U.S. patent application number 11/978519 was filed with the patent office on 2008-10-23 for hla binding motifs and peptides and their uses.
Invention is credited to Esteban Celis, Robert Chesnut, Howard M. Grey, W. Martin Kast, Ralph T. Kubo, Alessandro Sette, John Sidney, Scott Southwood.
Application Number | 20080260762 11/978519 |
Document ID | / |
Family ID | 46324974 |
Filed Date | 2008-10-23 |
United States Patent
Application |
20080260762 |
Kind Code |
A1 |
Grey; Howard M. ; et
al. |
October 23, 2008 |
HLA binding motifs and peptides and their uses
Abstract
The present invention provides the means and methods for
selecting immunogenic peptides and the immunogenic peptide
compositions capable of specifically binding glycoproteins encoded
by HLA alleles and inducing T cell activation in T cells restricted
by the allele. The peptides are useful to elicit an immune response
against a desired antigen.
Inventors: |
Grey; Howard M.; (La Jolla,
CA) ; Sette; Alessandro; (La Jolla, CA) ;
Sidney; John; (San Diego, CA) ; Southwood; Scott;
(Santee, CA) ; Kubo; Ralph T.; (Carlsbad, CA)
; Celis; Esteban; (Rochester, MN) ; Chesnut;
Robert; (Cardiff-by-the-Sea, CA) ; Kast; W.
Martin; (La Canada, CA) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C.
1100 NEW YORK AVENUE, N.W.
WASHINGTON
DC
20005
US
|
Family ID: |
46324974 |
Appl. No.: |
11/978519 |
Filed: |
October 30, 2007 |
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Current U.S.
Class: |
424/185.1 ;
530/324; 530/326; 530/327; 530/328; 530/329 |
Current CPC
Class: |
A61K 39/145 20130101;
A61K 39/02 20130101; C07K 2319/40 20130101; A61K 2039/5158
20130101; C07K 7/06 20130101; A61K 2039/55516 20130101; A61K 39/00
20130101; A61K 2039/572 20130101; C07K 2319/01 20130101; C12N
2710/20034 20130101; C12N 2770/24234 20130101; A61K 38/00 20130101;
A61K 39/12 20130101; A61K 39/0011 20130101; C12N 2730/10134
20130101; A61K 38/10 20130101; C12N 2740/16034 20130101; C12N
2710/00088 20130101; C07K 2319/33 20130101; C07K 14/70539 20130101;
A61K 2039/55566 20130101 |
Class at
Publication: |
424/185.1 ;
530/327; 530/328; 530/329; 530/326; 530/324 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07K 7/06 20060101 C07K007/06; C07K 14/00 20060101
C07K014/00 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
[0013] The Sequence Listing written in file "Sequence Listing.txt,"
2.9 megabytes, created on Jan. 24, 2005 on two identical copies of
compact discs for application Ser. No. 10/817,970, Grey et al., HLA
Binding Motifs and Peptides and Their Uses, is herein
incorporated-by-reference.
[0014] Subject matter disclosed herein was funded, in part, by the
United States government under grants from the National Institutes
of Health. The U.S. government may have certain rights in this
invention. This invention was funded, in part, by the U.S.
government under a contract from the National Institutes of Health.
The U.S. government may have certain rights in the invention.
Claims
1. (canceled)
2. A peptide selected from the group consisting of peptides found
in Tables 2-189.
3. An isolated peptide less than 15 amino acids in length
comprising the oligopeptide LLTFWNPPV (SEQ ID NO:11419).
4. A composition comprising the peptide of claim 3, wherein said
peptide is linked to a T helper peptide.
5. A composition comprising the peptide of claim 3, wherein said
peptide is linked to a spacer or linker, wherein said spacer or
linker is between one and six amino acids in length.
6. A composition comprising the peptide of claim 3, wherein said
peptide is linked to a carrier.
7. A composition comprising the peptide of claim 3, wherein said
peptide is linked to a lipid.
8. A composition comprising the peptide of claim 4, wherein said
T-helper peptide is the PADRE helper peptide having the sequence
aKXVAAWTLKAAa (SEQ ID NO:14635), wherein a is d-alanine and X is
cyclohexylalanine.
9. A homopolymer of the peptide of claim 3.
10. A heteropolymer of the peptide of claim 3 and at least one
different peptide, wherein said at least one different peptide is
not derived from influenza and is a cytotoxic T cell (CTL)-inducing
peptide or a helper T cell (HTL)-inducing peptide.
11. A composition comprising the peptide of claim 3 and a
carrier.
12. A composition comprising the peptide of claim 3 and a
pharmaceutically acceptable excipient.
13. A composition comprising the peptide of claim 3 and a
liposome.
14. A composition comprising the peptide of claim 3, and one or
more different isolated peptides, wherein said one or more
different isolated peptides is selected from the group consisting
of: a cytotoxic T cell (CTL)-inducing peptide and a helper T cell
(HTL)-inducing peptide.
15. The composition of claim 14, further comprising a carrier.
16. The composition of claim 14 further comprising a
pharmaceutically acceptable excipient.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is divisional of U.S. application
Ser. No. 10/817,970, filed Apr. 6, 2004, which is a
continuation-in-part of U.S. application Ser. No. 08/821,739, filed
Mar. 20, 1997; which claims the benefit of U.S. Provisional Appl.
No. 60/013,833, filed Mar. 21, 1996; said U.S. application Ser. No.
08/821,739 is also a continuation-in-part of U.S. application Ser.
No. 08/589,107, filed Jan. 23, 1996, abandoned; said U.S.
application Ser. No. 08/821,739 is also a continuation-in-part of
U.S. application Ser. No. 08/451,913, filed May 26, 1995,
abandoned; said U.S. application Ser. No. 08/821,739 is also a
continuation-in-part of U.S. application Ser. No. 08/186,266, filed
Jan. 25, 1994, now U.S. Pat. No. 5,662,907; which is a
continuation-in-part of U.S. application Ser. No. 08/159,339, filed
Nov. 29, 1993, now U.S. Pat. No. 6,037,135; which is a
continuation-in-part of U.S. application Ser. No. 08/103,396, filed
Aug. 6, 1993, abandoned; which is a continuation-in-part of U.S.
application Ser. No. 08/027,746, filed Mar. 5, 1993, abandoned;
which is a continuation-in-part of U.S. application Ser. No.
07/926,666, filed Aug. 7, 1992, abandoned;
said U.S. application Ser. No. 08/821,739 is also a
continuation-in-part of U.S. application Ser. No. 08/347,610, filed
Dec. 1, 1994; which is a continuation-in-part of U.S. application
Ser. No. 08/159,339, filed Nov. 29, 1993, now U.S. Pat. No.
6,037,135; which is a continuation-in-part of U.S. application Ser.
No. 08/103,396, filed Aug. 6, 1993, abandoned; which is a
continuation-in-part of U.S. application Ser. No. 08/027,746, filed
Mar. 5, 1993, abandoned; which is a continuation-in-part of U.S.
application Ser. No. 07/926,666, filed Aug. 7, 1992, abandoned; the
present application is also a continuation-in-part of U.S.
application Ser. No. 09/665,510, filed Sep. 19, 2000; which is a
continuation-in-part of U.S. application Ser. No. 08/347,610, filed
Dec. 1, 1994; which is a continuation-in-part of U.S. application
Ser. No. 08/159,339, filed Nov. 29, 1993, now U.S. Pat. No.
6,037,135; which is a continuation-in-part of U.S. application Ser.
No. 08/103,396, filed Aug. 6, 1993, abandoned; which is a
continuation-in-part of U.S. application Ser. No. 08/027,746, filed
Mar. 5, 1993, abandoned; which is a continuation-in-part of U.S.
application Ser. No. 07/926,666, filed Aug. 7, 1992, abandoned; the
present application is also a continuation-in-part of U.S.
application Ser. No. 09/017,524, filed Feb. 3, 1998; which is a
continuation-in-part of U.S. application Ser. No. 08/589,107, filed
Jan. 23, 1996, abandoned; said U.S. application Ser. No. 09/017,524
is also a continuation-in-part of U.S. application Ser. No.
08/758,409, filed Nov. 27, 1996, abandoned; said Ser. No.
09/017,524 application is also a continuation-in-part of U.S.
application Ser. No. 08/821,739, filed Mar. 20, 1997; which claims
the benefit of U.S. Provisional Appl. No. 60/013,833, filed Mar.
21, 1996; said U.S. application Ser. No. 08/821,739 is also a
continuation-in-part of U.S. application Ser. No. 08/589,107, filed
Jan. 23, 1996, abandoned; said U.S. application Ser. No. 08/821,739
is also a continuation-in-part of U.S. application Ser. No.
08/451,913, filed May 26, 1995, abandoned; said U.S. application
Ser. No. 08/821,739 is also a continuation-in-part of U.S.
application Ser. No. 08/347,610, filed Dec. 1, 1994; which is a
continuation-in-part of U.S. application Ser. No. 08/159,339, filed
Nov. 29, 1993, now U.S. Pat. No. 6,037,135; which is a
continuation-in-part of U.S. application Ser. No. 08/103,396, filed
Aug. 6, 1993, abandoned; which is a continuation-in-part of U.S.
application Ser. No. 08/027,746, filed Mar. 5, 1993, abandoned;
which is a continuation-in-part of U.S. application Ser. No.
07/926,666, filed Aug. 7, 1992, abandoned; said U.S. application
Ser. No. 08/821,739 is also is a continuation-in-part of U.S.
application Ser. No. 08/186,266, filed Jan. 25, 1994, now U.S. Pat.
No. 5,662,907; which is a continuation-in-part of U.S. application
Ser. No. 08/159,339, filed Nov. 29, 1993, now U.S. Pat. No.
6,037,135; which is a continuation-in-part of U.S. application Ser.
No. 08/103,396, filed Aug. 6, 1993, abandoned; which is a
continuation-in-part of U.S. application Ser. No. 08/027,746, filed
Mar. 5, 1993, abandoned; which is a continuation-in-part of U.S.
application Ser. No. 07/926,666, filed Aug. 7, 1992, abandoned; the
present application is also a continuation-in-part of U.S.
application Ser. No. 09/017,735, filed Feb. 3, 1998; which is a
continuation-in-part of U.S. application Ser. No. 08/205,713, filed
Mar. 4, 1994; which is a continuation-in-part of U.S. application
Ser. No. 08/159,184, filed Nov. 29, 1993, abandoned; which is a
continuation-in-part of U.S. application Ser. No. 08/073,205, filed
Jun. 4, 1993, abandoned; which is a continuation-in-part of U.S.
application Ser. No. 08/027,146, filed Mar. 5, 1993, abandoned;
said U.S. application Ser. No. 09/017,735 is also a
continuation-in-part of U.S. application Ser. No. 08/589,108, filed
Jan. 23, 1996, abandoned; said U.S. application Ser. No. 09/017,735
is also a continuation-in-part of pending U.S. application Ser. No.
08/454,033, filed May 26, 1995; said U.S. application Ser. No.
09/017,735 is also a continuation-in-part of U.S. application Ser.
No. 08/349,177, filed Dec. 2, 1994, abandoned; which is a
continuation-in-part of U.S. application Ser. No. 08/159,184, filed
Nov. 29, 1993, abandoned; which is a continuation-in-part of U.S.
application Ser. No. 08/073,205, abandoned, filed Jun. 4, 1993;
which is a continuation-in-part of U.S. application Ser. No.
08/027,146, filed Mar. 5, 1993, abandoned; said U.S. application
Ser. No. 09/017,735 application is also a continuation-in-part of
U.S. application Ser. No. 08/822,382, filed Mar. 20, 1997,
abandoned; which claims the benefit of U.S. Provisional Appl. No.
60/013,980, filed Mar. 21, 1996; said U.S. application Ser. No.
09/017,735 application is also a continuation-in-part of U.S.
application Ser. No. 08/753,622, filed Nov. 27, 1996, abandoned;
said U.S. application Ser. No. 09/017,735 application is also a
continuation-in-part of U.S. application Ser. No. 08/205,713, filed
Mar. 4, 1994; which is a continuation-in-part of U.S. application
Ser. No. 08/159,184, filed Nov. 29, 1993, abandoned; which is a
continuation-in-part of U.S. application Ser. No. 08/073,205, filed
Jun. 4, 1993, abandoned; which is a continuation-in-part of U.S.
application Ser. No. 08/027,146, filed Mar. 5, 1993, abandoned; the
present application is also a continuation-in-part of pending U.S.
application Ser. No. 08/454,033, filed May 26, 1995; which is a
continuation-in-part of U.S. application Ser. No. 08/349,177, filed
Dec. 2, 1994, abandoned; which is a continuation-in-part of U.S.
application Ser. No. 08/159,184, filed Nov. 29, 1993, abandoned;
which is a continuation-in-part of U.S. application Ser. No.
08/073,205, filed Jun. 4, 1993, abandoned; which is a
continuation-in-part of U.S. application Ser. No. 08/027,146, filed
Mar. 5, 1993, abandoned; the present application is also a
continuation-in-part of U.S. application Ser. No. 09/017,743, filed
Feb. 3, 1998; which is a continuation-in-part of U.S. application
Ser. No. 08/753,615, filed Nov. 27, 1996, abandoned; which is a
continuation-in-part of U.S. application Ser. No. 08/590,298, filed
Jan. 23, 1996, abandoned; which is a continuation-in-part of U.S.
application Ser. No. 08/452,843, filed May 30, 1995; which is a
continuation-in-part of U.S. application Ser. No. 08/344,824, filed
Nov. 23, 1994; which is a continuation-in-part of U.S. application
Ser. No. 08/278,634, filed Jul. 21, 1994, abandoned; said Ser. No.
09/017,743 is also a continuation-in-part of said application Ser.
No. 08/344,824, filed Nov. 23, 1994; and a continuation-in-part of
said application Ser. No. 08/452,843, filed May 30, 1995; the
present application is also a continuation-in-part of U.S.
application Ser. No. 08/452,843, filed May 30, 1995; which is a
continuation-in-part of U.S. application Ser. No. 08/344,824, filed
Nov. 23, 1994; which is a continuation-in-part of U.S. application
Ser. No. 08/278,634, filed Jul. 21, 1994, abandoned; the present
application is also a continuation-in-part of U.S. application Ser.
No. 08/344,824, filed Nov. 23, 1994; which is a
continuation-in-part of U.S. application Ser. No. 08/278,634, filed
Jul. 21, 1994, abandoned; the present application is also a
continuation-in-part of U.S. application Ser. No. 09/226,775, filed
Jan. 6, 1999; which is a continuation-in-part of U.S. application
Ser. No. 08/815,396, filed Mar. 10, 1997, abandoned; which claims
the benefit of U.S. Provisional Appl. No. 60/013,113, filed Mar.
11, 1996; U.S. application Ser. No. 09/226,775 is also a
continuation-in-part of U.S. application Ser. No. 08/485,218, filed
Jun. 7, 1995, abandoned; which is a continuation-in-part of U.S.
application Ser. No. 08/305,871, filed Sep. 14, 1994, now U.S. Pat.
No. 5,736,142; which is a continuation-in-part of U.S. application
Ser. No. 08/121,101, filed Sep. 14, 1993, abandoned; the present
application is also a continuation-in-part of U.S. application Ser.
No. 10/030,014, which is the national stage of International Appl.
No. PCT/US00/17842, filed Jun. 28, 2000; which claims the benefit
of U.S. Provisional Appl. No. 60/141,422, filed Jun. 29, 1999; the
present application is also a continuation-in-part of U.S.
application Ser. No. 10/121,415, filed Apr. 11, 2002; which is a
continuation-in-part of U.S. application Ser. No. 09/189,702, filed
Nov. 10, 1998; which is a continuation-in-part of U.S. application
Ser. No. 09/098,584, filed Jun. 17, 1998, abandoned; the present
application is also a continuation-in-part of International Appl.
No. PCT/US03/31308, filed Oct. 3, 2003; which claims the benefit of
U.S. Provisional Appl. No. 60/416,207, filed Oct. 3, 2002; said
International Appl. No. PCT/US03/31308 also claims the benefit of
U.S. Provisional Appl. No. 60/417,269, filed Oct. 8, 2002; the
present application is also a continuation-in-part of U.S.
application Ser. No. 09/260,714, filed Mar. 1, 1999, abandoned; the
present application is also a continuation-in-part of U.S.
application Ser. No. 10/470,364, which is the national stage of
International Appl. No. PCT/US02/02708, filed Jan. 29, 2002; which
is a continuation-in-part of U.S. application Ser. No. 09/935,476,
filed Aug. 22, 2001, which claims the benefit of U.S. Provisional
Appl. No. 60/264,969, filed Jan. 29, 2001; said U.S. application
Ser. No. 09/935,476 is also a continuation-in-part of U.S.
application Ser. No. 09/346,105, filed Jun. 30, 1999; the present
application is also a continuation-in-part of U.S. application Ser.
No. 10/469,201, which is the national stage of International Appl.
No. PCT/US01/51650, filed Oct. 18, 2001; which claims the benefit
of U.S. Provisional Appl. No. 60/285,624, filed Apr. 20, 2001; said
International Appl. No. PCT/US01/51650 also claims the benefit of
U.S. Provisional Appl. No. 60/242,350, filed Oct. 19, 2000.
[0002] The present application is a continuation-in-part of U.S.
Ser. No. 08/205,713 filed Mar. 4, 1994. The present application is
also related to U.S. Ser. No. 09/017,735, U.S. Ser. No. 08/753,622,
U.S. Ser. No. 08/822,382, U.S. Ser. No. 60/013,980, U.S. Ser. No.
08/589,108, U.S. Ser. No. 08/454,033, U.S. Ser. No. 08/349,177,
U.S. Ser. No. 08/073,205, and U.S. Ser. No. 08/027,146. The present
application is also related to U.S. Ser. No. 09/017,524, U.S. Ser.
No. 08/821,739, U.S. Ser. No. 60/013,833, U.S. Ser. No. 08/758,409,
U.S. Ser. No. 08/589,107, U.S. Ser. No. 08/451,913 and to U.S. Ser.
No. 08/347,610, U.S. Ser. No. 08/186,266, U.S. Ser. No. 08/159,339,
U.S. Ser. No. 09/116,061, U.S. Ser. No. 08/103,396, U.S. Ser. No.
08/027,746, and U.S. Ser. No. 07/926,666. The present application
is also related to U.S. Ser. No. 09/017,743; U.S. Ser. No.
08/753,615; U.S. Ser. No. 08/590,298; U.S. Ser. No. 08/452,843;
U.S. Ser. No. 09/115,400; U.S. Ser. No. 08/344,824; and U.S. Ser.
No. 08/278,634. The present application is also related to U.S.
Ser. No. 08/197,484 and U.S. Ser. No. 08/815,396. All of the above
applications are incorporated herein by reference.
[0003] This application is a continuation-in-part of application
U.S. Ser. No. 08/278,634 filed Jul. 21, 1994, which is incorporated
herein by reference.
[0004] This application is a continuation-in-part of application
U.S. Ser. No. 08/278,634 filed Jul. 21, 1994, which is incorporated
herein by reference.
[0005] This application is a continuation-in-part of U.S.
application Ser. No. 09/346,105, entitled "Consistent Immune
Responses in Diverse Genetic Populations," filed 30 Jun. 1999,
Sidney et al. This application also claims the benefit of the 29
Jan. 2001 filing date of U.S. Application Ser. No. 60/264,969,
entitled "Subunit Vaccines with A2 Supermotifs," Sidney, et al.,
each of which is incorporated by reference in its entirety.
[0006] The present application is related to U.S. Ser. No.
08/589,108, filed Jan. 23, 1996 and now abandoned, and to U.S. Ser.
No. 08/205,713 filed Mar. 4, 1994, which is a continuation-in-part
of U.S. Ser. No. 08/159,184, filed Nov. 29, 1993 and now abandoned,
which is a continuation-in-part of U.S. Ser. No. 08/073,205 filed
Jun. 4, 1993 and now abandoned, which is a continuation-in-part of
U.S. Ser. No. 08/027,146 filed Mar. 5, 1993 and now abandoned. The
application is also related to U.S. Ser. No. 60/013,980 filed Mar.
21, 1996 and now abandoned, U.S. Ser. No. 08/454,033 filed May 26,
1995, U.S. Ser. No. 08/349,177 filed Dec. 2, 1994, and U.S. Ser.
No. 08/753,622 filed Jan. 27, 1996 and now abandoned. Each of the
above-referenced applications is incorporated herein by
reference.
[0007] This application is a Continuation in Part ("CIP") of U.S.
Ser. No. 08/815,396, filed Mar. 10, 1997, which is a CIP of U.S.
Ser. No. 60/013,113, filed Mar. 11, 1996; and is a CIP of U.S. Ser.
No. 08/485,218 filed Jun. 7, 1995, which is a CIP of U.S. Ser. No.
08/305,871 filed Sep. 14, 1994, now U.S. Pat. No. 5,736,142 issued
Apr. 7, 1998, which is a CIP of abandoned application U.S. Ser. No.
08/121,101 filed Sep. 14, 1993. The present application is related
to U.S. Ser. No. 09/017,735, which is a CIP of abandoned U.S. Ser.
No. 08/589,108; U.S. Ser. No. 08/753,622, U.S. Ser. No. 08/822,382,
U.S. Ser. No. 60/013,980, U.S. Ser. No. 08/454,033, U.S. Ser. No.
09/116,424, U.S. Ser. No. 08/205,713, and U.S. Ser. No. 08/349,177,
which is a CIP of abandoned U.S. Ser. No. 08/159,184, which is a
CIP of abandoned U.S. Ser. No. 08/073,205, which is a CIP of
abandoned U.S. Ser. No. 08/027,146. The present application is also
related to U.S. Ser. No. 09/017,524, U.S. Ser. No. 08/821,739, U.S.
Ser. No. 60/013,833, U.S. Ser. No. 08/758,409, U.S. Ser. No.
08/589,107, U.S. Ser. No. 08/451,913, U.S. Ser. No. 08/186,266,
U.S. Ser. No. 09/116,061, and U.S. Ser. No. 08/347,610, which is a
CIP of U.S. Ser. No. 08/159,339, which is a CIP of abandoned U.S.
Ser. No. 08/103,396, which is a CIP of abandoned U.S. Ser. No.
08/027,746, which is a CIP of abandoned U.S. Ser. No. 07/926,666.
The present application is also related to U.S. Ser. No.
09/017,743, U.S. Ser. No. 08/753,615; U.S. Ser. No. 08/590,298,
U.S. Ser. No. 09/115,400, and U.S. Ser. No. 08/452,843, which is a
CIP of abandoned U.S. Ser. No. 08/344,824, which is a CIP of
abandoned U.S. Ser. No. 08/278,634. The present application is also
related to U.S. Ser. No. 60/087,192 and U.S. Ser. No. 09/009,953,
which is a CIP of 60/036,713 and 60/037,432. All of the above
applications are incorporated herein by reference.
[0008] The present application is a continuation in part of U.S.
Ser. No. 08/159,184, which is a continuation in part of U.S. Ser.
No. 08/073,205, which is a continuation in part of U.S. Ser. No.
08/027,146, all of which are incorporated herein by reference.
[0009] To identify peptides of the invention, MHC-peptide complex
isolation, and isolation and sequencing of naturally processed
peptides was carried out as described in the related applications.
This application may be relevant to U.S. Ser. No. 09/189,702 filed
Nov. 10, 1998, which is a CIP of U.S. Ser. No. 08/205,713 filed
Mar. 4, 1994, which is a CIP of Ser. No. 08/159,184 filed Nov. 29,
1993 and now abandoned, which is a CIP of Ser. No. 08/073,205 filed
Jun. 4, 1993 and now abandoned, which is a CIP of Ser. No.
08/027,146 filed Mar. 5, 1993 and now abandoned. The present
application is also related to U.S. Ser. No. 09/226,775, which is a
CIP of U.S. Ser. No. 08/815,396, which claims the benefit of U.S.
Ser. No. 60/013,113, now abandoned. Furthermore, the present
application is related to U.S. Ser. No. 09/017,735, which is a CIP
of abandoned U.S. Ser. No. 08/589,108; U.S. Ser. No. 08/753,622,
U.S. Ser. No. 08/822,382, abandoned U.S. Ser. No. 60/013,980, U.S.
Ser. No. 08/454,033, U.S. Ser. No. 09/116,424, and U.S. Ser. No.
08/349,177. The present application is also related to U.S. Ser.
No. 09/017,524, U.S. Ser. No. 08/821,739, abandoned U.S. Ser. No.
60/013,833, U.S. Ser. No. 08/758,409, U.S. Ser. No. 08/589,107,
U.S. Ser. No. 08/451,913, U.S. Ser. No. 08/186,266, U.S. Ser. No.
09/116,061, and U.S. Ser. No. 08/347,610, which is a CIP of U.S.
Ser. No. 08/159,339, which is a CIP of abandoned U.S. Ser. No.
08/103,396, which is a CIP of abandoned U.S. Ser. No. 08/027,746,
which is a CIP of abandoned U.S. Ser. No. 07/926,666. The present
application may also be relevant to U.S. Ser. No. 09/017,743, U.S.
Ser. No. 08/753,615; U.S. Ser. No. 08/590,298, U.S. Ser. No.
09/115,400, and U.S. Ser. No. 08/452,843, which is a CIP of U.S.
Ser. No. 08/344,824, which is a CIP of abandoned U.S. Ser. No.
08/278,634. The present application may also be related to
provisional U.S. Ser. No. 60/087,192 and U.S. Ser. No. 09/009,953,
which is a CIP of abandoned U.S. Ser. No. 60/036,713 and abandoned
U.S. Ser. No. 60/037,432. In addition, the present application may
be relevant to U.S. Ser. No. 09/098,584, and U.S. Ser. No.
09/239,043. The present application may also be relevant to
co-pending U.S. Ser. No. 09/583,200 filed May 30, 2000, U.S. Ser.
No. 09/260,714 filed Mar. 1, 1999, and U.S. Provisional Application
"Heteroclitic Analogs And Related Methods", Attorney Docket Number
018623-015810US filed Oct. 6, 2000. All of the above applications
are incorporated herein by reference.
[0010] The present application is a continuation in part of U.S.
Ser. No. 08/159,339, which is continuation in part of U.S. Ser. No.
08/103,396 which is a continuation in part of U.S. Ser. No.
08/027,746 which is a continuation in part of U.S. Ser. No.
07/926,666. It is related to U.S. Ser. No. 08/186,266. The
application of supermotifs and motifs and binding analysis to the
identification of epitopes is described in WO01/21189 and
co-pending U.S. application Ser. No. 09/239,043, filed Jan. 27,
1999; Ser. No. 09/350,401, filed Jul. 8, 1999; Ser. No. 09/412,863
filed Oct. 5, 1999; Ser. No. 09/390,061 filed Sep. 3, 1999; Ser.
No. 09/458,302 filed Dec. 10, 1999; Ser. No. 09,458,297 filed Dec.
10, 1999; Ser. No. 09/458,298 filed Dec. 10, 1999, Ser. No.
09/633,364 filed Aug. 7, 2000; Ser. No. 09/458,299 filed Dec. 10,
1999; and Ser. No. 09/641,528 filed Aug. 15, 2000.
[0011] This application is a Continuation in Part ("CIP") of U.S.
Ser. No. 08/815,396, filed Mar. 10, 1997, which is a CIP of U.S.
Ser. No. 60/013,113, filed Mar. 11, 1996; and is a CIP of U.S. Ser.
No. 08/485,218 filed Jun. 7, 1995, which is a CIP of U.S. Ser. No.
08/305,871 filed Sep. 14, 1994, now U.S. Pat. No. 5,736,142 issued
Apr. 7, 1998, which is a CIP of abandoned application U.S. Ser. No.
08/121,101 filed Sep. 14, 1993. The present application is related
to U.S. Ser. No. 09/017,735, which is a CIP of abandoned U.S. Ser.
No. 08/589,108; U.S. Ser. No. 08/753,622, U.S. Ser. No. 08/822,382,
U.S. Ser. No. 60/013,980, U.S. Ser. No. 08/454,033, U.S. Ser. No.
09/116,424, U.S. Ser. No. 08/205,713, and U.S. Ser. No. 08/349,177,
which is a CIP of abandoned U.S. Ser. No. 08/159,184, which is a
CIP of abandoned U.S. Ser. No. 08/073,205, which is a CIP of
abandoned U.S. Ser. No. 08/027,146. The present application is also
related to U.S. Ser. No. 09/017,524, U.S. Ser. No. 08/821,739, U.S.
Ser. No. 60/013,833, U.S. Ser. No. 08/758,409, U.S. Ser. No.
08/589,107, U.S. Ser. No. 08/451,913, U.S. Ser. No. 08/186,266,
U.S. Ser. No. 09/116,061, and U.S. Ser. No. 08/347,610, which is a
CIP of U.S. Ser. No. 08/159,339, which is a CIP of abandoned U.S.
Ser. No. 08/103,396, which is a CIP of abandoned U.S. Ser. No.
08/027,746, which is a CIP of abandoned U.S. Ser. No. 07/926,666.
The present application is also related to U.S. Ser. No.
09/017,743, U.S. Ser. No. 08/753,615; U.S. Ser. No. 08/590,298,
U.S. Ser. No. 09/115,400, and U.S. Ser. No. 08/452,843, which is a
CIP of abandoned U.S. Ser. No. 08/344,824, which is a CIP of
abandoned U.S. Ser. No. 08/278,634. The present application is also
related to U.S. Ser. No. 60/087,192 and U.S. Ser. No. 09/009,953,
which is a CIP of 60/036,713 and 60/037,432.
[0012] All of the above applications are incorporated herein by
reference. These applications are referred to herein as the "parent
applications".
BACKGROUND OF THE INVENTION
[0015] The present invention relates to compositions and methods
for preventing, treating or diagnosing a number of pathological
states such as viral diseases and cancers. In particular, it
provides novel peptides capable of binding selected major
histocompatibility complex (MHC) molecules and inducing an immune
response.
[0016] The genetic makeup of a given mammal encodes the structures
associated with the immune system of that species. Although there
is a great deal of genetic diversity in the human population, even
more so comparing humans and other species, there are also common
features and effects. In mammals, certain molecules associated with
immune function are termed the major histocompatibility
complex.
[0017] MHC molecules are classified as either Class I or Class II
molecules. Class II MHC molecules are expressed primarily on cells
involved in initiating and sustaining immune responses, such as T
lymphocytes, B lymphocytes, dendritic cells, macrophages, etc.
Class II MHC molecules are recognized by helper T lymphocytes and
induce proliferation of helper T lymphocytes and amplification of
the immune response to the particular immunogenic peptide that is
displayed. Complexes between a particular disease-associated
antigenic peptide and class II HLA molecules are recognized by
helper T lymphocytes and induce proliferation of helper T
lymphocytes and amplification of specific CTL and antibody immune
responses.
[0018] Class I MHC molecules are expressed on almost all nucleated
cells and are recognized by cytotoxic T lymphocytes (CTLs), which
then destroy the antigen-bearing cells. Complexes between a
particular antigenic peptide and class I MHC molecules are
recognized by CD8+ cytotoxic T lymphocytes (CTLs), which then
destroy the cells bearing antigens bound by the HLA class I
molecules expressed on those cells. CD8+ T lymphocytes frequently
mature into cytotoxic effector which can lyse cells bearing the
stimulating antigen. CTLs are particularly important in tumor
rejection and in fighting viral, fungal, and parasitic
infections.
[0019] A complex of an HLA molecule and a peptidic antigen acts as
the ligand recognized by HLA-restricted T cells (Buus, S. et al.,
Cell 47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985;
Townsend, A. and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989;
Germain, R. N., Annu. Rev. Immunol. 11:403, 1993).
[0020] Through the study of single amino acid substituted antigen
analogs and the sequencing of endogenously bound, naturally
processed peptides, critical residues that correspond to motifs
required for specific binding to HLA antigen molecules have been
identified (see also, e.g., Southwood, et al., J. Immunol.
160:3363, 1998; Rammensee, et al., Immunogenetics 41:178, 1995;
Rammensee et al., SYFPEITHI, access via web at:
http://134.2.96.221/scripts. hlaserver.dll/home.htm; Sette, A. and
Sidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H.,
Curr. Opin. Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr.
Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J. Curr.
Biol. 6:52, 1994; Ruppert et al., Cell 74:929-937, 1993; Kondo et
al., J. Immunol. 155:4307-4312, 1995; Sidney et al., J. Immunol.
157:3480-3490, 1996; Sidney et al., Human Immunol. 45:79-93, 1996;
Sette, A. and Sidney, J. Immunogenetics 50:201-212, 1999). The
presence of these residues correlates with binding affinity for HLA
molecules. The identification of motifs and/or supermotifs that
correlate with high and intermediate affinity binding is an
important issue with respect to the identification of immunogenic
peptide epitopes for the inclusion in a vaccine. Kast et al. (J.
Immunol. 152:3904-12, 1994) have shown that motif-bearing peptides
account for 90% of the epitopes that bind to allele-specific HLA
class I molecules. In this study all possible peptides of 9 amino
acids in length and overlapping by eight amino acids (240
peptides), which cover the entire sequence of the E6 and E7
proteins of human papillomavirus type 16, were evaluated for
binding to five allele-specific HLA molecules that are expressed at
high frequency among different ethnic groups. This unbiased set of
peptides allowed an evaluation of the predictive value of HLA class
I motifs. From the set of 240 peptides, 22 peptides were identified
that bound to an allele-specific HLA molecule with high or
intermediate affinity. Of these 22 peptides, 20 (i.e. 91%) were
motif-bearing. Thus, this study demonstrates the value of motifs
for the identification of peptide epitopes for inclusion in a
vaccine: application of motif-based identification techniques will
identify about 90% of the potential epitopes in a target antigen
protein sequence.
[0021] A relationship between binding affinity for HLA class I
molecules and immunogenicity of discrete peptide epitopes on bound
antigens was determined by the present inventors. As disclosed in
greater detail herein, higher HLA binding affinity is correlated
with greater immunogenicity.
[0022] Furthermore, x-ray crystallographic analysis of HLA-peptide
complexes has revealed pockets within the peptide binding cleft of
HLA molecules which accommodate, in an allele-specific mode,
specific residues of peptide ligands; these residues in turn
determine the HLA binding capacity of the peptides in which they
are present. (See, e.g., Madden, D. R. Annu. Rev. Immunol. 13:587,
1995; Smith, et al., Immunity 4:203, 1996; Fremont et al., Immunity
8:305, 1998; Stern et al., Structure 2:245, 1994; Jones, E. Y.
Curr. Opin. Immunol. 9:75, 1997; Brown, J. H. et al., Nature
364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci. USA
90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M.
L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science
257:927, 1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et
al., Science 257:919, 1992; Saper, M. A., Bjorkman, P. J. and
Wiley, D. C., J. Mol. Biol. 219:277, 1991).
[0023] Peptides of the present invention may also comprise epitopes
that bind to HLA class II DR molecules. A greater degree of
heterogeneity in both size and binding frame position of the motif,
relative to the N- and C-termini of the peptide, exists for class
II peptide ligands. This increased heterogeneity of HLA class II
peptide ligands is due to the structure of the binding groove of
the HLA class II molecule which, unlike its class I counterpart, is
open at both ends. Crystallographic analysis of HLA class II
DRB*0101-peptide complexes showed that the major energy of binding
is contributed by peptide residues complexed with complementary
pockets on the DRB*0101 molecules. An important anchor residue
engages the deepest hydrophobic pocket (see, e.g., Madden, D. R.
Ann. Rev. Immunol. 13:587, 1995) and is referred to as position 1
(P1). P1 may represent the N-terminal residue of a class II binding
peptide epitope, but more typically is flanked towards the
N-terminus by one or more residues. Other studies have also pointed
to an important role for the peptide residue in the sixth position
towards the C-terminus, relative to P1, for binding to various DR
molecules.
[0024] In the past few years evidence has accumulated to
demonstrate that a large fraction of HLA class I and class II
molecules can be classified into a relatively few supertypes, each
characterized by largely overlapping peptide binding repertoires,
and consensus structures of the main peptide binding pockets. Thus,
peptides of the present invention are identified by any one of
several HLA-specific amino acid motifs, or if the presence of the
motif corresponds to the ability to bind several allele-specific
HLA molecules, a supermotif. The HLA molecules that bind to
peptides that possess a particular amino acid supermotif are
collectively referred to as an HLA "supertype."
[0025] Accordingly, the definition of class I and class II
allele-specific HLA binding motifs, or class I or class II
supermotifs allows identification of regions within a protein that
have the potential of binding particular HLA molecules.
[0026] The MHC class I antigens are encoded by the HLA-A, B, and C
loci. HLA-A and HLA-B antigens are expressed at the cell surface at
approximately equal densities, whereas the expression of HLA-C is
significantly lower (perhaps as much as 10-fold lower). Each of
these loci have a number of alleles.
[0027] Specific motifs for several of the major HLA-A alleles
(copending U.S. patent application Ser. Nos. 08/159,339 and
08/205,713, referred to here as the copending applications) and
HLA-B alleles have been described. Several authors (Melief, Eur. J.
Immunol., 21:2963-2970 (1991); Bevan, et al., Nature, 353:852-955
(1991)) have provided preliminary evidence that class I binding
motifs can be applied to the identification of potential
immunogenic peptides in animal models. Strategies for
identification of peptides or peptide regions capable of
interacting with multiple MHC alleles have been described in the
literature.
[0028] Because human population groups, including racial and ethnic
groups, have distinct patterns of distribution of HLA alleles it
will be of value to identify motifs that describe peptides capable
of binding more than one HLA allele, so as to achieve sufficient
coverage of all population groups. The present invention addresses
these and other needs.
[0029] The recognition of foreign pathogens, foreign cells (i.e.,
tumor), or one's own cells by the immune system occurs largely
through major histocompatibility (MHC) molecules. MHC molecules
present unique molecular fragments of foreign and self molecules
that permit recognition and, when appropriate, stimulation of
various immune effectors, namely B and T lymphocytes. MHC molecules
are classified as either class I or class II. Class II MHC
molecules are expressed primarily on cells involved in initiating
and sustaining immune responses, such as T lymphocytes, B
lymphocytes, macrophages, etc. Class II MHC molecules are
recognized by helper T lymphocytes and induce proliferation of
helper T lymphocytes and amplification of the immune response to
the particular immunogenic peptide that is displayed. CD4+ T
lymphocytes are activated with recognition of a unique peptide
fragment presented by a class II MHC molecule, usually found on an
antigen presenting cell like a macrophage or dendritic cell. Often
known as helper T lymphocytes (HTL), CD4+ lymphocytes proliferate
and secrete cytokines that either support a antibody-mediated
response through the production of IL-4 and IL-10 or support a
cell-mediated response through the production of IL-2 and
IFN-.gamma..
[0030] T lymphocytes recognize an antigen in the form of a peptide
fragment bound to the MHC class I or class II molecule rather than
the intact foreign antigen itself. An antigen presented by a MHC
class I molecule is typically one that is endogenously synthesized
by the cell (i.e., an intracellular pathogen). The resulting
cytoplasmic antigens are degraded into small fragments in the
cytoplasm, usually by the proteosome (Niedermann et al., Immunity,
2: 289-99 (1995)). Some of these small fragments are transported
into the endoplasmic reticulum (a pre-Golgi compartment) where the
fragment interacts with class I heavy chains to facilitate proper
folding and association with the subunit .beta.2 microglobulin to
result in a stable complex formation between the fragment, MHC
class I chain and .beta.2 microglobulin. This complex is then
transported to the cell surface for expression and potential
recognition by specific CTLs. Antigens presented by MHC class II
molecules are usually soluble antigens that enter the antigen
presenting cell via phagocytosis, pinocytosis, or receptor-mediated
endocytosis. Once in the cell, the antigen is partially degraded by
acid-dependent proteases in endosomes. The resulting fragments or
peptide associate with the MHC class II molecule after the release
of the CLIP fragment to form a stable complex that is then
transported to the surface for potential recognition by specific
HTLs. See Blum, et al., Crit. Rev. Immunol., 17: 411-17 (1997);
Arndt, et al., Immunol. Res., 16: 261-72 (1997).
[0031] Investigations of the crystal structure of the human MHC
class I molecule, HLA-A2.1, indicate that a peptide binding groove
is created by the folding of the .alpha.1 and .alpha.2 domains of
the class I heavy chain (Bjorkman, et al., Nature 329:506 (1987).
In these investigations, however, the identity of peptides bound to
the groove was not determined.
[0032] Buus, et al., Science 242:1065 (1988) first described a
method for acid elution of bound peptides from MHC. Subsequently,
Rammensee and his coworkers (Falk, et al., Nature 351:290 (1991)
have developed an approach to characterize naturally processed
peptides bound to class I molecules. Other investigators have
successfully achieved direct amino acid sequencing of the more
abundant peptides in various HPLC fractions by conventional
automated sequencing of peptides eluted from class I molecules of
the B type (Jardetzky, et al., Nature 353:326 (1991) and of the
A2.1 type by mass spectrometry (Hunt, et al., Science 225:1261
(1992). A review of the characterization of naturally processed
peptides in MHC Class I has been presented by Rotzschke and Falk
(Rotzschke and Falk, Immunol. Today 12:447 (1991). PCT publication
WO97/34621, incorporated herein by reference, describes peptides
which have a binding motif for A2.1 alleles.
[0033] Peptides that bind a particular MHC allele frequently will
fit within a motif and have amino acid residues with particular
biochemical properties at specific positions within the peptide.
Such residues are usually dictated by the biochemical properties of
the MHC allele. Peptide sequence motifs have been utilized to
screen peptides capable of binding MHC molecules (Sette, et al.,
Proc. Natl. Acad. Sci. USA 86:3296 (1989)), and it has been
reported that class I binding motifs identified potential
immunogenic peptides in animal models (De Bruijn, et al., Eur. J.
Immunol. 21: 2963-70 (1991); Pamer, et al., Nature 353: 852-955
(1991)). Also, binding of a particular peptide to a MHC molecule
has been correlated with immunogenicity of that peptide (Schaeffer,
et al., Proc. Natl. Acad. Sci. USA 86:4649 (1989)).
[0034] Of the many thousand possible peptides that are encoded by a
complex foreign pathogen, only a small fraction ends up in a
peptide form capable of binding to MHC class I or class II antigens
and thus of being recognized by T cells. This phenomenon is known
as immunodominance (Yewdell et al., Ann. Rev. Immunol., 17: 51-88
(1997)). More simply, immunodominance describes the phenomenon
whereby immunization or exposure to a whole native antigen results
in an immune response directed to one or a few "dominant" epitopes
of the antigen rather than every epitope that the native antigen
contains. Immunodominance is influenced by a variety of factors
that include MHC-peptide affinity, antigen processing, and antigen
availability.
[0035] In general, CTL and HTL responses are not directed against
all possible epitopes. Rather, they are restricted to a few
immunodominant determinants. (Zinkernagel, et al., Adv. Immunol.
27:51-59 (1979); Bennink, et al., J. Exp. Med. 168:1935-1939
(1988); Rawle, et al., J. Immunol. 146:3977-84 (1991); Sercarz et
al. Ann. Rev. Immunol. 11:729-766 (1993)). It has been recognized
that immunodominance (Benacerraf, et al., Science 175:273-79
(1972)) could be explained by either the ability of a given epitope
to selectively bind a particular HLA protein (determinant selection
theory) (Vitiello, et al., J. Immunol. 131:1635 (1983); Rosenthal,
et al., Nature 267:156-58 (1977)), or being selectively recognized
by the existing TCR (T cell receptor) specificity (repertoire
theory) (Klein, J., IMMUNOLOGY, THE SCIENCE OF SELF-NONSELF
DISCRIMINATION, John Wiley & Sons, New York, pp. 270-310
(1982)). It has been demonstrated that additional factors, mostly
linked to processing events, can also play a key role in dictating,
beyond strict immunogenicity, which of the many potential
determinants will be presented as immunodominant (Sercarz, et al.,
Annu. Rev. Immunol. 11:729-66 (1993)).
[0036] The present understanding is that because T cells to
dominant epitopes may have been clonally deleted, selecting
subdominant epitopes may allow extant T cells to be recruited which
will then lead to a therapeutic response. However, the binding of
HLA molecules to subdominant epitopes is often less vigorous than
to dominant ones. Accordingly, there is a need to be able to
modulate the binding affinity of particular immunogenic epitopes
for one or more HLA molecules, and thereby to modulate the immune
response elicited by the peptide.
[0037] Accordingly, while some MHC binding peptides have been
identified, there is a need in the art to identify novel MHC
binding peptides from pathogens that can be utilized to generate an
immune response in vaccines against the pathogens from which they
originate. Further, there is a need in the art to identify peptides
capable of binding a wide array of different types of MHC molecules
such they are immunogenic in a large fraction a human outbred
population.
[0038] Sette et al., Proc. Natl. Acad. Sci. USA 86:3296 (1989)
showed that MHC allele specific motifs could be used to predict MHC
binding capacity. Schaeffer et al., Proc. Natl. Acad. Sci. USA
86:4649 (1989) showed that MHC binding was related to
immunogenicity. Several authors (De Bruijn et al., Eur. J.
Immunol., 21:2963-2970 (1991); Pamer et al., 991 Nature 353:852-955
(1991)) have provided preliminary evidence that class I binding
motifs can be applied to the identification of potential
immunogenic peptides in animal models. Class I motifs specific for
a number of human alleles of a given class I isotype have yet to be
described. It is desirable that the combined frequencies of these
different alleles should be high enough to cover a large fraction
or perhaps the majority of the human outbred population.
[0039] Thus a need exists, met for the first time herein, to
prepare analog peptides which elicit a more vigorous response. This
ability greatly enhances the usefulness of peptide-based vaccines
and therapeutic agents. The present invention provides these and
other advantages.
[0040] One of the most formidable obstacles to the development of
broadly efficacious peptide-based immunotherapeutics has been the
extreme polymorphism of HLA molecules. Effective coverage of a
population without bias would thus be a task of considerable
complexity if epitopes were used specific for HLA molecules
corresponding to each individual allele because a huge number of
them would have to be used in order to cover an ethnically diverse
population. There exists, therefore, a need to develop peptide
epitopes that are bound by multiple HLA antigen molecules at high
affinity for use in epitope-based vaccines. The greater the number
of HLA antigen molecules bound, the greater the breadth of
population coverage by the vaccine. Analog peptides may be
engineered based on the information disclosed herein and thereby
used to achieve such an enhancement in breadth of population
coverage.
[0041] Thus, the use of analoguing to modify peptide epitopes to
enhance population coverage and/or immunogenicity provides a
heretofore undisclosed advantage of the present invention in
creating effective, immunogenic vaccines for a broad segment of the
population.
[0042] Despite the developments in the art, the prior art has yet
to provide a useful human peptide-based vaccine or therapeutic
agent based on this work. The present invention provides these and
other advantages.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] FIG. 1. Preferred Motif Table.
[0044] FIG. 2. HLA superfamilies for HLA-A and HLA-B alleles.
Various alleles of HLA-A and HLA-B are classified according to
superfamily based on sequencing analysis or binding assays
(verified supertype members) or on the basis of B and F pocket
structure (predicted supertype members).
[0045] FIG. 3 shows binding motifs for peptides capable of binding
HLA alleles sharing the B7-like specificity.
[0046] FIG. 4 shows the B7-like cross-reactive motif.
[0047] FIGS. 5A and 5B. Position 2 and C-terminus define
specificity of HLA-A*0201. The preference for specific residues in
position 2(a) or at the C-terminus (b) is shown as a function of
the percent of peptides bearing a specific residue that bind A*0201
with IC.sub.50 of 500 nM or better. ARB values of peptides bearing
specific residues in position 2 (a) or at the C-terminus (b) were
calculated as described herein, and indexed relative to the residue
with the highest binding capacity. The average (geometric) binding
capacity of peptides with L in position 2 was 1991 nM. The average
(geometric) binding capacity of peptides with V at the C-terminus
was 2133 nM. Peptides included in the analysis had at least one
tolerated anchor residue, as described in the text, at either
position 2 or the C-terminus.
[0048] FIGS. 6A-6D. Map of the A*0201 motif. Summary map of the
A*0201 motif for 8-mer (b), 10-mer (c) and 11-mer (d) peptides. At
secondary anchor positions, residues shown as preferred (or
deleterious) are associated with an average binding capacity at
least 3-fold greater than (or 3-fold less than) peptides of the
same size carrying other residues at the same position. At the
primary anchor positions, preferred residues are those associated
with an average binding capacity within 10-fold of the optimal
residue at the same position. Tolerated primary anchor residues are
those associated with an average binding capacity between 10- and
100-fold of the optimal residue at the same position.
[0049] FIGS. 7A-7D. Position 2 fine specificity of HLA-A2-supertype
molecules. ARB values of peptides bearing specific residues in
position 2 were calculated for each A2-supertype molecule as
described in the text, and indexed relative to the residue with the
highest ARB for each specific molecule. The average (geometric)
binding capacity of the peptides bearing the residue with the
highest ARB were 55, 59, 89, and 41 mM for A*0202, A*0206, and
A*6802, respectively.
[0050] FIGS. 8A-8D. C-terminal fine specificity of HLA-A2-supertype
molecules. ARB values of peptides bearing specific residues at the
C-terminus were calculated for each A2-supertype molecule as
described in the text, and indexed relative to the residue with the
highest ARB for each specific molecule. The average (geometric)
binding capacity of the peptides bearing the residue with the
highest ARB were 291, 48, 250, and 553 nM for A*0202, A*0203,
A*0206, and A*6802, respectively.
[0051] FIG. 9. Map of the A*0202 motif. Summary map of A*0202 motif
for 9-mer (a) and 10-mer (b) peptides. At secondary anchor
positions, residues shown as preferred (or deleterious) are
associated with an average binding capacity at least 3-fold greater
than (or 3-fold less than) peptides of the same size carrying other
residues at the same position. At the primary anchor positions,
preferred residues are those associated with an average binding
capacity within 10-fold of the optimal residue at the same
position. Tolerated primary anchor residues are those associated
with an average binding capacity between 10- and 100-fold of the
optimal residue at the same position.
[0052] FIG. 10. Map of the A*0203 motif. Summary maps of A*0203
motif for 9-mer (a) and 10-mer (b) peptides. At secondary anchor
positions, residues shown as preferred (or deleterious) are
associated with an average binding capacity at least 3-fold greater
than (or 3-fold less than) peptides of the same size carrying other
residues at the same position. At the primary anchor positions,
preferred residues are those associated with an average binding
capacity within 10-fold of the optimal residue at the same
position. Tolerated primary anchor residues are those associated
with an average binding capacity between 10- and 100-fold of the
optimal residue at the same position.
[0053] FIG. 11. Map of the A*0206 motif. Summary maps of A*0206
motif for 9-mer (a) and 10-mer (b) peptides. At secondary anchor
positions, residues shown as preferred (or deleterious) are
associated with an average binding capacity at least 3-fold greater
than (or 3-fold less than) peptides of the same size carrying other
residues at the same position. At the primary anchor positions,
preferred residues are those associated with an average binding
capacity within 10-fold of the optimal residue at the same
position. Tolerated primary anchor residues are those associated
with an average binding capacity between 10- and 100-fold of the
optimal residue at the same position.
[0054] FIG. 12. Map of the A*6802 motif. Summary maps of A*6802
motif for 9-mer (a) and 10-mer (b) peptides. At secondary anchor
positions, residues shown as preferred (or deleterious) are
associated with an average binding capacity at least 3-fold greater
than (or 3-fold less than) peptides of the same size carrying other
residues at the same position. At the primary anchor positions,
preferred residues are those associated with an average binding
capacity within 10-fold of the optimal residue at the same
position. Tolerated primary anchor residues are those associated
with an average binding capacity between 10- and 100-fold of the
optimal residue at the same position.
[0055] FIG. 13. A2 supermotif consensus summary of secondary and
primary anchor influences on A2-supertype binding capacity of 9-(a)
and 10-mer (b) peptides. Residues shown significantly influence
binding to 3 or more A2-supertype molecules. The number of
molecules influenced are indicated in parentheses. At secondary
anchor positions, residues are considered preferred only if they do
not have a deleterious influence on more than one molecule.
Preferred residues which were deleterious in the context of one
molecule are indicated by reduced and italicized font. Assessment
at the primary anchor positions are based on single substitution
and peptide library analyses, as discussed in the text.
[0056] FIG. 14 is a flow diagram of an HLA-A purification
scheme.
[0057] FIG. 15 is an SDS-PAGE analysis of affinity purified.
HLA-A3.2 from the cell line EHM using an affinity column prepared
with the mAb GAP A3 coupled to protein A-Sepharose. [0058] Lane
1--Molecular weight standards [0059] Lane 2--A3.2 acid eluate
[0060] Lane 3--A3.2 a second acid eluate [0061] Lane 4--Base
elution #1 [0062] Lane 5--Base elution #2 [0063] Lane
6--Concentrated base elution 1 [0064] Lane 7--Concentrated base
elution 2 [0065] Lane 8--BSA--10 pg [0066] Lane 9--BSA--3 pg [0067]
Lane 10--BSA--1 pg
[0068] FIG. 16 shows reverse phase high performance liquid
chromatography (RP-HPLC) separation of HLA-A3 acid eluted 20
peptides.
[0069] FIG. 17 shows binding of a radioactively labeled peptide of
the invention to MHC molecules as measured by the bound
radioactivity.
[0070] FIG. 18 shows inhibition of binding of a peptide of the
invention to MHC molecules in the presence of three peptides (HBc
18-27 (92-4-07), a Prostate Specific Antigen peptide (939.01), and
HIV nef 73-82 (940.03)).
[0071] FIG. 19 shows the dependency of the binding on MHC
concentration in the presence or absence of .beta..sub.2
microglobulin.
[0072] FIG. 20 shows dose dependent inhibition of binding with the
addition of unlabeled peptide.
[0073] FIG. 21 Scatchard Analysis of binding to MHC All confirming
an apparent K.sub.D of 6 nM.
[0074] FIG. 22 shows the binding of a radioactively labeled peptide
of the invention to MHC A1 as measured by bound reactivity.
[0075] FIG. 23 shows dose dependent inhibition of binding with the
addition of unlabeled peptide.
[0076] FIG. 24. Scatchard Analysis of binding to MHC A1 confirming
an apparent K.sub.D of 21 nM.
[0077] FIG. 25 shows the binding of two peptides of this invention
as a function of MHC A24 concentration as measured by bound
reactivity.
[0078] FIG. 26 shows the dose dependent inhibition of binding to
MHC A24 with the addition of unlabeled peptides.
[0079] FIG. 27A and FIG. 27B show the Scatchard Analysis of binding
to MHC A24 of the two peptides confirming a K.sub.D of 30 and 60
nM, respectively.
[0080] FIG. 28 shows the effect on MHC class I molecules of
.beta..sub.2 Microglobulin and a peptide of choice on-acid-stripped
PHA blasts.
[0081] FIG. 29 shows CTL induction using GC43 A2.1 responders and
autologous acid-stripped PBMCs or PHA blasts loaded with the
777.03-924.07-927.32 peptide pool.
[0082] FIG. 30 shows CTL induction using X351 or X355 A2.1
responders and autologous acid stripped PBMCs or PHA blasts as
stimulators after loading with the 1044.04-1044.05-1044.06 peptide
pool.
[0083] FIG. 31 shows CTL induction using GC49 A2.1 responders and
Autologous Acid stripped PHA blasts as stimulators after loading
with 939.03 peptide.
[0084] FIG. 32 shows CTL induction using GC66 A1 responders and
autologous acid stripped PBMCs as stimulators after loading of
peptide 938.01.
[0085] FIG. 33 illustrates the lysis of peptide sensitized targets
and endogenous targets following stimulation with SAC-I activated
PBMCs loaded with a MAGE 3 peptide.
[0086] FIG. 34 shows a comparison of the acid strip-loading with
the cold temperature incubation.
[0087] FIG. 35 shows a CTL response to an immunogenic peptide for
MAGE/All.
[0088] FIG. 36 shows a CTL response to an immunogenic peptide for
HIV/A3.
[0089] FIG. 37 shows a CTL response to an immunogenic peptide for
HCV/A3.
[0090] FIG. 38 shows a CTL response to an immunogenic peptide for
HBV/A3.
[0091] FIG. 39 shows allele specific motifs of five A3 supertype
alleles: A*0301, A*1101, A*3101, A*3301, and A*6801. Individual
residues, or groups of residues, associated for each non-anchor
position with either good ("preferred") or poor ("deleterious")
binding capacities to each individual allele are shown.
[0092] FIG. 40 shows the A3 supermotif. Numbers in parenthesis
indicate the number of molecules for which the residue or residue
group was preferred or deleterious.
[0093] FIG. 41 summarizes the motifs for the B7 supertype alleles
(FIG. 41A-E) and for the B7 supermotif (FIG. 41F). The Figure and
corresponding motif/supermotif is as follows: a) B*0702, b) B*3501,
c) B51, d) B*5301, and e) B*5401. These maps were subsequently used
to define the B7 supermotif (f). Values in parenthesis indicate the
frequency that a residue or residue group was preferred or
deleterious.
[0094] FIG. 42 shows relative average binding capacity of the
A*0101 motif 9-mer peptides as a function of the different residues
occurring at each of the non-anchor positions. FIG. 42A and FIG.
42B depict data, and FIG. 42C and FIG. 42D depict graphics. Data
sets from either 2-9 motif (FIG. 42A), 3-9 motif (FIG. 42B) peptide
sets were analyzed and tabulated [as described in the Examples].
The 2-9 and 3-9 sets contained 101 and 85 different peptides,
respectively. Maps of secondary effects influencing the binding
capacity of 9-mer peptides carrying the 2-9 (FIG. 42C) 3-9 mer
(FIG. 42D) A*0101 motifs are also shown.
[0095] FIG. 43 shows relative average binding capacity of the
A*0101 motif 10 mer peptides as function of the different residues
occurring at each of the non-anchor positions. Data sets from
either 2-10 mer (FIG. 43A) or 3-10 (FIG. 43B) motif sets of
peptides were analyzed and tabulated. The 2-10 and 3-10 sets
contained 91 and 89 different peptides, respectively. Maps of
secondary effects influencing the binding capacity of 10 mer
peptides carrying the 2-10 (FIG. 43C) mer and (1) and or 3-10 mer
(FIG. 43D) A1 motifs are also shown.
[0096] FIG. 44 shows preferred and deleterious secondary anchor
residues for the refined A24 9-mer and 10-mer motifs.
[0097] FIG. 49 is a flow diagram of an HLA-A purification
scheme.
[0098] FIG. 45 shows a scattergram of the log of relative binding
plotted against the "Grouped Ratio" algorithm for 9 mer
peptides.
[0099] FIG. 46 shows a scattergram of the log of relative binding
plotted against the average "Log of Binding" algorithm score for 9
mer peptides.
[0100] FIG. 47 and FIG. 48 show scattergrams of a set of 10-mer
peptides containing preferred residues in positions 2 and 10 as
scored by the "Grouped Ratio" and "Log of Binding" algorithms.
BRIEF SUMMARY OF THE INVENTION
[0101] The present invention relates to compositions and methods
for preventing, treating or diagnosing a number of pathological
states such as viral diseases and cancers. Thus, provided herein
are novel peptides capable of binding selected major
histocompatibility complex (MHC) molecules and inducing or
modulating an immune response. Some of the peptides disclosed are
capable of binding human class II MHC (HLA) molecules, including
HLA-DR and HLA-DQ alleles. Other peptides disclosed herein are
capable of binding to human class I molecules, including one or
more of the following: HLA-A1, HLA-A2.1, HLA-A3.2, HLA-A11,
HLA-A24.1, HLA-B7, and HLA-B44 molecules. Other peptides disclosed
are capable of binding to murine class I molecules. Also provided
are compositions that include immunogenic peptides having binding
motifs specific for MHC molecules. The peptides and compositions
disclosed can be utilized in methods for inducing an immune
response, a cytotoxic T lymphocyte (CTL) response or helper T
lymphocyte (HTL) response in particular, when administered to a
system.
[0102] The present invention also provides a method of identifying
peptide epitopes comprising an HLA A3 supermotif. An A3 supermotif,
when present in a peptide, allows the peptide to bind more than one
HLA molecule that is a member of the HLA A3 supertype. An HLA
supertype describes a set of HLA molecules grouped on the basis of
shared peptide-binding specificities. Accordingly, HLA molecules
that share similar binding repertoires for peptides bearing the
HLA-A3 supermotif are grouped into the HLA A3 supertype. The HLA A3
supertype is comprised by HLA A3, A11, A31, A3301, and A6801.
[0103] The present invention provides compositions comprising
immunogenic peptides having binding motifs for HLA alleles. The
immunogenic peptides are about 9 to 10 residues in length and
comprise conserved residues at certain positions such as a proline
at position 2 and an aromatic residue (e.g., Y,W,F) or hydrophobic
residue (e.g., L,I,V,M, or A) at the carboxy terminus. In
particular, an advantage of the peptides of the invention is their
ability to bind to two or more different HLA alleles.
[0104] The present invention also provides compositions comprising
immunogenic peptides having binding motifs for MHC Class I
molecules. The immunogenic peptides are typically between about 8
and about 11 residues and comprise conserved residues involved in
binding proteins encoded by the appropriate MHC allele. A number of
allele specific motifs have been identified.
[0105] For instance, the motif for HLA-A3.2 comprises from the
N-terminus to C-terminus a first conserved residue of L, M, I, V,
S, A, T and F at position 2 and a second conserved residue of K, R
or Y at the C-terminal end. Other first conserved residues are C, G
or D and alternatively E. Other second conserved residues are H or
F. The first and second conserved residues are preferably separated
by 6 to 7 residues.
[0106] The motif for HLA-A1 comprises from the N-terminus to the
C-terminus a first conserved residue of T, S or M, a second
conserved residue of D or E, and a third conserved residue of Y.
Other second conserved residues are A, S or T. The first and second
conserved residues are adjacent and are preferably separated from
the third conserved residue by 6 to 7 residues. A second motif
consists of a first conserved residue of E or D and a second
conserved residue of Y where the first and second conserved
residues are separated by 5 to 6 residues.
[0107] The motif for HLA-A11 comprises from the N-terminus to the
C-terminus a first conserved residue of T or V at position 2 and a
C-terminal conserved residue of K. The first and second conserved
residues are preferably separated by 6 or 7 residues.
[0108] The motif for HLA-A24.1 comprises from the N-terminus to the
C-terminus a first conserved residue of Y, F or W at position 2 and
a C terminal conserved residue of F, I, W, M or L. The first and
second conserved residues are preferably separated by 6 to 7
residues.
[0109] The present invention also provides compositions comprising
immunogenic peptides having allele-specific binding motifs, such as
binding motifs for HLA-A2.1 molecules. For HLA class I epitopes,
which bind to the appropriate HLA Class I allele, the peptides
typically comprise epitopes from 8-11 amino acids in length, often
9 to 10 residues in length, that comprise conserved residues at
certain positions such as positions 2 and the C-terminal position.
Moreover, the peptides preferably do not comprise negative binding
residues as defined herein at other positions such as, in an
HLA-A2.1 motif-bearing epitope, positions 1, 3, 6 and/or 7 in the
case of peptides 9 amino acids in length and positions 1, 3, 4, 5,
7, 8 and/or 9 in the case of peptides 10 amino acids in length. For
HLA class II epitopes, the peptides typically comprise a motif of 6
to about 25 amino acids for a class II HLA motif, typically, 9 to
13 amino acids in length, which is recognized by a particular HLA
molecule. The present invention defines positions within a motif
enabling the selection of peptides which will bind efficiently to
HLA A2.1.
[0110] The invention also provides the parameters for the design of
vaccines which are expected to effectively target large portions of
the population. Following the guidance set forth herein, to prepare
vaccines with respect to a particular infectious organism or virus
or tumor, the relevant antigen is assessed to determine the
location of epitopes which are most likely to effect a cytotoxic T
response to an infection or tumor. By analyzing the amino acid
sequence of the antigen according to the methods set forth herein,
an appropriate set of epitopes can be identified. Peptides which
consist of these epitopes can readily be tested for their ability
to bind one or more HLA alleles characteristic of the A2 supertype.
In general, peptides which bind with an affinity represented by an
IC.sub.50 of 500 nM or less have a high probability of eliciting a
cytotoxic T lymphocyte (CTL) response. The ability of these
peptides to do so can also readily be verified. Vaccines can then
be designed based on the immunogenic peptides thus identified. The
vaccines themselves can consist of the peptides per se, precursors
which will be expected to generate the peptides in vivo, or nucleic
acids encoding these peptides for production in vivo.
[0111] Thus, in one aspect, the invention is directed to a method
for identifying an epitope in an antigen characteristic of a
pathogen or tumor. The epitope identified by this method is more
likely to enhance an immune response in an individual bearing an
allele of the A2 supertype than an arbitrarily chosen peptide. The
method comprises analyzing the amino acid sequence of the antigen
for segments of 8-11 amino acids, where the amino acid at position
2 is a small or aliphatic hydrophobic residue (L, I, V, M, A, T or
Q) and the amino acid at the C-terminus of the segment is also a
small or aliphatic hydrophobic residue (L, I, V, M, A or T). In
preferred embodiments, the residue at position 2 is L or M. In
other preferred embodiments, the segment contains 9-10 amino acids.
In another preferred embodiment, the segment contains Q or N at
position 1 and/or R, H or K at position 8, and lacks a D, E and G
at position 3 when the segment is a 10-mer. Also preferred is V at
position 2 and at the C-terminus.
[0112] The corresponding family of HLA molecules (i.e., the HLA-A2
supertype that binds these peptides) is comprised of at least:
A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*0209,
A*0214, A*6802, and A*6901.
[0113] Also described herein are compositions comprising
immunogenic peptides having binding motif subsequences for HLA-A2.1
molecules. The immunogenic epitopes in the peptides, which bind to
the appropriate MHC allele, are preferably 8-11 residues in length
and more preferably 9 to 10 residues in length and comprise
conserved residues at certain positions such as positions 2 and the
C-terminus (often position 9). Moreover, the peptides do not
comprise negative binding residues as defined herein at other
positions such as positions 1, 3, 6 and/or 7 in the case of
peptides 9 amino acids in length and positions 1, 3, 4, 5, 7, 8
and/or 9 in the case of peptides 10 amino acids in length. The
present invention defines positions within a motif enabling the
selection of peptides which will bind efficiently to HLA A2.1.
[0114] The HLA-A2.1 motif-bearing peptides comprise epitopes of
8-11 amino acids which typically have a first conserved residue at
the second position from the N-terminus selected from the group
consisting of L, M, I, V, A, T, and Q and a second conserved
residue at the C-terminal position selected from the group
consisting of V, L, I, A, M, and T. In a preferred embodiment, the
peptide may have a first conserved residue at the second position
from the N-terminus selected from the group consisting of V, A, T,
or Q; and a second conserved residue at the C-terminal position
selected from the group consisting of L, M, I, V, A, and T.
Secondary anchor specificities have also been defined for the
HLA-A2.1 binding motif.
[0115] The HLA-A1 motifs characterized by the presence in peptide
ligands of T, S, or M as a primary anchor residue at position 2 and
the presence of Y as a primary anchor residue at the C-terminal
position of the epitope. An alternative allele-specific A1 motif is
characterized by a primary anchor residue at position 3 rather than
position 2. This motifs characterized by the presence of D, E, A,
or S as a primary anchor residue in position 3, and a Y as a
primary anchor residue at the C-terminal position of the epitope
(see, e.g., DiBrino et al., J. Immunol., 152:620, 1994; Kondo et
al., Immunogenetics 45:249, 1997; and Kubo et al., J Immunol.
152:3913, 1994 for reviews of relevant data).
[0116] The motif for HLA-A1 comprises from the N-terminus to the
C-terminus a first conserved residue of T, S or M, a second
conserved residue of D or E, and a third conserved residue of Y.
Other second conserved residues are A, S or T. The first and second
conserved residues are adjacent and are preferably separated from
the third conserved residue by 6 to 7 residues. A second motif
consists of a first conserved residue of E or D and a second
conserved residue of Y where the first and second conserved
residues are separated by 5 to 6 residues.
[0117] The HLA-A3 motif is characterized by the presence in peptide
ligands of L, M, V, I, S, A, T, F, C, G, or D as a primary anchor
residue at position 2, and the presence of K, Y, R, H, F, or A as a
primary anchor residue at the C-terminal position of the epitope
(see, e.g., DiBrino et al., Proc. Natl. Acad. Sci. USA 90:1508,
1993; and Kubo et al., J Immunol. 152:3913-24, 1994).
[0118] For instance, the motif for HLA-A3.2 comprises from the
N-terminus to C-terminus a first conserved residue of L, M, I, V,
S, A, T and F at position 2 and a second conserved residue of K, R
or Y at the C-terminal end. Other first conserved residues are C, G
or D and alternatively E. Other second conserved residues are H or
F. The first and second conserved residues are preferably separated
by 6 to 7 residues.
[0119] The HLA-A11 motif is characterized by the presence in
peptide ligands of V, T, M, L, I, S, A, G, N, C, D, or F as a
primary anchor residue in position 2, and K, R, Y, or H as a
primary anchor residue at the C-terminal position of the epitope
(see, e.g., Zhang et al., Proc. Natl. Acad. Sci USA 90:2217-2221,
1993; and Kubo et al., J Immunol. 152:3913-3924, 1994). The first
and second conserved residues are preferably separated by 6 or 7
residues.
[0120] The HLA-A3 and HLA-A11 are members of the HLA-A3 supertype
family. The HLA-A3 supermotifs characterized by the presence in
peptide ligands of A, L, I, V, M, S, or, T as a primary anchor at
position 2, and a positively charged residue, R or K, at the
C-terminal position of the epitope, e.g., in position 9 of 9-mers
(see, e.g., Sidney et al., Hum. Immunol. 45:79, 1996). Exemplary
members of the corresponding family of HLA molecules (the HLA-A3
supertype) that bind the A3 supermotif include A*0301, A*1101,
A*3101, A*3301, and A*6801.
[0121] The invention further comprises an extended A3 supermotif,
which is based on a detailed map of the secondary anchor
requirements for binding to molecules of the HLA A3 supertype. The
extended supermotif allows for the efficient prediction of
cross-reactive binding of peptides to alleles of the A3 supertype
by screening the native sequence of a particular antigen. It is
also used to select analog options for peptides that bear amino
acids defined by the primary supermotif. Analoging can comprise
selection of desired residues at the primary and/or secondary
anchor positions, thereby altering the binding affinity and immune
modulating properties of the resulting analogs.
[0122] In order to identify A3 supermotif-bearing epitopes in a
target antigen, a native protein sequence, e.g., a tumor-associated
antigen, an infectious organism, or a donor tissue for
transplantation, is screened using a means for computing, such as
an intellectual calculation or a computer, to determine the
presence of an A3 supermotif within the sequence. The information
obtained from the analysis of native peptide can be used directly
to evaluate the status of the native peptide or may be utilized
subsequently to generate the peptide epitope.
[0123] The HLA-A24 motifs characterized by the presence in peptide
ligands of Y, F, W, or M as a primary anchor residue in position 2,
and F, L, I, or W as a primary anchor residue at the C-terminal
position of the epitope (see, e.g., Kondo et al., J: Immunol.
155:4307-4312, 1995; and Kubo et al., J. Immunol. 152:3913-3924,
1994). The motif for HLA-A24.1 comprises from the N-terminus to the
C-terminus a first conserved residue of Y, F or W at position 2 and
a C terminal conserved residue of F, I, W, M or L. The first and
second conserved residues are preferably separated by 6 to 7
residues.
[0124] The invention also comprises peptides comprising epitopes
containing an HLA-B7 supermotif. Following the methods described in
the copending applications noted above, certain peptides capable of
binding at multiple HLA alleles which possess a common motif have
been identified. The motifs of those peptides can be characterized
as follows: N-XPXXXXXX(A,V,I,L,M)-C (SEQ ID NO:14618);
N-XPXXXXXXX(A,V,I,L,M)-C (SEQ ID NO:14619); N-XPXXXXXX(F,W,Y)-C
(SEQ ID NO:14620); and N-XPXXXXXXX(F,W,Y)-C (SEQ ID NO:14621).
Motifs that are capable of binding at multiple alleles are referred
to here as "supermotifs." The particular supermotifs above are
specifically called "B7-like-supermotifs." The epitopes are 8-11
amino acids in length, often 9 or 10 amino acids in length, and
comprise conserved residues of a proline at position 2 and an
aromatic residue (e.g., Y, W, F) or hydrophobic residue (e.g., L,
I, V, M, A) at the C-terminal position of the epitope. Peptides
bearing an HLA-B7 supermotif bind to more than one HLA-B7 supertype
family member. The corresponding family of HLA molecules that bind
the B7 supermotif (i.e., the HLA-B7 supertype) is comprised of at
least twenty six HLA-B proteins comprising at least: B*0702,
B*0703, B*0704, B*0705, B*1508, B*3501, B*3502, B*3503, B*3504,
B*3505, B*3506, B*3507, B*3508, B*5101, B*5102, B*5103, B*5104,
B*5105, B*5301, B*5401, B*5501, B*5502, B*5601, B*5602, B*6701, and
B*7801 (see, e.g., Sidney, et al., J: Immunol. 154:247, 1995;
Barber, et al., Curro Bioi. 5:179, 1995; Hill, et aI., Nature
360:434, 1992; Ramannsee, et aI., Immunogenetics 41:178, 1995).
[0125] The present invention defines positions within a motif
enabling the selection of peptides that will bind efficiently to
more than one HLA-A, HLA-B or HLA-C alleles. Immunogenic peptides
of the invention are typically identified using a computer to scan
the amino acid sequence of a desired antigen for the presence of
the supermotifs. Examples of antigens include viral antigens and
antigens associated with cancer. An antigen associated with cancer
is an antigen, such as a melanoma antigen, that is characteristic
of (i.e., expressed by) cells in a malignant tumor but not normally
expressed by healthy cells. Epitopes on a number of immunogenic
target proteins can be identified using the sequence motifs
described herein. Examples of suitable antigens particularly
include hepatitis B core and surface antigens (HBVc, HBVs)
hepatitis C antigens, Epstein-Barr virus antigens, and human
immunodeficiency virus (HIV) antigens, and also include prostate
specific antigen (PSA), melanoma antigens (e.g., MAGE-1), and human
papilloma virus (HPV) antigens Lassa virus, p53 CEA, and Her2/neu;
this list is not intended to exclude other sources of antigens.
[0126] Epitopes on a number of immunogenic target proteins, i.e.,
target antigens, have been identified. Examples of suitable
antigens include tumor-associated antigens such as tyrosinase
related proteins 1 and 2 (TRP 1 and TRP), which are frequently
associated with melanoma; MART1; p53 and murine p53 (mp53),
carcinoembryonic antigen (CEA), Her2/neu; and MAGE, including
MAGE1, MAGE2, and MAGE3, which are expressed on a broad range of
tumors; prostate cancer-associated antigens such as prostate
specific antigen (PSA), human kallikrein (huK2), prostate specific
membrane antigen (PSM), and prostatic acid phosphatase (PAP);
antigens from viruses such as hepatitis B (e.g., HBV core and
surface antigens (HBVc, HBVs)) hepatitis C antigens, Epstein-Barr
virus, human immunodeficiency type-1 virus (HIV 1), Kaposi's
sarcoma herpes (KSHV), human papilloma virus (HPV), influenza
virus, and Lassa virus antigens, Mycobacterium tuberculosis (MT)
antigens, trypanosome, e.g., Trypansoma cruzi (T. cruzi), antigens
such as surface antigen (TSA), and malaria antigens.
[0127] The peptides are thus useful in pharmaceutical compositions
for both in vivo and ex vivo therapeutic and diagnostic
applications (e.g., tetramer reagents; Beckman Coulter).
[0128] The present invention also provides compositions comprising
immunogenic peptides having binding motifs for non-A2 HLA alleles.
The immunogenic peptides are preferably about 9 to 10 residues in
length and comprise conserved residues at certain positions such as
proline at position 2 and an aromatic residue (e.g., Y, W, F) or
hydrophobic residue (e.g., L, I, V, M, or A) at the carboxy
terminus. In particular, an advantage of the peptides of the
invention is their ability to bind to two or more different HLA
alleles.
[0129] Upon identification of epitopes comprising the HLA A3
supermotif, motif-bearing peptides can be isolated from a native
sequence or synthesized. Accordingly, epitope-based vaccine
compositions directed to a target antigen are prepared. These
epitope-based vaccines preferably have enhanced, typically
broadened, population coverage. The HLA-A3 supermotif-bearing
epitopes comprising the vaccine composition preferably bind to more
than one HLA A3 supertype molecule with a K.sub.D of less than 500
nM, and stimulate a CTL response in patients bearing an HLA A3
supertype allele to which the peptide binds.
[0130] Motif-bearing peptides may additionally be used as
diagnostic, rather than immunogenic, reagents to evaluate an immune
response. For example, an HLA-A3 supermotif-bearing peptide epitope
may be used prognostically to analyze an immune response for the
presence of specific CTL populations from patients who possess an
HLA A3 supertype allele bound by the peptide epitope.
[0131] Certain specific embodiments of the invention are summarized
below.
[0132] The present invention provides a method for identifying a
peptide epitope predicted to bind two or more allele-specific HLA
A3 supertype molecules. The peptide epitope of, for example, 8-15
amino acid residues, typically 8-11 amino acid residues, and
preferably 9-10 amino acid residues, is identified in an amino acid
sequence using a means for computing such as an intellectual
calculation, preferably a computer, to determine the presence of an
A3 supermotif within the sequence. As noted above, the A3
supermotif comprises a first primary amino acid anchor residue that
is V, S, M, A, T, L, or I at position two from the amino terminal
end of the epitope and a second primary amino acid anchor residue
that is R or K at the carboxyl terminal end of the epitope. We note
that the epitope may be comprised by a peptide or protein sequence
larger than the epitope itself and still fall within the bounds of
the invention.
[0133] Following identification, the peptide epitope may be
synthesized such that the first residue of the motif is at the
second position from the amino terminal residue of the peptide.
Further, a peptide may be synthesized that comprises at least two
epitopes, preferably at least two distinct epitopes.
[0134] The binding affinity of a peptide epitope in accordance with
the invention for at least one HLA A3 supertype molecule is
preferably determined. A preferred peptide epitope has a binding
affinity of less than 500 nM for the at least one HLA A3 supertype
molecule, and more preferably less than 50 nM.
[0135] Synthesis of an A3 supermotif-containing epitope may occur
in vitro or in vivo. In a preferred embodiment, the peptide is
encoded by a recombinant nucleic acid and expressed in a cell. The
nucleic acid may encode one or more peptides, at least one of which
is an epitope of the invention.
[0136] A peptide epitope of the invention, in the context of an HLA
A-3 supertype molecule to which it binds, can be contacted, either
in vitro or in vivo, with a cytotoxic T lymphocyte and thereby be
used to elicit a T cell response in an HLA-diverse population.
[0137] A CTL response against a target antigen may be induced,
preferably with peripheral blood mononuclear cells (PBMCs), from a
patient that has an allele-specific HLA-A3 molecule that is a
member of the A3 supertype. A CTL response can be induced by
contacting the PBMCs with an A3 supermotif-bearing peptide epitope
derived from the target antigen. Preferably, the supermotif-bearing
epitope binds the HLA molecule with a K.sub.D of less than 500 nM.
The CTLs or PBMCs may further be contacted with a helper T
lymphocyte (HTL) peptide epitope, whereby both a CTL and an HTL
response are induced. The CTL epitope and the HTL epitope may be
comprised by a single peptide. Further, the HTL epitope may be
lipidated, preferably with palmitic acid, and may be linked by a
spacer molecule to the CTL epitope. The epitope may be expressed by
a nucleotide sequence; in a preferred embodiment the nucleotide
sequence is comprised by an attenuated viral host.
[0138] As will be apparent from the discussion below, other
embodiments of methods and compositions are also within the scope
of the invention. Further, novel synthetic peptides produced by any
of the methods described herein are also part of the invention.
[0139] The present invention provides peptides and nucleic acids
encoding them for use in vaccines and therapeutics. The invention
provides methods of inducing a cytotoxic T cell response against a
preselected antigen in, a patient, the method comprising contacting
a cytotoxic T cell with an immunogenic peptide of the invention.
The peptides of the invention may be derived from a number of
antigens including viral antigens, tumor associated antigens,
parasitic antigens, fungal antigens and the like. The methods of
the invention can be carried out in vitro or in vivo. In a
preferred embodiment the peptides are contacted with the cytotoxic
T cell by administering to the patient a nucleic acid molecule
comprising a sequence encoding the immunogenic peptide.
[0140] In one embodiment, the peptide is of between about 9 and
about 15 residues and binds to at least two HLA-A3-like molecules
with a dissociation constant of less than about 500 nM and induces
a cytotoxic T cell response. The immunogenic peptides have a
sequence of 9 residues comprising a binding motif from the
N-terminus to the C-terminus as follows: [0141] a first primary
anchor residue at the second position selected from the group
consisting of A, L, I, V, M, S and T and a second primary anchor
residue at the ninth position selected from the group consisting of
R and K; and [0142] one or more secondary anchor residues selected
from the group consisting of Y, F, or W, at the third position, Y,
F, or W at the sixth position, Y, F, or W at the seventh position,
P at the eighth position, and any combination thereof.
[0143] The invention further provides immunogenic peptides which
bind to HLAA*0301 gene products. These peptides comprise a nine
residue binding motif from the N-terminus to the C-terminus as
follows: [0144] a first primary anchor residue at the second
position selected from the group consisting of A, L, I, V, M, S and
T and a second primary anchor residue at the ninth position
selected from the group consisting of R and K; and [0145] one or
more secondary anchor residues selected from the group consisting
of R, H, or K at the first position, Y, F, or W, at the third
position, P, R, H, K, Y, F, or W at the fourth position, A at the
fifth position, Y, F, or W at the sixth position, P at the eighth
position, and any combination thereof.
[0146] The invention also provides immunogenic peptides which bind
to HLAA*1101 gene products. These peptides comprise a nine residue
binding motif from the N-terminus to the C-terminus as follows:
[0147] a first primary anchor residue at the second position
selected from the group consisting of A, L, 1, V, M, S and T and a
second primary anchor residue at the ninth position selected from
the group consisting of R and K; and [0148] a secondary anchor
residue selected from the group consisting of A at the first
position, Y, F, or W, at the third position, Y, F, or W at the
fourth position, A at the fifth position, Y, F, or W at the sixth
position, Y, F, or W at the seventh position, P at the eighth
position, and any combination thereof.
[0149] The present invention is directed to methods of modulating
the binding of peptide epitopes to HLA class I molecules and HLA
class II molecules. The invention includes a method of modifying
binding of an original peptide epitope that bears a motif
correlated with binding to an HLA molecule, said motif comprising
at least one primary anchor position, said at least one primary
anchor position having specified therefore primary anchor amino
acid residues consisting essentially of two or more residues, said
method comprising exchanging the primary anchor residue of the
original peptide epitope for another primary anchor residue, with
the proviso that the original primary anchor residue is not the
same as the exchanged primary anchor residue. A preferred
embodiment of the invention includes a method where the original
primary anchor residue is a less preferred residue, and the
exchanged residue is a more preferred residue.
[0150] One alternative embodiment of the invention includes a
method of modifying binding of an original peptide epitope that
bears a motif correlated with binding to an HLA molecule, said
motif comprising at least one primary anchor position having
specified therefore at least one primary anchor residue, and at
least one secondary anchor position having specified therefore at
least one secondary residue, said method comprising exchanging the
secondary anchor residue of the original peptide epitope for
another secondary anchor residue, with the proviso that the
original secondary anchor residue is different than the exchanged
amino acid residue. In some cases the original secondary residue is
a deleterious residue and the exchanged residue is a residue other
than a deleterious residue and/or the original secondary anchor
residue is a less preferred residue and the exchanged residue is a
more preferred residue.
[0151] Another alternative embodiment is a method comprising
modifying binding of an epitope that bears an HLA B7 supermotif of
a primary anchor amino acid residue P at a position two and a
primary anchor amino acid residue which is V, I, L, F, M, W, Y or A
at a carboxyl terminus, wherein said residues are separated by at
least five residues and wherein the amino acid positions are
numbered consecutively from an amino to carboxyl orientation, said
method comprising: [0152] (a) exchanging a primary anchor residue
at the carboxyl terminus for a residue which is V, I, L, F, M, W, Y
or A, with the proviso that the original primary anchor residue at
the carboxyl terminus is not the same as the exchanged residue.
[0153] An alternative method of this embodiment includes a method
wherein the primary anchor residue at the carboxyl terminus is
separated from the primary anchor residue at position two by six
residues. The method further comprises: [0154] (a) exchanging a
secondary anchor residue at position one for a residue which is F,
Y, W, L, I, V, or M with the proviso that the original secondary
anchor residue at positions one is not the same as the exchanged
residue; or [0155] (b) exchanging a secondary anchor residue at
position three and/or eight for a residue which is F, Y or W with
the proviso that the original secondary anchor residue at positions
three, and/or eight is not the same as the exchanged residue.
[0156] A further alternative embodiment is a method comprising
modifying binding of an epitope that bears an HLA B7 supermotif of
a primary anchor amino acid residue P at a position two and a
primary anchor amino acid residue which is V, I, L, F, M, W, Y or A
at a carboxyl terminus, wherein said residues are separated by at
least six residues and wherein the amino acid positions are
numbered consecutively from an amino to carboxyl orientation, said
method comprising: [0157] (a) exchanging a secondary anchor residue
at position one for a residue which is F, Y, W, L, I, V, or M with
the proviso that the original secondary anchor residue at positions
one is not the same as the exchanged residue; or [0158] (b)
exchanging a secondary anchor residue at position three and/or
eight for a residue which is F, Y or W with the proviso that the
original secondary anchor residue at positions three, and/or eight
is not the same as the exchanged residue; [0159] (c) performing
steps (a) and (b).
[0160] Another embodiment of the invention comprises a method of
modifying binding of a peptide epitope that bears an HLA A2
supermotif of a primary anchor amino acid residue which is L, I, V,
M, A, T, or Q at a position two and a primary anchor amino acid
residue which is L, I, V, M, A, or T at a carboxyl terminus,
wherein said residues are separated by at least five residues, and
wherein the amino acid positions are numbered consecutively from an
amino to carboxyl orientation, said method comprising: [0161] (a)
exchanging an original primary anchor residue at position two for a
residue which is L, I, V, M, A, T, or Q with the proviso that the
original primary anchor residue at position two is not the same as
the exchanged residue at position two; or [0162] (b) exchanging a
primary anchor residue at the carboxyl terminus of the epitope for
a residue which is L, I, V, M, A, or T with the proviso that the
original primary anchor residue at the carboxyl terminus is not the
same as the exchanged residue; or, [0163] (c) performing steps (a)
and (b).
[0164] Also included is a method comprising modifying binding of a
peptide epitope that bears an HLA A3 supermotif of a primary anchor
amino acid residue which is V, S, M, A, T, L, or I at a position
two and a primary anchor amino acid residue which is R or K at a
carboxyl terminus, wherein said residues are separated by at least
five residues, and wherein the amino acid positions are numbered
consecutively from an amino to carboxyl orientation, said method
comprising: [0165] (a) exchanging an original primary anchor
residue at position two for a residue which is V, S, M, A, T, L, or
I with the proviso that the original primary anchor residue at
position two is not the same as the exchanged residue at position
two; or [0166] (b) exchanging a primary anchor residue at the
carboxyl terminus of the epitope for a residue which is R or K with
the proviso that the original primary anchor residue at the
carboxyl terminus is not the same as the exchanged residue; or,
[0167] (c) performing steps (a) and (b).
[0168] The preceding embodiment may be a method where the primary
anchor residue at the carboxyl terminus is separated from the
primary anchor residue at position two by six residues, said method
comprising: [0169] (a) exchanging an original secondary anchor
residue at position three, six or seven which is Y, F or W, with
the proviso that the original secondary anchor residue at position
three, six or seven, respectively, is not the same as the exchanged
residue; or [0170] (b) exchanging an original secondary anchor
residue at position eight for a residue which is P with the proviso
that the original secondary anchor residue at position eight is not
P.
[0171] Another embodiment of the invention includes a method
comprising modifying binding of a peptide epitope that bears an HLA
A3 supermotif of a primary anchor amino acid residue which is V, S,
M, A, T, L, or I at a position two and a primary anchor amino acid
residue which is R or K at a carboxyl terminus, wherein said
residues are separated by six residues, and wherein the amino acid
positions are numbered consecutively from an amino to carboxyl
orientation, said method comprising: [0172] (a) exchanging an
original secondary anchor residue at position three, six or seven
which is Y, F or W, with the proviso that the original secondary
anchor residue at position three, six or seven, respectively, is
not the same as the exchanged residue; or [0173] (b) exchanging an
original secondary anchor residue at position eight for a residue
which is P with the proviso that the original secondary anchor
residue at position eight is not P; or [0174] (c) where the epitope
bears at least one deleterious residue indicated for said
supermotif in Table 138, exchanging said deleterious residue for a
residue which is not deleterious; or [0175] (d) performing two or
more of steps (a)-(c).
[0176] Alternative embodiments of the invention include a method
comprising modifying binding of a peptide epitope that bears an HLA
A3 motif of a primary anchor amino acid residue which is A, L, I,
V, M, S, T, F, C, G, or D at a position two and a primary anchor
amino acid residue which is R, K, Y, H, F, or A at a carboxyl
terminus, wherein said residues are separated by at least five
residues, and wherein the amino acid positions are numbered
consecutively from an amino to carboxyl orientation, said method
comprising: [0177] (a) exchanging an original primary anchor
residue at position two for a residue which is A, L, I, V, M, S, T,
F, C, G, or D with the proviso that the original primary anchor
residue at position two is not the same as the exchanged residue at
position two; or [0178] (b) exchanging a primary anchor residue at
the carboxyl terminus of the epitope for a residue which is R, K,
Y, H, F, or A with the proviso that the original primary anchor
residue at the carboxyl terminus is not the same as the exchanged
residue; or, [0179] (c) performing steps (a) and (b).
[0180] Another embodiment of the invention is a method comprising
modifying binding of a peptide epitope that bears an HLA A3 motif
of a primary anchor amino acid residue which is A, L, I, V, M, S,
T, F, C, G, or D at a position two and a primary anchor amino acid
residue which is R, K, Y, H, F, or A at a carboxyl terminus,
wherein said residues are separated by six residues, and wherein
the amino acid positions are numbered consecutively from an amino
to carboxyl orientation, said method comprising: [0181] (a)
exchanging an original secondary anchor residue at position one for
a residue which is R, H, or K with the proviso that the original
secondary anchor residue at position one is not the same as the
exchanged residue of position one; or [0182] (b) exchanging a
secondary anchor residue at position three for a residue which is
Y, F or W with the proviso that the original secondary anchor
residue at position three is not the same as the exchanged residue
of position three; or, [0183] (c) exchanging an original secondary
anchor residue at position four for a residue which is P, R, H, K,
Y, F, or W with the proviso that the original secondary anchor
residue at position four is not the same as the exchanged residue
at position four; or [0184] (d) exchanging an original secondary
anchor residue at position five for a residue which is A with the
proviso that the original secondary anchor residue at position five
is not A; or [0185] (e) exchanging an original secondary anchor
residue at position six for a residue which is Y, F or W with the
proviso that the original secondary anchor residue at position six
is not the same as the exchanged residue at position six; or [0186]
(f) exchanging an original secondary anchor residue at position
eight for a residue which is P with the proviso that the original
secondary anchor residue at position eight is not P; or [0187] (g)
where the epitope bears at least one deleterious residue indicated
for said motif in Table 138, exchanging said deleterious residue
for a residue which is not a deleterious residue; or [0188] (h)
performing two or more of steps (a)-(g).
[0189] Further, the invention includes a method comprising
modifying binding of a peptide epitope that bears an HLA A11 motif
of a primary anchor amino acid residue which is V, T, M, L, I, S,
A, G, N, C, D, or F at a position two and a primary anchor amino
acid residue which is K, R, Y, or H at a carboxyl terminus, wherein
said residues are separated by at least five residues, and wherein
the amino acid positions are numbered consecutively from an amino
to carboxyl orientation, said method comprising: [0190] (a)
exchanging an original primary anchor residue at position two for a
residue which is V, T, M, L, I, S, A, G, N, C, D or F with the
proviso that the original primary anchor residue at position two is
not the same as the exchanged residue at position two; or [0191]
(b) exchanging a primary anchor residue at the carboxyl terminus of
the epitope for a residue which is K, R, Y, or H with the proviso
that the original primary anchor residue at the carboxyl terminus
is not the same as the exchanged residue; or, [0192] (c) performing
steps (a) and (b).
[0193] An alternative method comprises modifying binding of a
peptide epitope that bears an HLA A11 motif of a primary anchor
amino acid residue which is V, T, M, L, I, S, A, G, N, C, D or F at
a position two and a primary anchor amino acid residue which is K,
R, Y, or H at a carboxyl terminus, wherein said residues are
separated by six residues, and wherein the amino acid positions are
numbered consecutively from an amino to carboxyl orientation, said
method comprising: [0194] (a) exchanging an original secondary
anchor residue at position one for a residue which is A with the
proviso that the original secondary anchor residue at position one
is not the same as the exchanged residue; or [0195] (b) exchanging
a secondary anchor residue at position three, four, six, or seven
for a residue which is Y, F or W with the proviso that the original
secondary anchor residue at position three, four, six or seven
respectively is not the same as the exchanged residue at such
position; or [0196] (c) exchanging a secondary residue at position
five for a residue which is A with the proviso that the original
secondary anchor residue at position five is not the same as the
exchanged residue at position five; or [0197] (d) exchanging a
secondary anchor residue at position eight for a residue which is P
with the proviso that the original secondary anchor residue at
position eight is not the same as the exchanged residue at position
eight; or [0198] (e) where the epitope bears at least one
deleterious residue indicated for said motif in Table 138,
exchanging said deleterious residue for a residue which is not a
deleterious residue; or [0199] (f) performing two or more of steps
(a)-(e).
[0200] An additional embodiment of the invention comprises a method
for modifying binding of a peptide epitope that bears an HLA A2.1
motif of a primary anchor amino acid residue which is L, M, V, Q,
I, A, or T at a position two and a primary anchor amino acid
residue which is V, L, I, M, A, or T at a carboxyl terminus,
wherein said residues are separated by at least five residues, and
wherein the amino acid positions are numbered consecutively from an
amino to carboxyl orientation, said method comprising: [0201] (a)
exchanging an original primary anchor residue at position two for a
residue which is L, M, V, Q, I, A, or T with the proviso that the
original primary anchor residue at position two is not the same as
the exchanged residue at position two; or [0202] (b) exchanging a
primary anchor residue at the carboxyl terminus of the epitope for
a residue which is V, L, I, M, A, or T with the proviso that the
original primary anchor residue at the carboxyl terminus is not the
same as the exchanged residue; or, [0203] (c) performing steps (a)
and (b).
[0204] An alternative embodiment of the invention comprises a
method of modifying binding of a peptide epitope that bears an HLA
A2.1 motif of a primary anchor amino acid residue which is L, M, V,
Q, I, A, or T at a position two and a primary anchor amino acid
residue which is V, L, I, M, A, or T at a carboxyl terminus,
wherein said residues are separated by six residues, and wherein
the amino acid positions are numbered consecutively from an amino
to carboxyl orientation, said method comprising: [0205] (a)
exchanging an original secondary anchor residue at positions one,
three, and/or five for a residue which is Y, F or W with the
proviso that the original secondary anchor residue at positions
one, three, and/or five respectively is not the same as the
exchanged residue at such a position; or [0206] (b) exchanging an
original secondary anchor residue at position four for a residue
which is S, T or C with the proviso that the original secondary
anchor residue at position four is not the same as the exchanged
residue at position four; or [0207] (c) exchanging an original
secondary anchor residue at position seven for a residue which is A
with the proviso that the original secondary anchor residue at
position seven is not A; or [0208] (d) exchanging a secondary
anchor residue at position eight for a residue which is P with the
proviso that the original secondary anchor residue at position
eight is not P; or [0209] (e) where the epitope bears at least one
deleterious residue indicated for said motif in Table 138,
exchanging said deleterious residue for a residue which is not a
deleterious residue; or [0210] (f) performing two or more of steps
(a)-(e).
[0211] Further, an additional embodiment of the invention comprises
a method of modifying binding of a peptide epitope that bears an
HLA A2.1 motif of a primary anchor amino acid residue which is L,
M, V, Q, I, A, or T at a position two and a primary anchor amino
acid residue which is V, L, I, M, A, or T at a carboxyl terminus,
wherein said residues are separated by seven residues, and wherein
the amino acid positions are numbered consecutively from an amino
to carboxyl orientation, said method comprising: [0212] (a)
exchanging an original secondary anchor residue at position one for
a residue which is A, Y, F, or W with the proviso that the original
secondary anchor residue at position one is not the same as the
exchanged residue; or [0213] (b) exchanging an original secondary
anchor residue at position three for a residue which is L, V, I, or
M with the proviso that the original secondary anchor residue at
position three is not the same as the exchanged residue; or [0214]
(c) exchanging a secondary anchor residue at positions four and/or
six for a residue which is G with the proviso that the original
secondary anchor residue at position four and/or six respectively
is not G; or [0215] (d) exchanging an original secondary anchor
residue at position eight for a residue which is F, Y, W, L, V, I
or M with the proviso that the original secondary anchor residue at
position eight is not the same as the exchanged residue; or [0216]
(e) where the epitope bears at least one deleterious residue
indicated for said motif in Table 138, exchanging said deleterious
residue for a residue which is not a deleterious residue; or [0217]
(f) performing two or more of steps (a)-(e).
[0218] Another embodiment of the invention comprises a method of
modifying binding of a peptide epitope that bears an HLA A24 motif
of a primary anchor amino acid residue which is Y, F, W or M at a
position two and a primary anchor amino acid residue which is F, L,
I, or W at a carboxyl terminus, wherein said residues are separated
by at least five residues, and wherein the amino acid positions are
numbered consecutively from an amino to carboxyl orientation, said
method comprising: [0219] (a) exchanging an original primary anchor
residue at position two for a residue which is Y, F, W or M with
the proviso that the original primary anchor residue at position
two is not the same as the exchanged residue; or [0220] (b)
exchanging a primary anchor residue at the carboxyl terminus for a
residue which is F, L, I, or W with the proviso that the original
primary anchor residue at the carboxyl terminus is not the same as
the exchanged residue; or, [0221] (c) performing steps (a) and
(b).
[0222] A further embodiment comprises a method of modifying binding
of a peptide epitope that bears an HLA A24 motif of a primary
anchor amino acid residue which is Y, F, W or M at a position two
and a primary anchor amino acid residue which is F, L, I, or W at a
carboxyl terminus, wherein said residues are separated by six
residues, and wherein the amino acid positions are numbered
consecutively from an amino to carboxyl orientation, said method
comprising: [0223] (a) exchanging an original secondary anchor
residue at position one for a residue which is Y, F, W, R, H, or K
with the proviso that the original secondary anchor residue at
position one is not the same as the exchanged residue at position
one; or [0224] (b) exchanging a secondary anchor residue at
position four for a residue which is S, T, or C with the proviso
that the original secondary anchor residue at position four is not
the same as the exchanged residue; or [0225] (c) exchanging a
secondary anchor residue at positions seven and/or eight for a
residue which is Y, F or W with the proviso that the original
secondary anchor residue at positions seven and/or eight
respectively is not the same as the exchanged residue at position
seven or eight; or [0226] (d) where the epitope bears at least one
deleterious residue indicated for said motif in Table 138,
exchanging said deleterious residue for a residue which is not a
deleterious residue; or [0227] (e) performing two or more of steps
(a)-(d).
[0228] An alternative embodiment of the invention includes a method
comprises modifying binding of a peptide epitope that bears an HLA
A24 motif of a primary anchor amino acid residue which is Y, F, W,
or M at a position two and a primary anchor amino acid residue
which is F, L, I, or W at a carboxyl terminus, wherein said
residues are separated by seven residues, and wherein the amino
acid positions are numbered consecutively from an amino to carboxyl
orientation, said method comprising: [0229] (a) exchanging a
secondary anchor residue at position four for a residue which is P
with the proviso that the original secondary anchor residue at
position four is not P; or [0230] (b) exchanging an original
secondary anchor residue at position five for a residue which is Y,
F, W or P with the proviso that the original secondary anchor
residue at position five is not the same as the exchanged residue
at position five; or [0231] (c) exchanging a secondary anchor
residue at position seven for a residue which is P with the proviso
that the original secondary anchor residue at position seven is not
the same as the exchanged residue at position seven; or [0232] (d)
where the epitope bears at least one deleterious residue indicated
for said motif in Table 138, exchanging said deleterious residue
for a residue which is not a deleterious residue; or [0233] (e)
performing two or more of steps (a)-(d).
[0234] An alternative embodiment of the invention includes a method
comprising modifying binding of a peptide epitope that bears an HLA
A1 motif of a primary anchor amino acid residue which is S, T, or M
at a position two and a primary anchor amino acid residue which is
Y at a carboxyl terminus, wherein said residues are separated by at
least five residues, and wherein the amino acid positions are
numbered consecutively from an amino to carboxyl orientation, said
method comprising: [0235] (a) exchanging an original primary anchor
residue at position two for a residue which is S, T, or M with the
proviso that the original primary anchor residue at position two is
not the same as the exchanged residue at position two.
[0236] Another embodiment of the invention comprises a method of
modifying binding of a peptide epitope that bears an HLA A1 motif
of a primary anchor amino acid residue which is S, T, or M at a
position two and a primary anchor amino acid residue which is Y at
a carboxyl terminus, wherein said residues are separated by six
residues, and wherein the amino acid positions are numbered
consecutively from an amino to carboxyl orientation, said method
comprising: [0237] (a) exchanging a secondary anchor residue at
position one for a residue which is G, F, W, or Y with the proviso
that the original secondary anchor residue at position one is not
the same as the exchanged residue at position one; or [0238] (b)
exchanging an original secondary anchor residue at position three
for a residue which is D, E, or A with the proviso that the
original secondary anchor residue at position three is not the same
as the exchanged residue at position three; or [0239] (c)
exchanging a secondary anchor residue at position four and/or
position eight for a residue which is Y, F, or W with the proviso
that the original secondary anchor residue at position four and/or
eight is not the same as the exchanged residue at position four
and/or eight; or [0240] (d) exchanging a secondary anchor residue
at position six for a residue which is P with the proviso that the
original secondary anchor residue at position six is not P; or
[0241] (e) exchanging a secondary anchor residue at position seven
for a residue which is D, E, Q, or N with the proviso that the
original secondary anchor residue at position seven is not the same
as the exchanged residue at position seven; or [0242] (f) where the
epitope bears at least one deleterious residue indicated for said
motif in Table 138, exchanging said deleterious residue for a
residue which is not a deleterious residue; or [0243] (e)
performing two or more of steps (a)-(f).
[0244] An additional embodiment of the invention comprises a method
of modifying binding of a peptide epitope that bears an HLA A1
motif of a primary anchor amino acid residue which is S, T, or M at
a position two and a primary anchor amino acid residue which is Y
at a carboxyl terminus, wherein said residues are separated by
seven residues, and wherein the amino acid positions are numbered
consecutively from an amino to carboxyl orientation, said method
comprising: [0245] (a) exchanging a secondary anchor residue at
position one for a residue which is Y, F, or W with the proviso
that the original secondary anchor residue at position one is not
the same as the exchanged residue at position one; or [0246] (b)
exchanging an original secondary anchor residue at position three
for a residue which is D, E, A, Q, or N with the proviso that the
original secondary anchor residue at position three is not the same
as the exchanged residue at position three; or [0247] (c)
exchanging a secondary anchor residue at position four for a
residue which is A with the proviso that the original secondary
anchor residue at position four is not A; or [0248] (d) exchanging
a secondary anchor residue at position five for a residue which is
Y, F, W, Q, or N with the proviso that the original secondary
anchor residue at position five is not the same as the exchanged
residue at position five; or [0249] (e) exchanging a secondary
anchor residue at position seven for a residue which is P, A, S, T,
or C with the proviso that the original secondary anchor residue at
position seven is not the same as the exchanged residue at position
seven; or [0250] (f) exchanging a secondary anchor residue at
position eight for a residue which is G, D, or E with the proviso
that the original secondary anchor residue at position eight is not
the same as the exchanged residue at position seven; or [0251] (g)
exchanging a secondary anchor residue at position nine for a
residue which is P with the proviso that the original secondary
anchor residue at position nine is not P; or [0252] (h) where the
epitope bears at least one deleterious residue indicated for said
motif in Table 138, exchanging said deleterious residue for a
residue which is not a deleterious residue; or [0253] (i)
performing two or more of steps (a)-(h).
[0254] Further, a method of the invention comprises modifying
binding of a peptide epitope that bears an HLA A1 motif of a
primary anchor amino acid residue which is D, E, A, or S at a
position three and a primary anchor amino acid residue which is Y
at a carboxyl terminus, wherein said residues are separated by at
least five residues, and wherein the amino acid positions are
numbered consecutively from an amino to carboxyl orientation, said
method comprising: [0255] (a) exchanging an original primary anchor
residue at position three for a residue which is D, E, A, or S with
the proviso that the original primary anchor residue at position
three is not the same as the exchanged residue at position
three.
[0256] Another embodiment of the invention comprises a method of
modifying binding of a peptide epitope that bears an HLA A1 motif
of a primary anchor amino acid residue which is D, E, A, or S at a
position three and a primary anchor amino acid residue which is Y
at a carboxyl terminus, wherein said residues are separated by five
residues, and wherein the amino acid positions are numbered
consecutively from an amino to carboxyl orientation, said method
comprising: [0257] (a) exchanging a secondary anchor residue at
position one for a residue which is G, R, H, or K with the proviso
that the original secondary anchor residue at position one is not
the same as the exchanged residue at position one; or [0258] (b)
exchanging an original secondary anchor residue at position two for
a residue which is A, S, T, C, L, I, V, or M with the proviso that
the original secondary anchor residue at position two is not the
same as the exchanged residue at position two; or [0259] (c)
exchanging a secondary anchor residue at position four for a
residue which is G, S, T, or C with the proviso that the original
secondary anchor residue at position four is not the same as the
exchanged residue at position four; or [0260] (d) exchanging a
secondary anchor residue at position six for a residue which is A,
S, T, or C with the proviso that the original secondary anchor
residue at position six is not the same as the exchanged residue at
position six; or [0261] (e) exchanging a secondary anchor residue
at position seven for a residue which is L, I, V, or M with the
proviso that the original secondary anchor residue at position
seven is not the same as the exchanged residue at position seven;
or [0262] (f) exchanging a secondary anchor residue at position
eight for a residue which is D or E with the proviso that the
original secondary anchor residue at position eight is not the same
as the exchanged residue at position eight; or [0263] (g) where the
epitope bears at least one deleterious residue indicated for said
motif in Table 138, exchanging said deleterious residue for a
residue which is not a deleterious residue; or [0264] (h)
performing two or more of steps (a)-(g).
[0265] An alternative method of the invention comprises modifying
binding of a peptide epitope that bears an HLA A1 motif of a
primary anchor amino acid residue which is D, E, A, or S at a
position three and a primary anchor amino acid residue which is Y
at a carboxyl terminus, wherein said residues are separated by six
residues, and wherein the amino acid positions are numbered
consecutively from an amino to carboxyl orientation, said method
comprising: [0266] (a) exchanging a secondary anchor residue at
position one, position five, and/or position nine for a residue
which is Y, F, or W with the proviso that the original secondary
anchor residue at position one, position five, and/or position nine
is not the same as the exchanged residue at position one, position
five, or position nine; or [0267] (b) exchanging an original
secondary anchor residue at position two for a residue which is S,
T, C, L, I, V, or M with the proviso that the original secondary
anchor residue at position two is not the same as the exchanged
residue at position two; or [0268] (c) exchanging a secondary
anchor residue at position four for a residue which is A with the
proviso that the original secondary anchor residue at position four
is not A; or [0269] (d) exchanging a secondary anchor residue at
position seven for a residue which is P or G with the proviso that
the original secondary anchor residue at position seven is not the
same as the exchanged residue at position seven; or [0270] (e)
exchanging a secondary anchor residue at position eight for a
residue which is G with the proviso that the original secondary
anchor residue at position eight is not G; or [0271] (f) where the
epitope bears at least one deleterious residue indicated for said
motif in Table 138, exchanging said deleterious residue for a
residue which is not a deleterious residue; or [0272] (g)
performing two or more of steps (a)-(f).
[0273] An additional embodiment of the invention comprises a method
of modifying binding of an epitope that bears an HLA DR motif or
supermotif of a primary anchor amino acid residue which is L, I, V,
M, F, W, or Y at a position one and a primary anchor amino acid
residue which is C, S, T, P, A, L, I, V, or M at a position six,
wherein said residues are separated by 4 residues and wherein the
amino acid positions are numbered consecutively from an amino to
carboxyl orientation, said method comprising: [0274] (a) exchanging
an original primary anchor residue at position one for a residue
which is L, I, V, M, F, W, or Y, with the proviso that the original
primary anchor residue at position one is not the same as the
exchanged residue; or [0275] (b) exchanging an original primary
anchor residue at position six for a residue which is C, S, T, P,
A, L, I, V, or M, with the proviso that the original primary anchor
residue at position six is not the same as the exchanged residue;
or [0276] (c) performing steps (a) and (b).
[0277] Alternative embodiments of the invention also include a
method comprising of modifying binding of a peptide epitope that
bears an HLA DR4 motif of a primary anchor amino acid residue which
is F, M, Y, L, I, V, or W at a position one and a primary anchor
amino acid residue which is V, S, T, C, P, A, L, I, or M at a
position six, wherein said residues are separated by four residues,
and wherein the amino acid positions are numbered consecutively
from an amino to carboxyl orientation, said method comprising:
[0278] (a) exchanging an original secondary anchor residue at
position two for a residue which is M with the proviso that the
original secondary anchor residue at position two is not M; or
[0279] (b) exchanging an original secondary anchor residue at
position three for a residue which is T with the proviso that the
original secondary anchor residue at position three is not T; or
[0280] (c) exchanging an original secondary anchor residue at
position five for a residue which is I with the proviso that the
original secondary anchor residue at position five is not I; or
[0281] (d) exchanging an original secondary anchor residue at
position seven for a residue which is M or H with the proviso that
the original secondary anchor residue at position seven is not the
same as the exchanged residue at position seven; or [0282] (e)
where the epitope bears at least one deleterious residue indicated
for said motif in Table 139, exchanging said deleterious residue
for a residue which is not a deleterious residue; or [0283] (f)
performing two or more of steps (a)-(e).
[0284] An additional embodiment of the invention comprises a method
of modifying binding of a peptide epitope that bears an HLA DR1
motif of a primary anchor amino acid residue which is F, M, Y, L,
I, V, or W at a position one and a primary anchor amino acid
residue which is V, M, A, T, S, P, L, or I at a position six,
wherein said residues are separated by four residues, and wherein
the amino acid positions are numbered consecutively from an amino
to carboxyl orientation, said method comprising: [0285] (a)
exchanging an original secondary anchor residue at position four
for a residue which is P, A, M, or Q with the proviso that the
original secondary anchor residue at position four is not the same
as the exchanged residue at position four. [0286] (b) exchanging an
original secondary anchor residue at position seven for a residue
which is M with the proviso that the original secondary anchor
residue at position seven is not M; or [0287] (c) exchanging an
original secondary anchor residue at position nine for a residue
which is A, V, or M with the proviso that the original secondary
anchor residue at position nine is not the same as the exchanged
residue at position nine; or [0288] (d) where the epitope bears at
least one deleterious residue indicated for said motif in Table
139, exchanging said deleterious residue for a residue which is not
a deleterious residue; or [0289] (e) performing two or more of
steps (a)-(d).
[0290] Further, an embodiment of the invention comprises a method
of modifying binding of a peptide epitope that bears an HLA DR7
motif of a primary anchor amino acid residue which is F, M, Y, L,
I, V, or W at a position one and a primary anchor amino acid
residue which is I, V, M, S, A, C, T, P, or L at a position six,
wherein said residues are separated by four residues, and wherein
the amino acid positions are numbered consecutively from an amino
to carboxyl orientation, said method comprising: [0291] (a)
exchanging an original secondary anchor residue at position two
and/or position seven for a residue which M with the proviso that
the original secondary anchor residue at position two and/or seven
is not M; [0292] (b) exchanging an original secondary anchor
residue at position three for a residue which is W with the proviso
that the original secondary anchor residue at position three is not
M; or [0293] (c) exchanging an original secondary anchor residue at
position four for a residue which is A with the proviso that the
original secondary anchor residue at position four is not A; or
[0294] (d) exchanging an original secondary anchor residue at
position nine for a residue which is I or V with the proviso that
the original secondary anchor residue at position nine is not the
same as the exchanged residue; or [0295] (e) where the epitope
bears at least one deleterious residue indicated for said motif in
Table 139, exchanging said deleterious residue for a residue which
is not a deleterious residue; or [0296] (f) performing two or more
of steps (a)-(e).
[0297] An additional embodiment of the invention comprises a method
of modifying binding of an epitope that bears an HLA DR3 motif of a
primary anchor amino acid residue which is L, I, V, M, F, or Y at a
position one and a primary anchor amino acid residue which is D at
a position four, wherein said residues are separated by two
residues and wherein the amino acid positions are numbered
consecutively from an amino to carboxyl orientation, said method
comprising: [0298] (a) exchanging an original primary anchor
residue at position one for a residue which is L, I, V, M, F, or Y,
with the proviso that the original primary anchor residue at
position one is not the same as the exchanged residue.
[0299] Another embodiment of the invention comprises a method of
modifying binding of an epitope that bears an HLA DR3 motif of a
primary anchor amino acid residue which is L, I, V, M, F, A, or Y
at a position 1 and a primary anchor amino acid residue which is D,
N, Q, E, S, or T at a position four, and a primary anchor amino
acid residue which is K, R, or H at a position six, wherein the
amino acid positions are numbered consecutively from an amino to
carboxyl orientation, said method comprising: [0300] (a) exchanging
an original primary anchor residue at position one for a residue
which is L, I, V, M, F, A, or Y with the proviso that the original
primary anchor residue at position one is not the same as the
exchanged residue; or [0301] (b) exchanging an original primary
anchor residue at position four for a residue which is D, N, Q, E,
S, or T with the proviso that the original primary anchor residue
at position four is not the same as the exchanged residue; or
[0302] (c) exchanging an original primary anchor residue at
position six for a residue which is K, R, or H with the proviso
that the original primary anchor residue at position six is not the
same as the exchanged residue; or [0303] (d) performing two or more
of steps (a)-(c).
[0304] Lastly, an additional embodiment of the invention comprises
a method of modifying an epitope to alter its stability by
exchanging a C residue for a residue which is .alpha.-amino butyric
acid.
[0305] As will be apparent from the discussion below, other methods
and embodiments are also contemplated. Further, novel synthetic
peptides produced by any of the methods described herein are also
part of the invention.
DEFINITIONS
[0306] The following definitions are provided to enable one of
ordinary skill in the art to understand some of the preferred
embodiments of invention disclosed herein. It is understood,
however, that these definitions are exemplary only and should not
be used to limit the scope of the invention as set forth in the
claims. Those of ordinary skill in the art will be able to
construct slight modifications to the definitions below and utilize
such modified definitions to understand and practice the invention
disclosed herein. Such modifications, which would be obvious to one
of ordinary skill in the art, as they may be applicable to the
claims set forth below, are considered to be within the scope of
the present invention. If a definition set forth in this section is
contrary to or otherwise inconsistent with a definition set forth
in patents, published patent applications and other publications
and sequences from GenBank and other data bases that are herein
incorporated by reference, the definition set forth in this section
prevails over the definition that is incorporated herein by
reference.
[0307] An "HLA supertype or family", as used herein, describes sets
of HLA molecules grouped on the basis of shared peptide-binding
specificities, rather than serologic supertypes based on shared
antigenic determinants. HLA class I molecules that share somewhat
similar binding affinity for peptides bearing certain amino acid
motifs are grouped into HLA supertypes. The terms "HLA
superfamily," "HLA supertype family," "HLA family," and "HLA
xx-like molecules" (where xx denotes a particular HLA type), are
synonyms.
[0308] An "HLA-A3-like" HLA molecule (also referred to as an allele
as used herein refers to a group of HLA molecules encoded by HLA-A
alleles that share an overlapping peptide binding motif with the
HLA-A3 supermotif disclosed here. The 9 residue supermotif shared
by these alleles comprises the following primary anchor residues:
A, L, I, V, M, S, or, T at position 2 and positively charged
residues, such as R and K at position 9 (the C-terminus in 9-mers).
Exemplary members of this family, identified by either serology or
DNA typing, include: A3 (A*0301), A11 (A*1101, A31 (A*3101),
A*3301, and A*6801. Other members of the family include A34 A66 and
A*7401. As explained in detail below, binding to each of the
individual alleles can be finely modulated by substitutions at the
secondary anchor positions.
[0309] The "HLA-A2-like" supertype is characterized by a preference
for peptide ligands with small or aliphatic amino acids (L, 1, V, M
A and T at position 2 and the C-terminus. The family is comprised
of at least eight HLA-A alleles (A*0201, A*0202, A*0203, A*0204,
A*0205, A*0206, A*6802, and A*6901).
[0310] The "HLA-B7-like" supertype is comprised of products from at
least a dozen HLA-B alleles (B7, B*3501-3, B51 B*5301 B*5401 B*5501
B*5502 B*5601 BB*6701, and B*7801) (Sidney, et al., J Immunol
154:247 (1995); Barber, et al., Curr Biol 5:179 (1995); Hill, et
al., Nature 360:434 (1992); Rammensee et al. Immunogeneties 41:178
(1995)), and is characterized by molecules that recognize peptides
bearing proline in position 2 and hydrophobic or aliphatic amino
acids (L, I, V, W, and Y) at their C-terminus.
[0311] As used herein, the term "IC.sub.50" refers to the
concentration of peptide in a binding assay at which 50% inhibition
of binding of a reference peptide is observed. Depending on the
conditions in which the assays are run (i.e., limiting MHC proteins
and labeled peptide concentrations), these values may approximate
K.sub.D values. It should be noted that IC.sub.50 values can
change, often dramatically, if the assay conditions are varied, and
depending on the particular reagents used (e.g., HLA preparation,
etc.). For example, excessive concentrations of HLA molecules will
increase the apparent measured IC.sub.50 of a given ligand.
[0312] Alternatively, binding is expressed relative to a reference
peptide. As a particular assay becomes more, or less, sensitive,
the IC.sub.50's of the peptides tested may change somewhat.
However, the binding relative to the reference peptide will not
change. For example, in an assay run under conditions such that the
IC.sub.50 of the reference peptide increases 10-fold, the IC.sub.50
values of the test peptides will also shift approximately 10-fold.
Therefore, to avoid ambiguities, the assessment of whether a
peptide is a good, intermediate, weak, or negative binder is
generally based on its IC.sub.50, relative to the IC.sub.50 of a
standard peptide. The binding may be reported as a ratio or the
ratio may be used to normalize the IC.sub.50 value as described in
Example 1.
[0313] As used herein, "high affinity" with respect to peptide
binding to HLA class I molecules is defined as binding with an
K.sub.D (or IC.sub.50) of less than 50 nM. "Intermediate affinity"
is binding with a K.sub.D (or IC.sub.50) of between about 50 and
about 500 mM. As used herein, "high affinity" with respect to
binding to HLA class II molecules is defined as binding with an
K.sub.D (or IC.sub.50) of less than 100 nM. "Intermediate affinity"
is binding with a K.sub.D (or IC.sub.50) of between about 100 and
about 1000 nM. Assays for determining binding are described in
detail, e.g., in PCT publications WO 94/20127 and WO 94/03205.
[0314] Binding may also be determined using other assay systems
including those using: live cells (e.g., Ceppellini et al., Nature
339:392 (1989); Christnick et al., Nature 352:67 (1991); Busch et
al., Int. Immunol. 2:443 (1990); Hill et al., J Immunol. 147:189
(1991); del Guercio et al., J Immunol. 154:685 (1995)), cell free
systems using detergent lysates (e.g., Cerundolo et al., J Immunol.
21:2069 (1991)), immobilized purified MHC (e.g., Hill et al., J
Immunol. 152, 2890 (1994); Marshall et al., J Immunol. 152:4946
(1994)), ELISA systems (e.g., Reay et al., EMBO J 11:2829 (1992)),
surface plasmon resonance (e.g., Khilko et al., J Biol. Chem.
268:15425 (1993)); high flux soluble phase assays (Hammer et al.,
J. Exp. Med. 180:2353 (1994)), and measurement of class I MHC
stabilization or assembly (e.g., Ljunggren et al., Nature 346:476
(1990); Schumacher et al., Cell 62:563 (1990); Townsend et al.,
Cell 62:285 (1990); Parker et al., J Immunol. 149:1896 (1992))
[0315] The relationship between binding affinity for MHC class I
molecules and immunogenicity of discrete peptide epitopes has been
analyzed in two different experimental approaches (Sette, et al.,
J. Immunol., 153:5586-92 (1994)). In the first approach, the
immunogenicity of potential epitopes ranging in MHC binding
affinity over a 10,000-fold range was analyzed in HLA-A*0201
transgenic mice. In the second approach, the antigenicity of
approximately 100 different hepatitis B virus (HBV)-derived
potential epitopes, all carrying A*0201 binding motifs, was
assessed by using PBL of acute hepatitis patients. In both cases,
it was found that an affinity threshold of approximately 500 nM
(preferably 500 nM or less) determines the capacity of a peptide
epitope to elicit a CTL response. These data correlate well with
class I binding affinity measurements of either naturally processed
peptides or previously described T cell epitopes. These data
indicate the important role of determinant selection in the shaping
of T cell responses.
[0316] The term "peptide" is used interchangeably with
"oligopeptide" in the present specification to designate a series
of residues, typically L-amino acids, connected one to the other
typically by peptide bonds between the alpha-amino and carbonyl
groups of adjacent amino acids. In certain embodiments, the
oligopeptides of the invention are less than about 15 residues in
length and usually consist of between about 8 and about 11
residues, preferably 9 or 10 residues. In certain embodiments, the
oligopeptides are generally less than 250 amino acids in length,
and can be less than 150, 100, 75, 50, 25, or 15 amino acids in
length. Further, an oligopeptide of the invention can be such that
it does not comprise more than 15 contiguous amino acids of a
native antigen. The preferred CTL-inducing peptides of the
invention are 13 residues or less in length and usually consist of
between about 8 and about 11 residues, preferably 9 or 10
residues.
[0317] "Synthetic peptide" refers to a peptide that is not
naturally occurring, but is man-made using such methods as chemical
synthesis or recombinant DNA technology.
[0318] The nomenclature used to describe peptide compounds follows
the conventional practice wherein the amino group is presented to
the left (the N-terminus) and the carboxyl group to the right (the
C-terminus) of each amino acid residue. In the formulae
representing selected specific embodiments of the present
invention, the amino- and carboxyl-terminal groups, although not
specifically shown, are in the form they would assume at
physiologic pH values, unless otherwise specified. In the amino
acid structure formulae, each residue is generally represented by
standard three letter or single letter designations. The L-form of
an amino acid residue is represented by a capital single letter or
a capital first letter of a three-letter symbol, and the D-form for
those amino acids having D-forms is represented by a lower case
single letter or a lower case three letter symbol. Glycine has no
asymmetric carbon atom and is simply referred to as "Gly" or G.
Symbols for each amino acids are shown below:
TABLE-US-00001 TABLE 1 Amino acids with their abbreviations Amino
acid Three letter code Single letter code Alanine Ala A Arginine
Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine
Gln Q Glutamic acid Glu E Glycine Gly G Histidine His H Isoleucine
Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe
F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W
Tyrosine Tyr Y Valine Val V
[0319] In some embodiments, as used herein, the term "peptide" is
used interchangeably with "epitope" in the present specification to
designate a series of residues, typically L-amino acids, connected
one to the other, typically by peptide bonds between the
.alpha.-amino and carboxyl groups of adjacent amino acids, that
binds to a designated MHC allele.
[0320] With regard to a particular amino acid sequence, an
"epitope" is a set of amino acid residues which is involved in
recognition by a particular immunoglobulin, or in the context of T
cells, those residues necessary for recognition by T cell receptor
proteins and/or Major Histocompatibility Complex (MHC) receptors.
In an immune system setting, in vivo or in vitro, an epitope is the
collective features of a molecule, such as primary, secondary and
tertiary peptide structure, and charge, that together form a site
recognized by an immunoglobulin, T cell receptor or HLA molecule.
Throughout this disclosure epitope and peptide are often used
interchangeably.
[0321] It is to be appreciated that protein or peptide molecules
that comprise an epitope of the invention as well as additional
amino acid(s) are still within the bounds of the invention. In
certain embodiments, there is a limitation on the length of a
peptide of the invention. The embodiment that is length-limited
occurs when the protein/peptide comprising an epitope of the
invention comprises a region (i.e., a contiguous series of amino
acids) having 100% identity with a native sequence. In order to
avoid the definition of epitope from reading, e.g., on whole
natural molecules, there is a limitation on the length of any
region that has 100% identity with a native peptide sequence. Thus,
for a peptide comprising an epitope of the invention and a region
with 100% identity with a native peptide sequence, the region with
100% identity to a native sequence generally has a length of: less
than or equal to 600 amino acids, often less than or equal to 500
amino acids, often less than or equal to 400 amino acids, often
less than or equal to 250 amino acids, often less than or equal to
100 amino acids, often less than or equal to 85 amino acids, often
less than or equal to 75 amino acids, often less than or equal to
65 amino acids, and often less than or equal to 50 amino acids. In
certain embodiments, an "epitope" of the invention is comprised by
a peptide having a region with less than 51 amino acids that has
100% identity to a native peptide sequence, in any increment down
to 5 amino acids.
[0322] Accordingly, peptide or protein sequences longer than 600
amino acids are within the scope of the invention, so long as they
do not comprise any contiguous sequence of more than 600 amino
acids that have 100% identity with a native peptide sequence. For
any peptide that has five contiguous residues or less that
correspond to a native sequence, there is no limitation on the
maximal length of that peptide in order to fall within the scope of
the invention. It is presently preferred that a CTL epitope be less
than 600 residues long in any increment down to eight amino acid
residues.
[0323] A "dominant epitope" induces an immune response upon
immunization with whole native antigens which comprise the epitope.
(See, e.g., Sercarz, et al., Annu. Rev. Immunol. 11:729-766
(1993)). Such a response is cross-reactive in vitro with an
isolated peptide epitope.
[0324] A "cryptic epitope" elicits a response by immunization with
isolated peptide, but the response is not cross-reactive in vitro
when intact whole protein which comprises the epitope is used as an
antigen.
[0325] A "subdominant epitope" is an epitope which evokes little or
no response upon immunization with whole antigens which comprise
the epitope, but for which a response can be obtained by
immunization in vivo or in vitro with an isolated epitope, and this
response (unlike the case of cryptic epitopes) is detected when
whole protein is used to recall the response in vitro.
[0326] A "pharmaceutical excipient" comprises a material such as an
adjuvant, a carrier, pH-adjusting and buffering agents, tonicity
adjusting agents, wetting agents, preservatives, and the like.
[0327] As used herein, the term "pharmaceutically acceptable"
refers to a generally non-toxic, inert, and/or physiologically
compatible composition.
[0328] As used herein, the term "protective immune response" or
"therapeutic immune response" refers to a CTL and/or an HTL
response to an antigen derived from an infectious agent or a tumor
antigen, which in some way prevents or at least partially arrests
disease symptoms, side effects or progression. The immune response
may also include an antibody response that has been facilitated by
the stimulation of helper T cells.
[0329] In certain embodiments, an "immunogenic peptide" is a
peptide which comprises an allele-specific motif such that the
peptide will bind an MHC (HLA) molecule and induce a CTL response.
Immunogenic peptides of the invention are capable of binding to an
appropriate class I MHC molecule (e.g., HLA-A2.1) and inducing a
cytotoxic T cell response against the antigen from which the
immunogenic peptide is derived.
[0330] An "immunogenic response" includes one that stimulates a CTL
and/or HTL response in vitro and/or in vivo as well as modulates an
ongoing immune response through directed induction of cell death
(or apoptosis) in specific T cell populations.
[0331] In certain embodiments, an "immunogenic peptide" or "peptide
epitope" is a peptide which comprises an allele-specific motif or
supermotif such that the peptide will bind an HLA molecule and
induce a CTL or HTL response. Thus, immunogenic peptides of the
invention are capable of binding to an appropriate HLA molecule and
thereafter inducing a cytotoxic T cell response, or a helper T cell
response, to the antigen from which the immunogenic peptide is
derived.
[0332] Immunogenic peptides of the invention are capable of binding
to an appropriate HLA-A2 molecule and inducing a cytotoxic T-cell
response against the antigen from which the immunogenic peptide is
derived. The immunogenic peptides of the invention are less than
about 15 residues in length, often less than 12 residues in length
and usually consist of between about 8 and about 11 residues,
preferably 9 or 10 residues.
[0333] The term "derived" when used to discuss an epitope is a
synonym for "prepared." A derived epitope can be isolated from a
natural source, or it can be synthesized in accordance with
standard protocols in the art. Synthetic epitopes can comprise
artificial amino acids "amino acid mimetics," such as D isomers of
natural occurring L amino acids or non-natural amino acids such as
cyclohexylalanine. A derived/prepared epitope can be an analog of a
native epitope.
[0334] Immunogenic peptides are conveniently identified using the
algorithms of the invention. The algorithms are mathematical
procedures that produce a score which enables the selection of
immunogenic peptides. Typically one uses the algorithmic score with
a "binding threshold" to enable selection of peptides that have a
high probability of binding at a certain affinity and will in turn
be immunogenic. The algorithm is based upon either the effects on
MHC binding of a particular amino acid at a particular position of
a peptide or the effects on binding of a particular substitution in
a motif containing peptide.
[0335] The term "residue" refers to an amino acid or amino acid
mimetic incorporated into an oligopeptide by an amide bond or amide
bond mimetic.
[0336] A "conserved residue" is an amino acid which occurs in a
significantly higher frequency than would be expected by random
distribution at a particular position in a peptide. Typically a
conserved residue is one where the MHC structure may provide a
contact point with the immunogenic peptide. At least one to three
or more, preferably two, conserved residues within a peptide of
defined length defines a motif for an immunogenic peptide. These
residues are typically in close contact with the peptide binding
groove, with their side chains buried in specific pockets of the
groove itself. Typically, an immunogenic peptide will comprise up
to three conserved residues, more usually two conserved
residues.
[0337] Alternatively, a "conserved residue" is a conserved amino
acid occupying a particular position in a peptide motif typically
one where the MHC structure may provide a contact point with the
immunogenic peptide. One to three, typically two, conserved
residues within a peptide of defined length defines a motif for an
immunogenic peptide. These residues are typically in close contact
with the peptide binding groove, with their side chains buried in
specific pockets of the groove itself.
[0338] A "primary anchor residue" is an amino acid at a specific
position along a peptide sequence which is understood to provide a
contact point between the immunogenic peptide and the HLA molecule.
One to three, usually two, primary anchor residues within a peptide
of defined length generally defines a "motif" for an immunogenic
peptide. These residues are understood to fit in close contact with
peptide binding grooves of an HLA molecule, with their side chains
buried in specific pockets of the binding grooves themselves. For
example, analog peptides have been created by altering the presence
or absence of particular residues in these primary anchor
positions. Such analogs are used to finely modulate the binding
affinity of a peptide comprising a particular motif or supermotif.
Typically, the primary anchor residues are located in the 2 and 9
position of 9 residue peptide.
[0339] A "secondary anchor residue" is an amino acid at a position
other than a primary anchor position in a peptide. The secondary
anchor residues are said to occur at secondary anchor positions. A
secondary anchor residue occurs at a significantly higher frequency
than would be expected by random distribution of amino acids at one
position. A secondary anchor residue can be identified as a residue
which is present at a higher frequency among high affinity binding
peptides, or a residue otherwise associated with high affinity
binding. For example, analog peptides have been created by altering
the presence or absence of particular residues in these secondary
anchor positions. Such analogs are used to finely modulate the
binding affinity of a peptide comprising a particular motif or
supermotif.
[0340] As used herein, "negative binding residues" or "deleterious"
residues are amino acids which if present at certain positions (for
example, positions 1, 3 and/or 7 of a 9-mer) (typically not primary
anchor positions) will, in certain embodiments, result in decreased
binding affinity for its target HLA molecule, and in certain
embodiments, will result in a peptide being a nonbinder or poor
binder and in turn fail to be immunogenic (i.e., induce a CTL
response) or induce a CTL response despite the presence of the
appropriate conserved residues within the peptide. For
motif-bearing peptides, by definition negative residues will not be
at primary anchor positions.
[0341] The term "motif" refers to the pattern of residues in a
peptide of defined length, usually about 8 to about 11 amino acids,
which is recognized by a particular MHC allele (one or more HLA
molecules). The peptide motifs are typically different for each
human MHC allele and differ in the pattern of the highly conserved
residues and negative residues. Peptide motifs are often unique for
the protein encoded by each human HLA allele, differing in their
pattern of the primary and secondary anchor residues. Typically as
used herein, a "motif" refers to that pattern of residues which is
recognized by an HLA molecule encoded by a particular allele.
[0342] The binding motif for an allele can be defined with
increasing degrees of precision. In one case, all of the conserved
residues are present in the correct positions in a peptide and
there are no negative residues in positions 1, 3 and/or 7.
[0343] The designation of a residue position in an epitope as the
"carboxyl terminus" or the "carboxyl terminal position" refers to
the residue position at the end of the epitope which is nearest to
the carboxyl terminus of a peptide, which is designated using
conventional nomenclature as defined below. The "carboxyl terminal
position" of the epitope may or may not actually correspond to the
end of the peptide or polypeptide.
[0344] The designation of a residue position in an epitope as
"amino terminus" or "amino-terminal position" refers to the residue
position at the end of the epitope which is nearest to the amino
terminus of a peptide, which is designated using conventional
nomenclature as defined below. The "amino terminal position" of the
epitope may or may not actually correspond to the end of the
peptide or polypeptide.
[0345] The term "tolerated residue" is a synonym for a "less
preferred residue". A "tolerated" residue refers to an anchor
residue specific for a particular motif, the presence of which
residue is correlated with suboptimal, but acceptable, binding to
the particular HLA molecule.
[0346] A "motif bearing peptide" or "peptide which comprises a
motif" refers to a peptide that comprises primary anchors specified
for a given motif or supermotif.
[0347] In certain embodiments, a "supermotif" is a peptide binding
specificity shared by HLA molecules encoded by two or more HLA
alleles. Preferably, a supermotif-bearing peptide is recognized
with high or intermediate affinity (as defined herein) by two or
more HLA molecules or antigens.
[0348] Alternatively, the term "supermotif" refers to motifs that,
when present in an immunogenic peptide, allow the peptide to bind
more than one HLA antigen. The supermotif preferably is recognized
with high or intermediate affinity (as defined herein) by at least
one HLA allele having a wide distribution in the human population,
preferably recognized by at least two alleles, more preferably
recognized by at least three alleles, and most preferably
recognized by more than three alleles.
[0349] "Human Leukocyte Antigen" or "HLA" is a human class I or
class II Major Histocompatibility Complex (MHC) protein (see,
Stites, et al., IMMUNOLOGY, 8.sup.TH ED., Lange Publishing, Los
Altos, Calif. (1994).
[0350] "Major Histocompatibility Complex" or "MHC" is a cluster of
genes which plays a role in control of the cellular interactions
responsible for physiologic immune responses. In humans, the MHC
complex is also known as the HLA complex. For a detailed
description of the MHC and HLA complexes, see, Paul, FUNDAMENTAL
IMMUNOLOGY, 3.sup.RD ED., Raven Press, New York, 1993.
[0351] "Heteroclitic analogs" are defined herein as a peptide with
increased potency for a specific T cell, as measured by increased
responses to a given dose, or by a requirement of lesser amounts to
achieve the same response as a homologous native class I peptide.
Advantages of heteroclitic analogs include that the antigens can be
more potent, or more economical (since a lower amount is required
to achieve the same effect as a homologous class I peptide). In
addition, heteroclitic analogs are also useful to overcome
antigen-specific T cell unresponsiveness (T cell tolerance).
[0352] The phrases "isolated" or "biologically pure" refer to
material which is substantially or essentially free from components
which normally accompany it as found in its native state. Thus, the
peptides of this invention do not contain materials normally
associated with their in situ environment, e.g., MHC I molecules on
antigen presenting cells. Even where a protein has been isolated to
a homogenous or dominant band, there are trace contaminants in the
range of 5-10% of native protein which co-purify with the desired
protein. Isolated peptides of this invention do not contain such
endogenous co-purified protein.
[0353] "Peripheral blood mononuclear cells" (PBMCs) are cells found
in from the peripheral blood of a patient. PBMCs comprise, e.g.,
CTLs and HTLs and antigen presenting cells. These cells can contact
an antigen in vivo, or be obtained from a mammalian source and
contacted with an antigen in vitro.
[0354] "Cross-reactive binding" indicates that a peptide is bound
by more than one HLA molecule; a synonym is degenerate binding.
[0355] "Promiscuous recognition" is where the same peptide bound by
different HLA molecules is recognized by the same T cell clone. It
may also refer to the ability of a peptide to be recognized by a
single T cell receptor in the context of multiple HLA alleles.
[0356] "Link" or "join" refers to any method known in the art for
functionally connecting peptides, including, without limitation,
recombinant fusion, covalent bonding, disulfide bonding, ionic
bonding, hydrogen bonding, and electrostatic bonding.
[0357] A "non-native" sequence or "construct" refers to a sequence
that is not found in nature, i.e., is "non-naturally occurring".
Such sequences include, e.g., peptides that are lipidated or
otherwise modified, and polyepitopic compositions that contain
epitopes that are not contiguous in a native protein sequence.
[0358] As used herein, a "vaccine" is a composition that contains
one or more peptides of the invention, see, e.g., TABLE 2, TABLE
11, TABLE 12, TABLE 10, TABLE 11, TABLE 12, TABLE 13, TABLE 14,
TABLE 15, TABLE 16, TABLE 17, TABLE 18, TABLE 19, and TABLE 20.
There are numerous embodiments of vaccines in accordance with the
invention, such as by a cocktail of one or more peptides; one or
more peptides of the invention comprised by a polyepitopic peptide;
or nucleic acids that encode such peptides or polypeptides, e.g., a
minigene that encodes a polyepitopic peptide. The "one or more
peptides" can include any whole unit integer from 1-150, e.g., at
least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more
peptides of the invention. The peptides or polypeptides can
optionally be modified, such as by lipidation, addition of
targeting or other sequences. HLA class I-binding peptides of the
invention can be linked to HLA class II-binding peptides, to
facilitate activation of both cytotoxic T lymphocytes and helper T
lymphocytes. Vaccines can comprise peptide pulsed antigen
presenting cells, e.g., dendritic cells.
DETAILED DESCRIPTION OF THE INVENTION
[0359] Certain embodiments of the present invention relate in part
to an epitope-based approach for vaccine design. Such an approach
is based on the well-established finding that the mechanism for
inducing CTL immune response comprises the step of presenting a CTL
epitope as a peptide of about 8-11 amino acids bound to an HLA
molecule displayed on an antigen-presenting cell. The HLA molecule
is the product of a class I MHC wherein the product is expressed on
most nucleated cells.
[0360] Certain embodiments of the present invention relate to the
determination of allele-specific peptide motifs for human Class I
(and class II) MHC (sometimes referred to as HLA) allele subtypes.
These motifs are then used to define T cell epitopes from any
desired antigen, particularly those associated with human viral
diseases, cancers or autoimmune diseases, for which the amino acid
sequence of the potential antigen or auto-antigen targets is
known.
[0361] Certain embodiments of the present invention relate to
peptides comprising allele-specific peptide motifs and supermotifs
which bind to HLA class I and class II molecules, in particular,
HLA-A3-like alleles. Such motifs (i.e., allele-specific motifs or
supermotifs) are then used to identify, prepare and modify epitopes
from a source protein which are recognized and bound by HLA
molecules, e.g., to create analogs of any desired peptide antigen,
particularly those associated with human cancers and precancerous
conditions, and from infectious agents such as viruses, bacteria,
fungi, and protozoal parasite.
[0362] As noted above, high HLA binding affinity is correlated with
higher immunogenicity. Higher immunogenicity can be manifested in
several different ways. For instance, a higher binding peptide will
be immunogenic more often. Close to 90% of high binding peptides
are immunogenic, as contrasted with about 50% of the peptides which
bind with intermediate affinity. A higher binding peptide will also
lead to a more vigorous response. As a result, less peptide is
required to elicit a similar biological effect. Thus, in some
embodiments of the invention high binding epitopes are particularly
desired.
[0363] In some embodiments of the invention, the identification of
subdominant, as opposed to dominant epitopes is desired. In the
nomenclature adopted here (see, Sercarz, et al., (1993), supra), a
"dominant epitope" induces a response upon immunization with whole
native antigens. Such a response is cross-reactive in vitro with
the peptide epitope. A "cryptic epitope" elicits a response by
peptide immunization, but is not cross-reactive in vitro when
intact whole protein is used as an antigen. Finally, a "subdominant
epitope" is an epitope which evokes little or no response upon
immunization with whole antigens, but for which a response can be
obtained by peptide immunization, and this response (unlike the
case of cryptic epitopes) is detected when whole protein is used to
recall the response in vitro.
[0364] HLA class I alleles have historically been classified based
on serology or phylogenetic relationships, however, these alleles
can be (re)classified into supertypes on the basis of their ligand
specificity. At least two HLA class I supertypes, A2 and B7, have
been identified. In certain embodiments, the HLA class I A3
supertype is disclosed and claimed herein.
[0365] It remains unknown how many supertypes will be identified
and how inclusive they will be, data demonstrate that the
phenomenon of cross-reactivity of peptide-binding specificities,
previously thought to be restricted to HLA class II
(Panina-Bordignon, et al., Eur J Immunol 19:2237 (1989);
O'Sullivan, et al., J Immunol 145:1799 (1990); Busch, et al., Int
Immunol 2:443 (1990)), is also a feature of peptide binding to HLA
class I molecules. The availability of quantitative binding assays
along with the detailed supermotifs disclosed herein allows the
identification of highly cross-reactive peptides. This, in turn,
allows for broad population coverage with a cocktail of a few CTL
and/or HTL epitopes, a scenario of great significance for the use
of epitope-based vaccines (Vitiello, et al., J Clin Invest 95:341
(1995)).
[0366] The data presented herein demonstrate that products from at
least five different HLA alleles (A3, A11, A31, A*3301, and
A*6801), and likely at least three others (A*3401, A*6601, and
A*7401) predicted on the basis of pocket analysis (data not shown),
are properly grouped into a single functional HLA A3 supertype.
This determination was made on the basis of a number of
observations. As a group, these molecules: (a) share certain key
structural features within their peptide-binding regions; (b) have
similar preferences for the primary anchor residues in the peptides
they bind, i.e., a primary supermotif present in the peptides bound
by the HLA molecules of the superfamily; and (c) share largely
overlapping binding repertoires. Knowledge of the A3 supermotif
allows for identification of a cross-reactive peptide for a source,
and allows for production of peptide analogs by substituting at
primary anchor positions to alter the binding properties of the
peptides.
[0367] Furthermore, by examining the binding activity of a large
panel of peptides bearing the primary A3 supermotif, an extended A3
supermotif was defined. This extended supermotif was based on a
detailed map of the secondary anchor requirements for binding to
molecules of the A3 supertype. The extended supermotif allows for
the efficient prediction of cross-reactive binding of peptides to
alleles of the A3 supertype by screening the native sequence of a
particular antigen. This extended supermotif is also used to select
analog options for peptides which bear amino acids defined by the
primary supermotif.
[0368] By examining the binding activity of a large panel of
peptides bearing anchor residues preferred by these allelic
molecules, an A3-like supermotif was also defined. This supermotif,
which is based on a detailed map of the secondary anchor
requirements of each of the A3-like supertype molecules, allows for
the efficient prediction of A3-like degenerate binding peptides.
Finally, it was shown that the A3-like supertype, and supertypes in
general, are represented with remarkably high phenotypic
frequencies in all major ethnic groups. As such, HLA class I
supertypes based on peptide-binding specificities represent a
functional alternative to serologic and phylogenetic classification
for understanding the relationships between HLA class I molecules.
Besides their use for the generation of the A3-like supermotif, the
individual secondary anchor maps disclosed in this study represent
in themselves a significant contribution to the understanding of
peptide binding to class I molecules. Because these maps were
derived using peptides of homogeneous size, the preference
determinations at each of the secondary positions may be more
accurate than those derived from the sequencing of pools of
naturally processed peptides. Also, the motifs defined herein allow
the determination of residues which have deleterious effects on
peptide binding.
[0369] Barber and co-workers (Barber, et al., Curr Biol 5:179
(1995)) have demonstrated that peptides could be recognized in the
context of two molecules we have included in the HLA-B7-like
supertype, and two other peptides have been reported as being
recognized in the context of more than one A3-like allele (Missale,
et al., J Exp Med 177:751 (1993); Koenig, et al., J Immunol 145:127
(1990); Culmann, et al., J Immunol 146:1560 (1991)) (see TABLE
142). Using a method for in vitro induction of primary CTLs
(Wentworth, et al., Mol Immunol 32:603 (1995)) we observed several
instances in which peptides can be recognized in the context of
both A3 and A11. We tested the A3-like supertype restricted
epitopes for binding capacity to A3-like supertype molecules, and
noted relatively high levels of degeneracy. Of the seven epitopes
listed in TABLE 142, only one was a nonamer that could be analyzed
for the supermotif proposed in FIG. 40A (future studies will be
aimed at extending the supermotif to peptides longer than 15
nine-mers). This peptide was supermotif positive, and bound three
of five A3-like molecules. Nonetheless, it is important that each
of the epitopes conformed to the A3-like supertype primary anchor
specificities.
[0370] Comparison of the supertype classifications we have proposed
on the basis of peptide binding with the classification of HLA-A
alleles on the basis of DNA sequence (and serologic reactivity)
relationships (Ishikawa, et al., Hum Immonol 39:220 (1994);
Firgaira, et al., Immunogenetics 40:445 (1994); Karo, et al., J
Immunol 143:3371 (1989)) reveals both similarities and differences.
For example, HLA-A3 and A11 appear to be closely related and
derived from a common ancestral gene (48-50). A31 and A33, however,
derive from the ancient lineage comprising the A2/A10/A19 groups,
which is different from the lineage of A3 and A11. Finally,
HLA-A*6901 belongs to the A28 HLA evolutionary group
[Fernandez-Viiia, et al., Hum Immunol 33:163 (1992); Ishikawa, et
al., Hum Immonol 39:220 (1994); Lawlor, et al., Annu Rev Immunol
8:23 (1990)], which also contains the HLA-A*6802 and -A*6901
alleles. Yet, on the basis of their peptide-binding specificity,
HLA A*6801 is a member of the A3-like supertype, whereas A*6802 and
A*6901 have been demonstrated to belong to the A2-like supertype
[del Guercio, et al., J Immunol 154:685 (1995)]. Thus, based on the
available phylogenetic tree of HLA alleles [Ishikawa, et al., Hum
Immonol 39:220 (1994); Firgaira, et al., Immunogenetics 40:445
(1994); Karo, et al., Immunol 143:3371 (1989)], A3-like alleles are
found in both of the ancient HLA lineages: A11A9 which includes A3
and A11, and A21A 101A which includes A31, A33, and A*6801. If the
existence of the HLA-A3-like supertype is reflective of common
ancestry, then the A3-like motif might in fact represent primeval
human HLA class I peptide-binding specificity, and other
specificities may represent adaptations to changing pathogenic
environments.
[0371] The discovery of the individual residues of the secondary
anchor motif disclosed herein represents a significant contribution
to the understanding of peptide binding to class I molecules. These
secondary anchor maps were derived using peptides of homogeneous
size. Thus, the preference determinations at each of the secondary
positions may be more accurate than those derived from the
sequencing of pools of naturally processed peptides. Also, the
motifs defined herein allow the determination of residues which
have deleterious or other types of effects on peptide binding.
[0372] The definition of primary and secondary anchor specificities
for the A3 supertype provides guidance for modulating the binding
activity of peptides that bind to members of the A3 supertype
family. This information may be used to generate highly
cross-reactive epitopes by identifying residues within a native
peptide sequence that can be analogued to increase greater binding
cross-reactivity within a supertype, or analogued to increase
immunogenicity.
[0373] The phenomena of HLA supertypes may be related to optimal
exploitation of the peptide specificity of human transporter
associated with antigen processing (TAP) molecules (Androlewicz, et
al., Proc. Nat'l Acad. Sci. USA 90:9130 (1993); Androlewicz, et
al., Immunity 1:7 (1994); van Endert, et al., Immunity 1:491
(1994); Heemels, et al., Immunity 1:775 (1994); Momburg, et al.,
Curr. Opin. Immunol. 6:32 (1994); Neefjes, et al., Science 261:769
(1993)). The TAP molecules have been shown to preferentially
transport peptides with certain sequence features such as
hydrophobic, aromatic, or positively charged C-termini.
[0374] Recent studies, performed by van Endert and associates, in
collaboration with the present inventors, evaluated the relative
affinities for TAP of a large collection of peptides, and have
described an extended TAP binding motif (Van Endert et al. J. Exp.
Med. 182:1883 (1995)) Strikingly, this tap motif contains many of
the structural features associated with the A3 extended supermotif,
such as the preference for aromatic residues at positions 3 and 7
of nonamer peptides and the absence of negatively charged residues
at positions 1 and 3, and P at position 1.
[0375] The preparation and evaluation of motif-bearing peptides are
described in PCT publications WO 94/20127 and WO 94/03205. Briefly,
peptides from a particular antigen are synthesized and tested for
their ability to bind to HLA proteins in assays using, for example,
purified HLA class I molecules and radioiodinated peptides and/or
cells expressing empty class I molecules (which lack peptide in
their receptor) by, for instance, immunofluorescent staining and
flow microfluorimetry, peptide-dependent class I assembly assays,
and inhibition of CTL recognition by peptide competition. Those
peptides that bind to the class I molecule are further evaluated
for their ability to serve as targets for CTLs derived from
infected or immunized individuals, as well as for their capacity to
induce primary in vitro or in vivo CTL responses that can give rise
to CTL populations capable of reacting with selected target cells
associated with a disease.
[0376] The concept of dominance and subdominance is relevant to
immunotherapy of infectious diseases and cancer. For example, in
the course of chronic viral disease, recruitment of subdominant
epitopes can be crucial for successful clearance of the infection,
especially if dominant CTL specificities have been inactivated by
functional tolerance, suppression, mutation of viruses and other
mechanisms (Franco, et al., Curr. Opin. Immunol. 7:524-531,
(1995)). Furthermore, in the case of cancer and tumor antigens, it
appears that CTLs recognizing at least some of the highest binding
affinity peptides might have been functionally inactivated by
tolerance and suppression, and that lower binding affinity peptides
are preferentially recognized.
[0377] In particular, it has been noted that a significant number
of epitopes derived from known non-viral tumor associated antigens
(TAA) bind HLA Class I with intermediate affinity (IC.sub.50 in the
50-500 mM range). It has been found that 8 of 15 known TAA peptides
recognized by tumor infiltrating lymphocytes (TIL) or CTL bound in
the 50-500, mM range. These data are in contrast with estimates
that 90% of known viral antigens that were recognized as peptides
bound HLA with IC.sub.50 of 50 IM or less while only approximately
10% bound in the 50-500 mM range (Sette, et al., J. Immunol.,
153:5586-5592 (1994)). This phenomenon is probably due in the
cancer setting to elimination, or functional inhibition of the CTL
recognizing several of the highest binding peptides, presumably
because of T cell tolerization events.
[0378] The present invention provides methods for modulating
binding affinity of immunogenic peptides by selection of desired
residues in the primary and secondary anchor positions. As
explained in detail below, a supermotif for enhanced binding to A3
like alleles is provided here. Depending on the desired affect on
binding affinity, the anchor residues in a desired peptide are
substituted. Examples of modulations that may be achieved using the
present invention include increased affinity for a particular
allele (e.g., by substitution of secondary anchor residues specific
for the allele), increased cross-reactivity among different alleles
(e.g., by substitution of secondary anchor residues shared by more
than one allele), and production of a subdominant epitope (e.g., by
substitution of residues which increase affinity but are not
present on the immunodominant epitope).
[0379] Thus, in some embodiments of the invention, the
identification of subdominant, as opposed to dominant epitopes is
desired. In a preferred embodiment, these subdominant epitopes can
then be engineered to increase HLA binding affinity. As noted
herein, higher HLA binding affinity is correlated with greater
immunogenicity. Greater immunogenicity can be manifested in several
different ways. Close to 90% of "high" binding peptides have been
found to be immunogenic, as contrasted with about 50% of the
peptides which bind with "intermediate" affinity. Moreover, higher
binding affinity peptides lead to more vigorous immunogenic
responses. As a result, less peptide is required to elicit a
similar biological effect. Thus, in preferred embodiments of the
invention, high binding epitopes are particularly desired.
[0380] Epitope-bearing peptides in accordance with the invention
can be prepared synthetically, by recombinant DNA technology, or
from natural sources such as whole viruses or tumors. Although the
peptide will preferably be substantially free of other naturally
occurring host cell proteins and fragments thereof, in some
embodiments the peptides are synthetically conjugated to native
molecules or particles; the peptides can also be conjugated to
non-native molecules or particles.
[0381] The peptides in accordance with the invention can be a
variety of lengths, and either in their neutral (uncharged) forms
or in forms which are salts. The peptides in accordance with the
invention are either free of modifications such as glycosylation,
side chain oxidation, or phosphorylation; or they contain these
modifications.
[0382] Desirably, the epitope-bearing peptide will be as small as
possible while still maintaining relevant immunologic activity of
the large peptide; of course it is particularly desirable with
peptides from pathogenic organisms that the peptide be small in
order to avoid pathogenic function. When possible, it may be
desirable to optimize epitopes of the invention to a length of
about 8 to about 13, preferably 9 to 10 amino acid residues for a
class I molecule and about 6 to about 25 amino acid residues for a
class II molecules. Preferably, the peptides are commensurate in
size with endogenously processed viral peptides or tumor cell
peptides that are bound to HLA class I or class II molecules on the
cell surface. Nevertheless, the identification and preparation of
peptides of other lengths can be carried out using the techniques
described here such as the disclosures of primary anchor positions.
It is to be appreciated that peptide epitopes in accordance with
the invention can be present in peptides or proteins that are
longer than the epitope itself. Moreover, multiepitopic peptides
can comprise at least one epitope of the invention along with other
epitope(s).
[0383] In particular, the invention provides motifs that are common
to peptides bound by more than one HLA allele. By a combination of
motif identification and MHC-peptide interaction studies, peptides
useful for peptide vaccines have been identified.
[0384] Peptides comprising the epitopes from these antigens are
synthesized and then tested for their ability to bind to the
appropriate MHC molecules in assays using, for example, purified
class I molecules and radioiodinated peptides and/or cells
expressing empty class I molecules by, for instance,
immunofluorescent staining and flow microfluorometry,
peptide-dependent class I assembly assays, and inhibition of CTL
recognition by peptide competition. Those peptides that bind to the
class I molecule are further evaluated for their ability to serve
as targets for CTLs derived from infected or immunized individuals,
as well as for their capacity to induce primary in vitro or in vivo
CTL responses that can give rise to CTL populations capable of
reacting with virally infected target cells or tumor cells as
potential therapeutic agents.
[0385] The (HLA) MHC class I antigens (i.e., the products of the
MHC class I alleles) are encoded by the HLA-A, B, and C loci. HLA-A
and B antigens are expressed at the cell surface at approximately
equal densities, whereas the expression of HLA-C is significantly
lower (perhaps as much as 10-fold lower). Each of these loci have a
number of alleles (i.e., a multiplicity of allelic variants) in the
population. Indeed, there are believed to be well over 500 class I
and class II alleles. The peptide binding motifs of the invention
are relatively specific for each allelic subtype.
[0386] Since a cytotoxic T-cell response cannot be elicited unless
the epitope is presented by the class I HLA contained on the
surface of the cells of the individual to be immunized, it is
important that the epitope be one that is capable of binding the
HLA exhibited by that individual.
[0387] The starting point, therefore, for the design of effective
vaccines is to ensure that the vaccine will generate a large number
of epitopes that can successfully be presented. It may be possible
to administer the peptides representing the epitopes per se. Such
administration is dependent on the presentation of "empty" HLA
molecules displayed on the cells of the subject. In one approach to
use of the immunogenic peptides per se, these peptides may be
incubated with antigen-presenting cells from the subject to be
treated ex vivo and the cells then returned to the subject.
[0388] Alternatively, the 8-11 amino acid peptide can be generated
in situ by administering a nucleic acid containing a nucleotide
sequence encoding it. Means for providing such nucleic acid
molecules are described in WO99/58658, the disclosure of which is
incorporated herein by reference. Further, the immunogenic peptides
can be administered as portions of a larger peptide molecule and
cleaved to release the desired peptide. The larger peptide may
contain extraneous amino acids, in general the fewer the better.
Thus, peptides which contain such amino acids are typically 25
amino acids or less, more typically 20 amino acids or less, and
more typically 15 amino acids or less. The precursor may also be a
heteropolymer or homopolymer containing a multiplicity of different
or same CTL epitopes. Of course, mixtures of peptides and nucleic
acids which generate a variety of immunogenic peptides can also be
employed. The design of the peptide vaccines, the nucleic acid
molecules, or the hetero- or homo-polymers is dependent on the
inclusion of the desired epitope. Thus, in certain embodiments, the
present invention provides a paradigm for identifying the relevant
epitope which is effective across the broad population range of
individuals who are characterized by the A2 supertype. The
following pages describe the methods and results of experiments for
identification of the A2 supermotif, and other motifs and
supermotifs.
[0389] In certain embodiments, it is preferred that peptides
include an epitope that binds to an HLA-A2 supertype allele. These
motifs may be used to define T-cell epitopes from any desired
antigen, particularly those associated with human viral diseases,
cancers or autoimmune diseases, for which the amino acid sequence
of the potential antigen or autoantigen targets is known.
[0390] Epitopes on a number of potential target proteins can be
identified based upon HLA binding motifs. Examples of suitable
antigens include TRP1, prostate cancer-associated antigens such as
prostate specific antigen (PSA), human kallikrein (huK2), prostate
specific membrane antigen (PSM), and prostatic acid phosphatase
(PAP), antigens from viruses such as hepatitis B (e.g., hepatitis B
core and surface antigens (HBVc, HBVs)), hepatitis C antigens,
Epstein-Barr virus (EBV) antigens, human immunodeficiency virus
(HIV) antigens, human papilloma virus (HPV) antigens, Kaposi's
sarcoma virus (KSHV), influenza virus, Lassa virus, melanoma
antigens (e.g., MAGE-1, MAGE2, and MAGE3) Mycobacterium
tuberculosis (MT) antigens, p53, carcinoembryonic antigen (CEA),
trypanosome, e.g., Trypansoma cruzi (T. cruzi), antigens such as
surface antigen (TSA), Her2/neu, and malaria antigens. Examples of
suitable fungal antigens include those derived from Candida
albicans, Cryptococcus neoformans, Coccidoides spp., Histoplasma
spp, and Aspergillus fumigatis. Examples of suitable protozoal
parasitic antigens include those derived from Plasmodium spp.,
Trypanosoma spp., Schistosoma spp., Leishmania spp and the like.
Examples of suitable bacterial antigens include those derived from
Mycobacterium spp., Chlamydiaceae spp, and the like.
[0391] The peptides are thus useful in pharmaceutical compositions
for both in vivo and ex vivo therapeutic and diagnostic
applications.
[0392] Autoimmune associated disorders for which the peptides of
the invention may be employed to relieve the symptoms of, treat or
prevent the occurrence or reoccurrence of include, for example,
multiple sclerosis (MS), rheumatoid arthritis (RA), Sjogren
syndrome, scleroderma, polymyositis, dermatomyositis, systemic
lupus erythematosus, juvenile rheumatoid arthritis, ankylosing
spondylitis, myasthenia gravis (MG), bullous pemphigoid (antibodies
to basement membrane at dermal-epidermal junction), pemphigus
(antibodies to mucopolysaccharide protein complex or intracellular
cement substance), glomerulonephritis (antibodies to glomerular
basement membrane), Goodpasture's syndrome, autoimmune hemolytic
anemia (antibodies to erythrocytes), Hashimoto's disease
(antibodies to thyroid), pernicious anemia (antibodies to intrinsic
factor), idiopathic thrombocytopenic purpura (antibodies to
platelets), Grave's disease, and Addison's disease (antibodies to
thyroglobulin), and the like.
[0393] The autoantigens associated with a number of these diseases
have been identified. For example, in experimentally induced
autoimmune diseases, antigens involved in pathogenesis have been
characterized: in arthritis in rat and mouse, native type-II
collagen is identified in collagen-induced arthritis, and
mycobacterial heat shock protein in adjuvant arthritis;
thyroglobulin has been identified in experimental allergic
thyroiditis (EAT) in mouse; acetyl choline receptor (AChR) in
experimental allergic myasthenia gravis (EAMG); and myelin basic
protein (MBP) and proteolipid protein (PLP) in experimental
allergic encephalomyelitis (EAE) in mouse and rat. In addition,
target antigens have been identified in humans: type-II collagen in
human rheumatoid arthritis; and acetyl choline receptor in
myasthenia gravis.
[0394] Without wishing to be bound by theory, it is believed that
the presentation of antigen by HLA Class I mediates suppression of
autoreactive T cells by CD8.sup.+ suppressor T cells (see, e.g.,
Jiang, et al. Science, 256:1213 (1992)). Such suppressor T cells
release cytokines such as transforming growth factor-.beta.
(TGF-.beta.), which specifically inhibit the autoreactive T cells.
Miller, et al., Proc. Natl. Acad. Sci., USA, 89:421-425 (1992).
[0395] Peptides comprising the epitopes from these antigens may be
synthesized and then tested for their ability to bind to the
appropriate MHC molecules in assays using, for example, purified
class I molecules and radioiodonated peptides and/or cells
expressing empty class I molecules by, for instance,
immunofluorescent staining and flow microfluorometry,
peptide-dependent class I assembly assays, and inhibition of CTL
recognition by peptide competition. Those peptides that bind to the
class I molecule may be further evaluated for their ability to
serve as targets for CTLs derived from infected or immunized
individuals, as well as for their capacity to induce primary in
vitro or in vivo CTL responses that can give rise to CTL
populations capable of reacting with virally infected target cells
or tumor cells as potential therapeutic agents.
[0396] Recent evidence suggests however, that high affinity MHC
binders might be, in most instances, immunogenic, suggesting that
peptide epitopes might be selected on the basis of MHC binding
alone.
[0397] Peptides comprising the supermotif sequences can be
identified, as noted above, by screening potential antigenic
sources. Useful peptides can also be identified by synthesizing
peptides with systematic or random substitution of the variable
residues in the supermotif, and testing them according to the
assays provided. As demonstrated below, it is useful to refer to
the sequences of the target HLA molecule, as well.
[0398] For epitope-based vaccines, the peptides of the present
invention preferably comprise a supermotif and/or motif recognized
by an HLA I or HLA II molecule having a wide distribution in the
human population. TABLE 22 shows the distribution of certain HLA
alleles in human populations. Since the MHC alleles occur at
different frequencies within different ethnic groups and races, the
choice of target MHC allele may depend upon the target population.
TABLE 69 shows the frequency of various alleles at the HLA-A locus
products among different races. For instance, the majority of the
Caucasoid population can be covered by peptides which bind to four
HLA-A allele subtypes, specifically HLA-A2.1, A1, A3.2, and A24.1.
Similarly, the majority of the Asian population is encompassed with
the addition of peptides binding to a fifth allele HLA-A 1.2.
[0399] The nomenclature used to describe peptide compounds follows
the conventional practice wherein the amino group is presented to
the left (the N-terminus) and the carboxyl group to the right (the
C-terminus) of each amino acid residue. In the formulae
representing selected specific embodiments of the present
invention, the amino- and carboxyl-terminal groups, although not
specifically shown, are in the form they would assume at
physiologic pH values, unless otherwise specified. In the amino
acid structure formulae, each residue is generally represented by
standard three letter or single letter designations. The L-form of
an amino acid residue is represented by a capital single letter or
a capital first letter of a three-letter symbol, and the D-form for
those amino acids having D-forms is represented by a lower case
single letter or a lower case three letter symbol. Glycine has no
asymmetric carbon atom and is simply referred to as "Gly" or G. The
letter X in a motif represents any of the 20 amino acids found in
TABLE 72, as well non-naturally occurring amino acids or amino acid
mimetics. Brackets surrounding more than one amino acid indicates
that the motif includes any one of the amino acids. For example,
the supermotif "N-XPXXXXXX(A,V,I,L,M)-C (SEQ ID NO:______)"
includes each of the following peptides: N-XPXXXXXXA-C (SEQ ID
NO:______), N-XPXXXXXXV-C (SEQ ID NO:______), N-XPXXXXXXI-C (SEQ ID
NO:______), N-XPXXXXXXL-C (SEQ ID NO:______), and N-XPXXXXXXM-C
(SEQ ID NO:______).
[0400] The large degree of HLA polymorphism is an important factor
to be taken into account with the epitope-based approach to vaccine
development. To address this factor, epitope selection encompassing
identification of peptides capable of binding at high or
intermediate affinity to multiple HLA molecules is preferably
utilized, most preferably these epitopes bind at high or
intermediate affinity to two or more allele-specific HLA
molecules.
[0401] CTL-inducing peptides of interest for vaccine compositions
preferably include those that have an IC.sub.50 or binding affinity
value for a class HLA molecule(s) of 500 nM or better (i.e., the
value is 500 nM or less) or, for class II HLA molecules, 1000 mM or
better (i.e., the value is greater than or equal to 1000 nM). For
example, peptide binding is assessed by testing the capacity of a
candidate peptide to bind to a purified HLA molecule in vitro.
Peptides exhibiting high or intermediate affinity are then
considered for further analysis. Selected peptides are generally
tested on other members of the supertype family. In preferred
embodiments, peptides that exhibit cross-reactive binding are then
used in cellular screening analyses or vaccines.
[0402] The relationship between binding affinity for HLA class I
molecules and immunogenicity of discrete peptide epitopes on bound
antigens was determined for the first time in the art by the
present inventors. As disclosed in greater detail herein, higher
HLA binding affinity is correlated with greater immunogenicity.
[0403] Greater immunogenicity can be manifested in several
different ways. Immunogenicity corresponds to whether an immune
response is elicited at all, and to the vigor of any particular
response, as well as to the extent of a population in which a
response is elicited. For example, a peptide might elicit an immune
response in a diverse array of the population, yet in no instance
produce a vigorous response. In accordance with these principles,
close to 90% of high binding peptides have been found to elicit a
response and thus be "immunogenic," as contrasted with about 50% of
the peptides that bind with intermediate affinity (see, e.g.,
Schaeffer et al. PNAS (1988)). Moreover, not only did peptides with
higher binding affinity have an enhanced probability of generating
an immune response, the generated response tended to be more
vigorous than the response seen with weaker binding peptides. As a
result, less peptide is required to elicit a similar biological
effect if a high affinity binding peptide is used rather than a
lower affinity one. Thus, in preferred embodiments of the
invention, high affinity binding epitopes are used.
[0404] The correlation between binding affinity and immunogenicity
was analyzed by the present inventors by two different experimental
approaches (see, e.g., Sette, et al., J Immunol. 153:5586-5592
(1994)). In the first approach, the immunogenicity of potential
epitopes ranging in HLA binding affinity over a 10,000-fold range
was analyzed in HLA-A*0201 transgenic mice. In the second approach,
the antigenicity of approximately 100 different hepatitis B virus
(HBV)-derived potential epitopes, all carrying A*0201 binding
motifs, was assessed by using PBL from acute hepatitis patients.
Pursuant to these approaches, it was determined that an affinity
threshold value of approximately 500 nM (preferably 50 nM or less)
determines the capacity of a peptide epitope to elicit a CTL
response. These data are true for class I binding affinity
measurements for naturally processed peptides and for synthesized T
cell epitopes. These data also indicate the important role of
determinant selection in the shaping of T cell responses (see,
e.g., Schaeffer et al. Proc. Natl. Acad. Sci. USA 86:4649-4653
(1989)).
[0405] Peptides of the present invention may also comprise epitopes
that bind to HLA class II molecules (HLA class II molecules are
also referred to as MHC-DR molecules). An affinity threshold
associated with immunogenicity in the context of HLA class II DR
molecules has also been delineated (see, e.g., Southwood et al. J.
Immunology 160:3363-73, 1998, and WO99/61916). In order to define a
biologically significant threshold of DR binding affinity, a
database of the binding affinities of 32 DR-restricted epitopes for
their restricting element (i.e., the HLA molecule that binds the
motif) was compiled. In approximately half of the cases (15 of 32
epitopes), DR restriction was associated with high binding
affinities, i.e. binding affinity values of 100 nM or less (in some
embodiments, the binding affinity value was less than 100 nM). In
the other half of the cases (16 of 32), DR restriction was
associated with intermediate affinity (binding affinity values in
the 100-1000 nM range). In only one of 32 cases was DR restriction
associated with an IC.sub.50 of 1000 nM or greater. Thus, 1000 nM
can be defined as an affinity threshold associated with
immunogenicity in the context of DR molecules. Thus, as seen with
HLA class I molecules, an affinity threshold associated with
immunogenicity is defined for epitopes recognized by HLA class II
molecules.
[0406] Definition of motifs that are predictive of binding to
specific class I and class II alleles allows the identification of
potential peptide epitopes from an antigenic protein whose amino
acid sequence is known. Typically, identification of potential
peptide epitopes is initially carried out using a computer to scan
the amino acid sequence of a desired antigen for the presence of
motifs and/or supermotifs.
[0407] Definition of motifs specific for different class I alleles
allows the identification of potential peptide epitopes from an
antigenic protein whose amino acid sequence is known. Typically,
identification of potential peptide epitopes is initially carried
out using a computer to scan the amino acid sequence of a desired
antigen for the presence of motifs. The epitopic sequences are then
synthesized. The capacity to bind MHC Class I molecules is measured
in a variety of different ways. One means is a Class I molecule
binding assay as described in the related applications, noted
above. Other alternatives described in the literature include
inhibition of antigen presentation (Sette, et al., J Immunol.
141:3893 (1991), in vitro assembly assays (Townsend, et al., Cell
62:285 (1990), and FACS based assays using mutated cells, such as
RMA-S (Melief, et al., Eur. J. Immunol. 21:2963 (1991)).
[0408] In the typical case, immunoprecipitation is used to isolate
the desired allele. A number of protocols can be used to isolate
HLA molecules for use in in binding assays, depending upon the
specificity of the antibodies used. For example, allele-specific
mAb reagents can be used for the affinity purification of the
HLA-A, HLA-B1, and HLA-C molecules. Several mAb reagents for the
isolation of HLA-A molecules are available (see TABLE 4, TABLE 71,
and TABLE 24). The monoclonal BB7.2 is suitable for isolating
HLA-A2 molecules. Thus, for each of the targeted HLA-A alleles,
reagents are available that may be used for the direct isolation of
the HLA-A molecules. Affinity columns prepared with these mAbs
using standard techniques are successfully used to purify the
respective HLA-A allele products. In addition to allele-specific
mAbs, broadly reactive anti-HLA-A, B, C mAbs, such as W6/32 and
B9.12.1, and one anti-HLA-B, C mAb, B1.23.2, could be used in
alternative affinity purification protocols as described in
previous applications or in the examples section below.
[0409] The procedures used to identify peptides of the present
invention generally follow the methods disclosed in Falk et al.,
Nature 351:290 (1991), which is incorporated herein by reference.
Briefly, the methods involve large-scale isolation of MHC class I
molecules, typically by immunoprecipitation or affinity
chromatography, from the appropriate cell or cell line. Examples of
other methods for isolation of the desired MHC molecule equally
well known to the artisan include ion exchange chromatography,
lectin chromatography, size exclusion, high performance ligand
chromatography, and a combination of all of the above
techniques.
[0410] The peptides bound to the peptide binding groove of the
isolated MHC molecules are eluted typically using acid treatment.
Peptides can also be dissociated from class I molecules by a
variety of standard denaturing means, such as heat, pH, detergents,
salts, chaotropic agents, or a combination thereof.
[0411] Peptide fractions are further separated from the MHC
molecules by reversed-phase high performance liquid chromatography
(HPLC) and sequenced. Peptides can be separated by a variety of
other standard means well known to the artisan, including
filtration, ultrafiltration, electrophoresis, size chromatography,
precipitation with specific antibodies, ion exchange
chromatography, isoelectrofocusing, and the like.
[0412] Sequencing of the isolated peptides can be performed
according to standard techniques such as Edman degradation
(Hunkapiller, M. W., et al. Methods Enzymol. 91, 399 [1983]). Other
methods suitable for sequencing include mass spectrometry
sequencing of individual peptides as previously described (Hunt, et
al., Science 225:1261 (1992), which is incorporated herein by
reference). Amino acid sequencing of bulk heterogenous peptides
(e.g., pooled HPLC fractions) from different class I molecules
typically reveals a characteristic sequence motif for each class I
allele.
[0413] Upon identification of motif-bearing sequences, peptides
corresponding to the sequences are then synthesized and, typically,
evaluated for binding to the corresponding HLA allele. The capacity
to bind MHC Class molecules is measured in a variety of different
ways. One means is a Class I molecule binding assay as described in
the related applications, noted above. Other alternatives described
in the literature include inhibition of antigen presentation
(Sette, et al., J. Immunol. 141:3893 (1991), in vitro assembly
assays (Townsend, et al., Cell 62:285 (1990), and FACS based assays
using mutated ells, such as RMA-S (Melief, et al., Eur. J. Immunol.
21:2963 (1991)).
[0414] Next, peptides that test positive in the MHC class I binding
assay are assayed for the ability of the peptides to induce
specific CTL (or HTL, for class II motif-bearing peptides)
responses in vitro. For instance, antigen-presenting cells that
have been incubated with a peptide can be assayed for the ability
to induce CTL responses in responder cell populations.
Antigen-presenting cells can be normal cells such as peripheral
blood mononuclear cells or dendritic cells (Inaba, et al., J. Exp.
Med. 166:182 (1987); Boog, Eur. J. Immunol. 18:219 [1988]).
Alternatively, transgenic mice comprising an appropriate HLA
transgene can be used to assay the ability of a peptide to induce a
response in cytotoxic T lymphocytes essentially as described in
copending U.S. patent application Ser. No. 08/205,713.
[0415] Definition of motifs specific for different class I alleles
allows the identification of potential peptide epitopes from an
antigenic protein whose amino acid sequence is known. Typically,
identification of potential peptide epitopes is initially carried
out using a computer to scan the amino acid sequence of a desired
antigen for the presence of motifs.
[0416] Following identification of motif-bearing epitopes, the
epitopic sequences are then synthesized. The capacity to bind MHC
Class molecules is measured in a variety of different ways. One
means is a Class I molecule binding assay as described in the
related applications, noted below. Other alternatives described in
the literature include inhibition of antigen presentation (Sette,
et al., J. Immunol. 141:3893 (1991), in vitro assembly assays
(Townsend, et al., Cell 62:285 (1990), and FACS based assays using
mutated cells, such as RMA.S (Melief, et al., Eur. J. Immunol.
21:2963 (1991)).
[0417] As disclosed herein, higher HLA binding affinity is
correlated with greater immunogenicity. Greater immunogenicity can
be manifested in several different ways. Immunogenicity can
correspond to whether an immune response is elicited at all, and to
the vigor of any particular response, as well as to the extent of a
diverse population in which a response is elicited. For example, a
peptide might elicit an immune response in a diverse array of the
population, yet in no instance produce a vigorous response. In
accordance with the principles disclosed herein, close to 90% of
high binding peptides have been found to be immunogenic, as
contrasted with about 50% of the peptides which bind with
intermediate affinity. Moreover, higher binding affinity peptides
lead to more vigorous immunogenic responses. As a result, less
peptide is required to elicit a similar biological effect if a high
affinity binding peptide is used. Thus, in preferred embodiments of
the invention, high affinity binding epitopes are particularly
useful. Nevertheless, substantial improvements over the prior art
are achieved with intermediate or high binding peptides.
[0418] The relationship between binding affinity for HLA class I
molecules and immunogenicity of discrete peptide epitopes has been
determined for the first time in the art by the present inventors.
In these experiments, in which discrete peptides were referred to,
it is to be noted that cellular processing of peptides in vivo will
lead to such peptides even if longer fragments are used.
Accordingly, longer peptides comprising one or more epitopes are
within the scope of the invention. The correlation between binding
affinity and immunogenicity was analyzed in two different
experimental approaches (Sette, et al., J. Immunol. 153:5586-5592,
1994). In the first approach, the immunogenicity of potential
epitopes ranging in HLA binding affinity over a 10,000-fold range
was analyzed in HLA-A*0201 transgenic mice. In the second approach,
the antigenicity of approximately 100 different hepatitis B virus
(HBV)-derived potential epitopes, all carrying A*0201 binding
motifs, was assessed by using PBL (peripheral blood lymphocytes)
from acute hepatitis patients. Pursuant to these approaches, it was
determined that an affinity threshold value of approximately 500 nM
(preferably 50 nM or less) is correlated with the capacity of a
peptide epitope to elicit a CTL response. These data are true for
class I binding affinity measurements for naturally processed
peptides and for synthesized T-cell epitopes. These data also
indicate the important role of determinant selection in the shaping
of T-cell responses (see, e.g., Schaeffer, et al., Proc. Natl.
Acad. Sci. USA 86:4649-4653, 1989).
[0419] Accordingly, CTL-inducing peptides preferably include those
that have an IC.sub.50 for class I HLA molecules of 500 nM or less.
In the case of motif-bearing peptide epitopes from tumor associated
antigens, a binding affinity threshold of 200 nM has been shown to
be associated with killing of tumor cells by resulting CTL
populations.
[0420] In a preferred embodiment, following assessment of binding
activity for an HLA-A2 allele-specific molecule, peptides
exhibiting high or intermediate affinity are then considered for
further analysis. Selected peptides may be tested on other members
of the supertype family. In preferred embodiments, peptides that
exhibit cross-reactive binding are then used in vaccines or in
cellular screening analyses.
[0421] For example, peptides that test positive in the HLA-A2 (or
other MHC class I) binding assay, i.e., that have binding affinity
values of 500 nM or less, are assayed for the ability of the
peptides to induce specific CTL responses in vitro. For instance,
antigen-presenting cells that have been incubated with a peptide
can be assayed for the ability to induce CTL responses in responder
cell populations. Antigen-presenting cells can be normal cells such
as peripheral blood mononuclear cells or dendritic cells (Inaba, et
al., J. Exp. Med. 166:182 (1987); Boog, Eur. J. Immunol. 18:219
[1988]).
[0422] Alternatively, mutant mammalian cell lines that are
deficient in their ability to load class I molecules with
internally processed peptides, such as the mouse cell lines RMA-S
(Karre, et al. Nature, 319:675 (1986); Ljunggren, et al., Eur. J.
Immunol. 21:2963-2970 (1991)), and the human somatic T cell hybrid,
T-2 (Cerundolo, et al., Nature 345:449-452 (1990)) and which have
been transfected with the appropriate human class I genes are
conveniently used, when peptide is added to them, to test for the
capacity of the peptide to induce in vitro primary CTL responses.
Other eukaryotic cell lines which could be used include various
insect cell lines such as mosquito larvae (ATCC cell lines CCL 125,
126, 1660, 1591, 6585, 6586), silkworm (ATTC CRL 8851), armyworm
(ATCC CRL 1711), moth (ATCC CCL 80) and Drosophila cell lines such
as a Schneider cell line (see Schneider J. Embryol. Exp. Morphol.
27:353-65 [1927]). That have been transfected with the appropriate
human class I MHC allele encoding genes and the human B2
microglobulin genes.
[0423] Peripheral blood lymphocytes are conveniently isolated
following simple venipuncture or leukapheresis of normal donors or
patients and used as the responder cell sources of CTL precursors.
In one embodiment, the appropriate antigen-presenting cells are
incubated with 10-100 .mu.M of peptide in serum-free media for 4
hours under appropriate culture conditions. The peptide-loaded
antigen-presenting cells are then incubated with the responder cell
populations in vitro for 7 to 10 days under optimized culture
conditions. Positive CTL activation can be determined by assaying
the cultures for the presence of CTLs that kill radiolabeled target
cells, both specific peptide-pulsed targets as well as target cells
expressing the endogenously processed form of the relevant virus or
tumor antigen from which the peptide sequence was derived.
[0424] Specificity and MHC restriction of the CTL is determined by
testing against different peptide target cells expressing
appropriate or inappropriate human MHC class I. The peptides that
test positive in the MHC binding assays and give rise to specific
CTL responses are referred to herein as immunogenic peptides.
[0425] After determining their binding affinity, additional
confirmatory work can be performed to select, amongst these vaccine
candidates, epitopes with preferred characteristics in terms of
population coverage, antigenicity, and immunogenicity.
[0426] Thus, various strategies can be utilized to evaluate
immunogenicity, including:
[0427] 1) Evaluation of primary T cell cultures from normal
individuals (see, e.g., Wentworth, P. A. et al., Mol. Immunol.
32:603, 1995; Celis, E. et al., Proc. Natl. Acad. Sci. USA 91:2105,
1994; Tsai, V. et al., J. Immunol. 158:1796, 1997; Kawashima, I. et
al., Human Immunol. 59:1, 1998); This procedure involves the
stimulation of peripheral blood lymphocytes (PBL) from normal
subjects with a test peptide in the presence of antigen presenting
cells in vitro over a period of several weeks. T cells specific for
the peptide become activated during this time and are detected
using, e.g., a lymphokine-release or a .sup.51Cr cytotoxicity assay
involving peptide sensitized target cells.
[0428] 2) Immunization of HLA transgenic mice (see, e.g.,
Wentworth, P. A. et al., J. Immunol. 26:97, 1996; Wentworth, P. A.
et al., Int. Immunol. 8:651, 1996; Alexander, J. et al., J.
Immunol. 159:4753, 1997); In this method, peptides in incomplete
Freund's adjuvant are administered subcutaneously to HLA transgenic
mice. Several weeks following immunization, splenocytes are removed
and cultured in vitro in the presence of test peptide for
approximately one week. Peptide-specific T cells are detected
using, e.g., a .sup.51Cr-release assay involving peptide sensitized
target cells and target cells expressing endogenously generated
antigen.
[0429] Demonstration of recall T cell responses from patients who
have been effectively vaccinated or who have a tumor; (see, e.g.,
Rehermann, B. et al., J. Exp. Med. 181:1047, 1995; Doolan, D. L. et
al., Immunity 7:97, 1997; Bertoni, R. et al., J. Clin. Invest.
100:503, 1997; Threlkeld, S. C. et al., J. Immunol. 159:1648, 1997;
Diepolder, H. M. et al., J. Virol. 71:6011, 1997; Tsang et al., J.
Natl. Cancer Inst. 87:982-990, 1995; Disis et al., J. Immunol.
156:3151-3158, 1996). In applying this strategy, recall responses
are detected by culturing PBL from patients with cancer who have
generated an immune response "naturally", or from patients who were
vaccinated with tumor antigen vaccines. PBL from subjects are
cultured in vitro for 1-2 weeks in the presence of test peptide
plus antigen presenting cells (APC) to allow activation of "memory"
T cells, as compared to "naive" T cells. At the end of the culture
period, T cell activity is detected using assays for T cell
activity including .sup.51Cr release involving peptide-sensitized
targets, T cell proliferation, or lymphokine release.
[0430] Kast, et al. (J. Immunol. 152:3904-3912, 1994) have shown
that motif-bearing peptides account for 90% of the epitopes that
bind to allele-specific HLA class I molecules. In this study all
possible peptides of 9 amino acids in length and overlapping by
eight amino acids (240 peptides), which cover the entire sequence
of the E6 and E7 proteins of human papillomavirus type 16, were
evaluated for binding to five allele-specific HLA molecules that
are expressed at high frequency among different ethnic groups. This
unbiased set of peptides allowed an evaluation of the predictive
value of HLA class I motifs. From the set of 240 peptides, 22
peptides were identified that bound to an allele-specific HLA
molecules with high or intermediate affinity. Of these 22 peptides,
20, (i.e. 91%), were motif-bearing. Thus, this study demonstrated
the value of motifs for the identification of peptide epitopes for
inclusion in a vaccine: application of motif-based identification
techniques eliminates screening of 90% of the potential epitopes.
The quantity of available peptides, and the complexity of the
screening process would make a comprehensive evaluation of an
antigen highly difficult, if not impossible without use of
motifs.
[0431] An immunogenic peptide epitope of the invention may be
included in a polyepitopic vaccine composition comprising
additional peptide epitopes of the same antigen, antigens from the
same source, and/or antigens from a different source. Moreover,
class II epitopes can be included along with class I epitopes.
Peptide epitopes from the same antigen may be adjacent epitopes
that are contiguous in sequence or may be obtained from different
regions of the protein.
[0432] The relationship between binding affinity for HLA class I
molecules and immunogenicity of discrete peptide epitopes on bound
antigens has been analyzed in three different experimental
approaches (see, e.g. Sette, et al., J. Immunol. 153:5586 (1994)).
In the first approach, the immunogenicity of potential epitopes
ranging in MHC binding affinity over a 10,000-fold range was
analyzed in HLA-A*0201 transgenic mice. In the second approach, the
antigenicity of approximately 100 different hepatitis B virus
(HBV)-derived potential epitopes, all carrying A*0201 binding
motifs, was assessed by using PBL (peripheral blood lymphocytes) of
acute hepatitis patients (see, e.g. Sette, et al., J. Immunol.
153:5586 (1994)). In the third approach the binding affinity of
previously known antigenic peptides for the relevant HLA class I
was determined (Sette et al. Molec. Immunol. 31:813, 1994) In all
cases, it was found that an affinity threshold of approximately 500
nM (preferably 500 nM or less) determines the capacity of a peptide
epitope to elicit a CTL response. In the case of class II HLA a
relevant threshold of affinity was set at 1000 nM by similar
studies performed by Southwood and colleagues (Southwood et al. J.
Immunol. 160:3363-3373 (1998). These data also indicate the
important role of determinant selection in the shaping of T cell
responses.
[0433] Immunogenic peptides can be identified in relevant native
sequences with reference e.g., to one of the supermotifs or motifs
set out in TABLE 137, TABLE 138, and TABLE 139. A particular motif
is denoted in the tables and is defined by its primary anchor
residues, i.e. a motif bearing peptide must comprise at least one
of the specified residues at each primary anchor position. A
peptide may be analogued at any one or more of its primary anchor
residues by exchanging one of the specified primary anchor residues
with another primary anchor residue at the same position specified
for the same motif. The numeric positions within each motif are
designated in an amino to carboxyl orientation. Alternatively, a
peptide may be analogued at any one or more of the designated
secondary anchor residues described on TABLE 138 and TABLE 139 by
exchanging an existing residue with one of the designated secondary
anchor residues at the designated positions. In a preferred
embodiment, to enhance binding affinity, deleterious residues are
removed from native sequences; similarly deleterious residues are
not used to substitute for another residue at a designated
position. Modifications to a primary anchor position and/or a
secondary anchor position may be made at one position or multiple
positions.
[0434] Peptides that comprise epitopes and/or immunogenic peptides
of the invention can be prepared synthetically, or by recombinant
DNA technology or from natural sources such as whole viruses or
tumors. Although the peptide will preferably be substantially free
of other naturally occurring host cell proteins and fragments
thereof, in some embodiments the peptides can be synthetically
conjugated to native fragments or particles.
[0435] The present invention relates to allele-specific peptide
motifs and binding peptides for human and murine MHC allele. It is
contemplated that the peptide binding motifs of the invention are
relatively specific for each allele. In an embodiment of the
invention, the allele-specific motifs and binding peptides are for
human class I MHC (or HLA) alleles. HLA alleles include HLA-A,
HLA-B, and HLA-C alleles. In another embodiment of the invention
the allele-specific motifs and binding peptides are for human class
II MHC (or HLA) alleles. Such HLA alleles include HLA-DR and HLA-DQ
alleles. HLA molecules that share similar binding affinity for
peptides bearing certain amino acid motifs are grouped into HLA
supertypes. See, i.e., Stites, et al., IMMUNOLOGY, 8.sup.TH ED.,
Lange Publishing, Los Altos, Calif. (1994). Peptides that bind one
or more alleles in one or more supertypes are contemplated as part
of the invention. Examples of the supertypes within HLA-A and HLA-B
molecules are shown in FIG. 2. In yet another embodiment, the
allele-specific motifs and binding peptides are for murine class I
(or H-2) MHC alleles. Such H-2 alleles include H-2Dd, H-2 Kb, H-2
Kd, H-2 Db, H-2Ld, and H-2Kk. Exemplary tables describing
allele-specific motifs are presented below. Binding within a
particular supertype for murine MHC alleles is also
contemplated.
[0436] These peptides were then used to define specific binding
motifs for each of the following alleles A3.2, A1, A11, and A24.1.
These motifs are described previously. The motifs described in
TABLES 6-9, below, are defined from pool sequencing data of
naturally processed peptides as described in the related
applications. Preferred (i.e., canonical) and tolerated (i.e.,
extended) residues associated with anchor positions of the
indicated HLA supertypes are presented in FIG. 2 and TABLE 3.
[0437] In one embodiment, the motif for HLA-A3.2 comprises from the
N-terminus to C-terminus a first conserved residue of L, M, I, V,
S, A, T and F at position 2 and a second conserved residue of K, R
or Y at the C-terminal end. Other first conserved residues are C, G
or D and alternatively E. Other second conserved residues are H or
F. The first and second conserved residues are preferably separated
by 6 to 7 residues. In another embodiment, the motif for HLA-A1
comprises from the N-terminus to the C-terminus a first conserved
residue of T, S or M, a second conserved residue of D or E, and a
third conserved residue of Y. Other second conserved residues are
A, S or T. The first and second conserved residues are adjacent and
are preferably separated from the third conserved residue by 6 to 7
residues. A second motif consists of a first conserved residue of E
or D and a second conserved residue of Y where the first and second
conserved residues are separated by 5 to 6 residues.
[0438] In yet another embodiment, the motif for HLA-A 11 comprises
from the N-terminus to the C-terminus a first conserved residue of
T, V, M, L, I, S, A, G, N, C D, or F at position 2 and a C-terminal
conserved residue of K, R, Y or H. The first and second conserved
residues are preferably separated by 6 or 7 residues. In one
embodiment, the motif for HLA-A24.1 comprises from the N-terminus
to the C-terminus a first conserved residue of Y, F or W at
position 2 and a C terminal conserved residue of F, I, W, M or L.
The first and second conserved residues are preferably separated by
6 to 7 residues.
[0439] The MHC-binding peptides identified herein represent
epitopes of a native antigen. With regard to a particular amino
acid sequence, an epitope is a set of amino acid residues which is
recognized by a particular antibody or T cell receptor. Such
epitopes are usually presented to lymphocytes via the MHC-peptide
complex. An epitope retains the collective features of a molecule,
such as primary, secondary and tertiary peptide structure, and
charge, that together form a site recognized by an antibody, T cell
receptor or MHC molecule. It is to be appreciated, however, that
isolated or purified protein or peptide molecules larger than and
comprising an epitope of the invention are still within the bounds
of the invention. Moreover, it is contemplated that synthesized
peptides can incorporate various biochemical changes that enhance
their immunological effectiveness.
[0440] The epitopes present in the invention can be dominant,
sub-dominant, or cryptic. A dominant epitope is an epitope that
induces an immune response upon immunization with a whole native
antigen. See, i.e., Sercarz, et al., Ann. Rev. Immunol. 11: 729-766
(1993). Such a peptide is considered immunogenic because it elicits
a response against the whole antigen. A subdominant epitope, on the
other hand, is one that evokes little or no response upon
immunization with whole antigen that contains the epitope, but for
which a response can be obtained by immunization with an isolated
epitope. Immunization with a sub-dominant epitope will prime for a
secondary response to the intact native antigen. A cryptic epitope
elicits a response by immunization with an isolated peptide, but
fails to prime a secondary response to a subsequent challenge with
whole antigen.
[0441] An epitope present in the invention can be cross-reactive or
non-cross-reactive in its interactions with MHC alleles and alleles
subtypes. Cross-reactive binding of an epitope (or peptide) permits
an epitope to be bound by more than one HLA molecule. Such
cross-reactivity is also known as degenerate binding. A
non-cross-reactive epitope would be restricted to binding a
particular MHC allele or allele subtype.
[0442] Cross-reactive binding of HLA-A2.1 motif-bearing peptides
with other HLA-A2 allele-specific molecules can occur. Those
allele-specific molecules that share binding specificities with
HLA-A2.1 are deemed to comprise the HLA-A2.1 supertype. The B
pocket of A2 supertype HLA molecules is characterized by a
consensus motif including residues (this nomenclature uses single
letter amino acid codes, where the subscript indicates peptide
position) F/Y.sub.9, A.sub.24, M.sub.45, E/N.sub.63, K/N.sub.66,
V.sub.67, H/Q.sub.70 and Y/C.sub.99. Similarly, the A2-supertype F
pocket is characterized by a consensus motif including residues
D.sub.77, T.sub.80, L.sub.81 and Y.sub.116 (155). About 66% of the
peptides binding A*0201 will be cross-reactive amongst three or
more A2-supertype alleles.
[0443] The A2 supertype as defined herein is consistent with
cross-reactivity data, (Fruci, D. et al., Hum. Immunol. 38:187,
1993), from live cell binding assays (del Guercio, M.-F. et al., J.
Immunol. 154:685, 1995) and data obtained by sequencing naturally
processed peptides (Sudo, T., et al., J. Immunol. 155:4749, 1995)
bound to HLA-A2 allele-specific molecules. Accordingly the family
of HLA molecules (i.e., the HLA-A2 supertype that binds these
peptides) is comprised of at least nine HLA-A proteins: A*0201,
A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*6802, and
A*6901.
[0444] As described herein, the HLA-A2 supermotif comprises peptide
ligands with L, I, V, M, A, T, or Q as primary anchor residues at
position 2 and L, I, V, M, A, or T as a primary anchor residue at
the C-terminal position of the epitope. HLA-A2 motifs that are most
particularly relevant to the invention claimed here comprise V, A,
T, or Q at position two and L, I, V, M, A, or T at the C-terminal
anchor position. A peptide epitope comprising an HLA-A2 supermotif
may bind more than one HLA-A2 supertype molecule.
[0445] The epitopes of the present invention can be any suitable
length. Class I molecule binding peptides typically are about 8 to
13 amino acids in length, and often 9, 10, 11, or 12 amino acids in
length. These peptides include conserved amino acids at certain
positions such as the second position from the N-terminus and the
C-terminal position. Also, the peptides often do not include amino
acids at certain positions that negatively affect binding of the
peptide to the HLA molecules. For example, the peptides often do
not include amino acids at positions 1, 3, 6 and/or 7 for peptides
9 amino acid peptides in length or positions 1, 3, 4, 5, 7, 8
and/or 9 for peptides 10 amino acids in length. Further, defined
herein are positions within a peptide sequence that can be utilized
as criteria for selecting HLA-binding peptide. These defined
positions are often referred to herein as a binding "motif."
[0446] Definition of motifs specific for different MHC alleles
allows the identification of potential peptide epitopes from an
antigenic protein whose amino acid sequence is known. Typically,
identification of potential peptide epitopes is initially carried
out using a computer to scan the amino acid sequence of a desired
antigen for the presence of motifs. The epitopic sequences are then
synthesized.
[0447] In general, class I peptide binding motifs generally include
a first conserved residue at position two from the N-terminus
(wherein the N-terminal residue is position one) and a second
conserved residue at the C-terminal position (often position 9 or
10). As a specific example, the HLA A*0201 class I peptide binding
motifs include a first conserved residue at position two from the
N-terminus (wherein the N-terminal residue is position one)
selected from the group consisting of I, V, A and T and a second
conserved residue at the C-terminal position selected from the
group consisting of V, L, I, A and M. Alternatively, the peptide
may have a first conserved residue at the second position from the
N-terminus (wherein the N-terminal residue is position one)
selected from the group consisting of L, M, I, V, A and T; and a
second conserved residue at the C-terminal position selected from
the group consisting of A and M. If the peptide has 10 residues it
will contain a first conserved residue at the second position from
the N-terminus (wherein the N-terminal residue is position one)
selected from the group consisting of L, M, I, V, A, and T; and a
second conserved residue at the C-terminal position selected from
the group consisting of V, I, L, A and M; wherein the first and
second conserved residues are separated by 7 residues.
[0448] One embodiment of an HTL-inducing peptide is less than about
50 residues in length and usually consist of between about 6 and
about 30 residues, more usually between about 12 and 25, and often
between about 15 and 20 residues, for example 15, 16, 17, 18, 19,
or 20 residues. One embodiment of a CTL-inducing peptide is 13
residues or less in length and usually consists of about 8, 9, 10
or 11 residues, preferably 9 or 10 residues. In one embodiment,
HLA-DR3 a binding is characterized by an L, I, V, M, F or Y residue
at position 1 and a D or E residue at position 4. In another
embodiment, HLA-DR3 b binding is characterized by an L, I, V, M, F,
Y or A residue at position 1, a D, E, N, Q, S or T residue at
position 4, and a K, R or H residue at position 6. In another
embodiment, key anchor residues of a DR supertype binding motif are
an L, I, V, M, F, W or Y residue at position 1 and an L, I, V, M,
S, T, P, C or A residue at position 6. See, TABLE 3.
[0449] Moreover, in another embodiment, murine Db binding is
characterized by an N residue at position 5 and L, I, V or M
residue at the C-terminal position. In yet another embodiment,
murine Kb binding is characterized by a Y or F residue at position
5 and an L, I, V or M residue at the C-terminal position. In an
additional embodiment, murine Kd binding is characterized a Y or F
residue at position 2 and an L, I, V, or M residue at the
C-terminal position. In a further embodiment, murine Kk binding is
characterized by an E or D residue at position 2 and an L, I, M, V,
F, W, Y or A residue at the C-terminal position. In a further
embodiment, murine Ld binding is characterized by a P residue at
position 2 and an L, I, M, V, F, W or Y residue at the C-terminal
position. See, TABLE 5.
HLA Class I Motifs Indicative of CTL Inducing Peptide Epitopes:
[0450] The primary anchor residues of the HLA class I peptide
epitope supermotifs and motifs are delineated below. In some cases,
peptide epitopes may be listed in both a motif and a supermotif
Table. The relationship of a particular motif and respective
supermotif is indicated in the description of the individual
motifs.
[0451] The HLA-A1 supermotif is characterized by the presence in
peptide ligands of a small (T or S) or hydrophobic (L, I, V, or M)
primary anchor residue in position 2, and an aromatic (Y, F, or W)
primary anchor residue at the C-terminal position of the epitope.
The corresponding family of HLA molecules that bind to the A1
supermotif (i.e., the HLA-A1 supertype) is comprised of at least
A*0101, A*2601, A*2602, A*2501, and A*3201 (see, e.g., DiBrino, M.
et al., J. Immunol. 151:5930, 1993; DiBrino, M. et al., J. Immunol.
152:620, 1994; Kondo, A. et al., Immunogenetics 45:249, 1997).
Other allele-specific HLA molecules predicted to be members of the
A1 superfamily are shown in Table 137. Peptides binding to each of
the individual HLA proteins can be modulated by substitutions at
primary and/or secondary anchor positions, preferably choosing
respective residues specified for the supermotif.
[0452] Primary anchor specificities for allele-specific HLA-A2.1
molecules (see, e.g., Falk et al., Nature 351:290-296, 1991; Hunt
et al., Science 255:1261-1263, 1992; Parker et al., J. Immunol.
149:3580-3587, 1992; Ruppert et al., Cell 74:929-937, 1993) and
cross-reactive binding among HLA-A2 and -A28 molecules have been
described. (See, e.g., Fruci et al., Human Immunol. 38:187-192,
1993; Tanigaki et al., Human Immunol. 39:155-162, 1994; Del Guercio
et al., J. Immunol. 154:685-693, 1995; Kast et al., J. Immunol.
152:3904-3912, 1994 for reviews of relevant data.) These primary
anchor residues define the HLA-A2 supermotif; which presence in
peptide ligands corresponds to the ability to bind several
different HLA-A2 and -A28 molecules. The HLA-A2 supermotif
comprises peptide ligands with L, I, V, M, A, T, or Q as a primary
anchor residue at position 2 and L, I, V, M, A, or T as a primary
anchor residue at the C-terminal position of the epitope.
[0453] The corresponding family of HLA molecules (i.e., the HLA-A2
supertype that binds these peptides) is comprised of at least:
A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*0209,
A*0214, A*6802, and A*6901. Other allele-specific HLA molecules
predicted to be members of the A2 superfamily are shown in Table
137. As explained in detail below, binding to each of the
individual allele-specific HLA molecules can be modulated by
substitutions at the primary anchor and/or secondary anchor
positions, preferably choosing respective residues specified for
the supermotif.
[0454] The HLA-A3 supermotif is characterized by the presence in
peptide ligands of A, L, I, V, M, S, or, T as a primary anchor at
position 2, and a positively charged residue, R or K, at the
C-terminal position of the epitope, e.g., in position 9 of 9-mers
(see, e.g., Sidney et al., Hum. Immunol. 45:79, 1996). Exemplary
members of the corresponding family of HLA molecules (the HLA-A3
supertype) that bind the A3 supermotif include at least A*0301,
A*1101, A*3101, A*3301, and A*6801. Other allele-specific HLA
molecules predicted to be members of the A3 supertype are shown in
Table 1. As explained in detail below, peptide binding to each of
the individual allele-specific HLA proteins can be modulated by
substitutions of amino acids at the primary and/or secondary anchor
positions of the peptide, preferably choosing respective residues
specified for the supermotif.
[0455] The HLA-A24 supermotif is characterized by the presence in
peptide ligands of an aromatic (F, W, or Y) or hydrophobic
aliphatic (L, I, V, M, or T) residue as a primary anchor in
position 2, and Y, F, W, L, I, or M as primary anchor at the
C-terminal position of the epitope (see, e.g., Sette and Sidney,
Immunogenetics, in press, 1999). The corresponding family of HLA
molecules that bind to the A24 supermotif (i.e., the A24 supertype)
includes at least A*2402, A*3001, and A*2301. Other allele-specific
HLA molecules predicted to be members of the A24 supertype are
shown in Table 137. Peptide binding to each of the allele-specific
HLA molecules can be modulated by substitutions at primary and/or
secondary anchor positions, preferably choosing respective residues
specified for the supermotif.
[0456] The HLA-B7 supermotif is characterized by peptides bearing
proline in position 2 as a primary anchor, and a hydrophobic or
aliphatic amino acid (L, I, V, M, A, F, W, or Y) as the primary
anchor at the C-terminal position of the epitope. The corresponding
family of HLA molecules that bind the B7 supermotif (i.e., the
HLA-B7 supertype) is comprised of at least twenty six HLA-B
proteins including: B*0702, B*0703, B*0704, B*0705, B*1508, B*3501,
B*3502, B*3503, B*3504, B*3505, B*3506, B*3507, B*3508, B*5101,
B*5102, B*5103, B*5104, B*5105, B*5301, B*5401, B*5501, B*5502,
B*5601, B*5602, B*6701, and B*7801 (see, e.g., Sidney, et al., J.
Immunol. 154:247, 1995; Barber, et al., Curr. Biol. 5:179, 1995;
Hill, et al., Nature 360:434, 1992; Rammensee, et al.,
Immunogenetics 41:178, 1995 for reviews of relevant data). Other
allele-specific HLA molecules predicted to be members of the B7
supertype are shown in Table 137. As explained in detail below,
peptide binding to each of the individual allele-specific HLA
proteins can be modulated by substitutions at the primary and/or
secondary anchor positions of the peptide, preferably choosing
respective residues specified for the supermotif.
[0457] The HLA-B27 supermotif is characterized by the presence in
peptide ligands of a positively charged (R, H, or K) residue as a
primary anchor at position 2, and a hydrophobic (F, Y, L, W, M, I,
A, or V) residue as a primary anchor at the C-terminal position of
the epitope (see, e.g., Sidney and Sette, Immunogenetics, in press,
1999). Exemplary members of the corresponding family of HLA
molecules that bind to the B27 supermotif (i.e., the B27 supertype)
include at least B*1401, B*1402, B*1509, B*2702, B*2703, B*2704,
B*2705, B*2706, B*3801, B*3901, B*3902, and B*7301. Other
allele-specific HLA molecules predicted to be members of the B27
supertype are shown in Table 137. Peptide binding to each of the
allele-specific HLA molecules can be modulated by substitutions at
primary and/or secondary anchor positions, preferably choosing
respective residues specified for the supermotif.
[0458] The HLA-B44 supermotif is characterized by the presence in
peptide ligands of negatively charged (D or E) residues as a
primary anchor in position 2, and hydrophobic residues (F, W, Y, L,
I, M, V, or A) as a primary anchor at the C-terminal position of
the epitope (see, e.g., Sidney et al., Immunol. Today 17:261,
1996). Exemplary members of the corresponding family of HLA
molecules that bind to the B44 supermotif (i.e., the B44 supertype)
include at least: B*1801, B*1802, B*3701, B*4001, B*4002, B*4006,
B*4402, B*4403, and B*4006. Peptide binding to each of the
allele-specific HLA molecules can be modulated by substitutions at
primary and/or secondary anchor positions; preferably choosing
respective residues specified for the supermotif.
[0459] The HLA-B58 supermotif is characterized by the presence in
peptide ligands of a small aliphatic residue (A, S, or T) as a
primary anchor residue at position 2, and an aromatic or
hydrophobic residue (F, W, Y, L, I, V, M, or A) as a primary anchor
residue at the C-terminal position of the epitope (see, e.g.,
Sidney and Sette, Immunogenetics, in press, 1999 for reviews of
relevant data). Exemplary members of the corresponding family of
HLA molecules that bind to the B58 supermotif (i.e., the B58
supertype) include at least: B*1516, B*1517, B*5701, B*5702, and
B*5801. Other allele-specific HLA molecules predicted to be members
of the B58 supertype are shown in Table 137. Peptide binding to
each of the allele-specific HLA molecules can be modulated by
substitutions at primary and/or secondary anchor positions,
preferably choosing respective residues specified for the
supermotif.
[0460] The HLA-B62 supermotif is characterized by the presence in
peptide ligands of the polar aliphatic residue Q or a hydrophobic
aliphatic residue (L, V, M, I, or P) as a primary anchor in
position 2, and a hydrophobic residue (F, W, Y, M, I, V, L, or A)
as a primary anchor at the C-terminal position of the epitope (see,
e.g., Sidney and Sette, Immunogenetics, in press, 1999). Exemplary
members of the corresponding family of HLA molecules that bind to
the B62 supermotif (i.e., the B62 supertype) include at least:
B*1501, B*1502, B*1513, and B5201. Other allele-specific HLA
molecules predicted to be members of the B62 supertype are shown in
Table 137
[0461] Peptide binding to each of the allele-specific HLA molecules
can be modulated by substitutions at primary and/or secondary
anchor positions, preferably choosing respective residues specified
for the supermotif.
[0462] The HLA-A1 motif is characterized by the presence in peptide
ligands of T, S, or M as a primary anchor residue at position 2 and
the presence of Y as a primary anchor residue at the C-terminal
position of the epitope. An alternative allele-specific A1 motif is
characterized by a primary anchor residue at position 3 rather than
position 2. This motif is characterized by the presence of D, E, A,
or S as a primary anchor residue in position 3, and a Y as a
primary anchor residue at the C-terminal position of the epitope
(see, e.g., DiBrino et al., J. Immunol., 152:620, 1994; Kondo et
al., Immunogenetics 45:249, 1997; and Kubo et al., J. Immunol.
152:3913, 1994 for reviews of relevant data). An HLA-A1 extended
motif includes a D residue in position 3 and A, I, L, or F at the
C-terminus. Peptide binding to HLA A1 can be modulated by
substitutions at primary and/or secondary anchor positions,
preferably choosing respective residues specified for the motif.
Residues T, S, or M at position 2 and Y at the C-terminal position
are a subset of the A1 supermotif primary anchors.
[0463] An HLA-A2*0201 motif was characterized by the presence in
peptide ligands of L or M as a primary anchor residue in position
2, and L or V as a primary anchor residue at the C-terminal
position of a 9-residue peptide (see, e.g., Falk et al., Nature
351:290-296, 1991) and was further found to comprise an I at
position 2 and I or A at the C-terminal position of a nine amino
acid peptide (see, e.g., Hunt et al., Science 255:1261-1263, Mar.
6, 1992; Parker et al., J. Immunol. 149:3580-3587, 1992). The
A*0201 allele-specific motif has also been defined to additionally
comprise V, A, T, or Q as a primary anchor residue at position 2,
and M or T as a primary anchor residue at the C-terminal position
of the epitope (see, e.g., Kast et al., J. Immunol. 152:3904-3912,
1994). Thus, the HLA-A*0201 motif comprises peptide ligands with L,
I, V, M, A, T, or Q as primary anchor residues at position 2 and L,
I, V, M, A, or T as a primary anchor residue at the C-terminal
position of the epitope. The preferred and tolerated residues that
characterize the primary anchor positions of the HLA-A*0201 motif
are identical to the residues describing the A2 supermotif. (For
reviews of relevant data, see, e.g., Del Guercio et al., J.
Immunol. 154:685-693, 1995; Ruppert et al., Cell 74:929-937, 1993;
Sidney et al., Immunol. Today 17:261-266, 1996; Sette and Sidney,
Curr. Opin. in Immunol. 10:478-482, 1998). Secondary anchor
residues that characterize the A*0201 motif have additionally been
defined (see, e.g., Ruppert et al., Cell 74:929-937, 1993). Peptide
binding to HLA-A*0201 molecules can be modulated by substitutions
at primary and/or secondary anchor positions, preferably choosing
respective residues specified for the motif.
[0464] The HLA-A3 motif is characterized by the presence in peptide
ligands of L, M, V, I, S, A, T, F, C, G, or D as a primary anchor
residue at position 2, and the presence of K, Y, R, H, F, or A as a
primary anchor residue at the C-terminal position of the epitope
(see, e.g., DiBrino et al., Proc. Natl. Acad. Sci. USA 90:1508,
1993; and Kubo et al., J. Immunol. 152:3913-3924, 1994). Peptide
binding to HLA-A3 can be modulated by substitutions at primary
and/or secondary anchor positions, preferably choosing respective
residues specified for the motif.
[0465] The HLA-A11 motif is characterized by the presence in
peptide ligands of V, T, M, L, I, S, A, G, N, C, D, or F as a
primary anchor residue in position 2, and K, R, Y, or H as a
primary anchor residue at the C-terminal position of the epitope
(see, e.g., Zhang et al., Proc. Natl. Acad. Sci USA 90:2217-2221,
1993; and Kubo et al., J. Immunol. 152:3913-3924, 1994). Peptide
binding to HLA-A11 can be modulated by substitutions at primary
and/or secondary anchor positions, preferably choosing respective
residues specified for the motif.
[0466] The HLA-A24 motif is characterized by the presence in
peptide ligands of Y, F, W, or M as a primary anchor residue in
position 2, and F, L, I, or W as a primary anchor residue at the
C-terminal position of the epitope (see, e.g., Kondo et al., J.
Immunol. 155:4307-4312, 1995; and Kubo et al., J. Immunol.
152:3913-3924, 1994). Peptide binding to HLA-A24 molecules can be
modulated by substitutions at primary and/or secondary anchor
positions; preferably choosing respective residues specified for
the motif.
Motifs Indicative of Class II HTL Inducing Peptide Epitope
[0467] The primary anchor residues of the HLA class II supermotifs
and motifs are delineated below.
HLA DR-1-4-7 Supermotif
[0468] Motifs have also been identified for peptides that bind to
three common HLA class II allele-specific HLA molecules: HLA
DRB1*0401, DRB1*0101, and DRB1*0701 (see, e.g., the review by
Southwood et al. J. Immunology 160:3363-3373, 1998). Collectively,
the common residues from these motifs delineate the HLA DR-1-4-7
supermotif. Peptides that bind to these DR molecules carry a
supermotif characterized by a large aromatic or hydrophobic residue
(Y, F, W, L, I, V, or M) as a primary anchor residue in position 1,
and a small, non-charged residue (S, T, C, A, P, V, I, L, or M) as
a primary anchor residue in position 6 of a 9-mer core region.
Allele-specific secondary effects and secondary anchors for each of
these HLA types have also been identified (Southwood et al.,
supra). These are set forth in Table 139. Peptide binding to
HLA-DRB1*0401, DRB1*0101, and/or DRB1*0701 can be modulated by
substitutions at primary and/or secondary anchor positions,
preferably choosing respective residues specified for the
supermotif.
[0469] Two alternative motifs (i.e., submotifs) characterize
peptide epitopes that bind to HLA-DR3 molecules (see, e.g., Geluk
et al., J. Immunol. 152:5742, 1994). In the first motif (submotif
DR3A) a large, hydrophobic residue (L, I, V, M, F, or Y) is present
in anchor position 1 of a 9-mer core, and D is present as an anchor
at position 4, towards the carboxyl terminus of the epitope. As in
other class II motifs, core position 1 may or may not occupy the
peptide N-terminal position.
[0470] The alternative DR3 submotif provides for lack of the large,
hydrophobic residue at anchor position 1, and/or lack of the
negatively charged or amide-like anchor residue at position 4, by
the presence of a positive charge at position 6 towards the
carboxyl terminus of the epitope. Thus, for the alternative
allele-specific DR3 motif (submotif DR3B): L, I, V, M, F, Y, A, or
Y is present at anchor position 1; D, N, Q, E, S, or T is present
at anchor position 4; and K, R, or H is present at anchor position
6. Peptide binding to HLA-DR3 can be modulated by substitutions at
primary and/or secondary anchor positions, preferably choosing
respective residues specified for the motif.
[0471] As with HLA class I binding peptides, motifs have also been
defined for HLA class II-binding peptides. Several studies have
identified an important role for an aromatic or hydrophobic residue
(I, L, M, V, F, W, or Y) at position 1 of a 9-mer core region,
typically nested within a longer peptide sequence, in the binding
of peptide ligands to several HLA-class II alleles (Hammer et al.
Cell 74:197, (1993); Sette et al. J. Immunol. 151:3163-70 (1993);
O'Sullivan et al. J. Immunol. 147:2663 (1991); and Southwood et al.
J. Immunol. 160:3363-73 (1998)). A strong role has also been
demonstrated for the residue in position 6 of the 9-mer core, where
short and/or hydrophobic residues (S, T, C, A, P, V, I, L, or M)
are preferred. This position 1-position 6 motif has been described
as a DR-supermotif (Southwood et al. J. Immunol. 160:3363-3373
(1998)) and has been shown to efficiently identify peptides capable
of binding a large set of common HLA-class II alleles.
[0472] Peptides binding to class II molecules may also be analyzed
with respect to the identification of secondary preferred or
deleterious residues. For example, to derive a more detailed
DRB1*0401 motif to define secondary residues influencing peptide
binding, we employed a strategy similar to that performed with
class I peptides. For each peptide analyzed, nine-residue-long core
regions were aligned on the basis of the primary class II positions
P1 and P6 anchors. Then, the average binding affinity of a peptide
carrying a particular residue was calculated for each position,
relative to the remainder of the group. Following this method,
values showing average relative binding were compiled. These values
also present a map of the positive or negative effect of each of
the 20 naturally occurring amino acids in DRB1*0401 binding
capacity when occupying a particular position relative to the P1-P6
class II motif positions.
[0473] Variations in average relative binding of greater than or
equal to fourfold or less than or equal to 0.25 were arbitrarily
considered significant and indicative of secondary effects of a
given residue on HLA-peptide interactions. Most secondary effects
were associated with P4, P7, and P9. These positions correspond to
secondary anchors engaging shallow pockets on the DR molecule.
Similar studies defining secondary residues were also performed for
DRB1*0101 and DRB1*0701. The definitions of secondary residues of
motifs for DR1, DR4, and DR7 are shown in TABLE 139.
[0474] Upon definition of allele-specific secondary effects and
secondary anchors, allele-specific algorithms were derived and
utilized to identify peptides binding DRB1*0101, DRB1*0401, and
DRB*0701. Further experiments, identified a large set of HLA class
II molecules, which includes at least the DRB1*0101, DRB1*0401, and
DRB*0701, DRB1*1501, DRB1*0901 and DRB1*1302 allelic products
recognizing the DR supermotif, and is characterized by largely
overlapping peptide binding repertoires.
[0475] The data presented above confirm that several common HLA
class II types are characterized by largely overlapping peptide
binding repertoires. On this basis, in analogy to the case of HLA
class I molecules, HLA class II molecules can be grouped in a HLA
class II supertype, defined and characterized by similar, or
largely overlapping (albeit not identical) peptide binding
specificities.
[0476] Analogs of HLA class II binding peptides that bear HLA class
II motifs may be created in a manner similar to Class I molecules.
Peptides bearing motifs may be modified at primary anchor residues
to modulate binding affinity, at secondary residues or both primary
and secondary residues. Examples may be found in related
application U.S. Ser. No. 08/121,101. For example, the
TT.sub.830-843 peptide (QYIKANSKFIGITE (SEQ ID NO:______) is
capable of binding strongly, i.e. with an affinity of between
10-100 nM, to many DR alleles including DR1, DR2, DR5, and DR7.
However, the peptide binds 100-1000-fold less well to DR4w4. It was
predicted that the lower affinity of TT.sub.830-843 for DR4w4
correlated with the presence of a positive charge in position 7
(K.sub.837) in the DR binding motif. Positive charges in position 7
are allowed in the case of DR1 or DR7, but not in the case of
DR4w4. For this reason, it was predicted that TT analogs carrying a
non-charged residue in position 837 would be good DR4w4 binders.
Analysis of the binding characteristics of a peptide analog bearing
an S substitution for the charged K residue demonstrated that the
analog was capable of binding at much higher affinity to DR4w4
compared to the native peptide, i.e. the IC.sub.50 of the analog
was 13 nM compared to an IC.sub.50 of 15,000 nM for the native
sequence.
[0477] The peptides present in the invention can be identified by
any suitable method. For example, peptides are conveniently
identified using the algorithms of the invention described in the
co-pending U.S. patent application Ser. No. 09/894,018. These
algorithms are mathematical procedures that produce a score which
enables the selection of immunogenic peptides. Typically one uses
the algorithmic score with a binding threshold to enable selection
of peptides that have a high probability of binding at a certain
affinity and will in turn be immunogenic. The algorithm are based
upon either the effects on MHC binding of a particular amino acid
at a particular position of a peptide or the effects on binding MHC
of a particular substitution in a motif containing peptide.
[0478] Peptide sequences characterized in molecular binding assays
and capture assays have been and can be identified utilizing
various technologies. Motif-positive sequences are identified using
a customized application created at Epimmune. Sequences are also
identified utilizing matrix-based algorithms, and have been used in
conjunction with a "power" module that generates a predicted 50%
inhibitory concentration (PIC) value. These latter methods are
operational on Epimmune's HTML-based Epitope Information System
(EIS) database. All of the described methods are viable options in
peptide sequence selection for IC.sub.50 determination using
binding assays.
[0479] Additional procedures useful in identifying the peptides of
the present invention generally follow the methods disclosed in
Falk et al., Nature 351:290 (1991). Briefly, the methods involve
large-scale isolation of MHC class I molecules, typically by
immunoprecipitation or affinity chromatography, from the
appropriate cell or cell line. Examples of other methods for
isolation of the desired MHC molecule equally well known to the
artisan include ion exchange chromatography, lectin chromatography,
size exclusion, high performance liquid chromatography, and a
combination of some or all of the above techniques.
[0480] For example, isolation of peptides bound to MHC class I
molecules include lowering the culture temperature from 37.degree.
C. to 26.degree. C. overnight to destabilize .beta..sub.2
microglobulin and stripping the endogenous peptides from the cell
using a mild acid treatment. The methods release previously bound
peptides into the extracellular environment allowing new exogenous
peptides to bind to the empty class I molecules. The
cold-temperature incubation method enables exogenous peptides to
bind efficiently to the MHC complex, but requires an overnight
incubation at 26.degree. C. which may slow the cell's metabolic
rate. It is also likely that cells not actively synthesizing MHC
molecules (i.e., resting PBMC) would not produce high amounts of
empty surface MHC molecules by the cold temperature procedure.
[0481] Immunoprecipitation is also used to isolate the desired
allele. A number of protocols can be used, depending upon the
specificity of the antibodies used. For example, allele-specific
mAb reagents can be used for the affinity purification of the
HLA-A, HLA-B, and HLA-C molecules. Several mAb reagents for the
isolation of HLA-A molecules are available (TABLE 3). Monoclonal
antibody BB7.2 is suitable for isolating HLA-A2 molecules. Thus,
for each of the targeted HLA-A alleles, reagents are available that
may be used for the direct isolation of the HLA-A molecules.
Affinity columns prepared with these mAbs using standard techniques
are successfully used to purify the respective HLA-A allele
products.
[0482] In addition to allele-specific mAbs, broadly reactive
anti-HLA-A, B, C mAbs, such as W6/32 and B9.12.1, and one
anti-HLA-B, C mAb, B1.23.2, could be used in alternative affinity
purification protocols as described in patents and patent
applications described herein.
[0483] The peptides bound to the peptide binding groove of the
isolated MHC molecules are typically eluted using acid treatment.
Peptides can also be dissociated from MHC molecules by a variety of
standard denaturing means, such as, for example, heat, pH,
detergents, salts, chaotropic agents, or a combination acid
treatment and/or more standard denaturing means.
[0484] Peptide fractions are further separated from the MHC
molecules by reversed-phase high performance liquid chromatography
(HPLC) and sequenced. Peptides can be separated by a variety of
other standard means well known to the artisan, including
filtration, ultrafiltration, electrophoresis, size chromatography,
precipitation with specific antibodies, ion exchange
chromatography, isoelectrofocusing, and the like.
[0485] Sequencing of the isolated peptides can be performed
according to standard techniques such as Edman degradation
(Hunkapiller, M. W., et al., Methods Enzymol. 91, 399 (1983)).
Other methods suitable for sequencing include mass spectrometry
sequencing of individual peptides as previously described (Hunt, et
al., Science 225:1261 (1992)). Amino acid sequencing of bulk
heterogeneous peptides (i.e., pooled HPLC fractions) from different
MHC molecules typically reveals a characteristic sequence motif for
each MHC allele. For assays of peptide-HLA interactions (e.g.,
quantitative binding assays) cells with defined MHC molecules are
useful.
[0486] A large number of cells with defined MHC molecules,
particularly MHC Class I molecules, are known and readily
available. For example, human EBV-transformed B cell lines have
been shown to be excellent sources for the preparative isolation of
class I and class II MHC molecules. Well-characterized cell lines
are available from private and commercial sources, such as American
Type Culture Collection ("Catalogue of Cell Lines and Hybridomas,"
6th edition (1988) Manassas, Va., U.S.A.); National Institute of
General Medical Sciences 1990/1991 Catalog of Cell Lines (NIGMS)
Human Genetic Mutant Cell Repository, Camden, N.J.; and ASHI
Repository, Brigham and Women's Hospital, 75 Francis Street,
Boston, Mass. 02115. TABLE 3 and TABLE 23 list some B cell lines
suitable for use as sources for HLA alleles. All of these cell
lines can be grown in large batches and are therefore useful for
large scale production of MHC molecules. One of skill will
recognize that these are merely exemplary cell lines and that many
other cell sources can be employed. Similar EBV B cell lines
homozygous for HLA-B and HLA-C could serve as sources for HLA-B and
HLA-C alleles, respectively. Specific cell lines and antibodies
used to determine class II and murine peptides disclosed herein are
set forth in TABLES 6 and 7.
[0487] The peptides of the invention can be prepared synthetically,
or by recombinant DNA technology or from natural sources such as
whole viruses or tumors. Although the peptide will preferably be
substantially free of other naturally occurring host cell proteins
and fragments thereof, in some embodiments the peptides can be
synthetically or naturally conjugated to native protein fragments
or particles. The peptides of the invention can be prepared in a
wide variety of ways. Because of their relatively short size, the
peptides can be synthesized in solution or on a solid support in
accordance with conventional techniques. Various automatic
synthesizers are commercially available and can be used in
accordance with known protocols. See, for example, Stewart and
Young, Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co.
(1984), supra.
[0488] The capacity to bind MHC molecules is measured in a variety
of different ways. One means is a MHC binding assay as described in
the related applications, noted above. Other alternatives described
in the literature include inhibition of antigen presentation
(Sette, et al., J. Immunol. 141:3893 (1991), in vitro assembly
assays (Townsend, et al., Cell 62:285 (1990), and FACS based assays
using mutated cells, such as RMA.S (Melief, et al., Eur. J.
Immunol. 21:2963 (1991)).
[0489] Capture Assay: Unlike the HPLC-based molecular binding
assay, noted above, the high throughput screening ("HTS") Capture
assay does not utilize a size-exclusion silica column for
separation of bound from unbound radioactive marker. Instead, wells
of an opaque white 96-well Optiplate (Packard) are coated with 3
.mu.g (100 .mu.l @ 30 .mu.g/ml) of HLA-specific antibody (Ab) that
"capture" complexes of radiolabeled MHC and unlabeled peptide
transferred from the molecular binding assay plate in 100 .mu.l of
0.05% NP40/PBS. After a 3-hour incubation period, the supernatant
is decanted and scintillation fluid (Microscint 20) added. Captured
complexes are then measured on a microplate scintillation and
luminescence counter (TopCount NXTTM; Packard).
[0490] Additional assays for determining binding are described in
detail, i.e., in PCT publications WO 94/20127 and WO 94/03205.
Binding data results are often expressed in terms of IC.sub.50
value. IC.sub.50 is the concentration of peptide in a binding assay
at which 50% inhibition of binding of a reference peptide occurs.
Given the conditions in which the assays are preformed (i.e.,
limiting MHC proteins and labeled peptide concentrations), these
values approximate K.sub.D values. It should be noted that
IC.sub.50 values can change, often dramatically, if the assay
conditions are varied, and depending on the particular reagents
used (i.e., MHC preparation, etc.). For example, excessive
concentrations of MHC molecules will increase the apparent measured
IC.sub.50 of a given ligand. Alternatively, binding is expressed
relative to a reference peptide. Although as a particular assay
becomes more, or less, sensitive, the IC.sub.50's of the peptides
tested may change somewhat, the binding relative to the reference
peptide will not significantly change. For example, in an assay
preformed under conditions such that the IC.sub.50 of the reference
peptide increases 10-fold, the IC.sub.50 values of the test
peptides will also increase approximately 10-fold. Therefore, to
avoid ambiguities, the assessment of whether a peptide is a good,
intermediate, weak, or negative binder is generally based on its
IC.sub.50, relative to the IC.sub.50 of a standard peptide.
[0491] Binding may also be determined using other assay systems
including those using: live cells (i.e., Ceppellini et al., Nature
339:392, 1989; Christnick et al., Nature 352:67, 1991; Busch et
al., Int. Immunol. 2:443, 19990; Hill et al., J. Immunol. 147:189,
1991; del Guercio et al., J. Immunol. 154:685, 1995), cell free
systems using detergent lysates (i.e., Cerundolo et al., J.
Immunol. 21:2069, 1991), immobilized purified MHC (i.e., Hill et
al., J. Immunol. 152, 2890, 1994; Marshall et al., J. Immunol.
152:4946, 1994), ELISA systems (i.e., Reay et al., EMBO J. 11:2829,
1992), surface plasmon resonance (i.e., Khilko et al., J. Biol.
Chem. 268:15425, 1993); high flux soluble phase assays (Hammer et
al., J. Exp. Med. 180:2353, 1994), and measurement of class I MHC
stabilization or assembly (i.e., Ljunggren et al., Nature 346:476,
1990; Schumacher et al., Cell 62:563, 1990; Townsend et al., Cell
62:285, 1990; Parker et al., J. Immunol. 149:1896, 1992).
[0492] High affinity with respect to HLA class I molecules is
defined as binding with an IC.sub.50, or K.sub.D value, of 50 nM or
less; intermediate affinity with respect to HLA class I molecules
is defined as binding with an IC.sub.50 or K.sub.D value of between
about 50 and about 500 nM. High affinity with respect to binding to
HLA class II molecules is defined as binding with an IC.sub.50 or
K.sub.D value of 100 nM or less; intermediate affinity with respect
to binding to HLA class II molecules is defined as binding with an
IC.sub.50 or K.sub.D value of between about 100 and about 1000 nM.
These values are as previously defined in the related patents and
applications cited above.
[0493] The immunogenic peptides can be prepared synthetically, or
by recombinant DNA technology or from natural sources such as whole
viruses or tumors. Although the peptide will preferably be
substantially free of other naturally occurring host cell proteins
and fragments thereof, in some embodiments the peptides can be
synthetically conjugated to native fragments or particles.
[0494] The polypeptides or peptides can be a variety of lengths,
either in their neutral (uncharged) forms or in forms which are
salts, and either free of modifications such as glycosylation, side
chain oxidation, or phosphorylation or containing these
modifications, subject to the condition that the modification not
destroy the biological activity of the polypeptides as herein
described.
[0495] Desirably, the peptide will be as small as possible while
still maintaining substantially all of the biological activity of
the large peptide. In one embodiment, it may be desirable to
optimize peptides of the invention to a length of 8, 9, 10 or 11
amino acid residues, commensurate in size with endogenously
processed viral peptides or tumor cell peptides that are bound to
MHC class I molecules on the cell surface. In another embodiment,
it may be desirable to optimize peptides of the invention to about
15 to 20 amino acid residues, commensurate with peptides that are
bound to MHC class II molecules on the cell surface.
[0496] Peptides having the desired activity may be modified as
necessary to provide certain desired attributes, e.g., improved
pharmacological characteristics, while increasing or at least
retaining substantially all of the biological activity of the
unmodified peptide to bind the desired MHC molecule and activate
the appropriate T cell. For instance, the peptides may be subject
to various changes, such as substitutions, either conservative or
non-conservative, where such changes might provide for certain
advantages in their use, such as improved MHC binding. By
"conservative substitution" is meant replacing an amino acid
residue with another which is biologically and/or chemically
similar, e.g., one hydrophobic residue for another, or one polar
residue for another. The substitutions include combinations such as
Gly, Ala; Val, Ile, Leu, Met; Asp, Glu; Asn, Gln; Ser, Thr; Lys,
Arg; and Phe, Tyr. The effect of single amino acid substitutions
may also be probed using D-amino acids. Such modifications may be
made using well known peptide synthesis procedures, as described in
e.g., Merrifield, Science 232:341-347 (1986), Barany and
Merrifield, The Peptides, Gross and Meienhofer, eds. (N.Y.,
Academic Press), pp. 1-284 (1979); and Stewart and Young, Solid
Phase Peptide Synthesis, (Rockford, Ill., Pierce), 2d Ed. (1984),
incorporated by reference herein.
[0497] The peptides of the invention can also be modified by
extending or decreasing the compound's amino acid sequence, e.g.,
by the addition or deletion of amino acids. The peptides or analogs
of the invention can also be modified by altering the order or
composition of certain residues, it being readily appreciated that
certain amino acid residues essential for biological activity,
e.g., those at critical contact sites or conserved residues, may
generally not be altered without an adverse effect on biological
activity. The non-critical amino acids need not be limited to those
naturally occurring in proteins, such as L-.alpha.-amino acids, or
their D-isomers, but may include non-natural amino acids as well,
such as .beta.-.gamma.-.delta.-aminoacids, as well as many
derivatives of L-.alpha.-amino acids.
[0498] Typically, a series of peptides with single amino acid
substitutions are employed to determine the effect of electrostatic
charge, hydrophobicity, etc. on binding. For instance, a series of
positively charged (e.g., Lys or Arg) or negatively charged (e.g.,
Glu) amino acid substitutions are made along the length of the
peptide revealing different patterns of sensitivity towards various
MHC molecules and T cell receptors. In addition, multiple
substitutions using small, relatively neutral moieties such as Ala,
Gly, Pro, or similar residues may be employed. The substitutions
may be homo-oligomers or hetero-oligomers. The number and types of
residues which are substituted or added depend on the spacing
necessary between essential contact points and certain functional
attributes which are sought (e.g., hydrophobicity versus
hydrophilicity). Increased binding affinity for an MHC molecule or
T cell receptor may also be achieved by such substitutions,
compared to the affinity of the parent peptide. In any event, such
substitutions should employ amino acid residues or other molecular
fragments chosen to avoid, for example, steric and charge
interference which might disrupt binding.
[0499] Amino acid substitutions are typically of single residues.
Substitutions, deletions, insertions or any combination thereof may
be combined to arrive at a final peptide. Substitutional variants
are those in which at least one residue of a peptide has been
removed and a different residue inserted in its place. Such
substitutions generally are made in accordance with the following
TABLE 72 when it is desired to finely modulate the characteristics
of the peptide.
[0500] Substantial changes in function (e.g., affinity for MHC
molecules or T cell receptors) are made by selecting substitutions
that are less conservative than those in TABLE 70, i.e., selecting
residues that differ more significantly in their effect on
maintaining (a) the structure of the peptide backbone in the area
of the substitution, for example as a sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule at
the target site or (c) the bulk of the side chain. The
substitutions which in general are expected to produce the greatest
changes in peptide properties will be those in which (a)
hydrophilic residue, e.g. seryl, is substituted for (or by) a
hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or
alanyl; (b) a residue having an electropositive side chain, e.g.,
lysl, arginyl, or histidyl, is substituted for (or by) an
electronegative residue, e.g. glutamyl or aspartyl; or (c) a
residue having a bulky side chain, e.g. phenylalanine, is
substituted for (or by) one not having a side chain, e.g.,
glycine.
[0501] The peptides may also comprise isosteres of two or more
residues in the immunogenic peptide. An isostere as defined here is
a sequence of two or more residues that can be substituted for a
second sequence because the steric conformation of the first
sequence fits a binding site specific for the second sequence. The
term specifically includes peptide backbone modifications well
known to those skilled in the art. Such modifications include
modifications of the amide nitrogen, the .alpha.-carbon, amide
carbonyl, complete replacement of the amide bond, extensions,
deletions or backbone crosslinks. See, generally, Spatola,
Chemistry and Biochemistry of Amino Acids, peptides and Proteins,
Vol. VII (Weinstein ed., 1983).
[0502] Modifications of peptides with various amino acid mimetics
or unnatural amino acids are particularly useful in increasing the
stability of the peptide in vivo. Stability can be assayed in a
number of ways. For instance, peptidases and various biological
media, such as human plasma and serum, have been used to test
stability. See, e.g., Verhoef et al., Eur. J. Drug Metab.
Pharmacokin. 11:291-302 (1986). Half life of the peptides of the
present invention is conveniently determined using a 25% human
serum (v/v) assay. The protocol is generally as follows. Pooled
human serum (Type AB, non-heat inactivated) is delipidated by
centrifugation before use. The serum is then diluted to 25% with
RPMI tissue culture media and used to test peptide stability. At
predetermined time intervals a small amount of reaction solution is
removed and added to either 6% aqueous trichloracetic acid or
ethanol. The cloudy reaction sample is cooled (4.degree. C.) for 15
minutes and then spun to pellet the precipitated serum proteins.
The presence of the peptides is then determined by reversed-phase
HPLC using stability-specific chromatography conditions.
[0503] The peptides of the present invention or analogs thereof
which have CTL stimulating activity may be modified to provide
desired attributes other than improved serum half life. For
instance, the ability of the peptides to induce CTL activity can be
enhanced by linkage to a sequence which contains at least one
epitope that is capable of inducing a T helper cell response.
[0504] Substitutions, deletions, insertions or any combination
thereof may be combined to arrive at a final peptide.
Substitutional variants are those in which at least one residue of
a peptide has been removed and a different residue inserted in its
place. Such substitutions generally are made in accordance with the
following TABLE 70 when it is desired to finely modulate the
characteristics of the peptide.
[0505] The peptides may also comprise isosteres of two or more
residues in the MHC-binding peptide. An isostere as defined here is
a sequence of two or more residues that can be substituted for a
second sequence because the steric conformation of the first
sequence fits a binding site specific for the second sequence. The
term specifically includes peptide backbone modifications well
known to those skilled in the art. Such modifications include
modifications of the amide nitrogen, the .alpha.-carbon, amide
carbonyl, complete replacement of the amide bond, extensions,
deletions or backbone crosslinks. See, generally, Spatola,
Chemistry and Biochemistry of Amino Acids, Peptides and Proteins,
Vol. VII (Weinstein ed., 1983).
[0506] Modifications of peptides with various amino acid mimetics
or unnatural amino acids are particularly useful in increasing the
stability of the peptide in vivo. Stability can be assayed in a
number of ways. For instance, peptidases and various biological
media, such as human plasma and serum, have been used to test
stability. See, e.g., Verhoef et al., Eur. J. Drug Metab.
Pharmacokin. 11:291-302 (1986). Half life of the peptides of the
present invention is conveniently determined using a 25% human
serum (v/v) assay. The protocol is generally as follows. Pooled
human serum (Type AB, non-heat inactivated) is delipidated by
centrifugation before use. The serum is then diluted to 25% with
RPMI tissue culture media and used to test peptide stability. At
predetermined time intervals a small amount of reaction solution is
removed and added to either 6% aqueous trichloracetic acid or
ethanol. The cloudy reaction sample is cooled (4.degree. C.) for 15
minutes and then spun to pellet the precipitated serum proteins.
The presence of the peptides is then determined by reversed-phase
HPLC using stability-specific chromatography conditions.
[0507] Such analogs may also possess improved shelf-life or
manufacturing properties. More specifically, non-critical amino
acids need not be limited to those naturally occurring in proteins,
such as L-.alpha.-amino acids, or their D-isomers, but may include
non-natural amino acids as well, such as amino acids mimetics, e.g.
D- or L-naphylalanine; D- or L-phenylglycine; D- or
L-2-thieneylalanine; D- or L-1, -2,3-, or 4-pyreneylalanine; D- or
L-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- or
L-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or
L-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)-phenylglycine;
D-(trifluoromethyl)-phenylalanine; D-.rho.-fluorophenylalanine; D-
or L-.rho.-biphenylphenylalanine; D- or
L-.rho.-methoxybiphenylphenylalanine; D- or
L-2-indole(alkyl)alanines; and, D- or L-alkylalanines, where the
alkyl group can be a substituted or unsubstituted methyl, ethyl,
propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl,
iso-pentyl, or a non-acidic amino acids. Aromatic rings of a
nonnatural amino acid include, e.g., thiazolyl, thiophenyl,
pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl
aromatic rings.
[0508] Another embodiment for generating effective peptide analogs
involves the substitution of residues that have an adverse impact
on peptide stability or solubility in, e.g., a liquid environment.
This substitution may occur at any position of the peptide epitope.
Analogs of the present invention may include peptides containing
substitutions to modify the physical property (e.g., stability or
solubility) of the resulting peptide. For example, a cysteine (C)
can be substituted out in favor of .alpha.-amino butyric acid. Due
to its chemical nature, cysteine has the propensity to form
disulfide bridges and sufficiently alter the peptide structurally
so as to reduce binding capacity. Substituting .alpha.-amino
butyric acid for C not only alleviates this problem, but actually
improves binding and crossbinding capability in certain instances
(see, e.g., the review by Sette et al., In: Persistent Viral
Infections, Eds. R. Ahmed and I. Chen, John Wiley & Sons,
England, 1999). Substitution of cysteine with .alpha.-amino butyric
acid may occur at any residue of a peptide epitope, i.e. at either
anchor or non-anchor positions.
[0509] The binding activity, particularly modification of binding
affinity or cross-reactivity among HLA supertype family members, of
peptides of the invention can also be altered using analoging,
which is described in co-pending U.S. application Ser. No.
09/226,775 filed Jan. 6, 1999. In brief, the analoging strategy
utilizes the motifs or supermotifs that correlate with binding to
certain HLA molecules. Analog peptides can be created by
substituting amino acid residues at primary anchor, secondary
anchor, or at primary and secondary anchor positions. Generally,
analogs are made for peptides that already bear a motif or
supermotif. For a number of the motifs or supermotifs in accordance
with the invention, residues are defined which are deleterious to
binding to allele-specific HLA molecules or members of HLA
supertypes that bind the respective motif or supermotif (see, e.g.,
Rupert et al. Cell 74:929, 1993; Sidney, J. et al., Hu. Immunol.
45:79, 1996; and Sidney et al.; Sidney, et al., J. Immunol.
154:247, 1995). Accordingly, removal of such residues that are
detrimental to binding can be performed in accordance with the
present invention. For example, in the case of the A3 supertype,
when all peptides that have such deleterious residues are removed
from the population of peptides used in the analysis, the incidence
of cross-reactivity increased from 22% to 37% (see, e.g., Sidney,
J. et al., Hu. Immunol. 45:79, 1996).
[0510] Thus, one strategy to improve the cross-reactivity of
peptides within a given supermotif is simply to delete one or more
of the deleterious residues present within a peptide and substitute
a small "neutral" residue such as Ala (that may not influence T
cell recognition of the peptide). An enhanced likelihood of
cross-reactivity is expected if, together with elimination of
detrimental residues within a peptide, "preferred" residues
associated with high affinity binding to an allele-specific HLA
molecule or to multiple HLA molecules within a superfamily are
inserted.
[0511] To ensure that an analog peptide, when used as a vaccine,
actually elicits a CTL response to the native epitope in vivo, the
analog peptide may be used to induce T cells in vitro from
individuals of the appropriate HLA allele. Thereafter, the
immunized cells' capacity to lyse wild type peptide sensitized
target cells is evaluated. Alternatively, evaluation of the cells'
activity can be evaluated by monitoring IFN release. Each of these
cell monitoring strategies evaluate the recognition of the APC by
the CTL. It will be desirable to use as antigen presenting cells,
typically cells that have been either infected, or transfected with
the appropriate genes to establish whether endogenously produced
antigen is also recognized by the T cells induced by the analog
peptide. It is to be noted that peptide/protein-pulsed dendritic
cells can be used to present whole protein antigens for both HLA
class I and class II.
[0512] Another embodiment of the invention is to create analogs of
weak binding peptides, to thereby ensure adequate numbers of
cellular binders. Class I binding peptides exhibiting binding
affinities of 500-5000 nM, and carrying an acceptable but
suboptimal primary anchor residue at one or both positions can be
"fixed" by substituting preferred anchor residues in accordance
with the respective supertype. The analog peptides can then be
tested for binding and/or cross-binding capacity.
[0513] Another embodiment of the invention is to create analogs of
peptides that are already cross-reactive binders and are vaccine
candidates, but which bind weakly to one or more alleles of a
supertype. If the cross-reactive binder carries a suboptimal
residue (less preferred or deleterious) at a primary or secondary
anchor position, the peptide can be analoged by substituting out a
deleterious residue and replacing it with a preferred or less
preferred one, or by substituting out a less preferred reside and
replacing it with a preferred one. The analog peptide can then be
tested for cross-binding capacity.
[0514] The present invention provides methods for creating analogs
of immunogenic peptides, as well as the analogs themselves.
Analoging can comprise selection of desired residues at the primary
and/or secondary anchor positions, thereby altering the binding
affinity and immune modulating properties of the resulting analogs.
Examples of modulations that may be achieved using the present
invention include preparation of analogs with increased affinity
for a particular HLA molecule (e.g., adding by substitution
preferred secondary anchor residues specific for the molecule);
preparation of analogs with increased cross-reactivity among
different alleles (e.g., by substitution at a secondary or primary
anchor position with a residue shared by more than one HLA
molecule); or by production of a subdominant epitope (e.g., by
substitution of residues which increase affinity but are not
present on the immunodominant epitope). Peptides bearing epitopes
may be modified (e.g., having analogs created thereof) to provide
certain desired attributes, e.g., improved pharmacological
characteristics, while increasing or at least retaining the ability
to bind the desired HLA protein and, e.g., to activate the desired
T cell. Moreover, peptides which lack a desired activity can be
modified so as to thereby have that activity. In a presently
preferred embodiment, a deleterious or non-preferred residue is
removed and a preferred residue is substituted, preferred residues
having been defined on the basis of a correlation with an increased
binding affinity of the peptide that bears that particular motif or
supermotif for the HLA molecule to which the peptide is bound.
[0515] The peptides can also be modified by extending or decreasing
the compound's amino acid sequence, e.g., by the addition or
deletion of amino acids; for this embodiment it is generally
preferred to add amino acids. If amino acids are added for class I
restricted peptides, they are preferably added between the second
amino acid from the N terminus and the C terminus (for peptides
bearing a motif with primary anchors at position 2 and the
C-terminus). For class II restricted peptides, amino acids can
generally be added at the termini of the peptide. Peptides,
including analogs, of the invention can also be modified by
altering the order or composition of certain residues, it being
readily appreciated that certain amino acid residues essential for
biological activity, e.g., those at anchor positions, or conserved
residues, may generally not be altered without an adverse effect on
a biological activity. In certain contexts, however, it may be
desirable to produce peptides which lead to a biological activity
that might otherwise be deemed "adversely affected".
[0516] Heteroclitic analog peptides of the invention are
particularly useful to induce an immune response against antigens
to which a patient's immune system has become tolerant. Tolerance
refers to a specific immunologic nonresponsiveness induced by prior
exposure to an antigen. Thus, tolerance can be overcome in the
patient by identifying a particular class I peptide epitope to
which a patient is tolerant, modifying the peptide epitope sequence
according to the methods of the invention, and inducing an immune
response that cross-reacts against the tolerized epitope (antigen).
Overcoming tolerance is particularly desirable, for example, when a
patient's immune system is tolerant of a viral or tumor-associated
antigen, the latter antigens being often over-expressed
self-proteins as a consequence of cell transformation. Heteroclitic
analoging is described in co-pending U.S. provisional application
No. 60/166,529 filed Nov. 18, 1999 and US provisional application
for "Heteroclitic Analogs And Related Methods", Tangri et al.,
inventors, Attorney Docket number 018623-015810US, filed Oct. 6,
2000.
[0517] The peptides of the present invention or analogs thereof
which have CTL stimulating activity may be modified to provide
desired attributes other than improved serum half life. For
instance, the ability of the peptides to induce CTL activity can be
enhanced by linkage to a sequence which contains at least one
epitope that is capable of inducing a T helper cell response.
[0518] In some embodiments, the T helper peptide is one that is
recognized by T helper cells in the majority of the population.
This can be accomplished by selecting amino acid sequences that
bind to many, most, or all of the MHC class II molecules. These are
known as "loosely MHC-restricted" T helper sequences. Examples of
amino acid sequences that are loosely MHC-restricted include
sequences from antigens such as Tetanus toxin at positions 830-843
(QYIKANSKFIGITE (SEQ ID NO:______)), Plasmodium falciparum
circumsporozoite (CS) protein at positions 378-398
(DIEKKIAKMEKASSVFNVVNS (SEQ ID NO:______)), and Streptococcus 18 kD
protein at positions 1-16 (YGAVDSILGGVATYGAA (SEQ ID NO:______)).
Further examples of amino acid sequences that are recognized by HTL
present in a broad segments of the population are sequences that
bear the DR supermotif as shown in TABLE 139.
[0519] Alternatively, it is possible to prepare synthetic peptides
capable of stimulating T helper lymphocytes, in a loosely
MHC-restricted fashion, using amino acid sequences not found in
nature (see, e.g., PCT publication WO 95/07707). These synthetic
compounds, called Pan-DR-binding epitopes or PADRE.RTM. molecules
(Epimmune, San Diego, Calif.), are designed on the basis of their
binding activity to most HLA-DR (human MHC class II) molecules
(see, e.g., U.S. Ser. No. 08/121,101 (now abandoned) and related
U.S. Ser. No. 08/305,871 (now U.S. Pat. No. 5,736,142)). For
instance, a pan-DR-binding epitope peptide having the formula:
aKXVWANTLKAAa, where X is either cyclohexylalanine, phenylalanine,
or tyrosine, and "a" is either D-alanine or L-alanine, has been
found to bind to most HLA-DR alleles, and to stimulate the response
of T helper lymphocytes from most individuals, regardless of their
HLA type.
[0520] Particularly preferred immunogenic peptides/T helper
conjugates are linked by a spacer molecule. The spacer is typically
comprised of relatively small, neutral molecules, such as amino
acids or amino acid mimetics, which are substantially uncharged
under physiological conditions. The spacers are typically selected
from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino
acids or neutral polar amino acids. It will be understood that the
optionally present spacer need not be comprised of the same
residues and thus may be a hetero- or homo-oligomer. When present,
the spacer will usually be at least one or two residues, more
usually three to six residues. Alternatively, the CTL peptide may
be linked to the T helper peptide without a spacer.
[0521] The immunogenic peptide may be linked to the T helper
peptide either directly or via a spacer either at the amino or
carboxy terminus of the CTL peptide. The amino terminus of either
the immunogenic peptide or the T helper peptide may be acylated.
The T helper peptides used in the invention can be modified in the
same manner as CTL peptides. For instance, they may be modified to
include D-amino acids or be conjugated to other molecules such as
lipids, proteins, sugars and the like. Exemplary T helper peptides
include tetanus toxoid 830-843, influenza 307-319, malaria
circumsporozoite 382-398 and 378-389.
[0522] In some embodiments it may be desirable to include in the
pharmaceutical compositions of the invention at least one component
which primes CTL. Lipids have been identified as agents capable of
priming CTL in vivo against viral antigens. For example, palmitic
acid residues can be attached to the alpha and epsilon amino groups
of a Lys residue and then linked, e.g., via one or more linking
residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to an
immunogenic peptide. The lipidated peptide can then be injected
directly in a micellar form, incorporated into a liposome or
emulsified in an adjuvant, e.g., incomplete Freund's adjuvant. In a
preferred embodiment a particularly effective immunogen comprises
palmitic acid attached to alpha and epsilon amino groups of Lys,
which is attached via linkage, e.g., Ser-Ser, to the amino terminus
of the immunogenic peptide. Also in a preferred embodiment a
particularly effective immunogen comprises palmitic acid attached
to alpha and epsilon amino groups of Lys, which is attached via
linkage, e.g., Ser-Ser, to the amino terminus of a class I
restricted peptide having T cell determinants, such as those
peptides described herein as well as other peptides which have been
identified as having such determinants.
[0523] As another example of lipid priming of CTL responses, E.
coli lipoproteins, such as
tripalmitoyl-S-glycerylcysteinlyseryl-serine (P.sub.3CSS) can be
used to prime virus specific CTL when covalently attached to an
appropriate peptide. See, Deres et al., Nature 342:561-564 (1989),
incorporated herein by reference. Peptides of the invention can be
coupled to P.sub.3CSS, for example, and the lipopeptide
administered to an individual to specifically prime a CTL response
to the target antigen. Further, as the induction of neutralizing
antibodies can also be primed with P.sub.3CSS conjugated to a
peptide which displays an appropriate epitope, the two compositions
can be combined to more effectively elicit both humoral and
cell-mediated responses to infection.
[0524] In addition, additional amino acids can be added to the
termini of a peptide to provide for ease of linking peptides one to
another, for coupling to a carrier support, or larger peptide, for
modifying the physical or chemical properties of the peptide or
oligopeptide, or the like. Amino acids such as tyrosine, cysteine,
lysine, glutamic or aspartic acid, or the like, can be introduced
at the C- or N-terminus of the peptide or oligopeptide.
Modification at the C terminus in some cases may alter binding
characteristics of the peptide. In addition, the peptide or
oligopeptide sequences can differ from the natural sequence by
being modified by terminal-NH.sub.2 acylation, e.g., by alkanoyl
(C.sub.1-C.sub.20) or thioglycolyl acetylation, terminal-carboxyl
amidation, e.g., ammonia, methylamine, etc. In some instances these
modifications may provide sites for linking to a support or other
molecule.
[0525] The peptides of the invention can be prepared in a wide
variety of ways. Because of their relatively short size, the
peptides can be synthesized in solution or on a solid support in
accordance with conventional techniques. Various automatic
synthesizers are commercially available and can be used in
accordance with known protocols. See, for example, Stewart and
Young, Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co.
(1984), supra.
[0526] Another aspect of the present invention is directed to
vaccines which comprise an immunogenically effective amount of one
or more peptides as described herein. Peptides may be introduced
into a host using a variety of delivery vehicles known to those of
skill in the art including PLG microspheres with entrapped peptides
and virus-like particles. Furthermore, epitopes may be introduced
as multiple antigen peptides (MAPs) (see e.g., Mora and Tam, J.
Immunol. 161:3616-23 (1998)), or as immunostimulating complexes
(ISCOMS) (see e.g., Hu et al. Clin. Exp. Immunol. 113:235-43
(1998)) as known in the art.
[0527] Vaccines that contain an immunogenically effective amount of
one or more peptides as described herein are a further embodiment
of the invention. Once appropriately immunogenic epitopes have been
defined, they can be delivered by various means, herein referred to
as "vaccine" compositions. Such vaccine compositions can include,
for example, lipopeptides (e.g., Vitiello, A. et al., J: Clin.
Invest. 95:341, 1995), peptide compositions encapsulated in
poly(DL-lactide-co-glycolide) ("PLG") microspheres (see, e.g.,
Eldridge, et al., Molec. Immunol. 28:287-294, 1991: Alonso et al.,
Vaccine 12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995),
peptide compositions contained in immune stimulating complexes
(ISCOMS) (see, e.g., Takahashi et al., Nature 344:873-875, 1990; Hu
et al., Clin Exp Immunol. 113:235-24: 1998), multiple antigen
peptide systems (MAPs) (see e.g., Tam, J. P., Proc. Natl. Acaa Sci.
U.S.A. 85:5409-5413, 1988; Tam, J P., J Immunol. Methods 196:17-32,
1996), vir delivery vectors (Perkus, M. E. et al., In: Concepts in
vaccine development, Kaufmann H. E., ed., p. 379, 1996;
Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. et al.,
Nature 320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology
4:790, 1986; Top, F. et al., J Infect. Dis. 124:148, 1971; Chanda,
P. K. et al., Virology 175:535, 1990), particles of viral or
synthetic origin (e.g., Kofler, N. et al., J Immunol. Methods.
192:2-1996; Eldridge, J. H. et al., Sem. Bematol. 30:16, 1993;
Fa10, L. D., Jr. et al., Nature Med. 7:649, 1995), adjuvants
(Warren, H. S., Vogel, F. R., and Chedid, L. A. Annu. Re Immunol.
4:369, 1986; Gupta, R. K. et al., Vaccine 11:293, 1993), liposomes
(Reddy, R et al., J. Immunol. 148:1585, 1992; Rock, K. L., Immunol.
Today 17:131, 1996), or, naked or particle absorbed cDNA (Ulmer, J.
B. et al., Science 259:1745, 1993; Robinsol H. L., Hunt, L. A., and
Webster, R. G., Vaccine 11:957, 1993; Shiver, J. W. et al., In:
Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 423,
1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev. Immunol.
12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16,
1993). Toxin-targeted delivery technologies, also known as receptor
mediated targeting, such as those of Avant Immunotherapeutics, Inc.
(Needham, Mass.) may also be used.
[0528] Vaccine compositions of the invention include nucleic
acid-mediated modalities. DNA or RNA encoding one or more of the
peptides of the invention can also be administered to a patient.
This approach is described, for instance, in Wolff et. al., Science
247: 1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466;
5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720; and in
more detail below. Examples of DNA-based delivery technologies
include "naked DNA", facilitated (bupivicaine, polymers,
peptide-mediated) delivery, cationic lipid complexes, and
particle-mediated ("gene gun") or pressure-mediated delivery (see,
e.g., U.S. Pat. No. 5,922,687).
[0529] For therapeutic or prophylactic immunization purposes, the
peptides of the invention can be expression vectors include
attenuated viral hosts, such as vaccinia or fowlpox. This approach
involves the use of vaccinia virus, for example, as a vector to
express nucleotide sequences that encode the pep tides of the
invention. Upon introduction into an acutely or chronically
infected host or into a non-infected host, the recombinant vaccinia
virus expresses the immunogenic peptide, and thereby elicits a host
CTL and/or HTL response. Vaccinia vectors and methods useful in
immunization protocols are described in, e.g., U.S. Pat. No.
4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG
vectors are described in Stover et al., Nature 351:456-460 (1991).
A wide variety of other vectors useful for therapeutic
administration or immunization of the peptides of the invention,
e.g. adeno and adeno-associated virus vectors, retroviral vectors,
Salmonella typhi vectors, detoxified anthrax toxin vectors, and the
like, will be apparent to those skilled in the art from the
description herein.
[0530] Furthermore, vaccines in accordance with the invention can
encompass one or more of the peptides of the invention.
Accordingly, a peptide can be present in a vaccine individually.
Alternatively, the peptide can be individually linked to its own
carrier; alternatively, the peptide can exist as a homopolymer
comprising multiple copies of the same peptide, or as a
heteropolymer of various peptides. Polymers have the advantage of
increased immunological reaction and, where different peptide
epitopes are used to make up the polymer, the additional ability to
induce antibodies and/or CTLs that react with different antigenic
determinants of the pathogenic organism or tumor-related peptide
targeted for an immune response. The composition may be a naturally
occurring region of an antigen or may be prepared, e.g.,
recombinantly or by chemical synthesis.
[0531] Carriers that can be used with vaccines of the invention are
well known in the art, and include, e.g., thyroglobulin, albumins
such as human serum albumin, tetanus toxoid, polyamino acids such
as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B
virus core protein, and the like. The vaccines can contain a
physiologically tolerable (i.e., acceptable) diluent such as water,
or saline, preferably phosphate buffered saline. The vaccines also
typically include an adjuvant. Adjuvants such as incomplete
Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum
are examples of materials well known in the art. Additionally, CTL
responses can be primed by conjugating peptides of the invention to
lipids, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine
(P3CSS).
[0532] Upon immunization with a peptide composition in accordance
with the invention, via injection, aerosol, oral, transdermal,
transmucosal, intrapleural, intrathecal, or other suitable routes,
the immune system of the host responds to the vaccine by producing
large amounts of HTLs and/or CTLs specific for the desired antigen.
Consequently, the host becomes at least partially immune to later
infection, or at least partially resistant to developing an ongoing
chronic infection, or derives at least some therapeutic benefit
when the antigen was tumor-associated.
[0533] In certain embodiments, components that induce T cell
responses are combined with components that induce antibody
responses to the target antigen of interest. Thus, in certain
preferred embodiments of the invention, class I peptide vaccines of
the invention are combined with vaccines which induce or facilitate
neutralizing antibody responses to the target antigen of interest,
particularly to viral envelope antigens. A preferred embodiment of
such a composition comprises class I and class II epitopes in
accordance with the invention. An alternative embodiment of such a
composition comprises a class I epitope in accordance with the
invention, along with a PADRE.RTM. (Epimmune, San Diego, Calif.)
molecule (described, for example, in U.S. Pat. No. 5,736,142).
[0534] For therapeutic or immunization purposes, the peptides of
the invention can also be expressed by vectors. Examples of
expression vectors include attenuated viral hosts, such as vaccinia
or fowlpox. This approach involves the use of vaccinia virus as a
vector to express nucleotide sequences that encode the peptides of
the invention. Upon introduction into an acutely or chronically
infected host or into a non-infected host, the recombinant vaccinia
virus expresses the immunogenic peptide, and thereby elicits a host
CTL response. Vaccinia vectors and methods useful in immunization
protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another
vector is BCG (Bacille Calmette Guerin). BCG vectors are described
in Stover, et al. Nature 351:456-60 (1991). A wide variety of other
vectors useful for therapeutic administration or immunization of
the peptides of the invention, e.g., Salmonella typhi vectors,
retroviral vectors, adenoviral or adeno-associated viral vectors,
and the like will be apparent to those skilled in the art from the
description herein.
[0535] Alternatively, recombinant DNA technology may be employed
wherein a nucleotide sequence which encodes an immunogenic peptide
of interest is inserted into an expression vector, transformed or
transfected into an appropriate host cell and cultivated under
conditions suitable for expression. These procedures are generally
known in the art, as described generally in Sambrook et al.,
Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,
Cold Spring Harbor, N.Y. (1982) (also 1989), which is incorporated
herein by reference. Thus, fusion proteins which comprise one or
more peptide sequences of the invention can be used to present the
appropriate T cell epitope. For example, a coding sequence encoding
a peptide of the invention can be provided with appropriate linkers
and ligated into expression vectors commonly available in the art,
and the vectors used to transform suitable hosts to produce the
desired fusion protein. A number of such vectors and suitable host
systems are now available. Expression constructs, i.e., minigenes
are described in greater detail in the sections below. Such
methodologies are also used to present at least one peptide of the
invention along with a substance which is not a peptide of the
invention.
[0536] As the coding sequence for peptides of the length
contemplated herein can be synthesized by chemical techniques, for
example, using the phosphotriester method of Matteucci et al., J.
Am. Chem. Soc. 103:3185 (1981), with modification can be made
simply by substituting the appropriate base(s) for those encoding
the native peptide sequence. The coding sequence can then be
provided with appropriate linkers and ligated into expression
vectors commonly available in the art, and the vectors used to
transform suitable hosts to produce the desired fusion protein. A
number of such vectors and suitable host systems are now available.
For expression of the fusion proteins, the coding sequence will be
provided with operably linked start and stop codons, promoter and
terminator regions and usually a replication system to provide an
expression vector for expression in the desired cellular host. For
example, promoter sequences compatible with bacterial hosts are
provided in plasmids containing convenient restriction sites for
insertion of the desired coding sequence. The resulting expression
vectors are transformed into suitable bacterial hosts. Of course,
yeast or mammalian cell hosts may also be used, employing suitable
vectors and control sequences.
[0537] The peptides of the present invention and pharmaceutical and
vaccine compositions thereof are useful for administration to
mammals, particularly humans, to treat and/or prevent viral
infection and cancer. Examples of diseases which can be treated
using the immunogenic peptides of the invention include prostate
cancer, hepatitis B, hepatitis C, AIDS, renal carcinoma, cervical
carcinoma, lymphoma, CMV and condlyloma acuminatum.
[0538] For pharmaceutical compositions, the immunogenic peptides of
the invention are administered to an individual already suffering
from cancer or infected with the virus of interest. Those in the
incubation phase or the acute phase of infection can be treated
with the immunogenic peptides separately or in conjunction with
other treatments, as appropriate. In therapeutic applications,
compositions are administered to a patient in an amount sufficient
to elicit an effective CTL response to the virus or tumor antigen
and to cure or at least partially arrest symptoms and/or
complications. An amount adequate to accomplish this is defined as
"therapeutically effective dose" or "unit dose." Amounts effective
for this use will depend on, e.g., the peptide composition, the
manner of administration, the stage and severity of the disease
being treated, the weight and general state of health of the
patient, and the judgment of the prescribing physician, but
generally range for the initial immunization (that is for
therapeutic or prophylactic administration) from about 1.0 .mu.g to
about 5000 .mu.g of peptide for a 70 kg patient, followed by
boosting dosages of from about 1.0 .mu.g to about 1000 .mu.g of
peptide pursuant to a boosting regimen over weeks to months
depending upon the patient's response and condition by measuring
specific CTL activity in the patient's blood. In alternative
embodiments, generally for humans the dose range for the initial
immunization (that is for therapeutic or prophylactic
administration) is from about 1.0 .mu.g to about 20,000 .mu.g of
peptide for a 70 kg patient, preferably, 100 .mu.g-, 150 .mu.g-,
200 .mu.g-, 250 .mu.g-, 300 .mu.g-, 400 .mu.g-, or 500 .mu.g-20,000
.mu.g, followed by boosting dosages in the same dose range pursuant
to a boosting regimen over weeks to months depending upon the
patient's response and condition by measuring specific CTL activity
in the patient's blood. In embodiments where recombinant nucleic
acid administration is used, the administered material is titrated
to achieve the appropriate therapeutic response.
[0539] It must be kept in mind that the peptides and compositions
of the present invention may generally be employed in serious
disease states, that is, life-threatening or potentially life
threatening situations. In such cases, in view of the minimization
of extraneous substances and the relative nontoxic nature of the
peptides, it is possible and may be felt desirable by the treating
physician to administer substantial excesses of these peptide
compositions.
[0540] For therapeutic use, administration should begin at the
first sign of viral infection or the detection or surgical removal
of tumors or shortly after diagnosis in the case of acute
infection. This is followed by boosting doses until at least
symptoms are substantially abated and for a period thereafter. In
chronic infection, loading doses followed by boosting doses may be
required.
[0541] Treatment of an infected individual with the compositions of
the invention may hasten resolution of the infection in acutely
infected individuals. For those individuals susceptible (or
predisposed) to developing chronic infection the compositions are
particularly useful in methods for preventing the evolution from
acute to chronic infection. Where the susceptible individuals are
identified prior to or during infection, for instance, as described
herein, the composition can be targeted to them, minimizing need
for administration to a larger population.
[0542] The peptide compositions can also be used for the treatment
of chronic infection and to stimulate the immune system to
eliminate virus-infected cells in carriers. It is important to
provide an amount of immuno-potentiating peptide in a formulation
and mode of administration sufficient to effectively stimulate a
cytotoxic T cell response. Thus, for treatment of chronic
infection, a representative dose is in the range of about 1.0 .mu.g
to about 5000 .mu.g, preferably about 5 .mu.g to 1000 .mu.g for a
70 kg patient per dose. Immunizing doses followed by boosting doses
at established intervals, e.g., from one to four weeks, may be
required, possibly for a prolonged period of time to effectively
immunize an individual. In the case of chronic infection,
administration should continue until at least clinical symptoms or
laboratory tests indicate that the viral infection has been
eliminated or substantially abated and for a period thereafter.
[0543] The pharmaceutical compositions for therapeutic treatment
are intended for parenteral, topical, oral or local administration.
Preferably, the pharmaceutical compositions are administered
parenterally, e.g., intravenously, subcutaneously, intradermally,
or intramuscularly. Thus, the invention provides compositions for
parenteral administration which comprise a solution of the
immunogenic peptides dissolved or suspended in an acceptable
carrier, preferably an aqueous carrier. A variety of aqueous
carriers may be used, e.g., water, buffered water, 0.8% saline,
0.3% glycine, hyaluronic acid and the like. These compositions may
be sterilized by conventional, well known sterilization techniques,
or may be sterile filtered. The resulting aqueous solutions may be
packaged for use as is, or lyophilized, the lyophilized preparation
being combined with a sterile solution prior to administration. The
compositions may contain pharmaceutically acceptable auxiliary
substances as required to approximate physiological conditions,
such as pH adjusting and buffering agents, tonicity adjusting
agents, wetting agents and the like, for example, sodium acetate,
sodium lactate, sodium chloride, potassium chloride, calcium
chloride, sorbitan monolaurate, triethanolamine oleate, etc.
[0544] A pharmaceutical composition of the invention may comprise
one or more T cell stimulatory peptides of the invention. For
example, a pharmaceutical composition may comprise 1,2,3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30 or more T cell stimulatory peptides of
the invention. Moreover, a pharmaceutical composition of the
invention may comprise one or more T cell stimulatory peptides of
the invention in combination with one or more other T cell
stimulatory peptides. The concentration of each unique T cell
stimulatory peptide of the invention in the pharmaceutical
formulations can vary widely, e.g., from less than about 0.001%,
about 0.002%, about 0.003%, about 0.004%, about 0.005%, about
0.006%, 0.007%, 0.008%, 0.009%, about 0.01%, about 0.02%, about
0.025%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about
0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about
0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%,
about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about
1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%,
about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about
8%, about 9%, about 10%, about 20%, to about 50% or more by weight,
and will be selected primarily by fluid volumes, viscosities, etc.,
in accordance with the particular mode of administration selected.
In a preferred embodiment, the concentration of each unique T cell
stimulatory peptide of the invention in the pharmaceutical
formulations is about 0.001%, about 0.002%, about 0.003%, about
0.004%, about 0.005%, about 0.006%, 0.007%, 0.008%, 0.009%, about
0.01%, about 0.02%, about 0.025%, about 0.03%, about 0.04%, about
0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about
0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%,
about 0.7%, about 0.8%, about 0.9%, about 1% by weight. In a more
preferred embodiment, the concentration of each unique T cell
stimulatory peptide of the invention in the pharmaceutical
formulations is about 0.01%, about 0.02%, about 0.025%, about
0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about
0.08%, about 0.09%, about 0.1% by weight.
[0545] The concentration of CTL stimulatory peptides of the
invention in the pharmaceutical formulations can vary widely, i.e.,
from less than about 0.1%, usually at or at least about 2% to as
much as 20% to 50% or more by weight, and will be selected
primarily by fluid volumes, viscosities, etc., in accordance with
the particular mode of administration selected. A human unit dose
form of the peptide composition is typically included in a
pharmaceutical composition that comprises a human unit dose of an
acceptable carrier, preferably an aqueous carrier, and is
administered in a volume of fluid that is known by those of skill
in the art to be used for administration of such compositions to
humans.
[0546] The peptides of the invention may also be administered via
liposomes, which serve to target the peptides to a particular
tissue, such as lymphoid tissue, or targeted selectively to
infected cells, as well as increase the half-life of the peptide
composition. Liposomes include emulsions, foams, micelles,
insoluble monolayers, liquid crystals, phospholipid dispersions,
lamellar layers and the like. In these preparations the peptide to
be delivered is incorporated as part of a liposome, alone or in
conjunction with a molecule which binds to, e.g., a receptor
prevalent among lymphoid cells, such as monoclonal antibodies which
bind to the CD45 antigen, or with other therapeutic or immunogenic
compositions. Thus, liposomes either filled or decorated with a
desired peptide of the invention can be directed to the site of
lymphoid cells, where the liposomes then deliver the selected
therapeutic/immunogenic peptide compositions. Liposomes for use in
the invention are formed from standard vesicle-forming lipids,
which generally include neutral and negatively charged
phospholipids and a sterol, such as cholesterol. The selection of
lipids is generally guided by consideration of, e.g., liposome
size, acid lability and stability of the liposomes in the blood
stream. A variety of methods are available for preparing liposomes,
as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng.
9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and
5,019,369, incorporated herein by reference.
[0547] For targeting to the immune cells, a ligand to be
incorporated into the liposome can include, e.g., antibodies or
fragments thereof specific for cell surface determinants of the
desired immune system cells. A liposome suspension containing a
peptide may be administered intravenously, locally, topically, etc.
in a dose which varies according to, inter alia, the manner of
administration, the peptide being delivered, and the stage of the
disease being treated.
[0548] For solid compositions, conventional nontoxic solid carriers
may be used which include, for example, pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharin,
talcum, cellulose, glucose, sucrose, magnesium carbonate, and the
like. For oral administration, a pharmaceutically acceptable
nontoxic composition is formed by incorporating any of the normally
employed excipients, such as those carriers previously listed, and
generally 10-95% of active ingredient, that is, one or more
peptides of the invention, and more preferably at a concentration
of 25%-75%.
[0549] For aerosol administration, the immunogenic peptides are
preferably supplied in finely divided form along with a surfactant
and propellant. Typical percentages of peptides are 0.01%-20% by
weight, preferably 1%-10%. The surfactant must, of course, be
nontoxic, and preferably soluble in the propellant. Representative
of such agents are the esters or partial esters of fatty acids
containing from 6 to 22 carbon atoms, such as caproic, octanoic,
lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic
acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
Mixed esters, such as mixed or natural glycerides may be employed.
The surfactant may constitute 0.1%-20% by weight of the
composition, preferably 0.25-5%. The balance of the composition is
ordinarily propellant. A carrier can also be included, as desired,
as with, e.g., lecithin for intranasal delivery.
[0550] In another aspect the present invention is directed to
vaccines which contain as an active ingredient an immunogenically
effective amount of an immunogenic peptide as described herein. The
peptide(s) may be introduced into a host, including humans, linked
to its own carrier or as a homopolymer or heteropolymer of active
peptide units. Such a polymer has the advantage of increased
immunological reaction and, where different peptides are used to
make up the polymer, the additional ability to induce antibodies
and/or CTLs that react with different antigenic determinants of the
virus or tumor cells. Useful carriers are well known in the art,
and include, e.g., thyroglobulin, albumins such as human serum
albumin, tetanus toxoid, polyamino acids such as
poly(lysine:glutamic acid), influenza, hepatitis B virus core
protein, hepatitis B virus recombinant vaccine and the like. The
vaccines can also contain a physiologically tolerable (acceptable)
diluent such as water, phosphate buffered saline, or saline, and
further typically include an adjuvant. Adjuvants such as incomplete
Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum
are materials well known in the art. And, as mentioned above, CTL
responses can be primed by conjugating peptides of the invention to
lipids, such as P3CSS. Upon immunization with a peptide composition
as described herein, via injection, aerosol, oral, transdermal or
other route, the immune system of the host responds to the vaccine
by producing large amounts of CTLs specific for the desired
antigen, and the host becomes at least partially immune to later
infection, or resistant to developing chronic infection.
[0551] The peptides of the present invention and pharmaceutical and
vaccine compositions of the invention are useful for administration
to mammals, particularly humans, to treat and/or prevent infections
or cancer. Vaccine compositions containing the peptides of the
invention are administered to a patient susceptible to or otherwise
at risk of viral infection or cancer to elicit an immune response
against the antigen and thus enhance the patient's own immune
response capabilities. Such an amount is defined to be an
"immunogenically effective dose." In this use, the precise amounts
again depend on the patient's state of health and weight, the mode
of administration, the nature of the formulation, etc., but
generally range from about 1.0 .mu.g to about 5000 .mu.g per 70
kilogram patient, more commonly from about 10 .mu.g to about 500
.mu.g mg per 70 kg of body weight.
[0552] In some instances it may be desirable to combine the peptide
vaccines of the invention with vaccines which induce neutralizing
antibody responses to the virus of interest, particularly to viral
envelope antigens.
[0553] The peptides can be used to treat any number of infectious
diseases, such as viral, bacterial, fungal, and parasitic
infections. Suitable antigens are disclosed, for instance, in WO
94/20127 and WO 94/03205. Examples of diseases which can be treated
using the immunogenic peptides of the invention include neoplastic
disease such as prostate cancer, breast cancer, colon cancer, renal
carcinoma, cervical carcinoma, and lymphoma; and infectious
conditions such as hepatitis B, hepatitis C, AIDS, CMV,
tuberculosis, malaria, and condlyloma acuminatum.
[0554] As noted herein, the peptides of the invention induce CTL or
HTL immune responses when contacted with a CTL or HTL specific to
an epitope comprised by the peptide. The manner in which the
peptide is contacted with the CTL or HTL is not critical to the
invention. For instance, the peptide can be contacted with the CTL
or HTL either in vivo or in vitro. If the contacting occurs in
vivo, the peptide itself can be administered to the patient or
other vehicles, e.g., DNA vectors encoding one or more peptide,
viral vectors encoding the peptide(s), liposomes and the like, can
be used, as described herein.
[0555] For pharmaceutical compositions, the immunogenic peptides,
or DNA encoding them, are administered to an individual already
suffering from cancer or infected with a pathogen. The peptides or
DNA encoding them can be administered individually or as fusions of
one or more of the peptide sequences disclosed here. Those in the
incubation phase or the acute phase of infection can be treated
with the immunogenic peptides separately or in conjunction with
other treatments, as appropriate. In therapeutic applications,
compositions are administered to a patient in an amount sufficient
to elicit an effective CTL or HTL response to the pathogen or tumor
antigen and to cure or at least partially arrest or slow symptoms
and/or complications. An amount adequate to accomplish this falls
within the present definition of "therapeutically effective dose."
Amounts effective for this use will depend on, e.g., the particular
composition administered, the manner of administration, the stage
and severity of the disease being treated, the weight and general
state of health of the patient, and the judgment of the prescribing
physician. Generally the dosage range for an initial immunization
(i.e., therapeutic or prophylactic administration) is from about
1.0 .mu.g to about 5000 .mu.g of peptide for a 70 kg patient, more
typically 10 .mu.g to 500 .mu.g, followed by boosting dosages of
from about 1.0 .mu.g to about 1000 .mu.g of peptide pursuant to a
boosting regimen over weeks to months depending upon the patient's
response and condition by measuring specific CTL activity in the
patient's blood. The peptides and compositions of the present
invention are often employed in serious disease states, that is,
life-threatening or potentially life threatening situations. In
such cases, upon use of purified compositions of the invention, the
relative nontoxic nature of the peptides, it may be felt desirable
by the treating physician to administer substantial excesses of
these peptide compositions relative to these stated dosage
amounts.
[0556] For therapeutic use, administration should generally begin
at the first diagnosis of infection or cancer. This is followed by
boosting doses until at least symptoms are substantially abated and
for a period thereafter. In chronic infection, loading doses
followed by boosting doses may be required.
[0557] Treatment of an infected individual with a peptide or
composition of the invention may hasten resolution of the infection
in acutely infected individuals. For those individuals susceptible
(or predisposed) to developing chronic infection, the compositions
are particularly useful in methods for preventing the evolution
from acute to chronic infection. Where susceptible individuals are
identified prior to or during infection, the composition can be
targeted to them, minimizing need for administration to a larger
population.
[0558] The peptide or compositions in accordance with the invention
can also be used for the treatment of chronic infection and to
stimulate the immune system to eliminate pathogen-infected cells
in, e.g., persons who have not manifested symptoms of disease but
act as a disease vector. In this context, it is generally important
to provide an amount of immuno-potentiating peptide in a
formulation and mode of administration sufficient to effectively
stimulate a cytotoxic T cell response. Immunizing doses followed by
boosting doses at an interval, e.g., from three weeks to six
months, may be required (possibly for a prolonged period of time)
to effectively immunize an individual. In the case of chronic
infection, administration should continue until at least clinical
symptoms or laboratory tests indicate that the viral infection has
been eliminated or substantially abated and for a period
thereafter. The dosages, routes of administration, and dose
schedules are adjusted in accordance with methodologies known in
the art.
[0559] The pharmaceutical compositions for therapeutic treatment
are intended for parenteral, topical, oral or local administration.
Preferably, the pharmaceutical compositions are administered
parentally, e.g., intravenously, subcutaneously, intradermally, or
intramuscularly. Thus, the invention provides compositions for
parenteral administration which comprise a solution of the
immunogenic peptides dissolved or suspended in an acceptable
carrier, preferably an aqueous carrier. A variety of aqueous
carriers may be used, e.g., water, buffered water, 0.8% saline,
0.3% glycine, hyaluronic acid and the like. These compositions may
be sterilized by conventional, well known sterilization techniques,
or may be sterile filtered. The resulting aqueous solutions may be
packaged for use as is, or lyophilized, the lyophilized preparation
being combined with a sterile solution prior to administration. The
compositions may contain pharmaceutically acceptable auxiliary
substances as required to approximate physiological conditions,
such as pH-adjusting and buffering agents, tonicity adjusting
agents, wetting agents and the like, for example, sodium acetate,
sodium lactate, sodium chloride, potassium chloride, calcium
chloride, sorbitan monolaurate, triethanolamine oleate, etc.
[0560] The concentration of peptides of the invention in the
pharmaceutical formulations can vary widely, i.e., from less than
about 0.1%, usually at or at least about 2% to as much as 20% to
50% or more by weight, and will be selected primarily by fluid
volumes, viscosities, etc., in accordance with the particular mode
of administration selected.
[0561] For solid compositions, conventional nontoxic solid carriers
may be used which include, for example, pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharin,
talcum, cellulose, glucose, sucrose, magnesium carbonate, and the
like. For oral administration, a pharmaceutically acceptable
nontoxic composition is formed by incorporating any of the normally
employed excipients, such as those carriers previously listed, and
generally 10-95% of active ingredient, that is, one or more
peptides of the invention, and more preferably at a concentration
of 25%-75%.
[0562] For aerosol administration, the immunogenic peptides are
preferably supplied in finely divided form along with a surfactant
and propellant. Typical percentages of peptides are 0.01%-20% by
weight, preferably 1%-10%. The surfactant should generally be
nontoxic, and preferably soluble in the propellant. Representative
of such agents are the esters or partial esters of fatty acids
containing from 6 to 22 carbon atoms, such as caproic, octanoic,
lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic
acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
Mixed esters, such as mixed or natural glycerides may be employed.
The surfactant may constitute 0.1%-20% by weight of the
composition, preferably 0.25-5%. The balance of the composition is
ordinarily propellant. A carrier can also be included, as desired,
as with, e.g., lecithin for intranasal delivery.
[0563] In some instances it may be desirable to combine the peptide
vaccines of the invention with vaccines which induce neutralizing
antibody responses to the virus of interest, particularly to viral
envelope antigens.
[0564] For therapeutic or immunization purposes, nucleic acids
encoding one or more of the peptides of the invention can also be
administered to the patient. A number of methods are conveniently
used to deliver the nucleic acids to the patient. For instance, the
nucleic acid can be delivered directly, as "naked DNA". This
approach is described, for instance, in Wolff et. al., Science 247:
1465-68 (1990) as well as U.S. Pat. Nos. 5,580,859 and 5,589,466.
The nucleic acids can also be administered using ballistic delivery
as described, for instance, in U.S. Pat. No. 5,204,253. Particles
comprised solely of DNA can be administered. Alternatively, DNA can
be adhered to particles, such as gold particles.
[0565] The nucleic acids can also be delivered complexed to
cationic compounds, such as cationic lipids. Lipid-mediated gene
delivery methods are described, for instance, in WO 96/18372; WO
93/24640; Mannino and Gould-Fogerite (1988) BioTechniques 6(7):
682-691; Rose U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner et
al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-14.
[0566] For therapeutic or immunization purposes, the peptides of
the invention can also be expressed by attenuated viral hosts, such
as vaccinia or fowlpox. This approach involves the use of vaccinia
virus as a vector to express nucleotide sequences that encode the
peptides of the invention. Upon introduction into an acutely or
chronically infected host or into a noninfected host, the
recombinant vaccinia virus expresses the immunogenic peptide, and
thereby elicits a host CTL response. Vaccinia vectors and methods
useful in immunization protocols are described in, e.g., U.S. Pat.
No. 4,722,848, incorporated herein by reference. Another vector is
BCG (Bacille Calmette Guerin). BCG vectors are described in Stover
et al. (Nature 351:456-60 (1991)) which is incorporated herein by
reference. A wide variety of other vectors useful for therapeutic
administration or immunization of the peptides of the invention,
e.g., Salmonella typhi vectors and the like, will be apparent to
those skilled in the art from the description herein.
[0567] Nucleic acids encoding one or more of the peptides of the
invention can also be administered to the patient. This approach is
described, for instance, in Wolff, et. al., Science, 247:1465-68
(1990) as well as U.S. Pat. Nos. 5,580,859 and 5,589,466.
[0568] A preferred means of administering nucleic acids encoding
the peptides of the invention uses minigene constructs encoding
multiple epitopes of the invention. To create a DNA sequence
encoding the selected CTL epitopes (minigene) for expression in
human cells, the amino acid sequences of the epitopes are reverse
translated. A human codon usage table is used to guide the codon
choice for each amino acid. These epitope-encoding DNA sequences
are directly adjoined, creating a continuous polypeptide sequence.
To optimize expression and/or immunogenicity, additional elements
can be incorporated into the minigene design. Examples of amino
acid sequence that could be reverse translated and included in the
minigene sequence include: helper T lymphocyte epitopes, a leader
(signal) sequence, and an endoplasmic reticulum retention signal.
In addition, MHC presentation of CTL epitopes may be improved by
including synthetic (e.g. poly-alanine) or naturally-occurring
flanking sequences adjacent to the CTL epitopes.
[0569] The minigene sequence is converted to DNA by assembling
oligonucleotides that encode the plus and minus strands of the
minigene. Overlapping oligonucleotides (30-100 bases long) are
synthesized, phosphorylated, purified and annealed under
appropriate conditions using well known techniques. The ends of the
oligonucleotides are joined using T4 DNA ligase. This synthetic
minigene, encoding the CTL epitope polypeptide, can then cloned
into a desired expression vector.
[0570] Standard regulatory sequences well known to those of skill
in the art are included in the vector to ensure expression in the
target cells. Several vector elements are required: a promoter with
a down-stream cloning site for minigene insertion; a
polyadenylation signal for efficient transcription termination; an
E. coli origin of replication; and an E. coli selectable marker
(e.g. ampicillin or kanamycin resistance). Numerous promoters can
be used for this purpose, e.g., the human cytomegalovirus (hCMV)
promoter. See, U.S. Pat. Nos. 5,580,859 and 5,589,466 for other
suitable promoter sequences.
[0571] Additional vector modifications may be desired to optimize
minigene expression and immunogenicity. In some cases, introns are
required for efficient gene expression, and one or more synthetic
or naturally-occurring introns could be incorporated into the
transcribed region of the minigene. The inclusion of mRNA
stabilization sequences can also be considered for increasing
minigene expression. It has recently been proposed that
immunostimulatory sequences (ISSs or CpGs) play a role in the
immunogenicity of DNA vaccines. These sequences could be included
in the vector, outside the minigene coding sequence, if found to
enhance immunogenicity.
[0572] In some embodiments, a bicistronic expression vector, to
allow production of the minigene-encoded epitopes and a second
protein included to enhance or decrease immunogenicity can be used.
Examples of proteins or polypeptides that could beneficially
enhance the immune response if co-expressed include cytokines
(e.g., IL2, IL12, GM-CSF), cytokine-inducing molecules (e.g., LeIF)
or costimulatory molecules. Helper (HTL) epitopes could be joined
to intracellular targeting signals and expressed separately from
the CTL epitopes. This would allow direction of the HTL epitopes to
a cell compartment different than the CTL epitopes. If required,
this could facilitate more efficient entry of HTL epitopes into the
MHC class II pathway, thereby improving CTL induction. In contrast
to CTL induction, specifically decreasing the immune response by
co-expression of immunosuppressive molecules (e.g. TGF-.beta.) may
be beneficial in certain diseases.
[0573] Once an expression vector is selected, the minigene is
cloned into the polylinker region downstream of the promoter. This
plasmid is transformed into an appropriate E. coli strain, and DNA
is prepared using standard techniques. The orientation and DNA
sequence of the minigene, as well as all other elements included in
the vector, are confirmed using restriction mapping and DNA
sequence analysis. Bacterial cells harboring the correct plasmid
can be stored as a master cell bank and a working cell bank.
[0574] Therapeutic quantities of plasmid DNA are produced by
fermentation in E. coli, followed by purification. Aliquots from
the working cell bank are used to inoculate fermentation medium
(such as Terrific Broth), and grown to saturation in shaker flasks
or a bioreactor according to well known techniques. Plasmid DNA can
be purified using standard bioseparation technologies such as solid
phase anion-exchange resins supplied by Quiagen. If required,
supercoiled DNA can be isolated from the open circular and linear
forms using gel electrophoresis or other methods.
[0575] Purified plasmid DNA can be prepared for injection using a
variety of formulations. The simplest of these is reconstitution of
lyophilized DNA in sterile phosphate-buffer saline (PBS). A variety
of methods have been described, and new techniques may become
available. As noted above, nucleic acids are conveniently
formulated with cationic lipids. In addition, glycolipids,
fusogenic liposomes, peptides and compounds referred to
collectively as protective, interactive, non-condensing (PINC)
could also be complexed to purified plasmid DNA to influence
variables such as stability, intramuscular dispersion, or
trafficking to specific organs or cell types.
[0576] The nucleic acids can also be administered using ballistic
delivery as described, for instance, in U.S. Pat. No. 5,204,253.
Particles comprised solely of DNA can be administered.
Alternatively, DNA can be adhered to particles, such as gold
particles.
[0577] Target cell sensitization can be used as a functional assay
for expression and MHC class I presentation of minigene-encoded CTL
epitopes. The plasmid DNA is introduced into a mammalian cell line
that is suitable as a target for standard CTL chromium release
assays. The transfection method used will be dependent on the final
formulation. Electroporation can be used for "naked" DNA, whereas
cationic lipids allow direct in vitro transfection. A plasmid
expressing green fluorescent protein (GFP) can be co-transfected to
allow enrichment of transfected cells using fluorescence activated
cell sorting (FACS). These cells are then chromium-51 labeled and
used as target cells for epitope-specific CTL lines. Cytolysis,
detected by .sup.51Cr release, indicates production of MHC
presentation of minigene-encoded CTL epitopes.
[0578] In vivo immunogenicity is a second approach for functional
testing of minigene DNA formulations. Transgenic mice expressing
appropriate human MHC molecules are immunized with the DNA product.
The dose and route of administration are formulation dependent
(e.g. IM for DNA in PBS, IP for lipid-complexed DNA). Twenty-one
days after immunization, splenocytes are harvested and restimulated
for 1 week in the presence of peptides encoding each epitope being
tested. These effector cells (CTLs) are assayed for cytolysis of
peptide-loaded, chromium-51 labeled target cells using standard
techniques. Lysis of target cells sensitized by MHC loading of
peptides corresponding to minigene-encoded epitopes demonstrates
DNA vaccine function for in vivo induction of CTLs.
[0579] An embodiment of a vaccine composition in accordance with
the invention comprises ex vivo administration of a cocktail of
epitope-bearing peptides to PBMC, or isolated DC therefrom, from
the patient's blood. After pulsing the DC with peptides and prior
to reinfusion into patients, the DC are washed to remove unbound
peptides. In this embodiment, a vaccine comprises peptide-pulsed
DCs which present the pulsed peptide epitopes in HLA molecules on
their surfaces.
[0580] Dendritic cells can also be transfected, e.g., with a mini
gene comprising nucleic acid sequences encoding the epitopes in
accordance with the invention, in order to elicit immune responses.
Vaccine compositions can be created in vitro, following dendritic
cell mobilization and harvesting, whereby loading of dendritic
cells occurs in vitro.
[0581] Transgenic animals of appropriate haplotypes may
additionally provide a useful tool in optimizing the in vivo
immunogenicity of minigene DNA. In addition, animals such as
monkeys having conserved HLA molecules with cross reactivity to CTL
epitopes recognized by human MHC molecules can be used to determine
human immunogenicity of CTL epitopes (Bertoni, et al., J. Immunol.
161:4447-4455 (1998)).
[0582] Such in vivo studies are required to address the variables
crucial for vaccine development, which are not easily evaluated by
in vitro assays, such as route of administration, vaccine
formulation, tissue biodistribution, and involvement of primary and
secondary lymphoid organs. Because of their simplicity and
flexibility, HLA transgenic mice represent an attractive
alternative, at least for initial vaccine development studies,
compared to more cumbersome and expensive studies in higher animal
species, such as nonhuman primates.
[0583] Antigenic peptides are used to elicit a CTL response ex
vivo, as well. The resulting CTL cells, can be used to treat
chronic infections, or tumors in patients that do not respond to
other conventional forms of therapy, or will not respond to a
therapeutic vaccine peptide or nucleic acid in accordance with the
invention. Ex vivo CTL or HTL responses to a particular antigen
(infectious or tumor-associated antigen) are induced by incubating
in tissue culture the patient's (CTLp), or genetically compatible,
CTL or HTL precursor cells together with a source of
antigen-presenting cells (APC), such as dendritic cells, and the
appropriate immunogenic peptide. After an appropriate incubation
time (typically about 7-28 days (1-4 weeks)), in which the
precursor cells are activated and matured and expanded into
effector cells, the cells are infused back into the patient, where
they will destroy their specific target cell (an infected cell or a
tumor cell). Transfected dendritic cells may also be used as
antigen presenting cells. In order to optimize the in vitro
conditions for the generation of specific cytotoxic T cells, the
culture of stimulator cells is maintained in an appropriate
serum-free medium.
[0584] Antigenic peptides may be used to elicit CTL ex vivo, as
well. The resulting CTL, can be used to treat chronic infections
(viral or bacterial) or tumors in patients that do not respond to
other conventional forms of therapy, or will not respond to a
peptide vaccine approach of therapy. Ex vivo CTL responses to a
particular pathogen (infectious agent or tumor antigen) are induced
by incubating in tissue culture the patient's CTL precursor cells
(CTLp) together with a source of antigen-presenting cells (APC) and
the appropriate immunogenic peptide. After an appropriate
incubation time (typically 1-4 weeks), in which the CTLp are
activated and mature and expand into effector CTL, the cells are
infused back into the patient, where they will destroy their
specific target cell (an infected cell or a tumor cell).
Transfected dendritic cells are also useful for cellular delivery
of antigenic peptides.
[0585] The peptides may also find use as diagnostic reagents. For
example, a peptide of the invention may be used to determine the
susceptibility of a particular individual to a treatment regimen
which employs the peptide or related peptides, and thus may be
helpful in modifying an existing treatment protocol or in
determining a prognosis for an affected individual. In addition,
the peptides may also be used to predict which individuals will be
at substantial risk for developing chronic infection.
[0586] For example, a peptide of the invention may be used in a
tetramer staining assay to assess peripheral blood mononuclear
cells for the presence of antigen-specific CTLs following exposure
to a pathogen or immunogen. The HLA-tetrameric complex is used to
directly visualize antigen-specific CTLs (see, e.g., Ogg, et al.
Science 279:2103-2106, 1998; and Altman, et al. Science 174:94-96,
1996) and determine the frequency of the antigen-specific CTL
population in a sample of peripheral blood mononuclear cells. A
tetramer reagent using a peptide of the invention may be generated
as follows: A peptide that binds to an allele-specific HLA molecule
or supertype molecule is refolded in the presence of the
corresponding HLA heavy chain and .beta..sub.2-microglobulin to
generate a trimolecular complex. The complex is biotinylated at the
carboxyl terminal end of the heavy chain at a site that was
previously engineered into the protein. Tetramer formation is then
induced by the addition of streptavidin. By means of fluorescently
labeled streptavidin, the tetramer can be used to stain
antigen-specific cells. The cells may then be identified, for
example, by flow cytometry. Such an analysis may be used for
diagnostic or prognostic purposes.
[0587] In addition, the peptides may also be used to predict which
individuals will be at substantial risk for developing chronic
infection.
[0588] All publications, patents, and patent applications cited in
this specification are herein incorporated by reference as if each
individual publication, patent or patent application were
specifically and individually indicated to be incorporated by
reference.
[0589] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to one of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
EXAMPLES
[0590] The following examples are provided by way of illustration
only and not by way of limitation. Those of skill in the art will
readily recognize a variety of non-critical parameters that could
be changed or modified to yield essentially similar results.
Example 1
Peptide Synthesis
[0591] Peptides utilized were synthesized as previously described
by Ruppert, J., et al., "Prominent Role of Secondary Anchor
Residues in Peptide Binding to HLA-A2.1 Molecules," Cell,
74:929-937 (1993) or purchased as crude material from Chiron
Mimotopes (Chiron Corp., Australia). Synthesized peptides were
typically purified to >95% homogeneity by reverse phase HPLC.
Purity of synthesized peptides was determined using analytical
reverse-phase HPLC and amino acid analysis, sequencing, and/or mass
spectrometry. Lyophilized peptides were resuspended at 4-20 mg/ml
in 100% DMSO, then diluted to required concentrations in PBS+0.05%
(v/v) NP40 (Fluka Biochemika, Buchs, Switzerland).
Example 2
Class I Antigen Isolation
[0592] Class I antigen isolation was carried out as described in
the related applications, noted above. Naturally processed peptides
were then isolated and sequenced as described there. An
allele-specific motif and algorithms were determined and
quantitative binding assays were carried out.
[0593] Using the motifs identified above for HLA-A2.1 allele amino
acid sequences from a number of antigens were analyzed for the
presence of these motifs. TABLES 147-148, TABLES 150-151, and
TABLES 152-153 provide the results of these searches.
[0594] Isolated MHC molecules were used in a quantitative binding
assay to identify the specificity and avidity of peptide-HLA
interactions. Purification of HLA-A, HLA-B and HLA-C antigens were
carried out by essentially similar methods, using cells and
antibodies chosen as appropriate for the desired HLA molecule. A
flow diagram of an HLA-A antigen purification scheme is presented
in FIG. 14 and FIG. 49. Briefly, the cells bearing the appropriate
allele were grown in large batches (6-8 liters yielding
.about.5.times.10.sup.9 cells), harvested by centrifugation and
washed. All cell lines were maintained in RPMI 1640 media (Sigma)
supplemented with 10% fetal bovine serum (FBS) and antibiotics.
[0595] For large-scale cultures, cells were grown in roller bottle
culture in RPMI 1640 with 10% FBS or with 10% horse serum and
antibiotics. Cells were harvested by centrifugation at 1500 RPM
IEC-CRU5000 centrifuge with 259 rotor and washed three times with
phosphate-buffered saline (PBS) (0.01 M PO.sub.4, 0.154 M NaCl, pH
7.2). Cells were pelleted and stored at -70.degree. C. or treated
with detergent lysing solution to prepare detergent lysates. Cell
lysates were prepared by the addition of stock detergent solution
[1% NP-40 (Sigma) or Renex 30 (Accurate Chem. Sci. Corp., Westbury,
N.Y. 11590), 150 mM NaCl, 50 mM Tris, pH 8.0] to the cell pellets
(previously counted) at a ratio of 50-100.times.10.sup.6 cells per
ml detergent solution. A cocktail of protease inhibitors was added
to the premeasured volume of stock detergent solution immediately
prior to the addition to the cell pellet. Addition of the protease
inhibitor cocktail produced final concentrations of the following:
phenylmethylsulfonyl fluoride (PMSF), 2 mM; aprotinin, 5 .mu.g/ml;
leupeptin, 10 .mu.g/ml; pepstatin, 10 .mu.g/ml; iodoacetamide, 100
.mu.M; and EDTA, 3 ng/ml. Cell lysis was allowed to proceed at
4.degree. C. for 1 hour with periodic mixing. Routinely
5-10.times.10.sup.9 cells were lysed in 50-100 ml of detergent
solution. The lysate was clarified by centrifugation at
15,000.times.g for 30 minutes at 4.degree. C. and subsequent
passage of the supernatant fraction through a 0.2.mu. filter unit
(Nalgene). Cell lines used for HLA-B and -C isolations are provided
in TABLE 23.
[0596] The HLA antigen purification was achieved using affinity
columns prepared with mAb-conjugated Sepharose beads. For antibody
production, cells were grown in RPMI with 10% FBS in large tissue
culture flasks (Corning 25160-225). Antibodies were purified from
clarified tissue culture medium by ammonium sulfate fractionation
followed by affinity chromatography on protein-A-Sepharose (Sigma).
Briefly, saturated ammonium sulfate was added slowly with stirring
to the tissue culture supernatant to 45% (volume to volume)
overnight at 4.degree. C. to precipitate the immunoglobulins. The
precipitated proteins were harvested by centrifugation at
10,000.times.g for 30 minutes. The precipitate was then dissolved
in a minimum volume of PBS and transferred to dialysis tubing
(Spectro/Por 2, Mol. wt. cutoff 12,000-14,000, Spectum Medical
Ind.). Dialysis was against PBS (.gtoreq.20 times the protein
solution volume) with 4-6 changes of dialysis buffer over a 24-48
hour period at 4.degree. C. The dialyzed protein solution was
clarified by centrifugation (10,000.times.g for 30 minutes) and the
pH of the solution adjusted to pH 8.0 with 1N NaOH.
Protein-A-Sepharose (Sigma) was hydrated according to the
manufacturer's instructions, and a protein-A-Sepharose column was
prepared. A column of 10 ml bed volume typically binds 50-100 mg of
mouse IgG.
[0597] The protein sample was loaded onto the protein-A-Sepharose
column using a peristaltic pump for large loading volumes or by
gravity for smaller volumes (<100 ml). The column was washed
with several volumes of PBS, and the eluate was monitored at A280
in a spectrophotometer until base line was reached. The bound
antibody was eluted using 0.1 M citric acid at suitable pH
(adjusted to the appropriate pH with 1N NaOH). For mouse IgG-1 pH
6.5 was used for IgG2a pH 4.5 was used and for IgG2b and IgG3 pH
3.0 was used. 2 M Tris base was used to neutralize the eluate.
Fractions containing the antibody (monitored by A280) were pooled,
dialyzed against PBS and further concentrated using an Amicon
Stirred Cell system (Amicon Model 8050 with YM30 membrane).
Antibodies were used for affinity purification of HLA-B and HLA-C
molecules are provided in TABLE 24.
[0598] The HLA antigens were purified using affinity columns
prepared with mAb-conjugated Sepharose beads. The affinity columns
were prepared by incubating protein-A-Sepharose beads (Sigma) with
affinity-purified mAb as described above. Five to 10 mg of mAb per
ml of bead is the preferred ratio. The mAb bound beads were washed
with borate buffer (borate buffer: 100 mM sodium tetraborate, 154
mM NaCl, pH 8.2) until the washes show A280 at based line. Dimethyl
pimelimidate (20 mM) in 200 mM triethanolamine was added to
covalently crosslink the bound mAb to the protein-A-Sepharose
(Schneider, et al J. Biol. Chem. 257:10766 (1982). After incubation
for 45 minutes at room temperature on a rotator, the excess
crosslinking reagent was removed by washing the beads twice with
10-20 ml of 20 mM ethanolamine, pH 8.2. Between each one the slurry
was placed on a rotator for 5 minutes at room temperature. The
beads were washed with borate buffer and with PBS plus 0.02% sodium
azide.
[0599] The cell lysate (5-10.times.10.sup.9 cell equivalents) was
then slowly passed over a 5-10 ml affinity column (flow rate of
0.1-0.25 ml per minute) to allow the binding of the antigen to the
immobilized antibody. After the lysate was allowed to pass through
the column, the column was washed sequentially with 20 column
volumes of detergent stock solution plus 0.1% sodium dodecyl
sulfate, 20 column volumes of 0.5 M NaCl, 20 mM Tris, pH 8.0, and
10 column volumes of 20 mM Tris, pH 8.0. The HLA antigen bound to
the mAb was eluted with a basic buffer solution (50 mM diethylamine
in water). As an alternative, acid solutions such as 0.15-0.25 M
acetic acid were also used to elute the bound antigen. An aliquot
of the eluate (1/50) was removed for protein quantification using
either a colorimetric assay (BCA assay, Pierce) or by SDS-PAGE, or
both. SDS-PAGE analysis was performed as described by Laemmli
(Laemmli, U.K., Nature 227:680 (1970)) using known amounts of
bovine serum albumin (Sigma) as a protein standard.
[0600] Allele specific antibodies were used to purify the specific
MHC molecule. In the case of HLA-A2 and HLA-A3 mAbs BB7:2 and GAPA3
were used respectively. An example of SDS PAGE analysis of purified
HLA-A3.2 molecules is shown in FIG. 15.
[0601] FIG. 15 shows SDS-PAGE (12.5%) analysis of affinity purified
HLA-A3.2 from the cell line EHM. An affinity column (10 ml) was
prepared with protein A-sepharose beads coupled to the monoclonal
antibody GAPA3 which is specific for HLA-A3. A detergent lysate of
5.times.10.sup.9 cells was passaged over the column and the column
was washed extensively. The bound HLA-A3.2 molecules were eluted
from the column with 0.15M acetic acid, 50 ml. One ml of the eluate
was removed and lyophilized to concentrate the sample. The sample
was taken up to 50 .mu.l with Laemmli sample buffer and 20 .mu.l
were loaded in lane 2. Lane 1 contained molecular weight standards:
Myosin, 230 kD; .beta.-galactosidase, 116 kD; phosphorylase B, 97.4
kD; bovine serum albumin, 66.2 kD; ovalbumin, 45 kD; carbonic
anhydrase, 31 kD; soybean trypsin inhibitor, 21.5 kD; and lysozyme,
14.4 kD. Standard concentrations of bovine serum albumin were run
in lanes 8, 10 .mu.g, 9, 3 .mu.g, and 10, .mu.g to aid in the
estimation of protein yield. For this particular HLA-A3.2
preparation, the estimated yield was approximately 112 pg.
[0602] For HLA-A11, A24.1 and A1, an alternative protocol, was used
whereby anti-HIA-B and C monoclonal antibodies were used to deplete
HLA-B and C molecules. The remaining HLA-A molecules were
subsequently purified using the W6/32 mAb as described below.
[0603] Based on the density of class I expression as indicated by
the results of immunofluorescent staining analysis, it is
anticipated that average yields of class I antigen isolated from
the EBV B cell lines will range from 800-1200 pg per 1010 cell
equivalents.
Example 3
An Alternative Class I Purification Protocol
[0604] HLA-A2.1 molecules were isolated using the mAb B1.23.2 which
detects an epitope expressed by HLA-B and C allele molecules, but
not by HLA-A antigens. The mAb, W6/32, detects all human class I
molecules, including HLA-A, B and C. As mentioned above, these mAbs
react well with the B cell lines serving as sources of HLA-A
antigens. The B1.23.2 mAb reacts with the various human B cell
lines, but fails to react with a mouse cell line that expresses a
transfected HLA-A2.1 protein or a chimeric A2.1 mouse K.sup.b
molecule. It does react with the human cell line, CIR (Alexander,
J., et al., Immunogenetics, 29, 380 C19893), that lacks expression
of HLA-A and B molecules, but expresses low levels of. HLA-C
molecules. This pattern of reactivity illustrates how the 81.23.2
mAb can be used to deplete the B cell lysates of HLA-B and C
molecules.
[0605] Affinity columns were prepared using the affinity-purified
B1.23.2 and W6/32 mAbs, respectively, as described above. The
procedures for the preparation of the affinity columns are
essentially identical to the procedures described for the
preparation of the allele-specif is mAb columns described above.
The B1.23.2 mAb affinity column was used to deplete the detergent
lysates of HLA-B and C molecules using the protocol as described
above. The cell lysate depleted of HLA-B and C was then passed over
a W6/32 mAb affinity column. The MHC molecule that was eluted from
this second passage was the A allele product.
[0606] This alternative affinity purification is useful for the
purification of any HLA-A allele product, and does not rely on the
need for allele-specific mAbs. In addition, it could also be used
to isolate any class I molecule type from transfected cell
lines.
Example 4
MHC Purification
[0607] The EBV transformed cell lines JY (A*0201), M7B (A*0202),
FUN (A*0203), DAH (A*0205), CLA (A*0206), KNE (A*0207), AP
(A*0207), and AMAI (A*6802) were used as the primary source of MHC
molecules. Single MHC allele transfected 721.221 lines were also
used as sources of A*0202 and A*0207. Cells were maintained in
vitro by culture in RPMI 1640 medium (Flow Laboratories, McLean,
Va.), supplemented with 2 mM L-glutamine (GIBCO, Grand Island,
N.Y.), 100 U (100 .mu.g/ml) penicillin-streptomycin solution
(GIBCO), and 10% heat-inactivated FCS (Hazelton Biologics). Large
scale cultures were maintained in roller bottles.
[0608] HLA molecules were purified from cell lysates (Sidney, J.,
et al., "The Measurement of MHC/Peptide Interactions by Gel
Infiltration," Curr Prot Immunol 18.3.1-18.3.19 (1998)). Briefly,
cells were lysed at a concentration of 10.sup.8 cells/ml in 50 mM
Tris-HCL, pH 8.5, containing 1% (v/v) NP-40 150 mM NaCl, 5 mM EDTA,
and 2 mM PMSF. Lysates were then passed through 0.45 .mu.M filters,
cleared of nuclei and debris by centrifugation at 10,000.times.g
for 20 minutes and MHC molecules purified by monoclonal
antibody-based affinity chromatography.
[0609] For affinity purification, columns of inactivated Sepharose
CL4B and Protein A Sepharose were used as pre-columns. Class I
molecules were captured by repeated passage over Protein A
Sepharose beads conjugated with the anti-HLA (A, B, C) antibody
W6/32 (Sidney, J., et al., supra). HLA-A molecules were further
purified from HLA-B and -C molecules by passage over a B1.23.2
column. After 2 to 4 passages the W6/32 column was washed with
10-column volumes of 10 mM Tris-HCL, pH 8.0 with 1% (v/v) NP-40,
2-column volumes of PBS, and 2-column volumes of PBS containing
0.4% (w/v) n-octylglucoside. Class I molecules were eluted with 50
mM dimethylamine in 0.15 M NaCl containing 0.4% (w/v)
n-octylglucoside, pH 11.5.A 1/26 volume of 2.0 M Tris, pH 6.8, was
added to the eluate to reduce the pH to .about.8.0. The eluate was
then concentrated by centrifugation in Centriprep 30 concentrators
at 2000 rpm (Amicon, Beverly, Mass.). Protein purity,
concentration, and effectiveness of depletion steps were monitored
by SDS-PAGE and BCA assay.
Example 5
Isolation and Sequencing of Naturally Processed Peptides
[0610] For the HLA-A preparations derived from the base (50 mM
diethylamine) elution protocol, the eluate was immediately
neutralized with 1 N acetic acid to pH 7.0-7.5. The neutralized
eluate was concentrated to a volume of 1-2 ml in an Amicon stirred
cell [Model 8050, with YM3 membranes (Amicon)]. Ten ml of ammonium
acetate (0.01 M, pH 8.0) was added to the concentrator to remove
the non-volatile salts, and the sample was concentrated to
approximately 1 ml. A small sample (1/50) was removed for protein
quantitation as described above. The remainder was recovered into a
15 ml polypropylene conical centrifuge tube (Falcon, 2097) (Becton
Dickinson). Glacial acetic acid was added to obtain a final
concentration of 10% acetic acid. The acidified sample was placed
in a boiling water bath for 5 minutes to allow for the dissociation
of the bound peptides. The sample was cooled on ice, returned to
the concentrator and the filtrate was collected. Additional
aliquots of 10% acetic acid (1-2 ml) were added to the
concentrator, and this filtrate was pooled with the original
filtrate. Finally, 1-2 ml of distilled water was added to the
concentrator, and this filtrate was pooled as well.
[0611] The retentate contains the bulk of the HLA-A heavy chain and
a.sub.2-microglobulin, while the filtrate contains the naturally
processed bound peptides and other components with molecular
weights less than about 3000. The pooled filtrate material was
lyophilized in order to concentrate the peptide fraction. The
sample was then ready for further analysis.
[0612] For HPLC (high performance liquid chromatography) separation
of the peptide fractions, the lyophilized sample was dissolved in
50 .mu.l of distilled water, or into 0.1% trifluoracetic acid (TFA)
(Applied Biosystems) in water and injected to a C18 reverse-phase
narrow bore column (Beckman C18 Ultrasphere, 10.times.250 mm),
using a gradient system described by Stone and Williams (Stone, K.
L. and Williams K. R., in, Macromolecular Sequencing and Synthesis;
Selected Methods and Applications, A. R. Liss, New York, 1988, pp.
7-24. Buffer A was 0.06% TFA in water (Burdick-Jackson) and buffer
B was 0.052% TFA in 80% acetonitrile (Burdick-Jackson). The flow
rate was 0.250 ml/minute with the following gradient: 0-60 min.,
2-37.5% B; 60-95 min., 37.5-75% B; 95-105 min., 75-98% B. The
Gilson narrow bore HPLC configuration is particularly useful for
this purpose, although other configurations work equally well.
[0613] A large number of peaks were detected by absorbance at 214
nm, many of which appear to be of low abundance. Whether a given
peak represents a single peptide or a peptide mixture was not
determined. Pooled fractions were then sequenced to determine
motifs specific for each allele as described below.
[0614] Pooled peptide fractions, prepared as described above were
analyzed by automated Edman sequencing using the Applied Biosystems
Model 477A automated sequencer. The sequencing method is based on
the technique developed by Pehr Edman in the 1950s for the
sequential degradation of proteins and peptides to determine the
sequence of the constituent amino acids.
[0615] The protein or peptide to be sequenced was held by a 12-mm
diameter porous glass fiber filter disk in a heated, argon-purged
reaction chamber. The filter was generally pre-treated with
BioBrene Plus.TM. and then cycled through one or more repetitions
of the Edman reaction to reduce contaminants and improve the
efficiency of subsequent sample sequencing. Following the
pre-treatment of the filter, a solution of the sample protein or
peptide (10 pmol-5 nmol range) was loaded onto the glass filter and
dried. Thus, the sample was left embedded in the film of the
pre-treated disk. Covalent attachment of the sample to the filter
was usually not necessary because the Edman chemistry utilized
relatively apolar solvents, in which proteins and peptides are
poorly soluble.
[0616] Briefly, the Edman degradation reaction has three steps:
coupling, cleavage, and conversion. In coupling step,
phenylisothiocyanate (PITC) is added. The PITC reacts
quantitatively with the free amino-terminal amino acid of the
protein to form the phenylthiocarbamyl-protein in a basic
environment. After a period of time for the coupling step, the
excess chemicals are extracted and the highly volatile organic
acid, trifluoroacetic acid, TFA, is used to cleave the PITC-coupled
amino acid residue from the amino terminus of the protein yielding
the anilinothiazolinone (A-TZ) derivative of the amino acid. The
remaining protein/peptide is left with a new amino terminus and is
ready for the next Edman cycle. The ATZ amino acid is extracted and
transferred to a conversion flask, where upon addition of 25% TFA
in water, the ATZ amino acid is converted to the more stable
phenylthiohydantoin (PTH) amino acid that can be identified and
quantified following automatic injection into the Model 120 PTH
Analyzer which uses a microbore C-18 reverse-phase HPLC column for
the analysis.
[0617] In the present procedures, peptide mixtures were loaded onto
the glass filters. Thus, a single amino acid sequence usually does
not result. Rather, mixtures of amino acids in different yield are
found. When the particular residue is conserved among the peptides
being sequenced, increased yield for that amino acid is
observed.
Example 6
MHC-Peptide Binding Assays
[0618] Quantitative assays to measure the binding of peptides to
soluble Class I molecules are based on the inhibition of binding of
a radiolabeled standard peptide. These assays were performed as
previously described (Sidney, J., et al., supra.). Briefly, 1-10 nM
of radiolabeled peptide was co-incubated at room temperature with 1
.mu.M to 1 nM of purified MHC in the presence of 1 .mu.M human
.beta..sub.2-microglubulin (Scripps Laboratories, San Diego,
Calif.) and a cocktail of protease inhibitors. Following a two day
incubation, the percent of MHC bound radioactivity was determined
by size exclusion gel filtration chromatography using a TSK 2000
column. Alternatively, the percent of MHC bound radioactivity was
determined by capturing MHC/peptide complexes on W6/32 antibody
coated plates, and determining bound cpm using the TopCount
microscintillation counter (Packard Instrument Co., Meriden, Conn.)
(Southwood, et al., Epimmune Technical Report Epi 063-99).
[0619] The radio labeled standard peptide utilized for the A*0201,
A*0202, A*0203, A*0205, A*0206, and A*0207 assays was an
F.sub.6>Y analog of the HBV core 18-27 epitope (sequence
FLPSDYFPSV (SEQ ID NO:______)). The average IC.sub.50 of this
peptide for each molecule was 5.0, 4.3, 10, 4.3, 3.7, and 23 nM,
respectively. A C.sub.4>A analog of HBV pol 646 (sequence
FTQAGYPAL (SEQ ID NO:______)), or MAGE 1 282 (sequence YVIKVSARV
(SEQ ID NO:______)), was utilized as the label for the A*6802
assay. Their IC.sub.50s for A*6802 were 40 and 8 nM,
respectively.
[0620] In the case of competitive assays, the concentration of
peptide yielding 50% inhibition of the binding of the radiolabeled
peptide was calculated. Peptides were initially tested at one or
two high doses. The IC.sub.50 of peptides yielding positive
inhibition were then determined in subsequent experiments, in which
two to six further dilutions were tested. Under the conditions
utilized, where [label]<[MHC] and IC.sub.50.gtoreq.[MHC], the
measured IC.sub.50 values are reasonable approximations of the true
Kd values. Each competitor peptide was tested in two to four
independent experiments. As a positive control, the unlabeled
version of the radiolabeled probe was also tested in each
experiment.
Example 7
Alternative Binding Assay
[0621] Epstein-Barr virus (EBV)-transformed homozygous cell lines,
fibroblasts, CIR, or 721.22 transfectants were used as sources of
HLA class I molecules. These cells were maintained in vitro by
culture in RPMI 1640 medium supplemented with 2 mM L-glutamine
(GIBCO, Grand Island, N.Y.), 50 .mu.M 2-ME, 100 .mu.g/ml of
streptomycin, 100 U/ml of penicillin (Irvine Scientific) and 10%
heat-inactivated FCS (Irvine Scientific, Santa Ana, Calif.). Cells
were grown in 225-cm.sup.2 tissue culture flasks or, for
large-scale cultures, in roller bottle apparatuses. Cells were
harvested by centrifugation at 1500 RPM using an IEC-CRU5000
centrifuge with a 259 rotor and washed three times with
phosphate-buffered saline (PBS) (0.01 M PO.sub.4, 0.154 M NaCl, pH
7.2).
[0622] Cells were pelleted and stored at -70.degree. C. or treated
with detergent lysing solution to prepare detergent lysates. Cell
lysates were prepared by the addition of stock detergent solution
[1% NP-40 (Sigma) or Renex 30 (Accurate Chem. Sci. Corp., Westbury,
N.Y. 11590), 150 mM NaCl, 50 mM Tris, pH 8.0] to the cell pellets
(previously counted) at a ratio of 50-100.times.10.sup.6 cells per
ml detergent solution. A cocktail of protease inhibitors was added
to the premeasured volume of stock detergent solution immediately
prior to the addition to the cell pellet. Addition of the protease
inhibitor cocktail produced final concentrations of the following:
phenylmethylsulfonyl fluoride (PMSF), 2 mM; aprotinin, 5 .mu.g/ml;
leupeptin, 10 .mu.g/ml; pepstatin, 10 .mu.g/ml; iodoacetamide, 100
.mu.M; and EDTA, 3 ng/ml. Cell lysis was allowed to proceed at
4.degree. C. for 1 hour with periodic mixing. Routinely
5-10.times.10.sup.9 cells were lysed in 50-100 ml of detergent
solution. The lysate was clarified by centrifugation at
15,000.times.g for 30 minutes at 4.degree. C. and subsequent
passage of the supernatant fraction through a 0.2.mu. filter unit
(Nalgene).
[0623] The HLA-A antigen purification was achieved using affinity
columns prepared with mAb-conjugated Sepharose beads. For antibody
production, cells were grown in RPMI with 10% FBS in large tissue
culture flasks (Corning 25160-225). Antibodies were purified from
clarified tissue culture medium by ammonium sulfate fractionation
followed by affinity chromatography on protein-A-Sepharose (Sigma).
Briefly, saturated ammonium sulfate was added slowly with stirring
to the tissue culture supernatant to 45% (volume to volume)
overnight at 4.degree. C. to precipitate the immunoglobulins. The
precipitated proteins were harvested by centrifugation at
10,000.times.g for 30 minutes. The precipitate was then dissolved
in a minimum volume of PBS and transferred to dialysis tubing
(Spectro/Por 2, Mol. wt. cutoff 12,000-14,000, Spectrum Medical
Ind.). Dialysis was against PBS (.gtoreq.20 times the protein
solution volume) with 4-6 changes of dialysis buffer over a 24-48
hour period at 4.degree. C. The dialyzed protein solution was
clarified by centrifugation (10,000.times.g for 30 minutes) and the
pH of the solution adjusted to pH 8.0 with 1N NaOH.
Protein-A-Sepharose (Sigma) was hydrated according to the
manufacturer's instructions, and a protein-A-Sepharose column was
prepared. A column of 10 ml bed volume typically binds 50-100 mg of
mouse IgG.
[0624] The protein sample was loaded onto the protein-A-Sepharose
column using a peristaltic pump for large loading volumes or by
gravity for smaller volumes (<100 ml). The column was washed
with several volumes of PBS, and the eluate was monitored at A280
in a spectrophotometer until base line was reached. The bound
antibody was eluted using 0.1 M citric acid at suitable pH
(adjusted to the appropriate pH with 1N NaOH). For mouse IgG-1 pH
6.5 was used for IgG2a pH 4.5 was used and for IgG2b and IgG3 pH
3.0 was used. 2 M Tris base was used to neutralize the eluate.
Fractions containing the antibody (monitored by A280) were pooled,
dialyzed against PBS and further concentrated using an Amicon
Stirred Cell system (Amicon Model 8050 with YM30 membrane). The
anti-A2 mAb, BB7.2, was useful for affinity purification.
[0625] The HLA-A antigen was purified using affinity columns
prepared with mAb-conjugated Sepharose beads. The affinity columns
were prepared by incubating protein-A-Sepharose beads (Sigma) with
affinity-purified mAb as described above. Five to 10 mg of mAb per
ml of bead is the preferred ratio. The mAb bound beads were washed
with borate buffer (borate buffer: 100 mM sodium tetraborate, 154
mM NaCl, pH 8.2) until the washes show A280 at based line. Dimethyl
pimelimidate (20 mM) in 200 mM triethanolamine was added to
covalently crosslink the bound mAb to the protein-A-Sepharose
(Schneider, et al., J. Biol. Chem. 257:10766 (1982). After
incubation for 45 minutes at room temperature on a rotator, the
excess crosslinking reagent was removed by washing the beads twice
with 10-20 ml of 20 mM ethanolamine, pH 8.2. Between each one the
slurry was placed on a rotator for 5 minutes at room temperature.
The beads were washed with borate buffer and with PBS plus 0.02%
sodium azide.
[0626] The cell lysate (5-10.times.10.sup.9 cell equivalents) was
then slowly passed over a 5-10 ml affinity column (flow rate of
0.1-0.25 ml per minute) to allow the binding of the antigen to the
immobilized antibody. After the lysate was allowed to pass through
the column, the column was washed sequentially with 20 column
volumes of detergent stock solution plus 0.1% sodium dodecyl
sulfate, 20 column volumes of 0.5 M NaCl, 20 mM Tris, pH 8.0, and
10 column volumes of 20 mM Tris, pH 8.0. The HLA-A antigen bound to
the mAb was eluted with a basic buffer solution (50 mM
dimethylamine in water). As an alternative, acid solutions such as
0.15-0.25 M acetic acid were also used to elute the bound antigen.
An aliquot of the eluate (1/50) was removed for protein
quantification using either a colorimetric assay (BCA assay,
Pierce) or by SDS-PAGE, or both. SDS-PAGE analysis was performed as
described by Laemmli (Laemmli, U.K., Nature 227:680 (1970)) using
known amounts of bovine serum albumin (Sigma) as a protein
standard. Allele specific antibodies were used to purify the
specific MHC molecule. In the case of HLA-A2, the mAb BB7.2 was
used.
[0627] A detailed description of the protocol utilized to measure
the binding of peptides to Class I HLA molecules has been published
(Sette, et al., Mol. Immunol. 31:813, 1994; Sidney, et al., in
Current Protocols in Immunology, Margulies, Ed., John Wiley &
Sons, New York, Section 18.3, 1998). Briefly, purified MHC
molecules (5 to 500 nM) were incubated with various unlabeled
peptide inhibitors and 1-10 nM .sup.125I-radiolabeled probe
peptides for 48 h in PBS containing 0.05% Nonidet P-40 (NP40) (or
20% w/v digitonin for H-2 IA assays) in the presence of a protease
inhibitor cocktail. The final concentrations of protease inhibitors
(each from CalBioChem, La Jolla, Calif.) were 1 mM PMSF, 1.3 nM
1.10 phenanthroline, 73 .mu.M pepstatin A, 8 mM EDTA, 6 mM
N-ethylmaleimide, and 200 .mu.M N alpha-p-tosyl-L-lysine
chloromethyl ketone (TLCK). All assays were performed at pH
7.0.
[0628] Following incubation, MHC-peptide complexes were separated
from free peptide by gel filtration on 7.8 mm.times.15 cm TSK200
columns (TosoHaas 16215, Montgomeryville, Pa.), eluted at 1.2
mls/min with PBS pH 6.5 containing 0.5% NP40 and 0.1% NaN.sub.3.
The eluate from the TSK columns was passed through a Beckman 170
radioisotope detector, and radioactivity was plotted and integrated
using a Hewlett-Packard 3396A integrator, and the fraction of
peptide bound was determined.
[0629] Radiolabeled peptides were iodinated using the chloramine-T
method. A specific radiolabeled probe peptide was utilized in each
assay. Typically, in preliminary experiments, each MHC preparation
was titered in the presence of fixed amounts of radiolabeled
peptides to determine the concentration of HLA molecules necessary
to bind 10-20% of the total radioactivity. All subsequent
inhibition and direct binding assays were performed using these HLA
concentrations.
[0630] Since under these conditions [label]<[HLA] and
IC.sub.50>[HLA], the measured IC.sub.50 values are reasonable
approximations of the true K.sub.D values. Peptide inhibitors are
typically tested at concentrations ranging from 120 .mu.g/ml to 1.2
ng/ml, and are tested in two to four completely independent
experiments. To allow comparison of the data obtained in different
experiments, a relative binding figure is calculated for each
peptide by dividing the IC.sub.50 of a positive control for
inhibition, i.e. the reference peptide that is included in every
binding assay, by the IC.sub.50 for each tested peptide (typically
unlabeled versions of the radiolabeled probe peptide). For database
purposes, and inter-experiment comparisons, relative binding values
are compiled. These values can subsequently be converted into
normalized IC.sub.50 nM values by dividing the standard historical
IC.sub.50 of the reference peptide by the relative binding of the
peptide of interest. This method of data compilation has proven to
be the most accurate and consistent for comparing peptides that
have been tested on different days, or with different lots of
purified MHC.
[0631] For example, the standard reference peptide (or positive
control) for the HLA-A2.1 binding assays described herein is the
peptide having a sequence of FLPSDYFPSV (SEQ ID NO:______), which
has an average historical IC.sub.50 value of 5 nM in multiple,
repeated binding assays. This standard value is used to normalize
reported IC.sub.50 values for HLA-A2.1 binding as described herein.
Thus, a relative binding value of a test HLA-A2.1 motif-bearing
peptide can be converted into a normalized IC.sub.50 by dividing
the standard reference IC.sub.50 value, i.e., 5 nM, by the relative
binding value of the test HLA-A2.1 motif-bearing peptide.
Example 8
Sequence and Binding Analysis
[0632] Using the assay described in Example 4, a relative binding
value was calculated for each peptide by dividing the IC.sub.50 of
the positive control for inhibition by the IC.sub.50 for each
tested peptide. These values can subsequently be converted back
into IC.sub.50 nM values by dividing the IC.sub.50 nM of the
positive controls for inhibition by the relative binding of the
peptide of interest. This method of data compilation has proved to
be accurate and consistent for comparing peptides that have been
tested on different days or with different lots of purified MHC.
Standardized relative binding values also allow the calculation a
geometric mean, or average relative binding value (ARB), for all
peptides with a particular characteristic (Ruppert, J., et al.,
"Prominent Role of Secondary Anchor Residues in Peptide Binding to
HLA-A2.1 Molecules," Cell 74:929-937 (1993); Sidney, J., et al.,
"Definition of an HLA-A3-Like Supermotif Demonstrates the
Overlapping Peptide Binding Repertoires of Common HLA Molecules,"
Hum Immunol. 45:79-93 (1996); Sidney, J., et al., "Specificity and
Degeneracy in Peptide Binding to HLA-B7-Like Class I Molecules," J.
Immunol. 157:3480-3490 (1996); Kondo, A., et al., "Prominent Roles
of Secondary Anchor Residues in Peptide Binding to HLA-A24 Human
Class I Molecules," J. Immunol. 155:4307-4312 (1995); Kondo, A., et
al., "Two Distinct HLA-A*0101-Specific Submotifs Illustrate
Alternative Peptide Binding Modes," Immunogenetics 45:249-258
(1997); Gulukota, K., et al., "Two Complementary Methods for
Predicting Peptides Binding Major Histocompatibility Complex
Molecules," J. Mol. Biol. 267:1258-1267 (1997); Southwood, S., et
al., "Several Common HLA-DR Types Share Largely Overlapping Peptide
Binding Repertoires," J. Immunol 160:3363-3373 (1998)).
[0633] Maps of secondary interactions influencing peptide binding
to HLA-A2 supertype molecules based on ARB were derived as
previously described (Ruppert, J. et al., "Prominent Role of
Secondary Anchor Residues in Peptide Binding to HLA-A2.1
Molecules," Cell 74:929-937 (1993); Sidney, J., et al., "Definition
of an HLA-A3-Like Supermotif Demonstrates the Overlapping Peptide
Binding Repertoires of Common HLA Molecules," Hum Immunol. 45:79-93
(1996); Sidney, J., et al., "Specificity and Degeneracy in Peptide
Binding to HLA-B7-Like Class I Molecules," J. Immunol.
157:3480-3490 (1996); Kondo, A., et al., "Prominent Roles of
Secondary Anchor Residues in Peptide Binding to HLA-A24 Human Class
I Molecules," J. Immunol. 155:4307-4312 (1995); Kondo, A., et al.,
"Two Distinct HLA-A*0101-Specific Submotifs Illustrate Alternative
Peptide Binding Modes," Immunogenetics 45:249-258 (1997); Gulukota,
K., et al., "Two Complementary Methods for Predicting Peptides
Binding Major Histocompatibility Complex Molecules," J. Mol. Biol.
267:1258-1267 (1997)). Essentially, all peptides of a given size
(8, 9, 10 or 11 amino acids) and with at least one tolerated main
anchor residue were selected for analysis. The binding capacity of
peptides in each size group was analyzed by determining the ARB
values for peptides that contain specific amino acid residues in
specific positions. For determination of the specificity at main
anchor positions ARB values were standardized relative to the ARB
of peptides carrying the residue associated with the best binding.
For secondary anchor determinations, ARB values were standardized
relative to the ARB of the whole peptide set considered. That is,
for example, an ARB value was determined for all 9-mer peptides
that contain A in position 1, or F in position 7, etc. Because of
the rare occurrence of certain amino acids, for some analyses
residues were grouped according to individual chemical similarities
as previously described (Ruppert, J. et al., supra; Sidney, J., et
al., supra; Sidney, J., et al., supra; Kondo, A., et al., supra;
Kondo, A., et al., supra; Gulukota, K., et al., supra; Southwood,
S., et al., supra).
Frequencies of HLA-A2-Supertype Molecules
[0634] To select a panel of A2-supertype molecules representative
of the allelic forms most frequent in major ethnic groups,
unpublished population typing data from D. Mann and M.
Fernandez-Vina were utilized. These data were consistent with
published data (Sudo, T., et al., "DNA Typing for HLA Class I
Alleles: I. Subsets of HLA-A2 and of -A28," Hum. Immunol.
33:163-173 (1992); Ellis, J. M., et al., "Frequencies of HLA-A2
alleles in Five US Population Groups," Hum. Immunol. 61:334-340
(2000); Krausa, P., et al., "Genetic Polymorphism Within HLA-A*02:
Significant Allelic Variation Revealed in Different Populations,"
Tissue Antigens 45:233-231 (1995) and Imanishi, T., et al., "Allele
and Haplotype Frequencies for HLA and Complement Loci in Various
Ethnic Groups" Tsuji, K., et al., (eds): HLA 1991, Proceedings of
the Eleventh International Histo-Compatibility Workshop and
Conference, Vol. 1, Oxford University Press, Oxford, pp. 1065-1220
(1992)), and are shown in TABLE 38. For the four major ethnic
groups considered, it was apparent that seven HLA alleles represent
the vast majority of A2 supertype alleles. Included in this group
are A*0201, A*0202, A*0203, A*0205, A*0206, A*0207, and A*6802.
Each of these alleles is present in 2% or more of the general
population, and also occur with a frequency greater than 5% in at
least one major ethnicity. Other alleles are represented with only
minor frequencies of 1.3%, or less, in any one major ethnic group.
Furthermore, none of the minor alleles are present with a frequency
greater than 1% in the general population. Based on these
observations, A*0201, A*0202, A*0203, A*0205, A*0206, A*0207, and
A*6802 were selected for studies defining peptide binding
specificity and cross-reactivity in the A2-supertype.
Main Anchor Positions of A2 Supertype Molecules
[0635] Previous studies indicated a largely overlapping peptide
binding specificity for a set of Class I molecules designated as
the A2-supertype. Here, the main peptide binding specificity of
A2-supertype molecules was examined in more detail. Some of these
results have been published previously, and are shown here only for
reference purposes (Ruppert, J., et al., supra and Sidney, J., et
al., "The HLA-A*0207 Peptide Binding Repertoire is Limited to a
Subset of the A*0201 Repetoire," Hum. Immunol., 58:12-20
(1997)).
[0636] In a first series of studies, non-conservative lysine (K)
substitutions were introduced at every position of two peptides
previously noted to bind multiple A2-supertype molecules: 1) the
HCV NS3 590 9-mer peptide (sequence YLVAYQATV (SEQ ID NO:______)),
and 2) the HBV core 18 F.sub.6>Y10-mer analog peptide (sequence
FLPSDYFPSV (SEQ ID NO:______)). These peptides were tested for
their capacity to bind A*0201, A*0202, A*0203, A*0205, A*0206,
A*0207 and A*6802. In TABLE 39 and TABLE 40, binding capacities are
expressed as ratios relative to the parent peptide. Peptides whose
binding capacities are within 10-fold of the best binder are
considered preferred; those whose relative binding capacities are
10-100-fold less than the best binder are considered tolerated. A
dash ("-") indicates a relative binding of less than 0.01. In the
case of the HCV NS3 590 peptide (TABLE 39), K substitutions at
position 2 and the C-terminus resulted in greater than 100-fold
reduction in binding to each HLA molecule. Greater than 100-fold
decreases in binding were also noted in the context of A*6802 when
K was substituted in positions 1 and 5. Reductions in binding
capacity in the 10-100-fold range were noted when substitutions
were made at several other positions, notably positions 3 and 7.
When the 10-mer HBV core 18 F.sub.6>Y ligand (TABLE 40) was
investigated, greater than 100-fold reductions in binding capacity
were again noted when the peptide was substituted at position 2 and
the C-terminus. Significant reductions in binding were also
observed following substitution at position 7.
[0637] Together, these data suggest that A2-supertype molecules
bind both 9- and 10-mer peptide ligands via anchor residues in
position 2 and at the C-terminus. The presence of an additional
primary or secondary anchor towards the middle of the peptide is
demonstrated by the fact that the binding of both the 9-mer and
10-mer peptides was usually reduced by substitutions at position 7.
Specificity of the Position 2 and C-Terminal Anchor Residues.
[0638] Based on these results, the ligand specificity of
A2-supertype molecules at position 2 and the C-terminus was
analyzed using additional HCV NS3 590 and HBV core 18 F.sub.6>Y
single substitution analogs, and also single substitution analogs
of a poly-alanine peptide (peptide 953.01; sequence ALAKAAAAV (SEQ
ID NO:______)). For these analyses, preferred amino acids for
anchor residues were defined as those associated with a binding
capacity within 10-fold of the optimal residue. Amino acids whose
relative binding capacity was between 0.01 and 0.1 were defined as
tolerated, and those associated with a binding capacity less than
0.01 were considered as non-tolerated. In the accompanying tables,
a dash ("-") indicates a relative binding of less than 0.01.
Binding capacities are expressed as ratios relative to the related
analog with the highest binding affinity for each individual
molecule.
[0639] At position 2 small aliphatic and hydrophobic residues were
found to be generally tolerated, while other residues, including
large polar, aromatic, and charged residues were typically not well
tolerated (TABLE 41, TABLE 42, and TABLE 43). L, I, V, and M were
preferred as anchor residues in most (>80%) contexts (TABLE 44).
The allele/peptide combinations in Table 44 refer to the number of
instances in which a given residue was associated with a relative
binding in the 1-0.1 range (preferred) or 0.1-0.01 range
(tolerated). A, T, Q, and S were less frequently preferred as
anchor residues, but were either preferred or tolerated in >80%
of the contexts examined (TABLE 44). None of the other amino acids
examined were preferred in any context and only rarely tolerated
(residues, but were either preferred or tolerated in >80% of the
contexts examined. None of the other amino acids examined were
preferred in any context and only rarely tolerated).
[0640] At the C-terminus, V was found to be the optimal residue in
the context of all 3 parent peptides for A*0201, A*0206, and
A*6802, and in 2 out of 3 cases for A*0203 and A*0205 (TABLE 45,
TABLE 46, and TABLE 47). Overall, either V or L was the optimal
C-terminal residue for each molecule, regardless of the peptide
tested. The allele/peptide combinations in Table 48 refer to the
number of instances in which a given residue was associated with a
relative binding in the 1-0.1 range (preferred) or 0.1-0.01 range
(tolerated). The aliphatic/hydrophobic amino acids V, L, and I were
preferred as anchor residues in greater than 66.7% of the
MHC-peptide contexts. M, A, and T were tolerated approximately 50%
of the time. Other residues examined were either not accepted at
all, or were tolerated only rarely.
A Re-Evaluation of the Peptide Binding Specificity of A*0201
[0641] The fine specificity of A*0201 binding was investigated in
more detail using a database of over 4000 peptides between 8- and
11-residues in length. It was found that over 30% of the peptides
bearing L or M in position 2 bound A*0201 with affinities of 500
nM, or better (FIG. 1a). Between 5 and 15% of the peptides bearing
the aliphatic residues I, V, A, T, and Q bound with IC.sub.50s of
500 nM, or better. No other residue, including aromatic (F, W, and
Y), charged (R, H, K, D, and E), polar (S and N) and small (C, G,
and P) residues, was associated with IC.sub.50s of 500 nM, or
better.
[0642] Consistent with the single substitution analysis, V was
found to be the optimal A*0201 C-terminal anchor residue (FIG. 1b).
Overall, 31.9% of the peptides with V at the C-terminus were A*0201
binders. I, L, S, C, M, T and A were also tolerated, with 7.1 to
28.6% of the peptides binding with an IC.sub.50 of 500 nM, or
better.
[0643] The correlation between peptide length (between 8 and 11
residues) and binding capacity was analyzed next. It was found that
27.6% of the 9-mer peptides bound with IC.sub.50 of 500 nM, or
less, in good agreement with previous estimates (Ruppert, J., et
al., supra) (TABLE 49). ARB values are standardized to the peptide
set of optimal size and shown for reference purposes.
[0644] Longer peptides were also capable of binding, although
somewhat less well; 17.8% of 10-mer, and 14.5% of the 11-mer
peptides had affinities of 500 mM or better. Finally, it was noted
that 8-mer peptides bound A*0201 only rarely, with 3.5% of the
peptides having binding capacities better than 500 nM.
[0645] The A*0201 peptide binding database was further analyzed to
assess the stringency of most frequently (48.7%), and with higher
average relative binding capacity than other peptides in the
library (TABLE 50). Peptides with one preferred residue and one
tolerated residue also bound relatively frequently, in the 17.6 to
28.4% range. Finally, peptides with at least one non-tolerated
residue, or with tolerated residues at both main anchor positions,
bound only rarely, if at all, with frequencies of binding in the
0-7.1% range. No significant difference was detected in terms of
primary anchor preferences as a function of ligand size.
[0646] To identify secondary anchor effects, the A*0201 binding
capacity of peptides in each size group was further analyzed by
determining the ARB values for peptides that contain a particular
amino acid residue in a specific, but size dependent, position. The
resulting ARB values, by corresponding residue/position pairs, for
8-11-mer sequences are shown in TABLE 51, TABLE 52, TABLE 53, and
TABLE 54. All of the peptides in TABLE 51, TABLE 52, TABLE 53, and
TABLE 54 had at least 1 preferred and 1 tolerated residue at the
main anchor positions. At secondary anchor positions values
corresponding to a 3-fold or greater increase in binding capacity
are indicated by increased and bolded font. Negative effects,
associated with a three-fold decrease in binding affinity, are
identified by underlined and italicized font. Also, residues
determined to be preferred or tolerated anchors are indicated by
bold font. ARB values at the anchor positions were derived from the
analyses described in FIG. 5. To allow use of the values shown in
this table as coefficients for predictive algorithms, the values
for non-tolerated anchor residues have been set to 0.001,
equivalent to a 1000-fold reduction in binding capacity, to filter
out non-motif peptides.
[0647] In TABLE 51, TABLE 52, TABLE 53, and TABLE 54, the results
of the analysis of a panel of 93 8-mer peptides, 1389 9-mer
peptides, 953 10-mer peptides, and 95 11-mer peptides,
respectively, are based on naturally occurring sequences from
various viral, bacterial, or pathogen origin. ARB values shown were
calculated, for example, as described in Sidney et al., Human
Immunology 62: 1200 (2001) and Sidney et al., J. Immunology 157:
3480 (1996). For 9-mer and 10-mer peptides ARB values were derived
for each residue considered individually. For studies of 8-mer and
11-mer peptides (TABLE 51 and TABLE 54, respectively) ARB values
were based on the grouping of chemically similar residues, as
described in Ruppert et al., Cell 74: 929 (1993). The average
geometric binding capacity of the 8-mer, 9-mer, 10-mer, and 11-mer
panels was 14420 nM, 1581 nM, 3155 nM, and 3793 nM,
respectively.
[0648] Summary maps are shown in FIGS. 6A-6D. In most positions,
some secondary influence could be detected. The majority (55%) of
the negative influences involved the presence of acidic (D and E)
or basic (R, H, and K) residues. Proline (P) and large polar
residues (Q, and N) were also frequently disruptive. While each
particular size was associated with unique preferences, in most
instances (79%) preferred residues were aromatic (F, W, or Y) or
hydrophobic (L, I, V, or M). Most peptide lengths exhibited a
preference for F, Y and M in position 3. Similarly, all peptide
sizes shared a preference for aromatic or hydrophobic residues in
the C-2 position.
[0649] Several distinct preference patterns were also observed for
peptides of a given size. For example, 8-mer peptides did not have
any preference in either position 1 or position 3 for the
hydrophobic or aromatic residues preferred by 9-, 10-, and 11-mer
peptides. 11-mer peptides were unique in the preference for G in
multiple positions throughout the middle of the peptide.
Main Anchor Specificities of Other A2-Supertype Molecules
[0650] In the next set of analyses, the main anchor specificities
of A*0202, A*0203, A*0206, and A*6802, four of the most prevalent
A2-supertype alleles next to A*0201, was assessed. Peptides in the
A2-supertype binding database often reflect selection using an
A*0201-based bias, such as the selection of only A*0201 binding
peptides, or the selection of peptides scoring high in A*0201
algorithms. As a result, in most cases, peptide binding data for
non-A*0201 molecules is available for only peptides with supertype
preferred and tolerated residues. Despite this limitation, a
database of about 400 peptides was available for study. A database
of sufficient size was not available to allow analysis of A*0205
and A*0207, although an analysis of the specificity of A*0207 has
been published previously (Sidney, J., et. al., supra).
[0651] Analyses of the position 2 specificities are summarized in
FIG. 3a-d. In general, V, T, A, I, and M were tolerated in the
context of each molecule. Allele specific preferences were also
noted. In the case of A*0202 Q was the most preferred residue.
Other residues (L, I, V, A, T and M) were tolerated, and were
roughly equivalent, with ARB in the 0.08-0.30 range. By contrast,
A*0203 had a preference for L, M and Q. Residues V, A, I and T were
associated with lower overall binding affinities. A third pattern
was noted for A*0206, where Q, V, I, A, and T were all well
tolerated with ARB values between 0.47 and 1.0, while L and M were
less well tolerated. Finally, for A*6802 V and T were the optimal
residues, with ARB >0.45. A was also preferred, but with a lower
ARB (0.13). Significant decreases in binding were seen with I and
M, which had ARB between 0.050 and 0.020. L and Q were not
tolerated, with ARB <0.010.
[0652] At the C-terminus, I, V, L, A, M and T were tolerated by all
A2-supertype molecules tested, with ARB >0.060 (FIG. 4a-d). I
and V were the two residues most preferred by each allele; V was
the optimal residue for A*0203, A*0206, and A*6802. L was typically
the next most preferred residue. T, A, and M were usually
associated with lower ARB values.
[0653] In conclusion, the position 2 and C-terminal anchor residues
preferred or tolerated by A*0201 were also well tolerated by other
A2-supertype molecules. While each allele had a somewhat unique
pattern of preferences at position 2, the patterns of preferences
exhibited by each allele at the C-terminus were fairly similar.
Secondary Influences on Peptide Binding to A2-Supertype
Molecules
[0654] The same library of peptide ligands was analyzed to
determine the ligand size preferences of A*0202, A*0203, A*0206,
and A*6802. Fore each allele, ARB values are standardized to the
peptide set of optimal size. We found that for each molecule 9-11
mer peptides were well tolerated, with ARB >0.36 (TABLE 55,
TABLE 56, TABLE 57, and TABLE 58). For A*0203, A*0206, and A*6802,
9-mer peptides were optimal, but 10-mers were optimal in the case
of A*0202. For all alleles, 8-mer peptides were much less well
tolerated, with ARB in each case <0.11. 3
[0655] The influence of secondary anchor residues on the capacity
of peptides to bind A*0202, A*0203, A*0206, and A*6802 was examined
next. The number of peptides available only allowed analysis of 9-
and 10-mer ligands. The ARB values for 9-mer and 10-mer peptides as
a function of the presence of a particular residue in a specific
position are shown in TABLES 59-66, and summary maps in FIG. 9,
FIG. 10, FIG. 11, and FIG. 12. As noted above, positive and
negative effects are defined as associated with three-fold or
greater increases or decreases in binding affinity,
respectively.
[0656] In TABLE 59 and TABLE 60, a panel of 268 9-mer peptides and
a panel of 120 10-mer peptides, respectively, were tested for
binding to the A*0202 allele. In TABLE 61 and TABLE 62, a panel of
272 9-mer peptides and a panel of 122 10-mer peptides,
respectively, were tested for binding to the A*0203 allele. In
TABLE 63 and TABLE 64, a panel of 268 9-mer peptides and a panel of
120 10-mer peptides, respectively, were tested for binding to the
A*0206 allele. In TABLE 65 and TABLE 66, a panel of 268 9-mer
peptides and a panel of 120 10-mer peptides, respectively, were
tested for binding to the A*6802 allele. All peptides were based on
naturally occurring sequences from various viral, bacterial, or
pathogen origin and had at least 1 preferred and 1 tolerated
residue at the main anchor positions. ARB values are based on the
grouping of chemically similar residues, generally as described in
Ruppert et al., Cell 74: 929 (1993), for example. At secondary
anchor positions values corresponding to a 3-fold or greater
increase in binding capacity are indicated by bolded and increased
font. Negative effects, associated with a three-fold decrease in
binding affinity, are indicated by underlined and italicized font.
Also, residues determined to be preferred or tolerated anchors are
indicated by bold font. To allow use of the values shown in this
table as coefficients for predictive algorithms, the values for
non-tolerated anchor residues were set to 0.001, equivalent to a
1000-fold reduction in binding capacity, to filter out non-motif
peptides. The average geometric binding capacity of each panel in
TABLE 59, TABLE 60, TABLE 61, TABLE 62, TABLE 63, TABLE 64, TABLE
65, and TABLE 66 was 401 nM, 342 nM, 85 nM, 95 nM, 387 nM, 643 nM,
838 nM, and 1055 nM, respectively.
[0657] In general, deleterious effects were frequently (35%)
associated with charged residues (D, E, R, H, or K). An additional
35% of the deleterious influences could be attributed to G or P.
Positive influences were relatively evenly attributed to basic (R,
H, K), acid (D, E), hydrophobic (F, W, Y, L, I, V, M) or small (A,
P) residues.
[0658] While each molecule had a distinctive pattern of preferences
and aversions, some common trends could be noted in the case of
10-mer peptides. For example, for all molecules Q and N were
preferred in position 1, and R, H, and K were preferred in position
8. D, E, and G were uniformly deleterious for 10-mer peptides in
position 3. Consensus preferences or aversions were not noted for
9-mer peptides.
[0659] In summary, the data in this section describe detailed
motifs for 9- and 10-mer peptides binding to A*0202, A*0203,
A*0206, and A*6802. Each motif is characterized by specific
features associated with good, or poor, binding peptides.
A Consensus A2-Supermotif
[0660] How well A*0201 binders also bound to other A2-supertype
molecules was assessed next. It was found that peptides that bound
A*0201 with good affinity (IC.sub.50<500 nM) frequently bound
other A2-supertype molecules (TABLE 67). Between 36.1 and 73.6% of
A*0201 binding peptides bound other A2-supertype molecules.
Analysis of A2-supertype degeneracy as a function of A*0201
affinity also yielded interesting results. The motifs described
above for A2 supertype molecules are very similar and largely
overlapping. In this respect, a consensus motif can be identified
that incorporates features commonly shared by the molecule-specific
motifs (FIG. 9). The consensus motif specifies the presence of
hydrophobic and aliphatic residues in position 2 of peptide
ligands. At this position, V, L and M are preferred, while T, Q, A,
and I are all tolerated. On the basis of the preference rank of
each residue in the context of each A2-supertype molecule, V is the
most preferred residue. At the C-terminus the consensus motif
specifies the presence of hydrophobic and aliphatic residues L, I,
V, M, A, and T. V is most frequently the optimal residue, while L
and I are also considered preferred, typically being the next most
optimal residues. M, A, and T are considered as tolerated
residues.
[0661] The secondary anchor maps for A*0201, A*0202, A*0203,
A*0206, and A*6802 were utilized to derive a supertype consensus
secondary anchor motif for 9- and 10-mer peptides (FIG. 9).
Residues considered as preferred for 3 or more A2-supertype
molecules, without being deleterious for any molecule, were
considered as preferred for the supertype consensus motif.
Conversely, residues identified as deleterious for 3 or more
molecules were designated as deleterious in the consensus motif.
The consensus motif overlaps significantly with the detailed A*0201
motif, and includes a preference for aromatic residues in position
1 and/or 3, and a shared aversion for charged residues in position
3.
Correlation Between A*0201 Binding Affinity and A2-Supertype
Cross-Reactivity
[0662] Because of the dominance in four major ethnicities of A*0201
compared with other A2 supertype alleles (see, e.g., TABLE 38), it
was of interest to determine how well A*0201 binders also bound to
other A2-supertype molecules. It was found that peptides that bound
A*0201 with good affinity (IC.sub.50<500 nM) frequently bound
other A2-supertype molecules (TABLE 67). Between 36.1 and 73.6% of
A*0201 binding peptides bound other A2-supertype molecules.
Analysis of A2-supertype degeneracy as a function of A*0201
affinity also yielded interesting results. 72.8% of the peptides
that bound A*0201 with IC.sub.50<500 nM bound 3 or more
A2-supertype molecules (TABLE 68). As a general rule, the higher
the binding affinity of a peptide for A*0201, the higher the
likelihood that the peptide would also bind 3 or more supertype
molecules. Over 96% of the peptides that bound A*0201 with
affinities of 20 nM or better also bound 3 or more A2-supertype
molecules. By contrast, A2-supermotif peptides that did not bind
A*0201 with affinities better than 500 nM only rarely (10%) bound 3
or more A2 supermotif molecules, and never bound 4 or more
molecules.
[0663] In summary, this analysis of the cross-reactive binding of
peptides to A*0201 and other A2-supertype molecules confirms the
fact that this family of HLA molecules recognizes similar
structural features in their peptide ligands. It has also been
shown that A*0201 binding affinity correlates with the propensity
to bind multiple A2-supertype alleles.
Analysis.
[0664] The results of this analysis allow for the detailed
definition of the properties of peptides that bind to HLA-A*0201
and other A2-supertype molecules. The A2-supertype molecules share
not only largely overlapping peptide binding specificity, but also
significantly overlapping peptide binding repertoires. Specific
features of peptide ligands associated with degenerate A2-supertype
binding capacity were identified which provide a logical
explanation for the supertype relationship.
[0665] In a previous study the peptide binding specificity of
A*0201 was analyzed, and a detailed motif, including the
identification of secondary anchor features, was constructed. In
the present analyses, performed with a 10-fold larger database, we
confirmed that data and extended the analysis to include 8- and
11-mer peptides. Overall, the specificity of A*0201 for 8- and
11-mer peptides was largely similar to that for 9- and 10-mer
peptides. For example, regardless of peptide size, the majority of
negative influences on binding capacity were associated with the
presence of charged residues in secondary anchor positions, while
the majority of positive influences were associated with the
presence of hydrophobic residues. The definition of detailed motifs
for 8- and 11-mer peptides should allow for a more complete
identification of epitopes. Identification of A*0201 binders has
been greatly facilitated by the use of the algorithms based on ARB
values. In the present analyses a substantially larger database was
used than previously available, allowing for a refinement of
algorithm coefficients. Because the newer coefficients are based on
a significantly larger data set, they are statistically more
accurate and should afford more efficient and precise prediction of
epitopes. Indeed, recent analysis has shown that a revised A*0201
9-mer polynomial algorithm based on a larger data set is more
accurate than both an older algorithm based on a small data set,
and neural network prediction methodologies. In addition to
increasing the accuracy of epitope prediction (Ruppert, J., et al.,
supra; Sidney, J., et al., supra; Kondo, A., et al., supra;
Gulukota, K., et al., supra; Parker, K. C., et al., "Sequence
Motifs Important for Peptide Binding to the Human MHC Class I
Molecule, HLA-A2," J. Immunol. 149:3580-3587 (1992) and Milik, M.,
et al., "Application of an Artificial Neural Network to Predict
Specific Class I MHC Binding Peptide Sequences," Nature (Biotech)
16:753-756 (1998)), detailed peptide binding motifs defining both
primary and secondary anchor positions allow for the rational
design of optimized ligands. For example, natural sequences
carrying sub-optimal residues at primary and/or secondary positions
can be identified. The sub-optimal residues may be replaced with
optimal anchors, generating epitopes with increased binding
affinity (Sidney, J., et al., supra; Pogue, R. R., et al.,
"Amino-Terminal Alteration of the HLA-A*0201-Restricted Human
Immunodeficiency Virus Pol Peptide Increases Complex Stability and
in Vitro Immunogenicity," Proc. Nat'l. Acad. Sci., USA,
92:8166-8170 (1995) and Bakker, A. B., et al., "Analogues of CTL
epitopes With Improved MHC Class-I Binding Capacity Elicit
Anti-Melanoma CTL Recognizing the Wide-Type Epitope," Int. J.
Cancer, 70:302-309 (1997)). Following this type of modification,
wild type peptides that were unable to elicit responses, or were
poor immunogens, may become highly immunogenic Pogue, R. R., et
al., supra; Bakker, A. B., et al., supra; Parkhurst, M. R.,
"Improved Induction of Melanoma-Reactive CTL With Peptides From the
Melanoma Antigen gp100 Modified at HLA-A*0201-Binding Peptides," J.
Immunol. 157:2539-2548 (1996); Rosenberg, S. A., et al.,
"Immunologic and Therapeutic Evaluation of a Synthetic Peptide
Vaccine for the Treatment of Patients With Metastatic Melanoma,"
Nature (Med) 4:321-327 (1998); Sarobe, P., et al., "Enhanced in
vitro Potency and in vivo Immunogenicity of a CTL Epitope From
Hepatitis C Virus Core Protein Following Amino Acid Replacement at
Secondary HLA-A2.1 binding positions," J. Clin. Invest.
102:1239-1248 (1998) and Ahlers, J. D., et al., "Enhanced
Immunogenicity of HIV-1 Vaccine Construct by Modification of the
Native Peptide Sequence," Proc. Nat'l Acad. Sci., USA,
94:10856-10861 (1997)). The CTL induced by such analog peptides
have been shown to be capable, in most instances, of recognizing
target cells expressing wild type antigen sequences. This
phenomenon is likely to reflect less stringent epitope binding
requirements for target cell recognition compared to that needed
for stimulation of naive T-cells to induce differentiation into
effectors (Cho, B. K., et al., "Functional Differences Between
Memory and Naive CD8 T Cells," Proc. Nat'l. Acad. Sci. USA
96:2976-2981 (1999); Sykulev, Y., et al., "Evidence That A Single
Peptide--MHC Complex On A Target Cell Can Elicit Acytolytic T Cell
Response," Immunity 4:565-571 (1996)). Thus, the detailed motifs
described herein will facilitate not only in the identification of
naturally occurring CTL epitopes, but also in the design of
engineered epitopes with increased binding capacity and/or
immunogenic characteristics.
[0666] The peptide binding specificity for other A2-supertype
molecules was also investigated using single substitution analog
peptides and peptide libraries. In agreement with previous reports
(del Guercio, M-F, et al., "Binding of a Peptide Antigen to
Multiple HLA Alleles Allows Definition of an A2-Like Supertype," J.
Immunol. 154:685-693 (1995) and (Sidney, J., et al., "Practical,
Biochemical and Evolutionary Implications of the Discovery of HLA
Class I Supermotifs," Immunol Today 17:261-266 (1996)); see also
reports filed for NIH-NIAID contract NO1-AI-45241), we found that
the primary anchor motifs of A2-supertype molecules were remarkably
similar. The use of peptide libraries allowed detailed
characterization of the secondary anchor preferences and aversions
of each molecule. It was shown that, while each A2-supertype
molecule had a unique specificity, a supermotif based on consensus
patterns could be identified. Because the supermotif describes
features of peptide ligands that are shared amongst A2-supertype
molecules, it is expected to allow the efficient identification of
highly cross-reactive peptides, and indicate appropriate strategies
for anchor fixing, allowing modulation of the supertype degeneracy
of peptide ligands. A further result of the present analysis was
the derivation of coefficients that could be utilized in algorithms
for predicting peptide binding to A*0202, A*0203, A*0206, and
A*6802.
[0667] As HLA A*0201 is by far the most prevalent A2-supertype
allele, both in the general population and within major ethnic
groups, the peptide screening strategy that was utilized focused
first on the identification of A*0201 binders. It was determined
that over 70% of the peptides that bind to A*0201 also bind to at
least 2 additional A2-supertype molecules, and that the propensity
to bind other A2-supertype alleles correlated with A*0201 binding
affinity.
[0668] In conclusion, the data described herein provide formal
demonstration of the shared peptide binding specificity of a group
of HLA-A molecules designated as the A2-supertype. Not only do
these molecules recognize similar features at primary and secondary
anchor positions of their peptide ligands, they also share largely
overlapping peptide binding repertoires. The demonstration that
these molecules share largely overlapping repertoires has a
significant implication for the design of potential vaccine
constructs. Indeed, the concept that A2-supertype cross-reactivity
at the peptide binding level may be of immunological relevance has
been demonstrated in a number of studies, in both infectious
disease (Khanna R., et al., "Identification of Cytotoxic T-Cell
Epitopes Within Epstein-Barr Virus (EBV) Oncogene Latent Membrane
Protein 1 (LMP1): Evidence for HLA A2 Supertype-Restricted Immune
Recognition of EBV-Infected Cells by LMP 1-Specific Cytotoxic T
lymphocytes," Eur J Immunol, 28:451-458 (1998); Bertoletti, A., et
al., "Molecular Features of the Hepatitis B Virus Nucleocapsid
T-Cell Epitope 18-27: Interaction With HLA An T-Cell Receptor,"
Hepatology 26:1027-1034 (1997); Livingston, B. D., et al.,
"Immunization With the HBV Core 18-27 Epitope Elicits CTL Responses
in Humans Expressing Different HLA-A2 Supertype Molecules," Hum
Immunol 60:1013-1017, (1999); Bertoni, R., et al., "Human
Histocompatibility Leukocyte Antigen-Binding Supermotifs Predict
Broadly Cross-Reactive Cytotoxic T Lymphocyte Responses in Patients
With Acute Hepatitis," J Clin Invest 100:503-513 (1997); and
Doolan, D. L., et al., "Degenerate Cytotoxic T-Cell Epitopes from
P. falciparum Restricted by Multiple HLA-A and HLA-B Supertype
Alleles," Immunity 7:97-112 (1997)) and cancer (Fleischhauer, K.,
et al., "Multiple HLA-A Alleles Can Present an Immunodominant
Peptide of the Human Melanoma Antigen Melan-A/MART-1 To A
Peptide-Specific HLA-A*0201+Cytotoxic Cell Line," J Immunol, 157:
787-797 (1996); Rivoltini, L., et al., "Binding and Presentation of
Peptides Derived From Melanoma Antigens MART-1 and Glycoprotein-100
by HLA-A2 Subtypes: Implications for Peptide-Based Immunotherapy,"
J Immunol 156:3882-3891 (1996); Kawashima, I., "The Multi-Epitope
Approach for Immunotherapy for Cancer: Identification of Several
CTL Epitopes from Various Tumor-Associated Antigens Expressed on
Solid Epithelial Tumors," Hum Immunol 59:1-14 (1998)) settings.
Example 9
Peptide Composition for Prophylactic Uses
[0669] Vaccine compositions of the present invention are used to
prevent infection or treat cancer in persons. For example, a
polyepitopic peptide epitope composition containing multiple CTL
and HTL epitopes is administered to individuals at risk for HCV
infection. The composition is provided as a single lipidated
polypeptide that encompasses multiple epitopes. The vaccine is
administered in an aqueous carrier comprised of Freund's Incomplete
Adjuvant. The dose of peptide for the initial immunization is from
about 1 to about 50,000 .mu.g for a 70 kg patient administered in a
human dose volume. The initial administration of vaccine is
followed by booster dosages at 4 weeks followed by evaluation of
the magnitude of the immune response in the patient, by techniques
that determine the presence of epitope-specific CTL populations in
a PBMC sample. Additional booster doses are administered as
required. The composition is found to be both safe and efficacious
as a prophylaxis against HCV infection.
[0670] Alternatively, the polyepitopic peptide composition can be
administered as a nucleic acid in accordance with methodologies
known in the art and disclosed herein.
Example 10
Definition of an A3.2 Specific Motif
[0671] There is some ambiguity in the international nomenclature of
A3 alleles. The A3.2 allele herein is expressed by cell lines EHM,
H0301, and GM3107. This particular subtype is currently referred to
as the 3.2 allele (Yang, in Immunobiology of HLA, Vol. 1, Dupont
ed., Springer-Verlag, New York pp. 43-44 and 54-55, 1989), or the
product of the A*0301 gene (its sequence corresponds to the one
published by Strachan, et al., EMBO J., 3:887 (1984), and has been
verified by direct cloning and sequencing of the A3 gene found in
EHM cell line. The HLA-A3.2 encoded by the A*0301 gene referred to
in this document is the commonly expressed HLA-A3 allelic form.
[0672] In one case using MAT cells, pooled peptide fractions
prepared as described in Example 3 above were obtained from
HLA-A3.2 homozygous cell lines, for example, CM3107. The pooled
fractions were HPLC fractions corresponding to 7% to 19%
CH.sub.3CN. For this class I molecule, this region of the
chromatogram was most abundant in peptides. Data from independent
experiments were averaged as described below.
[0673] The amino acid sequence analyses from four independent
experiments were analyzed and the results are shown in TABLE 73.
For each position except the first, the data were analyzed by
modifying the method described by Falk et al. to allow for
comparison of experiments from different HLA types. This modified
procedure yielded quantitative yet standardized values while
allowing the averaging of data from different experiments involving
the same HLA type.
[0674] The raw sequenator data was converted to a simple matrix of
10 rows (each representing one Edman degradation cycle) and 16
columns (each representing one of the twenty amino acids; W, C, R
and H were eliminated for technical reasons. The data corresponding
to the first row (first cycle) was not considered further because,
this cycle is usually heavily contaminated by free amino acids).
The values of each row were summed to yield a total pmoles value
for that particular cycle. For each row, values for each amino acid
were then divided by the corresponding total yield value, to
determine what fraction of the total signal is attributable to each
amino acid at each cycle. By doing so, an "Absolute Frequency"
table was generated. This absolute frequency table allows
correction for the declining yields of each cycle.
[0675] The retentate contains the bulk of the HLA-A heavy chain and
132-microglobulin, while the filtrate contains the naturally
processed bound peptides and other components with molecular
weights less than about 3000. The pooled filtrate material was
lyophilized in order to concentrate the peptide fraction. The
sample was then ready for further analysis.
[0676] For HPLC (high performance liquid chromatography) separation
of the peptide fractions, the lyophilized sample was dissolved in
50 .mu.l of distilled water, or into 0.1% trifluoracetic acid (TFA)
(Applied Biosystems) in water and injected into a C18 reverse-phase
narrow bore column (Beckman C18 Ultrasphere, 10.times.250 mm),
using a gradient system described by Stone and Williams (Stone, K.
L. and Williams K. R., in, Macromolecular Sequencing and Synthesis;
Selected Methods and Applications, A. R. Liss, New York, 1988, pp.
7-24). Buffer A was 0.06% TFA in water (Burdick-Jackson) and buffer
B was 0.052% TFA in 80% acetonitrile (Burdick-Jackson). The flow
rate was 0.250 ml/minute with the following gradient: 0-60 min.,
2-37.5% B; 60-95 min., 37.5-75% B; 95-105 min., 75-98% B. The
Gilson narrow bore HPLC configuration is particularly useful for
this purpose, although other configurations work equally well.
[0677] A large number of peaks were detected by absorbance at 214
nm, many of which appear to be of low abundance (FIG. 16). Whether
a given peak represents a single peptide or a peptide mixture was
not determined. Pooled fractions were then sequenced to determine
motifs specific for each allele as described below.
[0678] Pooled peptide fractions, prepared as described above were
analyzed by automated Edman sequencing using the Applied Biosystems
Model 477A automated sequencer. The sequencing method is based on
the technique developed by Pehr Edman in the 1950s for the
sequential degradation of proteins and peptides to determine the
sequence of the constituent amino acids.
[0679] The protein or peptide to be sequenced was held by a 12-mm
diameter porous glass fiber filter disk in a heated, argon-purged
reaction chamber. The filter was generally pre-treated with
BioBrene Plus.TM. and then cycled through one or more repetitions
of the Edman reaction to reduce contaminants and improve the
efficiency of subsequent sample sequencing. Following the
pretreatment of the filter, a solution of the sample protein or
peptide (10 pmol-5 nmol range) was loaded onto the glass filter and
dried. Thus, the sample was left embedded in the film of the
pretreated disk. Covalent attachment of the sample to the filter
was usually not necessary because the Edman chemistry utilized
relatively apolar solvents, in which proteins and peptides are
poorly soluble.
[0680] Starting from the absolute frequency table, a "relative
frequency" table was then generated to allow comparisons among
different amino acids. To do so the data from each column was
summed, and then averaged. Then, each value was divided next by the
average column value to obtain relative frequency values. These
values quantitate, in a standardized manner, increases and
decreases per cycle, for each of the different sixteen amino acid
types. Tables generated from data from different experiments can
thus be added together to generate average relative frequency
values (and their standard deviations). All standard deviations can
then be averaged, to estimate a standard deviation value applicable
to the samples from each table. Any particular value exceeding 1.00
by more than two standard deviations is considered to correspond to
a significant increase.
[0681] The results of the foregoing analysis for HLA-A3.2 were as
follows: at position 2, a 2.2-fold increase in valine (V) with
lesser increases (1.5-1-7) for structurally similar residues
leucine (L) and methionine. My. At position 3, tyrosine (Y) and
aspartic acid (0) showed increases in frequency. At position 7
isoleucine (I) was increased, and at position 8 asparagine (N), and
glutamine (Q) were increased. At positions 9 and 10, lysine (K) was
increased more than 2-fold over the expected random yield.
[0682] Cysteine was not modified and thus not detected tryptophan
coeluted with diphenylurea, and in some experiments, PTH-arginine
coeluted with the major derivative of PTH-threonine. Therefore,
cysteine and tryptophan are not detectable and arginine is detected
only in the absence of threonine.
[0683] Previously described MHC structures showed instances of
critically conserved residues at position 2 (or 3) and at the C
terminus (either position 9 or 10}. These residues are referred to
as "conserved" residues. The modified data analysis of this
invention considered the conserved positions at the N and C
terminals.
[0684] Thus, the HLA-A3.2 motif should have position two occupied
by V, L or M, a length of 9 or 10 amino acids, and C-terminal
position occupied by K.
Example 11
Definition of an A2.1. Specific Motif
[0685] In one case, pooled peptide fractions prepared as described
in Example above were obtained from HLA-A2.1 homozygous cell lines,
for example, JY. The pooled fractions were HPLC fractions
corresponding to 7% to 45% CH.sub.3CN. For this class I molecule,
this region of the chromatogram was most abundant in peptides. Data
from independent experiments were averaged as described below.
[0686] The amino acid sequence analyses from four independent
experiments were analyzed and the results are shown in TABLE 148
and TABLE 149. For each position except the first, the data were
analyzed by modifying the method described by Falk et al., supra,
to allow for comparison of experiments from different HLA types.
This modified procedure yielded quantitative yet standardized
values while allowing the averaging of data from different
experiments involving the same HLA type.
[0687] The raw sequenator data was converted to a simple matrix of
10 rows (each representing one Edman degradation cycle) and 16
columns (each representing one of the twenty amino acids; W, C, R
and H were eliminated for technical reasons. The data corresponding
to the first row (first cycle) was not considered further because,
this cycle is usually heavily contaminated by free amino acids).
The values of each row were summed to yield a total pmoles value
for that particular cycle. For each row, values for each amino acid
were then divided by the corresponding total yield value, to
determine what fraction of the total signal is attributable to each
amino acid at each cycle. By doing so, an "Absolute Frequency"
table was generated. This absolute frequency table allows
correction for the declining yields of each cycle.
[0688] Starting from the absolute frequency table, a "relative
frequency" table was then generated to allow comparisons among
different amino acids. To do so the data from each column was
summed, and then averaged. Then, each value was divided next by the
average column value to obtain relative frequency values. These
values quantitate, in a standardized manner, increases and
decreases per cycle, for each of the different sixteen amino acid
types. Tables generated from data from different experiments can
thus be added together to generate average relative frequency
values (and their standard deviations). All standard deviations can
then be averaged, to estimate a standard deviation value applicable
to the samples from each table. Any particular value exceeding 1.00
by more than two standard deviations is considered to correspond to
a significant increase.
Example 12
HLA-A2.1. Binding Motif and Algorithm
[0689] The structural requirements for peptide binding to A2.1 have
been defined for both, 9-mer and 10-mer peptides. Two approaches
have been used. The first approach referred to as the "poly-A
approach" uses a panel of single amino acid substitutions of a
9-mer prototype poly-A binder (ALAKAAAAV (SEQ ID NO:______)) that
is tested for A2.1 binding using the methods of Example 4 above to
examine the degree of degeneracy of the anchor-positions and the
possible influence of non-anchor positions on A2.1 binding.
[0690] The second approach, the "Motif-Library approach", uses a
large library of peptides selected from sequences of potential
target molecules of viral and tumor origin and tested for A2.1
binding using the methods in Example 4 above. The frequencies by
which different amino-acids occurred at each position in good
binders and non-binders were analysed to further define the role of
non-anchor positions in 9-mers and 10-mers.
A2.1 Binding of Peptide 9-mers
[0691] Poly A Approach. A poly-A 9-mer peptide, containing the A2.1
motif L (Leu) in position 2 and V (Val) in position 9 was chosen as
a prototype binder. A K (Lys) residue was included in position 4 to
increase solubility. A panel of 91 single amino-acid substitution
analogues of the prototype parental 9-mer was synthesized and
tested for A2.1 binding (TABLES 150 and 151). Shaded areas mark
analogs with a greater than 10-fold reduction in binding capacity
relative to the parental peptide. A reduction in binding greater
than 100-fold is indicated by hyphenation.
[0692] Anchor-Positions 2 and 9 in poly-A Analogs. The effect of
single-amino-acid substitutions at the anchor positions 2 and 9 was
examined first. Most substitutions in these positions had profound
detrimental effects on binding capacity, thus confirming their role
for binding. More specifically, in position 2 only L and M bound
within a 10-fold range ("preferred residues"). Residues with
similar characteristics, such as I, V, A, and T were tolerated, but
bound 10 to 100-fold less strongly than the parental peptide. All
the remaining substitutions (residues S, N, D, F, C, K, G, and P)
were not tolerated and decreased binding by more than 100-fold.
Comparably stringent requirements were observed for position 9,
where V, L and I were preferred and A and M are tolerated, while
the residues T, C, N, F, and Y virtually abolished binding.
According to this set of peptides, an optimal 2-9 motif could be
defined with L, M in position 2 and V, I, or L in position 9.
[0693] Non-Anchor Positions 1 and 3-8 in poly-A Analogs. All
non-anchor positions were more permissive to different
substitutions than the anchor-positions 2 and 9, i.e most residues
were tolerated. Significant decreases in binding were observed for
some substitutions in distinct positions. More specifically, in
position 1 a negative charge (residues D and E) or a P greatly
reduced the binding capacity. Most substitutions were tolerated in
position 3 with the exception of the residue K. Significant
decreases were also seen in position 6 upon introduction of either
a negative charge (D, E) or a positively charged residue (R). A
summary of these effects by different single amino acid
substitutions is given in TABLES 152 and 153.
[0694] The Motif-Library Approach. To further evaluate the
importance of non-anchor positions for binding, peptides of
potential target molecules of viral and tumor origin were scanned
for the presence of sequences containing optimal 2-9 anchor motifs.
A set of 161 peptides containing a L or M in position 2 and a V, L
or I in position 9 was selected, synthesized and tested for binding
(see Example 6). Only 11.8% of these peptides bind with high
affinity (ratio .gtoreq.0.10; 22.4% were intermediate binders
(ratio .gtoreq.0.1). As many as 36% were weak binders (ratio
<0.01-0.0001), and 31% were non-binders (ratio <0.0001). The
high number of non-binders containing optimal anchor-motifs
indicates that in this set of peptides positions other than the 2-9
anchors influence A2.1 binding capacity. Appendix 1 sets forth all
of the peptides having the 2-9 motif used for this analysis and the
binding data for those peptides.
[0695] To define the influence on non-anchor positions more
specifically, the frequency of occurrence of each amino acid in
each of the non-anchor positions was calculated for the good and
intermediate binders on one hand and non-binders on the other hand.
Amino acids of similar chemical characteristic were grouped
together. Weak binders were not considered for the following
analysis. The frequency of occurrence of each amino acid in each of
the non-anchor positions was calculated for the good binders and
non-binders (TABLE 154).
[0696] Several striking trends become apparent. For example in
position 1, only 3.6% of the A2.1 binders and as much as 35% of the
non-binders carried a negative charge (residues D and E). This
observation correlates well with previous findings in the set of
poly-A analogs, where a D or E substitution greatly affected
binding. Similarly, the residue P was 8 times more frequent in
non-binders than in good binders. Conversely, the frequencies of
aromatic residues (Y, F, W) were greatly increased in A2.1 binders
as compared to non-binders.
[0697] Following this approach, amino acids of similar structural
characteristics were grouped together. Then, the frequency of each
amino acid group in each position was calculated for binders versus
non-binders (TABLE 155). Finally, the frequency in the binders
group was divided by the frequency in the non-binders to obtain a
"frequency ratio". This ratio indicates whether a given amino-acid
or group of residues occurs in a given position preferentially in
good binders (ratio >1) or in non-binders (ratio <1).
[0698] Different Residues Influence A2.1 Binding. In order to
analyse the most striking influences of certain residues on A2.1
binding, a threshold level was set for the ratios described in
TABLE 155. Residues showing a more than 4-fold greater frequency in
good binders were regarded as preferred residues (+). Residues
showing a 4-fold lower frequency in A2.1 binders than in
non-binders were regarded as disfavored residues (-). Following
this approach, residues showing the most prominent positive or
negative effects on binding are listed in TABLE 156.
[0699] This table identifies the amino acid groups which influence
binding most significantly in each of the non-anchor positions. In
general, the most negative effects were observed with charged amino
acids. In position 1, negatively, charged amino acids were not
observed in good binders, i.e., those amino acids were negative
binding residues at position 1. The opposite was true for position
6 where only basic amino acids were detrimental for binding i.e.,
were negative binding residues. Moreover, both acidic and basic
amino acids were not observed in A2.1 binders in positions 3 and 7.
A greater than 4-fold increased frequency of non-binders was found
when P was in position 1.
[0700] Aromatic residues were in general favored in several of the
non-anchor positions, particularly in positions 1, 3, and 5. Small
residues like S, T, and C were favored in position 4 and A was
favored in position 7.
[0701] An Improved A2.1 9-mer Motif. The data described above was
used to derive a stringent A2.1 motif. This motif is based in
significant part on the effects of the non-anchor positions 1 and
3-8. The uneven distribution of amino acids at different positions
is reflective of specific dominant negative binding effects of
certain residues, mainly charged ones, on binding affinity. A
series of rules were derived to identify appropriate anchor
residues in positions 2 and 9 and negative binding residues at
positions 1 and 3-8 to enable selection of a high affinity binding
immunogenic peptide. These rules are summarized in TABLE 157.
[0702] To validate the motif defined above and shown in TABLE 157
published sequences of peptides that have been naturally processed
and presented by A2.1 molecules were analysed (TABLE 158). Only
9-mer peptides containing the 2-9 anchor residues were
considered.
[0703] When the frequencies of these peptides were analysed, it was
found that in general they followed the rules summarized in TABLE
157. More specifically, neither acidic amino acids nor P were found
in position 1. Only one acidic amino acid and no basic amino acids
were found in position 3. Positions 6 and 7 showed no charged
residues. Acidic amino acids, however, were frequently found in
position 8, where they are tolerated, according to our definition
of the A2.1 motif. The analysis of the sequences of naturally
processed peptides therefore reveals that >90% of the peptides
followed the defined rules for a complete motif.
[0704] Thus the data confirms a role of positions other than the
anchor positions 2 and 9 for A2.1 binding. Most of the deleterious
effects on binding are induced by charged amino acids in non-anchor
positions, i.e. negative binding residues occupying positions 1, 3,
6 or 7.
A2.1 Binding of Peptide 10-mers
[0705] The "Motif-Library" Approach. Previous data clearly
indicated that 10-mers can also bind to HLA molecules even if with
a somewhat lower affinity than 9-mers. For this reason we expanded
our analysis to 10-mer peptides.
[0706] Therefore, a "Motif-Library" set of 170 peptide 10-mers
containing optimal motif-combinations was selected from known
target molecule sequences of viral and tumor origin and analysed as
described above for 9-mers. In this set we found 5.9% good binders,
17.1% intermediate binders, 41.2% weak binders and 35.9%
non-binders. The actual sequences, origin and binding capacities of
this set of peptides are included as TABLE 182. This set of 10-mers
was used to determine a) the rules for 10-mer peptide binding to
A2.1, b) the similarities or differences to rules defined for
9-mers, and c) if an insertion point can be identified that would
allow for a superimposable common motif for 9-mers and 10-mers.
[0707] Amino-acid frequencies and frequency ratios for the various
amino-acid groups for each position were generated for 10-mer
peptides as described above for 9-mer peptides and are also shown
in TABLE 159 and TABLE 160, respectively for grouped residues.
[0708] A summary of preferred versus disfavored residues and of the
rules derived for the 10-mers in a manner analogous to that used
for 9-mers, is also listed in TABLE 161 and TABLE 162,
respectively.
[0709] When the frequency-ratios of different amino-acid groups in
binders and non-binders at different positions were analysed and
compared to the corresponding ratios for the 9-mers, both striking
similarities and significant differences emerged (TABLES 163 and
164). At the N-terminus and the C-termini of 9-mers and 10-mers,
similarities predominate. In position 1 for example, in 10-mers
again the P residue and acidic amino acids were not-tolerated. In
addition at position 1 in 10-mers aromatic residues were frequently
observed in A2.1 binders. In position 3, acidic amino acids were
frequently associated with poor binding capacity in both 9-mers and
10-mers. Interestingly, however, while in position 3 aromatic
residues were preferred in 9-mers, aliphatic residues (L, V, I, M)
were preferred in 10-mers.
[0710] At the C-terminus of the peptides, basic amino acids are not
favored in position 7, and both acidic and basic amino acids are
not favored in position 8 for 10-mers. This is in striking
agreement with the observation that the same pattern was found in
9-mers at positions 6 and 7. Interestingly, again the favored
residues differ between two peptides sizes. Aromatic (Y, F, W) or
aliphatic (L, V, I, M) residues were preferred in 10-mers at
position 8, while the A residue was preferred by 9-mers in the
corresponding position 7.
[0711] By contrast, in the center of the peptide no similarities of
frequency preferences were observed at positions 4, 5, and 6 in
10-mers and positions 4 and 5 in the 9-mers.
[0712] Most interestingly, among the residues most favored in the
center of the tested peptides were G in position 4 and 6, P in
position 5 was not observed in binders. All of these residues are
known to dramatically influence the overall secondary structure of
peptides, and in particular would be predicted to strongly
influence the propensity of a 10-mer to adopt a "kinked" or
"bulged" conformation.
[0713] Charged residues are predominantly deleterious for binding
and are frequently observed in non-binders of 9-mers and
10-mers.
[0714] However, favored residues are different for 9-mers and
10-mers. Glycine is favored while Proline is disfavored in the
center of 10-mer peptides but this is not the case for 9-mers.
[0715] These data establish the existence of an "insertion area"
spanning two positions (4, 5) in 9-mers and 3 positions (4, 5, 6)
in 10-mers. This insertion area is a more permissive region where
few residue similarities are observed between the 9-mer and 10-mer
antigenic peptides. Furthermore, in addition to the highly
conserved anchor positions 2 and 9, there are "anchor areas" for
unfavored residues in positions 1 and 3 at the N-terminus for both
9-mer and 10-mer and positions 7-10 or 6-9 at the C-terminus for
10-mers and 9-mers, respectively.
Example 13
Algorithm to Predict Binding of 9-mer Peptides to HLA-A2.1
[0716] Within the population of potential A2.1 binding peptides
identified by the 2,9 motif, as shown in the previous example, only
a few peptides are actually good or intermediate binders and thus
potentially immunogenic. It is apparent from the data previously
described that the residues present in positions other than 2 and 9
can influence, often profoundly, the binding affinity of a peptide.
For example, acidic residues at position 1 for A2.1 peptides do not
appear to be tolerated. Therefore, a more exact predictor of
binding could be generated by taking into account the effects of
different residues at each position of a peptide sequence, in
addition to positions 2 and 9.
[0717] More specifically, we have utilized the data bank obtained
during the screening of our collection of A2.1 motif containing
9-mer peptides to develop an algorithm which assigns a score for
each amino acid, at each position along a peptide. The score for
each residue is taken as the ratio of the frequency of that residue
in good and intermediate binders to the frequency of occurrence of
that residue in non-binders.
[0718] In the present "Grouped Ratio" algorithm residues have been
grouped by similarity. This avoids the problem encountered with
some rare residues, such as tryptophan, where there are too few
occurrences to obtain a statistically significant ratio. TABLES 165
and 166 is a listing of scores obtained by grouping for each of the
twenty amino acids by position for 9-mer peptides containing
perfect 2/9 motifs. A peptide is scored in the "Grouped Ratio"
algorithm as a product of the scores of each of its residues. In
the case of positions other than 2 and 9, the scores have been
derived using a set of peptides which contain only preferred
residues in positions 2 and 9. To enable us to extend our "Grouped
Ratio" algorithm. to peptides which may have residues other than
the preferred ones at 2 and 9, scores for 2 and 9 have been derived
from a set of peptides which are single amino acid substitutions at
positions 2 and 9. FIG. 45 shows a scattergram of the log of
relative binding plotted against "Grouped Ratio" algorithm score
for our collection of 9-mer peptides from the previous example.
[0719] The present "Grouped Ratio" algorithm can be used to predict
a population of peptides with the highest occurrence of good
binders. If one were to rely, for example, solely on a 2(L,M) and
9(V) motif for predicting A2.1 binding 9-mer peptides, it would
have been predicted that all 160 peptides in our database would be
good binders. In fact, as has already been described, only 12% of
these peptides would be described as good binders and only 22% as
intermediate binders; 66% of the peptides predicted by such a 2,9
motif are either weak or non-binding peptides. In contrast, using
the "Grouped Ratio" algorithm described above, and selecting a
score of 1.0 as threshold, 41 peptides were selected. Of this set,
27% are good binders, and 49% are intermediate, while only 20% are
weak and 5% are non-binders (TABLE 167).
[0720] The present example of an algorithm has used the ratio of
binders/non-binders to measure the impact of a particular residue
at each position of a peptide. It is immediately apparent to one of
ordinary skill that there are alternative ways of creating a
similar algorithm.
[0721] An algorithm using the average binding affinity of all the
peptides with a certain amino acid (or amino acid type) at a
certain position has the advantage of including all of the peptides
in the analysis, and not just good/intermediate binders and
non-binders. Moreover, it gives a more quantitative measure of
affinity than the simpler "Grouped Ratio" algorithm. We have
created such an algorithm by calculating for each amino acid, by
position, the average log of binding when that particular residue
occurs in our set of 160 2,9 motif containing peptides. These
values are shown in TABLE 168. The algorithm score for a peptide is
then taken as the sum of the scores by position for each residues.
FIG. 46 shows a scattergram of the log of relative binding against
the average "Log of Binding" algorithm score. TABLE 167 shows the
ability of the two algorithms to predict peptide binding at various
levels, as a function of the cut-off score used. The ability of a
2,9 motif to predict binding in the same peptide set is also shown
for reference purposes. It is clear from this comparison that both
algorithms of this invention have a greater ability to predict
populations with higher frequencies of good binders than a 2,9
motif alone. Differences between the "Grouped Ratio" algorithm and
the "Log of Binding" algorithm are small in the set of peptides
analyzed here, but do suggest that the "Log of Binding" algorithm
is a better, if only slightly, predictor than the "Grouped Ratio"
algorithm.
[0722] The log of binding algorithm was further revised in two
ways. First, poly-alanine (poly-A) data were incorporated into the
algorithms at the anchor positions for residues included in the
expanded motifs where data obtained by screening a large library of
peptides were not available. Second, an "anchor requirement
screening filter" was incorporated into the algorithm. The poly-A
approach is described in detail, above. The "anchor requirement
screening filter" refers to the way in which residues are scored at
the anchor positions, thereby providing the ability to screen out
peptides which do not have preferred or tolerated residues in the
anchor positions. This is accomplished by assigning a score for
unacceptable residues at the anchor positions which are so high as
to preclude any peptide which contains them from achieving an
overall score which would allow it to be considered as a potential
binder.
[0723] The results for 9-mers and 10-mers are presented in TABLE
177 and TABLE 178, below. In these tables, values are group values
as follows: A; G; P; D,E; R,H,K; L,I,V,M; F,Y,W; S,T,C; and Q,N,
except where noted in the tables.
Example 14
Use of an Algorithm to Predict Binding of 10-mer Peptides to
HLA-A2.1
[0724] Using the methods described in the proceeding example, an
analogous set of algorithms has been developed for predicting the
binding of 10-mer peptides. TABLE 169 shows the scores used in a
"Grouped Ratio" algorithm, and TABLE 170 shows the "Log of Binding"
algorithm scores, for 10-mer peptides. TABLE 171 shows a comparison
of the application of the two different algorithmic methods for
selecting binding peptides. FIG. 47 and FIG. 48 show, respectively,
scattergrams of a set of 10-mer peptides containing preferred
residues in positions 2 and 10 as scored by the "Grouped Ratio" and
"Log of Binding" algorithms.
Example 15
Binding of A2.1 Algorithm Predicted Peptides
[0725] The results of Examples 6 and 7 indicate that an algorithm
can be used to select peptides that bind to HLA-A2.1 sufficiently
to have a high probability of being immunogenic.
[0726] To test this result, we tested our algorithm on a large
(over 1300) non-redundant, independent set of peptides derived from
various sources. After scoring this set with our algorithm, we
selected 41 peptides (TABLE 171) for synthesis, and tested them for
A2.1 binding. This set of peptides was comprised of 21 peptides
with high algorithm scores, and 20 peptides with low algorithm
scores.
[0727] The binding data and categorization profile are shown in
TABLE 172 and TABLE 173 respectively. The correlation between
binding and algorithm score was 0.69. It is immediately apparent
from TABLE 173 the striking difference between peptides with high
algorithm scores, and those with low algorithm scores.
Respectively, 76% of the high scorers and none of the low scorers
were either good or intermediate binders. This data demonstrates
the utility of the algorithm of this invention.
Example 16
HLA A2.1 Allele-Specific Motif and HLA A2 Supermotif Binding
[0728] We have also derived further information on the structural
requirements of A2.1 binding. To do this we first sought to
determine the degree of permissiveness of anchor positions 2 and 9.
For this purpose, a panel of analogs bearing single substitutions
at either position 2 or 9 of a model poly (A) 9-mer peptide
containing the previously reported A2.1 motif L in position 2 and V
in position 9 (Ruppert, et al, Cell 74:929-937 (1993) was
synthesized, and its binding capacity measured. Thirteen different
analogs were synthesized for both anchor positions 2 and 9.
[0729] The present invention also encompasses analogs of peptides
bearing the A2.1 allele-specific motif and the A2 supermotif.
Analog peptides can have amino acid substitution at primary and/or
secondary anchor positions of the A2.1 allele-specific motif or of
the A2 supermotif. The complete structural requirements of peptide
binding to the HLA A2.1 allele-specific motif are disclosed for the
first time herein. This information was developed by determining
the degree of permissiveness for amino acids at primary anchor
positions 2 and 9. For this purpose, a panel of analogs bearing
single substitutions at either position 2 or 9 of a model poly (A)
9-mer peptide containing the previously reported A2.1 motif, L in
position 2 and V in position 9 (Ruppert, et al, Cell 74:929 (1993)
was synthesized, and the peptides' binding capacity measured.
Thirteen different analogs were synthesized for both anchor
positions 2 and 9.
[0730] In good agreement with the previously reported A2.1 motif
allele-specific, the peptides carrying L or M in position 2 were
the best binders. Decreases in binding capacity (10- to 100-fold)
were apparent even with relatively conservative substitutions such
as isoleucine (I), valine (V), alanine (A), and threonine (T).
Similar data (not shown) were found for glutamine (Q) at positions
2. More radical changes (i.e., residues D, K, F, C, P, G, N, and S)
completely abolished binding capacity. Similar results were
obtained at position 9, where only conservative substitutions such
as L and I bound within 10-fold of the unsubstituted model poly A
A2.1 peptide. Analogs carrying A or M substitutions also bound, but
less strongly (10- to 100-fold decrease). Finally, all other
substitutions tested (C, N, F, S, G, P, and R) were associated with
complete loss of A2.1 binding capacity. Thus, based on these data
and in good agreement with previous studies (Falk et al. Nature
351:290 (1991) and Hunt et al. Science 255:1261 91992)), an A2.1
allele motif is now defined as set forth in Tables 137 and 138.
Thus, based on these data and in good agreement with previous
studies (19-20), a "canonical" A2.1 motif could be identified as L
or M in position 2 and L. V. or I in position 9.
[0731] Analogs to peptides bearing an HLA A2.1 allele-specific
motif may be created based on the substitution of specific residues
at primary anchor positions. For example, analog peptides with an
enhanced binding affinity for HLA A2.1 molecules may be engineered
by substituting preferred residues for tolerated residues at
primary anchor positions. Examples of such substitutions and the
effects on A2.1 binding are shown in TABLE 147. For this study, a
set of 25 HLA A2.1 peptides of different relative binding values
was selected. For each of the peptides a substitution of one, or in
some instances both, primary anchor positions was made. In the case
of the position 2 primary anchor residue, the analog peptide was
made with a leucine or methionine substitution. For the C-terminal
primary anchor position, a valine residue was substituted in the
analogued peptide. In all single substitution analogued peptides,
improved HLA A2.1 binding was observed. Significant improvement in
binding was also observed in several peptides that contained
substitutions at both primary anchor positions. These results
indicate that it is possible to improve the binding of a peptide
that bears tolerated or less preferred primary anchor residues by
substitution with preferred or optimal amino acid residues.
[0732] TABLE 147. Binding activities of analogs of A2.1
motif-bearing peptides. The "(a)" indicates an analogued peptide.
Relative binding to A2.1 HLA molecules is shown in the last column.
Binding is expressed as a ratio of binding of the test peptide
relative to a standard peptide. A higher value for the analog
relative to the native sequence indicates an increase in binding
affinity of the analog relative to the native sequence. The
standard A2.1 peptide (FLPSDYFPSV (SEQ ID NO:______)) binds to A2.1
molecules with an IC.sub.50 of 5.0. The ratio is converted to
IC.sub.50 by dividing the IC.sub.50 of the standard peptide, i.e.
5.0, by the ratio shown in the table.
[0733] Development of the HLA-A2 Supertype. Direct HLA binding
assays with radiolabeled peptides and mammalian cells which express
HLA class I molecules, such as EBV-transformed B cell lines and
PHA-activated blasts have been developed. Significant binding of
the radiolabeled probe could be obtained if the target cells were
preincubated overnight at 26.degree. C. in the presence of
.beta.2-microglobulin. Under these conditions, up to a few percent
of the HLA molecules expressed by either cell type could be bound
by the labeled peptides. With these assays, the degree of
cross-reactivity of the A*0201-restricted hepatitis B virus core
18-27 peptide with other A2 subtypes was examined. It was
determined that this peptide epitope also bound the A*0202, A*0205,
and A*0206 but not A*0207 allele-specific HLA molecules.
[0734] Inhibition experiments with panels of synthetic peptide
analogs underlined the similar ligand specificities of the
HLA-A*0201, A*0202, and A*0205 alleles. Furthermore, analysis of
the polymorphic residues that help form the polymorphic B and F
pockets of various HLA alleles allowed prediction of binding of the
hepatitis B virus core 18-27 epitope to two other HLA alleles
(HLA-A*6802 and A*6901). The B and F pockets are the pockets on the
HLA molecules that come into contact with positions 2 and the
C-terminus of a peptide, respectively. Thus, it appears that a
family of at least six different HLA-A molecules (A*0201, A*0202,
A*0205, A*0206, A*6802, A*6901) collectively defined as the A2
supertype, share overlapping ligand specificities. Furthermore, use
of purified HLA molecules in binding assays have demonstrated that
A*0203 and A*0207 are also properly included in the A2
supertype.
[0735] Therefore, based on these results for the HLA A2.1
allele-specific motif, findings are extrapolated to the HLA A2
supermotif. The A2 supertype binding of any peptide which carries a
"non-canonical" (but still acceptable) residue in position 2 or 9
(or 10) (for example A, T, or Q in 2; or L, A, M or T in 9 or 10)
is increased by creating an analog which replaces the acceptable
residue with a more "canonical" or preferred anchor. For example,
the FHV Env 2181 peptide with sequence (LWVTVYYGV (SEQ ID
NO:______)) bind A2.1 with a IC50% of 12,500 nM, while the position
2 anchor substituted analog LMVTVYYGV (SEQ ID NO:______) binds with
IC50% of 3.3 nM. The HBVc 18-27 naturally occurring sequence
FLPSDFFPSI (SEQ ID NO:______) binds A2.1 with IC50% 22 nM, but its
C-terminal anchor substituted V.sub.lo variant binds A2.1 with a Kd
of 2.5 nM. For example, the HBV pol 538 peptide (YMDDVVLGA (SEQ ID
NO:______)) binds A2.1 with an IC.sub.50 of 200 nM, while an analog
containing a V substitution at position 9 binds with an IC.sub.50
of 5.1 nM. Other examples of fixed anchor peptides are shown in
TABLE 145. Some of the fixed peptides were tested for their ability
to induct CTL responses. For example, the HIV Env 2181 peptide and
the HBV pol 721 peptides were tested in primary CTL assays (21),
and found to be positive. Positive CTL recognition data exists also
for the HBV 18-27 and HBV pol 538 peptides.
[0736] The binding activity of a peptide that does not bear a
motif, but that is selected on the basis of similarity to a known
peptide epitope that has the ability to bind an HLA molecule, may
also be modulated. Such a peptide may be engineered to enhance
binding to HLA molecules by substituting primary anchor residues,
as designated for the particular motif, for non-anchor residues
such that a motif-bearing peptide is created. For example, the HIV
Env 2181 peptide with sequence (LWVTVYYGV (SEQ ID NO:______)),
which does not bear an A2.1 motif or A2 supermotif primary anchor
residue at position 2, binds A2.1 with an IC.sub.50 of 12,500 nM,
while the position 2 anchor substituted analog LMVTVYYGV (SEQ ID
NO:______) binds with an IC.sub.50 of 3.3 nM.
[0737] Analogoued peptides may also be tested for their ability to
induce CTL responses. For example, the analog HIV Env 2181 peptide
was tested in primary CTL assays, and found to be positive
(Wentworth et al. Molec. Immunol. 32: 603-612 91995)). Positive CTL
recognition data also exist for the HBV pol 538 peptide.
[0738] The A2 supermotif may also be used to create substituted
analogs, enhancing the binding affinity of such a peptide for
several members of the A2 supertype. An example of how such
analoguing was accomplished was demonstrated with the peptide HPV
16 EF.86-93 TLGIVCPI (SEQ ID NO:______) and its analog TLGIVXPI
(SEQ ID NO:______) (where X stands for .alpha. amino butyric acid).
The binding patterns of these two peptides for A2 alleles, i.e.,
for the A2 supertype, was then tested. It was found that the X
substitution greatly increased binding affinity for all A2 alleles,
resulting in a more useful peptide, characterized by increased
binding capacity and broader crossreactivity than its original
parent sequence. Furthermore, the peptide was more stable and less
subject to oxidation. Subsequent experiments utilizing A2/Kb
transgenic mice demonstrated that CTLs induced by the X-substituted
peptide were fully crossreactive with the wild type sequence, and
that the X peptide, as a result of its higher binding affinity, was
a more potent immunogen.
[0739] In conclusion, from the data shown herein, analogs are
created of A2.1 peptides (9-mers or 10-mers) at primary and/or
secondary residues. Such analogs exhibit higher binding affinity by
substituting out "negative" or neutral residues from a native
sequence, and inserting either neutral or preferred residues. These
peptides are understood to have unique immunological properties in
that they, while still crossreactive with the wild type sequences,
may not be subject to tolerance, deletion or suppressive
mechanisms, which serve to inactivate a CTL response to the wild
type sequence, present as a result of cancer or infection.
Determination of Secondary Residues and an Extended A2.1 Motif for
9-mer and 10-mer Peptides.
[0740] Analysis of 9-mer Peptide Binding to HLA A2.1 Molecules.
Data have revealed a prominent role for residues that are not
primary anchors in determining binding capacity of 9-mer peptides
for A*0201. The results of these analyses are described in Ruppert
et al., Cell 74:929 (1993). Accordingly, the frequency of a given
amino acid group in A2.1 binding peptides was divided by the
frequency of nonbinding peptides to obtain a frequency ratio. This
ratio indicates whether a residue occurs at a given position
preferentially in binding (ratio >1) or nonbinding peptides
(ratio <1). To facilitate the analysis, a threshold level was
set for the ratios, such that residues present at more than 4-fold
greater frequency in binding peptides compared with nonbinding
peptides were regarded as favored or preferred residues, and
residues present at less than 4-fold lower frequency in binding
peptides than in nonbinding peptides were regarded as unfavored or
deleterious residues. Following this approach, groups of residues
showing prominent associations as having favored or unfavored
binding, respectively, were identified.
[0741] In general, the most detrimental effects were observed with
charged amino acids. At position 1, both P and acidic (E and D)
residues were infrequent in A2.1-binding peptides. At position 6,
basic (H, R, and K) residues were associated with nonbinding
peptides, whereas both acidic and basic residues were infrequent in
good binding peptides at positions 3 and 7. Conversely, aromatic
residues were associated with high affinity binding in positions 1,
3, and 5. Furthermore, residues with OH-- or SH-- containing side
chains, such as S, T, or C, were favored at position 4, while A was
favored in position 7 and P in position 8. In conclusion, these
frequency analyses allowed for the definition of an extended A2.1
motif that takes into account the impact of secondary anchor
positions (other than primary anchor positions 2 and C-terminus)
for peptide binding to HLA A2.1 molecules. The extended A2.1 9-mer
motif is set forth in TABLE 138.
[0742] Analysis of 10-mer Peptide Binding to HLA A2.1 Molecules.
The same approach described above for 9-mer peptides was also used
to analyze the data obtained with a set of 10-mer peptides. At the
N- and C-termini of the peptides, the pattern observed was rather
similar to the one observed with 9-mers. For instance, in the
10-mer set, as in the case 10 of the 9-mer peptides, position 1 was
characterized by an increased frequency of aromatic residues in the
binder set, while negative charges and P were again associated with
poor binding. Again at position 3, amino acids with negative charge
were associated with poor binding. Interestingly, at this position,
aliphatic (rather than aromatic) residues were associated with high
affinity binding. At the C-termini of the peptides, certain
similarities were also observed. In the 10-mer, the penultimate
residue at position 9 (corresponding to position 8 in the 9-mer)
was quite permissive, with only basic residues being found more
frequently in nonbinding peptides. Similar to the situation at
position 7 in the 9-mer, neither positive nor negative charges were
tolerated in the antepenultimate position 8 of the 10-mers. Also,
position 7 did not favor positive-residues in the 10-mers, as
previously observed for position 6 in the 9-mers. In comparison to
what was observed at position 3 (for both 9-mers and 10-mers), the
residues associated with good binding were, however, different.
Aromatic and hydrophobic residues were frequent in high affinity
binders at position 8 (as opposed to only A being frequent at
position 7 in the 9-mers).
[0743] Finally, a rather distinctive pattern was observed in the
middle of the peptide. At position 4, G was favored in high
affinity binding peptides, while both A and positive charges were
very frequent in nonbinding peptides. P, in position 5, was
completely absent in peptides that bind to HLA A2.1 molecules. It
is noteworthy that none of the trends observed in positions 4 and 5
in the 10-mer set have any counterpart in position 3 or 4 in the
9-mer set.
[0744] In summary, an extended motif has been generated for A2.1
binding 10-mer peptides, following a strategy similar to the one
described for 9-mer peptides above. The extended A2.10-mer motif is
set forth in TABLE 138. Both important differences and striking
similarities were noted in comparing the 9-mer and 10-mer sets at
these nonanchor positions.
Example 17
HLA Class I A3 Supertype Binding
[0745] This example provides supermotif data useful for the
preparation of analogs of supermotif-bearing peptides as well as
for determination of native sequences with particular properties.
The supermotif data were derived by calculating at each non-anchor
position along the peptide sequence the average relative binding
capacity of peptides carrying each of the 20 common amino acids,
grouped according to individual chemical similarities.
A-3 Supermotif
[0746] HLA class I protein purification. The following Epstein-Barr
virus (EBV)-transformed homozygous cell lines were used as sources
of class I molecules: GM3107 (A3, B7; Human Genetic Mutant
Repository); BVR (A11, B35.3, Cw4; Human Genetic Mutant
Repository); SPACH (A31, B62, Cw1/3; ASHI Repository Collection);
and LWAGS (A*3301, B14, Cw8; ASHI Repository Collection) (Bodmer,et
al, Hum. Immunol. 43:149 (1995)). A C1R transfectant characterized
by Dr. Walter Storkus (University of Pittsburgh) was used for the
isolation of A*6801. Cell lines were maintained as previously
described (Sidney, et al., J. Immunol. 154:247 (1995); Sette, et
al., Mol. Immunol. 31:813 (1994)).
[0747] Cell lysates were prepared and HLA class I molecules
purified as previously described (Sidney, et al., J. Immunol.
154:247 (1995); Sette, et al., Mol. Immunol. 31:813 (1994)).
Briefly, cells were lysed at a concentration of 10.sup.8 cells/ml
in 50 mM Tris-HCl, pH 8.5, containing 1% Nonidet P-40 (Fluka
Biochemika, Buchs, Switzerland), 150 mM NaCl, 5 mM EDTA, and 2 mM
PMSF. The lysates were passed through 0.45 .mu.M filters and
cleared of nuclei and debris by centrifugation at 10,000 g for 20
minutes. HLA proteins were then purified by affinity
chromatography. Columns of inactivated Sepharose CL 4B and Protein
A Sepharose were used as precolumns. The cell lysate was depleted
of HLA-B and HLA-C proteins by repeated passage over Protein A
Sepharose beads conjugated with the anti-HLA(B,C) antibody B1.23.2
(Rebai, et al., Tissue Antigens 22:107 (1983)). Typically two to
four passages were required for effective depletion. Subsequently,
the anti HLA(A,B,C) antibody W6/32 (Barnstable, et al., Cell 14:9
(1978)) was used to capture HLA-A molecules. Protein purity,
concentration, and effectiveness of depletion steps were monitored
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE).
[0748] Binding assays. Quantitative assays for the binding of
peptides to soluble class I molecules on the basis of the
inhibition of binding of a radiolabeled standard probe peptide to
detergent solubilized HLA molecules were performed as previously
described (Kubo, et al., J. Immunol. 152:3913 (1994); Kast, et al.,
J. Immunol. 152:3904 (1994); Sidney, et al., J. Immunol. 154:247
(1995); Sette, et al., Mol. Immunol. 31:813 (1994); Ruppert, et
al., Cell 74:929 (1993)). Briefly, 1-10 nM of radiolabeled probe
peptide, iodinated by the Chloramine-T method (Greenwood, et al.,
Biochem. J. 89:114 (1963)), was co-incubated at room temperature
with various amounts of HLA in the presence of 1 .mu.M human
.beta..sub.2-microglobulin (Scripps Laboratories, San Diego,
Calif., USA) and a cocktail of protease inhibitors. At the end of a
two day incubation period, the percent of HLA-bound radioactivity
was determined by size exclusion gel filtration chromatography on a
TSK 2000 column.
[0749] The A3CON1 peptide (sequence KVFPYALINK (SEQ ID NO:______))
(Kubo, et al., J. Immunol. 152:3913 (1994)) was used as the
radiolabeled probe for the A3, A11, A31, and A*6801 assays. A T7Y
analog of HBVc 141-151 (sequence STLPETYVVRR (SEQ ID NO:______))
(Missale, et al., J. Exp. Med. 177:751 (1993)) was used as the
radiolabeled probe for the A*3301 assay. In the case of competitive
assays, the concentration of peptide yielding 50% inhibition of the
binding of the radiolabeled probe peptide (IC.sub.50) was
calculated. Peptides were usually tested at one or two high doses,
and the IC.sub.50 of peptides yielding positive inhibition were
determined in subsequent experiments, in which two to six further
dilutions were tested, as necessary. HLA concentrations yielding
approximately 15% binding of the radiolabeled probe peptide were
used for all competitive inhibition assays. Under these conditions
the concentration of the labeled peptide is less than the
concentration of the HLA molecule and the IC.sub.50 is less than
the concentration of the HLA molecule, accordingly the measured
IC.sub.50s are reasonable approximations of the true K.sub.D
values. Each competitor peptide was tested in two to four
completely independent experiments. As a positive control, in each
experiment, the unlabeled version of the relevant radiolabeled
probe was tested and its IC.sub.50 measured. The average IC.sub.50s
of A3CON1 for the A3, A11, A31, and A*6801 assays were 11, 6, 18,
and 8 nM, respectively. The average IC.sub.50 of the HBVc 141-151
peptide in the A*3301 assay was 29 nM.
[0750] Definition of secondary anchor positions for five HLA-A3
supertype alleles (A3, A11, A31, A3301, A6801). A modification of
the procedure used by Ruppert, et al., Cell 74:929 (1993) to define
A*0201 secondary anchor motifs was utilized. Briefly, HLA-specific
secondary anchor position motifs were defined by assessing the
effect on HLA binding of the 20 commonly occurring amino acids at
each non-primary anchor position of 9-mer sequences. Assessment was
made by calculating the average relative binding values for each
position-amino acid combination (e.g., position 1, alanine;
position 2, alanine, etc.). To overcome problems with the low
occurrence of certain amino acids, some residues were grouped as
previously described (Ruppert, et al., Cell 74:929 (1993)). Residue
types associated at a particular position with average binding
capacities fourfold higher or lower than the overall average
binding capacity of a 200-peptide set were considered to be
associated with good or poor binding capacity, respectively.
[0751] Peptide synthesis. Peptides were either synthesized as
previously described (Ruppert, et al., Cell 74:929 (1993)), or
purchased as crude material from Chiron Mimotopes (Chiron Corp.,
Australia). Peptides that were synthesized were purified to >95%
homogeneity by reversed-phase high-pressure liquid chromatography
(HPLC). The purity of these synthetic peptides was assayed on an
analytical reversed-phase column and their composition ascertained
by amino acid analysis, sequencing, and/or mass spectrometry
analysis.
[0752] Structural analysis of the peptide-binding pockets of
various HLA A3 supertype molecules. Previous studies indicated that
the HLA molecules A3, A11, and A*6801 are associated with
specificity for ligands carrying small or hydrophobic residues in
position 2, and positively charged C-termini (Kubo, et al., J.
Immunol. 152:3913 (1994); Guo, et al., Nature 360:364 (1994); Falk,
et al., Immunogenetics 40:238 (1994); Dibrino, et al., J. Immunol.
151:5930 (1993); DiBrino, et al., Proc. Nat'l Acad. Sci. USA
90:1508 (1993); Zhang, et al., Proc. Nat'l Acad. Sci. USA 90:2217
(1993); Sette, et al., Mol. Immunol. 31:813 (1994)).
[0753] The side chains of the residue in position 2 and at the
C-termini of antigenic peptides are known to contact the residues
forming the B and F pockets of HLA class I molecules (Madden, et
al., Cell 75:693 (1993); Saper, et al., J. Mol. Biol. 219:277
(1991)), the residues of the HLA molecule that form these
polymorphic pockets were tabulated for various putative HLA class I
A3 supertype molecules. It was found that the HLA types which are
known to recognize peptides with small or hydrophobic residues in
position 2 (e.g., A*0101, A*0201, A*0301, A*1101, A*6801, and
A*6802), and HLA types which recognize positively charged residues
at the C-terminus (e.g., A*0301, A*1101, A*6801, and B*2705) of
their peptide ligands shared certain key structural features. In
particular, for HLA molecules that bind peptides that have small
and hydrophobic residues at position 2, it was found that the HLA
molecule carried aliphatic residues (M or V) at positions 45 and
67, and potential hydrogen-bond-forming residues such as N and K,
or H and Q at positions 66 and 70, respectively. All of these HLA
molecules also carried a Y residue at position 99. In contrast,
class I A3 supertype molecules that exhibited different binding
specificities differed in one or more of these positions.
Similarly, only class I molecules that prefer positively charged
C-termini carried D, T, L, and D at positions 77, 80, 81, and 116,
respectively.
[0754] In short, this analysis established that a set of HLA class
I molecules (A3, A11, and A*6801), designated as the A3 supertype,
share binding repertoires for peptides comprising a motif
characterized by small or hydrophobic residues in position 2 and
positively charged residues at their C-terminal positions, and
share certain key structural features in their B and F pockets. The
A3 supertype molecules bind to peptides having a corresponding
motif designated as the A3 supermotif.
[0755] Analysis of other class I HLA molecules for which motifs
were unknown, revealed that A*3101, A*3301, A*3401, A*6601, and
A*7401 also shared these same consensus sequences in their B and F
pockets. Accordingly, these molecules were also designated to be
part of the A3 supertype. Falk, et al., Immunogenetics 40:238
(1994) subsequently verified that A31 and A33 are indeed
characterized by a repertoire for peptides with an A3 peptide
motif.
[0756] A3 molecules exhibit overlapping primary anchor
specificities. To compare the range of motifs recognized by some of
the most frequent A3 HLA molecules (A3, A11, A31, A*3301, and
A*6801), more detailed molecular analysis of the main anchor
residues (position 2 and C-terminal) of the peptides bound by these
molecules was undertaken. A3- and A11-specific peptide-binding
assays measuring the capacity of unlabeled synthetic peptides to
inhibit the binding of a radiolabeled peptide to affinity-purified
HLA class I molecules have been previously described (Kubo, et al.,
J. Immunol. 152:3913 (1994); Kast, et al., J. Immunol. 152:3904
(1994); Sette, et al., Mol. Immunol. 31:813 (1994)). Binding assays
specific for A31, A*3301, and A*6801 were developed using similar
approaches (Kubo, et al., J. Immunol. 152:3913 (1994); Kast, et
al., J. Immunol. 152:3904 (1994); del Guercio, et al., J. Immunol.
154:685 (1995); Sidney, et al., J. Immunol. 154:247 (1995); Sette,
et al., Mol. Immunol. 31:813 (1994); Ruppert, et al., Cell 74:929
(1993)).
[0757] Primary anchor specificities of peptides bound by the A3
supertype HLA molecules were subsequently explored by preparing a
panel of peptides carrying substitutions at position 2 or 9, 9
being the C terminus, of a prototype poly-alanine 9-mer peptide
AXAAAAAX (SEQ ID NO:______). These peptides were tested to evaluate
their inhibitory capacity for A3, A11, A31, A*3301, and A*6801.
Inhibitory capacity was determined by detecting whether the binding
of a labeled probe was inhibited in the presence of a peptide from
the panel. Each HLA molecule expressed individual preferences, but
in the majority of instances, significant peptide binding was
obtained when the peptide's position 2 was occupied by either A, I,
L, M, S, T, or V, and the C-terminus was either R or K. These data
were found to be in good agreement with pool sequencing data
previously generated (Kubo, et al., J. Immunol. 152:3913 (1994);
Falk, et al., Immunogenetics 40:238 (1994); Dibrino, et al., J.
Immunol. 151:5930 (1993); DiBrino, et al., Proc. Nat'l Acad Sci.
USA 90:1508 (1993)), and also extended in the cases of A31, A*3301,
and A*6801, the definition of the primary anchor motifs.
[0758] In conclusion, these data indicate that a primary anchor
supermotif for the A3 supertype is defined as A, I, L, M, S, T, or
V in position 2, and either R or K at the C-terminus.
[0759] A3 supertype molecules share overlapping peptide-binding
repertoires. The extent to which peptides which have the A3
supermotif exhibit cross-reactivity binding amongst the HLA A3
supertype molecules was examined. A set of 200 naturally occurring
9-mer peptide sequences carrying residues A, I, L, M, S, T, or V in
position 2 and K or R in the C terminus (i.e., peptides with an A3
supermotif) was assembled. Other than the constraint that each
possible anchor combination be represented in proportion to the
natural frequency of the individual amino acids, the peptides
comprising the set were randomly selected from viral and tumor
antigen sequences. When each peptide was tested for its capacity to
bind purified A3, A11, A31, A*3301, and A*6801 HLA molecules, it
was apparent that a unique binding pattern was associated with each
allelic type. For example, some peptides were rather selective,
binding only one class I type, whereas certain other peptides
cross-reacted rather extensively, binding four or five of the
molecules tested.
[0760] It was found that, in general, about 10% (5%-16%) of the
peptide-HLA combinations were associated with good binding
(IC.sub.50.ltoreq.50 nM), and about 17% (11%-24%) with intermediate
binding (IC.sub.50 50-500 nM) to any given allele. These
frequencies of high and intermediate binding are similar to those
previously noted for A*0201 pool-sequencing-motif-containing
peptides (Ruppert, et al., Cell 74:929 (1993)).
[0761] Most notable, however, was the relatively high degree of
cross-reactivity observed. Of the 127 peptides that were capable of
binding to at least one A3 molecule, 43 of them (34%) bound three
or more of the A3 supertype molecules. Four peptides bound all five
of the A3 molecules tested. In contrast, in a set of 39 peptides
which were tested for binding to five unrelated class 1 molecules
(A*0101, A3, A24, and B7), only three (8%) bound to two molecules,
and none bound to three or more molecules. The peptides identified
as high or intermediate binders for at least four of the five A3
molecules tested are listed in Table 141. In Table 141, good or
intermediate binding capacities are defined as IC.sub.50.ltoreq.500
nM, and are highlighted by shading. Taken together, these data
demonstrate significant overlap in the binding repertoires of the
A3 supertype molecules, and validate the A3 primary anchor
supermotif. From the set of peptides used in this evaluation, 10
additional peptides binding with high or intermediate affinity to
at least four of the five A3 molecules tested were identified (see,
TABLE 141 and below).
[0762] The peptides identified as high or intermediate binders for
at least four of the five A3 molecules tested are listed in TABLE
141. In TABLE 141, good or intermediate binding capacities are
defined as IC.sub.50.ltoreq.500 nM, and are highlighted by
shading.
[0763] Taken together, these data demonstrate significant overlap
in the binding repertoires of the A3 supertype molecules, and
validate the A3 primary anchor supermotif. From the set of peptides
used in this evaluation, 10 additional peptides binding with high
or intermediate affinity to at least four of the five A3 molecules
tested were identified (see, TABLE 141 and below).
[0764] Secondary Anchor Residues Which Confer Additional Properties
to A3 Supermotif-bearing Peptide Ligands. As stated above, although
the overlap in the binding repertoires of A3 supertype molecules is
significant, each A3 HLA molecule also retains a substantial degree
of binding specificity. To understand the basis of the observed
cross-reactivities, an extended supermotif that defines molecules
having the A3 supermotif primary anchors and further specificities
at other positions was defined. The amino acid patterns determined
at these non-primary anchor positions are designated as secondary
anchor positions.
[0765] First, refined motifs for each of the A3-like alleles
analyzed herein (A3, A11, 30 A31, A*3301, and A*6801), outlining
secondary anchor-binding specificities, were derived as described
in the Materials and Methods. This approach is similar to the one
previously used to define a refined A*0201 motif (Ruppert, et al.,
Cell 74:929 (1993)). The motifs were derived by calculating at each
nonanchor position along the peptide sequence the average relative
binding capacity of peptides carrying each of the 20 common amino
acids, grouped according to individual chemical similarities.
Representative of the data generated by this procedure, the values
calculated for A3 are shown in TABLE 140. Following this as an
example, 21 different peptides were tested which possessed an
aromatic residue (F, W, Y) in position 3 of their sequence. These
peptides had an average relative binding capacity to A3 31.7-fold
higher than the overall average of the 200-peptide set. By analogy
to what was previously described in the case of A*0201, preferred
and deleterious residues were defined as residues associated with
average binding capacities that were fourfold greater than or
fourfold less than, respectively, the overall average. Accordingly,
aromatic residues in position 3 were considered "preferred"
residues for A3 binding.
[0766] The extended A3 supermotif including both primary and
secondary anchor positions is referred to as an extended A3
supermotif and is employed to identify molecules in a native
sequence that possess certain desired properties. Alternatively,
the secondary anchors of the supermotif are employed to develop
analogs of peptides that possess residues in accordance with the
definition of the primary A3 supermotif.
[0767] The A3 supermotif including both primary and secondary
anchor positions is referred to as the extended A3 supermotif, and
is employed to identify molecules in a native sequence that possess
certain desired properties. Alternatively, the secondary anchors of
the supermotif are employed to develop analogs of peptides that
possess residues in accordance with the definition of the primary
A3 supermotif.
[0768] Of course, analogs can also be prepared by utilizing the
primary supermotif. For example, native peptide sequences that fall
within the primary A3 supermotif can be analogued by substitution
of another supermotif defined amino acid at a position where
another supermotif defined primary anchor amino acid existed in the
native sequence. Although presently less preferred, an analog of a
native sequence that does not fall within the definition of the
primary supermotif is prepared. Accordingly, one or more amino
acids within the definition of the primary supermotif is
substituted for one or more amino acids of the native sequence
which do not fit the supermotif.
[0769] Accordingly, extended motifs for each of the A3 supertype
alleles analyzed herein (A3, A11, A31, A*3301, and A*6801) were
derived as described in accordance with the methodology used to
define the supermotif for the primary anchor residues. This
approach was similar to the one previously used to define an
extended A*0201 motif (Ruppert, et al., Cell 74:929 (1993)). The
extended motifs were derived by calculating at each nonanchor
position along the peptide sequence, the average relative binding
capacity of peptides carrying each of the 20 common amino acids,
grouped according to individual chemical similarities.
Representative of the data generated by this procedure, the values
calculated for A3 supertype alleles are shown in TABLE 143.
[0770] For example, 21 different peptides were tested which
possessed an aromatic residue (F, W, Y) in position 3 of their
sequence. These peptides had an average relative binding capacity
to A3 31.7-fold higher than the overall average of the 200-peptide
set. By analogy to what was previously described in the case of
A*0201, preferred and deleterious residues were defined as residues
associated with average binding capacities that were four-fold
greater than or four-fold less than, respectively, the overall
average. Accordingly, aromatic residues in position 3 were
considered "preferred" residues for A3 binding. A similar analysis
was performed for each allele (A3, A11, A31, A*3301) and was used
to derive maps of allele-specific secondary anchor requirements for
each position. See TABLE 143.
[0771] Summaries of the extended motifs obtained for peptides which
binds to each HLA protein of the A3 supertype examined are shown in
FIG. 39.
[0772] As depicted in FIG. 39, each protein exhibited its own
unique secondary anchor requirements. For example, positively
charged residues (R, H, K) at position 4 were preferred by the A3
allele, but not by any other A3 supertype molecule. Similarly at
position 8, glycine (G) was associated with poor binding capacity
only for A11, whereas negative charges (D, E) were deleterious only
for A31. Besides these types of unique protein-specific features,
certain residues were associated with either poor or good binding
in a majority of the molecules of the A3 supertype. For example,
proline (P) in position 1 was deleterious for all five of the A3
supertype molecules tested. Aromatic residues (F, W, Y) in position
7 and proline in position 8 were preferred by four of the five
molecules tested (FIG. 39).
[0773] A similar analysis was performed for each allele (A3, A11,
A31, A*3301) and was used to derive maps of allele-specific
secondary anchor requirements for each position (TABLE 140).
Summaries of the modified motifs obtained for each allele of the
A3-like supertype examined are shown in FIG. 39. Each molecule
exhibited its own unique secondary anchor requirements. For
example, positively charged residues(R, H, K) at position 4 were
preferred by A3, but not by any other A3-like molecule. Similarly
at position 8, glycine (G) was associated with poor binding
capacity only for A11, whereas negative charges (D, E) were
deleterious only for A31. Besides these types of unique
allele-specific features, certain residues were associated with
either poor or good binding in a majority of the molecules of the
A3 supertype. For example, proline (P) in position 1 was
deleterious for all five of the A3-like molecules tested. Aromatic
residues (F, W, Y) in position 7 and proline in position 8 were
preferred by four of the five molecules tested (FIG. 39).
[0774] On the basis of the various individual extended motifs, an
extended A3 supermotif was constructed. Residues deleterious for at
least three of the five alleles considered were defined as
"deleterious residues" in the supermotif. Conversely, residues
preferred by at least three of the five alleles considered, but
also not deleterious for any allele, were defined as "preferred
residues." The extended A3 supermotif derived following this
approach is shown in FIG. 40.
[0775] Efficacy of the A3-Extended Supermotif in Predicting Highly
Cross-Reactive Peptides. To test the validity of the extended A3
supermotif defined above, an additional set of 108 peptides not
previously included in the analysis of supermotifs was tested for
binding to HLA molecules encoded by A3, A11, A31, A*3301, and
A*6801 alleles. This set included 30 peptides which had at least
one preferred supermotif residue and no supermotif deleterious
residues, 43 peptides with at least one supermotif deleterious
residue (supermotif negative), and 35 peptides with neither
supermotif preferred nor deleterious residues (supermotif
neutral).
[0776] Of the 30 supermotif positive peptides, 27 (90%) bound to
two or more molecules within the A3 supertype and 16 (53%) bound to
three or more molecules. By contrast, 18 (51%) of 35 extended
supermotif neutral peptides bound two or more A3 types, and eight
(23%) bound three or more molecules. Finally, the supermotif
negative peptides were much less capable of binding multiple
alleles, with six (14%) peptides binding two A3 supertype
molecules, and no peptides binding three or more molecules.
[0777] These results are qualitatively similar to those obtained
when the original set of peptides used to define the primary anchor
residues in the supermotifs was subjected to the same type of
analysis, and are in striking contrast with the level of
cross-reactivity observed in the case of the previously mentioned
binding of a control set of peptides to unrelated HLA proteins, in
which only a few peptides (8%) bound to a protein other than their
intended original protein.
[0778] Calculation of phenotypic frequencies of HLA supertypes in
various ethnic backgrounds and projected population coverage. Gene
frequencies for each HLA allele were calculated from antigen or
allele frequencies in accordance with principles in the art (see
e.g. Imanishi, et al., Proc. of the Eleventh International
Histocompatibility Workshop and Conference, Vol. 1, Tokyo, Oxford
University Press (1992) and Fernandez-Vina, et al., Hum. Immunol.
33:163 (1992)) utilizing the binomial distribution formula:
gf=1-(SQRT(1-af))
(Tiwari, et al., The HLA complex, In HLA AND DISEASE ASSOCIATES,
NY, Springer-Verlag (1985)).
[0779] To obtain overall phenotypic frequencies, cumulative gene
frequencies were calculated and the cumulative antigen frequencies
derived by the use of the inverse formula:
af=1-(1-Cgf).sup.2
[0780] As discussed below, where frequency data was not available
at the level of DNA typing, correspondence to the serologically
defined antigen frequencies was assumed. To obtain total population
coverage no linkage disequilibrium was assumed and only alleles
confirmed as belonging to each of the supertypes were included
(minimal estimates). Estimates of total coverage achieved by
interloci combinations were made by adding to the A coverage the
proportion of the non-A covered population that could be expected
to be covered by the B alleles considered (e.g.,
total=A+B*(1-A)).
[0781] Confirmed members of the A3 supertype are A3, A11, A31,
A*3301, and A*6801. Although the A3 supertype may potentially
include A32, A66, and A*7401, these alleles were not included in
overall frequency calculations.
[0782] High Phenotypic Frequencies of HLA Supertypes are Conserved
in All Major Ethnic Groups. Thus, to evaluate HLA supertypes in
general, and the A3 supertype in particular, the incidence of
various HLA class I alleles or antigens was examined. To date, much
of the available HLA-A and -B population data are based on
serologic typing. These data do not have resolution at the level of
alleles as defined by DNA sequences, and thus do not distinguish
between subtypes. However, comparison of the peptide-binding
specificities of subtypes, either through peptide-binding studies
(del Guercio, et al., J. Immunol. 154:685 (1995); Tanigaki, et al.,
Hum. Immunol. 39:155 (1994)), pool sequencing analysis (Fleischer,
et al., Tissue Antigens 44:311 (1994); Rotzschke, et al., Eur. J.
Immunol. 22:2453 (1992)), or analysis of pocket structure based on
primary sequence, suggest that in most instances subtypes will have
very similar, if not identical, peptide main anchor specificities.
Thus in the following analysis, if population data at the DNA
subtype level were not available, but either binding data,
published motifs, or sequence analysis suggested that subtypes will
have overlapping peptide binding specificities, a one-to-one
correspondence between subtype alleles and the serologically
defined antigens was assumed.
[0783] When the incidence of the various A3 supertype alleles or
antigens in different ethnic backgrounds was examined, it became
apparent that while the frequency of each individual allele or
antigen can vary drastically between ethnic groups (Imanishi, et
al., Proceedings of the Eleventh International Histocompatibility
Workshop and Conference, Vol. 1, Tokyo, Oxford University Press
(1992)), the cumulative frequency of the five A3 supertype alleles,
viewed collectively, is remarkably constant (between 37% to 53%
depending on the ethnic population studied). For example, the
individual A3 allele is common in Caucasians, African-Americans,
and Hispanics, but almost absent in Japanese. Conversely, the A31
allele is frequent in Japanese but rare in Caucasians and
African-Americans. By contrast, in each of the five populations
examined, the A3 HLA supertype was present in at least 37%, and as
high as 53%, of the individuals.
[0784] Notably, the existence of an A3 HLA supertype is not an
isolated incident, as the existence of A2 (del Guercio, et al., J.
Immunol. 154:685 (1995)) and B7 (Sidney, et al., J. Immunol.
154:247 (1995)) supertypes are reported. These additional
supertypes are also very prominent, with remarkably constant
cumulative frequencies (in the 40% to 60% range) amongst different
ethnic backgrounds. These supertypes are discussed in greater
detail in the following Examples. In fact, at the gene level, at
least one half of the total copies of HLA-A or -B genes in
existence appear to belong to one or another of these three HLA
supertypes.
[0785] As pointed out in the Background section, the existence of
an A3 HLA supertype is not an isolated incident, as the A2
supertype (del Guercio, et al., J. Immunol. 154:685 (1995)) and B7
supertype (Sidney, et al., J. Immunol. 154:247 (1995)) are
reported. These A2 and B7 supertypes are also very prominent, with
remarkably constant cumulative frequencies (in the 40% to 60%
range) amongst different ethnic backgrounds. In fact, at the gene
level, at least one half of the total copies of HLA-A or -B genes
in existence appear to belong to one or another of these three HLA
supertypes.
[0786] T Cell Recognition of Supermotif Peptides When Bound by HLA
Molecules. To better gauge the biologic relevance of these
observations, we investigated whether supertype cross-reactive
peptides are recognized by CTLs, when the peptides are bound by
various supertype molecules. Two peptides have been reported as
being recognized by CTLs in the context of more than one A3
supertype allele [see, e.g., Missale, et al, J. Exp. Med. 177:751
(1993); Koenig, et al, J Immunol 145:127 (1990); Culmann, et al, J.
Immunol. 146:1560 (1991)] (see Table 145). Using a method for in
vitro induction of primary CTLs [Wentworth, et al, Mol. Immunol.
32:603 (1995)] we observed several instances in which peptides can
be recognized in the context of both A3 and A11 [P. Wentworth and
A. Sette, unpublished observations] (see TABLE 145). We tested the
A3 supermotif epitopes for binding capacity to A3 supertype
molecules, and noted relatively high levels of degeneracy.
[0787] Of the seven epitopes listed in TABLE 145, only one was a
nonamer that could be analyzed for the extended supermotif proposed
in FIG. 40 (the secondary anchors are presently understood to be
unique to a given epitope length). The sole nonamer peptide was
supermotif positive, and bound three of five A3-like molecules.
Nonetheless, it is important to note that each of the epitopes in
TABLE 145 conformed to the A3-like supertype primary anchor
specifications.
[0788] Identification of A3 Supermotif-Bearing Epitopes in a
Peptide Antigen. A native protein sequence, e.g., a
tumor-associated antigen, an infectious organism or a donated
tissue, is screened to identify sequences that bear the A3
supermotif. In a presently preferred embodiment, the native
sequence is screened using computer-based programs; such programs
are written in accordance with procedures known in the art based on
the A3 supermotif definition disclosed herein. The information
gleaned from this analysis is used directly to evaluate the status
of the native peptide, or may be utilized to subsequently generate
the peptide epitope.
[0789] The information gleaned from analysis of a native peptide
can be used directly to ascertain a number of characteristics. The
characteristics to be ascertained will depend, as appreciated by
one of ordinary skill in the art, on the type of native peptide.
For example, a donor tissue for potential transplantation into a
patient who bears an HLA allele of the A3 supertype can be screened
to identify the prevalence of A3 supermotif epitopes in a target
antigen that has been observed to be immunogenic
post-transplantation; if alternative donor tissues are available,
the tissue having the lowest prevalence of A3 supermotif epitopes
would be chosen based on this parameter. If an infectious organism
has more than one strain, an A3 epitope can be located in one
strain, and then other strains of the same organism can be
evaluated to determine the conservancy of that epitope throughout
the strains. A given infectious organism can be evaluated
sequentially; e.g., an epitope from a viral organism can be
evaluated in an initial screening, and its presence tracked over
time to determine if that epitope is being mutated. If a
therapeutic response has been directed to the epitope this
mutagenic phenomenon is referred to as viral escape and methods of
identification of epitopes can be used to track this phenomenon. If
a therapeutic composition is designed to be directed to an epitope,
non-diseased tissues from the potential recipient can be biopsied
and evaluated to determine whether the composition has potential
for inducing as adverse autoimmune-type response in the recipient.
Furthermore, upon identification of an epitope in a native peptide,
that epitope sequence can be evaluated in accordance with the
analoging disclosures presented herein and in applications from
which priority is claimed.
[0790] Upon identification of an epitope in a native peptide, that
epitope can be synthesized by any number of procedures in the art.
The epitope can be synthesized directly, such as by chemical means,
or indirectly such as by use of nucleic acids that encode the
epitope. The synthesized epitope can be used to induce a
therapeutic or prophylactic immune response in a recipient.
[0791] Selection of A3 Supertype Epitopes For Inclusion in a
Disease-Specific Vaccine. This example illustrates the procedure
for the selection of A3 peptide epitopes for a vaccine composition
of the invention.
[0792] The following principles are utilized when selecting an
array of epitopes from a particular disease-related antigen,
whether the epitopes are discrete in a composition, are embedded or
overlapping in a native sequence, and/or to be encoded by a
minigene. Such embodiments are used to create a vaccine to
prophylax or treat the disease in patients who bear an HLA allele
from the A3 supertype. Each of the following principles are
balanced in order to make the selection.
[0793] 1.) Epitopes are selected which, upon administration, mimic
immune responses that have been observed to be correlated with
disease clearance. For HLA Class I this includes, as an example,
3-4 epitopes that come from at least one antigen of a disease
causing organism or cancer-associated antigen. In other words, this
comports with a scenario where it has been observed that in
patients who spontaneously clear the disease, that they had
generated an immune response to at least 3 epitopes on at least one
disease antigen. For HLA Class II a similar rationale is employed;
again 3-4 epitopes are selected from at least one disease
antigen.
[0794] 2.) Epitopes are selected that have the requisite binding
affinity established to be correlated with immunogenicity: for HLA
Class I an IC.sub.50 of 500 nM or less, or for Class II an
IC.sub.50 of 1000 nM or less.
[0795] 3.) In this example A3 epitopes are employed that provide
population coverage among patients who bear an A3 supertype allele.
The following example discusses selection of epitopes to achieve
even broader coverage.
[0796] When selecting epitopes for disease-related antigens it can
be preferable to select native epitopes; although not always the
case a patient may have developed tolerance to tumor-associated
antigens, whereby analogs of native epitopes may be useful, analogs
are also useful for infectious disease antigens.
[0797] Therefore, of relevance as a vaccine, particularly for
infectious disease vaccines, are epitopes referred to as "nested
epitopes." Nested epitopes occur where at least two epitopes
overlap in a given peptide sequence. A peptide comprising
"transcendent nested epitopes" is a peptide that has both HLA class
I and HLA class II epitopes in it.
[0798] When providing nested epitopes, a sequence that has the
greatest number of epitopes per provided sequence is provided. A
correlate to this principle is to avoid providing a peptide that is
any longer than the amino terminus of the amino-terminal epitope
and the carboxyl terminus of the carboxyl-terminal epitope in the
peptide. When providing a longer peptide sequence, such as a
sequence comprising nested epitopes, the sequence is screened in
order to insure that it does not have pathological or other
deleterious biological properties.
[0799] 5.) When creating a minigene, as disclosed in greater detail
in other Examples, an objective is to generate the smallest peptide
possible that encompasses the epitopes of interest. The principles
employed are similar, if not the same as those employed when
selecting a peptide comprising nested epitopes. Thus, upon
determination of the nucleic acid sequence to be provided as a
minigene, the peptide encoded thereby is analyzed to determine
whether any "junctional epitopes" have been created. A junctional
epitope is an actual binding epitope, as predicted, e.g., by motif
analysis. Junctional epitopes are to be avoided because the
recipient may generate an immune response to that epitope, i.e., an
epitope not found in the native disease-related antigen. Of
particular concern is a junctional epitope that is a "dominant
epitope." A dominant epitope may lead to such a zealous response
that immune responses to other epitopes are diminished or
suppressed.
[0800] A vaccine composition comprised of selected peptides, when
administered, is safe, efficacious, and elicits an immune response
similar in magnitude of an immune response that clears an acute HBV
infection.
[0801] Selection of A3 Supertype Epitopes and Additional HLA
Epitopes, to Achieve Broadened Population Coverage in a
Disease-Specific Vaccine. This example exemplifies the procedures
to use to prepare a vaccine that covers a patient population that
bears an A3 supertype as well as one or more patient population(s)
that bear another HLA type or HLA supertype. To select such an
array of epitope, a protocol such as set forth in Example 14 is
employed, with the exception of a variation at parameter 3.) of
that example.
[0802] In order to achieve population coverage beyond a population
that bears A3 supertype alleles, A3 supermotif peptides along with
peptides that bear another supermotif, or a sufficient array of
allele-specific motif bearing peptides, are selected to give
broadened population coverage. For example, epitopes are selected
to provide at least 80% population coverage. A Monte Carlo
analysis, a statistical evaluation known in the art, is employed to
assess population coverage. Upon combining epitopes from, e.g.,
several supertypes a vaccine directed to a disease has more than
98% population coverage for 5 prevalent worldwide ethnic
groups.
[0803] A Polyepitopic Vaccine Composition Derived From A
Disease-Associated Peptide Antigen. A native protein sequence,
e.g., a tumor associated antigen or an infectious organism, is
screened, preferably using computer programs defined to identify
the presence of epitopes bearing the A3 supermotif, and optionally
epitope(s) bearing one or more HLA class I and/or class II
supermotif or motif, to identify "relatively short" regions of the
polyprotein that comprise multiple epitopes. This relatively short
sequence that contains multiple distinct, even overlapping,
epitopes is selected and used to generate a minigene construct or
for peptide synthesis. The minigene construct is engineered to
express the peptide, which corresponds to the native protein
sequence. The "relatively short" peptide is less than 100 amino
acids in length, preferably less than 75 amino acids in length, and
more preferably less than 50 amino acids in length. The protein
sequence of the vaccine composition is selected because it has
maximal number of epitopes contained within the sequence. As noted
herein, epitope motifs may be overlapping (i.e., frame shifted
relative to one another) with frame shifted overlapping epitopes,
e.g. two 9-mer epitopes can be present in a 10 amino acid peptide.
Such a vaccine composition is administered for therapeutic or
prophylactic purposes.
[0804] The vaccine composition will preferably include, for
example, three CTL epitopes, at least one of which is an A3
supermotif epitope, and at least one HTL epitope from the source
antigen. This polyepitopic native sequence is administered either
as a peptide or as a nucleic acid sequence which encodes the
peptide. Alternatively, an analog can be made of this native
sequence.
[0805] The embodiment of this example provides for the possibility
that an as yet undiscovered aspect of immune system processing will
apply to the native nested sequence and thereby facilitate the
production of therapeutic or prophylactic immune response-inducing
vaccine compositions. Additionally such an embodiment provides for
the possibility of motif-bearing epitopes for an HLA makeup that is
presently unknown. Furthermore, this embodiment directs the immune
response to sequences that are present in native HBV antigens.
Lastly, the embodiment provides an economy of scale when producing
nucleic acid vaccine compositions.
[0806] Related to this embodiment, computer programs are used which
identify, in a target sequence, the greatest number of epitopes per
sequence length.
[0807] Polyepitopic Vaccine Compositions Directed To Multiple
Diseases. Peptide epitopes bearing the A3 supermotif from a first
disease-related source are used in conjunction with A3
supermotif-bearing peptide epitopes from target antigens related to
one or more other diseases, to create a vaccine composition that is
used to prevent or treat a first disease as well as at least one
other disease. Examples of infectious diseases include, but are not
limited to, HIV, HBV, HCV, and HPV; examples of cancer-related
antigens are CEA, HER2, MAGE and p53.
[0808] In a preferred embodiment, not only are two or more diseases
targeted, but epitope(s) that bear the A3 supermotif and at least
one other motif are comprised by the composition. In this preferred
embodiment, the composition is used to treat multiple diseases
across a genetically diverse HLA patient population.
[0809] For example, a polyepitopic peptide composition comprising
multiple CTL and HTL epitopes that target greater than 98% of the
population may be created for administration to individuals at risk
for both HBV and HIV infection. The composition can be provided as
a single polypeptide that incorporates the multiple epitopes from
the various disease-associated sources.
[0810] Use Of Peptides To Evaluate An Immune Response. Peptides of
the invention may be used to analyze an immune response for the
presence of specific CTL populations corresponding to HBV from
patients whom possess an HLA allele in the A3 supertype. Such an
analysis may be performed as described by Ogg et al., Science
279:2103-2106, 1998. In the following example, peptides in
accordance with the invention are used as a reagent for diagnostic
or prognostic purposes, not as an immunogen.
[0811] In this example highly sensitive human leukocyte antigen
tetrameric complexes ("tetramers") may be used for a
cross-sectional analysis of, for example, HBV Env-specific CTL
frequencies from untreated HLA A3 supertype-positive individuals at
different stages of infection using an HBV Env peptide containing
an A2.1 extended motif. Tetrameric complexes are synthesized as
described (Musey et al., N. Engl. J. Med. 337:1267, 1997). Briefly,
purified heavy chain from an HLA molecule from the A3 supertype,
and .beta.2-microglobulin are synthesized by means of a prokaryotic
expression system. The heavy chain is modified by deletion of the
transmembrane-cytosolic tail and COOH-terminal addition of a
sequence containing a BirA enzymatic biotinylation site. The heavy
chain, .beta.2-microglobulin, and peptide are refolded by dilution.
The 45-kD refolded product is isolated by fast protein liquid
chromatography and then biotinylated by BirA in the presence of
biotin (Sigma, St. Louis, Mo.), adenosine 5'triphosphate and
magnesium. Streptavidin-phycoerythrin conjugate is added in a 1:4
molar ratio, and the tetrameric product is concentrated to 1 mg/ml.
The resulting product is referred to as tetramer-phycoerythrin.
[0812] Approximately one million PBMCs are centrifuged at 300 g for
5 minutes and resuspended in 50 ul of cold phosphate-buffered
saline. Tri-color analysis is performed with the
tetramer-phycoerythrin, along with anti-CD8-Tricolor, and
anti-CD38. The PBMCs are incubated with tetramer and antibodies on
ice for 30 to 60 min and then washed twice before formaldehyde
fixation. Gates are applied to contain >99.98% of control
samples. Controls for the tetramers include both A3
supertype-negative individuals and A3 supertype-positive uninfected
donors. The percentage of cells stained with the tetramer is then
determined by flow cytometry. The results indicate the number of
cells in the PBMC sample that contain epitope-restricted CTLs,
thereby readily indicating the stage of infection with HBV or the
status of exposure to HBV or to a vaccine that elicits a protective
response.
[0813] Use Of Peptide Epitopes To Evaluate Recall Responses. The
peptide epitopes of the invention are used as reagents to evaluate
T cell responses such as acute or recall responses, in patients
whom bear an allele from the HLA A3 supertype. Such an analysis may
be performed on patients who have recovered from infection, who are
chronically infected with the disease, or who have been vaccinated
with a disease-protective vaccine.
[0814] For example to evaluate HBV immune status, the class I
restricted CTL response of persons at risk for HBV infection who
have been vaccinated may be analyzed. The vaccine may be any HBV
vaccine. PBMC are collected from vaccinated individuals and HLA
typed. Appropriate peptide reagents that are both highly conserved
and, bear the A3 supermotif to provide cross-reactivity with
multiple HLA A3 supertype family members are then used for analysis
of samples derived from individuals who bear the HLA supertype.
[0815] PBMC from vaccinated individuals are separated on
Ficoll-Histopaque density gradients (Sigma Chemical Co., St. Louis,
Mo.), washed three times in HBSS (GIBCO Laboratories), resuspended
in RPMI-1640 (GIBCO Laboratories) supplemented with L-glutamine (2
mM), penicillin (50 U/ml), streptomycin (50 .mu.g/ml), and Hepes
(10 mM) containing 10% heat-inactivated human AB serum (complete
RPMI) and plated using microculture formats. Synthetic peptide is
added at 10 .mu.g/ml to each well and recombinant HBc Ag is added
at 1 .mu.g/ml to each well as a source of T cell help during the
first week of stimulation.
[0816] In the microculture format, 4.times.10.sup.5 PBMC are
stimulated with peptide in 8 replicate cultures in 96-well round
bottom plate in 100 .mu.l/well of complete RPMI. On days 3 and 10,
100 ml of complete RPMI and 20 U/ml final concentration of rIL-2
are added to each well. On day 7 the cultures are transferred into
a 96-well flat-bottom plate and restimulated with peptide, rIL-2
and 10.sup.5 irradiated (3,000 rad) autologous feeder cells. The
cultures are tested for cytotoxic activity on day 14. A positive
CTL response requires two or more of the eight replicate cultures
to display greater than 10% specific .sup.51Cr release, based on
comparison with uninfected control subjects as previously described
(Rehermann, et al., Nature Med. 2:1104,1108, 1996; Rehermann et
al., J. Clin. Invest. 97:1655-65, 1996; and Rehermann et al. J.
Clin. Invest. 98:1432-40, 1996).
[0817] Target cell lines are autologous and allogeneic
EBV-transformed B-LCL that are either purchased from the American
Society for Histocompatibility and Immunogenetics (ASHI, Boston,
Mass.) or established from the pool of patients as described
(Guilhot, et al. J. Virol. 66:2670-78, 1992).
[0818] Cytotoxicity assays are performed in the following manner.
Target cells consist of either allogeneic HLA-matched or autologous
EBV-transformed B lymphoblastoid cell line that are incubated
overnight with synthetic peptide at 10 .mu.M and labeled with 100
.mu.Ci of .sup.51Cr (Amersham Corp., Arlington Heights, Ill.) for 1
hour after which they are washed four times with HBSS. Cytolytic
activity is determined in a standard 4-h, split well .sup.51Cr
release assay using U-bottomed 96 well plates containing 3,000
targets/well. Stimulated PBMC are tested at E/T ratios of 20-50:1
on day 14. Percent cytotoxicity is determined from the formula:
100.times.[(experimental release-spontaneous release)/maximum
release-spontaneous release)]. Maximum release is determined by
lysis of targets by detergent (2% Triton X-100; Sigma Chemical Co.,
St. Louis, Mo.). Spontaneous release is <25% of maximum release
for all experiments.
[0819] The results of such an analysis will indicate to what extent
HLA-restricted CTL populations have been stimulated with the
vaccine. Of course, this protocol can also be used to monitor prior
HBV exposure.
[0820] The above examples are provided to illustrate the invention
but not to limit its scope. For example, the human terminology for
the Major Histocompatibility Complex, namely HLA, is used
throughout this document. It is to be appreciated that these
principles can be extended to other species as well. Moreover,
peptide epitopes have been disclosed in the related application
U.S. Ser. No. 08/820,360, which was previously incorporated by
reference. Thus, other variants of the invention will be readily
apparent to one of ordinary skill in the art and are encompassed by
the appended claims. All publications, patents, and patent
application cited herein are hereby incorporated by reference for
all purposes.
[0821] HLA Binding of Supermotifs and Extended Supermotifs. The A3
supertype restricted epitopes were tested for binding capacity to
A3 supertype molecules, and relatively high levels of
cross-reactivity were noted. Of the seven epitopes listed in TABLE
145, only one was a nonamer that could be analyzed for the
supermotif proposed in FIG. 40. This peptide was supermotif
positive, and bound three of five A3 molecules. Nonetheless, it is
important that each of the epitopes conformed to the A3 supertype
primary anchor specificities.
[0822] The phenomena of HLA super types may be related to optimal
exploitation of the peptide specificity of human transporter
associated with antigen processing (TAP) molecules (Androlewicz, et
al., Proc. Nat'l Acad. Sci. USA 90:9130 (1993); Androlewicz, et
al., Immunity 1:7 (1994); van Endert, et al., Immunity 1:491
(1994); Heemels, et al., Immunity 1:775 (1994); Momburg, et al.,
Curr. Opin. Immunol. 6:32 (1994); Neefjes, et al., Science 261:769
(1993)). The TAP molecules have been shown to preferentially
transport peptides with certain sequence features such as
hydrophobic, aromatic, or positively charged C-termini.
[0823] Recent studies, performed by van Endert and associates, in
collaboration with the present inventors, evaluated the relative
affinities for TAP of a large collection of peptides, and have
described an extended TAP binding motif (Van Endert et al. J. Exp.
Med. 182:1883 (1995)) Strikingly, this tap motif contains many of
the structural features associated with the A3 extended supermotif,
such as the preference for aromatic residues at positions 3 and 7
of nonamer peptides and the absence of negatively charged residues
at positions 1 and 3, and P at position 1.
HLA A3 Supertype Findings
[0824] The data from this Example demonstrate that products from at
least five different HLA alleles (A3, A11, A31, A*3301, and
A*6801), and likely at least three others (A*3401, A*6601, and
A*7401) predicted on the basis of pocket analysis (data not shown),
are properly grouped into a single functional HLA A3 supertype.
This determination was made on the basis of a number of
observations. As a group, these molecules: (a) share certain key
structural features within their peptide-binding regions; (b) have
similar preferences for the primary anchor residues in the peptides
they bind, i.e., a primary supermotif present in the peptides bound
by the HLA molecules of the superfamily; and (c) share largely
overlapping binding repertoires. Knowledge of the A3 supermotif
allows for identification of a cross-reactive peptide for a source,
and allows for production of peptide analogs by substituting at
primary anchor positions to alter the binding properties of the
peptides.
[0825] Furthermore, by examining the binding activity of a large
panel of peptides bearing the primary A3 supermotif, an extended A3
supermotif was defined. This extended supermotif was based on a
detailed map of the secondary anchor requirements for binding to
molecules of the A3 supertype. The extended supermotif allows for
the efficient prediction of cross-reactive binding of peptides to
alleles of the A3 supertype by screening the native sequence of a
particular antigen. This extended supermotif is also used to select
analog options for peptides which bear amino acids defined by the
primary supermotif.
[0826] The discovery of the individual residues of the secondary
anchor motif disclosed herein represents a significant contribution
to the understanding of peptide binding to class I molecules. These
secondary anchor maps were derived using peptides of homogeneous
size. Thus, the preference determinations at each of the secondary
positions may be more accurate than those derived from the
sequencing of pools of naturally processed peptides. Also, the
motifs defined herein allow the determination of residues which
have deleterious or other types of effects on peptide binding.
[0827] The definition of primary and secondary anchor specificities
for the A3 supertype provides guidance for modulating the binding
activity of peptides that bind to members of the A3 supertype
family. This information may be used to generate highly
cross-reactive epitopes by identifying residues within a native
peptide sequence that can be analogued to increase greater binding
cross-reactivity within a supertype, or analogued to increase
immunogenicity.
Example 18
Definition of HLA-A1-Specific Peptide Motifs
[0828] HLA-A1 molecules were isolated and their naturally processed
peptides characterized, as described in Example 3 above. In one
case using MAT cells, pooled fractions corresponding to 19% to 50%
CH.sub.3CN were used. As in the preceding example, residues showing
at any given position except the first position, at least a two
standard deviation increase over the random expected yield were
identified and shown in TABLE 74. On the basis of these data, only
Serine (S) and Threonine (T) were increased at position two. At
position 3, aspartic acid (D) and glutamic acid (E) were elevated
and at position 9 and 10 tyrosine (Y) showed a marked increase.
Other increases noted were proline (P) at position 4 and leucine
(L) at position 7. Therefore, the motifs for HLA-A1 based on these
data would have residues at position 2 occupied by S or T, a
peptide length of 9 or 10 amino acids and a C-terminal residue of
Y. Alternatively, another motif would comprise a D or E at position
3 together with a C terminal residue of Y.
Example 19
Definition of HLA-A11-Specific Peptide Motifs
[0829] HLA-A11 motifs were defined by amino acid sequence analysis
of pooled HPLC fractions, in one case corresponding to 7% to 45%
CH.sub.3CN of fractionated peptides eluted from HLA-A11 molecules
purified from the cell line BVR. On the basis of the data presented
in TABLE 75, a motif for A11 consists of a conserved residue at
position 2 of threonine (T) or valine (V), a peptide length of 9 or
10 amino acids, and a C-terminal conserved residue of lysine (K).
At position 3 increases in methionine (M) and phenylalanine (F)
were also seen and at position 8 glutamine (Q) was increased.
Example 20
Definition of HLA-A24.1-Specific Peptide Motifs
[0830] HLA-A24.1 allele-specific motifs were defined by amino acid
sequence analysis of pooled fractions in one case corresponding to
7% to 19% CH.sub.3CN of HPLC fractionated peptides eluted from
HLA-A24.1 molecules purified from the cell line KT3. On the basis
of the data presented in TABLE 76 a motif for HLA-A24.1 consists of
a conserved residue at position 2 occupied by tyrosine (Y), a
peptide length of 9 or 10 amino acids, and a C-terminal conserved
residue of phenylalanine (F) or leucine (L). Increases were also
observed at several other positions: isoleucine (I) and methonine
(M) at position 3; aspartic acid (D), glutamic acid (E), glycine
(G), lysine (K) and proline (P) at position 4; lysine (K),
methonine (M) and asparagine (N) at position 5; valine (V) at
position 6; asparagine (N) and valine (V) at position 7; and,
alanine (A), glutamic acid (E), lysine (K), glutamine (Q) and
serine (S) at position 8.
Example 21
B7Supertype Binding
[0831] Data indicated (Sidney, et al., J. Immunol. 154, 247 (1995);
Hill, et al., Nature 360:434 (1992); Falk, et al., Immunogenetics
38:161 (1993); Barber, et al., Curr. Biol. 5:179 (1995); Schonbach,
et al. J. Immunol. 154:5951 (1995)) that there is a relatively
large family of HLA B specificities, collectively defined as the B7
supertype. In this Example the molecular binding assays as
described in Example 1 are used to examine, in detail, the primary
anchor specificities (position 2 and C-terminus) of the five most
frequent B7 supertype HLA alleles (B*0702, B*3501, B51, B*5301, and
B*5401). The B7 supermotif was found to be characterized by
peptides that have a P in position 2, and a hydrophobic or aromatic
residue at the C-terminus (referred to as the B7 supermotif).
[0832] Characterization of the primary anchor specificity of B7
supertype alleles was performed utilizing a panel of single
substitution analogs of the HIV nef 84-92 peptide (sequence
FPVRPQVPL (SEQ ID NO:______). HIV nef 84-92 binds HLA molecules
encoded by the B*0702, B*3501, B51, B*5301, and B*5401 alleles with
either high (IC.sub.50.ltoreq.50 nM) or intermediate (IC.sub.50
50-500 nM) affinity.
[0833] It was found that all five B7 supertype molecules share a
rather stringent position 2 specificity for proline. With only one
exception (A in the case of B*3501), all of the substitutions
eliminating P at position 2 were associated with greater than
10-fold decreases in binding affinity as compared to the parent
peptide. By contrast, each HLA-B type expressed a rather unique
specificity pattern at the C-terminus. For example, B*0702
preferred M, F and L, while B 5101 preferred L, I, and V. Despite
these differences, the overall C-terminal specificity patterns
exhibited a large degree of overlap. All alleles shared a
specificity for residues of a hydrophobic chemical nature. The
aliphatic residues I and V were preferred by at least four of the
five molecules, and A, L, M, F, and W were preferred or tolerated
in a majority of instances. Other residues, such as Y or T, were
tolerated in only isolated instances, while some (e.g., K or D)
were not tolerated at all.
[0834] This primary anchor specificity data is in agreement with
data of Sidney, et al., J. Immunol. 154:247 (1995). Thus, peptides
capable of cross-reactive B7 supertype binding should have proline
in position 2 and a hydrophobic or aromatic (V, I, L, M, F, W, A)
residue at their C-terminus. In formally defining the B7 supertype
primary anchor motifs, Y has been conservatively included despite
its relative lack of cross-reactivity, because Y constitutes the
dominant signal in pool sequencing analyses of B*3501 (Hill, et
al., Nature 360:434 (1992); Falk, et al., Immunogenetics 38:161
(1993), Schonbach, et al. J. Immunol. 154:5951 (1995)). In summary,
the primary anchor motif of the B7 supertype is defined as P at
position 2, and A, I, L, M, V, F, W, and Y at the C-terminus.
[0835] Preferred Amino Acid Length of Ligands Bound by the B 7
Supertype HLA Molecules. Class I molecules usually prefer peptides
between 8 and 10 residues in length (Falk, et al., Nature 351:290
(1991)), although longer peptides have been shown to bind (Massale,
et al., J. Exp. Med. 177:751 (1993); Chen, et al., J. Immunol.
152:2874 (1994); Collins, et al. Nature 371:626 (1994)). To
determine the optimal peptide length for binding to molecules of
the B7 supertype, panels of 8-, 9-, 10- and 11-mer peptides
representing naturally occurring viral, tumor, or bacterial
sequences, (each peptide bore the B7 primary anchor supermotif)
were synthesized and tested in binding assays.
[0836] It was concluded that 9 amino acid residues represent the
optimal peptide length for all of the B7 supertype molecules
examined. This assessment was true both in terms of the percent of
peptides of each size bound by any molecule, but also in terms of
the degree of crossreactivity observed (data not shown). Thus, this
information is relevant when preparing analogs that are longer or
shorter than a starting native peptide.
[0837] Extended Supermotif (Secondary Anchor Motifs) of Peptides
that bind B7 Supertype HLA Molecules. Other residues can act as
secondary anchors, thus providing supplemental binding energy to
the peptides (Ruppert, et al, Cell 74:929-37 (1993); Madden, et al.
Cell 75, 693-708 (1993); Saito, et al., J. Biol. Chem. 268, 21309
(1993); Sidney, et al., Hu. Immunol. 45, 79-93 (1996); Kondo, et
al, J. Immunol. 155:4307-12 (1995); Parker, et al., J. Immunol.
152, 163-75 (1994)). It has also been shown that certain residues
can have negative effects on peptide binding to class I molecules
(Ruppert, et al, Cell 74:929-37 (1993); Sidney, et al., Hu.
Immunol., 45, 79-93 (1996); Kondo, et al, J. Immunol. 155:4307-12
(1995), Boehncke, et al., J. Immunol. 150, 331-41 (1993)).
[0838] To develop an extended B7 supermotif allowing the efficient
selection of peptides with cross-reactive B7 supertype binding,
secondary anchors and secondary effects involved in peptide binding
to B7 supertype molecules were defined in accordance with the
methods described herein.
[0839] The binding capacity of 199 nonamer peptides for the five
most common B7 supertype molecules, B*0702, B*3501, B51, B*5301,
and B*5401 was determined, and the data analyzed. The 199 nonamer
peptides represented naturally occurring viral sequences containing
the B7 supertype primary anchors (proline in position 2, and A, V,
I, L, M, F, and W at the C-terminus). For each position the average
relative binding capacity (ARBC) of peptides carrying each of the
20 amino acids was calculated and compared to the ARBC of the
entire peptide set. The occurrence of certain amino acids is very
infrequent, thus, residues were grouped according to individual
chemical similarities as previously described (Ruppert, et al, Cell
74:929 (1993)). This analysis was performed separately for B*0702,
B*3501, B51, B*5301, and B*5401.
[0840] It was found that the patterns of preferences and aversions,
in terms of secondary anchors, exhibited by each molecule were
unique. For example, in the panel tested, 18 peptides had
positively charged residues (R, H or K) in position 1. These
peptides, as a group, were very good B*0702 binders, having an ARBC
of 21. For B51, however, the same peptides were relatively poor
binders, with an ARBC of 0.25. However, profound similarities in
preferences were noted. For example, peptides bearing aromatic
residues (F, W, and Y) in position one were, as a group, very good
binders across the set of B7 supertype molecules, with ARBC of 4.2,
17, 16, 20, and 70 for B*0702, B*3501, B51, B*5301, and B*5401,
respectively.
[0841] The values discussed above were subsequently used to derive
maps of allele-specific secondary anchor requirements for each
position. To do this, preferred and deleterious residues were
defined as residues associated with ARBCs that were 3-fold greater
than, or 3-fold less than, respectively, the overall average. These
preferred and deleterious effects are summarized in FIG. 41. These
secondary anchor maps reveal that while each molecule exhibited its
own unique secondary anchor requirements, certain features were
highly conserved amongst the B7 supertype molecules. For example,
as indicated above, aromatic residues (F, W, and Y) at position 1
were preferred by all five of the B7 supertype molecules.
Conversely, at position 8, acidic residues (D, E) were associated
with poor binding capacity for four of five molecules.
[0842] Secondary effects preferred by three or more of the five B7
supertype molecules considered, were defined as shared. Shared
positive (preferred) effects were defined only if not deleterious
for any molecule. Conversely shared deleterious effects could not
be positive for any molecule. These shared features were
incorporated into an extended B7 supermotif which defined residues
associated with either poor or good binding in a majority of the
molecules of the B7 supertype.
[0843] Following this rationale, it was found that peptides bearing
supermotif preferred secondary residues exhibited a greater degree
of B7 supertype cross-reactivity than those which bear none, or
which bear deleterious residues. This finding was established by
determining the binding cross-reactivity of an independent set of
peptides bearing the B7 supertype primary anchor specificity. As
predicted, peptides which were extended supermotif positive (i.e.,
peptides with at least one extended supermotif preferred secondary
residues, and no deleterious residues) exhibited a substantially
greater degree of crossreactivity within the B7 supertype than
supermotif negative peptides (peptides with one or more supermotif
deleterious residues).
[0844] Implementation of the B7Supermotif to Improve the Binding
Capacity of Native Peptides by Creating Analogs. HLA supermotifs
(both primary and extended) are of value in predicting highly
cross-reactive native peptides, as demonstrated herein. Moreover,
definition of HLA supermotifs also allows one to engineer highly
crossreactive "degenerate" epitopes by identifying residues within
a native peptide sequence which can be analogued, or "fixed", to
confer upon a peptide certain characteristics, e.g., greater
binding cross-reactivity within the supertype.
[0845] To assess this possibility, six peptides which had been
shown to have a high degree of degeneracy within the B7-like
supertype were selected (TABLE 144). Each peptide already bound at
least three of the five most common B7-like molecules with either
high (IC.sub.50<50 nM) or intermediate (IC.sub.50 50-500 nM)
affinities. These peptides were analyzed in the context of both the
B7-like supermotif and the allele specific secondary anchor motifs
described above to determine if particular residues within their
sequences could be "fixed" to further increase their binding to the
B7-like supertype molecules. This assessment found that none of the
particular peptides considered contained a supermotif negative
residue. Three peptides (HCV core 168, MAGE 2170, and MAGE 3 196)
each had one residue which was deleterious for a single B7-like
molecule (TABLE 144).
[0846] Next, a panel of single substituted analogs was synthesized.
Some analogs contained secondary anchor substitutions which were
either supermotif positive, or positive in the context of a
particular allele without being deleterious for any other
substitutions were selected on the basis of the values disclosed
here. Because the preferences for the C-terminal primary anchors
were unique for each allele, substitutions at this position were
also considered. Thus, for example, to test if degeneracy could be
increased a number of analogs were made by substituting the
supermotif positive F for the native residue in position 1. Other
substitutions, such as the C-terminal L for Y in HBV pol 541 were
made to address poor binding of the parent peptide to B*0702 and
B*5401. When this panel was tested for its binding capacity to
molecules of the B7-like supertype, the data shown in TABLE 144 was
generated. In every case, an F substitution in position one
exhibited increased binding and/or degeneracy compared to the
parent sequence. For example, MAGE 2 170 bound with high affinity
to B*0702, intermediate affinity to B*3501, B51, and B*5301, but
only weakly to B*5401. The F1 analog of this peptide bound all five
of these molecules with high affinity.
[0847] The success of substitutions aimed at specific molecules
were much harder to generalize. For example, the substitution of L
at the C-terminus of HBV pol 541 for the native Y was successful in
conferring binding to B*0702 while increasing the binding affinity
to other molecules (significantly in the cases of B51 and B*5401).
In other instances, the effect observed was not as anticipated, as
demonstrated by the case of HBV env 313. This peptide bound with
high affinity to B*0702, B*3501, B51, and B*5301, but only weakly
to B*5401. An M in 5 analog was made to increase B*5401 binding
based on the observation that the aliphatic residues (L, I, V, and
M) in position 5 were positive for B*5401, and relatively neutral
for other molecules. As shown in TABLE 144, however, the
significantly increased B*5401 binding capacity achieved with the
M5 analog was at the expense of lowered binding to B*0702, B51, and
B*5301. While the success of individual analogs was variable, it is
notable that for each case at least one analog was capable of
either improving the binding affinity, or extending the degeneracy
of the parent peptide. Thus, already degenerate peptides can be
discretely "fixed" to improve their binding capacity and extend
their degeneracy.
[0848] For example, analogs representing primary anchor single
amino acid substitutions to I at the C-terminus of two different
B7-like peptides (HBV env 313 and HB pol 541) were synthesized and
tested for their B7-supertype binding capacity. It was found that
the I substitution had an overall positive effect on binding
affinity and/or cross-reactivity in both cases. In the case of HBV
env 313 the 19 replacement was effective in increasing
cross-reactivity from 4 to 5 alleles bound by virtue of an almost
400-fold increase B*5401 binding affinity. In the case of HBV pol
541, increased cross-reactivity was similarly achieved by a
substantial increase in B*5401 binding. Also, significant gains in
binding affinity for B*0702, B51, and B*5301 were observed with the
HBV pol 541 I9 analog.
[0849] Thus, HLA supermotifs are of value in engineering highly
cross-reactive peptides by identifying particular residues at
secondary anchor positions that are associated with such
cross-reactive properties. To demonstrate this, the capacity of a
second set of peptides representing discreet single amino acid
substitutions at positions one and three of five different
B7-supertype binding peptides were synthesized and tested for their
B-7 supertype binding capacity. In 4/4 cases the effect of
replacing the native residue at position 1 with the aromatic
residue F (an "F1" substitution) resulted in an increase in
cross-reactivity, compared to the parent peptide, and, in most
instances, binding affinity was increased three-fold or better
(TABLE 146). More specifically, for HBV env 313, MAGE2 170, and HCV
core 168 complete supertype cross-reactivity was achieved with the
F1 substitution analogs. These gains were achieved by dramatically
increasing B*5401 binding affinity. Also, gains in affinity were
noted for other alleles in the cases of HCV core 168 (B*3501 and
B*5301) and MAGE2 170 (B*3501, B51 and B*5301). Finally, in the
case of MAGE3 196, the F1 replacement was effective in increasing
cross-reactivity because of gains in B*0702 binding. An almost
70-fold increase in B51 binding capacity was also noted.
[0850] Two analogs were also made using the supermotif positive F
substitution at position three (an "F3" substitution). In both
instances increases in binding affinity and cross-reactivity were
achieved. Specifically, in the case of HBV pol 541, the F3
substitution was effective in increasing cross-reactivity by virtue
of its effect on B*5401 binding. In the case of MAGE3 196, complete
supertype cross-reactivity was achieved by increasing B*0702 and
B*3501 binding capacity. Also, in the case of MAGE3 196, it is
notable that increases in binding capacity between 40 and 5000-fold
were obtained for B*3501, B51, B*5301, and B*5401.
[0851] In conclusion, these data demonstrate that by the use of
single amino acid substitutions it is possible to increase the
binding affinity and/or cross-reactivity of peptide ligands for HLA
B7 supertype molecules.
Example 22
Identification of Immunogenic Peptides
[0852] To identify peptides of the invention, class I antigen
isolation, and isolation and sequencing of naturally processed
peptides was carried out as described above and in the parent
applications. These peptides were then used to define specific
binding motifs for each of the following alleles A3.2, A1, A11, and
A24.1. These motifs are described above. The motifs described in
TABLE 73, TABLE 74, TABLE 75, and TABLE 76, below, are defined from
pool sequencing data of naturally processed peptides as described
in the related applications. These motifs are described in the
parent applications and summarized in TABLE 73, TABLE 74, TABLE 75,
and TABLE 76, below.
[0853] Using the motifs identified above for various MHC class I
allele amino acid sequences from various pathogens and
tumor-related proteins were analyzed for the presence of these
motifs. Screening was carried out described in the related
applications. TABLE 11 provides the results of searches of the
antigens.
[0854] The peptides listed in TABLES 81-84 were identified as
described above and are grouped according to pathogen or antigen
from which they were derived.
[0855] Using the B7-like-supermotifs identified in the parent
applications described above, sequences from potential antigenic
sources including Hepatitis B Virus (HBV), Hepatitis C Virus (HCV),
Human Papilloma Virus (HPV), Human Immunodeficiency Virus (HIV),
MAGE2/3, and Plasmodium were analyzed for the presence of these
motifs.
[0856] In some embodiments, sequences for the target antigens were
obtained from the current GenBank data base. In certain
embodiments, sequences for the target antigens were obtained from
the GenBank database (Release No. 71.0; 3/92). The identification
of motifs was done using the "FINDPATTERNS" program (Devereux, et
al., Nucleic Acids Research, 12:387-95 (1984)). A computer search
was carried out for antigen proteins comprising the
B7-like-supermotif. TABLES 77-80, TABLES 81-84, and TABLES 85-86
provide the results of searches of the antigens. TABLES 77-80 and
TABLES 85-86 shows results of screening a number of antigens.
TABLES 81-84 shows results of screening MAGE antigens.
[0857] TABLE 25 and TABLE 34 list peptides identified in this
search. Accordingly, a preferred embodiment of the invention
comprises a composition comprising a peptide of TABLE 25 and/or
TABLE 34.
[0858] Other viral and tumor-related proteins can also be analyzed
for the presence of these motifs. The amino acid sequence or the
nucleotide sequence encoding products is obtained from the GenBank
database in the cases of Prostate Specific antigen (PSA), p53
oncogene, Epstein Barr Nuclear Antigen-1 (EBNA-1), and c-erb2
oncogene (also called HER-2/neu).
[0859] In the cases of Human Papilloma Virus (HPV), Prostate
Specific Antigen (PSA), p53 oncogene, Epstein Barr Nuclear
Antigen-1 (EBNA-1), and c-erb2 oncogene (also called HER-2/neu),
and Melanoma Antigen-1 (MAGE-1), a single sequence exists.
[0860] In the cases of Hepatitis B Virus (HBV), Hepatitis C Virus
(HCV), and Human immunodeficiency Virus (HIV) several
strains/isolates exist and many sequences have been placed in
GenBank.
[0861] For HBV, binding motifs are identified for the adr, adw and
ayw types. In order to avoid replication of identical sequences,
all of the adr motifs and only those motifs from adw and ayw that
are not present in adr are added to the list of peptides.
[0862] In the case of HCV, a consensus sequence from residue 1 to
residue 782 is derived from 9 viral isolates. Motifs are identified
on those regions that have no or very little (one residue)
variation between the 9 isolates. The sequences of residues 783 to
3010 from 5 viral isolates were also analyzed. Motifs common to all
the isolates are identified and added to the peptide list.
[0863] Finally, a consensus sequence for HIV type 1 for North
American viral isolates (10-12 viruses) was obtained from the Los
Alamos National Laboratory database (May 1991 release) and analyzed
in order to identify motifs that are constant throughout most viral
isolates. Motifs that bear a small degree of variation (one
residue, in 2 forms) were also added to the peptide list.
[0864] Using the B7-like supermotifs identified in the related
applications described above, sequences from various pathogens and
tumor-related proteins were analyzed for the presence of these
motifs. Screening was carried out described in the related
applications. TABLES 12 and 13_provide the results of searches of
the antigens.
[0865] Using the A3 supermotif described above, sequences from
various pathogens and tumor-related proteins were analyzed for the
presence of these motifs. Screening was carried out described in
the related applications. TABLE 8 provides the results of searches
of the antigens.
[0866] Using the A24 motif described above, sequences from various
pathogens and tumor-related proteins were analyzed for the presence
of these motifs. Screening was carried out described in the related
applications. TABLE 9 provides the results of searches of t1
antigens.
[0867] Several motifs for each allele shown below were used to
screen several antigens. Protein E6 of human papilloma virus (HPV)
type 16 using motifs from all of the alleles disclosed above are
shown (TABLES 77-80). Protein E7 of HPV type 18 was also searched
for motifs from all alleles (TABLES 77-80) Melanoma antigens MAGE
1, 2 and 3 were searched for motifs from all alleles (TABLES
81-84). The antigen PSA was searched for motifs from all alleles
(TABLES 85-86). Finally, core and envelope proteins from hepatitis
C virus were also searched (TABLE 87). In the tables and the
description of the motifs, the conventional symbol letter for each
amino acid was used. The letter "X" represents a wild card
character (any amino acid).
[0868] The following motifs were screened in the present
search:
TABLE-US-00002 For HLA-A1 (A*0101) 1 XSXXXXXXY (SEQ ID NO:_) 2
XSXXXXXXXY (SEQ ID NO:_) 3 XTXXXXXXY (SEQ ID NO:_) 4 XTXXXXXXXY
(SEQ ID NO:_) 5 XXDXXXXXY (SEQ ID NO:_) 6 XXDXXXXXXY (SEQ ID NO:_)
7 XXEXXXXXY (SEQ ID NO:_) 8 XXEXXXXXXY (SEQ ID NO:_) For HLA-A3.2
(A*0301) 1 XVXXXXXXK (SEQ ID NO:_) 2 XVXXXXXXXK (SEQ ID NO:_) 3
XLXXXXXXK (SEQ ID NO:_) 4 XLXXXXXXXK (SEQ ID NO:_) 5 XMXXXXXXK (SEQ
ID NO:_) 6 XMXXXXXXXK (SEQ ID NO:_) For HLA-A11 (A*1101) 1
XTXXXXXXK (SEQ ID NO:_) 2 XTXXXXXXXK (SEQ ID NO:_) 3 XVXXXXXXK (SEQ
ID NO:_) 4 XVXXXXXXXK (SEQ ID NO:_) For HLA-A24.1 (A*2401) 1
XYXXXXXXF (SEQ ID NO:_) 2 XYXXXXXXXF (SEQ ID NO:_) 3 XYXXXXXXL (SEQ
ID NO:_) 4 XYXXXXXXXL (SEQ ID NO:_)
Brief Description of TABLES 9-21
[0869] TABLES 9 and 10. Identified HLA-AI allele-binding peptides.
Peptides are identified by amino acid sequence, SEQ ID NO., number
of amino acids in peptide (AA), origin of peptide (organism),
identity of originating protein, position of peptide within protein
sequence, and analog status, wherein an analog is a peptide of the
invention where the amino acid sequence of any naturally-occurring
peptide sequence has been modified by substitution of one or more
amino acid residues.
[0870] TABLE 11. Binding affinity of HLA-A1 binding peptides.
Peptides are identified by amino acid sequence, SEQ ID NO., and
binding affinity to the designated HLA-A1 alleles (expressed as an
IC.sub.50).
[0871] TABLE 12. Identified HLA-A2 allele-binding peptides.
Peptides are identified by amino acid sequence, SEQ ID NO., number
of amino acids in peptide (AA), origin of peptide (organism),
identity of originating protein, position of peptide within protein
sequence, and analog status, wherein an analog is a peptide of the
invention where the amino acid sequence of any naturally-occurring
peptide sequence has been modified by substitution of one or more
amino acid residues.
[0872] TABLE 13. Binding affinity of HLA-A2 binding peptides.
Peptides are identified by amino acid sequence, SEQ ID NO., and
binding affinity to the designated HLA-A2 alleles (expressed as an
ICso).
[0873] TABLE 14. Identified HLA-A3 allele-binding peptides.
Peptides are identified by amino acid sequence, SEQ ID NO., number
of amino acids in peptide (AA), origin of peptide (organism),
identity of originating protein, position of peptide within protein
sequence, and analog status, wherein an analog is a peptide of the
invention where the amino acid sequence of any naturally-occurring
peptide sequence has been modified by substitution of one or more
amino acid residues. Binding affinity of HLA-A3 binding peptides.
Peptides are identified by amino acid sequence, SEQ ID NO., and
binding affinity to the designated HLA-A3 alleles (expressed as an
IC.sub.50).
[0874] TABLE 15. Identified HLA-A24 allele-binding peptides.
Peptides are identified by amino acid sequence, SEQ ID NO., number
of amino acids in peptide (AA), origin of peptide (organism),
identity of originating protein, position of peptide within protein
sequence, and analog status, wherein an analog is a peptide of the
invention where the amino acid sequence of any naturally-occurring
peptide sequence has been modified by substitution of one or more
amino acid residues. Binding affinity of HLA-A24 binding peptides.
Peptides are identified by amino acid sequence, SEQ ID NO., and
binding affinity to the designated HLA-A24 alleles (expressed as an
IC.sub.50).
[0875] TABLE 16. Identified HLA-B7 allele-binding peptides.
Peptides are identified by amino acid sequence, SEQ ID NO., number
of amino acids in peptide (AA), origin of peptide (organism),
identity of originating protein, position of peptide within protein
sequence, and analog status, wherein an analog is a peptide of the
invention where the amino acid sequence of any naturally-occurring
peptide sequence has been modified by substitution of one or more
amino acid residues. Binding affinity of HLA-B7 binding peptides.
Peptides are identified by amino acid sequence, SEQ ID NO., and
binding affinity to the designated HLA-B7 alleles (expressed as an
IC.sub.50).
[0876] TABLE 17. Identified HLA-B44 allele-binding peptides.
Peptides are identified by amino acid sequence, SEQ ID NO., number
of amino acids in peptide (AA), origin of peptide (organism),
identity of originating protein, position of peptide within protein
sequence, and analog status, wherein an analog is a peptide of the
invention where the amino acid sequence of any naturally-occurring
peptide sequence has been modified by substitution of one or more
amino acid residues. Binding affinity of HLA-B44 binding peptides.
Peptides are identified by amino acid sequence, SEQ ID NO., and
binding affinity to the designated HLA-B44 alleles (expressed as an
IC.sub.50).
[0877] TABLE 18. Identified HLA-DQ allele-binding peptides.
Peptides are identified by amino acid sequence, SEQ ID NO., number
of amino acids in peptide (AA), origin of peptide (organism),
identity of originating protein, position of peptide within protein
sequence, and analog status, wherein an analog is a peptide of the
invention where the amino acid sequence of any naturally-occurring
peptide sequence has been modified by substitution of one or more
amino acid residues. Binding affinity of HLA-DQ binding peptides.
Peptides are identified by amino acid sequence, SEQ ID NO., and
binding affinity to the designated HLA-DQ alleles (expressed as an
IC.sub.50).
[0878] TABLES 19 and 186. Identified HLA-DR allele-binding
peptides. Peptides are identified by amino acid sequence, SEQ ID
NO., number of amino acids in peptide (AA), origin of peptide
(organism), identity of originating protein, position of peptide
within protein sequence, and analog status, wherein an analog is a
peptide of the invention where the amino acid sequence of any
naturally-occurring peptide sequence has been modified by
substitution of one or more amino acid residues. Binding affinity
of HLA-DR binding peptides. Peptides are identified by amino acid
sequence, SEQ ID NO., and binding affinity to the designated HLA-DR
alleles (expressed as an IC.sub.50).
[0879] TABLE 20. Identified murine MHC class I allele-binding
peptides. Peptides are identified by amino acid sequence, SEQ ID
NO., number of amino acids in peptide (AA), origin of peptide
(organism), identity of originating protein, position of peptide
within protein sequence, and analog status, wherein an analog is a
peptide of the invention where the amino acid sequence of any
naturally-occurring peptide sequence has been modified by
substitution of one or more amino acid residues.
[0880] TABLE 21. Binding affinity of muring MHC class I-binding
peptides. Peptides are identified by amino acid sequence, SEQ ID
NO., and binding affinity to the designated murine MHC class I
alleles (expressed as an IC.sub.50).
Example 23
Additional Identification of Immunogenic Peptides
[0881] Using the motifs identified above for HLA-A2.1 allele amino
acid sequences from a tumor-related protein, Melanoma Antigen-1
(MAGE-1), were analyzed for the presence of these motifs. Sequences
for the target antigen are obtained from the GenBank data base
(Release No. 71.0; 3/92). The identification of motifs is done
using the "FINDPATTERNS" program (Devereux et al., Nucleic Acids
Research 12:387-395 (1984)).
[0882] Other viral and tumor-related proteins can also be analyzed
for the presence of these motifs. The amino acid sequence or the
nucleotide sequence encoding products is obtained from the GenBank
database in the cases of Human Papilloma Virus (HPV), Prostate
Specific antigen (PSA), p53 oncogene, Epstein Barr Nuclear
Antigen-1 (EBNA-1), and c-erb2 oncogene (also called
HER-2/neu).
[0883] In the cases of Hepatitis B Virus (HBV), Hepatitis C Virus
(HCV), and Human Immunodeficiency Virus (HIV) several
strains/isolates exist and many sequences have been placed in
GenBank.
[0884] For HBV, binding motifs are identified for the adr, adw and
ayw types. In order to avoid replication of identical sequences,
all of the adr motifs and only those motifs from adw and ayw that
are not present in adr are added to the list of peptides.
[0885] In the case of HCV, a consensus sequence from residue 1 to
residue 782 is derived from 9 viral isolates. Motifs are identified
on those regions that have no or very little (one residue)
variation between the 9 isolates. The sequences of residues 783 to
3010 from 5 viral isolates were also analyzed. Motifs common to all
the isolates are identified and added to the peptide list.
[0886] Finally, a consensus sequence for HIV type 1 for North
American viral isolates (10-12 viruses) was obtained from the Los
Alamos National Laboratory database (May 1991 release) and analyzed
in order to identify motifs that are constant throughout most viral
isolates. Motifs that bear a small degree of variation (one
residue, in 2 forms) were also added to the peptide list.
[0887] TABLES 181 and 182 provide the results of searches of the
following antigens cERB2, EBNA1, HBV, HCV, HIV, HPV, MAGE, p53, and
PSA. Only peptides with binding affinity of at least 1% as compared
to the standard peptide in assays described in Example 5 are
presented. Binding as compared to the standard peptide is shown in
the far right column. The column labeled "Pos." indicates the
position in the antigenic protein at which the sequence occurs.
[0888] Using the motifs disclosed here, amino acid sequences from
various antigens were screened for further motifs. Screening was
carried out as described above. TABLES 176 and TABLE 177 provide
the results of searches of the following antigens cERB2, CMV,
Influenza A, HBV, HIV, HPV, MAGE, p53, PSA, Hu S3 ribosomal
protein, LCMV, and PAP. Only peptides with binding affinity of at
least 1% as compared to the standard peptide in assays described in
Example 5 are presented. Binding as compared to the standard
peptide is shown for each peptide.
Example 24
Identification of Immunogenic Peptides in Autoantigens
[0889] As noted above, the motifs of the present invention can also
be screened in antigens associated with autoimmune diseases. Using
the motifs identified above for HLA-A2.1 allele amino acid
sequences from myelin proteolipid (PLP), myelin basic protein
(MBP), glutamic acid decarboxylase (GAD), and human collagen types
II and IV were analyzed for the presence of these motifs. Sequences
for the antigens were obtained from Trifilieff et al., C.R.
Sceances Acad. Sci. 300:241 (1985); Eyler at al., J. Biol. Chem.
246:5770 (1971); Yamashita et al. Biochiem. Biophys. Res. Comm.
192:1347 (1993); Su et al., Nucleic Acids Res. 17:9473 (1989) and
Pihlajaniemi et al. Proc. Natl. Acad. Sci. USA 84:940 (1987). The
identification of motifs was done using the approach described in
Example 5 and the algorithms of Examples 6 and 7. TABLE 178
provides the results of the search of these antigens.
[0890] Using the quantitative binding assays of Example 4, the
peptides are next tested for the ability to bind MHC molecules. The
ability of the peptides to suppress proliferative responses in
autoreactive T cells is carried out using standard assays for T
cell proliferation. For instance, methods as described by Miller et
al. Proc. Natl. Acad. Sci. USA, 89:421 (1992) are suitable.
[0891] For further study, animal models of autoimmune disease can
be used to demonstrate the efficacy of peptides of the invention.
For instance, in HLA transgenic mice, autoimmune model diseases can
be induced by injection of MBP, PLP or spinal cord homogenate (for
MS), collagen (for arthritis). In addition, some mice become
spontaneously affected by autoimmune disease (e.g., NOD mice in
diabetes). Peptides of the invention are injected into the
appropriate animals, to identify preferred peptides.
Example 25
Comparative Treatment of Data Obtained in Different Binding
Analysess
[0892] HLA class I supermotif and motif analysis of antigens of
interest was performed as described herein and in the related
applications, noted above. Peptides comprising the appropriate HLA
I motif or supermotif were then synthesized and assayed for binding
activity. A detailed description of the protocol utilized to
measure the binding of peptides to Class I and Class II MHC has
been published (Sette et al., Mol. Immunol. 31:813, 1994; Sidney et
al., in Current Protocols in Immunology, Margulies, Ed., John Wiley
& Sons, New York, Section 18.3, 1998).
[0893] Since under these conditions [label]<[HLA] and
IC.sub.50.gtoreq.[HLA], the measured IC.sub.50 values are
reasonable approximations of the true K.sub.D values. To allow
comparison of the data obtained in different experiments, a
relative binding figure is calculated for each peptide by dividing
the IC.sub.50 of a positive control for inhibition by the IC.sub.50
for each tested peptide (typically unlabeled versions of the
radiolabeled probe peptide). For inter-experiment comparisons,
relative binding values are compiled. These values can subsequently
be converted back into IC.sub.50 nM values by dividing the
IC.sub.50 nM of the positive controls for inhibition by the
relative binding of the peptide of interest. This method of data
compilation has proven to be the most accurate and consistent for
comparing peptides that have been tested on different days, or with
different lots of purified MHC.
[0894] HLA class I supermotif and motif-bearing peptides from HIV
regulatory proteins, e.g., nef, rev, vif, tat, and vpr, are shown
in TABLE 70, TABLE 71, TABLE 72, TABLE 73, TABLE 74, TABLE 75,
TABLE 76, TABLES 77-80, TABLES 81-84, and TABLES 85-86. In these
tables, "% conserv" refers to percent conservance, which is the
degree to which the sequences are conserved in the strains
evaluated to identify the sequences. The "A" designation indicates
that the peptide is an analog of the native sequence. In the motif
column, the designation "i" refers to individual motif and "s"
refers to supermotif.
[0895] HLA class I supermotif and motif-bearing peptides from other
antigens, e.g., cancer antigens such as CEA, p53, Her2/neu, MART1,
MAGE2, MAGE3, tyrosinase, flu, gp100, HBV, HCV, HIV, HPV (including
the strain designation), Epstein Barr Virus (EBV), prostate
cancer-associated antigens, gliadin, Mycobacterium leprae,
Mycobacterium tuberculosis, T. cruzi, Candida antigens, and malaria
(Plasmodium falciparum) antigens are shown in TABLE 87, TABLE 88,
TABLE 89, TABLE 90, TABLES 91-92, TABLES 93-94, TABLE 95, TABLE 96,
TABLES 97-102, TABLES 103-107, TABLES 108-110, TABLES 111-122 and
TABLES 123-124.
[0896] TABLE 87, TABLE 88 and TABLE 187 show peptides bearing an
HLA-A1 supermotif and/or motif.
[0897] TABLE 89, TABLE 90, TABLES 91-92, TABLES 93-94 and TABLE 188
show peptides bearing an HLA-A2 supermotif.
[0898] TABLE 95, TABLE 96 and TABLE 189 show peptides bearing an
HLA-A3 supermotif and/or motif.
[0899] TABLES 97-102 and TABLES 103-107 show peptides bearing an
HLA-A24 supermotif and/or motif.
[0900] TABLES 108-110 and TABLES 111-122 show peptides bearing an
HLA-B7 supermotif.
[0901] TABLE 123-124 shows peptides bearing an HLA-B44
supermotif.
[0902] Peptide binding data for the designated HLA molecules are
provided as IC.sub.50 values unless otherwise indicated. The "A"
designation indicates that the peptide is an analog of the native
sequence.
[0903] Using the HLA class II supermotif and motifs identified in
related applications and as described above, sequences from various
pathogens and tumor-related proteins were analyzed for the presence
of these motifs. Screening and binding assays was carried out as
described in the related applications designated herein.
[0904] HLA class II DR supermotif and DR3 motif-bearing peptides
from HIV regulatory proteins, e.g., nef, rev, vif, tat, and vpr,
are shown in TABLES 125-127 and TABLE 136. The term "% conserv"
refers to percent conservance, which is the degree to which the
sequences are conserved in the strains evaluated to identify the
sequences. In TABLE 136, in the "sequence" column, the core
sequence of the motif-bearing peptide is in lower case.
[0905] TABLES 128, TABLES 129-130, TABLES 131-132, TABLES 133-134,
TABLES 135, and TABLES 136 show HLA class II DR supermotif and DR 3
motif bearing peptides and the antigens from which they are
derived. The peptide reference number, sequence, antigen
protein/position of the sequence in the antigen, and binding data
are shown in the tables. TABLE 129 shows binding data for
DRB1*0101, *0301, *0401, *0404, and *0405. TABLE 130 shows binding
data for DRB1*0701, *0802, *0901, *1101, *1201, *1302, *1501,
DRB3*0101, DRB4*0101, DRB5*0101, and DQB1*0301. TABLE 131 and TABLE
133 provide the peptide reference number sequence and protein
antigen/position of sequence in antigen for the peptides. Binding
data are provided in TABLE 132 and TABLE 134.
[0906] Peptide binding data for the designated HLA molecules are
provided as IC.sub.50 values unless otherwise indicated. The "A"
designation indicates that the peptide is an analog of the native
sequence.
Example 26
Quantitative Binding Assays
[0907] To verify that motif-containing peptide sequences are indeed
capable of binding to the appropriate class I molecules,
quantitative binding assays were performed as described in the
parent applications. Binding affinities are expressed in reference
to standard peptides as described in those applications. In
addition, these applications describe algorithms to provide a more
exact predictor of binding based upon the effects of different
residues at each position of a peptide sequence, in addition to the
anchor or conserved residues.
[0908] Using isolated MHC molecules prepared as described in
Example 2, supra, quantitative binding assays were performed.
Briefly, indicated amounts of MHC as isolated above were incubated
in 0.05% NP40-PBS with .about.5 nM of radiolabeled peptides in the
presence of 1-3 .mu.M .beta..sub.2M and a cocktail of protease
inhibitors (final concentrations 1 mM PMSF, 1.3 mM 1.10
Phenanthroline, 73 .mu.M Pepstatin A, 8 mM EDTA, 200 .mu.M
N-.alpha.-p-tosyl-L-Lysine Chloromethyl ketone). After various
times, free and bound peptides were separated by TSK 2000 gel
filtration, as described previously in Sette, et al., J. Immunol.,
148:844 (1992). Peptides were labeled by the use of the Chloramine
T method Buus et al., Science, 235:1352 (1987), which is
incorporated herein by reference.
[0909] The various candidate HLA binding peptides were radiolabeled
and offered (5-10 nM) to 1 .mu.M purified HLA molecules. The HBc
18-27 peptide HLA binding peptide was radiolabeled and offered
(5-10 nM) to 1 .mu.M purified HLA A2.1. After two days at
23.degree. C. in presence of a cocktail of protease inhibitors and
1-3 .mu.M purified human .beta..sub.2M, the percent of MHC class I
bound radioactivity was measured by size exclusion chromatography,
as previously described for class II peptide binding assays in
Sette, et al., in Seminars in Immunology, Vol. 3, Gefter, ed. (W.B.
Saunders, Philadelphia, 1991), pp 195-202, which is incorporated
herein by reference. Using this protocol, high binding (30-95% of
standard peptide binding) was detected in all cases in the
presence, but not in the absence, of the relevant HLA allele. Also
using this protocol, high binding (95%) was detected in all cases
in the presence of purified HLA A2.1 molecules.
[0910] To explore the specificity of binding, we determined whether
the binding was inhibitable by excess unlabeled peptide, and if so,
what the 50% inhibitory concentration (IC.sub.50%) might be. The
rationale for this experiment was threefold. First, such an
experiment is crucial in order to demonstrate specificity. Second,
a sensitive inhibition assay is the most viable alternative for a
high throughput quantitative binding assay. Third, inhibition data
subjected to Scatchard analysis can give quantitative estimates of
the K of interaction and the fraction of receptor molecules capable
of binding ligand (% occupancy).
[0911] Results of binding assays described here may be expressed in
terms of IC.sub.50's. Given the conditions in which our assays are
run (i.e., limiting MHC and labeled peptide concentrations), these
values approximate K.sub.D values. It should be noted that
IC.sub.50 values can change, often dramatically, if the assay
conditions are varied, and depending on the particular reagents
used (e.g., Class I preparation, etc.). For example, excessive
concentrations of MHC will increase the apparent measured IC.sub.50
of a given ligand.
[0912] As a specific example to verify that motif-containing
peptide sequences are indeed capable of binding to the appropriate
class I molecules, specific binding assays were established.
HLA-A3.2 molecules were purified from GM3107 EBV cells by affinity
chromatography using the GAPA3 mAb (anti-A3) to isolate A3.2. Prior
to the step, the lysate was depleted of HLA B and C molecules by
repeated passages over a B1.23.2 column (this antibody is B, C
specific) generally as described in Example 2, above.
[0913] As a radiolabeled probe, the peptide 941.12 (KVFPYALINK (SEQ
ID NO:______)), containing an A3.2 motif, was used. This peptide
contains the anchor residues V.sub.2 and K.sub.10, associated with
A3.2-specific binders, described above. A Y residue was inserted at
position 5 to allow for radiolodination. Peptides were labeled by
the use of the Chloramine T method Buus et al., Science 235:1352
(1987), which is incorporated herein by reference.
[0914] A dose range of purified A3.2 was incubated with 10 nM of
941.12 at pH 7.0 and 23.degree. C., in presence of a protease
inhibitor cocktail (1 mM PMSF, 1.3 mM 1.10 phenanthroline, 73 .mu.M
pepstatin A, 8 mM EDTA, and 200 .mu.M N a.sub.p-tosyl-L-lysine
chloromethyl ketone (TLCK)), in presence of 1 .mu.M purified human
.beta.2 microglobulin. After two days, the % bound radioactivity
was measured by gel filtration over TSK 2000 columns as previously
described for class II peptide binding assays in Sette et al., in
Seminars in Immunology, Vol. 3, Gefter, ed. (W.B. Saunders,
Philadelphia, 1991), pp 195-202, which is incorporated herein by
reference. (see, FIG. 17). Good binding (in the 60 to 100% range)
was observed for A3.2 concentrations ranging between 35 and 300 nM.
30% binding was observed at 15 nM A3.2.
[0915] To minimize A3.2 usage and to increase the sensitivity of
the assay, a concentration of 5-10 nM A3.2 was selected for further
assays. In the experiment shown in FIG. 18, 7 nM A3.2 and an
equivalent concentration of radiolabeled 941.12 were incubated
using the conditions described above and in the presence of a dose
range of three peptides (HBc 18-27 (924.07), a Prostate Specific
Antigen peptide (939.01), and HIV nef 73-82 (940.03)). It was found
that peptide 940.03 inhibited strongly, with a 50% inhibitory
concentration (IC50%) of 22 nM, while a weaker inhibition was
observed with peptide 939.01 (IC50% 940 nM). Finally, peptide
924.07 did not show any inhibition up to the 30 .mu.M level. Thus,
it is concluded that peptides 940.03 and 939.01 are high and
intermediate affinity binders, respectively, while peptide 924.07
is classified as a low affinity or negative binder.
[0916] For instance, in analysis of an inhibition curve for the
interaction of the peptide HBc 18-27 with A2.1, the IC.sub.50% was
determined to be 25 nM. Further experiments were conducted to
obtain Scatchard plots. For HBc 18-27/A2.1, six different
experiments using six independent MHC preparations yielded a
K.sub.D of 15.5.+-.9.9.times.10.sup.-9 M and occupancy values of
6.2% (.+-.1.4).
[0917] Several reports have demonstrated that class I molecules,
unlike class II, are highly selective with regard to the size of
the peptide epitope that they recognize. The optimal size varies
between 8 and 10 residues for different peptides and different
class I molecules, although MHC binding peptides as long as 13
residues have been identified. To verify the stringent size
requirement, a series of N- and C-terminal truncation/extension
analogs of the peptide HBc 18-27 were synthesized and tested for
A2.1 binding. Previous studies had demonstrated that the optimal
size for CTL recognition of this peptide was the 10-mer HBc18-27
(Sette et al. supra). It was found that removal or addition of a
residue at the C terminus of the molecule resulted in a 30 to
100-fold decrease in binding capacity. Further removal or addition
of another residue completely obliterated binding. Similarly, at
the N-terminus of the molecule, removal or deletion of one residue
from the optimal HBc 18-27 peptide completely abrogated A2.1
binding.
[0918] Throughout this disclosure, results have been expressed in
terms of IC.sub.50's. Given the conditions in which the assays are
run (i.e., limiting MHC and labeled peptide concentrations), these
values approximate K.sub.D values. It should be noted that
IC.sub.50 values can change, often dramatically, if the assay
conditions are varied, and depending on the particular reagents
used (e.g., Class I preparation, etc.). For example, excessive
concentrations of MHC will increase the apparent measured IC.sub.50
of a given ligand.
[0919] An alternative way of expressing the binding data, to avoid
these uncertainties, is as a relative value to a reference peptide.
The reference peptide is included in every assay. As a particular
assay becomes more, or less, sensitive, the IC.sub.50's of the
peptides tested may change somewhat. However, the binding relative
to the reference peptide will not change. For example, in an assay
run under conditions such that the IC.sub.50 of the reference
peptide increases 10-fold, all IC.sub.50 values will also shift
approximately ten-fold. Therefore, to avoid ambiguities, the
assessment of whether a peptide is a good, intermediate, weak, or
negative binder should be based on its IC.sub.50, relative to the
IC.sub.50 of the standard peptide.
[0920] The reference peptide for the HLA-A2.1 assays described
herein is referred to as 941.01 having a sequence of FLPSDYFPSV
(SEQ ID NO:______). An average IC.sub.50 of 5 (nM) was observed
under the assay conditions utilized.
[0921] Other reference peptides used in the assays include the
following: A1CON1 (YLEPAIAKY (SEQ ID NO:______)), 25 nM for A*0101;
HBV core 18-27 F6->Y (FLPSDYFPSV (SEQ ID NO:______)), 4.6 nM for
A*0201; A3CON1 (KVFPYALINK (SEQ ID NO:______)), 10 nM for A*0301;
A3CON1 (KVFPYALINK (SEQ ID NO:______)), 5.9 nM for A*1101; A24CON1
(AYIDNYNKF (SEQ ID NO:______)), 12 nM for A82401; A2.1 signal
sequence 5-13 L.sub.7->Y (APRTLVYLL (SEQ ID NO:______)) 4.7 nM
for B*0701; HIV gp 586-593 Y.sub.1>F, Q.sub.5>Y (FLKDYQLL
(SEQ ID NO:______)), 14 nM for B*0801; Rat 60S (FRYNGLIHR (SEQ ID
NO:______)), 6.4 nM for B*2705; B35CON2 (FPFKYAAAF (SEQ ID
NO:______)), 4.4 nM B*3503.
[0922] If the IC.sub.50 of the standard peptide measured in a
particular-assay is different than that reported in the table then
it should be understood that the threshold values used to determine
good, intermediate, weak, and negative binders should be modified
by a corresponding factor. For example, if in an A2.1 binding
assay, the IC.sub.50 of the A2.1 standard (941.01) were to be
measured as 8 nM instead of 5 nM, then a peptide ligand would be
called a good binder only if it had an IC.sub.50 of less than 80 nM
(i.e., 8 nM.times.0.1), instead of the usual cut-off value of 50
nM.
[0923] The experimental system herein described can be used to test
binding of large numbers of synthetic peptides to a variety of
different class I specificities. Specific binding assays can be
performed as follows.
HLA-A11-Specific Assay
[0924] The cell line BVR was used as a source of HLA. The
dependency of the binding on MHC concentration in presence or
absence of .beta..sub.2M are shown in FIG. 19, while FIG. 20
depicts the dose dependency of the inhibition by excess unlabeled
ligand. Finally, FIG. 21 shows a Scatchard analysis experiment.
Values of apparent kD of -6 nM and of 10% active receptor were
obtained, and were remarkable for their similarity to the values
obtained for A2.1 and A3.2. The sequence of the peptide used as a
radiolabeled probe (940-06) is AVDLYHFLK (SEQ ID NO:______).
HLA-A1-Specific Assay
[0925] In this case, the EBV cell line Steinlin was used as a
source of purified HLA. The same protocol previously applied to
purification of other HLA alleles (i.e., depletion of B, C
molecules by a B1.23.2 mAb column, followed by purification of A
molecules by means of a W632 mAb column) was utilized. On the basis
of the pool sequencing data, consensus peptides were synthesized,
directly radiolabeled, and tested for HLA binding using the
standard protocol (1 mM .beta..sub.2M, 2 days RT incubation in
presence of protease inhibitors). A graph illustrating the
relationship between % binding and iM input HLA A1 is shown in FIG.
22. From the data, it was concluded that in analogy with what was
observed for HLA A2, 3, and 11, as little as 30 nM are sufficient
to obtain -10% binding. The sequence of the peptide used as a
radiolabeled probe (944.02) is YLEPAIAKY (SEQ ID NO:______). In the
next set of experiments, the specificity of the assay established
was verified by its inhabitability by excess unlabeled peptide. The
IC50% was measured (FIG. 23) as -20 nM. Further Scatchard analysis
(FIG. 24) verified that the apparent K.sub.D of the interaction
corresponded to 21 nM, with a % of active receptor corresponding to
5.1%.
HLA-A24 Specific Assay
[0926] HLA A24 molecules were purified from the KT3 EBV cell line.
In this case, two consensus peptides whose sequences were based on
the pool sequencing data have been synthesized. Their sequences
are: 979-01, AYIDNVYKF (SEQ ID NO:______) and 979.02, AYIDNYNKF
(SEQ ID NO:______). The results of experiments in which the % bound
of these two peptides as a function of input MHC was measured are
shown in FIG. 25. In both cases, 10-15% binding was obtained with
as little as 20-50 nM MHC. Cold inhibition experiments (FIG. 26),
limiting MHC concentrations, revealed that the binding was readily
inhibitable by excess unlabeled peptide, with an apparent K.sub.D
of 30 and 60 nM, respectively. Further Scatchard experiments
verified values of 136 nM and 28 nM, respectively. The apparent %
of available receptor (active MHC) were 8.3% and 7.4%, respectively
(FIG. 13A and FIG. 13B). On the basis of these data, peptide 979.02
was arbitrarily selected as standard label indicator for A24
assays. Furthermore, on the basis of the data herein described, we
also conclude that the goal of establishing an A24-specific binding
assay has been accomplished. In conclusion, specific assays for the
five major HLA alleles have been described.
Example 27
Expansion of HLA A Motifs
[0927] Establishing in vitro binding assays allows one to readily
quantitate in vitro the binding capacity of various synthetic
peptides to the various alleles of interest (HLA A1, A2, A3, A11,
and A24). This allows verification of the correctness of the motifs
by means of peptides carrying the various HLA A motifs for their
capacity to bind purified HLA molecules. Typically, peptides were
synthesized with specific HLA motifs embedded in a neutral backbone
composed of only alanine residues. In some cases, a K residue was
also introduced within the sequence, with the purpose of increasing
solubility. The use of such "neutral" poly A backbones, as applied
to the case of class II molecules, has been described in detail,
for example, by Jardetzky et al., (Jardetzky et al., EMBO J.,
9(6):1797, 1990)
[0928] For example, in the case of A3.2, a motif has been defined
with a hydrophobic residue in position 2 and a positive charge (K)
in position 9. Thus, to verify that the presence of these two
anchor residues would allow, in the context of a poly A backbone,
for A3.2 binding, the poly A analog with the sequence AMAAAAAK (SEQ
ID NO:______) was synthesized (TABLE 88).
[0929] Similarly, other peptides carrying other HLA motifs were
also synthesized and tested for HLA binding. It was found that in
all cases, the presence of the specific HLA motifs was conducive to
binding to the relevant HLA allele, with estimated K.sub.D
comprised of between 125 and 2.8 nM. In most cases, the binding was
also absolutely specific, in that no binding was detected to
irrelevant alleles. Only two exceptions to this general rule were
observed. Firstly, A3 and A11 peptides crossreacted extensively
with each other, perhaps as could have been expected by the fact
that the motifs for these two alleles are remarkably similar.
Second, some A1 peptides crossreacted, albeit with much lower
affinities, on A11 and A3.2.
[0930] To further define the structural requirements for the
interaction between peptide epitopes and various class I alleles of
interest, analogs of 10 residues in length of some of the 9 residue
peptides shown in TABLE 88 were synthesized (TABLE 89). These
analogs were generated by inserting an additional Ala residue
within the poly A backbone, so that the anchor residues are not
located in positions 2 and 10 (as opposed to 2 and 9 in the
previous table). The results obtained illustrate that motifs of 10
residues are also capable of specifically binding to the relevant
class I alleles, albeit with a slightly lower efficiency.
[0931] In summary, these data confirm that both 9-mer and 10-mer
peptides which contain the appropriate motifs can bind HLA. On the
basis of these data, 8-mer or 11-mer peptides should also be
capable of binding, even if perhaps with lower affinities.
[0932] The data described above show that the presence of certain
residues in the anchor positions does allow (at least in a
"neutral" poly A backbone) for HLA binding. To investigate to what
degree other amino acids (for example, chemically related amino
acids) might be tolerated in these crucial anchor positions,
analogs of some of the poly A peptides from TABLE 88 were
synthesized, in which the residue present in position 2 (or 3) or 9
was varied. The results of this analysis are shown in TABLE 88,
TABLE 89, TABLE 90, TABLES 91-92, TABLES 93-94, TABLE 95, and TABLE
96.
[0933] In the case of A3.2 (TABLE 90), in position 2, L, M, I, V,
S, A, T, and F were found to be preferred (binding >0.1 relative
to previously defined anchor residues), while C, G, and D were
permitted (binding >0.01 to 0.1 relative to previously defined
anchor residues). The substitution of E, because of its similarity
to D, in this position should also be tolerated. In position 9, K,
R, and Y were preferred. Because of a similarity in nature, that H
and F should also be preferred. No other residue was tolerated in
position 9 for A3 binding.
[0934] In the case of A11 (TABLES 91-92), the preferred residues in
position 2 were L, M, I, V, A, S, T, G, N (L and Q by similarity).
Tolerated were C, F, D (and E by similarity). In position 9, K was
preferred and R was tolerated. H should also be tolerated by
similarity.
[0935] In the case of A24 (TABLES 93-94), Y and F were preferred in
position 2 (and W by similarity); no other residue was tolerated.
In position 9, F, I, and L were preferred (and W and M by
extension). No other residue was tolerated.
[0936] In the case of A1, three different anchor residues had
previously been defined. The results shown in the preceding section
show that they act independently of each other (i.e., that two out
of three anchors would be sufficient for binding). This is indeed
the case. For this reason, analogs containing two anchors were
synthesized to define what residues might be preferred or tolerated
in each position. The data shown in Table 18 show that in position
2, T, S, and M are preferred, and no other residue is tolerated. In
position 3 (TABLE 96), D and E are preferred, and A, S (and T by
similarity) are tolerated. Finally, in position 9, only Y is
preferred, and no other residue appears to be tolerated (TABLE
96).
[0937] Thus, on the basis of the data, it is concluded that
peptides carrying any combination of two preferred residues can
bind. Peptides containing "imperfect" motifs, i.e., carrying a
preferred residue at one position and a tolerated one at the other
anchor position, should also be capable of binding, even if with
somewhat lower affinity. Using the motifs of this invention for
various MHC class I alleles amino acid-sequences from various viral
and tumor-related proteins were analyzed for the presence of
motifs. The results of this motif analysis are shown in TABLES
112-122.
Example 28
Validation of HLA Peptide Binding Motifs with an Unbiased Set of
HPV 16 Peptides
[0938] Human Papillomaviruses (HPVs) are implicated in the etiology
of cervical cancer (Pfister, H. (1974) Biology and biochemistry of
papillomaviruses, Rev. Physiol. Biochem., 99:111; zur Hausen, H.
(1991). Human papillomaviruses in the pathogenesis of anogenital
cancer. Virology. 184:9) and in up to 10% of total mortality due to
cancer worldwide (zur Hausen, H. (1991). Viruses in Human Cancers,
Science 254:1167). Cervical cancer is the second most common cause
of cancer-related death in females worldwide (Parkin, D. M., Laara,
E., and Muir, C. S. (1988), Estimates of the worldwide frequency of
sixteen major cancers in (1980), Int. J. Cancer, 41:184). HPV DNA
is present in more than 90% of the cervical carcinomas and
predominantly of the HPV 16 genotype (Resnick, R. M., Cornelissen,
M. T., Wright, D, K., Eichinger, G. H., Fox, H. S., ter Schegget,
J., and Manos, M. M. (1990). Detection and typing of human
papillomavirus in archival cervical cancer specimens by DNA
amplification with consensus primers. J. Natl. Cancer Inst.; Van
den Brule, A. J. C., Walboomers, J. M. M., du Maine, M., Kenemans,
P., and Meijer, C. J. L. M. (1991). Difference in prevalence of
human papillomavirus genotypes in cytomorphologically normal smears
is associated with a history of cervical intraepithetal neoplasia,
Int. J. Cancer, 48:404). The ability of HPV 16 early region 6 and 7
(E6, E7) open reading frames to in vitro immortalize rodent cells
(Yasumoto, S., Burkhardt, A. L., Doniger, J., and DiPaolo, J. A.
(1986). Human Papillomaviruses type 16 DNA induced malignant
transformation of NIH3T3 cells. J. Virol., 57:572) and human
keratinocytes (Pirisi. L., Yasumoto, S., Feller, M., Doniger, J.,
and DiPaolo, J. A. (1987). Transformation of human fibroblasts and
keratinocytes with human papillomavirus type 16 DNA. J. Virol.
61:1061) and to transform human fibroblasts (Smits, H. L.,
Raadsheer, E., Rood, I., Mehendale, S., Slater, R. M., van der
Noordaa, J., and ter Schegget, J. (1988). Induction of
anchorage-independent growth of human embryonic fibroblasts with a
deletion in the short arm of chromosome 11, J. Virol. 62:4538)
suggests direct involvement of HPV 16 in the multi-step process of
cervical carcinogenesis.
[0939] In general T cell immunity, in particular mediated by
cytotoxic T lymphocytes (CTL) is important in the defense against
virus-induced tumors (Melief, C. J. (1992). Tumor eradication by
adoptive transfer of cytotoxic T lymphocytes, Adv. Cancer Res.,
58:143; Melief, C. J., and Kast, W. M. (1992). Lessons from T cell
responses to virus induced tumors for cancer eradication in
general, Cancer Surv., 13:81). Recently in a mouse model, it was
reported that some degree of protection against HPV 16 E7
expressing tumors can be obtained with CTL after immunization with
HPV 16 E7 expressing cells (Chen. L., Thomas, E, K., Hu, S. L.,
Hellstrom, I., and Hellstrom, K. E. (1991). Human papillomavirus
type 16 nucleoprotein E7 is a tumor rejection antigen, Proc. Natl.
Acad. Sci., 88:110; Chen, L., Ashe, S., Brady, W. A., Hellstrom,
I., Hellstrom, K. E., Ledbetter, J. A., McGowan, P., and Linsley,
P. S. (1992). Costimulation of Antitumor immunity by the B7
counterreceptor for the T lymphocyte molecules CD28 and CTLA-4.
Cell. 71:1093). In vivo protection by CTL was recently shown in
mouse models in which synthetic peptides containing CTL epitopes
were used for efficient priming of mice against virus infections
(Schulz, M., Zinkernagel, R. M., and Hengarter, H. (1991).
Peptide-induced antiviral protection by cytotoxic T cells, Proc.
Natl. Acad. Sci., USA, 88:991; Kast, W. M., Roux, L., Curren, J.,
Blom, H. J. J., Voordouw, A. C., Meleon, R. H., Kolakofski, D., and
Melief, C. J. M. (1991). Protection against lethal Sendai virus
infection by in vivo priming of virus-specific cytotoxic T
lymphocytes with an unbound peptide, Proc. Natl. Acad. Sci., USA,
88:2283). Moreover in a mouse model it has now been shown that
complete protection against HPV 16 induced tumors can be achieved
by peptide vaccination with a CTL epitope derived from the viral
oncogene E7.
[0940] The HPV 16 E6 and E7 gene products are the most desirable
target antigens for vaccination against HPV 16 induced tumors. Both
are retained and highly expressed in HPV 16-transformed cancer
cells in vivo (Baker, C. J., Phelps, W. C., Lindgren, V., Braun, M.
J., Gonda, M. A., and Howley, P. M. [1987]. Structural and
transcriptional analysis of human papillomavirus type 16 sequences
in cervical carcinoma cell lines, J. Virol., 61:962; Smotkin, D.,
and Wettstein, F. O. [1986]. Transcription of human papillomavirus
type 16 early genes in a cervical cancer and cancer-derived cell
line and identification of the E7 protein, Proc. Natl. Acad. Sci.,
USA, 83:4680) and involved in the induction and maintenance of
cellular transformation in vitro (Crook, T., Morgenstern, J. P.,
Crawford, L., and Banks, L. [1989]. Continued expression of HPV-16
E7 protein is required for maintenance of the transformed phenotype
of cells co-transformed by HPV-16 plus EJ ra., EMBO J., 8:513;
Hawley-Nelson, P., Vousden, K. H., Hubbert, N. L., Lowy, D. R., and
Schiller, J. T. [1989]. HPV 16 E6 and E7 proteins cooperate to
immortalize human foreskin keratinocytes, EMBO J., 8:3905).
Dependence of in vitro growth of cell lines derived from cervical
cancers on the expression of E6 and E7 emphasizes involvement of
these oncogenes in maintenance of the phenotype of cervical
carcinoma cell lines (Von Knebel Doeberitz, M., Bauknect, T.,
Bartch, D., and zur Hausen, H. [1991]. Influence of chromosomal
integration on glucocorticoid-regulated transcription of
growth-stimulation papillomavirus genes E6 and E7 in cervical
carcinoma cells, Proc. Natl. Acad. Sci., USA, 88:1411). To
determine the CTL epitopes and potential vaccine candidates of HPV
16 for humans, we screened peptides spanning the HPV 16 E6 and E7
protein sequences for their ability to bind to the most frequent
human MHC molecules, namely HLA-A1, A3.2, A11.2 and A24. Combined
these five alleles will cover about 90% of the world population
(Dupont, B., ed. [1987]. Immunology of HLA Vol. I
Histocompatibility Testing, Springer-Verlag, New York).
[0941] A complete set of 240 overlapping synthetic peptides of 9 aa
length and 8 aa overlap covering the entire HPV 16 E6 and E7
oncogene sequences were synthesized. The peptides were tested for
their ability to bind the aforementioned HLA molecules in the
binding assay described above. The results of this analysis show
the relative affinity of all peptides for the respective HLA
alleles and reveal the possible candidate CTL epitopes for use in
peptide based vaccines for humans in TABLE 98, TABLE 99, TABLE 100,
and TABLE 101.
[0942] The results confirm that peptide binding motif described in
this invention for the aforementioned HLA alleles predict which
peptide of a protein is likely to bind into the groove of a
specified HLA molecule. Since we used a large and unbiased set of
peptides, the results of the peptide binding analyses were used to
evaluate the value of these motifs both for their predictive
capacities and the necessity to have particular anchor aa residues
on positions 2, (3) and 9 in a peptide.
[0943] Peptides. Peptides were generated by solid phase strategies
on a multiple peptide synthesizer (Abimed AMS 422) by repeated
cycles in which addition of Fmoc protected amino acids to a resin
of polystyrene was alternated with a Fmoc-deprotection procedure
(Gausepohl, H., Kraft, M., Boulin, Ch., and Frank, R. W. [1990].
Automated multiple peptide synthesis with BOP activation. in Proc.
of the 11th American peptide symposium. J. E. Rivier and G. R.
Marshall, Ed. ESCOM, Leiden. 1003-1004). The peptides all carrying
a COOH group at the C-terminal end, were cleaved from the resin and
side chain protective groups were removed by treatment with aqueous
TFA. Peptides were analyzed by reversed phase HPLC lyophilized and
dissolved at a concentration of 1 mg/ml in phosphate-buffered
saline with 3% DMSO (Sigma, St. Louis, Mo. 63175) before use. Once
dissolved, the peptides were stored at -70.degree. C. Since
cysteine containing peptides are susceptible to (air) oxidation
during synthesis and handling, these peptides were synthesized with
an alanine instead of a cysteine.
[0944] Identification of peptides from HPV 16 E6 and E7 proteins
that bind to different HLA-A alleles. A complete set of 240
peptides of 9 aa in length and overlapping by 8 aa, covering the
sequences of the entire HPV 16 E6 and E7 proteins, was tested for
binding to 5 different HLA-A molecules.
[0945] The results of this analysis are depicted in TABLE 98, TABLE
99, TABLE 100, and TABLE 101. TABLE 98 describes the peptides of
HPV 16 that bound to HLA-A1 molecules. All peptides were tested.
Listed are only peptides yielding ratio values of .gtoreq.0.001. It
can be seen that 2 peptides bound with high affinity to this
molecule (>0.1), 6 with intermediate affinity (0.1-0.01) and 1
with low affinity (0.01-0.001). Peptides were ranked by ratio value
to allow comparison of data obtained in different experiments. To
calculate the concentration of a peptide necessary to yield a 50%
inhibition dose (IC.sub.50) one has to divide the value of the
standard IC.sub.50 by the ratio. For example, peptide E6-80 has an
IC.sub.50 of 23 nM (81/3.5).
[0946] TABLE 99 describes the peptides that bound to HLA-A3.2
molecules. Seven peptides were identified as high affinity binders,
6 as intermediate affinity binders and 13 as low affinity binders.
TABLE 100 describes the peptides that bound to HLA-A11.2 molecules.
Six high affinity peptides were identified, 4 intermediate affinity
binders and 10 low affinity binders. Two high affinity binding
peptides (E6-59 IYRDGNPY (SEQ ID NO:______) and E6-80 ISEYRHYAY
(SEQ ID NO:______)) and two weak affinity binding peptides with a Y
at the 9th position (E6-42 QQLLRREVY (SEQ ID NO:______), E6-69
VADKALKFY (SEQ ID NO:______)) were identified for HLA-A11.2
Considering the high binding strength of the first two peptides and
the similarity between the HLA-A11.2 motif and the HLA-A3.2 motif
in which Y's are preferred at the 9th aa position, tyrosines should
be included at the 9th position in the HLA-A 11.2 motif. Comparing
TABLE 105 and TABLE 106, it is clear that there is a large overlap
of peptides that bound to both A3.2 and A11.2 molecules. Eighteen
out of 28 E6 and E7 peptides binding to these two HLA molecules
overlapped and only 8 peptides were unique for HLA-A3.2 and 2
peptides unique for HLA-A11.2.
[0947] Finally, TABLE 107 describes the peptides that bound to
HLA-A24 molecules. Here 2 peptides were identified as high affinity
binding peptides, 5 as intermediate affinity binding peptides and 5
as low binding peptides. One high affinity peptide (E6-72 KALKFYSKI
(SEQ ID NO:______)) and one intermediate affinity peptide (E7-49
RAHYNIVTF (SEQ ID NO:______)) were identified, indicating that an A
at the second position should be allowed in the HLA-A24 motif. All
these inclusions are indicated in TABLE 102. In analyzing TABLE 69,
TABLE 70, TABLE 71, TABLE 72, and TABLE 73, it can be concluded
that between 2 and 7 high affinity binding peptides were identified
for all of the tested HLA-A molecules. Occasionally some peptides
were binding to more alleles. Three peptides (E6-7, E6-37 and
E6-79), bound to HLA-A2.1, A3.2 and A11.2. One peptide (E6-38)
bound to HLA-A3.2, A11.2 and A24 and two peptides (E6-69 and E6-80)
bound to HLA-A1, A3.2 and A11.2. But these crossreactive peptides
bound only weakly to one or more of the different HLA molecules. In
general, however, it can be concluded that, except for HLA-A3.2 and
HLA-A11.2 molecules, almost all HLA molecules bind unique
peptides.
[0948] Validation of HLA-A peptide binding motifs with an unbiased
set of HPV 16 E6 and E7 peptides. We analyzed how well the motifs
for anchor positions described in this invention predicted the
binding of a peptide, and also the reverse: how well binding
peptides followed the identified motifs. For this, peptides were
ranked as high binders, intermediate binders, weak binders, and
negative binders and for each peptide the motif prediction based on
the anchor motif rules of Table 74 were analyzed. The overall
efficiency of the 2, (3), and 9 anchor motifs was then calculated
and this is summarized in TABLE 102. It can be concluded that the
motifs described above for the different HLA-A molecules are quite
accurate. One hundred percent of the HLA-A1, A3.2, and A24 high
binders would be predicted as well as 67% of the HLA-11.2. Even for
the intermediate binders between 40 and 100% would be predicted
depending on the HLA-A molecule analyzed. Furthermore, the percent
of weak binding peptides that would be predicted is low and the
percent of those peptides that were predicted to bind but actually
did not bind is very low for all these alleles.
[0949] Analyzed differently, of the 12 peptides predicted to bind
to HLA-A1 actually 5 bound with high or intermediate affinity. This
indicates that only a few peptides would have to be made to find
these potential CTL epitopes. The figures for HLA-A3.2, A11.2, and
A24 were 10/32, 7/26, and 4/7, respectively. This implies that the
predictive value for all of these alleles is good. Besides a small
number of peptides that had not been predicted by the recently
described motifs, the (-) in TABLE 104, TABLE 105, TABLE 106, and
TABLE 107, a number of peptides that were predicted by the 2, (3)
and 9 anchor motifs did not bind, indicating that having the right
anchor residues is not always sufficient for binding and
implicating that non-anchor residues can make negative
contributions to the binding of a peptide.
Example 29
Presence of a Motif is Necessary but not Sufficient for High
Affinity Class I Binding
[0950] To investigate further how the presence of different motifs
might influence the capacity of different peptides to bind to the
relevant HLA alleles, the sequences of various potential target
molecules were scanned for the presence of motif-containing
peptides. The peptides thus identified were synthesized and tested
for binding. It was found (TABLE 97) that in the case of A3.2, only
39 (19%) of the 205 peptides bound with high affinity in the 1 to
50 nM range. 22.4% of them bound with intermediate affinities (in
the 50 to 500 nM range), while 34.6% bound weakly (in the 500 nM to
50 .mu.M range). Finally, 23.9% of them did not bind at all, at
least up to the 50 .mu.M level. In the case of A11, 33 (33%) of the
100 peptides bound with high affinity in the 1 to 50 nM range. 35%
of them bound with intermediate affinities (in the 50 nM range),
while 24% bound weakly (in the 500 nM to 50 .mu.M range). Finally,
8% of them did not bind at all, at least up to the 50 .mu.M
level.
[0951] Similar results were also obtained (data not shown) in the
case of A1 and A24.
[0952] The same type of analyses were also performed in the case of
10-mer peptides carrying either the A3.2, and A11 motifs (TABLE 109
and TABLE 110). It was found that in these cases, the frequency of
good binders was even lower (17.5%, and 29.8%, respectively). These
data confirm the fact that motif-containing 10-mer peptides can
indeed bind, albeit with, in general, reduced affinity.
[0953] In summary, the data shown in this section clearly show that
the presence of the correct anchor residues is not sufficient per
se to allow for good HLA binding. It is thus apparent that the
nature of the residues contained in positions other than 2(3) and 9
(or 10) can influence binding. The most likely explanation of this
observation is that the presence of certain residues (in positions
other than 2 and 9) can negate or increase the binding potential of
a peptide determinant.
[0954] The data shown in the preceding sections describe how
specific binding assays can be used to identify, within
motif-containing peptides, peptides that are immunogenic. We also
wanted to devise an alternative strategy, namely to derive
procedures that would be able to predict, within motif-containing
peptides, which peptides might be good or intermediate binders and
thereby might be immunogenic. In other experiments not shown
intermediate or good binders have been shown to be immunogenic. In
particular, to identify residues that have a negative impact on
binding an analysis of all positions for A3.2, A11, and all
motif-containing peptides, both 9-mers and 10-mers is carried out.
In the case of A11, because of the small occurrence of nonbinding
peptides, a different cutoff was used such that the analysis
compares good and intermediate binders on the one hand to weak and
nonbinders on the other.
Example 30
Specificity and Cross-Reactivity of HLA Binding
[0955] Peptide sequences capable of binding the most common HLA
alleles have been identified in previous studies. However, a large
number of monospecific epitopes would be required to provide
substantial coverage of all ethnic groups. In contrast, the
alternative approach of identifying broadly crossreactive motifs
(supermotifs) has the potential of covering a similar proportion of
the population using just two or three motifs. TABLE 28 shows a
hypothetical population coverage achieved by each of the different
motif types or combinations of motif types, using known and
predicted motifs.
[0956] To explore specificity and cross-reactivity of HLA binding
in more detail, a panel of HLA-A and B restricted T cell epitopes
was tested for binding in the assays described in Examples 1 &
2, above. It was found (TABLE 26) that the majority of the peptides
were good or intermediate binders to the appropriate restriction
element. The binding, in general, was allele-specific. Similar data
were obtained with a panel of HLA-B naturally processed peptides
(TABLE 27), in which it was found that 12 of 12 peptides were good
binders to the relevant restriction element. In addition, however,
some cross-reactivities were detected, particularly in the case of
alleles which had overlapping motifs.
[0957] For example, a high degree of cross-reactivity was noted
between A3.2 and A11 (shaded areas, TABLE 26). The cross-reactivity
seen between B7 and B8 with the B8 epitope 1054.05 can be explained
by the fact that this peptide has the motif for both B7 and B8. The
B7 motif is proline in position 2 and small hydrophobics at the
C-terminal. B8 recognized residues with basic charges (R,K) in
positions 3 and 5, and small hydrophobics at the C-terminal. These
data demonstrate that 1) in general, for both the A and B isotypes,
the binding is rather specific; and 2) occasional
cross-reactivities exist and can usually be explained by either
shared motifs or the presence within a single peptide of more than
one motif.
[0958] The data available thus far have defined a set of motifs
which are summarized in Table 28. Three motifs are shared by
multiple alleles (identified as types C, D, and F in TABLE 28).
Alleles of type C have hydrophobic residues at position 2 and at
the C-terminus; alleles of type D have hydrophobic residues at
position 2, with positively charged residues (R,K) at the
C-terminus; and alleles of type F have proline at position 2, with
hydrophobic residues at the C-terminus. Coverage of a significant
fraction of the population is achieved by identifying peptides
which bind to the alleles listed in TABLE 28 for the C, D, and F
"supermotifs."
Example 31
Prediction of Alleles Binding the Major Motif Supermotifs
[0959] Further analysis of the crossreactivity observed between A3,
A11, A31, and Aw68 was made by assessing the similarities of these
HLA molecules in the residues that make up the B and F binding
pockets involved in the interactions with position 2 and the C
terminal residue of the peptides which bind these molecules. When
this analysis was performed, a high degree of similarity between
these alleles becomes evident (see, Matsumura, M., et al., Science,
257:927 1992 for a discussion of the structure of the peptide
binding pockets in the groove of MHC Class I molecules). TABLES 29
and 30 shows the residues which constitute the F or C-terminal
pocket for these alleles. The residues are completely conserved in
all four alleles, and experimental data have indicated that each of
these alleles recognized basic residues (R,K) at the C-terminus of
peptides. B27, an allele which also recognizes basic residues at
the C-termini of peptides, differs from A3, A11, A31, and Aw68 by
only a single residue, a conservative isoleucine to leucine
difference.
[0960] These striking similarities can be contrasted with the
sequences of HLA molecules which do not share the basic charge
C-terminal motif. Further similarities between A3, A11, A31 and
Aw68 are also seen in the B pocket (TABLE 31), where they also
share overlapping motifs (hydrophobics and threonine).
[0961] Remarkable motif similarities are demonstrated by the
preference of many HLA-B (B7, B14, B35, B51, B53, and B54) and
HLA-C (Cw4, Cw6, and Cw7) alleles for proline in position 2. An
analysis of the B pocket of the HLA-B alleles is shown in TABLE 33,
and reveals that they all share similar B pockets, having the same
or conservatively different (i.e., N/Q) residues in positions 9,
63, 66, and 70. Interestingly, in addition to sharing a motif based
on proline in position 2, all of these alleles prefer hydrophobic
residues (F of LIV) in position 9. If further alleles could be
identified which have motifs fitting the three basic patterns (C,
D, and F), it would allow exploitation of crossreactivity using
peptides already developed. Crossreactive alleles could be
identified by two different approaches. In the first approach, one
could establish assays for a large panel of different alleles and
empirically determine which motifs fit the various supermotifs. In
the second approach, one could attempt to predict a priori
crossreactivity based on pocket structure. The analysis discussed
above, which compared and contrasted the binding pockets of alleles
which share similar B pockets and motifs, or similar F pockets and
motifs with alleles which have different motifs, supports the
notion that sharing similar pockets will result in the sharing of
similar motifs. If this assumption is true, a number of assays for
which cell lines are readily available could be explored (TABLE
32). These alleles all have B and F pockets, which suggests that
their motifs might fit into one of the motif types defined in TABLE
28.
Example 32
Peptide Binding to B54
[0962] To experimentally address the feasibility of increasing
allele coverage by a priori selecting alleles which are likely to
crossreact, we have examined B54, which is present in about 10% of
the Asian population. Sequence analysis of the B pocket of B54
suggested a close similarity to B35, B51, and B53 (TABLE 33), B54
differing from the other alleles fairly conservatively at three
positions. Most interestingly, the polar residues at positions 9,
63, and 70, which are invariable amongst Pro.sub.2 preferring
alleles (i.e., alleles to which peptides comprising the
B7-like-supermotif bind) and, we speculate, may be crucial for
"proline-ness," were completely invariant. The F pocket of B54
shares the S,N,L triplet at positions 77, 80, and 81 with B7, B8,
and B35, and carries a pair of hydrophobic residues at positions 95
and 116, as do these other B alleles. B7, B8, and B35 all prefer
peptides with hydrophobic C-terminals.
[0963] The analysis discussed above suggested that B54 might
recognize peptides carrying a Pro.sub.2-hydrophobic-c-terminal
motif (i.e., a B7-like-supermotif). To test this hypothesis, we
analyzed whether the B35 binding B35CON2 peptide (Cytel number
1021.05; sequence FPFKYAAAF (SEQ ID NO:______)) could bind to B54.
Indeed, excellent binding was detected, with an estimated Kd in the
5 nM range. Thus, a high affinity ligand was selected for B54 based
on B and F pocket structural analysis without any previous
knowledge of a specific motif. These data illustrate how it may be
possible to select, a priori, alleles which have the potential for
extensive crossreactivity and thus cover a large segment of the
population.
Example 33
Binding of Peptides to B7-Like Supermotif HLA Alleles
[0964] Peptides bearing the B7-like supermotif were tested for
binding to purified HLA molecules of some of the alleles sharing
the B7-like specificity. The binding assay was performed as
described in Example 2. TABLE 35 shows the binding to HLA-B*0701,
B*3501, B*3502, B*3503, and B*5401 of a set of peptides reported in
the literature to be restricted or naturally bound to various HLA-B
alleles.
[0965] TABLE 36 shows the binding of a set of 124 9-mer and 124
10-mer B7-like supermotif bearing peptides of various viral and
bacterial origin to HLA-B*0701, B*3501, B*5301, and B*5401. In
general, immunogenicity is correlated with binding affinity in that
peptides which bind MHC with affinities of 500 nM or less show
greater immunogenicity.
[0966] As shown in TABLE 35 and TABLE 36, there are peptides which
are capable of binding to more than one allele, demonstrating that
molecules of the defined B7-like supermotif family are indeed
capable of binding overlapping sets of peptides. To date,
approximately 10 peptides capable of over 25% (at minimum)
population coverage, as defined through its binding to any B7-like
allele(s), have been identified (Table 106). HBV, HIV, HCV, Mage 2,
Mage 3, and P. falciparum are each represented by at least one
cross-reactive binder.
[0967] The basis for the observed cross-reactivity was examined by
first establishing for four alleles, B*0701, B*3501, B*5301, and
B*5401, their individual secondary anchor motifs (FIG. 3). From the
individual motifs, a B7-like cross reactive motif is comprised of
all residues which are positive secondary anchors for at least 2 of
the four alleles examined. In its negative aspect, the motif
excludes peptides bearing residues at certain positions which are
detrimental influences on binding for at least 2 of the four
alleles examined. As shown in TABLE 37, the B7-like cross-reactive
supermotif allows the improved prediction of peptides which will be
capable of binding to 2 or more alleles of the B7-like
superfamily.
Example 34
Ex Vivo Induction of Cytotoxic T Lymphocytes (CTL)
[0968] Peripheral blood mononuclear cells (PBMC) are isolated from
an HLA-typed patient by either venipuncture or apheresis (depending
upon the initial amount of CTLp required), and purified by gradient
centrifugation-using Ficoll-Paque (Pharmacia). Typically, one can
obtain one million PBMC for every ml of peripheral blood, or
alternatively, a typical apheresis procedure can yield up to a
total of 1-10.times.10.sup.10 PBMC.
[0969] The isolated and purified PBMC are co-cultured with an
appropriate number of antigen presenting cell (APC), previously
incubated ("pulsed") with an appropriate amount of synthetic
peptide (containing the HLA binding motif and the sequence of the
antigen in question). PBMC are usually incubated at
1-2.times.10.sup.6 cells/ml in culture medium such as RPMI-1640
(with autologous serum or plasma) or the serum-free medium AIM-V
(Gibco).
[0970] APC are usually used at concentrations ranging from
1.times.10.sup.4 to 2.times.10.sup.5 cells/ml, depending on the
type of cell used. Possible sources of APC include: 1) autologous
dendritic cells (DC), which are isolated from PBMC and purified as
described (Inaba, et al., J. Exp. Med. 166:182 (1987)); and 2)
mutant and genetically engineered mammalian cells that express
"empty" HLA molecules (which are syngeneic [genetically identical]
to the patient's allelic HLA form), such as the, mouse RMA-S cell
line or the human T2 cell line. APC containing empty HLA molecules
are known to be potent inducers of CTL responses, possibly because
the peptide can associate more readily with empty MHC molecules
than with MHC molecules which are occupied by other peptides
(DeBruijn, et al., Eur. J. Immunol. 21:2963-70 (1991)).
[0971] In those cases when the APC used are not autologous, the
cells will have to be gamma irradiated with an appropriate dose
(using, e.g., radioactive cesium or cobalt) to prevent their
proliferation both ex vivo, and when the cells are re-introduced
into the patients.
[0972] The mixture cultures, containing PBMC, APC and peptide are
kept in an appropriate culture vessel such as plastic T-flasks,
gas-permeable plastic bags, or roller bottles, at 37.degree.
centigrade in a humid air/CO.sub.2 incubator. After the activation
phase of the culture, which usually occurs during the first 3-5
days, the resulting effector CTL can be further expanded, by the
addition of recombinant DNA-derived growth factors such as
interleukin-2 (IL-2), interleukin-4 (IL-4), or interleukin-7 (IL-7)
to the cultures. An expansion culture can be kept for an additional
5 to 12 days, depending on the numbers of effector CTL required for
a particular patient. In addition, expansion cultures may be
performed using hollow fiber artificial capillary systems (Cellco),
where larger numbers of cells (up to 1.times.10.sup.11) can be
maintained.
[0973] Before the cells are infused into the patient, they are
tested for activity, viability, toxicity and sterility. The
cytotoxic activity of the resulting CTL can be determined by a
standard .sup.51Cr-release assay (Biddison, W. E. 1991, Current
Protocols in Immunology, p7, 17.1-7.17.5, Ed. J. Coligan et al., J.
Wiley and Sons, New York), using target cells that express the
appropriate HLA molecule, in the presence and absence of the
immunogenic peptide. Viability is determined by the exclusion of
trypan blue dye by live cells. Cells are tested for the presence of
endotoxin by conventional techniques. Finally, the presence of
bacterial or fungal contamination is determined by appropriate
microbiological methods (chocolate agar, etc.). Once the cells pass
all quality control and safety tests, they are washed and placed in
the appropriate infusion solution (Ringer/glucose lactate) and
infused intravenously into the patient.
Example 35
Assays for CTL Activity
[0974] 1. Peptide synthesis. Peptide syntheses were carried out by
sequential coupling of N-a-Fmoc-protected amino acids on an Applied
Biosystems (Foster City, Calif.) 430A peptide synthesizer using
standard Fmoc coupling cycles (software version 1.40). All amino
acids, reagents, and resins were obtained from Applied Biosystems
or Bachem. Solvents were obtained from Burdick & Jackson.
Solid-phase synthesis was started from an appropriately substituted
Fmoc-amino acid-Sasrin resin. The loading of the starting resin was
0.5-0.7 mmol/g polystyrene, and 0.1 or 0.25 meq were used in each
synthesis. A typical reaction cycle proceeded as follows: 1) The
N-terminal Fmoc group was removed with 25% piperidine in
dimethylformamide (DMF) for 5 minutes, followed by another
treatment with 25% piperidine in DMF for 15 minutes. The resin was
washed 5 times with DMF. An N-methylpyrolidone (NMP) solution of a
4 to 10 fold excess of a pre-formed 1-hydroxybenzotriazole ester of
the appropriate Fmoc-amino acid was added to the resin and the
mixture was allowed to react for 30-90 min. The resin was washed
with DMF in preparation for the next elongation cycle. The fully
protected, resin bound peptide was subjected to a piperidine cycle
to remove the terminal Fmoc group. The product was washed with
dichloromethane and dried. The resin was then treated with
trifluoroacetic acid in the presence of appropriate scavengers
[e.g. 5% (v/v) water] for 60 minutes at 20.degree. C. After
evaporation of excess trifluoroacetic acid, the crude peptide was
washed with dimethyl ether, dissolved in water and lyophilized. The
peptides wee purified to >95% homogeneity by reverse-phase HPLC
using H.sub.2O/CH.sub.3CN gradients containing 0.2% TFA modifier on
a Vydac, 300 .ANG. pore-size, C-18 preparative column. The purity
of the synthetic peptides was assayed on an analytical
reverse-phase column, and their composition ascertained by amino
acid analysis and/or sequencing. Peptides were routinely dissolved
in DMSO at the concentration of 20 mg/ml.
[0975] 2. Media. RPMI-1640 containing 10% fetal calf serum (FCS) 2
mM Glutamine, 50 ig/ml Gentamicin and 5.times.10.sup.-5M
2-mercaptoethanol served as culture medium and will be referred to
as R10 medium.
[0976] RPMI-1640 containing 25 mM Hepes buffer and supplemented
with 2% FCS was used as cell washing medium.
[0977] 3. Rat Concanavalin A supernatant. The spleen cells obtained
from Lewis rats (Sprague-Dawley) were resuspended at a
concentration of 5.times.10.sup.6 cells/ml in R10 medium
supplemented with 5 .mu.g/ml of ConA in 75 cm2 tissue culture
flasks. After 48 hr at 37.degree. C., the supernatants were
collected, supplemented with 1% ______-methyl-D-mannoside and
filter sterilized (0.45 .mu.m filter). Aliquots were stored frozen
at -20.degree. C.
[0978] 4. LPS-activated lymphoblasts. Murine splenocytes were
resuspended at a concentration of 1-1.5.times.10.sup.6/ml in R10
medium supplemented with 25 .mu.g/ml LPS and 7 .mu.g/ml dextran
sulfate in 75 cm.sup.2 tissue culture flasks. After 72 hours at
37.degree. C., the lymphoblasts were collected for use by
centrifugation.
[0979] 5. Peptide coating of lymphoblasts. Coating of the LPS
activated lymphoblasts was achieved by incubating 30.times.10.sup.6
lymphoblasts with 100 .mu.g of peptide in 1 ml of R10 medium for 1
hr at 37.degree. C. Cells were then washed once and resuspended in
R10 medium at the desired concentration for use in in vitro CTL
activation.
[0980] 6. Peptide coating of Jurkat A2/K.sup.b cells. Peptide
coating was achieved by incubating 10.times.10.sup.6 irradiated
(20,000 rads) Jurkat A2.1/K.sup.b cells with 20 .mu.g of peptide in
1 ml of R10 medium for 1 hour at 37.degree. C. Cells were washed
three times and resuspended at the required concentration in R10
medium.
[0981] 7. In Vitro CTL activation. One to four weeks after priming
spleen cells (5.times.10.sup.6 cells/well or 30.times.10.sup.6
cells/T25 flask) were concultured at 37.degree. C. with syngeneic,
irradiated (3,000 rads), peptide coated lymphoblasts
(2.times.10.sup.6 cells/well or 10.times.10.sup.6 cells/T25 flask)
in R10 medium to give a final volume of 2 ml in 24-well plates or
10 ml in T25 flasks.
[0982] Restimulation of effector cells. Seven to ten days after the
initial in vitro activation, described in paragraph 7 above, a
portion of the effector cells were restimulated with irradiated
(20,000 rads), peptide-coated Jurkat A2/K.sup.b cells
(0.2.times.10.sup.6 cells/well) in the presence of 3.times.10.sup.6
"feeder cells"/well (C57Bl/6 irradiated spleen cells) in R10 medium
supplemented with 5% rat ConA supernatant to help provide all of
the cytokines needed for optimal effector cell growth.
[0983] 9. Assay for cytotoxic activity. Target cells
(3.times.10.sup.6) were incubated at 37.degree. C. in the presence
of 200 .mu.l of sodium .sup.51Cr chromate. After 60 minutes, cells
were washed three times and resuspended in R10 medium. Peptides
were added at the required concentration. For the assay, 10.sup.4
51Cr-labeled target cells wee added to different concentrations of
effector cells (final volume of 200 .mu.l) in U-bottom 96-well
plates. After a 6-hour incubation period at 37.degree. C., 0.1 ml
aliquots of supernatant were removed from each well and
radioactivity was determined in a Micromedic automatic gamma
counter. The percent specific lysis was determined by the formula:
percent specific release=100.times.(experimental
release-spontaneous release)/(maximum release-spontaneous release).
Where peptide titrations wee performed, the antigenicity of a given
peptide (for comparison purposes) was expressed as the peptide
concentration required to induce 40% specific .sup.51Cr release at
a given E:T.
[0984] Transgenic mice were injected subcutaneously in the base of
the tail with an incomplete Freund's adjuvant emulsion containing
50 nM of the putative CTL epitopes containing the A2.1 motifs, and
50 nM of a hepatitis B core T helper epitope. Eight to 20 days
later, animals were sacrificed and spleen cells were restimulated
in vitro with syngeneic LPS lymphoblasts coated with the putative
CTL epitope. A source of IL-2 (rat con A supernatant) was added at
day 6 of the assay to a final concentration of 5% and CTL activity
was measured on day 7. The capacity of these effector T cells to
lyse peptide-coated target cells that express the A2 KB molecule
(Jurkat A2 KB) was measured as lytic units. The results are
presented in Tables 147 and 148.
[0985] The results of this experiment indicate that those peptides
having a binding of at least 0.01 are capable of inducing CTL. All
of the peptides in TABLES 181 and 182 having a binding of at least
about 0.01 would be immunogenic.
Example 36
Algorithms to Identify Immunogenic Peptides
[0986] In light of results presented in the examples above,
algorithms were developed to provide a more exact predictor of
binding based upon the effects of different residues at each
position of a peptide sequence, in addition to the anchor or
conserved residues. More specifically, we utilize the data bank
obtained during the screening of our collection of A1, 3, 11 or 24
motif-containing peptides to develop an algorithm for each
particular allele which assigns a score for each amino acid at each
position along a peptide. The score for each residue is taken as
the ratio of the frequency of that residue in good and intermediate
binders to the frequency of occurrence of that residue in
nonbinders.
[0987] In the present algorithm residues have been grouped by
similarity. This avoids the problem encountered with some rare
residues, such as tryptophan, where there are too few occurrences
to obtain a statistically significant ratio. A listing is made of
scores obtained by grouping for each of the twenty amino acids by
position for 9-mer peptides containing conserved residues that
define their motif (2/9 motifs). A peptide is scored in the
algorithm as a product of the scores of each of its residues.
[0988] The power of an algorithm to correlate with binding is
further underlined by its ability to predict a population of
peptides with the highest occurrence of good binders. If one were
to rely, for example, solely on the 2/9 motif for predicting 9-mer
peptides which bind to a specific MHC allele the large number of
peptides containing the motif would be predicted to be good
binders. In fact only a relatively small percentage of these
peptides are good binders and a somewhat larger percentage are
intermediate binders, while a still larger percentage of the
peptides predicted by the motif are either weak or nonbinding
peptides. In contrast, using the grouped algorithm of this
invention a population of peptides are created with a greater
percentage of good binders, a still greater percentage of
intermediate binders, and a smaller percentage, relative to that
predicted by motif-containing peptides, are weak and
nonbinders.
[0989] The present example of an algorithm uses the ratio of the
frequency of occurrence of an amino acid in binders and nonbinders
to measure the impact of a particular residue at each position of a
peptide. It is immediately apparent to one of ordinary skill in the
art that there are alternative ways of creating a similar
algorithm. For example, one could use average binding affinity
values, or relative binding of single amino acid substitutions in a
motif containing peptide with a poly-alanine backbone to generate
an algorithm table.
[0990] An algorithm using average binding affinity has the
advantage of including all of the peptides in the analysis, and not
just good/intermediate binders and nonbinders. Moreover, it gives a
more quantitative measure of affinity than the simpler group ratio
algorithm. We have created such an algorithm by calculating for
each amino acid, by position, the average log of binding when that
particular residue occurs in our set of motif containing peptides.
The algorithm score for a peptide is then taken as the sum of the
scores by position for each of its residues.
Example 37
Analysis of the Immunogenicity of CTL and HTL Peptides
[0991] Class I and II antigen isolation was carried out as
described in the related applications, noted above. Naturally
processed peptides were then isolated and sequenced as described
there. An allele-specific motif and algorithms were determined and
quantitative binding assays were carried out.
[0992] Using the motifs identified above for HLA-A2.1 and other
allele amino acid sequences from a number of antigens were analyzed
for the presence of these motifs. TABLE 2 provides the results of
these searches. The letter "J" represents norleucine.
[0993] Analyses of CTL and HTL responses against the immunogen, as
well as against common recall antigens are commonly used and are
known in the art. Assays employed included chromium release,
lymphokine secretion and lymphoproliferation assays. Assays useful
in these determinations are described in Current Protocols in
Immunology, J. E. Coligan, et al., eds., John Wiley & Sons
Press (2000), chapters 3, 4, 6, and 7.
[0994] In one embodiment, the appropriate antigen-presenting cells
are incubated with 10-100 .mu.M of peptide in serum-free media for
4 hours under appropriate culture conditions. The peptide-loaded
antigen-presenting cells are then incubated with the responder cell
populations in vitro for 7 to 10 days under optimized culture
conditions. If screening for MHC class I presented peptides,
positive CTL activation can be determined by assaying the cultures
for the presence of CTLs that kill radiolabeled target cells, both
specific peptide-pulsed targets as well as target cells expressing
the endogenously processed form of the relevant virus or tumor
antigen from which the peptide sequence was derived. If screening
for MHC class II-presented peptides, positive HTL activation can be
determined by assaying cultures for cytokine production or
proliferation.
[0995] In one embodiment, prior to incubation of the stimulator
cells with the cells to be activated, i.e., precursor CD8+ cells,
an amount of antigenic peptide is added to the stimulator cell
culture, of sufficient quantity to become loaded onto the human
Class I molecules to be expressed on the surface of the stimulator
cells. In the present invention, a sufficient amount of peptide is
an amount that will allow about 200, and preferably 200 or more,
human Class I MHC molecules loaded with peptide to be expressed on
the surface of each stimulator cell. Preferably, the stimulator
cells are incubated with >20 .mu.g/ml peptide.
[0996] Resting or precursor CD8+ cells are then incubated in
culture with the appropriate stimulator cells for a time period
sufficient to activate the CD8+ cells. Preferably, the CD8+ cells
are activated in an antigen-specific manner. The ratio of resting
or precursor CD8+ (effector) cells to stimulator cells may vary
from individual to individual and may further depend upon variables
such as the amenability of an individual's lymphocytes to culturing
for which the within-described treatment modality is used.
Preferably, however, the lymphocyte:stimulator cell ratio is in the
range of about 30:1 to 300:1. The effector/stimulator culture may
be maintained for as long a time as is conditions and the nature
and severity of the disease condition or other condition necessary
to stimulate a therapeutically useable or effective number of CD8+
cells.
[0997] The induction of CTL in vitro requires the specific
recognition of peptides that are bound to allele specific MHC class
I molecules on APC. The number of specific MHC/peptide complexes
per APC is crucial for the stimulation of CTL, particularly in
primary immune responses. While small amounts of peptide/MHC
complexes per cell are sufficient to render a cell susceptible to
lysis by CTL, or to stimulate a secondary CTL response, the
successful activation of a CTL precursor (pCTL) during primary
response requires a significantly higher number of MHC/peptide
complexes. Peptide loading of empty major histocompatability
complex molecules on cells allows the induction of primary
cytotoxic T lymphocyte responses. Peptide loading of empty major
histocompatability complex molecules on cells enables the induction
of primary cytotoxic T lymphocyte responses.
[0998] Since mutant cell lines do not exist for every human MHC
allele, it is advantageous to use a technique to remove endogenous
MHC-associated peptides from the surface of APC, followed by
loading the resulting empty MHC molecules with the immunogenic
peptides of interest. The use of non-transformed (non-tumorigenic),
non-infected cells, and preferably, autologous cells of patients as
APC is desirable for the design of CTL induction protocols directed
towards development of ex vivo CTL therapies. This application
discloses methods for stripping the endogenous MHC-associated
peptides from the surface of APC followed by the loading of desired
peptides.
[0999] A stable MHC class I molecule is a trimeric complex formed
of the following elements: 1) a peptide usually of 8-10 residues,
2) a transmembrane heavy polymorphic protein chain which bears the
peptide-binding site in its .alpha.1 and .alpha.2 domains, and 3) a
non-covalently associated non-polymorphic light chain, .beta..sub.2
microglobulin. Removing the bound peptides and/or dissociating the
.beta..sub.2 microglobulin from the complex renders the MHC class I
molecules nonfunctional and unstable, resulting in rapid
degradation. All MHC class I molecules isolated from PBMCs have
endogenous peptides bound to them. Therefore, the first step is to
remove all endogenous peptides bound to MHC class I molecules on
the APC without causing their degradation before exogenous peptides
can be added to then.
[1000] Two possible ways to free up MHC class I molecules of bound
peptides include lowering the culture temperature from 37.degree.
C. to 26.degree. C. overnight to destablize .beta.2 microglobulin
and stripping the endogenous peptides from the cell using a mild
acid treatment. The methods release previously bound peptides into
the extracellular environment allowing new exogenous peptides to
bind to the empty class I molecules. The cold-temperature
incubation method enables exogenous peptides to bind efficiently to
the MHC complex, but requires an overnight incubation at 26.degree.
C. which may slow the cell's metabolic rate. It is also likely that
cells not actively synthesizing MHC molecules (e.g., resting PBMC)
would not produce high amounts of empty surface MHC molecules by
the cold temperature procedure.
[1001] Harsh acid stripping involves extraction of the peptides
with trifluoroacetic acid, pH 2, or acid denaturation of the
immunoaffinity purified class I-peptide complexes. These methods
are not feasible for CTL induction, since it is important to remove
the endogenous peptides while preserving APC viability and an
optimal metabolic state which is critical for antigen presentation.
Mild acid solutions of pH 3 such as glycine or citrate-phosphate
buffers have been used to identify endogenous peptides and to
identify tumor associated T cell epitopes. The treatment is
especially effective, in that only the MHC class I molecules are
destabilized (and associated peptides released), while other
surface antigens remain intact, including MHC class II molecules.
Most importantly, treatment of cells with the mild acid solutions
does not affect the cell's viability or metabolic state. The mild
acid treatment is rapid since the stripping of the endogenous
peptides occurs in two minutes at 4.degree. C. and the APC is ready
to perform its function after the appropriate peptides are loaded.
The technique is utilized herein to make peptide-specific APCs for
the generation of primary antigen-specific CTL. The resulting APC
are efficient in inducing peptide-specific CD8+ CTL.
[1002] Activated CD8+ cells may be effectively separated from the
stimulator cells using one of a variety of known methods. For
example, monoclonal antibodies specific for the stimulator cells,
for the peptides loaded onto the stimulator cells, or for the CD8+
cells (or a segment thereof) may be utilized to bind their
appropriate complementary ligand. Antibody-tagged molecules may
then be extracted from the stimulator-effector cell admixture via
appropriate means, e.g., via well-known immunoprecipitation or
immunoassay methods.
[1003] Effective, cytotoxic amounts of the activated CD8+ cells can
vary between in vitro and in vivo uses, as well as with the amount
and type of cells that are the ultimate target of these killer
cells. The amount will also vary depending on the condition of the
patient and should be determined via consideration of all
appropriate factors by the practitioner. Preferably, however, about
1.times.10.sup.6 to about 1.times.10.sup.12, more preferably about
1.times.10.sup.8 to about 1.times.10.sup.11, and even more
preferably, about 1.times.10.sup.9 to about 1.times.10.sup.10
activated CD8+ cells are utilized for adult humans, compared to
about 5.times.10.sup.6-5.times.10.sup.7 cells used in mice.
[1004] Preferably, as discussed above, the activated CD8+ cells are
harvested from the cell culture prior to administration of the CD8+
cells to the individual being treated. It is important to note,
however, that unlike other present and proposed treatment
modalities, the present method uses a cell culture system that is
not tumorigenic. Therefore, if complete separation of stimulator
cells and activated CD8+ cells is not achieved, there is no
inherent danger known to be associated with the administration of a
small number of stimulator cells, whereas administration of
mammalian tumor-promoting cells may be extremely hazardous.
[1005] Methods of re-introducing cellular components are known in
the art and include procedures such as those exemplified in U.S.
Pat. No. 4,844,893 to Honsik, et al., and U.S. Pat. No. 4,690,915
to Rosenberg. For example, administration of activated CD8+ cells
via intravenous infusion is appropriate.
[1006] The peptides of the invention can be identified and tested
for in vivo immunogenicity using HLA transgenic mice. The utility
of HLA transgenic mice for the purpose of epitope identification
(Sette et al., J Immunol, 153:5586-92 (1994); Wentworth et al., Int
Immunol, 8:651-9 (1996); Engelhard et al., J Immunol, 146:1226-32
(1991); Man et al., Int Immunol, 7:597-605 (1995); Shirai et al., J
Immunol, 154:2733-42 (1995)), and vaccine development (Ishioka et
al., J Immunol, 162:3915-25 (1999)) has been established. Most of
the published reports have investigated the use of HLA A2.1/Kb mice
but it should be noted that B*27, and B*3501 mice are also
available. Furthermore, HLA A*11/K.sup.b mice (Alexander et al., J.
Immunol., 159:4753-61 (1997)), and HLA B7/Kb and HLA A1/K.sup.b
mice have also been generated. Data from 38 different potential
epitopes was analyzed to determine the level of overlap between the
A2.1-restricted CTL repertoire of A2.1/K.sup.b-transgenic mice and
A2.1+ humans (Wentworth et al., Eur J Immunol, 26:97-101 (1996)).
In both humans and mice, an MHC peptide binding affinity threshold
of approximately 500 nM correlates with the capacity of a peptide
to elicit a CTL response in vivo. A high level of concordance
between the human data in vivo and mouse data in vivo was observed
for 85% of the high-binding peptides, 58% of the intermediate
binders, and 83% of the low/negative binders. Similar results were
also obtained with HLA A11 and HLA B7 transgenic mice (Alexander et
al., J Immunol, Vol. 159(10):4753-61 (1997)). Thus, because of the
extensive overlap that exists between T cell receptor repertoires
of HLA transgenic mouse and human CTLs, transgenic mice are
valuable for assessing immunogenicity of the multi-epitope
constructs described herein. Peptides binding to MHC class II
alleles can be examined using HLA-DR transgenic mice. See, i.e.,
Taneja V., David C. S., Immunol Rev, 169:67-79 (1999)).
[1007] More sensitive techniques such as the ELISPOT assay,
intracellular cytokine staining, and tetramer staining have become
available in the art to determine lymphocyte antigen
responsiveness. It is estimated that these newer methods are 10- to
100-fold more sensitive than the common CTL and HTL assays
(Murali-Krishna et al., Immunity, 8:177-87 (1998)), because the
traditional methods measure only the subset of T cells that can
proliferate in vitro, and may, in fact, be representative of only a
fraction of the memory T cell compartment (Ogg G. S., McMichael A.
J., Curr Opin Immunol, 10:393-6 (1998)). Specifically in the case
of HIV, these techniques have been used to measure antigen-specific
CTL responses from patients that would have been undetectable with
previous techniques (Ogg et al., Science, 279:2103-6 (1998); Gray
et al., J Immunol, 162:1780-8 (1999); Ogg et al., J Virol,
73:9153-60 (1999); Kalams et al., J Virol, 73:6721-8 (1999);
Larsson et al., AIDS, 13:767-77 (1999); Corne et al., J Acquir
Immune Defic Syndr Hum Retrovirol, 20:442-7 (1999)).
[1008] The peptides of the present invention and pharmaceutical and
vaccine compositions thereof are useful for administration to
mammals, particularly humans, to treat and/or prevent viral
infection and cancer. Examples of diseases which can be treated
using the immunogenic peptides of the invention include prostate
cancer, hepatitis B, hepatitis C, AIDS, renal carcinoma, cervical
carcinoma, lymphoma, CMV and chondyloma acuminatum. A protective
(or prophylatic) vaccine includes one that will protect against
future exposure to pathogen or cancer. A therapeutic vaccine
includes one that will ameriolate, attenuate, or ablate symptoms or
disease state induced by or related to a pathogen or
malignancy.
[1009] In circumstances in which efficacy of a prophylactic vaccine
is primarily correlated with the induction of a long-lasting memory
response, restimulation assays can be the most appropriate and
sensitive measures to monitor vaccine-induced immunological
responses. Conversely, in the case of therapeutic vaccines, the
main immunological correlate of activity can be the induction of
effector T cell function, most aptly measured by primary assays.
Thus, the use of sensitive assays allows for the most appropriate
testing strategy for immunological monitoring of vaccine
efficacy.
[1010] The induction of CTL in vitro requires the specific
recognition of peptides that are bound to allele specific MHC class
I molecules on APC. The number of specific MHC/peptide complexes
per APC is crucial for the stimulation of CTL, particularly in
primary immune responses. While small amounts of peptide/MHC
complexes per cell are sufficient to render a cell susceptible to
lysis by CTL, or to stimulate a secondary CTL response, the
successful activation of a CTL precursor (pCTL) during primary
response requires a significantly higher number of MHC/peptide
complexes. Peptide loading of empty major histocompatability
complex molecules on cells allows the induction of primary
cytotoxic T lymphocyte responses. Peptide loading of empty major
histocompatability complex molecules on cells enables the induction
of primary cytotoxic T lymphocyte responses.
[1011] Since mutant cell lines do not exist for every human MHC
allele, it is advantageous to use a technique to remove endogenous
MHC-associated peptides from the surface of APC, followed by
loading the resulting empty MHC molecules with the immunogenic
peptides of interest. Antigen-presenting cells can be normal cells
such as peripheral blood mononuclear cells or dendritic cells
(Inaba, et al., J. Exp. Med. 166:182 (1987); Boog, Eur. J. Immunol.
18:219 (1988)). The use of non-transformed (non-tumorigenic),
non-infected cells, and preferably, autologous cells of patients as
APC is desirable for the design of CTL induction protocols directed
towards development of ex vivo CTL therapies. This application
discloses methods for stripping the endogenous MHC-associated
peptides from the surface of APC followed by the loading of desired
peptides.
[1012] A stable MHC class I molecule is a trimeric complex formed
of the following elements: 1) a peptide usually of 8-10 residues,
2) a transmembrane heavy polymorphic protein chain which bears the
peptide-binding site in its .alpha.1 and .alpha.2 domains, and 3) a
non-covalently associated non-polymorphic light chain, .beta..sub.2
microglobulin. Removing the bound peptides and/or dissociating the
.beta..sub.2 microglobulin from the complex renders the MHC class I
molecules nonfunctional and unstable, resulting in rapid
degradation. All MHC class I molecules isolated from PBMCs have
endogenous peptides bound to them. Therefore, the first step is to
remove all endogenous peptides bound to MHC class I molecules on
the APC without causing their degradation before exogenous peptides
can be added to them.
[1013] Two possible ways to free up MHC class I molecules of bound
peptides include lowering the culture temperature from 37.degree.
C. to 26.degree. C. overnight to destabilize .beta..sub.2
microglobulin and stripping the endogenous peptides from the cell
using a mild acid treatment. The methods release previously bound
peptides into the extracellular environment allowing new exogenous
peptides to bind to the empty class I molecules. The
cold-temperature incubation method enables exogenous peptides to
bind efficiently to the MHC complex, but requires an overnight
incubation at 26.degree. C. which may slow the cell's metabolic
rate. It is also likely that cells not actively synthesizing MHC
molecules (e.g., resting PBMC) would not produce high amounts of
empty surface MHC molecules by the cold temperature procedure.
[1014] Harsh acid stripping involves extraction of the peptides
with trifluoroacetic acid, pH 2, or acid denaturation of the
immunoaffinity purified class I-peptide complexes. These methods
are not feasible for CTL induction, since it is important to remove
the endogenous peptides while preserving APC viability and an
optimal metabolic state which is critical for antigen presentation.
Mild acid solutions of pH 3 such as glycine or citrate-phosphate
buffers have been used to identify endogenous peptides and to
identify tumor associated T cell epitopes. The treatment is
especially effective, in that only the MHC class I molecules are
destabilized (and associated peptides released), while other
surface antigens remain intact, including MHC class II molecules.
Most importantly, treatment of cells with the mild acid solutions
do not affect the cell's viability or metabolic state. The mild
acid treatment is rapid since the stripping of the endogenous
peptides occurs in two minutes at 4.degree. C. and the APC is ready
to perform its function after the appropriate peptides are loaded.
The technique is utilized herein to make peptide-specific APCs for
the generation of primary antigen-specific CTL. The resulting APC
are efficient in inducing peptide-specific CD8+ CTL.
[1015] Activated CD8+ cells may be effectively separated from the
stimulator cells using one of a variety of known methods. For
example, monoclonal antibodies specific for the stimulator cells,
for the peptides loaded onto the stimulator cells, or for the CD8+
cells (or a segment thereof) may be utilized to bind their
appropriate complementary ligand. Antibody-tagged molecules may
then be extracted from the stimulator-effector cell admixture via
appropriate means, e.g., via well-known immunoprecipitation or
immunoassay methods.
[1016] Effective, cytotoxic amounts of the activated CD8+ cells can
vary between in vitro and in vivo uses, as well as with the amount
and type of cells that are the ultimate target of these killer
cells. The amount will also vary depending on the condition of the
patient and should be determined via consideration of all
appropriate factors by the practitioner. Preferably, however, about
1.times.10.sup.6 to about 1.times.10.sup.12, more preferably about
1.times.10.sup.8 to about 1.times.10.sup.11, and even more
preferably, about 1.times.10.sup.9 to about 1.times.10.sup.10
activated CD8+ cells are utilized for adult humans, compared to
about 5.times.10.sup.6-5.times.10.sup.7 cells used in mice.
[1017] Preferably, as discussed above, the activated CD8+ cells are
harvested from the cell culture prior to administration of the CD8+
cells to the individual being treated. It is important to note,
however, that unlike other present and proposed treatment
modalities, the present method uses a cell culture system that is
not tumorigenic. Therefore, if complete separation of stimulator
cells and activated CD8+ cells is not achieved, there is no
inherent danger known to be associated with the administration of a
small number of stimulator cells, whereas administration of
mammalian tumor-promoting cells may be extremely hazardous.
[1018] Methods of re-introducing cellular components are known in
the art and include procedures such as those exemplified in U.S.
Pat. No. 4,844,893 to Honsik, et al. and U.S. Pat. No. 4,690,915 to
Rosenberg. For example, administration of activated CD8+ cells via
intravenous infusion is appropriate.
Example 38
Use of Peptide Epitopes as Diagnostic Agents for Evaluating Immune
Responses
[1019] In one embodiment of the invention, HLA class I and class II
binding peptides can be used as reagents to evaluate an immune
response. The evaluated immune response can be induced by any
immunogen. For example, the immunogen may result in the production
of antigen-specific CTLs or HTLs that recognize the peptide
epitope(s) employed as the reagent. Thus, a peptide of the
invention mayor may not be used as the immunogen. Assay systems
that can be used for such analyses include tetramer-based
protocols, staining for intracellular lymphokines, interferon
release assays, or ELISPOT assays.
[1020] For example, following exposure to a putative immunogen, a
peptide of the invention can be used in a tetramer staining assay
to assess peripheral blood mononuclear cells for the presence of
any antigen-specific CTLs. The HLA-tetrameric complex is used to
directly visualize antigen-specific CTLs and thereby determine the
frequency of such antigen-specific CTLs in a sample of peripheral
blood mononuclear cells (see, e.g., Ogg et al., Science
279:2103-2106, 1998; and Altman et al., Science 174:94-96,
1996).
[1021] A tetramer reagent comprising a peptide of the invention is
generated as follows: A peptide that binds to an HLA molecule is
refolded in the presence of the corresponding HLA heavy chain and
P2-microglobulin to generate a trimolecular complex. The complex is
biotinylated at the carboxyl terminal end of the HLA heavy chain,
at a site that was previously engineered into the protein. Tetramer
formation is then induced by adding streptavidin. When
fluorescently labeled streptavidin is used, the tetrameric complex
is used to stain antigen-specific cells. The labeled cells are then
readily identified, e.g., by flow cytometry. Such procedures are
used for diagnostic or prognostic purposes; the cells identified by
the procedure can be used for therapeutic purposes.
[1022] Peptides of the invention are also used as reagents to
evaluate immune recall responses. (see, e.g., Bertoni et al., J
Clin. Invest. 100:503-513, 1997 and Penna et al., J Exp. Med.
174:1565-1570, 1991). For example, a PBMC sample from an individual
expressing a disease-associated antigen (e.g. a tumor-associated
antigen such as CEA, p53, MAGE2/3,HER2/neu, or an organism
associated with neoplasia such as HPV or HSV) can be analyzed for
the presence of antigen-specific CTLs or HTLs using specific
peptides. A blood sample containing mononuclear cells may be
evaluated by cultivating the PBMCs and stimulating the cells with a
peptide of the invention. After an appropriate cultivation period,
the expanded cell population may be analyzed, for example, for CTL
or for HTL activity.
[1023] Thus, the peptides can be used to evaluate the efficacy of a
vaccine. PBMCs obtained from a patient vaccinated with an immunogen
may be analyzed by methods such as those described herein. The
patient is HLA typed, and peptide epitopes that are bound by the
HLA molecule(s) present in that patient are selected for analysis.
The immunogenicity of the vaccine is indicated by the presence of
CTLs and/or HTLs directed to epitopes present in the vaccine.
[1024] The peptides of the invention may also be used to make
antibodies, using techniques well known in the art (see, e.g.
CURRENTPROTOCOLSINIMMUNOLOGY, Wiley/Greene, NY; and Antibodies A
Laboratory Manual Harlow, Harlow and Lane, Cold Spring Harbor
Laboratory Press, 1989). Such antibodies are useful as reagents to
determine the presence of disease-associated antigens or may be
used therapeutically. Antibodies in this category include those
that recognize a peptide when bound by an HLA molecule, i.e.,
antibodies that bind to a peptide-MHC complex.
[1025] The immunogenic peptides of this invention may also be used
to make monoclonal antibodies. Such antibodies may be useful as
potential diagnostic or therapeutic agents.
[1026] Epitopes in accordance with the present invention were
successfully used to induce an immune response. Immune responses
with these epitopes have been induced by administering the epitopes
in various forms. The epitopes have been administered as peptides,
as nucleic acids, and as viral vectors comprising nucleic acids
that encode the epitope(s) of the invention. Upon administration of
peptide-based epitope forms, immune responses have been induced by
direct loading of an epitope onto an empty HLA molecule that is
expressed on a cell, and via internalization of the epitope and
processing via the HLA class I pathway; in either event, the HLA
molecule expressing the epitope was then able to interact with and
induce a CTL response. Peptides can be delivered directly or using
such agents as liposomes. They can additionally be delivered using
ballistic delivery, in which the peptides are typically in a
crystalline form. When DNA is used to induce an immune response, it
is administered either as naked DNA, generally in a dose range of
approximately 1-5 mg, or via the ballistic "gene gun" delivery,
typically in a dose range of approximately 10-100 .mu.g. The DNA
can be delivered in a variety of conformations, e.g., linear,
circular etc. Various viral vectors have also successfully been
used that comprise nucleic acids which encode epitopes in
accordance with the invention.
[1027] Accordingly compositions in accordance with the invention
exist in several forms. Embodiments of each of these composition
forms in accordance with the invention have been successfully used
to induce an immune response.
[1028] One composition in accordance with the invention comprises a
plurality of peptides. This plurality or cocktail of peptides is
generally admixed with one or more pharmaceutically acceptable
excipients. The peptide cocktail can comprise multiple copies of
the same peptide or can comprise a mixture of peptides. The
peptides can be analogs of naturally occurring epitopes. The
peptides can comprise artificial amino acids and/or chemical
modifications such as addition of a surface active molecule, e.g.,
lipidation; acetylation, glycosylation, biotinylation,
phosphorylation etc. The peptides can be CTL or HTL epitopes. In a
preferred embodiment the peptide cocktail comprises a plurality of
different CTL epitopes and at least one HTL epitope. The HTL
epitope can be naturally or non-naturally (e.g., PADRE.RTM.,
Epimmune Inc., San Diego, Calif.). The number of distinct epitopes
in an embodiment of the invention is generally a whole unit integer
from one through one hundred fifty (e.g., 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,
77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, or, 100).
[1029] An additional embodiment of a composition in accordance with
the invention comprises a polypeptide multi-epitope construct,
i.e., a polyepitopic peptide. Polyepitopic peptides in accordance
with the invention are prepared by use of technologies well-known
in the art. By use of these known technologies, epitopes in
accordance with the invention are connected one to another. The
polyepitopic peptides can be linear or non-linear, e.g.,
multivalent. These polyepitopic constructs can comprise artificial
amino acids, spacing or spacer amino acids, flanking amino acids,
or chemical modifications between adjacent epitope units. The
polyepitopic construct can be a heteropolymer or a homopolymer. The
polyepitopic constructs generally comprise epitopes in a quantity
of any whole unit integer between 2-150 (e.g., 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,
77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, or, 100). The polyepitopic construct can
comprise CTL and/or HTL epitopes. One or more of the epitopes in
the construct can be modified, e.g., by addition of a surface
active material, e.g. a lipid, or chemically modified, e.g.,
acetylation, etc. Moreover, bonds in the multiepitopic construct
can be other than peptide bonds, e.g., covalent bonds, ester or
ether bonds, disulfide bonds, hydrogen bonds, ionic bonds etc.
[1030] Alternatively, a composition in accordance with the
invention comprises construct which comprises a series, sequence,
stretch, etc., of amino acids that have homology to (i.e.,
corresponds to or is contiguous with) to a native sequence. This
stretch of amino acids comprises at least one subsequence of amino
acids that, if cleaved or isolated from the longer series of amino
acids, functions as an HLA class I or HLA class II epitope in
accordance with the invention. In this embodiment, the peptide
sequence is modified, so as to become a construct as defined
herein, by use of any number of techniques known or to be provided
in the art. The polyepitopic constructs can contain homology to a
native sequence in any whole unit integer increment from 70-100%,
e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or, 100
percent.
[1031] A further embodiment of a composition in accordance with the
invention is an antigen presenting cell that comprises one or more
epitopes in accordance with the invention. The antigen presenting
cell can be a "professional" antigen presenting cell, such as a
dendritic cell. The antigen presenting cell can comprise the
epitope of the invention by any means known or to be determined in
the art. Such means include pulsing of dendritic cells with one or
more individual epitopes or with one or more peptides that comprise
multiple epitopes, by nucleic acid administration such as ballistic
nucleic acid delivery or by other techniques in the art for
administration of nucleic acids, including vector-based, e.g. viral
vector, delivery of nucleic acids.
[1032] Further embodiments of compositions in accordance with the
invention comprise nucleic acids that encode one or more peptides
of the invention, or nucleic acids which encode a polyepitopic
peptide in accordance with the invention. As appreciated by one of
ordinary skill in the art, various nucleic acids compositions will
encode the same peptide due to the redundancy of the genetic code.
Each of these nucleic acid compositions falls within the scope of
the present invention. This embodiment of the invention comprises
DNA or RNA, and in certain embodiments a combination of DNA and
RNA. It is to be appreciated that any composition comprising
nucleic acids that will encode a peptide in accordance with the
invention or any other peptide based composition in accordance with
the invention, falls within the scope of this invention.
[1033] It is to be appreciated that peptide-based forms of the
invention (as well as the nucleic acids that encode them) can
comprise analogs of epitopes of the invention generated using
principles already known, or to be known, in the art. Principles
related to analoging are now known in the art, and are disclosed
herein; moreover, analoging principles are disclosed in co-pending
application serial number U.S. Ser. No. 09/226,775 filed 6 Jan.
1999. Generally the compositions of the invention are isolated or
purified.
[1034] The peptides may also find use as diagnostic reagents. For
example, a peptide of the invention may be used to determine the
susceptibility of a particular individual to a treatment regimen
which employs the peptide or related peptides, and thus may be
helpful in modifying an existing treatment protocol or in
determining a prognosis for an affected individual. In addition,
the peptides may also be used to predict which individuals will be
at substantial risk for developing chronic infection.
[1035] To identify peptides of the invention, class I antigen
isolation, and isolation and sequencing of naturally processed
peptides was carried out as described in the related applications.
These peptides were then used to define specific binding motifs for
each of the following alleles A3.2, A1, A11, and A24.1. These
motifs are described on page 3, above. The motifs described in
TABLES 6-9, below, are defined from pool sequencing data of
naturally processed peptides as described in the related
applications.
Example 39
Ex Vivo Induction of Cytotoxic T Lymphocytes (CTL)
[1036] Peripheral blood mononuclear cells (PBMC) are isolated from
an HLA-typed patient by either venipuncture or apheresis (depending
upon the initial amount of CTLp required), and purified by gradient
centrifugation using Ficoll-Paque (Pharmacia). Typically, one can
obtain one million PBMC for every ml of peripheral blood, or
alternatively, a typical apheresis procedure can yield up to a
total of 1-10.times.10.sup.10 PBMC.
[1037] The isolated and purified PBMC are co-cultured with an
appropriate number of antigen presenting cell (APC), previously
incubated ("pulsed") with an appropriate amount of synthetic
peptide (containing the HLA binding motif and the sequence of the
antigen in question). PBMC are usually incubated at
1-2.times.10.sup.6 cells/ml in culture medium such as RPMI-1640
(with autologous serum or plasma) or the serum-free medium AIM-V
(Gibco).
[1038] APC are usually used at concentrations ranging from
1.times.10.sup.4 to 2.times.10.sup.5 cells/ml, depending on the
type of cell used. Possible sources of APC include: 1) autologous
dendritic cells (DC), which are isolated from PBMC and purified as
described (Inaba, et al., J. Exp. Med., 166:182 (1987)); and 2)
mutant and genetically engineered mammalian cells that express
"empty" HLA molecules (which are syngeneic [genetically identical]
to the patient's allelic HLA form), such as the, mouse RMA-S cell
line or the human T2 cell line. APC containing empty HLA molecules
are known to be potent inducers of CTL responses, possibly because
the peptide can associate more readily with empty MHC molecules
than with MHC molecules which are occupied by other peptides
(DeBruijn, et al., Eur. J. Immunol., 21:2963-2970 (1991)).
[1039] In those cases when the APC used are not autologous, the
cells will have to be gamma irradiated with an appropriate dose
(using, e.g., radioactive cesium or cobalt) to prevent their
proliferation both ex vivo, and when the cells are re-introduced
into the patients.
[1040] The mixture cultures, containing PBMC, APC and peptide are
kept in an appropriate culture vessel such as plastic T-flasks,
gas-permeable plastic bags, or roller bottles, at 37.degree.
centigrade in a humid air/CO.sub.2 incubator. After the activation
phase of the culture, which usually occurs during the first 3-5
days, the resulting effector CTL can be further expanded, by the
addition of recombinant DNA-derived growth factors such as
interleukin-2 (IL-2), interleukin-4 (IL-4), or interleukin-7 (IL-7)
to the cultures. An expansion culture can be kept for an additional
5 to 12 days, depending on the numbers of effector CTL required for
a particular patient. In addition, expansion cultures may be
performed using hollow fiber artificial capillary systems (Cellco),
where larger numbers of cells (up to 1.times.10.sup.11) can be
maintained.
[1041] Before the cells are infused into the patient, they are
tested for activity, viability, toxicity and sterility. The
cytotoxic activity of the resulting CTL can be determined by a
standard .sup.51Cr-release assay (Biddison, W.E. 1991, Current
Protocols in Immunology, p7, 17.1-7.17.5, Ed. J. Coligan, et al.,
J. Wiley and Sons, New York), using target cells that express the
appropriate HLA molecule, in the presence and absence of the
immunogenic peptide. Viability is determined by the exclusion of
trypan blue dye by live cells. Cells are tested for the presence of
endotoxin by conventional techniques. Finally, the presence of
bacterial or fungal contamination is determined by appropriate
microbiological methods (chocolate agar, etc.). Once the cells pass
all quality control and safety tests, they are washed and placed in
the appropriate infusion solution (Ringer/glucose lactate) and
infused intravenously into the patient.
Example 40
Preparation of Effective HLA Allele-Specific Antigen Presenting
Cells
[1042] This example demonstrates the use of cold temperature
incubation or acid stripping/peptide loading method to prepare
effective HLA-allele-specific antigen presenting cells (APC). The
APC were used to sensitize precursor cytotoxic T lymphocytes which
led to the development of antigen-specific cytotoxic cells. This
was accomplished using either phytohemaglutinin (PHA) T-cell blasts
or peripheral blood mononuclear cells (PBMC) or staphylococcus
aureus Cowan I (SAC-I) activated PBMC as APC. The results are
applicable to other APC and to the other MHC alleles.
[1043] The following describes sources for materials used in the
following examples: [1044] L-Ascorbic acid, Cat #B582, J. T. Baker,
Phillipsburg, N.J. [1045] Anti-HLA A2 (BB7.2), Cat #HB82, ATCC,
Rockville, Md. [1046] Anti-HLA DR (LB3.1), from J. Gorga,
Children's Hospital, Pittsburgh, Pa. [1047] Anti-HLA Alpha chain
pan ABC (9.12.1), from R. [1048] DeMars, University of Wisconsin,
Madison, Wis. [1049] Anti-mouse IgG FITC conjugate, Cat #F2883,
Sigma, St. Louis, Mo. [1050] .beta..sub.2 microglobulin, Cat
#MO114, Scripps Labs, San Diego, Calif. [1051] BSA Fraction V, Cat
#A9418, Sigma, St. Louis, Mo. [1052] 50 cc conical centrifuge
tubes, Cat #2070, Falcon, Lincoln, Park, N.J. [1053] Cryo 1.degree.
C. freezing container, Cat #5100-0001, Nalge, Rochester, N.Y.
[1054] Cryovial, Cat #5000-0012, Nalge, Rochester, N.Y. [1055]
Dimethyl sulfoxide (DMSO), Cat #D2650, Sigma, St. Louis, Mo. [1056]
DNAse, Cat #260912, Calbiochem, San Diego, Calif. [1057] Dynabeads
M-450 goat anti-mouse IgG, Cat #110.06, Dynal, Great Neck, N.Y.
[1058] EDTA tetrasodium salt, Cat #ED4SS, Sigma, St. Louis, Mo.
[1059] FACScan, Becton Dickinson, San Jose, Calif. [1060] Fetal
calf serum (FCS), Cat #3000, Irvine Scientific, Irvine, Calif.
[1061] Ficoll-Paque, Cat #17-0840-03, Pharmacia, Piscataway, N.J.
[1062] Gentamicin, Cat #600-5750AD, Gibco, Grand Island, N.Y.
[1063] L-Glutamine, Cat #9317, Irvine Scientific, Irvine, Calif.
[1064] GS-6KR centrifuge, Beckman Instruments, Palo Alto, Calif.
[1065] Human AB serum (HS), Cat #100-112, Gemini Bioproducts,
Calabasas, Calif. [1066] Human rIL-2, Sandoz, Base1, Switzerland.
[1067] Human rIL-7, Cat #F1-1587-1, Genzyme, Cambridge, Mass.
[1068] Isopropanol, Cat #A464-4, Fisher Scientific, Pittsburgh, Pa.
[1069] MicroCELLector T-150 culture flask for selection of CD4+
cells, Cat #8030, Applied Immune Sciences, Menlo Park, Calif.
[1070] Micromedic automatic gamma counter, ICN Micromedics Systems,
Huntsville, Ala. [1071] OKT4 hybridoma supernatant, Cat #CRL 8002,
ATCC, Rockville, Md. [1072] Paraformaldehyde, Cat #T-353, Fisher,
Pittsburgh, Pa. [1073] PBS calcium and magnesium free (CMF), Cat
#17-516B, BioWhittaker, Walkersville, Md. [1074] Peptides used in
this study were synthesized at Cytel and described in TABLE 123.
[1075] Phytohemagglutinin (PHA), Cat #HA-16, Wellcome, Dartford,
England. [1076] RPMI 1640+Hepes+glutamine, Cat #12-115B,
BioWhittaker, Walkersville, Md. [1077] RPMI 1640+Hepes+glutamine,
Cat #380-24OOAJ, [1078] Gibco, Grand Island, N.Y. [1079] Sodium
chloride (NaCl), Cat #3624-05, J. T. Baker, Phillipsburg, N.J.
[1080] Sodium (.sup.51Cr) chromate, Cat #NEZ 030, NEN, Wilmington,
Del. [1081] Sodium phosphate monobasic, Cat #S9638, Sigma, St.
Louis, Mo. [1082] Triton X-100, Cat #X-100, Sigma, St. Louis, Mo.
[1083] 24 well tissue culture plate, Cat #3047, Falcon, Becton
Dickinson, San Jose, Calif. [1084] 96 well U-bottomed cluster
plate, Cat #3799, Costar, Cambridge, Mass.
[1085] Culture Medium. PHA blasts and CTL inductions were done in
RPMI 1640+Hepes+glutamine (Gibco) supplemented with 2 mM
L-glutamine (Irvine Scientific), 50 .mu.g/ml gentamicin (Gibco),
and 5% heat inactivated pooled human Type AB serum (Gemini
Bioproducts) [RPMI/5% HS]. EBV transformed lymphoblastoid cell
lines (LCL) were maintained in RPMI 1640+Hepes+glutamine
(BioWhittaker) supplemented with L-glutamine and gentamicin as
above and 10% heat inactivated fetal calf serum (Irvine Scientific)
[RPMI/10% FCS]. Chromium release assays were performed in RPMI/10%
FCS.
[1086] Cytokines. Recombinant human interleukin-2 (rIL-2) (Sandoz)
was used at a final concentration of 10 U/ml. Recombinant human
interleukin-7 (rIL-7) (Genzyme) was used at a final concentration
of 10 ng/ml.
[1087] Isolation of Peripheral Blood Mononuclear Cells (PBMC).
Whole blood was collected in heparin (10 U/ml) containing syringes
and spun in 50 cc conical centrifuge tubes (Falcon) at 1600 rpm
(Beckman GS-6KR) 15 min. The plasma layer was then removed and 10
ml of the buffy coat collected with a 10 ml pipette using a
circular motion. The buffy coat was mixed thoroughly and diluted
with an equal volume of serum free RPMI 1640. The diluted buffy
coat was then layered over 20 ml Ficoll-Paque (Pharmacia) in a 50
cc conical tube and centrifuged 400.times.g for 20 min at room
temperature with the brake off. The Ficoll-plasma interface
containing the PBMCs was collected using a transfer pipet (two
interfaces per 50 cc tube) and washed three times with 50 ml RPMI
(1700, 1500, and 1300 rpm for 10 min.
[1088] Freezing and Thawing PBMC. PBMC were frozen at
30.times.10.sup.6 cells/ml of 90% FCS+10% DMSO (Sigma), in 1 ml
aliquots using cyrovials (Nalge). Cryovials were placed in Cryo
1.degree. C. freezing containers (Nalge) containing isopropanol
(Fisher) and placed at -70.degree. C. from 4 hr (minimum) to
overnight (maximum). Isopropanol was changed after every 5 uses.
Cryovials were transferred to liquid nitrogen for long term
storage. PBMC were thawed by continuous shaking in a 37.degree. C.
water bath until the last crystal was nearly thawed. Cells were
immediately diluted into serum free RPMI medium containing DNAse 30
.mu.g/ml (to avoid clumping) (Calbiochem), and washed twice.
[1089] Depletion of Lymphocyte Subpopulations. CD4 lymphocyte
depletion was performed using antibody-coated flasks:
MicroCELLector T-150 flasks for the selection of CD4+ cells
(Applied Immune Sciences) were washed according to the
manufacturer's instructions with 25 ml PBS CMF+1 mM EDTA (Sigma) by
swirling flasks for 30 sec followed by incubation for 1 hr at room
temperature on a flat surface. Buffer was aspirated and flasks were
washed 2 additional times by shaking the flasks for 30 sec and
maintaining coverage of the binding surface. To each washed flask,
25 ml culture medium+5% HS were added and incubated for 20 min at
room temperature on a flat surface. Media was left in the flask
until it was ready to receive the cells. PBMC were thawed in
RPMI/5% HS containing 30 .mu.g/ml DNAse, and washed twice. HS in
the wash blocks Fc receptors on PBMCS. For one flask a maximum of
12.times.10.sup.7 cells were resuspended in 25 ml culture medium.
Culture medium was aspirated from the flask and then the cell
suspension was gently added to the MicroCELLector. Flasks
containing the cells were incubated for 1 hr at room temperature on
a flat surface. At the end of the incubation, the flask was gently
rocked from side to side for 10 sec to resuspend the nonadherent
cells. Nonadherent CD4 depleted cells were harvested, and then
flasks were washed twice with PBS CMF to collect the nonadherent
cells. Harvested CD4-depleted cells were pelleted by centrifugation
and resuspended in complete culture medium (RPMI/5%/HS).
[1090] Generation of PHA Blasts. PBMC were isolated using the
standard Ficoll-Paque protocol. Frozen cells were washed twice
before use. Cells were cultured at 2.times.10.sup.6/ml in RPMI/5%
HS containing 1 .mu.g/ml PHA (Wellcome) and 10 U/ml rIL-2. PHA
blasts were maintained in culture medium containing 10 U/ml rIL-2
with feeding and splitting as needed. PHA blasts were used as APC
on day 6 of culture. Generation of empty class 1 molecules and
peptide loading were only performed by the acid strip method when
using these APC.
[1091] Acid Stripping/Peptide Loading of PBMC and PHA Blasts. PBMC
were isolated using the Ficoll-Paque protocol. When using frozen
cells, PBMC were washed twice before using. PHA blasts were
prepared as previously described and washed twice before using.
Once cells were prepared, they were washed once in cold sterile
0.9% NaCl (J. T. Baker)+1% BSA. In a 50 cc conical centrifuge tube,
the cells were resuspended at 10.sup.7/ml in cold sterile
citrate-phosphate buffer [0-13 M L-ascorbic acid (J. T. Baker),
0.06 M sodium phosphate monobasic (Sigma) pH 3, 1% BSA, 3 .mu.g/ml
.beta..sub.2 microglobulin (Scripps Labs)] and incubated for 2 min
on ice. Immediately, 5 volumes of cold sterile neutralizing buffer
#1 [0.15 M sodium phosphate monobasic pH 7.5, 1% BSA, 3 .mu.g/ml
.beta..sub.2 microglobulin, 10 .mu.g/ml peptide] were added, and
the cells were pelleted at 1500 rpm, 5 min at 4.degree. C. Cells
were resuspended in 1 volume cold sterile neutralizing buffer #2
[PBS CMF, 1% BSA, 30 .mu.g/ml DNAse, 3 .mu.g/ml .beta..sub.2
microglobulin, 40 .mu.g/ml peptide] and incubated for 4 hrs at
20.degree. C. Cells were diluted with culture medium to
approximately 5.times.10.sup.6/ml and irradiated with 6000 rads.
Cells were then centrifuged at 1500 rpm for 5 min at room
temperature and resuspended in culture medium. The acid
stripped/peptide loaded cells were used immediately in the CTL
induction cultures (below).
[1092] Induction of Primary CTL using Acid Stripped/Peptide Loaded
Autologous PBMCs or PHA Blasts as Stimulators. Acid
stripping/peptide loading of PBMC and PHA blasts are described
above. During the last 4 hr incubation of stimulator cells with
peptide, the responder cell population was prepared: Responders
were PBMC that were depleted of CD4+ cells (described above).
Responder cells were resuspended in culture medium at
3.times.10.sup.6/ml. 1 ml of the responder cell suspension was
dispensed into each well of a 24-well tissue culture plate (Falcon,
Becton Dickinson). The plates were placed in the incubator at
37.degree. C., 5% CO.sub.2 until the stimulator population was
ready. Once irradiated, stimulator APC were resuspended in culture
medium containing 20 ng/ml rIL-7 at 10.sup.6/ml for the PBMC, or at
3.times.10.sup.5/ml for the PHA blasts. 1 ml of stimulator cell
suspension was added per well to the plates containing the
responders. On day 7 after induction, a 100 .mu.l culture medium
containing 200 ng/ml rIL-7 was added to each well (20 ng/well rIL-7
final). On day 10 after induction, 100 .mu.l of culture medium
containing 200 U/ml rIL-2 was added to each well (20 U/well rIL-2
final).
[1093] Antigen Restimulation of CTL. On day 12-14 after the
induction, the primary CTL were restimulated with peptide using
adherent APC. Autologous PBMC were thawed and washed as described
above. Cells were irradiated at 6000 rads. Cells were pelleted and
resuspended in culture medium at 4.times.10.sup.6/ml. 1 ml of cell
suspension was added to each well of a 24-well tissue culture
plate, and incubated for 2 hrs at 37.degree. C., 5% CO.sub.2.
Non-adherent cells were removed by washing each well three times
with serum free RPMI. After this step, a 0.5 ml culture medium
containing 3 .mu.g/ml .beta..sub.2 microglobulin and 20 .mu.g/ml
total peptide was added to each well. APC were incubated for 2 hrs
at 37.degree. C., under 5% CO.sub.2 with the peptide and
.beta..sub.2 microglobulin. Wells were aspirated and 1 ml of
responder cells at 1.5.times.10.sup.6/ml in culture medium was
added to each well. After 2 days, 1 ml of culture medium containing
20 U/ml rIL-2 was added to each well.
[1094] Cytotoxicity Chromium Release Assay. Seven days following
restimulation of primary induction, the cytotoxic activity of the
cultures was assessed.
[1095] a. Effector Cell Preparation: the responders, which at this
stage are renamed "effectors", were centrifuged and resuspended at
10.sup.7/ml in RPMI/10% FCS. Three-fold serial dilutions of
effectors were performed to yield effector to target ratios of
100:1, 33:1, 11:1, and 3:1. Effector cells were aliquoted at 100
.mu.l/well on 96 well U-bottomed cluster plates (Costar), in
duplicate.
[1096] b. Target Cell Preparation: Approximately 16-20 hrs prior to
the assay, target cells were resuspended at 3.times.10.sup.5/ml in
RPMI/10% FCS in the presence or absence of 3 .mu.g/ml .beta..sub.2
microglobulin and 10 .mu.g/ml total peptide. After preincubation,
target cells were centrifuged and pellets were resuspended in 200
.mu.l (300 .mu.Ci) sodium (.sup.51Cr) chromate (NEN). Cells were
incubated at 37.degree. C. for 1 hr with agitation. Labeled target
cells were washed 3 times with RPMI/10% FCS.
[1097] c. Setting Up the Assays: Target cell concentration was
adjusted to 10.sup.5/ml in RPMI/10% FCS and 100 .mu.l aliquots were
added to each well containing responders. K562 cells (cold targets,
to block NK, and LAK activity) were washed and resuspended in
RPMI/10% FCS at 10.sup.7/ml. Aliquots of 20 .mu.l were added per
well, yielding a 20:1 of cold K562 target:labeled target. For the
determination of the spontaneous .sup.51Cr release, 100 .mu.l/well
of RPMI/10% FCS were added to 100 .mu.l/well of labeled target
cells, and 20 .mu.l/well of K562. For maximum .sup.51Cr release,
100 .mu.l 1% Triton X-100 (Sigma) in PBS CMF, was added to the 100
.mu.l/well labelled target cells, and 20 .mu.l/well K562. Plates
were centrifuged for 2 min at 1200 rpm to accelerate cell conjugate
formation. Assays were incubated for 5 hr at 37.degree. C., 5%
CO.sub.2. Assays were harvested by centrifuging plates for 5 min at
1200 rpm and collecting 100 .mu.l/well of supernatant. Standard
gamma counting techniques were used to determine percent specific
lysis (Micromedic automatic gamma counter, 0.5 min per tube).
[1098] Cultured Cell Lines. JY, a HLA A2.1 expressing human
EBV-transformed B-cell line, was grown in RPMI/10% FCS. K562, a NK
cell sensitive erythroblastoma line was grown in RPMI/0% FCS. K562
was used to reduce background killing by NK and LAK cells in the
chromium release assays.
[1099] Peptides. The peptides used in these studies were
synthesized at Cytel and their sequences are described in TABLE
123. Peptides were routinely diluted in 100% DMSO at 20 mg/ml,
aliquoted, and stored at -20.degree. C.
[1100] FACS Analysis. Approximately 10.sup.6 cells were used for
each antibody that was to be tested. Cells were washed twice with
PBS CNU+0.1% BSA. To each sample, 100 .mu.l PBS CMF+0.1%
BSA+primary antibody at 2 .mu.g/ml (BB7.2, ATCC) or (9.12.1,
Inserm-CNRS, Marseille, France) or (LB3.1, Children's Hospital
Pittsburgh) were added. A negative control was always included.
Cells were incubated on ice for 20 min and washed twice with PBS
CMF+0.1% BSA. Cells were resuspended in 100 .mu.l anti-mouse IgG
FITC conjugate (Sigma), diluted 1:50 in PBS CMF+0.1% BSA, and
incubated 20 min on ice. Cells were washed twice with PBS CMF+0.1%
BSA, and resuspended in PBS for FACScan (Becton Dickinson)
analysis. When it was necessary to postpone analysis to the
subsequent days, the cells were fixed with PBS/1% paraformaldehyde
(Fisher) and analyzed within one week.
[1101] Binding Assays Using Intact Cells and Radiolabelled Peptide.
JY cells were treated with citrate-phosphate buffer and
neutralizing buffer #1 as described above. JY control cells were
left untreated in tissue culture media. After treatment both cell
populations were washed twice with serum free RPMI and loaded with
.sup.125I-radiolabelled 941.01 (HBc15-27) peptide (standard
chloramine T iodination). To determine binding specificity,
2.times.10.sup.6 cells were resuspended in 200 .mu.l neutralizing
buffer #2 (described above) containing .sup.125I-941.01 (10.sup.5
cpms)+/-100 .mu.g unlabelled 941.01. Cells were incubated for 4 hrs
at 20.degree. C. and washed twice with serum free RPMI to remove
free peptide. Cells were resuspended in 200 .mu.l of serum free
RPMI. In .alpha. microfuge tube the cell suspension was layered
over an 800 .mu.l FCS and pelleted by centrifugation for 5 sec.
Supernatants were aspirated and the radioactivity remaining in the
pellet was measured (Micromedic automatic gamma counter, 1 min per
tube).
Example 41
Class I MHC Molecule Peptide Stripping/Loading by Mild Acid
Treatment
[1102] Mild acid solutions of pH 3 such as glycine or
citrate-phosphate buffers have been used by various groups to
identify endogenous peptides and to identify tumor associated T
cell epitopes. The treatment is unique in that only the MHC class I
molecules are destabilized (and peptides released), while all other
surface antigens remain intact including MHC class II molecules.
Most importantly, treatment of cells with the mild acid solutions
of this example do not affect the cell's viability or metabolic
state. The mild acid treatment is rapid since the stripping of
endogenous peptides occurs in two minutes at 4.degree. C. and the
APC is ready to perform its function after the appropriate peptides
are loaded. In this example we utilized the technique to make
peptide specific APCs for the generation of primary
antigen-specific CTL. The resulting APC were efficient in inducing
peptide-specific CD8+ CTL.
[1103] Measurements by FACS Analysis. PHA-induced T-cell blasts
were acid stripped/peptide loaded according to the methods
described in Example 15. The resulting cells were stained for FACS
analysis using anti-HLA-A2 (BB7.2) and anti-HLA alpha
chain-specific (9.12.1) monoclonal antibodies. Controls for this
experiment included the same cell population which was not treated
at pH 3 (but treated with PBS buffer at pH 7.2), and with cells
treated with citrate-phosphate buffer (to strip the MHC) but
neutralized in the absence of .beta..sub.2 microglobulin and
peptide. The results presented in FIG. 28, indicate that treatment
of these cells with the citrate-phosphate (pH 3) buffer
significantly reduced (10-fold) the reactivity of the cells toward
both anti-HLA class I antibodies alone (anti-HLA-A2 and the alpha
chain specific), but not towards a monoclonal antibody specific for
class II MHC molecules (anti-HLA-DR). Most importantly,
neutralization of the acid-stripped cells in the presence of
.beta..sub.2 microglobulin and peptide resulted in preservation of
a significant amount of class I MHC antibody-reactive sites, with
only a 2.5-fold decrease in fluorescence intensity. Importantly,
the acid-treated cells remained viable, as measured by trypan blue
exclusion and forward/lateral FACS scatter analysis. Similar
results were obtained using EBV-transformed B cell lines, fresh (or
frozen) PBMC and other peptides (which bind to either HLA-A2.1 or
HLA-A1) (data not shown).
[1104] Binding of Radiolabeled Peptides to Empty MHC Molecules. To
determine the efficiency of peptide loading using the cold
temperature incubation or acid stripping/peptide loading protocol,
JY cells (an HLA-A2.1 EBV-transformed B cell line) were
preincubated at 26.degree. C. overnight or acid-stripped to remove
the endogenous MHC-associated peptides and the loading of exogenous
peptide was determined using a .sup.125I-radiolabelled HLA-A2.1
binding peptide. The specificity of this reaction was determined by
measuring the inhibition of labelled peptide binding using a cold
peptide of the same sequence. Results presented in TABLES 123-124
demonstrate that acid-treatment of the cells increased
significantly (approximately 10-fold) the amount of labeled peptide
binding to the JY cells. Furthermore, the binding of labelled
peptide was completely blocked by the addition of the cold peptide,
demonstrating specific binding (data not shown).
[1105] In Vitro Induction of Primary Antigen-Specific CTL Using
Acid Stripped/Peptide Loaded APCS. Additional critical parameters
for the induction of primary CTL using both the cold temperature
incubation and acid strip protocol are: 1) enrichment of CD8+
T-cells in the responder cell population (or depletion of CD4+
T-cells), 2) addition of rIL-7 to the CTL induction cultures from
day 0, and 3) restimulation of the cultures with antigen on day
12-14 using autologous adherent cells pulsed with peptide.
Example 42
Screening Peptides to Identify CTL Epitopes
[1106] In order to identify CTL epitopes, CTL was stimulated by
SAC-I activated PBMCs as APC. Cold temperature expression of the
MHC in which class I .beta.2 microglobulin complex is unstable was
utilized in addition to acid stripping to generate PBMC APC.
[1107] Complete Culture Medium. The tissue culture medium used in
this study consisted of RPMI 1640 with Hepes and L-glutamine
(Gibco) supplemented with 2 mM L-glutamine (Irvine Scientific), 0.5
mM sodium pyruvate (Gibco), 100 U/100 ug/ml penicillin/streptomycin
(Irvine), and 5% heat-inactivated Human Serum Type AB (RPMI/5% HS;
Gemini Bioproducts). Culture media used in the growth of
EBV-transformed lines contained 10% heat-inactivated fetal calf
serum (RPMI/10% FCS, Irvine) instead of human serum.
[1108] Cytokines. Recombinant human Interleukin-2 (rIL-2) and
Interleukin-4 (rIL-4) were obtained from Sandoz and used at a final
concentration of 10 U/ml and 10 ng/ml, respectively. Human
interferon-.gamma. (IFN-.gamma.) and recombinant human
Interleukin-7 (rIL-7) were obtained from Genzyme and used at 20
U/ml and 10 ng/ml, respectively.
[1109] Peptides. Peptides were synthesized at Cytel and are
described in TABLES 123-124. Peptides were routinely diluted in
100% DMSO at 20 mg/ml, aliquoted, and stored at -70.degree. C.
until use.
[1110] Cell Lines. JY, Steinlin, EHM, BVR, and KT3 are homozygous
human EBV-transformed B cell lines expressing HLA A.sub.2.1,
A.sub.1, A.sub.3, A.sub.11, and A.sub.24, respectively. They are
grown in RPMI/10% FCS. K562, an NK cell sensitive, erythoblastoma
line grown in RPMI/10% FCS, was used for reduction of background
killing in CTL assays. Melanoma cell lines either expressing the
MAGE antigen, mel 397 and mel 938, or not expressing the MAGE
antigen; mel 888, were also grown in RPMI/10% FCS.
[1111] Isolation of Peripheral Blood Mononuclear Cells (PBMCs).
Whole blood was collected into heparin containing syringes and spun
in 50 cc tubes at 1600 RPM (Beckman GS-6KR) for 15 minutes. The
plasma layer was then removed and 10 ml of buffy coat was collected
with a pipette using a circular motion. The buffy coat was mixed
well and diluted with an equal volume of RPMI. The buffy coat (30
ml) was then layered on 20 ml of Ficoll-Paque (Pharmacia) and
centrifuged at 1850 RPM (400.times.g) for 20 minutes, 25.degree.
C., with the brake off. The interface between the ficoll and the
plasma containing the PBMCs was recovered with a transfer pipet
(two interfaces per 50 ml tube) and washed three times with 50 ml
of RPMI (1700, 1500, and 1300 RPM for 10 minutes). Cells were
resuspended in 10-20 ml of culture medium, counted, and adjusted to
the appropriate concentration.
[1112] Freezing PBMCs. 30 million cells/tube (90% FCS/10% DMSO;
Sigma) were inserted into a Nalgene Cryo 1.degree. C. Freezing
Container containing isopropanol (Fisher) and placed at -70.degree.
C. from 4 hrs (minimum) to overnight (maximum). The isopropanol was
changed every five times. Tubes were transferred to liquid nitrogen
for long term storage. To thaw, PBMCs were continuously shaken in a
37.degree. C. water bath until the last crystal was almost thawed
(tubes were not allowed to sit in the water bath or at room
temperature for any period of time). Cells were diluted into
serum-free RPMI containing 30 .mu.g/ml DNase to prevent clumping by
dead cell DNA and washed twice.
[1113] Induction of Primary CTL Using SAC-I Activated PBMCs as
APCs
[1114] a. Preparation of APCs: PBMCs were purified using the
standard Ficoll-Paque protocol and resuspended at
1.times.10.sup.6/ml in RPMI/5% FCS containing 0.005% Pansorbin
cells (SAC-I cells expressing Protein A; Calbiochem), 20 .mu.g/ml
Immunobeads (Rabbit anti-Human IgM; Biorad), and 20 ng/ml of human
rIL-4. Two ml of cells per well were plated in a 24-well plate
(Falcon, Becton Dickinson) and cultured at 37.degree. C. After 3
days, the medium was removed and the cells were washed three times
followed by addition of RPMI/10% HS. The cells were used after
culturing for an additional 2 days in RPMI/10% HS.
[1115] b. Expression of Empty Class I Molecules on the Surface of
APCs and Peptide Loading of APCs.
[1116] 1. Cold Temperature Incubation: [1117] a. Expression of
empty MHC in APCs: The APCs were adjusted to a concentration of
2.times.10.sup.6/ml in complete culture medium containing 10 ng/ml
rIL-4, 20 U/ml human IFN-.gamma., and 3 .mu.g/ml 132 microglobulin
(.beta..sub.2m; Scripps Lab). The cells were then incubated
overnight at 26.degree. C. in the presence of 5% CO.sub.2. It
should be noted that these cells only express a fraction of Class I
molecules in the empty state (.about.10%). [1118] b. Peptide
loading of APC stimulator cells: Empty Class I expressing APCs were
washed 1-2 times with serum free RPMI (+L-glutamine and Hepes) and
resuspended at 1.times.10.sup.7 in serum-free RPMI containing 50
.mu.g/ml total of the peptide pool (i.e., 16.7 .mu.g/ml of each
peptide in a pool of three; 25 .mu.g/ml of each peptide in a pool
of two; 50 .mu.g/ml of individual peptide), 30 .mu.g/ml DNAse, and
3 .mu.g/ml .beta..sub.2m. Following a 4 hour incubation at
20.degree. C., the cells were irradiated at 6100 rads
(5.times.10.sup.6/ml; 25 million cells/tube), washed and adjusted
to the appropriate concentration for addition to the induction
culture (see below).
[1119] 2. Acid stripping: This was used as an alternative method
for generating empty MHC on the surface of the APCs. The SAC-I
activated PBMCs were washed once in cold 0.9% sodium chloride (J.
T. Baker) containing 1% BSA. The cells were resuspended at
10.sup.7/ml in cold citrate-phosphate buffer (0.13M L-ascorbic acid
[J. T. Baker], 0.06M sodium phosphate monobasic [Sigma], pH3)
containing 1% BSA and 3 .mu.g/ml .beta..sub.2m and incubated on
ice. After 2 minutes, 5 volumes of cold 0.15M sodium phosphate
monobasic buffer, pH7.5, containing 1% BSA, 3 .mu.g/ml
.beta..sub.2m, and 10 .mu.g/ml peptide [neutralizing buffer #1] was
added and the cells centrifuged at 1500 RPM for 5 minutes at
4.degree. C. The cells were resuspended in 1 ml of cold PBS
containing 1% BSA, 30 .mu.g/ml DNase, 3 .mu.g/ml .beta.2
microglobulin, and 50 .mu.g/ml peptide [neutralizing buffer #2] and
incubated for 4 hours at 20.degree. C. As above, subsequent to the
four hour incubation at 20.degree. C., the cells were irradiated at
6100 rads (5.times.10.sup.6/ml; 25 million cells/tube), washed,
then adjusted to the appropriate concentration for addition to the
induction culture (see below). [1120] c. Preparation of the CD4+
depleted PBMC responder cell population (depletion of lymphocyte
sub-populations using AIS flasks). AIS MicroCellector T-150 flasks
(specific for the depletion of CD4+ T cells; Menlo Park, Calif.)
were primed by adding 25 ml of PBS/1 mM EDTA, swirling for 30
seconds so that all surfaces were moistened, and then incubating
with the binding surface down at room temperature for 1 hour.
Following this incubation, flasks were shaken vigorously for 30
seconds, washed 1 time with PBS/EDTA, 2 additional times with PBS
and then incubated with 25 ml of culture medium for 15 minutes.
PBMCs were thawed in serum-free RPMI (+L-glutamine+Hepes)
containing 30 .mu.g/ml DNAse, washed once, and incubated for 15
minutes in culture medium. Following aspiration of culture medium
from the flasks, up to 180 million PBMCs were added in 25 ml of
culture medium containing 30 .mu.g/ml DNAse. After 1 hour at room
temperature, the flasks were rocked gently for 10 seconds to
resuspend the nonadherent cells. The nonadherent cell suspension
containing the CD8+ T cells was collected and the flasks were
washed 2 times with PBS. The CD4+ T cell depleted PBMCs were
centrifuged and counted for addition to the induction culture. The
CD4+ and CD8+ phenotype of the CD4+ depleted cell population was
determined by FACS analysis (see below). In general, this technique
resulted in a two-fold enrichment for CD8+ T cells with an average
of approximately 40-50% CD8+ T cells and 15-20% remaining CD4+ T
cells following depletion of CD4+ T cells. Depletion of CD4+ T
cells can also be accomplished by antibody and complement or
antibody coated magnetic beads (Dynabeads). Depletion of CD4+ T
cells served the purpose of enriching CTLp and removing cells which
would complete for cell nutrients and may interfere with CTLp
expansion. [1121] d. Induction of primary CTL. During the 4 hour
peptide loading of the stimulator APCs, CD4+ depleted PBMC to be
used as the responder population were prepared utilizing AIS flasks
for selection of CD8+ T cells through the depletion of CD4+ T cells
(above). The responder cells were plated at 3.times.10.sup.6/ml in
a 1 ml volume (24 well plate) and placed at 37.degree. C. until the
peptide loaded stimulator APCs were prepared. The irradiated,
peptide loaded APCs were washed 1 time in serum-free RPMI
(+L-glutamine and Hepes), adjusted to 1.times.10.sup.6/ml in
complete medium, and plated into a 24 well plate at 1 ml/plate: For
PBMC, 1.times.10.sup.6 stimulator cells (1 ml volume) were plated
into the wells containing the responder cells; For SAC-I activated
PBMC and PHA blasts, 1 ml of 3.times.10.sup.5/ml stimulator cells
were plated in each well. A final concentration of 10 .mu.g/ml of
additional peptide was added in addition to 10 ng/ml final
concentration of rIL-7 (2 ml total volume). On day 7 an additional
10 .mu.g/ml rIL-7 was added to the culture and 10 U/ml rIL-2 was
added every 3 days thereafter. On day 12, the cultures were
restimulated with peptide pulsed adherent cells and tested for
cytolytic activity 7 days later (below).
[1122] Protocol for Restimulation of Primary CTL Using Adherent
APC. PBMCs were thawed into serum-free RPMI (+L-glutamine and
Hepes) containing 30 .mu.g/ml DNAse, washed 2 times, and adjusted
to 5.times.10.sup.6/ml in culture medium containing DNAse. PBMCs
(25 million cells/tube in 5 ml) were irradiated at 6100R. After 1
wash, the PBMCs were resuspended in culture medium and adjusted to
4.times.10.sup.6/ml. 1 ml of irradiated PBMCs was added per well of
a 24-well plate. The PBMC were incubated for 2 hours at 37.degree.
C., washed 3 times to remove non-adherent cells, and cultured in
medium containing 20 .mu.g/ml total peptide and 3 .mu.g/ml
.beta..sub.2 microglobulin added in a 0.5 ml volume and again
incubated for 2 hours at 37.degree. C. The peptide was aspirated
and 1.5.times.10.sup.6 responder cells resuspended in culture
medium were added in a 1 ml volume. After 2 days, 1 ml of culture
medium containing 20 U/ml rIL-2 was added.
[1123] FACS Analysis. One million cells/tube were centrifuged,
resuspended in 100 .mu.l/tube PBS/0.1% BSA/0.02% sodium azide
(Sigma) plus 10 .mu.l/tube directly conjugated antibody (Becton
Dickinson), and incubated on ice 15-20 minutes. Cells were then
washed 2 times with PBS/0.1% BSA/0.02% sodium azide and resuspended
in PBS to analyze on FACScan (Beckton Dickinson). When it was not
possible to analyze samples within 1-2 days, cells were fixed with
PBS containing 1% paraformaldehyde (Fisher) and analyzed within one
week.
[1124] Cytotoxicity Assay
[1125] a. Target cell preparation. Approximately 16-20 hours prior
to the CTL assay, target cells (Class I matched EBV-transformed
lines) were washed once and resuspended in a 10 ml volume at
3.times.10.sup.5/ml in RPMI/5% FCS in the presence or absence of 10
.mu.g/ml total peptide.
[1126] b. Labeling of target cells: Target cells were centrifuged
and resuspended in 200 .mu.l/tube sodium .sup.51Cr chromate (NEN),
then incubated at 37.degree. C. for 1 hour on a shaker. Targets
were washed 3 times (10 ml/wash) with RPMI/10% FCS and resuspended
in 10 ml (to determine the efficiency of labelling, 50 .mu.l/target
was counted on the Micromedic automatic gamma counter).
[1127] c. CTL assay. Target cells were adjusted to
2.times.10.sup.5/ml and 50 .mu.l of the cell culture was added to
each well of a U-bottomed 96-well plate (Costar Corp.) for a final
concentration of 1.times.10.sup.4/well. K562 cells were washed
once, resuspended at 4.times.10.sup.6/ml, and 50 .mu.l/well was
added for a final concentration of 2.times.10.sup.5/well (ratio of
cold K562 to target was 20:1). Responder cells were washed once,
resuspended at 9.times.10.sup.6/ml, and three fold serial dilutions
were performed for effector to target ratios of 90:1, 30:1, 10:1,
and 3:1. Responder cells were added in a volume of 100 .mu.l in
duplicate wells. For spontaneous release, 50 .mu.l/well of labelled
target cells, 50 .mu.L/well K562, and 100 .mu.l/well of medium was
added. For maximum release, 50 .mu.l/well target, 50 .mu.l/well
K562, and 100 .mu.L/well of 0.1% Triton-X100 (Sigma) was added.
Plates were centrifuged for 5 minutes at 1200 RPM. Following a 5
hour incubation at 37.degree. C., plates were centrifuged again for
5 minutes at 1200 RPM, and 100 .mu.l/well of supernatant was
collected. Standard gamma counting techniques (Micromedic automatic
gamma counter; 0.5 minutes/tube) were used to determine the percent
specific lysis according to the formula: % specific lysis=cpm
experimental-cpm spontaneous release/cpm maximum release-cpm
spontaneous release.times.100. A cytotoxicity assay (CTL assay) was
considered positive if the lysis by CTL of targets sentized with a
specific peptide at the two highest effector to target (E:T) ratios
was 15% greater than lysis of control targets (i.e., target cells
without peptide). A cytotoxicity assay (CTL assay) was considered
borderline if the lysis by CTL of targets sensitized with a
specific peptide at the two highest effector to target (E:T ratios
was 6% greater than lysis of control targets (i.e., target cells
without peptide).
[1128] d. Results. Of the peptides that bind to the indicated
alleles, 9 of the 49 MAGE peptides, 10 of the 45 HIV peptides, 3 of
the 25 HCV peptides, and 2 of the 20 HBV peptides tested to date
induced primary CTL in vitro. Representative graphs illustrating
CTL responses to various immunogenic peptides are shown for MAGE
(FIG. 35), HIV (FIG. 36), HCV (FIG. 37), and HBV (FIG. 38). The CTL
induction data are summarized in TABLE 123-124 which lists the
immunogenic peptides which bind to the appropriate MHC and induce
primary CTL in vitro. Indicated is the peptide's sequence,
corresponding antigen and HLA allele to which it binds. Results
shown in FIG. 33 illustrate lysis of peptide sensitized targets and
endogenous targets following stimulation with SAC-I activated PBMCs
loaded with a MAGE 3 peptide, 1044.07 by the cold temperature and
incubation technique. FIG. 34 shows a comparison of the acid strip
loading technique (Panel a) with the cold temperature incubation
technique (panel b).
Example 43
Analog Peptides with Substitutions at Primary and Secondary Anchor
Positions and Effects on A24 Binding
[1129] A model poly alanine 9-mer peptide containing the A24-allele
specific motif of Y in position 2 and F in position 9 was used to
evaluate the possibility that there are other residues that can
serve as primary anchors for peptide binding to HLA-A24 molecules.
It was found that in position 2 not only Y, but also F, M, and
possibly W, were accepted. The acceptability of W at position 2 was
confirmed by data in this Example. At the C-termini of 9 or amino
acid ligands, F and W were most preferred, but also L and I were
accepted. From these results, it was concluded that A24 binding of
any peptide which carries a tolerated residue in position 2 or the
C-terminal position (for example, M in position 2) should be
increased by creating an analog peptide by replacing the acceptable
residue with a more canonical anchor.
[1130] The results of further experiments describing the prominent
role of amino acids which were not primary anchors as determinants
of A24 binding capacity have been determined (see, e.g., Kondo, et
al, J. Immunol. 155:4307 (1995)). Thus, an overall A24 binding data
was compiled, and for each position the relative average binding
affinity of peptides carrying particular residues was calculated.
Based on this calculation, preferred and deleterious residues were
identified; these are shown in FIG. 44.
[1131] Secondary Residues of 9-mer Peptides and A24 Binding. In the
case of 9-mer peptides it was found, for example, that peptides
carrying G or negatively charged residues (D, E) at position 1
tended to bind poorly, with an average affinity 10-fold lower than
the average affinity of a sample panel of 141 different 9-mer
peptides analyzed. By contrast, peptides carrying aromatic residues
(F, Y, W) at position 1 bound very well with an average affinity
11.8-fold higher than the overall average. Peptides with positively
charged residues (R, K, M in position one also tended to bind well,
with average affinity 4.6-fold higher than the overall average.
[1132] Negative effects on A24 binding capacity were also detected
when certain residues were present at several other positions: D or
E at positions 3 and 6, G at positions 4 and 7, positive charges
(K, R, H) at position 6, A at position 8, P at position 5, and
amides (Q and N) at positions 5 and 8. Conversely, it was found
that aromatic (Y, F, W) residues favored A24 binding when found at
position 7 or 8, and small hydrogen bonding residues such as (S, T,
C) had a positive effect when present at position 4.
[1133] Thus, it was found that every single position along the
9-mer sequence can influence A24 binding. It was also interesting
that hydrophobic residues (F, W, Y, L, I, V, and M) were never
associated with poor binding.
[1134] 10-mer Peptides and A24 Binding. A similar analysis was also
performed with 10-mer peptides. Analogous to the preceding section
concerning 9-mers, several secondary effects were also discerned
when analogs were prepared of 10 mer peptides.
[1135] As was the case for 9-mer peptides, negative residues (D, E)
in position 3 and 6 were associated with poor binding. In general,
however, the map of secondary effects for 10-mers was quite
distinct from that of 9-mers. For example, P, in the case of 9-mer
peptides, was not associated with significantly increased binding
at any position and was even associated with decreased binding at
position 5. However, for 10-mers, P was associated with increased
binding capacity when found at positions 4, 5, or 7 of 10-mer
peptide ligands.
[1136] In 10-mer peptides, position 5 appears to be most important
in terms of secondary effects, with (besides the already mentioned
P) Y, F, and W associated with good A24 binding and R, H, and K
associated with poor binding capacity. The presence of A at
positions 7 and 9, and amide (Q, N) residues at positions 4 and 8
were also associated with poor binding capacity. Thus, in
accordance with the principles for preparing peptide analogs
disclosed herein, this information provides guidance for the
preparation of 9-mer and 10-mer analogs of peptides that bind HLA
A24 molecules.
Example 44
Immunogenicity of HPV Peptides in A2.1 Transgenic Mice
[1137] A group of 14 HPV peptides, including 9 potential epitopes
plus 3 low binding and one non-binding peptides as controls was
screened for immunogenicity in HLA-A2.1 transgenic mice using the
methods described in Example 10. To test the immunogenic potential
of the peptides, HLA A2.1 transgenic mice were injected with 50
ig/mouse of each HPV peptide together with 140 .mu.g/mouse of
helper peptide (HBV core 128-140 (TPPAYRPPNAPIL (SEQ ID
NO:______)). The peptides were injected in the base of the tail in
a 1:1 emulsion IFA. Three mice per group were used. As a positive
control, the HBV polymerase 561-570 peptide, which induced a strong
CTL response in previous experiments, was utilized.
[1138] Based on these results (TABLE 179), four unrelated peptides
were considered to be the most immunogenic: TLGIVCPI (SEQ ID
NO:______), LLMGTLGIV (SEQ ID NO:______), YMLDLQPETT (SEQ ID
NO:______), and TIHDIILECV (SEQ ID NO:______). TLGIVCPI (SEQ ID
NO:______) and YMLDLQPETT (SEQ ID NO:______) were found to be good
HLA-A2.1 binders, while LLMGTLGIV and TIHDIILECV were found to be
intermediate binders in previous binding assays.
Mixtures of Selected HPV Epitopes
[1139] A combination of CTL peptides and a helper peptide were
tested for the ability to provide an increased immune response. The
four single peptides were injected separately in order to compare
their immunogenicity to injections containing only the two good
binders or only the two intermediate binders. In addition all four
peptide were injected together. To further evaluate the
immunogenicity of a combination of peptides with different binding
affinity decreases, another control was introduced in this
experiment. A mixture of the two good binders was injected in a
different site than the mixture of the two intermediate binders
into the base of the tail of the same mouse. All groups of CTL
epitopes were injected together with the HBVc helper epitope, with
the exception of two groups in which all four HPV coinjected with
two different doses of a PADRE helper peptide (aKXVAAWTLKAAa, where
a is d-alanine and X is cyclohexylalanine) either 1 .mu.g or 0.05
.mu.g per mouse.
[1140] All four peptides induced a strong CTL response when
injected alone and tested using target cells labeled with the
appropriate peptide (TABLE 180). TLGIVCPI (SEQ ID NO:______) proved
to be the strongest epitope, an observation confirming the results
described above. When mixtures of all four peptides were injected
and the responses were stimulated in vitro and tested with target
cells pulsed with each single peptide, all combinations showed a
strong CTL response. No significant difference was observed when
the two helper epitopes were compared. This might in part be due to
the fact that the highest dose of PADRE used in this experiment was
140-fold lower than the one for the HBV helper peptide.
[1141] Injection of mixtures of the two good binders together or
the two intermediate binders resulted in a very low CTL response in
both cases even though the single peptides were highly effective.
These results, however, are due to a very low number of cell
recovery after splenocyte culture of 6 days and are therefore
regarded as preliminary.
[1142] TABLE 176 provides the results of searches of the following
antigens cERB2, EBNA1, HBA, HCV, HIV, HPV, MAGE, p53, and PSA. Only
peptides with binding affinity of at least 1% as compared to the
standard peptide is shown in the far right column. The column
labeled "Pos." indicates that position in the antigenic protein at
which the sequence occurs.
[1143] TABLE 177 also provides the results of these searches.
Binding affinities are expressed as percentage of binding compared
to standard peptide in the assays as described in Example 5.
Example 45
Effects of Secondary Anchor Residues on A1 Binding
[1144] An analysis similar to that described above for A24 was also
described for peptides that bear a motif correlated to binding to
the HLA-A*0101 allele molecules. Briefly, previous studies have
defined two different peptide binding motifs specific for
HLA-A*0101: A motif defining anchors at position 2 and the
C-terminus, and a motif with anchors at position 3 and the
C-terminus. Such motifs for binding to the same HLA allele are
referred to as "submotifs."
[1145] Thus, 9-mer and 10-mer maps of secondary interactions were
derived for both A*0101 submotifs. To derive such maps of secondary
interactions, the relevant A*0101 binding data of peptide sets
corresponding to each of the two motifs were compiled. For each
position, the relative average binding affinity of peptides
carrying each particular residue was calculated. To compensate for
the low occurrence of certain residues, and to obtain a more
significant sampling, amino acids carrying chemically similar side
chains were combined, as suggested by Ruppert et al., supra.
[1146] The results obtained by this type of analysis for 9-mer
peptides are shown in FIG. 42A and FIG. 42B for the 2-9 and 3-9
motifs, respectively; diagrams illustrating the secondary effects
detected by this analysis are also shown as FIG. 42C and FIG. 42D
(for the 2-9 and 3-9 motifs, respectively). Increases or decreases
in average affinity greater than four-fold are defined as
significant, as described herein, and were used to determine
preferred or deleterious residues.
[1147] In general, for most positions binding capacity was
affected, either negatively or positively, by the presence of
particular residue types. For example, in the case of the 2-9
motif, it was found that peptides carrying either D or E at
position 1 bound poorly to A*0101 molecules, with an average
relative binding capacity (ARBC) of 0.20. Conversely, peptides
carrying aromatic residues (Y, F, or W) at the same position
(position 1) bound with an affinity, on average, four-fold higher
(ARBC 4-0) than the overall average binding capacity of the entire
peptide set.
[1148] Inspection of the diagrams reveals some interesting features
of peptide binding to A*0101. First, as noted above, the anchors at
positions 2 and 3 act synergistically with each other. The affinity
of peptides carrying the M, S or T anchors in position 2 is
dramatically increased by the presence of D or E in 3 (and to a
lesser extent by A). Conversely, the affinity of peptides carrying
the D or E anchors at position 3 was dramatically increased by the
presence of S, T, and M (but also other hydrophobic or short chain
molecules such as L, V, I, C and A) at position 2.
[1149] The degree to which peptides bearing either the 9-mer or 1
0-mer motifs differ in binding to the A*0101 HLA molecule is
revealed by examining other positions. Comparing the values in FIG.
42A and FIG. 42B, it is clear that there are numerous examples
where residues neutral in the context of one motif had positive or
negative effects in the context of the other motif. At position
one, for example, in the 2-9 motif G and aromatic (Y, F, and W)
residues are preferred (ARBC >4.0), A and positively charged (R,
H, and K) residues are relatively neutral (ARBCs between 4.0 and
0.25), and negatively charged (D and E) residues are deleterious
(ARBC <0.25). In the case of peptides carrying the 3-9 motif, a
different pattern is noted for position one and, with the exception
of G, which is still preferred, the preferences are shuffled.
Positively charged residues at position one have a significant
positive influence on peptide binding (ARBC of 8.3), negatively
charged and aromatic residues are neutral (ARBCs of 1.3 and 0.61,
respectively), and A is deleterious (ARBC of 0.15). Similar types
of modulation are observed at each position along the motif.
[1150] Overall, the shifts in secondary anchor preference from
motif to motif are set forth in the summary diagrams shown in FIG.
42A, FIG. 42B, FIG. 42C, and FIG. 42D. In this context, it can be
seen that, with the lone exception of the shared preference for G
in position one, and excluding the position 2 and 3 co-anchors, the
extended motifs of the two A*0101 9-mer motifs are in fact
completely different. Thus, in a quantitative sense, the two 9-mer
motifs have only one secondary effect out of 27 (3.7%) in common.
The degree to which these A*0101 motifs differ is in striking
contrast to the multiple similarities noted between the extended
motifs of A24, A*0201, and A3 molecules (Kondo, et al., supra,
where it was observed that between 3 and 5 (13-26%) secondary
effects were shared between any two extended motifs.
[1151] Effects of Secondary Residues on A1 Binding for 10-mer
Peptides. Analogous to what was described in the section above for
9-mer ligands, secondary anchor residues and secondary effects were
also defined for the 2-10 and 3-10 submotifs for peptides that bind
to HLA A1 molecules. The results of these analyses are presented in
FIG. 43A and FIG. 43B, FIG. 43C, and FIG. 43D. Once again, it
appeared the anchors present in position 2 and 3 could act
synergistically with each other. The presence of D, E (and to a
much lesser extent A, Q and N) in position 3, in the context of the
2-10 motif, and of hydrophobic (L, I, V, M) or short chain (S, T,
C) residues in position 2, in the context of the 3-10 submotif,
were associated with significant increases in average binding
affinity.
[1152] Comparison of the two 10-mer motifs at positions other than
2, 3 and the C-termini indicates that, as was the case with 9-mer
peptides, modulation in secondary anchor specificity occurs
dependent on what the primary anchor residues are. For example, at
position 7, A and S, T, and C are preferred in the 2-10 motif, but
are neutral in the 3-10 motif. Conversely, G is preferred in the
3-10 motif, but is neutral in the 2-10 motif. However, it is also
evident that, in contrast to the 9-mer motifs, these differences
observed in 10-mers are much less striking. In fact, the two 10-mer
motifs share a number of preferences. For example, Y, F, and W in
positions 1 and 5, A in 4, P in 7, and G in 8 had positive effects
for both motifs. Similarly, R, H, and K in 8 were deleterious in
both 10-mer motifs (FIGS. 5c and 5d). In total, the two 10-mer
motifs shared 6 secondary effects out of 25 (24%).
[1153] In accordance with the principles for preparing peptide
analogs disclosed herein, this information provides guidance for
the preparation of 9-mer and 10-mer peptides that bind to HLA A1
molecules.
[1154] These example and equivalents thereof will become more
apparent to those skilled in the art in light of the present
disclosure and the accompanying claims. It should be understood,
however, that the examples are designed for the purpose of
illustration only and not limiting of the scope of the invention in
any way. All patents and publications cited herein are fully
incorporated by reference herein in their entirety.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20080260762A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20080260762A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References