to the candidate antigenic peptide expressed by the given baculovirus via the peptide linker, and wherein the candidate antigenic peptide is bound to the peptide binding groove of the MHC molecule, thereby forming a library of MHC-peptide molecules displayed on the surface of baculoviruses, the library representing multiple different candidate antigenic peptides.

20. The method of claim 20, further comprising infecting cells with the recombinant baculoviruses, so that an MHC-peptide molecule from the library of MHC-peptide molecules is displayed on the surface of each of the cells infected by the baculovirus..

21. The method of claim 20, wherein the fifth nucleic acid sequence encodes at least the transmembrane portion of baculovirus envelope protein gp64.

22. The method of claim 20, wherein each of the peptides in the library comprises between about 4 and 5 conserved amino acids in a specific sequence sufficient to enable the peptide to bind to the MHC molecule.

23. The method of claim 20, wherein the nucleic acid sequences are introduced into the baculovirus genome using an E. coli transfer plasmid.

24. The method of claim 20, wherein the nucleic acid sequences are introduced into the baculovirus genome by direct cloning of the sequences into the genome.

25. The method of claim 20, wherein the library of candidate antigenic peptides represents from about 10³ to about 10⁹ different candidate antigenic peptides.

26. A library of functional MHC-peptide molecules displayed on the surface of baculovirus or baculovirus-infected cells produced by the method of claim 20.

27. A population of cells infected with the recombinant baculoviruses produced by the method of claim 20, wherein an MHC-peptide molecule from the library of MHC-peptide molecules is displayed on the surface of each of the cells infected by the baculovirus.

28. A method for identifying baculovirus or baculovirus-infected cells that display an MHC-peptide complex that is recognized by a specific T cell receptor, comprising:

a) providing baculoviruses or baculovirus-infected cells that display on the baculoviral surface or cell surface, respectively, at least one MHC-peptide complex, wherein the complex comprises;

i) at least a portion of an MHC molecule sufficient to form a peptide binding groove; and

ii) a candidate antigenic peptide that is covalently linked to the MHC molecule by a peptide linker and which is bound to the peptide binding groove of the MHC molecule, wherein the candidate antigenic peptide is from a library of candidate antigenic peptides, wherein each of the peptides in the library comprises conserved amino acids in a specific sequence sufficient to enable the peptide to bind to the MHC molecule;

b) contacting the baculoviruses or baculovirus-infected cells with a target T cell receptor; and

c) selecting baculoviruses or baculovirus-infected cells that bind to the target T cell receptor.

29. The method of claim 28, further comprising:

d) isolating the selected baculoviruses or baculoviruses from the selected baculovirus-infected cells of step (c);

e) infecting previously uninfected host cells with the isolated baculoviruses of (d) to produce baculoviruses or baculovirus-infected cells enriched for MHC-peptide complexes that bind to the target T cell receptor;

f) contacting the baculoviruses or baculovirus-infected cells from (e) with the target T cell receptor; and

g) selecting baculoviruses or baculovirus-infected cells that bind to the target T cell receptor.

30. The method of claim 29, further comprising isolating the selected baculoviruses or the baculoviruses from the selected baculovirus-infected cells of step (g) and repeating steps (e)-(g) at least one additional time to isolate and identify an MHC-peptide complex that binds to the target T cell receptor.

31. The method of claim 28, wherein the target T cell receptor is labeled with a detectable label.

32. The method of claim 28, wherein the target T cell receptor is expressed on the surface of a cell.

33. The method of claim 28, wherein the target T cell receptor is soluble and immobilized on a substrate.

34. The method of claim 28, wherein the library of candidate antigenic peptides represents from about 10³ to about 10⁹ different candidate antigenic peptides.

35. The method of claim 28, wherein the target T cell receptor is from a patient with a T cell-mediated disease.