U.S. patent application number 11/156369 was filed with the patent office on 2006-03-16 for epitope analogs.
Invention is credited to Adrian Bot, David C. Diamond, Liping Liu.
Application Number | 20060057673 11/156369 |
Document ID | / |
Family ID | 35198081 |
Filed Date | 2006-03-16 |
United States Patent
Application |
20060057673 |
Kind Code |
A1 |
Liu; Liping ; et
al. |
March 16, 2006 |
Epitope analogs
Abstract
Some embodiments relate to analogs of peptides corresponding to
class I MHC-restricted T cell epitopes and methods for their
generation. These analogs can contain amino acid substitutions at
residues that directly interact with MHC molecules, and can confer
improved, modified or useful immunologic properties. Additionally
classes of analogs, in which the various substitutions comprise the
non-standard residues norleucine and/or norvaline, are
disclosed.
Inventors: |
Liu; Liping; (Manassas,
VA) ; Bot; Adrian; (Valencia, CA) ; Diamond;
David C.; (West Hills, CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
35198081 |
Appl. No.: |
11/156369 |
Filed: |
June 17, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60581001 |
Jun 17, 2004 |
|
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60580962 |
Jun 17, 2004 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/302; 530/350; 536/23.5 |
Current CPC
Class: |
A61P 37/04 20180101;
A61P 31/12 20180101; A61P 35/02 20180101; C07K 7/06 20130101; A61K
38/00 20130101; C07K 14/4748 20130101; A61P 35/00 20180101 |
Class at
Publication: |
435/069.1 ;
530/302; 530/350; 435/320.1; 435/325; 536/023.5 |
International
Class: |
C07K 14/74 20060101
C07K014/74; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06 |
Claims
1. A peptide having an amino acid sequence comprising at least one
difference from a sequence of a segment of a target-associated
antigen, the segment having known or predicted affinity for the
peptide binding cleft of a MHC protein, wherein the at least one
difference is a Nle or Nva residue replacing a residue at an
MHC-binding motif anchor position in said segment.
2. The peptide of claim 1 wherein the anchor position is a primary
anchor position.
3. The peptide of claim 2 wherein said anchor position is P2.
4. The peptide of claim 2 wherein said anchor position is
P.OMEGA..
5. The peptide of claim 1 wherein the anchor position is an
auxiliary anchor position.
6. The peptide of claim 1 wherein said difference comprises a Nle
or Nva residue replacing a hydrophobic residue in said segment.
7. The peptide of claim 1 wherein I, L, or V is a preferred residue
in said MHC-binding motif anchor position.
8. The peptide of claim 1 having a length of about 8 to about 14
amino acids.
9. The peptide of claim 8 having a length of 9 to 10 amino
acids.
10. The peptide of claim 1 wherein said MHC protein is a human MHC
protein.
11. The peptide of claim 1 wherein said MHC protein is a class I
MHC protein.
12. The peptide of claim 11 wherein said MHC protein is of a type
selected from the group consisting of HLA-A2, A3, A24, A30, A66,
A68, A69, B7, B8, B15, B27, B35, B37, B38, B39, B40, B48, B51, B52,
B53, B60, B61, B62, B63, B67, B70, B71, B75, B77, C4, Cw1, Cw3,
Cw4, Cw6, Cw7, and Cw10.
13. The peptide of claim 12 said MHC protein is selected from the
group consisting of HLA-A2 and A24.
14. The peptide of claim 1, said MHC having an anchor residue
binding pocket, wherein the pocket is homologous to the B-- or
F-pocket of HLA-A*0201.
15. The peptide of claim 1 having at least one binding
characteristic that is substantially the same as, or better than, a
corresponding characteristic of said segment for said MHC.
16. The peptide of claim 15, wherein said binding characteristic is
elevated compared with that of said segment.
17. The peptide of claim 15 wherein said binding characteristic is
affinity.
18. The peptide of claim 15 wherein said binding characteristic is
stability of binding.
19. The peptide of claim 1 having an immunogenicity that is
substantially the same as, or better than, the immunogenicity of
said segment.
20. The peptide of claim 19 wherein said immunogenicity is
increased.
21. The peptide of claim 19 wherein said immunogenicity evokes an
immune response that is cross-reactive to said segment.
22. The peptide of claim 19 wherein said immunogenicity evokes a
CTL response.
23. The peptide of claim 19 wherein said immunogenicity is assessed
using an MHC-tetramer assay.
24. The peptide of claim 19 wherein said immunogenicity is assessed
using a cytokine assay.
25. The peptide of claim 19 wherein said immunogenicity is assessed
using a cytotoxicity assay.
26. The peptide of claim 19 wherein said immunogenicity is assessed
by measuring an immune response recognizing the peptide.
27. The peptide of claim 19 wherein said immunogenicity is assessed
by measuring an immune response recognizing said segment.
28. The peptide of claim 19 wherein said immunogenicity is assessed
using an in vitro immunizations system.
29. The peptide of claim 28 wherein said immunization system
comprises human cells.
30. The peptide of claim 19 wherein said immunogenicity is assessed
using an in vivo immunization system.
31. The peptide of claim 30 wherein said immunization system
comprises a transgenic mouse.
32. The peptide of claim 1 that is immunologically cross-reactive
with said segment.
33. The peptide of claim 32 wherein the cross-reactivity is
assessed by immunizing with said segment and assaying recognition
of the peptide.
34. The peptide of claim 32 wherein the cross-reactivity is
assessed by immunizing with the peptide and assaying recognition of
said segment.
35. The peptide of claim 1 comprising two differences.
36. The peptide of claim 35 wherein each difference independently
comprises a Nle or Nva residue.
37. The peptide of claim 35 wherein one difference does not
comprise a Nle or Nva residue.
38. The peptide of claim 1 comprising three or more
differences.
39. The peptide of claim 1 wherein the target-associated antigen is
a tumor-associated antigen.
40. The peptide of claim 1 wherein the target-associated antigen is
a pathogen-associated antigen.
41. An immunogenic composition comprising the peptide of claim
1.
42. A method of immunization comprising administering the
composition of claim 22 to a mammal.
43. The method of claim 42 comprising administering directly to the
lymphatic system.
44. A method of making a T cell epitope analogue comprising,
providing an amino acid sequence of a segment of a
target-associated antigen, the segment having known or predicted
affinity for the peptide binding cleft of a MHC protein, changing
at least one amino acid of the sequence corresponding to an anchor
position of a MHC binding motif to Nle or Nva, and synthesizing a
peptide comprising the changed sequence.
45. A T cell epitope peptide analogue wherein the analogue differs
from a native epitope peptide by replacement of at least one native
residue corresponding to an anchor position of a MHC binding motif
with a Nle or Nva residue.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. .sctn.
119(e) to U.S. Provisional Application No. 60/581,001, filed on
Jun. 17, 2004, entitled SSX-2 PEPTIDE ANALOGS, and to U.S.
Provisional Application No. 60/580,962, filed on Jun. 17, 2004,
entitled NY-ESO PEPTIDE ANALOGS; each of which is incorporated
herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] In certain embodiments, the invention disclosed herein
relates to analogs of peptides corresponding to class I
MHC-restricted T cell epitopes and methods for their generation.
These analogs can contain amino acid substitutions at residues that
directly interact with MHC molecules, and can confer improved,
modified or useful immunologic properties. In particular, epitope
analogs from the tumor-associated antigens SSX-2, NY-ESO-1, PRAME,
PSMA, tyrosinase, and melan-A are identified. Additionally classes
of analogs, in which the various substitutions comprise the
non-standard residues norleucine and/or norvaline, are
disclosed.
[0004] 2. Description of the Related Art
The Major Histocompatibility Complex and T Cell Target
Recognition
[0005] T lymphocytes (T cells) are antigen-specific immune cells
that function in response to specific antigen signals. B
lymphocytes and the antibodies they produce are also
antigen-specific entities. However, unlike B lymphocytes, T cells
do not respond to antigens in a free or soluble form. For a T cell
to respond to an antigen, it requires the antigen to be bound to a
presenting complex known as the major histocompatibility complex
(MHC).
[0006] MHC proteins provide the means by which T cells
differentiate native or "self" cells from foreign cells. MHC
molecules are a category of immune receptors that present potential
peptide epitopes to be monitored subsequently by the T cells. There
are two types of MHC, class I MHC and class II MHC. CD4+ T cells
interact with class II MHC proteins and predominately have a helper
phenotype while CD8+ T cells interact with class I MHC proteins and
predominately have a cytolytic phenotype, but each of them can also
exhibit regulatory, particularly suppressive, function. Both MHC
are transmembrane proteins with a majority of their structure on
the external surface of the cell. Additionally, both classes of MHC
have a peptide binding cleft on their external portions. It is in
this cleft that small fragments of proteins, native or foreign, are
bound and presented to the extracellular environment.
[0007] Cells called antigen presenting cells (APCs) display
antigens to T cells using the MHC. T cells can recognize an
antigen, if it is presented on the MHC. This requirement is called
MHC restriction. If an antigen is not displayed by a recognizable
MHC, the T cell will not recognize and act on the antigen signal. T
cells specific for the peptide bound to a recognizable MHC bind to
these MHC-peptide complexes and proceed to the next stages of the
immune response.
SUMMARY OF THE INVENTION
SSX-2.sub.41-49 Analog Embodiments
[0008] Embodiments include analogs of the MHC class I-restricted T
cell epitope SSX-2.sub.41-49, KASEKIFYV (SEQ ID NO. 1),
polypeptides comprising these analogs that can be processed by pAPC
to present the epitope analogs, and nucleic acids that express the
analogs. The analogs can have similar or improved immunological
properties compared to the wild-type epitope.
[0009] One embodiment relates to an isolated SSX-2 peptide having a
sequence comprising 1 or more amino acid substitutions of the
sequence KASEKIFYV (SEQ ID NO: 1), in an amount sufficient to
elicit cytokine production from a T cell line generated by
immunization against an epitope with the sequence KASEKIFYV (SEQ ID
NO:1). In one aspect, the amount sufficient can be not more than 10
uM. In a further aspect, the amount is not more than 3 uM. In a
further is not more than 1 uM. In a further aspect, the amount can
be not more than 0.3 uM. In one aspect, the substitutions can
include a standard amino acid. In a further aspect, the amount can
be not more than 0.3 uM. In one aspect, the substitutions can
include a standard amino acid, for example Tyr, Val, Leu, Ala, Ile,
Met, Trp, Phe, Asp, Asn, or Ser. In a further aspect, the
substitutions can include a non-standard amino acid. In one aspect,
the non-standard amino acid can be, for example, Nle, Nva, Abu, or
a D-stereoisomer of a standard amino acid. In a further aspect, the
substitutions can include a modified terminal amino acid. In one
aspect, the modified terminal amino acid can be an amidated
C-terminal amino acid. In a further aspect at least one of the
substitutions can be the addition of an amino acid, wherein the
addition is a C-terminal addition. In a further aspect, the peptide
further can include the substitution of conserved amino acids at
any site, but preferably at the P3, P5, or P7 sites which are not
expressly involved in any MHC interactions.
[0010] A further embodiment relates to an isolated peptide of 9
amino acids, P1 to P9, which can include one amino acid at each
site. For example, P1 can be K, F, Y. W, Phg, Phe(4-F),
Phe(4-NO.sub.2), MeTyr, .beta.-(3-benzothienyl)-Ala, or D-Lys; P2
can be A, L, V, I, M, D-Ala, Nal-2, Abu, Aib, Nle, or Nva; P3 can
be S; P4 can be E, Q, Nle, or Nva; P5 can be K; P6 can be I, L, V,
Nle, or Nva; P7 can be F; P8 can be Y, F, Phe(4-F); and P.OMEGA.
(P-omega) at P9 can be V, I, A, Nva, MeVal, or Abu. In some
instances, the sequence is not KASEKIFYV.
[0011] A further embodiment relates to an isolated peptide of 9
amino acids, P1 to P9, which can include one amino acid at each
site. For example, P1 can be K, F, Y, W, Phg, Phe(4-F),
Phe(4-NO.sub.2), MeTyr, .beta.-(3-benzothienyl)-Ala, or D-Lys; P2
can be V, L, M, Abu, Nle, or Nva; P3 can be S; P4 can be E, Q, Nle,
or Nva; P5 can be K: P6 can be I, L, V, Nle, or Nva; P7 can be F;
P8 can be Y, F, Phe(4-F); and P.OMEGA. at P9 can be V, I, A, Nva,
MeVal, Abu, or V--NH.sub.2.
[0012] A further embodiment relates to an isolated peptide of 9
amino acids, P1 to P9, which can include one amino acid at each
site. For example, P1 can be K, F, Y, W, Phg, Phe(4-F),
Phe(4-NO.sub.2), MeTyr, .beta.-(3-benzothienyl)-Ala, or D-Lys; P2
can be A, L, V, M, Abu, Nle, or Nva; P3 can be S; P4 can be E, Q,
Nle, or Nva; P5 can be K; P6 can be I, L, V, Nle, or Nva; P7 can be
F; P8 can be Y, F, Phe(4-F); P9 can be V; and P.OMEGA. at P10 can
be I or L.
[0013] A further embodiment relates to an isolated peptide of 9
amino acids, P1 to P9, which can include one amino acid at each
site. For example, P1 can be K, F, Y, W, Phg, Phe(4-F),
Phe(4-NO.sub.2), MeTyr, .beta.-(3-benzothienyl)-Ala, or D-Lys; P2
can be V; P3 can be S; P4 can be E, Q, Nle, or Nva; P5 can be K: P6
can be I, L, V, Nle, or Nva; P7 can be F; P8 can be Y, F, Phe(4-F);
P9 can be V; and PE at P.OMEGA. can be I, L, V, or Nle.
[0014] A further embodiment relates to an isolated peptide of 9
amino acids, P1 to P9, which can include one amino acid at each
site. For example, P1 can be K, F, Y, W, Phg, Phe(4-F),
Phe(4-NO.sub.2), MeTyr, .beta.-(3-benzothienyl)-Ala, or D-Lys; P2
can be L; P3 can be S; P4 can be E, Q, Nle, or Nva; P5 can be K: P6
can be I, L, V, Nle, or Nva; P7 can be F; P8 can be Y, F, Phe(4-F);
P9 can be V; and P.OMEGA. at P10 can be I, L, V, Nle or Nva.
[0015] A further embodiment relates to an isolated peptide having
the sequence: K{L, V, M, I, D-Ala, D-Val, Nal-2, Aib, Abu, Nle, or
Nva}SEKIFYV; or {F, Phg, Y, Phe(4-F), Phe(4-NO.sub.2),
O-methyl-Tyr, or .beta.-(3-benzothienyl-Ala}ASEKIFYV; or {Y, F, or
W}{V, M, or I}SEKIFYV; or {F or W}LSEKIFYV; or K{A, V, or
L}SEKIFYI; or K{L or V}SEKIFYV-NH.sub.2; or FVSEKIFY{I, A, Nva,
Abu, or MeVal}; or FVS{Q, Nle, Nva}KIFYV; or FVSEK{L, V, Nle, or
Nva}FYV; or FVSEKIF{F, Phe(4-F)}V; or KASEKIFYV{I, L,}; or
KVSEKIFYV{I, L, V, or Nle}; or KLSEKIFYV{L, V, Nle, or Nva}.
[0016] A further embodiment relates to an isolated peptide having
the sequence: K{L, V, M, Abu, Nle, or Nva}SEKIFYV; or {F or Phg}A
SEKIFYV; or YVSEKIFYV; or F{L, V, or I}SEKIFYV; or W{L or
I}SEKIFYV; or K{V or L}SEKIFYI.; or FVSEKIFY{I or Nva}.
[0017] A further embodiment relates to an isolated peptide having
the sequence: K{V or L}SEKIFYV; or {F or Y}ASEKIFYV; or
FVSEKIFYI.
[0018] A further embodiment relates to a class I MHC/peptide
complex wherein the peptide has the sequence of any of the peptides
in the embodiments described above and elsewhere herein. In one
aspect, the complex can be cross-reactive with a TCR that
recognizes a class I MHC/SSX-2.sub.41-49 complex. In a further
aspect, the complex can be an HLA-A2/SSX-2.sub.41-49 complex.
[0019] A further embodiment relates an immunogenic composition that
can include any of the peptide embodiments described above and
elsewhere herein. In one aspect the peptide can have, for example,
the sequence: K{L, V, M, Abu, Nle, or Nva}SEKIFYV; or {F or Phg}A
SEKIFYV; or YVSEKIFYV; or F{L, V, or I}SEKIFYV; or W{L or
I}SEKIFYV; or K{V or L}SEKIFYI; or FVSEKIFY{I or Nva}, or K{V or
L}SEKIFYV; or {F or Y}ASEKIFYV; or FVSEKIFYI.
[0020] Some further embodiments relate to analogs of the MHC class
I-restricted T cell epitope NY-ESO-1.sub.157-165, SLLMWITQC (SEQ ID
NO. 1), polypeptides that include these analogs that can be
processed by pAPC to present the epitope analogs, and nucleic acids
that express the analogs. The analogs can have similar or improved
immunological properties compared to the wild-type epitope.
[0021] One embodiment relates to an isolated NY-ESO-1.sub.157-165
peptide having a sequence comprising 1 or more amino acid
substitutions of the sequence SLLMWITQC (SEQ ID NO:1), in an amount
sufficient to elicit cytokine production from a T cell line
generated by immunization against an epitope with the sequence
SLLMWITQC (SEQ ID NO:1). For example, in one aspect, the amount
sufficient can be not more than 10 uM. In a further aspect, the
amount can be not more than 3 uM. Also, in a further aspect, the
amount can be not more than 1 uM. In a further aspect, the amount
is not more than 0.3 uM. In one aspect, the substitutions can
include a standard amino acid. In a further aspect, the
substitutions can include a non-standard amino acid. In one aspect,
the non-standard amino acid can be, for example, Tyr, Val, Leu,
Ala, Ile, Met, Nle, Nva, Trp, Phe, Asp, Asn, Ser, Abu, and a
D-stereoisomer of a standard amino acid. In a further aspect, the
substitutions can include a modified terminal amino acid. In one
aspect, the modified terminal amino acid can be an amidated
C-terminal amino acid. In a further aspect at least one of the
substitutions can be the addition of an amino acid, wherein the
addition is a C-terminal addition.
[0022] One embodiment relates to an isolated peptide having a
sequence in which: [0023] P1 is S, F, K, or W; [0024] P2 is L, I,
V, Nle, or Nva; [0025] P3 is L; [0026] P4 is M, L, or N; [0027] P5
is W; [0028] P6 is I, A, L, V, or N; [0029] P7 is T; [0030] P8 is
Q, E, D, or T; [0031] P.OMEGA. at P9 is C, V, I, L, A, Nva, Nle,
V--NH.sub.2, or L-NH.sub.2; and [0032] wherein the sequence is not
SLLMWITQ{C, V, I, L, A}, FVLMWITQA, or FILMWITQ{L, I}.
[0033] Another embodiment relates to an isolated peptide having a
sequence in which: [0034] P1 is Y; [0035] P2 is L, V, I, Nle, or
Nva; [0036] P3 is L; [0037] P4 is M, L, or N; [0038] P5 is W;
[0039] P6 is I, A, L, V, or N; [0040] P7 is T; [0041] P8 is Q, E,
D, or T; [0042] P.OMEGA. at P9 is V, I, L, Nva, Nle, V,
V--NH.sub.2, or L-NH.sub.2; and [0043] wherein the sequence is not
YVLMWITL or YLLMWIT{I, L}.
[0044] A further embodiment relates to an isolated decamer peptide
having a sequence {S,Y}LLMWITQ{C, V}{L, I, Nle}.
[0045] Yet another embodiment relates to an isolated peptide having
a sequence SILMWITQ{C, V, L, A}, YLLMWITQ{Nva, Nle}, F{L,
V}LMWITQ{V, L, I}, Y{I, Nva, Nle}LMWITQV, YLLLWITQV, or
TVLMWITQV.
[0046] A further embodiment relates to an isolated peptide having a
sequence {S, F}VLMWITQV, SLMWITQNva, or SNvaLMWITQV
[0047] Still another embodiment relates to an isolated peptide
having a sequence SNvaLMWITQV.
[0048] Some embodiments relate to an isolated peptide. The peptide
can include or consist essentially of a sequence in which: [0049]
P0 is X, XX, or XXX, wherein X specifies any amino acid or no amino
acid; and [0050] P1 is K, F, Y, W, Phg, Phe(4-F), Phe(4-NO.sub.2),
MeTyr, .beta.-(3-benzothienyl)-Ala, or D-Lys; and [0051] P2 is A,
L, V, I, M, D-Ala, Nal-2, Abu, Aib, Nle, or Nva; and [0052] P3 is
S; and [0053] P4 is E, Q, Nle, or Nva; and [0054] P5 is K: and
[0055] P6 is I, L, V, Nle, or Nva; and [0056] P7 is F; and [0057]
P8 is Y, F, Phe(4-F); and [0058] P.OMEGA. at P9 is V, I, A, Nva,
MeVal, Abu, or V--NH.sub.2, or P9 is V, and P.OMEGA. at P10 is I,
L, V, Nle or Nva; and [0059] P.OMEGA.+1 is X, XX, or XXX, wherein X
specifies any amino acid or no amino acid; and [0060] wherein the
sequence is not KASEKIFYV;
[0061] The isolated peptide can include or consist essentially of
the sequence: [0062] K{L, V, M, I, D-Ala, D-Val, Nal-2, Aib, Abu,
Nle, or Nva}SEKIFYV; or [0063] {F, Phg, Y, Phe(4-F),
Phe(4-NO.sub.2), O-methyl-Tyr, or
.beta.-(3-benzothienyl-Ala}ASEKIFYV; or [0064] {Y, F, or W}{V, M,
or I}SEKIFYV; or [0065] {F or W}LSEKIFYV; or [0066] K{A, V, or
L}SEKIFYI; or [0067] K{L or V}SEKIFYV--NH.sub.2; or [0068]
FVSEKIFY{I, A, Nva, Abu, or MeVal}; or [0069] FVS{Q, Nle,
Nva}KIFYV; or [0070] FVSEK{L, V, Nle, or Nva}FYV; or [0071]
FVSEKIF{F, Phe(4-F)}V; or [0072] KASEKIFYV{I, L,}; or [0073]
KVSEKIFYV{I, L, V, or Nle}; or [0074] KLSEKIFYV{L, V, Nle, or
Nva}.
[0075] The isolated peptide can include or consist essentially of
the sequence: [0076] K{L, V, M, Abu, Nle, or Nva}SEKIFYV; or [0077]
{F or Phg}A SEKIFYV; or [0078] YVSEKIFYV; or [0079] F{L, V, or
I}SEKIFYV; or [0080] W{L or I}SEKIFYV; or [0081] K{V or L}SEKIFYI;
or [0082] FVSEKIFY{I or Nva}.
[0083] Also, the isolated peptide can include or consist
essentially of the sequence: [0084] K{V or L}SEKIFYV; or [0085] {F
or Y}ASEKIFYV; or [0086] FVSEKIFYI; or [0087] KVSEKIFYV.
[0088] Further, the isolated peptide can include or consist
essentially of the sequence KVSEKIFYV.
[0089] The isolated peptide can have affinity for a class I MHC
peptide binding cleft. The MHC can be, for example, HLA-A2.
[0090] Some embodiments relate to a class I MHC/peptide complex
wherein the peptide can have the sequence of the peptide of claim
1. The class I MHC/peptide complex can be cross-reactive with a TCR
that recognizes a class I MHC/SSX-2.sub.41-49 complex. The class I
MHC/peptide complex can be an HLA-A2/SSX-2.sub.41-49 complex.
[0091] Other embodiments relate to a polypeptide that includes a
polypeptide as described above and elsewhere herein, in association
with a liberation sequence.
[0092] Still further embodiments relate to immunogenic compositions
that include a peptide as described above or elsewhere herein.
[0093] Others relate to nucleic acids encoding or nucleic acid
means for expressing a polypeptide as described above or elsewhere
herein. Also, some relate to immunogenic compositions that include
such nucleic acids or nucleic acid means.
[0094] Some embodiments relate to methods of inducing, maintaining,
or amplifying a CTL response. The methods can include intranodal
administration of a composition as described above and elsewhere
herein.
[0095] Other embodiments relate to methods of entraining a class I
MHC-restricted T cell response, which methods can include
intranodal administration a composition as described above or
elsewhere herein plus an immunopotentiating agent.
[0096] Further embodiments relate to methods of inducing,
maintaining, or entraining a CTL response, which methods can
include intranodal administration of a composition as described
above and elsewhere herein.
[0097] Some embodiments relate to isolated peptides that include 1
to 3 substitutions in the sequence KASEKIFYV having an affinity for
a class I MHC binding cleft that is similar to or greater than the
affinity of KASEKIFYV for said class I MHC binding cleft. The
halftime of dissociation can be similar to or greater than the
halftime of dissociation of KASEKIFYV from said class I MHC binding
cleft. The isolated peptide can be recognized by T cells with
specificity for the peptide KASEKIFYV.
[0098] Still further embodiments relate to isolated peptides that
include or consisting essentially of a sequence in which: [0099] P1
is K, F, Y, W, Phg, Phe(4-F), Phe(4-NO.sub.2), MeTyr,
.beta.-(3-benzothienyl)-Ala, or D-Lys; and [0100] P2 is A, L, V, I,
M, D-Ala, Nal-2, Abu, Aib, Nle, or Nva; and [0101] P3 is S; and
[0102] P4 is E, Q, Nle, or Nva; and [0103] P5 is K: and [0104] P6
is I, L, V, Nle, or Nva; and [0105] P7 is F; and [0106] P8 is Y, F,
Phe(4-F); and [0107] P.OMEGA. at P9 is V, I, A, Nva, MeVal, or Abu;
[0108] wherein the sequence is not KASEKIFYV; [0109] or [0110] P1
is K, F, Y, W, Phg, Phe(4-F), Phe(4-NO.sub.2), MeTyr,
.beta.-(3-benzothienyl)-Ala, or D-Lys; and [0111] P2 is V, L, M,
Abu, Nle, or Nva; and [0112] P3 is S; and [0113] P4 is E, Q, Nle,
or Nva; and [0114] P5 is K: and [0115] P6 is I, L, V, Nle, or Nva;
and [0116] P7 is F; and [0117] P8 is Y, F, Phe(4-F); and [0118]
P.OMEGA. at P9 is V, I, A, Nva, MeVal, Abu, or V--NH.sub.2; [0119]
or [0120] P1 is K, F, Y, W, Phg, Phe(4-F), Phe(4-NO.sub.2), MeTyr,
.beta.-(3-benzothienyl)-Ala, or D-Lys; and [0121] P2 is A, L, V, M,
Abu, Nle, or Nva; and [0122] P3 is S; and [0123] P4 is E, Q, Nle,
or Nva; and [0124] P5 is K: and [0125] P6 is I, L, V, Nle, or Nva;
and [0126] P7 is F; and [0127] P8 is Y, F, Phe(4-F); and [0128] P9
is V; and [0129] P.OMEGA. at P10 is I or L; [0130] or [0131] P1 is
K, F, Y, W, Phg, Phe(4-F), Phe(4-NO.sub.2), MeTyr,
.beta.-(3-benzothienyl)-Ala, or D-Lys; and [0132] P2 is V; and
[0133] P3 is S; and [0134] P4 is E, Q, Nle, or Nva; and [0135] P5
is K: and [0136] P6 is I, L, V, Nle, or Nva; and [0137] P7 is F;
and [0138] P8 is Y, F, Phe(4-F); and [0139] P9 is V; and [0140]
P.OMEGA. at P10 is I, L, V, or Nle; [0141] or [0142] P1 is K, F, Y,
W, Phg, Phe(4-F), Phe(4-NO.sub.2), MeTyr,
.beta.-(3-benzothienyl)-Ala, or D-Lys; and [0143] P2 is L; and
[0144] P3 is S; and [0145] P4 is E, Q, Nle, or Nva; and [0146] P5
is K: and [0147] P6 is I, L, V, Nle, or Nva; and [0148] P7 is F;
and [0149] P8 is Y, F, Phe(4-F); and [0150] P9 is V; and [0151]
P.OMEGA. at P10 is I, L, V, Nie or Nva.
[0152] Some embodiments relate isolated peptides that include or
consist essentially of a sequence in which: [0153] P0 is X, XX or
XXX, wherein X specifies any amino acid or no amino acid; and
[0154] P1 is S, F, K, W or Y; and [0155] P2 is L, I, V, Nle, or
Nva; and [0156] P3 is L; and [0157] P4 is M, L, or N; and [0158] P5
is W; and [0159] P6 is I, A, L, V, or N; and [0160] P7 is T; and
[0161] P8 is Q, E, D, or T; and [0162] P.OMEGA. at P9 is C, V, I,
L, A, Nva, Nle, V--NH.sub.2, or L-NH.sub.2; and [0163] P.OMEGA.+1
is X, XX, XXX, wherein X specifies any amino acid or no amino acid;
and [0164] wherein the sequence is not SLLMWITQ{C, V, I, L, A},
FVLMWITQA, FILMWITQ{L, I}, YVLMWITL or YLLMWIT{I, L}. [0165] P1 is
S, F, K, or W; [0166] P2 is L, I, V, Nle, or Nva; [0167] P3 is L;
[0168] P4 is M, L, or N; [0169] P5 is W; [0170] P6 is I, A, L, V,
or N; [0171] P7 is T; [0172] P8 is Q, E, D, or T; [0173] P.OMEGA.
at P9 is C, V, I, L, A, Nva, Nle, V--NH.sub.2, or L-NH.sub.2; and
[0174] wherein the sequence is not SLLMWITQ{C, V, I, L, A},
FVLMWITQA, or FILMWITQ{L, I}; [0175] or [0176] P1 is Y; [0177] P2
is L, V, I, Nle, or Nva; [0178] P3 is L; [0179] P4 is M, L, or N;
[0180] P5 is W; [0181] P6 is I, A, L, V, or N; [0182] P7 is T;
[0183] P8 is Q, E, D, or T; [0184] P.OMEGA. at P9 is V, I, L, Nva,
Nle, V, V--NH.sub.2, or L-NH.sub.2; and [0185] wherein the sequence
is not YVLMWITL or YLLMWIT{I, L}.
[0186] A further embodiment relates to a class I MHC/peptide
complex wherein the peptide can have the sequence of any of the
peptides in the embodiments described above or elsewhere herein. In
one aspect, the complex can be cross-reactive with a TCR that
recognizes a class I MHC/NY-ESO-1.sub.157-165 complex. In a further
aspect, the complex can be an HLA-A2/NY-ESO-1.sub.157-165
complex.
[0187] In one aspect of the above embodiments, the peptide can have
affinity for a class I MHC peptide binding cleft, such as
HLA-A2.
[0188] A further embodiment relates to a polypeptide comprising the
peptide sequence of any of the embodiments in association with a
liberation sequence.
[0189] A further embodiment relates to an immunogenic composition
that includes any of the peptide embodiments. In one aspect the
peptide can have a sequence as set forth herein.
[0190] A further embodiment relates to a nucleic acid encoding any
of the peptide embodiments, but preferably those which do not have
non-standard amino acid substitutions. In a further aspect, the
nucleic acid can be encoded in a vector.
[0191] A further embodiment relates to an immunogenic composition
that includes the nucleic acid encoding any of the peptide
embodiments.
[0192] A further embodiment relates to a method of inducing a CTL
response by intranodal administration of any of the compositions or
peptides of the embodiments. In a further aspect, the method can
allow for maintaining a CTL. In a further aspect, the method can
allow for amplifying a class I MHC-restricted T cell response. In a
further aspect, the method can allow for entraining a class I
MHC-restricted T cell response. In a further aspect, the method
also can include an immunopotentiating agent.
[0193] Some embodiments relate to isolated peptides having a
sequence comprising 1 to 3 or 4 amino acid substitutions in a
native epitope sequence, wherein a concentration of the peptide
required to elicit cytokine production from a T cell line generated
by immunization against an epitope with the sequence is not more
than a particular concentration, for example, 10 uM, 1 uM, 0.3 uM,
and the like. The substitutions can include a standard amino acid,
a non-standard amino acid, and the like. The non-standard amino
acid can be any of those described herein, for example, a
D-stereoisomer of a standard amino acid, Nva, or Nle. The
substitutions can include a modified terminal amino acid, and the
modified terminal amino acid can be an amidated C-terminal amino
acid. One of the substitutions can be the addition of an amino
acid, for example, the addition can be a C-terminal addition.
[0194] Other embodiments relate to peptides having an amino acid
sequence that includes at least one difference from a sequence of a
segment of a target-associated antigen, the segment having known or
predicted affinity for the peptide binding cleft of a MHC protein,
wherein the at least one difference can be a Nle or Nva residue
replacing a residue at an MHC-binding motif anchor position in said
segment. The anchor position can be a primary anchor position, for
example, P2 or P.OMEGA.. The anchor position can be an auxiliary
anchor position. The difference can include a Nle or Nva residue
replacing a hydrophobic residue in said segment. In some aspects I,
L, or V can be a preferred residue in the MHC-binding motif anchor
position. In some aspects the peptide can have a length of about 8
to about 14 amino acids or more preferably a length of 9 to 10
amino acids, for example.
[0195] The protein can be a human MHC protein, for example, class I
MHC protein. The MHC protein can be, for example, a type such as
HLA-A2, A3, A24, A30, A66, A68, A69, B7, B8, B15, B27, B35, B37,
B38, B39, B40, B48, B51, B52, B53, B60, B61, B62, B63, B67, B70,
B71, B75, B77, C4, Cw1, Cw3, Cw4, Cw6, Cw7, and Cw10. In some
aspects, the MHC protein can be HLA-A2 or A24. The MHC can have an
anchor residue binding pocket, wherein the pocket is homologous to
the B-- or F-pocket of HLA-A*0201. The MHC residues responsible for
forming binding pockets, and which binding pockets accommodate
epitope anchor residues and thus define the binding specificity of
the MHC molecule, are well understood in the art. One compilation
of such information is found at the FIMM (Functional Immunology)
web site at the hypertext transfer protocol (http://)
"sdmc.lit.org.sg:8080/fimm/." See also Schonbach C., Koh J. L. Y.,
Sheng X., Wong L., and V. Brusic. FIMM, a database of functional
molecular immunology. Nucleic Acids Research, 2000, Vol. 28, No. 1
222-224; and Schonbach C., Koh J L, Flower D R, Wong L., and Brusic
V. FIMM, a database of functional molecular immunology; update
2002. Nucleic Acids Research, 2002. Vol. 30. No. 1 226-229; each of
which is hereby incorporated by reference in its entirety. Also,
the peptide can have at least one binding characteristic that is
substantially the same as, or better than, a corresponding
characteristic of said segment for said MHC. For example, the
binding characteristic can be elevated compared with that of said
segment. Also, the binding characteristic can be affinity or
stability of binding for example.
[0196] The peptide can have an immunogenicity that is substantially
the same as, or better than, the immunogenicity of the segment. The
immunogenicity can be increased. The immunogenicity can evoke an
immune response that is cross-reactive to said segment or can evoke
a CTL response. The immunogenicity can be assessed, for example,
using an MHC-tetramer assay, a cytokine assay, a cytotoxicity
assay, by measuring an immune response recognizing the peptide, by
measuring an immune response recognizing said segment, using an in
vitro immunizations system, or any other suitable method. The
immunization system can include human cells. The immunogenicity can
be assessed using an in vivo immunization system, for example, one
that includes a transgenic mouse. The peptide can have an at least
similar binding characteristic as said segment for said MHC. For
example, in some aspects what is considered to be "similar" can be
determined based upon the instant disclosure. In some particular
aspects "similarity" can be based upon, for example, peptide
concentration for half-maximal binding, relative affinity,
stability (half time of dissociation) and
cross-reactivity/functional avidity. As an example, a peptide can
be considered similar if it has results or characteristics that are
within twofold, even threefold, four, five or 10 fold of the value
for the native peptide. Also, as an example, for
cross-reactivity/functional avidity a similar result can be one
where the data are within three and 10-fold of the native peptide.
As another example, percentage of binding values can be considered
similar when within 2, 3, 4, 5, 6, 7, 10, 15 or 20% of the native
peptide. Also, ED50 values can be considered similar in some
aspects when within 2- or 3-fold of native sequence. Similar
halftime of dissociation can be for example within 2- or 3-fold. As
still another example, for cross-reactivity a value that is about
2-fold different from wild-type can be considered similar. These
similar values are exemplary only and given in the context of some
aspects of some embodiments. Other "similar" values can be
determined based upon the other experiments and teachings
herein.
[0197] The peptides can be immunologically cross-reactive with the
segment. The cross-reactivity can be assessed by immunizing with
the segment and assaying recognition of the peptide. The
cross-reactivity can be assessed by immunizing with the peptide and
assaying recognition of the segment.
[0198] The peptide as described above and elsewhere herein can be
modified to include two differences, for example. In some instances
each difference independently can include a Nle or Nva residue. In
some instances one difference can not include a Nle or Nva residue.
Also, the peptide as described above and elsewhere herein can
include three or more differences.
[0199] The target-associated antigen can be a tumor-associated
antigen. The target-associated antigen can be a pathogen-associated
antigen.
[0200] Other embodiments relate to immunogenic composition that
include the instant peptides as described above and elsewhere
herein. Further embodiments relate to methods of immunization that
include administering such compositions to a mammal, for example,
administering directly to the lymphatic system.
[0201] Still other embodiments relate to methods of making a T cell
epitope analogue. The methods can include providing an amino acid
sequence of a segment of a target-associated antigen, the segment
can have known or predicted affinity for the peptide binding cleft
of a MHC protein; changing at least one amino acid of the sequence
corresponding to an anchor position of a MHC binding motif to Nle
or Nva; and synthesizing a peptide comprising the changed sequence.
The synthesis can be for example, chemical synthesis or any other
synthetic method.
[0202] Some embodiments relate to T cell epitopes peptide analogue
wherein the analogue differs from a native epitope peptide by
replacement of at least one native residue corresponding to an
anchor position of a MHC binding motif with a Nle or Nva
residue.
[0203] Further embodiments relate to methods to generate and
resulting compositions representing peptides that are immune active
and carry unnatural amino acids at one or multiple MHC anchor
residues.
BRIEF DESCRIPTION OF THE DRAWINGS
[0204] FIGS. 1A and B summarize the substitutions that have been
explored for SSX-2.sub.41-49 analogs at each individual amino acid
position for nonamers and decamers, respectively.
[0205] FIG. 2 is a schematic diagram of the methodology of a
preferred embodiment for identifying analogs.
[0206] FIG. 3 is a table showing the cross-reactivity and
functional avidity of SSX-2.sub.41-49 analogs substituted at a
single position.
[0207] FIG. 4 is a table showing the cross-reactivity and
functional avidity of SSX-2.sub.41-49 analogs substituted at two
positions.
[0208] FIG. 5 is a table showing the cross-reactivity and
functional avidity of SSX-2.sub.41-49 analogs substituted at more
than two positions.
[0209] FIG. 6 is a table showing the cross-reactivity and
functional avidity of SSX-2.sub.41-49 decamer analogs encompassing
the nominal 41-49 peptide.
[0210] FIG. 7 is a diagram showing the injection schedule of the
SSX-2.sub.41-49 analogs.
[0211] FIG. 8 shows the activity of the SSX-2.sub.41-49 A42V, A42L
analogs and wild-type in lysis of tumor cells.
[0212] FIG. 9 shows the injections schedule for in vivo
cytotoxicity studies and ex vivo cytotoxicity studies as well as
which SSX-2.sub.41-49 analog peptide was used for the boost.
[0213] FIG. 10 is a table showing the in vivo specific lysis
results for a number of the analogs as compared to a control
(wild-type peptide) and EAA (Melan A 26-35).
[0214] FIG. 11 is a table showing the in vivo specific lysis
results for a number of the SSX-2.sub.41-49 analogs as compared to
a control (wild-type peptide) and EAA as well as MHC binding and
MHC stability.
[0215] FIG. 12 shows the percent specific lysis of tumor cells
(624.38 human tumor cells) achieved following immunization with a
number of analogs as compared to a wild-type control.
[0216] Figure
[0217] FIGS. 13A-C summarize the substitutions that have been
explored at each individual amino acid position for nonamers and
decamers, respectively, as well as the results that were
obtained.
[0218] FIG. 14 is a diagram showing the injection schedule used for
analysis and testing of the NY-ESO-1 analogs.
[0219] FIGS. 15A-C show the specific elimination of target cells as
measured by removing the spleens and PBMC from challenged animals
and measuring CFSE fluorescence by flow cytometry.
[0220] FIGS. 16A and B show the in vivo cytotoxicity against target
cells coated with wild-type peptide after boost with NY-ESO-1
analogs.
[0221] FIGS. 17A and B show an ex vivo analysis of the ability of
the analogs to trigger enhanced immunity against the wild-type
epitope as assessed by cytokine production.
[0222] FIG. 18 illustrates a protocol for validating the
antigenicity of the PSMA.sub.288-297 epitope, as well as the
results of the testing.
[0223] FIG. 19 is a table showing the cross-reactivity and
functional avidity of PSMA.sub.238-297 analogs substituted at a
single position.
[0224] FIG. 20 is a table showing the cross-reactivity and
functional avidity of PSMA.sub.288-297 analogs substituted at two
positions.
[0225] FIG. 21 is a table showing the cross-reactivity and
functional avidity of PSMA.sub.288-297 analogs substituted at more
than two positions.
[0226] FIG. 22 shows the immunogenicity of various PSMA.sub.288-297
analogs measured by Elispot.
[0227] FIG. 23 shows the results of an assay regarding the
amplification of anti-PSMA.sub.288-297 response by the I297V analog
measured by Elispot.
[0228] FIG. 24 shows the results of boosting with the I297V analog.
The assay showed that the boosting resulted in cytotoxic immunity
against a PSMA.sup.+ human tumor line.
[0229] FIG. 25 illustrates a protocol for validating the
antigenicity of the PRAME.sub.425-433 epitope, as well as the
results of the testing.
[0230] FIG. 26 is a table showing the cross-reactivity and
functional avidity of PRAME.sub.425-433 analogs substituted at a
single position.
[0231] FIG. 27 includes two tables (A and B), which show the
cross-reactivity and functional avidity of PRAME.sub.425-433
analogs substituted at two positions.
[0232] FIG. 28 is a table showing the cross-reactivity and
functional avidity of PRAME.sub.425-433 analogs substituted at more
than two positions.
[0233] FIG. 29 shows the immunogenicity of a PRAME.sub.425-433
analog measured by Elispot.
[0234] FIG. 30 shows the results of boosting with the L426Nva
L433Nle analog. The assay showed that the boosting resulted in
cytotoxic immunity against native epitope coated cells.
[0235] FIG. 31 shows a protocol for the in vivo evaluation of PRAME
analogs, as well as the results of the evaluation.
[0236] FIG. 32 shows a protocol for the ex vivo stimulation of
cytokine production in analog induced, native epitope re-stimulated
T cells.
[0237] FIG. 33 shows the results of boosting with the L426Nva
L433Nle analog. The assay showed that the boosting resulted in
cytotoxic immunity against a human tumor cell line.
[0238] FIG. 34 depicts a protocol for in vitro immunization to
PRAME.sub.425-433.
[0239] FIG. 35 shows the tetramer analysis results after in vitro
immunization with PRAME.sub.425-433 analogs.
[0240] FIG. 36 depicts the structure of the plasmid, pCTLR2, a
plasmid that expresses the PRAME.sub.425-433 epitope.
[0241] FIG. 37 shows the assay results for an experiment in which
humor tumor cells were lysed by T cells primed with plasmid DNA and
boosted with peptides.
[0242] FIG. 38 shows the tetramer analysis results after plasmid
prime with Tyr369-377 and peptide boost with the V377Nva
analog.
[0243] FIG. 39
[0244] FIG. 40 shows a schematic representation of an Tyrosinase
analog immunogenicity evaluation protocol.
[0245] FIG. 41 shows the immune response results against 624.38
cells contacted with effector cells from HHD primed with plasmid
and boosted with Tyr369-377 analogs.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0246] Peptides encompassing T cell epitopes are usually poor
immunogens or immune modulators due to one of multiple factors: a
suboptimal pharmacokinetics profile, limited binding to MHC
molecules (reduced K.sub.on and increased K.sub.off), decreased
intrinsic recognition by T cells present in the normal immune
repertoire (e.g , through various forms of tolerance). Various
strategies have been pursued to improve the immunologic properties
of peptides, particularly the screening and use of peptides in
which the sequence differs from the natural epitope. Such analogs
are known by various names in the art, such as heteroclytic
peptides and altered peptide ligands (APL). The generation of such
analogs has most often utilized amino acids from the standard set
of genetically encoded residues (see for example Valmori, D. et
al., J. Immunol. 160:1750-1758, 1998). Use of non-standard amino
acids has typically been associated with efforts to improve the
biochemical stability of the peptide (see, for example, Blanchet,
J.-S. et al., J. Immunol. 167:5852-5861, 2001).
[0247] Generally, analogs can be categorized into the following two
main classes: (1) modification of peptide anchor residues to
achieve better HLA binding profiles and higher immune responses,
and (2) modification of peptide anchor residues and TCR contact
residues to circumvent T cell tolerance for self-antigens.
[0248] Some embodiments relate to analogs that have at least one of
the following retained or improved properties, including but not
limited to: [0249] 1. Cross-reactivity and functional avidity to
TCR [0250] 2. Affinity for and stability of binding to MHC class I
[0251] 3. In vivo effect on immunity assessed by cytotoxicity
[0252] 4. In vivo effect on immunity assessed by ex vivo production
of IFN-gamma [0253] 5. Increased resistance to proteolysis.
[0254] Some embodiments relate to peptide sequences, including
analogs, where the amino acids of the sequence are referred to with
a position designator, for example P1, P2, P3, P.OMEGA., etc. In
addition, the peptide sequences can be referred to as including a
P0 and/or P.OMEGA.+1 designator. In some aspects, P0 can be X, XX,
or XXX, where X is any amino acid or no amino acid. Similarly, in
some aspects, P.OMEGA.+1 can be X, XX, or XXX, where X is any amino
acid or no amino acid. Thus, for example, XXX can mean any
combination of any amino acid residues or no amino acid. Thus,
these embodiments can encompass polypeptides having up to three
additional amino acids (with any combination of amino acid
residues) on the N-terminus or C-terminus of the specified
sequence. Also, in some aspects, the embodiments can encompass no
additional amino acids on the N-terminus or the C-terminus.
[0255] The MHC residues responsible for forming binding pockets,
and which binding pockets accommodate epitope anchor residues and
thus define the binding specificity of the MHC molecule, are well
understood in the art. One compilation of such information is found
at the FIMM (Functional Immunology) web site at the hypertext
transfer protocol (http://) "sdmc.lit.org.sg:8080/fimm/." See also
Schonbach C., Koh J. L. Y., Sheng X., Wong L., and V. Brusic. FIMM,
a database of functional molecular immunology. Nucleic Acids
Research, 2000. Vol. 28. No. 1 222-224; and Schonbach C., Koh J L,
Flower D R, Wong L., and Brusic V. FIMM, a database of functional
molecular immunology; update 2002. Nucleic Acids Research, 2002,
Vol. 30, No. 1 226-229; each of which is hereby incorporated by
reference in its entirety.
[0256] The phrase "liberation sequence" refers to a peptide
comprising or encoding an epitope or an analog, which is embedded
in a larger sequence that provides a context allowing the epitope
or analog to be liberated by immunoproteasomal processing, directly
or in combination with N-terminal trimming or other physiologic
processes. In some aspects, the analog or epitope can be designed
or engineered.
[0257] Other embodiments relate to epitope arrays and other
polypeptides that include the epitope analog sequences that can be
processed to liberate the analog. Further embodiments relate to
nucleic acids, particularly DNA plasmids, encoding such
polypeptides, or simply an analog, and their expression therefrom.
The analogs, the polypeptides comprising them, and the encoding
nucleic acids can all be components of immunogenic compositions,
particularly compositions suitable for intralymphatic delivery, all
of which relate to further embodiments.
[0258] Peptide analogs with improved immunologic properties can be
designed by modifying the anchor residues involved in the
interaction with MHC molecules, so as to increase the binding and
stabilize the formation of MHC-peptide complexes. Such
modifications can be guided by knowledge of the binding motif or
preferred anchor residues of the restricting MHC molecule. There
further exist various rules, indexes and algorithms that can be
used to predict the properties of analogs bearing various
substitutions with the limitation that the substitution is selected
from the standard set of genetically encodable amino acids.
[0259] However, there are no databases or algorithms to predict the
outcome of replacing anchor residues with non-standard amino acids
and their usefulness is previously not well explored. It is herein
disclosed that the non-standard amino acids norleucine (Nle) and
norvaline (Nva) can be advantageously substituted into the anchor
residue positions of MHC-binding peptides. It is preferred that
they be placed in a position favorably occupied by a hydrophobic or
a large amino acid, especially I, L, or V.
[0260] MHC-binding motifs are generally defined in terms of
preferred residue side chains at nominal positions within a span of
8 to 10 amino acids (see for example Rammensee et al., "MHC Ligands
and Peptide Motifs," (Molecular Biology Intelligence Unit),
Springer-Verlag, Germany, 1997 Landes Bioscience, Austin, Tex.; and
Parker, et al., "Scheme for ranking potential HLA-A2 binding
peptides based on independent binding of individual peptide
side-chains," J. Immunol. 152:163-175. Website algorithms are also
available which can be used to predict MHC binding. See for
example, the world wide web page of Hans-Georg Rammensee, Jutta
Bachmann, Niels Emmerich, Stefan Stevanovic: SYFPEITHI: An Internet
Database for MHC Ligands and Peptide Motifs (hypertext transfer
protocol access via:
syfpeithi.bmi-heidelberg.com/scripts/MHCServer.dll/home.htm) and
another is "bimas.dcrt.nih.gov/molbio/hla_bind." For class
I-restricted epitopes the C-terminal position, P.OMEGA., is
typically a primary anchor. The 2.sup.nd position, P2, is also
often a primary anchor or, alternatively, P3 and/or P5 can serve
this role. Positions P2 through P7 have all been recognized as
secondary or auxiliary anchor positions for one or another MHC (see
Rammensee et al., and see Table 6 from U.S. patent application
Publication No. 2003-0215425 (U.S. patent application Ser. No.
10/026,066, filed on Dec. 7, 2001, entitled EPITOPE SYNCHRONIZATION
IN ANTIGEN PRESENTING CELLS; which is incorporated herein by
reference in its entirety for all of its disclosure). For class
II-restricted epitopes P1, P4, P6, P7, and P9 have been recognized
as anchor positions. The foregoing is intended as a general guide
and should be considered exemplary and not exhaustive or limiting.
Many analyses and compilations of binding motifs, anchor residues,
and the like are available in the scientific and patent literature
and over the internet. Their conventions and results further
provide those of skill in the art useful guide to the design of
epitope analogs, when coupled with the teaching herein.
[0261] The length of the peptide actually bound to the presenting
MHC molecule can be longer than the nominal motif sequence. The
ends of the binding cleft for class II MHC molecules are open so
that the bound peptide can be extended at either end of the core
motif. In contrast the binding cleft is closed at both ends in
class I MHC molecules so that the ends of the bound peptide must
generally correspond to the motif, however significant variation in
length can be accommodated through bulging and folding of the
central region of the bound peptide, so that peptides of up to at
least about 14 amino acids in length can be presented (see for
example Probst-Kepper, M. et al., J. Immunol. 173:5610-5616,
2004).
[0262] Epitope analogs can have improved K.sub.on and K.sub.off
related to the interaction with class I MHC molecules, as well as
preserved or increased interaction with T cell receptors
recognizing the original epitope, modified or improved in vivo or
ex vivo activity reflected in enhanced expansion of specific T cell
populations, improved cytokine production by specific T cells, or
in vivo or in vitro cytotoxicity against targets carrying natural
epitopes, mediated by T cells that reacted with the peptide. In
addition, such analogs may interact in a more optimal fashion with
multiple distinct MHC class I molecules.
[0263] Such peptide analogs with improved immune properties may
encompass one or multiple substitutions, including one or multiple
non-standard amino acids. Among non-standard amino acids,
substitutions for primary anchor residues consisting of norvaline
or norleucine are preferred since, as exemplified below, they can
not only improve on the interaction with MHC class I, but can also
preserve cross-reactivity with TCR specific for the native epitope
and show improved in vivo immune profile. More specifically,
mutating the P2 amino acid residue from A, L or V to norvaline or
norleucine improved immune properties and is thus preferred. In
addition, modifying the C terminal residue to norvaline or
preferably norleucine, improved immune features of the analogs. In
addition, analogs that encompass multiple substitutions at primary
and/or secondary anchor residues including norvaline and/or
norleucine at P2 or P.OMEGA., can be associated with improved
immune properties.
[0264] Certain uses of norvaline (Nva) and norleucine (Nle) are
mentioned in U.S. Pat. No. 6,685,947, PCT Publication Nos. WO
03/076585 A2 and WO 01/62776 A1 and U.S. patent Publication No.
20040253218A1. None of these references teaches the general
usefulness of Nva or Nle substituted at an anchor position of a
MHC-biding peptide to improve an immunological property. The '218
publication teaches that the substituted residues should be
incorporated at TCR-interacting positions and not at
MHC-interacting positions:
[0265] In still another embodiment of the invention, the peptide is
an analog of a peptide derived from an NS-specific antigen that is
immunogenic but not encephalitogenic. The most suitable peptides
for this purpose are those in which an encephalitogenic
self-peptide is modified at the T-cell receptor (TCR) binding site
and not at the MHC binding site(s), so that the immune response is
activated but not anergized (Karin et al, 1998; Vergelli et al,
1996).
[0266] HLA-A2.1-restricted peptides incorporating Nle disclosed in
the '776 publication are derived from CEA, p53, and MAGE-3. In the
CEA peptide I(Nle)GVLVGV and the p53 peptide S(Nle)PPPGTRV, Nle is
present at the P2 position. No teaching about the general
usefulness of norleucine is provided and no disclosure is provided
indicating how or if these substitutions altered the properties of
the analogs as compared to the native sequence.
[0267] Some of the instant embodiments relate to epitope analogs
that incorporate Nva and/or Nle at a position promoting binding to
MHC. Some embodiments specifically exclude the use Nle and/or Nva
in HLA-A2.1-restricted epitopes, HLA-A2.1 epitopes from CEA, p53,
and/or MAGE-3, or other peptides derived from MAGE-3, CEA, and/or
p53. In some embodiments, one or more of the specific sequences as
disclosed in the above referenced patent references are
specifically excluded. Other exemplary embodiments include the use
of Nle and/or Nva at P3, P5, and/or P.OMEGA. anchor positions, at
an auxiliary anchor position, to make an analog of a non-A2- or
non-A2.1-HLA restricted epitope, in an anchor position of a peptide
that is not derived from an oncogene or oncofetal protein, and in
an anchor position of a peptide derived from a CT antigen.
[0268] In general, such analogs may be useful for immunotherapy
and/or prophylaxis of various diseases such as infectious,
cancerous or inflammatory, as single agents or in combination
therapies, due to their optimized interaction with MHC molecules
and T cell receptors, key to onset and regulation of immune
responses.
Analog Production
[0269] The analogs may be produced using any method known to one of
skill in the art, including manufacturing the peptides using a
method of peptide synthesis or expressing nucleic acids that code
for the desired peptide analogs. Thus, when the analogs include one
or more non-standard amino acids, it is more likely that they will
be produced by a method of peptide production. When the analogs
include only one or more substitutions with standard amino acids,
they may be expressed from an expression vector using any method
known to one of skill in the art. Alternatively, the peptides may
be expressed using a method of gene therapy.
Analog Testing
[0270] The usefulness and/or the activity of the analogs was
identified. In this way useful and/or improved analogs can be
identified. To be useful, an analog may not necessarily be found to
be improved in the identified assays. Thus, a useful peptide may
contain other properties such as being useful in a tolerized
patient or resistant to proteolysis. To be improved, a peptide can
be found to have a clear improvement in binding to the TCR, binding
to the MHC molecule, and an improved immune response or any other
biological activity. To be useful, the peptide may be found not to
be improved when using a murine test system, but because of the
differences in the human immune system, may be improved when tested
in a human. Alternatively, the usefulness may stem from a potential
to break tolerance in a tolerized human. Alternatively, the
usefulness may stem from the ability to use the peptide as a base
for further substitutions to identify improved analogs.
[0271] In order to evaluate usefulness, improved properties and to
compare the analogs in any way to the wild-type, one or more of the
following assays were conducted: peptide binding affinity for
HLA-A*0201; peptide-HLA-A*0201 complex stability assay;
cross-reactivity assay (recognition of peptide analogs by wild-type
peptide specific CTL or recognition of wild-type peptide by CTL
generated using peptide analogs); immunogenicity assays, such as an
IFN-.gamma. secretion assay, a cytotoxicity assay, and/or an
Elispot assay; antigenicity assays such as an in vitro tumor cell
lysis assay, an ex vivo tumor cell lysis, and an in vivo tumor cell
lysis; and proteolysis assays to identify increased resistance to
proteolysis. Details of exemplary assays are presented in the
Examples.
Uses of the Analogs
[0272] Useful methods for using the disclosed analogs in inducing,
entraining, maintaining, modulating and amplifying class I
MHC-restricted T cell responses, and particularly effector and
memory CTL responses to antigen, are described in U.S. patent
application Ser. Nos. 09/380,534 and 09/776,232 both entitled A
METHOD OF INDUCING A CTL RESPONSE; U.S. Provisional Application No.
60/479,393, filed on Jun. 17, 2003, entitled METHODS TO CONTROL MHC
CLASS I-RESTRICTED IMMUNE RESPONSE; and U.S. patent application
Ser. No. 10/871,707 (Pub. No. 2005 0079152) and Provisional U.S.
Patent Application No. 60/640,402 filed on Dec. 29, 2004, both
entitled METHODS TO ELICIT, ENHANCE AND SUSTAIN IMMUNE RESPONSES
AGAINST MHC CLASS I-RESTRICTED EPITOPES, FOR PROPHYLACTIC OR
THERAPEUTIC PURPOSE. The analogs can also be used in research to
obtain further optimized analogs. Numerous housekeeping epitopes
are provided in U.S. application Ser. Nos. 10/117,937, filed on
Apr. 4, 2002 (Pub. No. 20030220239 A1), and Ser. No. 10/657,022
(2004-0180354), and in PCT Application No. PCT/US2003/027706 (Pub.
No. WO04022709A2), filed on Sep. 5, 2003; and U.S. Provisional
Application Nos. 60/282,211, filed on Apr. 6, 2001; 60/337,017,
filed on Nov. 7, 2001; 60/363,210 filed on Mar. 7, 2002; and
60/409,123, filed on Sep. 5, 2002; each of which applications is
entitled EPITOPE SEQUENCES. The analogs can further be used in any
of the various modes described in those applications. Epitope
clusters, which may comprise or include the instant analogs, are
disclosed and more fully defined in U.S. patent application Ser.
No. 09/561,571, filed on Apr. 28, 2000, entitled EPITOPE CLUSTERS.
Methodology for using and delivering the instant analogs is
described in U.S. patent applications Ser. Nos. 09/380,534 and
09/776,232 (Pub. No. 20020007173 A1), and in PCT Application No.
PCTUS98/14289 (Pub. No. WO9902183A2) each entitled A METHOD OF
INDUCING A CTL RESPONSE. Beneficial epitope selection principles
for such immunotherapeutics are disclosed in U.S. patent
application Ser. No. 09/560,465, filed on Apr. 28, 2000, Ser. No.
10/026,066 (Pub. No. 20030215425 A1), filed on Dec. 7, 2001, and
Ser. No. 10/005,905 filed on Nov. 7, 2001, all entitled EPITOPE
SYNCHRONIZATION IN ANTIGEN PRESENTING CELLS; Ser. No. 09/561,074
entitled METHOD OF EPITOPE DISCOVERY; Ser. No. 09/561,571, filed
Apr. 28, 2000, entitled EPITOPE CLUSTERS; Ser. No. 10/094,699 (Pub.
No. 20030046714 A1), filed Mar. 7, 2002, entitled
ANTI-NEOVASCULATURE PREPARATIONS FOR CANCER; application Ser. Nos.
10/117,937 (Pub. No. 20030220239 A1) and PCTUS02/11101 (Pub. No.
WO02081646A2), both filed on Apr. 4, 2002, and both entitled
EPITOPE SEQUENCES; and application Ser. Nos. 10/657,022 and PCT
Application No. PCT/US2003/027706 (Pub. No. W004022709A2), both
filed on Sep. 5, 2003, and both entitled EPITOPE SEQUENCES. Aspects
of the overall design of vaccine plasmids are disclosed in U.S.
patent application Ser. Nos. 09/561,572, filed on Apr. 28, 2000,
entitled EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATED
ANTIGENS and Ser. No. 10/292,413 (Pub. No.20030228634 A1), filed on
Nov. 7, 2002, entitled EXPRESSION VECTORS ENCODING EPITOPES OF
TARGET-ASSOCIATED ANTIGENS AND METHODS FOR THEIR DESIGN; Ser. No.
10/225,568 (Pub No. 2003-0138808) filed on Aug. 20, 2002, PCT
Application No. PCT/US2003/026231 (Pub. No. WO 2004/018666) filed
on Aug. 19, 2003, both entitled EXPRESSION VECTORS ENCODING
EPITOPES OF TARGET-ASSOCIATED ANTIGENS; and U.S. Pat. No.
6,709,844, entitled AVOIDANCE OF UNDESIRABLE REPLICATION
INTERMEDIATES IN PLASMID PROPAGATION. Specific antigenic
combinations of particular benefit in directing an immune response
against particular cancers are disclosed in Provisional U.S. patent
Application No. 60/479,554 filed on Jun. 17, 2003 and U.S. patent
application Ser. No. 10/871,708 filed on Jun. 17, 2004 and PCT
Patent Application No. PCT/US2004/019571 (Pub. No. WO 2004/112825),
all entitled COMBINATIONS OF TUMOR-ASSOCIATED ANTIGENS IN VACCINES
FOR VARIOUS TYPES OF CANCERS. Antigens associated with tumor
neovasculature (e.g., PSMA, VEGFR2, Tie-2) are also useful in
connection with cancerous diseases, as is disclosed in U.S. patent
application Ser. No. 10/094,699 (Pub. No. 20030046714 A1), filed
Mar. 7, 2002, entitled ANTI-NEOVASCULATURE PREPARATIONS FOR CANCER.
Methods to trigger maintain and manipulate immune responses by
targeted administration of biological response modifiers are
disclosed U.S. Provisional Application No. 60/640,727, filed on
Dec. 29, 2004. Methods to bypass CD4+ cells in the induction of an
immune response are disclosed in U.S. Provisional Application No.
60/640,821, filed on Dec. 29, 2004. Exemplary diseases, organisms
and antigens and epitopes associated with target organisms, cells
and diseases are described in U.S. application Ser. No. 09/776,232
(Pub. No. 20020007173), filed Feb. 2, 2001 and entitled METHOD OF
INDUCING A CTL RESPONSE. Exemplary methodology is found in U.S.
Provisional Application No. 60/580,969, filed on Jun. 17, 2004, and
U.S. patent application Ser. No. ______ (Attorney Docket:
MANNK.050A), filed on Jun. 17, 2005, both entitled COMBINATIONS OF
TUMOR-ASSOCIATED ANTIGENS IN DIAGNOTISTICS FOR VARIOUS TYPES OF
CANCERS. Methodology and compositions are also disclosed in U.S.
Provisional Application No. 60/640,598, filed on Dec. 29, 2004,
entitled COMBINATIONS OF TUMOR-ASSOCAITED ANTIGENS IN COMPOSITIONS
FOR VARIOUS TYPES OF CANCER. The integration of diagnostic
techniques to assess and monitor immune responsiveness with methods
of immunization including utilizing the instant analogs is
discussed more fully in Provisional U.S. Patent Application No.
60/580,964 filed on Jun. 17, 2004 and U.S. patent application Ser.
No. ______ (Atty. Docket No. MANNK.040A), filed on Jun. 17, 2005,
both entitled IMPROVED EFFICACY OF ACTIVE IMMUNOTHERAPY BY
INTEGRATING DIAGNOSTIC WITH THERAPEUTIC METHODS. The immunogenic
polypeptide encoding vectors are disclosed in U.S. patent
application Ser. No. 10/292,413 (Pub. No. 20030228634 A1) filed on
Nov. 7, 2002 entitled EXPRESSION VECTORS ENCODING EPITOPES OF
TARGET-ASSOCIATED ANTIGENS AND METHODS FOR THEIR DESIGN, and in
U.S. Provisional Application No. ______, filed on Jun. 17, 2005,
entitled (Attorney Docket No. MANNK.053PR), entitled METHODS AND
COMPOSITIONS TO ELICIT MULTIVALENT IMMUNE RESPONSES AGAINST
DOMINANT AND SUBDOMINANT EPITOPES, EXPRESSED ON CANCER CELLS AND
TUMOR STROMA. Additional useful disclosure, includes methods and
compositions of matter is found in U.S. Provisional Application No.
______, filed on Jun. 17, 2005 (Attorney Docket No. MANNK.054PR),
entitled MULTIVALENT ENTRAIN-AND-AMPLIFY IMMUNOTHERAPEUTICS FOR
CARCINOMA. Further methodology, compositions, peptides, and peptide
analogs are disclosed in U.S. Provisional Application Nos.
60/581,001 and 60/580,962, both filed on Jun. 17, 2004, and
respectively entitled "SSX-2 PEPTIDE ANALOGS" and "NY-ESO PEPTIDE
ANALOGS." All of these applications mentioned in this paragraph are
hereby incorporated by reference in their entirety for all that
they teach. Additional analogs, peptides and methods are disclosed
in U.S. patent application Ser. No. ______ (Attorney Docket:
MANNK.038A) entitled SSX-2 PEPTIDE ANALOGS; and Prov. Application
No. ______ (MANNK.052PR), entitled EPITOPE ANALOGS; all filed on
Jun. 17, 2005. As an example, without being limited thereto the
references are incorporated by reference for what they teach about
class I MHC-restricted epitopes, analogs, the design of analogs,
uses of epitopes and analogs, methods of using and making epitopes,
and the design and use of nucleic acid vectors for their
expression.
Antigens
[0273] There are many antigens, epitopes of which can be recognized
by T cells in an MHC-restricted manner, for which manipulation of
an immune response directed against them has therapeutic or
prophylactic potential. The principles for making analogs of
MHC-binding peptides described herein are generally applicable to
any of these antigens and their epitopes. A particular focus of the
present disclosure is epitopes from the tumor-associated antigens
(TuAA) SSX-2, NY-ESO-1, PRAME, PSMA, tyrosinase, and Melan-A.
[0274] SSX-2, also know as Hom-Mel-40, is a member of a family of
highly conserved cancer-testis antigens (Gure, A. O. et al. Int. J.
Cancer 72:965-971, 1997, which is hereby incorporated by reference
in its entirety). Its identification as the TuAA antigen is taught
in U.S. Pat. No. 6,025,191 entitled ISOLATED NUCLEIC ACID MOLECULES
THAT ENCODE A MELANOMA SPECIFIC ANTIGEN AND USES THEREOF, which is
hereby incorporated by reference in its entirety. Cancer-testis
antigens are found in a variety of tumors, but are generally absent
from normal adult tissues except testis. SSX-2 is expressed in many
different types of tumors, including synoviai sarcomas, melanoma,
head and neck cancers, breast, colon and ovarian cancers. In
addition to its widespread expression in a variety of cancers, it
is also immunogenic in patients with late stage disease. Further,
there is evidence of spontaneous humoral and cellular immune
responses towards this antigen in metastatic tumor patients (Ayyoub
M, et al., Cancer Res. 63(17): 5601-6, 2003; Ayyoub M, et al. J
Immunol. 168(4): 1717-22, 2002), which is incorporated herein by
reference in its entirety. Two HLA-A2 restricted T cell epitopes
have been identified recently using reverse T-cell immunology,
namely SSX-2.sub.41-49 (Ayyoub M, et al. J Immunol. 168(4):
1717-22, 2002; U.S. Pat. No.6,548,064, entitled ISOLATED PEPTIDES
CONSISTING OF AMINO ACID SEQUENCES FOUND IN SSX OR NY-ESO-1
MOLECULES, THAT BIND TO HLA MOLECULE; U.S. patent application Ser.
No. 10/117,937, entitled EPITOPE SEQUENCES) and SSX-2.sub.103-111
(Wagner C, et al. Cancer Immunity 3:18, 2003), each of which is
incorporated herein by reference in its entirety. The C-termini of
both epitopes can be efficiently generated by in vitro proteasome
digestion. Isolated HLA-A*0201/SSX-2.sub.41-49 multimer.sup.+
CD8.sup.+ T cells from tumor-infiltrated lymph nodes of SSX-2
positive patients exhibited high functional avidity and can
effectively recognize SSX-2 positive tumors; however, the
spontaneously occurring immunological responses were not sufficient
for stopping tumor growth, possibly because these immune response
did not develop until fairly late in the disease progression, and
the activated T cells were not numerous enough. U.S. Pat. No.
6,548,064 (which is incorporated herein by reference in its
entirety) further describes substituting a T or A residue at both
the P2 and P.OMEGA. position of an SSX-2 epitope.
[0275] NY-ESO-1 is a cancer-testis antigen found in a wide variety
of tumors and is also known as CTAG-1 (Cancer-Testis Antigen-1) and
CAG-3 (Cancer Antigen-3). NY-ESO-1 as a tumor-associated antigen
(TuAA) is disclosed in U.S. Pat. No. 5,804,381 entitled ISOLATED
NUCLEIC ACID MOLECULE ENCODING AN ESOPHAGEAL CANCER ASSOCIATED
ANTIGEN, THE ANTIGEN ITSELF, AND USES THEREOF that is hereby
incorporated by reference in its entirety. A paralogous locus
encoding antigens with extensive sequence identity, LAGE-1a/s and
LAGE-1b/L, have been disclosed in publicly available assemblies of
the human genome, and have been concluded to arise through
alternate splicing. Additionally, CT-2 (or CTAG-2, Cancer-Testis
Antigen-2) appears to be either an allele, a mutant, or a
sequencing discrepancy of LAGE-1b/L. Due to the extensive sequence
identity, many epitopes from NY-ESO-1 can also induce immunity to
tumors expressing these other antigens. The proteins are virtually
identical through amino acid 70. From 71-134 the longest run of
identities between NY-ESO-1 and LAGE is 6 residues, but potentially
cross-reactive sequences are present. And from 135-180 NY-ESO and
LAGE-1a/s are identical except for a single residue, but LAGE-1b/L
is unrelated due to the alternate splice. The CAMEL and LAGE-2
antigens appear to derive from the LAGE-1 mRNA, but from alternate
reading frames, thus giving rise to unrelated protein sequences.
More recently, GenBank Accession AF277315.5 (which is incorporated
herein by reference in its entirety), Homo sapiens chromosome X
clone RP5-865E18, RP5-1087L19, complete sequence, reports three
independent loci in this region that are labeled as LAGE1
(corresponding to CTAG-2 in the genome assemblies), plus LAGE2-A
and LAGE2-B (both corresponding to CTAG-1 in the genome
assemblies).
[0276] NY-ESO-1.sub.157-165 is identified as an HLA-A2 restricted
epitope in U.S. Pat. No. 6,274,145 entitled ISOLATED NUCLEIC ACID
MOLECULE ENCODING CANCER ASSOCIATED ANTIGEN, THE ANTIGEN ITSELF,
AND USES THEREOF, and U.S. patent application Ser. No. 10/117,937
(Pub. No. 20030220239) entitled EPITOPE SEQUENCES reports that this
C-terminus is generated by the housekeeping proteasome in an in
vitro assay. Analogs substituting A,V,L,I,P,F,M,W, or G at
P.OMEGA., alone or in combination with A at another position, are
disclosed in U.S. Pat. Nos. 6,417,165 and 6,605,711, both entitled
NY-ESO-1-PEPTIDE DERIVATIVES, AND USES THEREOF. The references
described in this paragraph are incorporated herein by reference in
their entirety.
[0277] PRAME, also know as MAPE, DAGE, and OIP4, was originally
observed as a melanoma antigen. Subsequently, it has been
recognized as a CT antigen, but unlike many CT antigens (e.g.,
MAGE, GAGE, and BAGE) it is expressed in acute myeloid leukemias.
PRAME is a member of the MAPE family which consists largely of
hypothetical proteins with which it shares limited sequence
similarity. The usefulness of PRAME as a TuAA is taught in U.S.
Pat. No. 5,830,753 entitled ISOLATED NUCLEIC ACID MOLECULES CODING
FOR TUMOR REJECTION ANTIGEN PRECURSOR DAGE AND USES THEREOF, which
is hereby incorporated by reference in its entirety. U.S. patent
application Ser. No. 10/181,499, entitled METHODS FOR SELECTING AND
PRODUCING T CELL PEPTIDE EPITOPES AND VACCINES INCORPORATING SAID
SELECTED EPITOPES (which is incorporated herein by reference in its
entirety) identifies a variety of potential epitopes, including
PRAME.sub.425-433, using in vitro digestion with
immunoproteasome.
[0278] PSMA (prostate-specific membranes antigen), a TuAA described
in U.S. Pat. No. 5,538,866 entitled "PROSTATE-SPECIFIC MEMBRANES
ANTIGEN" which is hereby incorporated by reference in its entirety,
is expressed by normal prostate epithelium and, at a higher level,
in prostatic cancer. It has also been found in the neovasculature
of non-prostatic tumors. PSMA can thus form the basis for vaccines
directed to both prostate cancer and to the neovasculature of other
tumors. This later concept is more fully described in U.S. patent
Publication No. 20030046714; PCT Publication No. WO 02/069907; and
a provisional U.S. Patent application No. 60/274,063 entitled
ANTI-NEOVASCULAR VACCINES FOR CANCER, filed Mar. 7, 2001, and U.S.
application Ser. No. 10/094,699, filed on Mar. 7, 2002, entitled
"ANTI-NEOVASCULAR PREPARATIONS FOR CANCER," each of which are
hereby incorporated by reference in its entirety. The teachings and
embodiments disclosed in said publications and applications are
contemplated as supporting principals and embodiments related to
and useful in connection with the present invention. Briefly, as
tumors grow they recruit ingrowth of new blood vessels. This is
understood to be necessary to sustain growth as the centers of
unvascularized tumors are generally necrotic and angiogenesis
inhibitors have been reported to cause tumor regression. Such new
blood vessels, or neovasculature, express antigens not found in
established vessels, and thus can be specifically targeted. By
inducing CTL against neovascular antigens the vessels can be
disrupted, interrupting the flow of nutrients to (and removal of
wastes from) tumors, leading to regression.
[0279] Alternate splicing of the PSMA mRNA also leads to a protein
with an apparent start at Met.sub.58, thereby deleting the putative
membrane anchor region of PSMA as described in U.S. Pat. No.
5,935,818 entitled "ISOLATED NUCLEIC ACID MOLECULE ENCODING
ALTERNATIVELY SPLICED PROSTATE-SPECIFIC MEMBRANES ANTIGEN AND USES
THEREOF" which is hereby incorporated by reference in its entirety.
A protein termed PSMA-like protein, Genbank accession number
AF261715, which is hereby incorporated by reference in its
entirety, is nearly identical to amino acids 309-750 of PSMA and
has a different expression profile. Thus the more preferred
epitopes are those with an N-terminus located from amino acid 58 to
308. PSMA.sub.288-297 was identified as possessing an HLA-A2
binding motif in WO 01/62776, entitled HLA BINDING PEPTIDES AND
THEIR USES, which is hereby incorporated by reference in its
entirety. Its production in vitro by digestion with housekeeping
proteasome and actual binding to HLA-A2 was disclosed in U.S.
patent application Publication No. 2003-0220239 entitled EPITOPE
SEQUENCES.
[0280] Tyrosinase is a melanin biosynthetic enzyme that is
considered one of the most specific markers of melanocytic
differentiation. Tyrosinase is expressed in few cell types,
primarily in melanocytes, and high levels are often found in
melanomas. The usefulness of tyrosinase as a TuAA is taught in U.S.
Pat. No. 5,747,271 entitled "METHOD FOR IDENTIFYING INDIVIDUALS
SUFFERING FROM A CELLULAR ABNORMALITY SOME OF WHOSE ABNORMAL CELLS
PRESENT COMPLEXES OF HLA-A2/TYROSINASE DERIVED PEPTIDES, AND
METHODS FOR TREATING SAID INDIVIDUALS" which is hereby incorporated
by reference in its entirety.
[0281] Melan-A, also called MART-1 (Melanoma Antigen Recognized by
T cells), is another melanin biosynthetic protein expressed at high
levels in melanomas. The usefulness of Melan-A/MART-1 as a TuAA is
taught in U.S. Pat. Nos. 5,874,560 and 5,994,523 both entitiled
MELANOMA ANTIGENS AND THEIR USE IN DIAGNOSTIC AND THERAPEUTIC
METHODS, as well as U.S. Pat. No. 5,620,886, entitled ISOLATED
NUCLEIC ACID SEQUENCE CODING FOR A TUMOR REJECTION ANTIGEN
PRECURSOR PROCESSED TO AT LEAST ONE TUMOR REJECTION ANTIGEN
PRESENTED BY HLA-A2, all of which are hereby incorporated by
reference in their entirety. The immunodominant HLA-A2 restricted
epitope from this TuAA is Melan-A.sub.26-35. It has been shown to
be produced by the housekeeping proteasome (Morel, S. et al.
Immunity 12:107-117, 2000), which is hereby incorporated by
reference in its entirety. Various analogs incorporating standard
amino acids, including an improved analog substituting L at P2, are
disclosed in U.S. Pat. No. 6,025,470, entitled ISOLATED NONA- AND
DECAPEPTIDES WHICH BIND TO HLA MOLECULES, AND THE USE THEREOF,
which is hereby incorporated by reference in its entirety. The use
of analogs incorporating non-standard amino acids with a primary
goal of improving biochemical stability is reported by Blanchet,
J.-S. et al., J. Immunol. 167:5852-5861, 2001, which is hereby
incorporated by reference in its entirety.
SSX-2 41-49 Analogs
[0282] As noted above, the natural immune response to SSX-2 in
cancer patients, including the response to SSX-2.sub.41-49, may not
be effective in controlling cancer. Additionally, wild-type
SSX-2.sub.41-49 is only a moderately immunogenic peptide that can
further limit its clinical potential. Stronger SSX-2 specific
immune responses induced by the use of superagonist analogs results
in clinical benefits for patients with SSX-2 positive tumor.
[0283] Thus, in one embodiment, the analogs can be used in
compositions to stimulate the immune response of a subject to mount
an immune response against a target cell displaying the target
antigen. The embodiment is contemplated to have utility in the
treatment and prevention of neoplastic and viral disease.
[0284] Since the wild-type SSX-2.sub.41-49 is only a moderately
immunogenic peptide that may prevent it from eliminating tumors
effectively in vivo, a method was used to de novo design
SSX-2.sub.41-49 variants that were more potent or had a variety of
improved properties. By using a more immunogenic SSX-2 analog
peptide, it was possible to stimulate a stronger immune response
and/or to amplify the naturally occurring immune response to
achieve a better chance of clinical response. Thus, the binding
properties (affinity and HLA-A*0201/peptide complexes stability),
immunogenicity, antigenicity and cross-reactivity to the wild-type
epitope were analyzed for each of the analogs to identify an
improved property. In some embodiments, by improved property it is
meant generally, that the analog can be better used for some
purpose than the wild-type. Thus, the analog need not exhibit
improved binding, stability, or activity to be improved and may
even show a reduced ability to mediate certain parts of the
process, but still be improved for use in another way. For example,
analogs that retain some activity, but not all activity may be
better in human systems that are tolerized to the wild-type
antigen.
[0285] Previously, modifications of natural tumor-associated
peptide epitopes by incorporating favorable anchor residues have
generated analogs with improved binding profiles with HLA molecules
and enhanced immunogenicity. One of the most successful examples is
the A27L peptide analog of Melan-A 26-35 epitope. Valmori et al.,
"Enhanced generation of specific tumor-reactive CTL in vitro by
selected Melan-A/MART-1 immunodominant peptide analogs," J Immunol.
1998, 160(4): 1750-8; which is hereby incorporated by reference in
its entirety. The original epitope failed to form a stable complex
with HLA-A2 molecules since it lacks optimum anchor amino acid
residue at position 2. The modified A27L Melan A 26-35 peptide
analog has demonstrated unequivocally increased binding profiles
with HLA-A2 molecules and greater immunogenicity than its wild-type
counterpart. Immunizing patients with this analog can generate
strong T cell immune responses that were able to recognize the
wild-type epitope presented at the cell surfaces. Similar
modifications have been obtained successfully with many other
tumor-associated epitopes such as GP100 209-217 (Parkhurst et al.,
"Improved induction of melanoma-reactive CTL with peptides from the
melanoma antigen gp100 modified at HLA-A*0201-binding residues," J
Immunol. 1996, 157(6): 2539-48; which is hereby incorporated by
reference in its entirety), Her-2 369-377 (Vertuani et al.,
"Improved immunogenicity of an immunodominant epitope of the
HER-2/neu protooncogene by alterations of MHC contact residues," J
Immunol. 2004, 172(6): 3501-8; which is hereby incorporated by
reference in its entirety).
[0286] Up to this point no SSX-2.sub.41-49 analogs have been
designed and studied even though they hold great promise for
development of SSX-2-based vaccines to treat a variety of cancers,
particularly SSX-2 positive cancers and/or tumors. Thus, methods
are disclosed herein that can be used for the identification and
production of analogs to a Synovial sarcoma X breakpoint 2 (SSX-2)
wild-type sequence. Using the methods, a panel of 95 novel
SSX-241-49 analogs based on the wild-type sequence from amino acids
241-249 were identified with a variety of improved properties. The
improved properties include, but are not limited to, binding to
class I MHC and T cell receptor (TCR) molecules, and biological
responses such as IFN-.gamma. secretion, cytotoxicity, and tumor
cell lysis. Peptides with improved potency that retained
cross-reactivity with the wild-type epitope were identified. Among
these analogs, some have been demonstrated to be the superagonist
variants of the wild-type SSX-241-49 peptide, some of which analogs
have been shown to have much higher affinity with HLA-A*0201
molecule, and the peptide-HLA complex possessed extended stability.
When the mice were immunized with these analogs, they were able to
induce enhanced CTL immune responses in HHD transgenic mice. The
resulting CTLs could effectively lyse A2+ and SSX-2+ tumor cell
lines both in vivo and in vitro, which indicated that the CTLs
generated using the analogs were able to recognize the wild-type
SSX-241-49 epitope that naturally presented at the cell surfaces.
In comparison with the wild-type SSX-241-49 epitope, the analogs
are better candidates for the development of cancer vaccines.
[0287] Embodiments include families of one or more peptides of 9 or
10 amino acids in length related by sequence to amino acids 41-49
of the human cancer testis (CT) antigen SSX-2 (SSX-2.sub.41-49).
The individual peptide embodiments have one to several defined
amino acid substitutions in the wild-type sequence. The substituted
amino acids are, variously, other members of the standard set of
amino acids commonly genetically encoded, derivatives thereof,
their D-stereoisomers, or other non-standard L-amino acids. These
analogs are useful for investigating the interaction of the
wild-type epitope with class I MHC and TCR molecules and other
components of the immune response, and for designing additional
analogs with further optimized immunologic properties. Some
embodiments of the analogs have at least similar immunologic
properties to the wild-type epitope in the HLA-transgenic mouse
model in which they have been tested. Such peptides can be useful
in humans, as SSX-2 is a self-antigen to which a degree of
tolerance may be expected, and the amino acid differences of the
analogs can help to stimulate populations of T cells that have
avoided negative selection but are cross-reactive with the
wild-type epitope. Various peptide embodiments can have one or more
improved immunologic properties in that they possess greater
affinity for MHC or greater stability of binding to MHC, elicit
greater cytokine production or require lower peptide concentrations
to elicit similar cytokine production from T cells that recognize
the wild-type epitope, are more immunogenic, can induce or amplify
a cross-reactive cytolytic response to the wild-type epitope, or
can break tolerance.
[0288] In one embodiment, the analogs can have at least one
substitution at a residue selected from the group consisting of,
P1, P2, P4, P6, P8, P9 and P10. In a further embodiment, the
analogs can have at least two substitutions at residues selected
from the group consisting of: P1, P2, P4, P6, P8, P9 and P10. In a
further embodiment, the analogs can have at least three
substitutions at residues selected from the group consisting of:
P1, P2, P4, P6, P8, P9 and P10. In a further embodiment, the
analogs can have substitutions at positions P2 and P9. In a further
embodiment, the peptides can have substitutions at residues P1, P2,
and P9. In a further embodiment, the peptide analogs can have
substitutions at residues P1, P2, and P4. In a further embodiment,
the peptide analogs can have substitutions at residues P1, P2, and
P6. In a further embodiment, the peptide analogs can have
substitutions at residues P1, P2, and P8. In one embodiment, two
substitutions can produce improved properties. In a further
embodiment, one substitution can produce improved properties. In a
further embodiment, three substitutions can produce improved
properties. In a further embodiment, the one or more substitutions
can produce improved properties but are still recognized by a TCR
that recognizes the wild-type sequence (still cross-react with the
wild-type sequence).
[0289] One embodiment relates to epitope arrays and other
polypeptides comprising the epitope analog sequences that can be
processed to liberate the analog. Further embodiments relate to
nucleic acids, particularly DNA plasmids, encoding such
polypeptides, or simply an analog, and their expression therefrom.
The analogs, the polypeptides comprising them, and the encoding
nucleic acids can all be components of immunogenic compositions,
particularly compositions suitable for intralymphatic delivery,
that constitute further embodiments.
Analog Design
[0290] Embodiments relate to the SSX-2.sub.41-49 peptide which
contain substitutions of the sequence KASEKIFYV (See FIG. 1). In a
further embodiment, the analog can be generally an analog of the
SSX-2.sub.41-50 decamer peptide with the sequence KASEKIFYVY. The
residues or amino acids that make up the peptide are referred to
herein as P1 -P9 or P1-P 10 to designate the position within the
peptide as numbered from the N-- to the C-terminus, P1
corresponding to the N-terminal Lysine and P9 corresponding to the
C-terminal Valine in the nonamer. Alternatively, the residues may
be referred to by the primary activity of the molecule that they
are involved in. For example, residue P2 is described as the
N-terminal primary anchor molecule, while P9 (or P10 in the
decamer) is described as the primary C-terminal anchor. Residues
P4, P6 and P8 are primarily involved in TCR interactions.
Substitutions can use any amino acids, including standard and
non-standard amino acids, known to one of skill in the art. A
number of exemplary amino acids are disclosed herein, however, the
substitutions disclosed herein are not meant to be a list that
includes all imagined substitutions, but are exemplary of the
substitutions that are possible. One of skill in the art may find a
number of other non-standard amino acids in catalogs and references
that may be purchased or chemically produced for use with the
analogs herein.
[0291] A number of possible analogs were produced by modification
of peptide anchor residues to achieve better HLA binding profiles
and higher immune responses, including at the N-terminal primary
anchor (P2 position), at the N-terminal secondary anchor (PI
position), at the N-terminal primary and secondary anchor (P1 and
P2 positions), and at the N-terminal primary/secondary anchor (P1
and P2 positions) and C-terminal primary anchor (P9 position).
Further, peptides with modifications at the anchor residues and TCR
contact residues were produced to circumvent T cell tolerance for
self-antigens, these modifications included modifications at the
N-terminal primary/secondary anchor (P1 and P2 positions) and
secondary TCR recognition sites (P4, P6 and/or P8 positions),
modifications at the N-terminal primary/secondary anchors (P1 and
P2 position), and modifications at the C-terminal primary anchor
(P9) and at secondary TCR recognition sites (P4, P6 and/or P8
positions). Further, decamer analogs were produced.
[0292] The choice of which residues would best produce analogs with
improved properties involved analysis of studies of MHC peptide
interactions, studies of TCR peptide interactions and previous
analogs that were known in the art. Some residues are primarily
involved in a specific interaction and some are secondarily or even
tertiarily involved. Thus, the knowledge of how the residues are
involved in the binding to these molecules was involved in the
analysis. Further some of the wild-type residues are preferred,
meaning that they work well for the intended interaction, while
others are non-preferred, meaning that they work poorly for the
interaction. Thus, in one embodiment, the non-preferred residues
can be substituted. For example, the valine at the C-terminus is
generally a preferred anchor residue because it produces a strong
interaction with the HLA molecule and, thus, it was less preferred
to substitute this residue. However, modifications of wild-type
tumor-associated peptide epitopes by incorporating favorable anchor
residues have generated analogs with improved binding profiles with
HLA molecules and enhanced immunogenicity. One of the most
successful examples is the A27L peptide analog of Melan-A 26-35
epitope (Valmori D, et al. J Immunol. 160(4): 1750-8, 1998; which
is hereby incorporated by reference in its entirety). The original
epitope failed to form a stable complex with HLA-A2 molecules since
it lacked an optimal anchor residue at position 2. In contrast the
modified Melan A.sub.26-35 A27L peptide analog demonstrated
unequivocally increased binding profiles with HLA-A2 molecules and
greater immunogenicity than its wild-type counterpart. Immunizing
patients with this analog generated strong T cell immune responses
that were able to recognize the wild-type epitope presented at the
cell surfaces. Similar modifications were obtained successfully
with many other tumor-associated epitopes such as GP100 209-217
(Parkhurst M R, et al. J Immunol. 157(6): 2539-48, 1996; which is
hereby incorporated by reference in its entirety), Her-2 369-377
(Vertuani S, et al. J Immunol. 172(6): 3501-8, 2004; which is
hereby incorporated by reference in its entirety).
[0293] The choice of how many residues to substitute involves a
desire to substitute better residues while still retaining enough
of the qualities of the epitope that it will still be recognized by
T cells which recognize the wild-type epitope. Thus, in one
embodiment, one or two substitutions can be made to the wild-type
peptide. In a further embodiment, more than two substitutions can
be made to the wild-type peptide, while still retaining
cross-reactivity with the wild-type peptide.
[0294] Thus, generally, the part of the peptide that is involved in
TCR recognition is desirably substituted to produce improved
immunogenicity while still cross-reacting with the wild-type
epitope. For example, a peptide that shows increased immunogenicity
is preferred. Because the P2 or second amino acid at the N-terminal
end is believed to be primarily involved in the process of
producing improved immunogenicity, primarily through improved
binding properties, it is a preferred substitution site and a
number of modifications were made in the exemplary analogs to
identify desired substitutions. Similar considerations apply to the
carboxy-terminal position, PS2 which also can be important for MHC
binding.
[0295] Thus, in one embodiment, the analog can include a
substitution at the P2 residue that substitutes a more hydrophobic
residue for the wild-type alanine. In a further embodiment, the
hydrophobic residue also can possess a more bulky side chain. In a
further embodiment, the residue at P1 can be substituted with a
more hydrophobic residue. In a further embodiment, residues P1 and
P2 both can be substituted with more hydrophobic residues. In
further embodiments at least one residue at P1, P2, and P9 can be
substituted. In a further embodiment, at least two residues at P1,
P2 and P9 can be substituted. In a further embodiment at least two
residues at P1, P2, P9 P4 and P6 can be substituted including one
or more residues involved in TCR binding.
[0296] Further, substitutions of those residues only secondarily
involved in binding to TCR or the MHC molecule can be advantageous.
For example, substitution of secondary TCR binding amino acids can
generate analogs that still bind and produce a response and do not
interfere with the binding to the MEC molecule, but preferably
overcome the tolerance issues of self-antigens. This is useful
because a patient who has cancer may be partially tolerized to the
antigen. Thus, in order to overcome that tolerance, an analog that
retains some activity can be preferable to an analog with more
improved immunogenicity, because it will be less likely to be
recognized as "self" by the immune system.
[0297] 1. N-Terminal Proximal Primary Anchor Modification(P2)
[0298] The N-terminal primary anchor is the 2.sup.nd N-terminal
amino acid of the peptide and is the N-terminal proximal primary
anchor. It is primarily involved in the interaction with the MHC
molecule and substitutions can result in improved binding and
stability. However, it may be secondarily involved in TCR
interactions also. Thus, substitutions at this site can result in a
peptide with improved interaction with MHC molecules as well as
improved interaction with the TCR.
[0299] The alanine found at this position in the wild-type sequence
is generally believed to be non-preferred for the interaction with
the MHC molecule. Thus, preferred embodiments of the analogs have a
substitution at this position. In one embodiment, the original Ala
42 found in the wild-type sequence can be substituted with a more
hydrophobic amino acid. Any more hydrophobic amino acid may be used
including any that is available or known to one of skill in the
art, including standard amino acids and non-standard amino acids.
In a further embodiment, the original Ala 42 is substituted with a
more hydrophobic amino acid also possessing a bulky side chain.
Examples of more hydrophobic amino acids includes, but are not
limited to: Leu, Val, Ile, Met, .alpha.-aminobutyric acid,
Norleucine and Norvaline. TABLE-US-00001 TABLE 1 N-TERMINAL
PROXIMAL PRIMARY ANCHOR MODIFICATION Cross-reactivity Predictive
Relative and fct avidity Scores Half-maximal affinity Stability
(native to Category Peptide name Sequence (R/NIH) Binding (mM)
(1/RA) (T1/2) (Hrs) analogs)* Native SSX2 41-49 KASEKIFYV 22/101711
14.64 1.0 11 1 peptide N-terminal SSX2 41-49 (A42L) KLSEKIFYV
28/73228 8.89 1.6 19 0.03 Primary Anchor SSX2 41-49 (A42V)
KVSEKIFYV 22/6407 5.2 2.8 20 0.03 SSX2 41-49 (A42I) KISEKIFYV
26/10068 8.8 1.7 22.5 3 SSX2 41-49 (A42M) KMSEKIFYV 26/52887 8.8
1.7 22.5 0.1 SSX2 41-49 (A42(D-Ala)) K(D-Ala)SEKIFYV NA N/B N/B N/B
10 SSX2 41-49 (A42(D-Leu)) K(D-Leu)SEKIFYV NA N/B N/B N/B N/T SSX2
41-49 (A42(D-Val)) K(D-Val)SEKIFYV NA N/B N/B N/B 3 SSX2 41-49
(A42(Nal-1)) KNal-1SEKIFYV NA N/B N/B N/B >10 SSX2 41-49
(A42(Nal-2)) KNal-2SEKIFYV NA 13.9 1.1 N/A 3 SSX2 41-49 (A42(Abu))
KAbuSEKIFYV NA 7.56 1.9 N/A 0.3 SSX2 41-49 (A42(Nle)) KNleSEKIFYV
NA 5.82 2.5 24 0.1 SSX2 41-49 (A42(Nva)) KNvaSEKIFYV NA 11.4 1.3
N/A 0.1 SSX2 41-49 (A42(Aib)) KAibSEKIFYV NA 18.4 0.8 N/A 3
[0300] 2. N-Terminal Secondary Anchor Modification (P1)
[0301] The N-terminal secondary anchor is the first amino acid at
the N-terminus. This residue is Lys 41 and is defined as a
secondary anchor residue in interacting with the HLA-A*0201
molecule. However, it is also engaged in the interaction with the T
cell receptors to a certain degree. Therefore, modifications of
this position can generate some heteroclitic analogs that are more
immunogenic and more suitable for the development of tumor
vaccines. Although the lysine at this position is generally
considered to be favored, substitutions can result in highly
improved properties.
[0302] Thus, in one embodiment, the original Lys 43 found in the
wild-type sequence can be substituted with a more hydrophobic amino
acid. Any more hydrophobic amino acid can be used, including any
that is available or known to one of skill in the art, including
standard amino acids and non-standard amino acids. In a further
embodiment, the Lys 43 can be substituted with an aromatic amino
acid. Examples of more hydrophobic amino acids include, but are not
limited to: Phe, Tyr, Trp, and D-Lys. TABLE-US-00002 TABLE 2
N-TERMINAL SECONDARY ANCHOR MODIFICATIONS Cross- reactivity Half-
and Predictive maximal Relative Stability fct avidity Scores
Binding affinity (T1/2) (native to Category Peptide name Sequence
(R/NIH) (mM) (1/RA) (Hrs) analogs)* Native SSX2 41-49 KASEKIFYV
22/1017 14.64 1.0 11 1 N-terminal SSX2 41-49 (K41F) FASEKIFYV
23/1336 9.55 1.5 >24 0.3 Secondary Anchor SSX2 41-49 (K41W)
WASEKIFYV 22/1336 27.07 0.5 N/A >10 SSX2 41-49 (K41Y) YASEKIFYV
21/1336 8.74 1.7 >24 3 SSX2 41-49(K41(D-Lys)) (D-Lys)ASEKIFYV NA
N/B N/B N/B >10 SSX2 41-49 (K41(Phg)) PhgASEKIFYV NA 5.83 2.5
>24 0.1 SSX2 41-49 (K41(Cha)) ChaASEKIFYV NA N/B N/B N/B >10
SSX2 41-49 (K41(Phe-4F)) Phe(4-F)ASEKIFYV NA 6.72 2.2 >24 3 SSX2
41-49 (K41(Phe-4NO2)) Phe(4-NO2)ASEKIFYV NA 12.8 1.1 N/A 3 SSX2
41-49 (K41(O-methyl O-methyl-TyrASEKIFYV NA 19.5 0.8 20 3 Tyr))
SSX2 41-49 (K41(b-(3- b-(3- NA 24.1 0.6 N/A 10 benzothienyl)Ala))
benzothienyl)AlaASEKIFYV
[0303] 3. N-Terminal Primary and Secondary Modifications (P2 and
P1)
[0304] In one embodiment, both primary and secondary anchor
residues were substituted to result in improved binding affinity to
the HLA molecule. In a further embodiment, the double substitution
produced improved stability of binding to the HLA molecule. In
further embodiments, the binding and/or stability was not improved
and may have even been reduced, but other properties of the
molecule were improved, such as activity or recognition by a
tolerized individual. TABLE-US-00003 TABLE 3 N-TERMINAL PRIMARY AND
SECONDARY ANCHOR MODIFICATION Half- Cross-reactivity maximal
Relative and fct avidity Predictive Binding affinity Stability
(native to Catergory Peptide name Sequence Scores (R/NIH) (mM)
(1/RA) (T1/2) (Hrs) analogs)* Native SSX2 41-49 KASEKIFYV 22/1017
14.64 1.0 11 1 N-terminal SSX2 41-49 (K41Y, A42L) YLSEKIFYV
29/96243 11.8 1.2 >24 N/T Primary/Secondary Anchor SSX2 41-49
(K41Y, A42V) YVSEKIFYV 23/8421 14.6 1.0 >24 0.1 SSX2 41-49
(K41Y, A42M) YMSEKIFYV 27/69508 25 0.6 >24 3 SSX2 41-49 (K41Y,
A42I) YISEKIFYV 27/13233 6.5 2.3 N/A 1 SSX2 41-49 (K41F, A42L)
FLSEKIFYV 28/96243 4.9 3.0 >24 0.3 SSX2 41-49 (K41F, A42V)
FVSEKIFYV 22/8421 4.675 3.1 24 0.1 SSX2 41-49 (K41F, A42M)
FMSEKIFYV 26/69508 6.58 2.2 >24 3 SSX2 41-49 (K41F, A42I)
FISEKIFYV 26/13233 5.368 2.7 >24 0.3 SSX2 41-49 (K41W, A42L)
WLSEKIFYV 27/96243 4.472 3.3 >24 0.3 SSX2 41-49 (K41W, A42V)
WVSEKIFYV 21/8421 4.82 3.0 >24 1 SSX2 41-49 (K41W, A42M)
WMSEKIFYV 25/69508 5.13 2.9 >24 1 SSX2 41-49 (K41W, A42I)
WISEKIFYV 25/13233 6.98 2.1 >24 0.1 SSX2 41-49 (K41(D-Lys),
A42L) (D-Lys)LSEKIFYV N/A 2.5 5.9 15 10 SSX2 41-49 (K41(D-Lys),
A42V) (D-Lys)VSEKIFYV N/A 24.5 0.6 N/A 10
[0305] 4. N-Terminal Primary/Secondary Anchor and C-Terminal
Primary Modification (P2, P1 and P9)
[0306] The C-terminal Val of the wild-type peptide is generally a
preferred anchor residue and primarily involved in the interaction
with the MHC molecule. However, substitutions were carried out to
identify which amino acids improve the analogs having primary and
secondary N-terminal modifications. These C-terminal substitutions
can be used in the absence of one or more N-terminal modifications
also.
[0307] These modifications were shown to improve binding affinity
and stability and in some cases resulted in analogs with decreased
cross-reactivity. Thus, in some embodiments, the substitution to
the C-terminus resulted in a peptide with improved binding and/or
stability without decreased cross-reactivity. However, in other
embodiments the substitution to the C-terminus resulted in a
peptide with improved binding and/or stability with equal or
decreased cross-reactivity. Each of the molecules can be of use in
certain cases or in certain patients. In one embodiment, the valine
at the C-terminus is substituted with a large aliphatic amino acid.
TABLE-US-00004 TABLE 4 N-TERMINAL PRIMARY/SECONDARY ANCHOR AND
C-TERMINAL PRIMARY MODIFICATIONS Cross- reactivity Half- and
Predictive maximal Relative Stability fct avidity Scores Binding
affinity (T1/2) (native to Catergory Peptide name Sequence (R/NIH)
(mM) (1/RA) (Hrs) analogs)* Native SSX2 41-49 KASEKIFYV 22/1017
14.64 1.0 11 1 N-terminal SSX2 41-49 (K41F, A42V, V49L) FVSEKIFYL
22/2586 10.7 1.4 17 >10 Primary/Secondary Anchor, C-terminal
Primary Anchor SSX2 41-49 (K41F, A42V, V49I) FVSEKIFYI 20/1263 9
1.6 24 0.3 SSX2 41-49 (K41F, A42V, V49A) FVSEKIFYA 16/601 6.9 2.1
16 1 SSX2 41-49 (K41F, A42V, V49M) FVSEKIFYM 16/601 17.8 0.8 22
>10 SSX2 41-49 (K41F, A42V, V49Nle) FVSEKIFY(Nle) N/A 5.59 2.6
>24 >10 SSX2 41-49 (K41F, A42V, FVSEKIFY(Nva) N/A 1.89 7.7 20
0.1 V49Nva) SSX2 41-49 (K41F, A42V, FVSEKIFY(MeVal) N/A 17.9 0.8 22
10 V49MeVal) SSX2 41-49 (K41F, A42V, V49Aib) FVSEKIFY(Aib) N/A N/A
N/A N/A >10 SSX2 41-49 (K41F, A42V, FVSEKIFY(Abu) N/A 3.43 4.3
20 1 V49Abu) N-terminal Primary SSX2 41-49 (A42V, V49I) KVSEKIFYI
20/961 13.9 1.1 N/A 0.3 Anchor, C-terminal Primary Anchor SSX2
41-49 (A42L, V49I) KLSEKIFYI 26/10984 5.682 2.6 N/A 0.03 SSX2 41-49
(A42a, V49v) K(D- N/A N/B N/B N/B >10 Ala)SEKIFY(D- Val)
C-terminal Primary SSX2 41-49 (V49I) KASEKIFYI 20/152.56 14 1.0 N/A
10 Anchor
[0308] 5. N-Terminal Primary/Secondary Anchor and TCR Residues
Modification
[0309] The TCR sites are generally recognized as residues P4, P6,
and P8 and are the primary residues involved in the binding to the
TCR. However, other residues may also be involved in the
interaction to a lesser extent. In one embodiment, one or more of
the sites primarily involved in TCR interaction can be substituted
to increase the interaction. Preferably, these substitutions can
generate heteroclitic analogs that do not interfere with binding to
the MHC molecule, but overcome the tolerance issues of the
wild-type peptides. In a further embodiment, at least one TCR
substitution can be included with at least one substitution at
position P1, P2, and/or P9. In a further embodiment, the
substitution at any one or more of the P4, P6, and P8 positions can
be a polar amino acid. In a further embodiment, the substitution
can be an aromatic amino acid at position P8. In a further
embodiment, the substitution can be an amino acid with a large
aliphatic side chain at position P6. In a further embodiment, the
substitution can be an amino acid which has a larger side chain to
preserve the interaction. TABLE-US-00005 TABLE 5 N-TERMINAL
PRIMARY/SECONDARY ANCHOR AND TCR SITES MODIFICATION Cross-
reactivity Half- and fct Predictive maximal Relative Stability
avidity Scores Binding affinity (T1/2) (native to Catergory Peptide
name Sequence (R/NIH) (mM) (1/RA) (Hrs) analogs)* Native SSX2 41-49
KASEKIFYV 22/1017 14.64 1.0 11 1 N-terminal SSX2 41-49 (K41F, A42V,
E44D) FVSDKIFYV 21/8421 13.18 1.1 N/A >10 Primary/Secondary
Anchor, TCR sites SSX2 41-49 (K41F, A42V, E44N) FVSNKIFYV 20/2054
8.97 1.6 N/A >10 SSX2 41-49 (K41F, A42V, E44S) FVSSKIFYV 20/2054
17.5 0.8 N/A >10 SSX2 41-49 (K41F, A42V, E44T) FVSTKIFYV 20/2054
12.94 1.1 N/A >10 SSX2 41-49 (K41F, A42V, E44O) FVSOKIFYV
20/2054 40.8 0.4 N/A 10 SSX2 41-49 (K41F, A42V, E44Nle)
FVS(Nle)KIFYV N/A 13 1.1 N/A 10 SSX2 41-49 (K41F, A42V, E44Nva)
FVS(Nva)KIFYV N/A 3.8 3.9 >24 3 SSX2 41-49 (K41F, A42V, I46L)
FVSEKLFYV 22/8421 7.8 1.9 24 3 SSX2 41-49 (K41F, A42V, I46V)
FVSEKVFYV 22/8421 N/A N/A 24 1 SSX2 41-49 (K41F, A42V, I46M)
FVSEKMFYV 18/8421 9.2 1.6 22 >10 SSX2 41-49 (K41F, A42V, I46Nle)
FVSEK(Nle)FYV N/A 12.8 1.1 19 10 SSX2 41-49 (K41F, A42V, I46Nva)
FVSEK(Nva)FYV N/A 6.21 2.4 >24 1 SSX2 41-49 (K41F, A42V, Y48T)
FVSEKIFTV 24/1531 3.9 3.8 24 >10 SSX2 41-49 (K41F, A42V, Y48F)
FVSEKIFFV 22/8421 8.8 1.7 20 10 SSX2 41-49 (K41F, A42V, Y48S)
FVSEKIFSV 24/1531 3.8 3.9 20 >10 SSX2 41-49 (K41F, A42V,
Y48(Phe-4F)) FVSEKIF(Phe- N/A 10.6 1.4 24 10 4F)V SSX2 41-49 (K41F,
A42V, Y48Phg) FVSEKIF(Phg)V N/A 5.85 2.5 >24 >10 SSX2 41-49
(K41F, A42V, I46L, Y48T) FVSEKLFTV 24/1531 5.67 2.6 24 >10 SSX2
41-49 (K41F, A42V, I46L, Y48S) FVSEKLFSV 24/1531 N/A N/A N/A N/T
N-terminal SSX2 41-49 (K41F, A42V, I46L, Y48T, V49A) FVSEKLFTA
18/109 6.3 2.3 12 >10 Primary/Secondary Anchor, C-terminal
Primary Anchor, TCR sites SSX2 41-49 (K41F, A42V, I46L, Y48S, V49A)
FVSEKLFSA 18/109 6.2 2.4 N/A >10
[0310] 6. C-Terminal Amide
[0311] In some embodiments, the C-terminal residue can be modified
to contain an amide in the place of the free carboxylic acid. Thus,
for example, if the peptide is a 9-mer (nonamer) the P9 residue can
be modified. If the peptide is a 10-mer (decamer) the P 10 residue
can be modified. Preferably this results in a peptide or analog
that has increased stability in biological media, including but not
limited to blood, lymph, and CNS. Preferably, the peptides can
retain the other necessary activities to result in an analog usable
for vaccination or as an immunogen. TABLE-US-00006 TABLE 6
C-TERMINAL AMIDE Predictive Relative Stability Cross-reactivity and
Scores Half-maximal affinity (T1/2) fct avidity (native to
Catergory Peptide name Sequence (R/NIH) Binding (uM) (1/RA) (Hrs)
analogs)* Native SSX2 41-49 KASEKIFYV 22/1017 14.64 1.0 11 1
C-terminal SSX2 41-49-NH2 KASEKIFYV-NH2 N/A N/B N/B N/T >10
amide SSX2 41-49-NH2 (A42L) KLSEKIFYV-NH2 N/A N/B N/B N/T 3 SSX2
41-49-NH2 (A42V) KVSEKIFYV-NH2 N/A N/B N/B N/T 10
[0312] 7. Decamers
[0313] The length of typical MHC binding peptides can vary from
about 8 to about 11 amino acids in length. However, most of the
previously used HLA-A*0201 are 9-mers (nonamers) or 10-mers
(decamers). Thus, in one embodiment, the analog can be an analog of
the wild-type sequence SSX-2.sub.41-50. However, because the
wild-type 10-mer does not have the correct binding motif and showed
no immunological activity, a 10-mer was created by substituting
amino acids at the P10 position and identifying the effect of
various wild-type and analogs (see FIG. 1).
[0314] 8. Remaining Residues
[0315] With reference to FIGS. 1A and 1B, any residues can also be
substituted with conservative amino acids. Conservative
substitutions can be paired with any of the above substitutions
that can produce an effect. Alternatively, conservative
substitutions can be specifically at residues that are not believed
to be involved in any of the activities at a primary, secondary, or
even tertiary level. Such residues include P3, P5 and P7. For
example, the Serine at position P3 can be substituted with an
alanine or threonine to produce an analog. Typically, such
conservative substitutions do not significantly affect the activity
of the analog, however, in some embodiments they can increase
certain activities or decrease certain activities.
NY-ESO-1.sub.157-165 Analogs
[0316] Many features regarding a variety of embodiments and aspects
of analog design are disclosed above, either generally or as
applied to the SSX-2 epitope. It is to be understood that such
disclosure is also applicable to this and subsequent epitopes.
Explicit restatement of such disclosure will be minimized for the
sake of brevity.
[0317] Embodiments relate to analogs of the MHC class I-restricted
T cell epitope NY-ESO-1.sub.157-165, SLLMWITQC (SEQ ID NO. 1),
polypeptides comprising these analogs that can be processed by pAPC
to present the epitope analogs, and nucleic acids that express the
analogs. The analogs can have similar or improved immunological
properties compared to the wild-type epitope.
[0318] One embodiment relates to methods to derivatize and improve
analogs of NY-ESO-1.sub.157-165, along with specific sequences that
encompass substitutions. The analogs can contain at least one
substitution, but can have multiple substitutions comprising
standard or non-standard amino acids singly or in various
combinations. The analogs can result in peptides with retained or
improved properties.
[0319] The epitope NY-ESO-1.sub.157-165 has been shown to be
presented by NY-ESO-1 expressing cell lines, by measuring the
epitope specific T cell activity against such cells (Jaeger, E. et
al., J. Exp. Med. 187:265-270, 1998; U.S. Pat. No. 6,274,145
entitled ISOLATED NUCLEIC ACID MOLECULE ENCODING CANCER ASSOCIATED
ANTIGEN, THE ANTIGEN ITSELF, AND USES THEREOF), which is
incorporated herein by reference in its entirety. Methodologies to
improve the physico-chemical properties of the peptide
NY-ESO-1.sub.157-165 have been described (U.S. Pat. No. 6,417,165
entitled NY-ESO-1-PEPTIDE DERIVATIVES, AND USES THEREOF), which is
incorporated herein by reference in its entirety, and can consist
of replacement of the terminal cysteine with other amino acids that
preserve or enhance the interaction with MHC and are devoid of the
deleterious property of disulfide C--C bond formation interfering
with the activity. However, sole manipulation of the C terminal
cysteine residue ignores the advantages of optimizing multiple
residues throughout the peptide for major histocompatibility (MHC)
and/or T cell Receptor (TCR) binding. Thus, beyond the practicality
of mutating the Cys residue, there is considerable opportunity in
mutating additional amino acids throughout the peptide. For
example, substitutions can be used to further optimize the binding
to MHC and/or TCR in a fashion that enables more effective
application in clinics.
[0320] Embodiments relate to families of one or more peptides of 9
or 10 amino acids in length related by sequence to amino acids
157-165 of the human cancer testis (CT) antigen NY-ESO-1
(NY-ESO-1.sub.157-165).
Analog Design
[0321] The analog is generally an analog of the
NY-ESO-1.sub.157-165, with the sequence SLLMWITQC (SEQ ID NO:1).
Analysis of whether wild-type amino acids are preferred or
non-preferred used previous analyses of other peptide-MHC or TCR
interactions. For example, the Cysteine at the C-terminus is
generally a non-preferred anchor residue because it does not
produce a strong interaction with the HLA molecule and, thus, it
was highly preferred to substitute this residue. However, although
the Serine at position P1 is generally preferred, it was found that
substituting an aromatic could produce a peptide with improved
properties. Further the Leucine at position P2 is generally
acceptable, but substituting a hydrophobic and/or bulky amino acid
resulted in a peptide with improved properties. The residues which
are primarily involved in the interaction with the TCR (P4, P6 and
P8) showed a preference generally for some polarity, and in the
case of P8 an aromatic generally produced peptides with favorable
properties.
[0322] One preferred embodiment relates to an analog that has a
substitution at the P2 position. Preferably, the substitution can
be a hydrophobic residue. More preferably, the substitution can be
a bulky hydrophobic residue. In a further embodiment, the residue
at P1 can be substituted with a more hydrophobic residue. In a
further embodiment, residues P1 and P2 can be both substituted with
more hydrophobic residues. In further embodiments at least one
residue at P1, P2, and P9 can be substituted. In a further
embodiment, at least two residues at P1, P2 and P9 can be
substituted. In a further embodiment at least two residues at P1,
P2, P9, P4, and P6 can be substituted, including one or more
residues involved in TCR binding. In a further embodiment, the
residue at P8 can be substituted with an aromatic. Examples of the
following substitutions are shown in FIGS. 13A-13C.
[0323] 1. N-Terminal Proximal Primary Anchor Modification(P2)
[0324] The N-terminal primary anchor is the 2.sup.nd N-terminal
amino acid of the peptide, thus, it is the N-terminal proximal
primary anchor. Although the original Leucine 158 is not considered
"non-preferred" for binding to the MHC molecule, substitutions can
produce a peptide with improved binding. Thus, in one embodiment,
the original Leu 158 found in the wild-type sequence can be
substituted with a similarly or more hydrophobic amino acid. Any
hydrophobic amino acid may be used, including one that is available
to or that is known to one of skill in the art, including standard
amino acids and non-standard amino acids. In a further embodiment,
the original Leu 158 can be substituted with a more hydrophobic
amino acid also possessing a bulky side chain. Examples of more
hydrophobic amino acids include, but are not limited to: Leu, Val,
Ile, Met, .alpha.-aminobutyric acid, Norleucine and Norvaline.
Further, a naphthal side chain can also be substituted. Preferably,
the substitution results in improved binding and stability with the
HLA molecule. However, this residue may be secondarily or
tertiarily involved in TCR interactions, and substitutions may also
result in improved recognition by the TCR.
[0325] 2. N-Terminal Secondary Anchor Modification (P1)
[0326] The N-terminal secondary anchor is the first amino acid at
the N-terminus or P1. This residue is involved in a number of
interactions. The residue of Ser 157 was defined as a secondary
anchor residue in interacting with HLA-A*0201 molecule, it also
engaged in the interaction with the T cell receptors to a certain
degree. Therefore, modifications of this position generate some
heteroclitic analogs that are more immunogenic and more suitable
for the development of tumor vaccines. Thus, substitutions can
result in a variety of improved qualities.
[0327] Although the Serine is not considered "non-preferred," a
number of substitutions can result in improved qualities of the
peptide. Thus, in one embodiment, the original Ser 157 found in the
wild-type sequence can be substituted with a more hydrophobic amino
acid. Any more hydrophobic amino acid can be used, including one
that is available to or that is known to one of skill in the art,
including standard amino acids and non-standard amino acids.
Examples of more hydrophobic amino acids include, but are not
limited to: Phe, Tyr, Trp, and D-Lys.
[0328] 3. N-Terminal Primary and Secondary Modifications (P2 and
P1)
[0329] In one embodiment, both primary and secondary anchor
residues were substituted to result in improved binding affinity to
the HLA molecule. In a further embodiment, the double substitution
produced improved stability of binding to the HLA molecule. In
further embodiments, the binding and/or stability was not improved
and may have even been reduced, but other properties of the
molecule were improved, such as activity or recognition by a
tolerized individual.
[0330] 4. N-Terminal Primary/Secondary Anchor and C-Terminal
Primary Modification (P2, P1 and P9)
[0331] The C-terminal cysteine of the wild-type peptide is
generally a non-preferred anchor residue. Because this residue is
generally primarily involved in the interaction with the MHC
molecule, it can be preferred to substitute residues that result in
a stronger interaction with the MHC molecule. Thus, substitutions
were shown to improve binding affinity and stability and in some
cases resulted in analogs with decreased cross-reactivity. In some
embodiments, the substitution to the C-terminus can result in a
peptide with improved binding and/or stability without decreased
cross-reactivity. However, in other embodiments the substitution to
the C-terminus can result in a peptide with improved binding and/or
stability with equal or decreased cross-reactivity. Because
substitution of this residue have been previously shown to provide
improved peptides, it can be preferable produce peptides that were
more improved in the interaction with the MHC molecule as well as
other interactions, such as the recognition by the TCR. Thus, in
some embodiments, the C-terminal substitution can be paired with at
least one other substitution. Examples of amino acid substitutions
to the C-terminus include, but are not limited to, valine, lysine,
alanine, and isoleucine.
[0332] 5. N-Terminal Primary/Secondary Anchor and TCR Residue
Modifications
[0333] The primary residues involved in the interaction with the
TCR are generally recognized as residues P4, P6, and P8. However,
other residues may also be involved in the interaction to a lesser
extent. In one embodiment, one or more of the sites primarily
involved in TCR interaction can be substituted to result in an
improved interaction. Preferably, these substitutions generate
heteroclitic analogs that do not interfere with binding to the MHC
molecule, but overcome the tolerance issues of the wild-type
peptides. In one embodiment, at least one TCR substitution can be
included with at least one substitution at position P1, P2, and/or
P9. In one embodiment, amino acids with some polarity can be
substituted at P4, P6, and P8. In a further embodiment, amino acids
which are aromatic can be substituted at the P8 position.
[0334] 6. C-Terminal Amide
[0335] In some embodiments, the C-terminal residue can be modified
to contain an amide in the place of the free carboxylic acid. Thus,
for example if the peptide is a 9-mer (nonamer) the P9 residue can
be modified. If the peptide is a 10-mer (decamer) the P10 residue
can be modified. Preferably this results in a peptide or analog
that has increased stability in biological media, including but not
limited to blood, lymph, and CNS. Preferably, the peptides retain
the other activities to result in an analog usable for vaccination
or as an immunogen.
[0336] 7. Decamers
[0337] The length of typical MHC binding peptides varies from about
8 to about 11 amino acids in length. However, most of the
previously used HLA-A*0201 are 9-mers (nonamers) or 10-mers
(decamers). Thus, in one embodiment, the analog can be a 10-mer of
the wild-type sequence NY-ESO-1.sub.157-166. However, because the
wild-type 10-mer does not have the correct binding motif and showed
no immunological activity, a 10-mer was created by substituting
amino acids at the P10 position and identifying the effect of
various wild-type and analogs (see FIGS. 13A-13C). In one
embodiment, the residues that were added or substituted for the
wild-type at the C-terminus can be selected from the group
consisting of norvaline, leucine, isoleucine, valine, and
alanine.
[0338] 8. Remaining Residues
[0339] With reference to FIGS. 13A and 13C, any residues can also
be substituted with conservative amino acids. Conservative
substitutions can be paired with any of the above substitutions
that can produce an effect. Alternatively, conservative
substitutions can be specifically at residues that are not believed
to be involved in any of the activities at a primary, secondary, or
even tertiary level. Such residues can include P3, P5 and/or P7.
Conservative substitutions are known to those of skill in the art,
but, for example, the Leucine at position P3 can be substituted
with an alanine or threonine to produce an analog. Typically, such
conservative substitutions do not significantly affect the activity
of the analog. However, in some embodiments they may increase
certain activities or decrease certain activities. Because of the
known interactions, it is unlikely that such conservative
substitutions will have a significant effect on any of the
activities.
PSMA.sub.288-299 Analogs
[0340] Many features regarding the variety of embodiments and
aspects of analog design are disclosed above, either generally or
as applied to particular epitopes. It is to be understood that such
disclosure is also applicable to this and subsequent epitopes.
Explicit restatement of such disclosure will be minimized for the
sake of brevity.
[0341] Some embodiments relate to analogs of the MHC class
I-restricted T cell epitope PSMA.sub.288-297, GLPSIPVHPI,
polypeptides comprising these analogs that can be processed by pAPC
to present the epitope analogs, and nucleic acids that express the
analogs. The analogs can have similar or improved immunological
properties compared to the wild-type epitope. Evidence validating
the presentation of this epitope by human cancer cells is presented
in Example 32 below.
[0342] One embodiment relates to methods to derivatize and improve
analogs of PSMA.sub.288-297, along with specific sequences that
encompass substitutions. The analogs can contain at least one
substitution, but can have multiple substitutions comprising
standard or non-standard amino acids singly or in various
combinations. The analogs may result in peptides with retained or
improved properties.
[0343] Embodiments relate to families of one or more peptides of 9
or 10 amino acids in length related by sequence to amino acids
288-297 of the human PSMA.
Analog Design
[0344] In some embodiments, the PSMA.sub.288-297 analog can contain
substitutions of the sequence GLPSIPVHPI. Reference to binding
motif data, such as presented in table 7 in example 2 below,
indicates that the P2 anchor residue can make the largest
individual contribution to affinity of any position in an
A2.1-restricted epitope. In this case the amino acid at the P2
position is the optimally preferred leucine. The P.OMEGA. anchor
residue, isoleucine, is favorable. In vitro binding studies using
the T2 cell assay system (not shown) have indicated that the native
peptide has generally superior binding characteristics,
particularly as compared to the SSX-2 and NY-ESO-1 epitopes. The
epitope exhibited significant binding at relatively low
concentrations, although this was paired with a relatively shallow
rise toward saturation. The wild-type epitope can be improved.
Analyses such as that represented by tables 7 and 8 are averages
and the behavior of a given residue in a particular sequence may
diverge from the average. Consistent with the favorable results
obtained with Nle and Nva for the SSX-2 and NY-ESO-1 epitopes
discussed above, Nle and Nva also can be successfully used for the
instant PSMA epitope. Finally, even similar binding
characteristics, if paired with alterations that help circumvent
whatever tolerance to the epitope may exist, can increase the
effective immunogenicity of the peptide. In the transgenic mouse
model the native peptide is poorly immunogenic (see Example 35 for
instance) which may reflect tolerance to the epitope; the region of
PSMA from which this epitope is derived is identical between mouse
and human PSMA.
[0345] 1. N-Terminus Proximal Primary Anchor Modification (P2)
[0346] As noted above, although the native residue at the P2
position of this epitope is generally the optimal residue among
genetically encoded amino acids, the effect of substituting other
preferred or bulky hydrophobic residues were examined for potential
improvement of binding, tolerance breaking and cross-reactive
immunity. Exemplary substitutions can include Met, Ile, Gln, Val,
Nva, Nle, and aminobutyric acid (Abu).
[0347] 2. N-Terminal Secondary Anchor Modification (P1)
[0348] The N-terminal secondary anchor is the first amino acid at
the N-terminus. The native Gly is only marginally preferred at this
position. Various observations (see tables 7 and 8 for example)
show that amino acids with potential to improve the epitope include
Ala, Ser, Abu and sarkosine (Sar, that is, N-methylglycine).
[0349] 3. C-Terminal Primary Anchor Modification (PE)
[0350] The native Ile at this position is generally a preferred but
not optimal residue. Substitution at this position can improve
binding. Exemplary substitutions can include Val, Leu, Nva, and
Nle.
[0351] 4. Secondary Anchors and TCR Exploration
[0352] The penultimate position (P.OMEGA.-1) can serve both as a
secondary anchor and a TCR interacting position. Substitution of
Ala, Leu, Ser, and Thr can be have their primary effect on TCR
interaction, though they can also contribute to improved binding.
P3 is another position that can effect both binding and
immunogenicity. Substitution of Trp at this position can improve
both.
[0353] Further embodiments relate to combinations of substitutions
at multiple positions in order to combine, synergize, and
counteract the various effects obtained with the single
substitutions.
PRAME.sub.425-433 Analogs
[0354] Many features regarding the variety of embodiments and
aspects of analog design are disclosed above, either generally or
as applied to particular epitopes. It is to be understood that such
disclosure is also applicable to this and subsequent epitopes.
Explicit restatement of such disclosure will be minimized for the
sake of brevity.
[0355] Embodiments include analogs of the MHC class I-restricted T
cell epitope PRAME.sub.425-433, SLLQHLIGL, polypeptides comprising
these analogs that can be processed by pAPC to present the epitope
analogs, and nucleic acids that express the analogs. The analogs
can have similar or improved immunological properties compared to
the wild-type epitope. Evidence validating the presentation of this
epitope by human cancer cells is presented in Example 39 below.
[0356] One embodiment relates to methods to derivatize and improve
analogs of PRAME.sub.425-433, along with specific sequences that
encompass substitutions. The analogs can contain at least one
substitution, but can have multiple substitutions comprising
standard or non-standard amino acids singly or in various
combinations. The analogs can result in peptides with retained or
improved properties.
[0357] Some embodiments relate to families of one or more peptides
of 9 or 10 amino acids in length related by sequence to amino acids
425-433 of the human PRAME sequence.
Analog Design
[0358] Some embodiments relate to analogs of the PRAME.sub.425-433
which can contain substitutions of the sequence SLLQHLIGL.
Reference to binding motif data, such as presented in table 7 in
Example 2 below, indicates that the P2 anchor residue can make the
largest individual contribution to affinity of any position in an
A2.1-restricted epitope. In this case the amino acid at the P2
position is the optimally preferred leucine. The P.OMEGA. anchor
residue, leucine, is favorable, though not as strongly preferred.
Analyses such as that represented by tables 7 and 8 are averages
and the behavior of a given residue in a particular sequence can
diverge from the average, nor is the wild type P.OMEGA. residue
necessarilly the most preferred for that position. Consistent with
the favorable results obtained with Nle and Nva for the other
epitopes, similar improvements can be obtained substituting Nle and
Nva with this sequence. Finally, even similar binding
characteristics, if paired with alterations that help circumvent
whatever tolerance to the epitope may exist, can increase the
effective immunogenicity of the peptide.
[0359] The rationale for various substitutions has been set forth
above. The particular substitutions investigated for the
PRAME.sub.425-433 epitope follow the same logic and are disclosed
in the examples 40-42 and FIGS. 25-27. Substitutions were made at
the primary anchor positions P2 and P.OMEGA. (P9), the secondary
anchor positions P1 and P.OMEGA.-1 (P8). Substitutions were also
made in the TCR interacting positions (in addition to secondary
anchor positions) P3 and P6. Selected substitutions have impact on
binding and/or stability of MHC class I--peptide complexes, key
features in determining the immunological properties of peptides.
In addition, due to T cell repertoire considerations and to
circumvent mechanisms responsible for the limited immunity to
native epitopes, substitutions that retain the capability of
analogs to interact with T cell receptors recognizing native
peptides, can be of practical value.
EXAMPLES
[0360] The following examples provide analogs and methods of
identifying analogs. The analogs can be used, for example, as
immunogens, vaccines, and/or treatment of a variety of cancers. The
analogs were produced as in Example 1. SSX-2.sub.41-49 analogs were
identified as shown in Example 2, those produced listed in Example
3 and tested for improved properties as in Examples 4-21. The
testing of NY-ESO-1.sub.157-165 analogs were tested for improved
properties as in Examples 22-30.
Example 1
Peptide Synthesis, Purification and Characterization
[0361] Peptides were synthesized on either a Symphony multiple
peptide synthesizer (PTI technologies, MA) or an ABI 433A peptide
synthesizer (Applied Biosystems, Foster City, Calif.) at 0.05-0.1
mmole scale using standard Fmoc solid phase chemistry. C-terminal
free acid peptides were synthesized using pre-load PEG-PS resins
(on Symphony) or Wang resin (on ABI). C-terminal amidated peptides
were synthesized on Fmoc-PAL-PEG-PS resin. All resins were
purchased from Applied Biosystems (Foster City, Calif.). The
Fmoc-amino acids used in peptide syntheses were purchased from
Novabiochem (San Diego, Calif.) and AnaSpec (San Jose, Calif.).
Post-synthesis cleavage was carried on by the standard
protocol.
[0362] Peptide purification was carried out on either
semi-preparative HPLC columns or SPE cartridges (Phenomenex,
Torrance, Calif.). The purity of all peptides was .gtoreq.90%. The
identity of each peptide was verified by Maldi-TOF MS (Voyager DE,
Applied Biosystems) and analytical HPLCs (Varian or Shimazu) using
a Synergi C12 column (Phenomenex, Torrance, Calif.).
Example 2
De Novo Designed SSX-241-49 Analogs
[0363] Structural modification of a moderately antigenic peptide
can considerably improve peptide-MHC binding, CTL recognition,
and/or immunogenicity. General guidelines regarding how to modify a
wild-type epitope in order to achieve a peptide analog with
enhanced potency are known in the art. An appreciated strategy is
to optimize the residues at the so-called anchor positions for
binding to the particular MHC molecule at issue. In the case of
HLA-A2 a marked preference for hydrophobic residues at the P2 and
P.OMEGA. positions has been observed, particularly L, and M at P2,
and V at P.OMEGA.. (P.OMEGA. denotes the C-terminal residue of the
epitope. For HLA-A2 that is P9 or P10 depending on the length of
the peptide.) Replacing the P1 position with aromatic residues,
such as F, Y and W can also be advantageous. TABLE-US-00007 TABLE 7
Coefficients used by the BIMAS algorithm (Algorithm available by
hypertext transfer protocol: //bimas.cit.nih.gov/molbio/hla_bind/)
9-mer Coefficient Table for HLA_A_0201 Amino Acid Position Type 1st
2nd 3rd 4th 5th 6th 7th 8th 9th A 1.000 1.000 1.000 1.000 1.000
1.000 1.000 1.000 1.000 C 1.000 0.470 1.000 1.000 1.000 1.000 1.000
1.000 1.000 D 0.075 0.100 0.400 4.100 1.000 1.000 0.490 1.000 0.003
E 0.075 1.400 0.064 4.100 1.000 1.000 0.490 1.000 0.003 F 4.600
0.050 3.700 1.000 3.800 1.900 5.800 5.500 0.015 G 1.000 0.470 1.000
1.000 1.000 1.000 0.130 1.000 0.015 H 0.034 0.050 1.000 1.000 1.000
1.000 1.000 1.000 0.015 I 1.700 9.900 1.000 1.000 1.000 2.300 1.000
0.410 2.100 K 3.500 0.100 0.035 1.000 1.000 1.000 1.000 1.000 0.003
L 1.700 72.000 3.700 1.000 1.000 2.300 1.000 1.000 4.300 M 1.700
52.000 3.700 1.000 1.000 2.300 1.000 1.000 1.000 N 1.000 0.470
1.000 1.000 1.000 1.000 1.000 1.000 0.015 P 0.022 0.470 1.000 1.000
1.000 1.000 1.000 1.000 0.003 Q 1.000 7.300 1.000 1.000 1.000 1.000
1.000 1.000 0.003 R 1.000 0.010 0.076 1.000 1.000 1.000 0.200 1.000
0.003 S 1.000 0.470 1.000 1.000 1.000 1.000 1.000 1.000 0.015 T
1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.500 V 1.700 6.300
1.000 1.000 1.000 2.300 1.000 0.410 14.000 W 4.600 0.010 8.300
1.000 1.000 1.700 7.500 5.500 0.015 Y 4.600 0.010 3.200 1.000 1.000
1.500 1.000 5.500 0.015 final 0.069 constant
[0364] TABLE-US-00008 TABLE 8 Scoring Pattern for HLA-A*0201 used
by the SYFPEITHI Algorithm (9-mers) (Algorithm available by
hypertext transfer protocol:
//syfpeithi.bmi-heidelberg.com/scripts/MHCServer.dll/home.htm) AA
P1 P2 P3 P4 P5 P6 P7 P8 P9 A 2 6 2 0 0 0 2 1 6 C 0 0 0 0 0 0 0 0 0
D -1 0 0 1 0 0 0 0 0 E -1 0 -1 2 0 0 0 2 0 F 1 0 1 -1 1 0 0 0 0 G 1
0 0 2 2 0 0 1 0 H 0 0 0 0 0 0 1 0 0 I 2 8 2 0 0 6 0 0 8 K 1 0 -1 0
1 0 -1 2 0 L 2 10 2 0 1 6 1 0 10 M 0 8 1 0 0 0 0 0 6 N 0 0 1 0 0 0
1 0 0 P 0 0 0 2 1 0 1 0 0 Q 0 0 0 0 0 0 0 0 0 R 0 0 0 0 0 0 0 0 0 S
2 0 0 0 0 0 0 2 0 T 0 6 -1 0 0 2 0 2 6 V 1 6 0 0 0 6 2 0 10 W 0 0 1
0 0 0 0 0 0 X 0 0 0 0 0 0 0 0 0 Y 2 0 1 -1 1 0 1 0 0
[0365] Adapted from: Rammensee, Bachmann, Stevanovic: MHC ligands
and peptide motifs. Landes Bioscience 1997
Example 3
[0366] The following analogs were produced using the predictions in
Example 1. TABLE-US-00009 TABLE 9 SEQ ID Catergory Number Peptide
name Sequence wild-type 1 SSX-2 41-49 KASEKIFYV N-terminal Primary
Anchor 2 SSX-2 41-49 (A42L) KLSEKIFYV 3 SSX-2 41-49 (A42V)
KVSEKIFYV 4 SSX-2 41-49 (A42I) KISEKIFYV 5 SSX-2 41-49 (A42M)
KMSEKIFYV 6 SSX-2 41-49 (A42(D-Ala)) K(D-Ala)SEKIFYV 7 SSX-2 41-49
(A42(D-Leu)) K(D-Leu)SEKIFYV 8 SSX-2 41-49 (A42(D-Val))
K(D-Val)SEKIFYV 9 SSX-2 41-49 (A42(Nal-1)) KNal-1SEKIFYV 10 SSX-2
41-49 (A42(Nal-2)) KNal-2SEKIFYV 11 SSX-2 41-49 (A42(Abu))
KAbuSEKIFYV 12 SSX-2 41-49 (A42(Nle)) KNleSEKIFYV 13 SSX-2 41-49
(A42(Nva)) KNvaSEKIFYV 14 SSX-2 41-49 (A42(Aib)) KAibSEKIFYV
N-terminal Secondary 15 SSX-2 41-49 (K41F) FASEKIFYV Anchor 16
SSX-2 41-49 (K41W) WASEKIFYV 17 SSX-2 41-49 (K41Y) YASEKIFYV 18
SSX-2 41-49 (K41(D-Lys)) (D-Lys)ASEKIFYV 19 SSX-2 41-49 (K41(Phg))
PhgASEKIFYV 20 SSX-2 41-49 (K41(Cha)) ChaASEKIFYV 21 SSX-2 41-49
(K41(Phe-4F)) Phe(4-F)ASEKIFYV 22 SSX-2 41-49 (K41(Phe-4NO2))
Phe(4-NO.sub.2)ASEKIFYV 23 SSX-2 41-49 (K41(O-methyl Tyr))
O-methyl-TyrASEKIFYV 24 SSX-2 41-49
(K41(.beta.-(3-benzothienyl)Ala))
.beta.-(3-benzothienyl)AlaASEKIFYV N-terminal 25 SSX-2 41-49 (K41Y,
A42L) YLSEKIFYV Primary/Secondary Anchor 26 SSX-2 41-49 (K41Y,
A42V) YVSEKIFYV 27 SSX-2 41-49 (K41Y, A42M) YMSEKIFYV 28 SSX-2
41-49 (K41Y, A42I) YISEKIFYV 29 SSX-2 41-49 (K41F, A42L) FLSEKIFYV
30 SSX-2 41-49 (K41F, A42V) FVSEKIFYV 31 SSX-2 41-49 (K41F, A42M)
FMSEKIFYV 32 SSX-2 41-49 (K41F, A42I) FISEKIFYV 33 SSX-2 41-49
(K41W, A42L) WLSEKIFYV 34 SSX-2 41-49 (K41W, A42V) WVSEKIFYV 35
SSX-2 41-49 (K41W, A42M) WMSEKIFYV 36 SSX-2 41-49 (K41W, A42I)
WISEKIFYV 37 SSX-2 41-49 (K41(D-Lys), A42L) (D-Lys)LSEKIFYV 38
SSX-2 41-49 (K41(D-Lys), A42V) (D-Lys)VSEKIFYV N-terminal 39 SSX-2
41-49 (K41F, A42V, V49L) FVSEKIFYL Primary/Secondary Anchor,
C-terminal Primary Anchor 40 SSX-2 41-49 (K41F, A42V, V49I)
FVSEKIFYI 41 SSX-2 41-49 (K41F, A42V, V49A) FVSEKIFYA 42 SSX-2
41-49 (K41F, A42V, V49M) FVSEKIFYM 43 SSX-2 41-49 (K41F, A42V,
V49Nle) FVSEKIFY(Nle) 44 SSX-2 41-49 (K41F, A42V, V49Nva)
FVSEKIFY(Nva) 45 SSX-2 41-49 (K41F, A42V, V49MeVal) FVSEKIFY(MeVal)
46 SSX-2 41-49 (K41F, A42V, V49MeLeu) FVSEKIFY(MeLeu) 47 SSX-2
41-49 (K41F, A42V, V49Aib) FVSEKIFY(Aib) 48 SSX-2 41-49 (K41F,
A42V, V49Abu) FVSEKIFY(Abu) N-terminal 49 SSX-2 41-49 (K41F, A42V,
E44D) FVSDKIFYV Primary/Secondary Anchor, TCR sites 50 SSX-2 41-49
(K41F, A42V, E44N) FVSNKIFYV 51 SSX-2 41-49 (K41F, A42V, E44S)
FVSSKIFYV 52 SSX-2 41-49 (K41F, A42V, E44T) FVSTKIFYV 53 SSX-2
41-49 (K41F, A42V, E44Q) FVSQKIFYV 54 SSX-2 41-49 (K41F, A42V,
E44(Nle)) FVS(Nle)KIFYV 55 SSX-2 41-49 (K41F, A42V, E44(Nva))
FVS(Nva)KIFYV 56 SSX-2 41-49 (K41F, A42V, I46L) FVSEKLFYV 57 SSX-2
41-49 (K41F, A42V, I46V) FVSEKVFYV 58 SSX-2 41-49 (K41F, A42V,
I46M) FVSEKMFYV 59 SSX-2 41-49 (K41F, A42V, I46(Nle)) FVSEK(Nle)FYV
60 SSX-2 41-49 (K41F, A42V, I46(Nva)) FVSEK(Nva)FYV 61 SSX-2 41-49
(K41F, A42V, Y48T) FVSEKIFTV 62 SSX-2 41-49 (K41F, A42V, Y48F)
FVSEKIFFV 63 SSX-2 41-49 (K41F, A42V, Y48S) FVSEKIFSV 64 SSX-2
41-49 (K41F, A42V, Y48(Phe-4F)) FVSEKIF(Phe4-F)V 65 SSX-2 41-49
(K41F, A42V, Y48(Phg)) FVSEKIF(Phg)V 66 SSX-2 41-49 (K41F, A42V,
I46L, Y48T) FVSEKLFTV 67 SSX-2 41-49 (K41F, A42V, I46L, Y48S)
FVSEKLFSV N-terminal 68 SSX-2 41-49 (K41F, A42V, I46L, Y48T,
FVSEKLFTA Primary/Secondary V49A) Anchor, C-terminal Primary
Anchor, TCR sites 69 SSX-2 41-49 (K41F, A42V, I46L, Y48S, FVSEKLFSA
V49A) N-terminal Primary Anchor, 70 SSX-2 41-49 (A42V, V49I)
KVSEKIFYI C-terminal Primary Anchor 71 SSX-2 41-49 (A42L, V49I)
KLSEKIFYI 72 SSX-2 41-49 (A42(D-Ala), V49(D-Val))
K(D-Ala)SEKIFY(D-Val) 73 SSX-2 41-49 (A42(D-Leu), V49(D-Val))
K(D-Leu)SEKIFY(D-Val) 74 SSX-2 41-49 (A42(D-Val), V49(D-Val))
K(D-Val)SEKIFY(D-Val) C-terminal Primary Anchor 75 SSX-2 41-49
(V49I) KASEKIFYI C-terminal amide 76 SSX-2 41-49-NH2 KASEKIFYV-NH2
77 SSX-2 41-49-NH2 (A42L) KLSEKIFYV-NH2 78 SSX-2 41-49-NH2 (A42V)
KVSEKIFYV-NH2 Decamers 79 SSX-2 41-50 KASEKIFYVY 80 SSX-2 41-50
(Y50I) KASEKIFYVI 81 SSX-2 41-50 (Y50L) KASEKIFYVL 82 SSX-2 41-50
(Y50V) KASEKIFYVV 83 SSX-2 41-50 (Y50 (Nle)) KASEKIFYV(Nle) 84
SSX-2 41-50 (Y50 (Nva)) KASEKIFYV(Nva) 85 SSX-2 41-50 (A42V, Y50I)
KVSEKIFYVI 86 SSX-2 41-50 (A42L, Y50I) KLSEKIFYVI 87 SSX-2 41-50
(A42V, Y50L) KVSEKIFYVL 88 SSX-2 41-50 (A42L, Y50L) KLSEKIFYVL 89
SSX-2 41-50 (A42V, Y50V) KVSEKIFYVV 90 SSX-2 41-50 (A42L, Y50V)
KLSEKIFYVV 91 SSX-2 41-50 (A42V, Y50(Nle)) KVSEKIFYV(Nle) 92 SSX-2
41-50 (A42L, Y50(Nle)) KLSEKIFYV(Nle) 93 SSX-2 41-50 (A42V,
Y50(Nva)) KVSEKIFYV(Nva) 94 SSX-2 41-50 (A42L, Y50(Nva))
KLSEKIFYV(Nva) 95 SSX-2 41-50 (A42V, V49I, Y50I) KVSEKIFYII 96
SSX-2 41-50 (A42L, V49I, Y50I) KLSEKIFYII 97 SSX-2 41-50 (V49I,
Y50I) KASEKIFYII
[0367] Abbreviations for non-standard amino acids: Nle, norleucine;
Nva, norvaline; Phg, phenylglycine; Phe(4-F),
4-fluorophenylalanine; Phe(4-NO.sub.2), 4-nitrophenylalanine; Abu,
.alpha.-aminobutyric acid; Aib, .alpha.-aminoisobutyric acid;
MeLeu, methyl-leucine; MeVal, methylvaline;
.beta.-(3-benzothienyl)Ala, .beta.-(3-benzothienyl)-alanine;
O-methyl-Tyr, O-methyltyorosine; Cha, cyclohexylalanine; Nal-1,
.beta.-(1-napthyl)-alanine; Nal-2, .beta.-(2-napthyl)-alanine;
--NH2 indicates that the carboxy terminus has been modified to the
amide.
Examples 4-21
Testing of SSX-2.sub.41-49 Analogs
[0368] The analogs produced in Example 3 were tested for activity,
such as binding and biological effect as follows in Examples
4-21:
Example 4
Peptide Binding Using T2 Cells
[0369] The affinity of peptide analogs and the wild-type epitope to
HLA-A*0201 was evaluated using a T2 cell based assay (Regner M, et
al., Exp Clin Immunogenet. 1996;13(1):30-5; which is hereby
incorporated by reference in its entirety).
[0370] For the binding assay, in brief, the T2 cells that lack
expression of TAP and thus do not assemble stable MHC class I on
the cell surface, were pulsed with different concentrations of
peptides (controls or analogs) overnight at 37.degree. C., washed
extensively, stained with fluorescently tagged antibody recognizing
MHC class I (A2 allele) and run through a FacsScan analyzer. The
difference between the MFI (mean fluorescence intensity)
corresponding to a given concentration of analog and the negative
control (non-MHC binder) is a function of how many stabilized
complexes between MHC and peptide are displayed on the surface of
T2 cells. Thus, at limiting concentrations of peptide, this is a
measurement of K.sub.on mostly and at saturation levels of peptide
that is a measurement of both K.sub.on and K.sub.off. The binding
was quantified by two factors that are mathematically related: half
maximal binding (the peptide concentration giving 50% of the signal
corresponding to saturation) and relative affinity (1/RA). Relative
affinity RA is binding normalized to a reference (wild-type
peptide); for example, the ratio between half max binding of
control relative to peptide analog. The higher the 1/RA index and
the lower the half maximal binding, the higher the K.sub.on of the
interaction between the analog and the MHC. Fifty three analogs
were identified with these binding parameters improved relative to
the wild-type peptide. These improved binders carry one, two, three
or multiple substitutions (including standard and/or non-standard
amino acids) involving positions that are known to participate in
the interaction with MHC and/or TCR. However, the overall effect on
MHC binding was dependent on the modification. Such peptide analogs
can be useful in therapeutic compositions or as a platform to
further derive therapeutic compositions.
Example 5
Peptide Stability Using T2 Cells
[0371] Peptide stability (K.sub.off) on MHC generally cannot be
solely inferred from binding (K.sub.on). In addition, along with
binding, the stability of peptides on MHC class I is notoriously
important in regard to the immunological properties of such
peptides, since the activation of T cells depends on the duration
of "signal 1" (MHC peptide complex interaction with T cell
receptor). For the stability assay, in brief, the T2 cells that
lack expression of TAP and thus do not assemble stable MHC class I
on the cell surface, were pulsed with a concentration of peptide
(controls or analogs) known to achieve maximal loading of MHC class
I ("saturation") overnight at 37.degree. C., washed extensively,
and chased for different intervals in the presence of emetine,
which blocks endogenous protein synthesis. After extensive washing,
the cells were stained with fluorescently tagged antibody
recognizing MHC class I (A2 allele) and run through a FacsScan
analyzer. The difference between the MFI (mean fluorescence
intensity) corresponding to a given concentration of analog and the
negative control (non-MHC binder) is a function of how many
stabilized complexes between MHC and peptide are displayed on the
surface of T2 cells. The decay of the signal over time was
mathematically expressed as stability index 50% relative to the
binding at 0 hours (at the beginning of the chase interval).
[0372] Such improved analogs can carry one, two, three or multiple
substitutions (including standard and/or non-standard amino acids)
involving positions that are known to participate in the
interaction with MHC and/or TCR, with an overall effect on MHC
stability that is dependent on the modification. Such peptide
analogs can be useful in therapeutic compositions or as a platform
to further derive therapeutic compositions. Forty three of the
analogs have increased stability relative to the natural
peptide.
[0373] The analogs that showed both improved binding and stability
are useful in improved compositions or as a platform to generate
improved compositions of therapeutic benefit.
Example 6
Evaluation of Immunologic Properties of Analogs: Cross-Reactivity
and Functional Avidity
[0374] The immunologic properties of peptides can be described as a
function of binding to MHC molecules (K.sub.on and K.sub.off) and
TCR (affinity of interaction between TCR and MHC-peptide
complexes). Modifications of primary MHC anchor residues generally
have a significant degree of predictability in regard to overall
impact on binding to MHC molecules.
[0375] Modifications of secondary MHC anchor residues may impact
the affinity of interaction of the MHC-peptide complex to TCR along
with the K.sub.on and K.sub.off relative to peptide-MHC
interaction.
[0376] A methodology was devised that allowed rapid and rational
screening of peptide analogs in a fashion coherent with proposed
methods of use and modeling the overall immunologic properties
(K.sub.on and K.sub.off relative to MHC interaction and TCR binding
properties in an integrated fashion). This method can include
generating T cell lines against a natural (non-mutated) epitope
(SSX-2.sub.41-49) using an immunization strategy potent enough to
generate a useful response in transgenic mice carrying human MHC
(such as the A2 allele). Peptide analogs were interrogated ex vivo
in the presence of competent APCs and the functional impact of T
cells specific for natural (non-mutated) epitope measured. The
evaluation was done at various concentrations of analog, since the
expected effect was biphasic in the case of cross reactive peptides
(activating at limited concentrations and inhibiting at higher
concentrations, due to antigen-induced cell death, AICD).
Measurement of the following three parameters can define basic and
useful characteristics of peptide analogs: [0377] 1. Minimal
required concentration of peptide analog to trigger effects
indicative of T cell activation (e.g. cytokine production); [0378]
2. Maximal (peak value) effect (e.g. cytokine production) at any
analog concentration; [0379] 3. Analog concentration at peak value
of activating effect (e.g., cytokine concentration)
[0380] For example, analogs that result in reduced values
associated with parameters #1 and 3 but increased #2, can be
useful. Use of natural epitope and unrelated non-cross reactive
peptides as references is valuable in identifying classes of
analogs of potential value. Analogs that display properties
quantitatively comparable to or even modestly attenuated from those
of natural epitopes are still deemed useful in light of the fact
that while they retain cross-reactivity, they may display
immunologic properties that are distinct from those of the natural
peptide--for example lower propensity to induce AICD or ability to
break tolerance or restore responsiveness in vivo.
[0381] Some advantages of this screening strategy include the
practicality and rapidity, use of more relevant polyclonal T cell
lines instead of potentially biased T cell clones as a read out,
and the composite value, integrating parameters such as K.sub.on,
K.sub.off and TCR affinity that may translate into cross-reactivity
and functional avidity of peptide-MHC complexes relative to TCR.
These parameters can be predictive of the in vivo immunologic
properties and thus can delineate useful panels of peptide analogs
to undergo further evaluation, optimization and practical
applications. Analogs that bind to MHC and retain cross-reactivity
against TCR specific for the nominal wild-type peptide are
predicted to trigger a measurable effect in this assay. The overall
methodology is presented in FIG. 2.
[0382] The method used for the generation of T cell lines was the
following: HHD transgenic mice carrying an A2 human allele (Pascolo
et al. J. Exp Med. 185(12):2043-51, 1997, which is hereby
incorporated herein by reference in its entirety) were immunized
with 50 ug of SSX-2 natural epitope (41-49) admixed with 25 ug of
pIpC at day 0, 4, 14 and 18 by bilateral administration into the
inguinal lymph nodes. At 7 days after the last boost, the mice were
sacrificed and a suspension of splenocytes prepared at
5.times.10.sup.6 million cells/ml in complete HL-1 medium. Cells
were incubated with different concentrations of peptide for 48
hours in flat-bottomed 96-well plates (200 ul/well) and for an
additional 24 hours with rIL-2 at 10 U/ml added to the wells. The
supernatant was harvested and the concentration of IFN-gamma
assessed by standard methods such as ELISA.
Example 7
Cross-Reactivity and Functional Avidity of Analogs Substituted at
Single Position
[0383] The strategy from above (Example 6, FIG. 2) was applied to
scan through a library of analogs bearing single substitutions
relative to the natural SSX-2.sub.41-49 epitope (KASEKIFYV) in its
wild-type version (FIG. 3). Strong inverse correlation was found
between the minimal required amount of analog to elicit IFN-gamma
production ex vivo and the maximal amount of cytokine production at
any concentration of analog.
[0384] Substitution of A42 with L, V or M improved on the
immunologic properties of the peptide, assessed in this assay. L
and V mutants were active. M was more active than the natural
epitope. The I mutant retained cross-reactivity to the TCR
recognizing the wild-type epitope.
[0385] Replacement of the A at position 42 with non-standard amino
acids Abu, Nle or Nva improved on the immunologic properties of the
peptide relative to the wild-type epitope, both in terms of the
minimal amount of analog required to trigger cytokine production
and the peak amount of cytokine produced. Mutants encompassing
D-Ala, D-Val, Nal-2 or Aib display retained cross-reactivity and
reduced immune activity in this assay relative to the natural
peptide, but can still be useful for further derivitization to
adjust or enhance their properties. An Nal-1 at position 42
abrogated the activity.
[0386] Changes of the first residue K.sub.41 showed that, while
replacement with F or Phg improved on the activity, W, D-Lys, and
Cha obliterated the immunologic properties in this assay.
Replacement of K with Y, Phe-4F, Phe(4-NO2), O-methyl-Tyr or
beta-(3-benzothienyl-Ala) retained activity.
[0387] Modification of position V.sub.49 (C-terminal residue) by
replacement with I retained the activity at a lower level compared
to the original epitope. Modification of the last residue by
addition of an --NH2 moiety obliterated the activity of the peptide
that was subsequently rescued by modifying the A at position 42
with L or V. This illustrates directly that analogs with activity
that is lower than that of the wild-type peptide are still useful
for further derivatization.
Example 8
Cross-Reactivity and Functional Avidity of Analogs Substituted at
Two Positions
[0388] The strategy from above (Example 6, FIG. 2) has been applied
to scan through a library of analogs bearing two substitutions,
relative to the wild-type SSX-2.sub.41-49 epitope in its wild-type
version (FIG. 4).
[0389] Coordinated modifications at position 1 and 2 have a
variable effect on the activity of analogs. For example,
substitution of K41 with Y, F or W corroborated with substitution
of A42 with V, M or I, and resulted in preserved or enhanced
activity of the analogs relative to the wild-type peptide. Such
doubly mutated peptides offer an increased opportunity to impact
the interaction with TCR in a fashion that results in tolerance
breaking (thus being useful for practical application), since the
P1 residue participates to a certain extent in binding to TCR.
Combinations between the following: Y (position 41) with V (at
position 42), W (position 41) with I or I (at position 42), and F
(position 41) with L, V, I (at position 42) resulted in analogs
that were more active relative to the wild-type peptide.
Combinations between Y at position 41 and I at position 42, or W at
position 41 and V or M at 42, conferred an activity similar to that
of wild-type peptide. Replacement of K with D-lysine at position 41
reduced resulted in analogs with retained activity in this assay.
Such peptides can be very useful since the metabolic degradation of
such peptides encompassing non-standard amino acids is decreased in
vivo.
[0390] Combinations between V or L at position 42 and I at position
49 resulted in increased activity over the natural peptide.
Example 9
Cross-Reactivity and Functional Avidity of Analogs Substituted at
Multiple Positions
[0391] The strategy from above (Example 6, FIG. 2) has been applied
to scan through a library of analogs bearing three or more
substitutions relative to the natural SSX-2.sub.41-49 epitope in
its wild-type version (FIG. 5).
[0392] F and V at positions 41 and 42 respectively, combined with I
or A at position 49 resulted in improved or similar activity
relative to the wild-type epitope. In contrast, L or M at position
49 resulted in heavily diminished activity.
[0393] Triple mutants comprising the non-standard amino acids Nva,
Abu or MeVal at the last position resulted in retention or
improvement of immune activity. Such peptides are extremely useful
due to increased in vivo stability and resistance to enzymatic
degradation.
[0394] Modification of amino acid residues within the putative TCR
binding region can result in peptides of considerable value, that
retain binding to MHC along with cross-reactivity and thus be
useful for restoration of immune responsiveness or tolerance
breaking since their conformation in the MHC groove is slightly
different from that of natural peptides. Additional substitutions
at position 44 (Q, Nva or Nle), position 46 (L, V, Nle or Nva) or
48 (F or Phe-4F) resulted in active analogs, whereas D, N, S or T
at position 44, M at 46 or T, S, Phg at position 48 or L at
position 46 with T at 48, resulted in analogs devoid of activity.
Finally, two analogs with 5 substitutions showed no activity (FIG.
5).
Example 10
Cross-Reactivity and Functional Avidity of Decamers Encompassing
the Natural Peptide and Mutated at Various Positions
[0395] The strategy from above (Example 6, FIG. 2) has been applied
to scan through a library of analogs of a decamer encompassing the
nominal SSX-2.sub.41-49 peptide (FIG. 6).
[0396] The decamer SSX-2 41-50 was significantly less active in
stimulating the T cell line specific for the 41-49 nonamer,
relative to the latter. Modification of the Y residue at position
50 to I or L, but to a lesser or no extent to V, Nle or Nva,
resulted in restoration of activity in this assay. Further
optimization of the activity of decameric analogs can be obtained
by modification of the A at position 2 with L or V. The A42L
substitution rescued the activity of the Y5ONva decamer. Peptide
analogs of similar or reduced activity in vitro (but retained
cross-reactivity) compared with the natural peptide are still
useful for induction or boost of immune responses due to: i) more
limited AICD; ii) potentially higher in vivo activity due to
increased stability on class I MHC and/or slightly modified
interaction with TCR which is can be important for tolerance
breaking.
Example 11
Use of Analogs to Trigger Enhanced Immunity Against Natural
Epitope, Assessed Ex Vivo
[0397] Three groups of mice (n=4) were immunized with a plasmid
expressing SSX-2.sub.41-49 natural epitope, by direct inoculation
into the inguinal lymph nodes with 25 ug in 25 ul of PBS/each lymph
node at day 0, 3, 14 and 17. This was followed by two additional
peptide boosts (similar amount) at day 28 and 31. The schedule of
immunization is shown in FIG. 7. One week after the boost,
splenocytes were stimulated ex vivo with SSX-2.sub.41-49 natural
peptide and tested against .sup.51Cr-labeled target cells (T2
cells) at various E:T ratios (FIG. 8). The results showed that the
analog A42V triggered a higher response against target cells
expressing the natural peptide, compared to the analog A42L or the
wild-type peptide itself, as boost agents. This correlated with the
binding and stability parameters determined by ex vivo
experimentation.
Example 12
Use of Analogs to Trigger Enhanced Immunity Against Natural
Epitoper, Assessed In Vivo
[0398] Eight groups of mice (n=4) were immunized with plasmid
expressing SSX-2.sub.41-49 natural epitope, by direct inoculation
into the inguinal lymph nodes with 25 ug in 25 ul of PBS/each lymph
node at day 0, 3, 14 and 17. This was followed by two additional
peptide boosts (similar amount) at day 28 and 31, using a negative
control peptide (Melan A 26-35 "EAA"), natural peptide or analogs
as shown in FIG. 9.
[0399] To evaluate the in vivo response against natural peptide,
splenocytes were isolated from littermate control HHD mice and
incubated with 20 g/mL or 1 ug/ml of natural peptide for 2 hours.
These cells were then stained with CFSE.sup.hi fluorescence (4.0
.mu.M or 1 .mu.M for 15 minutes) and intravenously co-injected into
immunized mice with an equal number of control splenocytes stained
with CFSE.sup.lo fluorescence (0.4 .mu.M). Eighteen hours later the
specific elimination of target cells was measured by removing
spleen and PBMC from challenged animals and measuring CFSE
fluorescence by flow cytometry. The relative depletion of the
populations corresponding to peptide loaded splenocytes was
calculated relative to the control (unloaded) population and
expressed as % specific lysis. FIG. 10 (spleen) and 11 (blood) show
the in vivo cytotoxicity elicited by the regimens described in FIG.
7. Three of the tested peptides (A42V, K41F and K41Y) showed
increased activity relative to the natural peptide, both in spleen
and blood, against target cells coated with 20 as well as lug/ml of
natural peptide. Interestingly, there was only limited correlation
between binding, stability of analogs in regard to the interaction
with MHC, and the capability to generate in vivo immunity against
natural peptide (FIG. 11).
Example 13
Use of Analogs to Trigger Enhanced Responses Against Tumor
Cells
[0400] Eight groups of mice (n=4) were immunized with plasmid
expressing SSX-2.sub.41-49 natural epitope, by direct inoculation
into the inguinal lymph nodes with 25 ug in 25 ul of PBS/each lymph
node at day 0, 3, 14 and 17. This was followed by two additional
peptide boosts (similar amount) at day 28 and 31, using a negative
control peptide (Melan A 26-35 "EAA"), natural peptide or analogs
as shown in FIG. 9.
[0401] One week after the boost, splenocytes were stimulated ex
vivo with SSX-2.sub.41-49 wild-type peptide and tested against
.sup.51Cr-labeled human tumor cells (624.38 melanoma cells) at
various E:T ratios (FIG. 12). Analog A42V and K41F A42V V491
elicited immune responses that mediated increased cytotoxicity
against human tumor cells expressing the natural SSX-2.sub.41-49
epitope.
Example 14
N-Terminal Proximal Primary Anchor Modification (2.sup.nd AA)
[0402] When the substituted analogs shown in Table 3 were tested,
the analogs showed improved binding and stability profiles in
comparison with the wild-type peptide epitope. However, the
magnitude of improvement for each analog varied, and the
substitution of A42V showed the highest improvement in terms of
binding affinity with HLA-A*0201 molecule. Further, the stability
of the A42V-HLA-A*0201 complex was better than the complex formed
between wild-type peptide and HLA-A*0201: the T1/2 extended from
11.5 hrs to 20 hrs. The peptides with 42 A to L, V and M
substitutions were able to induce the IFN-.gamma. secretion of
wild-type peptide specific CTL at remarkable lower concentrations.
The 42A to I substitution generated an analog with improved binding
and stability profile. The residue at the P2 position can also be
engaged in the interaction with TCR to a certain degree. This
observation was also supported by the results with the 42 A to Aib
analog, which possessed a similar binding affinity with HLA-A*0201
relative to the wild-type epitope.
Example 15
N-Terminal Secondary Anchor Modification (1.sup.st AA)
[0403] The N-terminal secondary anchor is the first amino acid at
the N-terminus. Thus, in one embodiment, the original Lys 43 found
in the wild-type sequence is substituted with a more hydrophobic
and bulky amino acid. Any more hydrophobic and bulky amino acid
also can be used, including any available to or that is known to
one of skill in the art, including standard amino acids and
non-standard amino acids. Examples of more hydrophobic amino acids
include, but are not limited to: Phe, Tyr, Trp, and D-Lys.
[0404] The residue of Lys 41 was defined as a secondary anchor
residue in interacting with HLA-A*0201 molecule, and it also
engaged in the interaction with the T cell receptors to a certain
degree. Therefore, modifications of this position can generate some
heteroclitic analogs that are more immunogenic and more suitable
for the development of tumor vaccines.
[0405] From Table 3, one could see that by replacing Lys 41 to Tyr,
Phe or Phe derivatives (Phenylglycine, Para-fluorophenylalanine,
Para-nitrophenylalanine), the resulting analogs have higher
affinity with the HLA-A*0201 molecule and form more stable
complexes. On the other hand, the Lys to Trp or Trp derivatives
analogs have shown significantly decreased affinity with the
HLA-A*0201 molecule although based on the predicted algorithms, the
Trp analog should have a similar affinity to that of the Tyr and
Phe analogs. The experimental data have demonstrated the limitation
of the predicted algorithms. For examples: Lys 41 to Phg
substitution has resulted in an analog with improved affinity and
extended stability with the HLA-A*0201 molecule, however, its
cross-reactivity with wild-type peptide specific CTL was fairly
poor. On the other hand, the para-nitrophenylalanine analog was
shown to induce the IFN-.gamma. secretion of the wild-type peptide
specific CTL at a much lower concentration, although its affinity
with the HLA-A*0201 molecule was about the same as that of
wild-type peptide.
Example 16
N-Terminal Primary/Secondary Anchor Modification
[0406] When both primary and secondary anchor residues at the
N-terminal were modified, a general trend was that resulting
analogs demonstrated improved affinity and extended stability with
the HLA-A*0201 molecule (Table 3), with only a few exceptions:
(K41Y, A42V), (K41Y, A42M) and (K41(D-Lys), A42V). Additionally,
they had very good cross-reactivity with the wild-type peptide
specific CTL. Combining the K41W substitution with A42V or A42L
improved the binding/stability profile, these analogs and also had
desirable cross-reactivity activity with the wild-type peptide. The
combination modifications of N-terminal primary anchor and
secondary anchor changed the peptide structure and conformation to
a greater degree.
Example 17
N-Terminal Primary/Secondary Anchor and C-Terminal Primary
Modification
[0407] The C-terminal Val of the wild-type peptide was a preferred
anchor residue. However, improved potency was observed when it was
mutated to Ile, having one additional --CH2 group; similar
improvement was also observed with a Val to Abu substitution.
Although the other analogs showed improved binding affinity and
stability with the MHC molecule, their cross-reactivity results
were poor. The results of these analogs indicated that the peptide
C-terminal anchor residue also plays a critical role in the
recognition of T cells. (Table 4).
Example 18
N-Terminal Primary/Secondary Anchor and TCR Sites Modification
[0408] Substitutions of secondary TCR binding amino acid residues
preferably generate heteroclitic analogs that did not interfere
with the binding to the MHC molecule, but overcame the tolerance
issues of self-antigens. By combining the substitutions of
N-terminal primary/secondary anchor residues (K41F and A42V) and
the TCR sites, analogs were generated with improved binding
affinity and stability (Table 6). Some of these analogs induced the
IFN-.gamma. production of the wild-type peptide specific CTL at
lower concentrations, such as K41F, A42V, E44(Nva) /(Nle) mutants
and K41F, A42V, 146L/(Nva) /(Nle) mutants.
Example 19
N-Terminal Amide
[0409] Replacing the peptide's free carboxylic acid C-terminus with
an amide improved the peptide's stability in biological media by
conferring stability to proteolysis and conferred dipeptidyl
carboxypeptidase resistance to the peptide. However, some of the
resultant analogs lost a significant amount of their affinity with
MHC molecules, as well as immunogenicity and antigenicity.
Interestingly, although the three analogs (Table 7) disclosed in
this application lost their binding capability with MHC molecule,
SSX-2.sub.41-49 --NH2 (A42V) retained its reactivity with wild-type
peptide specific CTLs as indicated by its capability of inducing
the secretion of IFN-.gamma. at a similar concentration to that of
the wild-type peptide. SSX-2.sub.41-49 --NH2 (A42L) was, however,
able to stimulate the IFN-.gamma. production at a lower
concentration.
Example 20
Decamers
[0410] The length of typical MHC binding peptides varies from 8-mer
to 11-mer, and most HLA-A*0201 binding peptides are 9-mers or
10-mers. In previous observations, a 9-mer and 10-mer from a
natural sequence were both found to possess a binding motif for the
same MHC, and had the same N-terminus. From the standpoint of
proteasomal processing they are distinct epitopes, but were
nonetheless antigenically cross-reactive. In the case of the
wild-type epitope SSX-2.sub.41-49, the epitope is a 9-mer peptide
and the 10-mer peptide, SSX-2.sub.41-50, lacks the appropriate MHC
binding motif and showed no immunological activity. Thus, the
wild-type epitope was therefore lengthened to a 10-mer with amino
acids that could create the appropriate binding motif. As shown in
Table 8, while many 10-mer analogs have a lower binding affinity
with the HLA-A*0201 molecule, analogs SSX-2.sub.41-50 (A42L,
Y50L/V/Nle/Nva) showed improved binding affinity with the
HLA-A*0201 molecule. Two 10-mer analogs in particular, A42L and Y50
Nle/Nva, were able to induce IFN-.gamma. production from T cells
immunized against the wild-type peptide at lower concentrations
than the wild-type peptide.
Example 21
Use of Analogs to Overcome Tolerization
[0411] One aspect in which the analogs can represent an improvement
over the wild-type epitope is in increased immunogenicity in a
human system and tolerance breaking. Differences in the TCR
repertoire, whether due to germ line differences or differences in
negative selection, have the potential to give anomalous results.
To address such issues the analogs are used in an in vitro
immunization of HLA-A2.sup.+ blood to generate CTL. Techniques for
in vitro immunization, even using naive donors, are known in the
field, for example, Stauss et al., Proc. Natl. Acad. Sci. USA
89:7871-7875, 1992; Salgaller et al. Cancer Res. 55:4972-4979,
1995; Tsai et al., J. Immunol. 158:1796-1802, 1997; and Chung et
al., J. Immunother. 22:279-287, 1999; each of which is hereby
incorporated by reference in their entirety.
[0412] Specifically, PBMCs from normal donors were purified by
centrifugation in Ficoll-Hypaque from buffy coats. All cultures
were carried out using autologous plasma (AP) to avoid exposure to
potential xenogeneic pathogens and recognition of FBS peptides. To
favor the in vitro generation of peptide-specific CTL, autologous
dendritic cells (DCs) were employed as APCs. DCs were generated and
the CTLs were induced with DCs and peptides from PBMCs as described
in Keogh et al., 2001, which is incorporated herein by reference in
its entirety. Briefly, monocyte-enriched cell fractions were
cultured for 5 days with GM-CSF and IL-4 and were cultured for 2
additional days in culture media with 2 .mu.g/ml CD40 ligand to
induce maturation. 2.times.10.sup.6 CD8+-enriched T
lymphocytes/well and 2.times.10.sup.5 peptide-pulsed DCs/well were
co-cultured in 24-well plates in 2 ml RPMI supplemented with 10%
AP, 10 ng/ml IL-7 and 20 IU/ml IL-2. Cultures were restimulated on
days 7 and 14 with autologous irradiated peptide-pulsed DCs.
Immunogenicity was assayed using the in vitro cytotoxicity and
cytokine production assays described herein.
Examples 22-30
Testing of NY-ESO-1.sub.157-165 Analogs
[0413] The analogs listed in FIG. 13 were tested for activity, such
as binding and biological effect as follows in Examples 22-30:
Example 22
Cross-Reactivity and Functional Avidity of Analogs Substituted at a
Single Position (FIGS. 13A-C).
[0414] The strategy from above (Example 6) was applied to scan
through a library of analogs bearing single substitutions relative
to the wild-type NY-ESO-1.sub.157-165 epitope in its native (or
wild-type) version (FIG. 13). Strong inverse correlation was found
between the minimal required amount of analog to elicit IFN-gamma
production ex vivo and the maximal amount of cytokine production at
any concentration of analog.
[0415] Substitution of S157 with F or K, resulted in analogs that
partially retained MHC binding and cross-reactivity with the T
cells specific for the nominal epitope. Substitution of L158 with I
improved the immunologic features of the peptide as assessed by
this methodology; whereas L158V resulted in partial retention of
activity. Modification of C165 with any of the amino acids V, L, A,
or I resulted in improved immune properties.
[0416] Peptides that have substitutions in the N-terminal position
or elsewhere, and present with retained but not increased activity
in this assay relative to the wild-type peptide, can be useful in
humans. In addition, they are material for further derivatization
to improve on their properties, as described below.
Example 23
Cross-Reactivity and Functional Avidity of Analogs Substituted at
Two Positions (FIG. 13A-C)
[0417] The strategy from above (Example 6) was applied to scan
through a library of analogs bearing two substitutions relative to
the wild-type NY-ESO-1.sub.157-165 epitope. Simultaneous
semi-conservative modifications at position 2 and 9 were shown to
have profound effects on the immune properties of analogs,
depending on the precise identity of the analogs. Combining L158I
with C165V or C165L further increased their activity relative to
the wild-type peptide. Similarly, L158V improved on the activity of
the C165V or C165L analogs, further increasing such activity
relative to wild-type peptide. L158V partially retained the
activity of C165A or C165I analogs, showing an interesting effect
of double mutation of primary anchor residues. Similarly, L158I
partially retained the activity of the C165A analog.
[0418] Simultaneous modifications at positions 1 and 9 had profound
effects on the immune properties of analogs, depending on the
precise identity of the analogs. S157Y combined with C165Nva
(norvaline) or Nle (norleucine) at position 9 resulted in
substantially improved activity over S157Y alone or the wild-type
peptide. The C165V mutant also rescued the activity of the S157Y
mutant. V--NH2 or L-NH2 at position 9 partially rescued the
activity of the S157Y analog--however, A-NH2 failed to do so.
Combinations between S157F and V, L, I, and to a lesser extent A at
the 9.sup.th position retained strong activity of the analog and
may be more useful than single mutants at position 9 due to the
participation of the first residue in the interaction with TCR.
Combinations between S157K and V, L, I and to a lesser extent A at
the 9.sup.th position, retained strong activity of the analog and
may be more useful than single mutants at position 9 due to the
participation of the first residue in the interaction with TCR and
the overall beneficial effect on the peptide solubility of K at
position 1.
Example 24
Cross-Reactivity and Functional Avidity of Analogs Substituted at
Multiple Positions (FIG. 13A-C)
[0419] The strategy from above (Example 6) was applied to scan
through a library of analogs bearing multiple substitutions
relative to the wild-type NY-ESO-1 epitope.
[0420] L158Nva or L158Nle considerably improved on the activity of
the S157Y C165V mutant. Combinations between V or I at position 158
and V, L, A or I at 165 can partially restore the potency of
analogs relative to the wild-type peptide. S157Y L158I C165V
displayed increased activity relative to the wild-type peptide and
S157V with C165V or C165I; and S157I with C165L or I, retained MHC
binding and cross-reactivity with T cells specific for the
wild-type peptide.
[0421] Triple substitutions comprising Y and V at positions 157 and
165, respectively, in addition to L or N at 160; A, L, V, or N at
162; or E, D or T at 164, retained the activity of the peptide in
this cross-reactivity assay, making these analogs useful compounds
for breaking T cell tolerance in vivo since positions 160, 162 and
164 participate in the interaction with TCR.
[0422] Triple substitutions comprising 157F and 158V plus V, L, I
at the position 165 showed activity in the assay described in
Example 2. In addition, triple mutants encompassing S157F and L1581
plus V or A at position 165 retained activity. Together, these data
underline the complex interactive and non-linear nature of multiple
substitutions.
[0423] Finally, triple mutants comprising S157W and to a higher
extent S157T together with 158V and 165V, showed retained or
increased activity respectively, relative to the wild-type
peptide.
Example 25
Cross-Reactivity and Functional Avidity of Decamers Encompassing
the Wild-Type Peptide and Mutated at Various Positions (FIG.
13A-C)
[0424] The strategy from above (Example 6) was applied to scan
through a library of analogs of a decamer encompassing the nominal
NY-ESO-1.sub.157-165 peptide. While the decamer itself lacked
significant in vitro activity, various substitutions at this
position partially rescued activity, such as L at 166, or L, I, Nle
at 166 combined with Y at 157 and V at 165.
[0425] Peptide analogs with similar or reduced activity in vitro
(but with retained cross-reactivity) compared to the wild-type
peptide are still useful for induction or boost of immune responses
due to: i) more limited AICD (antigen-induced cell death); ii)
higher in vivo activity due to increased stability on class I MHC
and/or slightly modified interaction with TCR. Thus, these analogs
are useful for breaking tolerance.
Example 26
Evaluation of Immunologic Properties of Analogs: Peptide Binding to
MHC Class I Molecules (FIG. 13A-C)
[0426] The affinity of peptide analogs and the wild-type epitope to
HLA-A*0201 was evaluated by T2 cell based assay (ref. Regner M, et
al., Exp Clin Immunogenet. 1996;13(1):30-5), which is incorporated
herein by reference in its entirety. For the binding assay, in
brief, T2 cells, that lacked expression of TAP and thus do not
assemble stable MHC class I on the cell surface, were pulsed with
different concentrations of peptides (controls or analogs)
overnight at 37.degree. C., washed extensively, stained with
fluorescently tagged antibody recognizing MHC class I (A2 allele)
and run through a FacsScan analyzer. Peptides that bind A2
stabilize its presence at the cell surface. The difference between
the MFI (mean fluorescence intensity) corresponding to a given
concentration of analog and the negative control (a non-MHC binding
peptide) is a function of how many stabilized complexes between MHC
and peptide are displayed on the surface of T2 cells. Thus, at
limiting concentrations of peptide, this is a measurement of
K.sub.on mostly and at saturation levels of peptide that is a
measurement of both K.sub.on and K.sub.off. In FIG. 13 the binding
is quantified by two factors that are mathematically related: Half
maximal binding (the peptide concentration giving 50% of the signal
corresponding to saturation) and relative affinity (1/RA), that is
binding normalized to a reference (wild-type peptide)--i.e. the
ratio between half maximal binding of control relative to peptide
analog. The higher the 1/RA index and the lower the half maximal
binding, the higher the K.sub.on of the interaction between an
analog and the MHC molecules. In FIG. 13, there are 39 analogs
described with such binding parameters improved relative to the
wild-type peptide. Such improved binders carry one, two, three, or
more substitutions of standard and/or non-standard amino acids at
positions that are known to participate in the interaction with MHC
and/or TCR, with an overall effect on MHC binding that is dependent
on precise/conjugated modification. Such peptide analogs are useful
in therapeutic compositions or as a platform to further derive
therapeutic compositions.
Example 27
Method of Immunization (FIG. 14)
[0427] Eight groups of mice (n=4) were immunized with a plasmid
expressing the wild-type NY-ESO-1.sub.157-165 epitope, by direct
inoculation into the inguinal lymph nodes with 25 ug in 25 ul of
PBS into each lymph node at days 0, 3, 14 and 17. This was followed
by two peptide boosts (similar amount) at day 28 and 31, using a
negative control peptide (HBVc), wild-type peptide or analog as
shown in FIG. 14.
Example 28
Use of Analogs to Trigger Enhanced Immunity Against Wild-Type
Epitope, Assessed In Vivo (FIGS. 15A-C)
[0428] To evaluate the in vivo responses obtained against the
wild-type epitope, splenocytes were isolated from littermate
control HHD mice and incubated with 20 .mu.g/mL or 1 .mu.g/ml of
wild-type peptide for 2 hours. These cells were then stained with
CFSE.sup.hi and CFSE.sup.med fluorescence (4.0 .mu.M or 1 .mu.M,
respectively, for 15 minutes) and intravenously co-injected into
immunized mice with an equal number of control splenocytes stained
with CFSE.sup.lo fluorescence (0.4 .mu.M). Eighteen hours later the
specific elimination of target cells was measured by removing the
spleens and PBMC from challenged animals and measuring CFSE
fluorescence by flow cytometry. The relative depletion of the
populations corresponding to peptide loaded splenocytes was
calculated relative to the control (unloaded) population and
expressed as % specific lysis. FIG. 15A shows the lack of in vivo
cytotoxicity in mice receiving the negative control peptide. FIG.
15B shows the variable cytotoxicity in mice immunized with plasmid
and amplified with wild-type peptide. FIG. 15C shows the
substantial, constant cytotoxicity in mice immunized with plasmid
and amplified with the analog L158Nva C165V.
Example 29
Comparison of Various Analogs in Triggering Enhanced Immunity
Against the Wild-Type Epitope, Assessed In Vivo (FIGS. 16A-B)
[0429] In the context of the immunization protocol described above
(Example 8) and using the methodology described in the Example 9,
in vivo cytotoxicity against target cells coated with limited (1
.mu.M; FIG. 16A) or supraoptimal amounts of wild-type peptide (20
.mu.M, FIG. 16B) was evaluated subsequent to the entrain and
amplify protocol using plasmid and peptide analog respectively for
the two stages. Results expressed as average % specific lysis.+-.SE
showed that the analog L158V C165Nva induced the highest activity
and that the analogs L158V C165V, L158V C165Nva and S157K L158V
C165V showed an effect in the same range with wild-type peptide or
the C165V mutant. Since multiple substitutions may alter the TCR
binding site, such analogs can be more useful than the wild-type
peptide in breaking tolerance against a self epitope. In addition,
the S157K triple mutant can ameliorate the poor solubility of the
wild-type peptide or other analogs, with direct practical
implications.
Example 30
Use of Analogs to Trigger Enhanced Immunity Against the Wild-Type
Epitope, Assessed Ex Vivo by Cytokine Production (FIGS. 17A-B)
[0430] In the context of the immunization protocol described above
(Example 27), and following the challenge described in Example 28,
splenocytes were isolated, pooled and stimulated in vitro with 10
.mu.M of wild-type peptide NY-ESO-1.sub.157-165 for 3 and 6 days
respectively. Supernatants were harvested and the concentration of
IFN-.gamma. measured by ELISA. Analog L158Nva C165V induced T cells
that produced large levels of IFN-gamma more rapidly upon ex vivo
stimulation (FIG. 17A). Other analogs such as S157F L158V C165V,
L158V C165Nva, and L158V C165V induced T cells that produced
increased amounts of IFN-gamma upon ex vivo re-stimulation with
wild-type peptide (FIG. 17B). In contrast, C165V failed to induce
increased capability of T cells to produce IFN-.gamma., relative to
the wild-type peptide following the protocols described in Examples
27-28.
Example 31
Characterization of binding and Stability by ELISA (iTopia
Testing)
[0431] Avidin-coated microtiter plates containing class I monomer
loaded with a so-called placeholder peptide were used to evaluate
peptide binding, affinity and off-rate. The monomer-coated plates
were supplied as part of the iTopia Epitope Discovery System Kit
(Beckman Ciulter, Inc., San Diego, Calif., USA). Assay buffers,
anti-MHC-FITC mAb and beta2-microglobulin and control peptides were
also supplied with the kits.
Binding Assay:
[0432] Native peptide and analogs were first evaluated for their
ability to bind each MHC molecule by binding assay. This assay
measures the ability of individual peptides to bind HLA molecules
under standardized optimal binding conditions. Monomer-coated
plates were first stripped, releasing the placeholder peptide and
leaving only the MHC heavy chain bound to the plate. Test peptides
were then introduced under optimal folding conditions, along with
the anti-MRC-FITC mAb. Plates were incubated for 18hrs at
21.degree. C. The anti-MHC-FITC mAb binds preferentially to a
refolded MHC complex. Therefore, the fluorescence intensity
resulted from each peptide was related to the peptide's ability to
form complex with MHC molecule. Each peptide's binding was
evaluated relative to the positive control peptide provided in the
kit, and the results were expressed `% binding`. The analogs
identified as `better-binders` in relative to the native peptide
were subsequently analyzed in the affinity and off-rate assays.
Affinity Assay:
[0433] For the affinity assay, after the initial stripping of the
placeholder peptide, increasing concentrations (range 10.sup.-4 to
10.sup.-8 M) of each test peptides for a given allele were added to
a series of wells and incubated under the conditions described
previously. Plates were read on the fluorometer. Sigmoidal dose
response curves were generated using Prism software. The amount of
peptide required to achieve 50% of the maximum was recorded as
ED.sub.50 value.
Off-Rate Assay:
[0434] For the off-rate assay, the plates were washed after 18 hrs
incubation at 21.degree. C. to remove the excess amount peptide.
The plates were then incubated on the allele-specific monomer
plates at 37 C. The plates were measured at multiple time points
(0, 0.5, 1, 1.5, 2, 4, 6 and 8 hrs) for relative fluorescence
intensity. The time required for 50% of the peptide to dissociate
from the MHC monomer is defined as the T1/2 value (hrs).
iScore Calculation:
[0435] The iScore is a multi-parameter calculation provided within
the iTopia software. Its value was calculated based on the binding,
affinity and stability data.
Example 32
Validation of the Antigenicity of PSMA.sub.288-297
[0436] HHD transgenic mice (n=4) were immunized with
PSMA.sub.288-297 peptide (25 .mu.g in 25 .mu.l of PBS, plus 12.5
.mu.g of pI:C to each lymph node) at day 0, 3, 14 and 17. One week
after the boost, splenocytes were stimulated ex vivo with the
native PSMA.sub.288-297 peptide and tested against
.sup.51Cr-labeled human tumor cells (PSMA.sup.+ A2.sup.+ LnCap
cells, or as negative control, LnCap cells coated with MHC class
I-blocking antibody) at various E:T ratios. The results expressed
as % specific lysis (mean.+-.SEM), showed that PSMA--specific T
cells were able to lyse human tumor cells in a fashion dependent on
MHC class I availability, confirming display of the PSMA epitope on
MHC class I of tumor cells, in a fashion allowing immune mediated
attack (FIG. 18).
Examples 33-38
Testing of PSMA.sub.288-297 Analogs
[0437] The analogs listed in FIGS. 19 and 20 were tested for
various properties such as improved affinity and stability of
binding, cross-reactivity with the native epitope, and
immunogenicity as follows in Examples 33-38.
Example 33
Cross-Reactivity and Functional Avidity of Analogs Substituted at
Single Position
[0438] Using the procedures described in Example 31 the binding
characteristics of PSMA.sub.288-297 and analogs were assessed in
comparison to each other (see FIG. 19). The positive control for
binding was melan-A.sub.26-35 A27L. Cross reactivity with the
native epitope was assessed by using the analog peptides to
stimulate IFG-gamma secretion from a T cell lines specific for the
native epitope, essentially as described in example 6. The data
shown in FIG. 19 was generated by stimulating with 10 .mu.g/ml of
analog (approximately 10 .mu.M). This concentration generally
resulted in maximal or near-maximal IFN-gamma production for the
analogs and thus was chosen to represent cross-reactivity.
[0439] The observed affinities of the analogs are reported in FIG.
19 as ED50s. Met, Ile, Gln, Val, Nva, Nle, and Abu were substituted
at the P2 position. These generally resulted in similar affinity.
The Nle and Met substitutions also maintained similar stability of
binding, measured as half-time of dissociation in hours. The Val,
Nva, and Abu analogs elicited a similar level of IFN-gamma
production.
[0440] Val, Leu, Nva, and Nle were substituted for the Ile at the
P.OMEGA. primary anchor position. All four had similar binding
affinity. The Val and Nva substitutions improved the stability of
binding and increased the amount of IFN-gamma produced, indicating
cross-reactivity and that the analogs can have improved
immunogenicity.
[0441] The Ser, Sar, and Abu substitutions at P1 maintained similar
binding characteristics but had marginally similar
cross-reactivity. The Ala, Leu, Ser, and Thr substitutions at the
P.OMEGA.-1 position also maintained similar binding
characteristics. Finally the Trp substitution at P3 exhibited
affinity and stability of binding that were both increased about
twofold and IFN-gamma production that was within twofold of the
native peptide, all generally similar values.
Example 34
Cross-Reactivity and Functional Avidity of Analogs Substituted at
Two Positions
[0442] The pattern seen above, that substitutions in this epitope
did not greatly impair binding affinity, continued with the double
substitutions examined (FIG. 20) which uniformly displayed similar
or improved binding affinity compared to the native peptide. Among
the analogs with substitutions at both primary anchor positions
those with Nva of Nle at P2 and Val at P.OMEGA., and Val at P2 and
Nva at P.OMEGA. displayed improved binding stability and the former
two increased IFN-gamma production (data not available for the
3.sup.rd analog). The Val and Nva substitutions at P.OMEGA. were
also paired with Ala and Abu substitutions at P1. These analogs all
had robust binding stability and IFN-gamma production that was
improved compared to the single P.OMEGA. substitutions, thus
further improving the P1 substitutions. The P.OMEGA. Nva
substitution was also able to restore similar cross-reactivity to
the P3 Trp substitution.
Example 35
Cross-Reactivity and Functional Avidity of Analogs Substituted at
Three Positions
[0443] Triple substitutions as P1, P2, and P3; P1, P2, and
P.OMEGA.; P2, P3, and P.OMEGA.; and P1, P3, and P.OMEGA. were made
(FIG. 21). In all cases the P1 substitution was Ala, the P3
substitution was Trp, and the P.OMEGA. substitution Val or Nva. As
above affinity at least similar to the native peptide was
maintained. For the P1, P2, P3 class Nva and Nle at P2 improved the
stability of binding. This P2 Nva analog elicited a similar amount
of IFN-gamma while the Nle analog showed a substantial
increase.
[0444] For the P1, P2, P.OMEGA. class, Nva and Val at P2 and
P.OMEGA. in either combination improved binding stability. This P2
Nva P.OMEGA. Val analog also showed a substantial increase in
IFN-gamma production. (No data on the other). Val at both P2 and
P.OMEGA. in this triple substitution showed binding stability and
IFN-gamma production that was nearly halved from that of the native
peptide.
[0445] For the P2, P3, P.OMEGA. group only the Nva/WNV analog
showed improved binding or IFN-gamma production. For the two P1,
P3, P.OMEGA. analogs examined P.OMEGA. of Val or Nva improved
binding stability but poor cross-reactivity.
Example 36
Cross-Reactive Immunogenicity of Various Analogs
[0446] Groups of HHD transgenic mice (n=8) were immunized with
peptide (natural epitope PSMA.sub.288-297, or analogs bearing
substitutions at primary or secondary anchor residues), by direct
inoculation into the inguinal lymph nodes, with 25 .mu.g in 25
.mu.l of PBS+12.5 .mu.g of pI:C to each lymph node at day 0, 3, 14
and 17.
[0447] Mice were sacrificed at 10 days after the last boost,
splenocytes prepared and assessed for IFN-.gamma. production by
ELISPOT analysis. Various numbers of splenocytes/well were
stimulated with 10 g/ml of native peptide in ELISPOT plates coated
with anti-IFN-.gamma. antibody. At 48 hours after incubation, the
assay was developed and the frequency of cytokine-producing T cells
that recognized native PSMA.sub.288-297 peptide was automatically
counted. The data were represented in FIG. 22 as the number of spot
forming colonies/well (mean of triplicates.+-.SD). The data show
increased priming of immune responses against the native epitope
achieved by the 1297V and P290W analogs, with the other analogs
showing slightly higher (but significant) activity than the native
peptide (1297Nva or G288Abu or L289Nle 1297Nva). To the extent that
the poor immunogenicity of the native epitope reflects tolerance,
the improved activity of these analogs represents tolerance
breaking.
Example 37
Amplification by the 1297V Analog of the Response to
PSMA.sub.288-297 Induced by Plasmid
[0448] Two groups of HHD transgenic mice (n=8) were immunized with
plasmid expressing PSMA.sub.288-297, by direct inoculation into the
inguinal lymph nodes with 25 .mu.g in 25 .mu.l of PBS to each lymph
node at day 0, 3, 14 and 17. This was followed by two peptide
boosts (25 .mu.g) at day 28 and 31 with either the natural peptide
or the I297V analog.
[0449] Mice were sacrificed at 10 days after the last boost,
splenocytes prepared and assessed for IFN-.gamma. production by
ELISPOT analysis. Various numbers of splenocytes/well were
stimulated with 10 .mu.g/ml of native peptide in ELISPOT plates
coated with anti-IFN-.gamma. antibody. At 48 hours after
incubation, the assay was developed and the frequency of
cytokine-producing T cells that recognized the PSMA.sub.288-297
peptide was automatically counted. The data were represented in
FIG. 23 as frequency of specific T cells normalized to 0.5 million
responder cells (mean of triplicates+SD). The data show that
irrespective of the number of splenocytes/well, the frequency of
native epitope--specific T cells was considerably higher in the
mouse group immunized with the 1297V analog.
Example 38
Ex Vivo Cytotoxicity Against Human Tumor Cells
[0450] HHD transgenic mice (n=4) were immunized with plasmid
expressing the PSMA.sub.288-297 epitope, by direct inoculation into
the inguinal lymph nodes with 25 .mu.g in 25 .mu.l of PBS to each
lymph node at day 0, 3, 14 and 17. This was followed by two peptide
boosts (same amount) at day 28 and 31, with the analog I297V. One
week after the boost, splenocytes were stimulated ex vivo with the
native PSMA.sub.288-297 peptide and tested overnight against
.sup.51Cr-labeled human tumor cells (Lncap, A2.sup.+ PSMA.sup.+; or
624.38 A2.sup.+ PSMA.sup.- or control 624.28 cells A2.sup.-
PSMA.sup.-) at various E:T ratios. The resulting immunity was
effective in mediating cytotoxicity against Lncap (FIG. 24).
Example 39
Validation of the Antigenicity of PRAME.sub.425-433
[0451] HHD transgenic mice (n=4) were immunized with
PRAME.sub.425-433 peptide (25 .mu.g in 25 .mu.l of PBS, plus 12.5
.mu.g of pI:C to each lymph node) at day 0, 3, 14 and 17. One week
after the boost, splenocytes were stimulated ex vivo with the
native PRAME.sub.425-433 peptide and tested against
.sup.51Cr-labeled human tumor cells (PRAME.sup.+ A2.sup.+ 624.38
melanoma cells; or negative control 624.38 cells, deficient in A2
expression) at various E:T ratios. The results expressed as %
specific lysis (mean.+-.SEM), showed that PRAME-specific T cells
were able to lyse human tumor cells, confirming display of the
PRAME.sub.425-433 epitope on MHC class I of tumor cells, in a
fashion allowing immune mediated attack (FIG. 25).
Examples 40-48
Testing of PRAME.sub.425-433 Analogs
[0452] The analogs listed in FIGS. 26-28 were tested for various
properties such as improved affinity and stability of binding,
cross-reactivity with the native epitope, and immunogenicity as
follows in Examples 40-48. Using the procedures described in
Example 31 the HLA-A*0201 binding characteristics of
PRAME.sub.425-433 and 69 analogs were assessed in comparison to
each other. The positive control for binding was melan-A.sub.26-35
A27L. The observed affinities of the analogs are reported as %
binding (compared to the positive control) and ED50s. Stability of
binding as half time of dissociation. Cross reactivity with the
native epitope was assessed by using the analog peptides to
stimulate IFG-gamma secretion from a T cell lines specific for the
native epitope, essentially as described in Example 6. The data
shown in FIGS. 26-28 were generated by stimulating with analog
peptide at approximately 0.3 .mu.M. The results were collected from
three separate experiments and were normalized to the amount of
IFN-.gamma. elicited by the native peptide in each. In some cases
the reported values are the average of two determinations. An
asterisk "*" indicates that IFN-.gamma. production was not
distinguishable from background.
Example 40
Cross-Reactivity and Functional Avidity of Analogs Substituted at a
Single Position (FIG. 26)
[0453] Single substitutions of Val, Met, Ile, Nie, Nva, and Abu
were made for the Leu at the P2 primary anchor position. All of
these analogs exhibited % binding within 20% of the native peptide.
The ED50 was determined for the Met and Nva analogs. The former had
an affinity somewhat improved but comparable to the native peptide
while the latter's affinity was reduced about 3-fold, but was
still-comparable to the PSMA.sub.288-287 epitope. All of the P2
substitutions maintained binding stability at least similar to the
native peptide. The Met, Nle, and Nva analogs elicited IFN-.gamma.
production within twofold of the native peptide and the Val analog
somewhat less.
[0454] Single substitution of Lys, Phe, Tyr, Thr, Orn (ornithine),
and Hse (homoserine) were made for the Ser at the P1 position. All
of these analogs exhibited % binding within 20% of the native
peptide except for the Phe analog which exceeded that range on the
high side. The ED50 for the Lys analog has not been determined, but
the other five analogs had affinities within threefold of the
native peptide. Stability of binding was generally similar to the
native peptide with the Phe P1 analog showing greatest binding
stability in this group with a half time of dissociation of 17.7
hours compared to 12.2 hours for the native peptide. With the
exception of the Lys P1 analog, which elicited 40% of the
IFN-.gamma. of the native peptide, all of these analogs were
considered cross-reactive as they elicited IFN-.gamma. production
within twofold of the native peptide.
[0455] Single substitutions of Val, Ile, Ala, Nle, Nva, Abu, were
made to the P.OMEGA. anchor position, as well as modifying the
carboxy-terminus by the addition of an amide group. Measured
binding affinities were at least similar to native peptide.
Stability of binding was also generally similar with outliers of
the Nva analog at the high end, t1/2 of 17.2 hours, and the
C-terminal amide at the low end with a significantly reduced t1/2
of only 3 hours. The Val, Ile, Ala, and Abu P.OMEGA. analogs
exhibit less preferred cross-reactivity, but the others elicited
IFN-.gamma. production within twofold of the native peptide.
[0456] Single substitutions at positions primarily affecting TCR
interactions were also made: Nle, Nva, and Abu at P3 and P6, and
Ala, Ser, and Sar at P8. The P6 Nva analog produced IFN-.gamma.
within twofold of that of the native peptide, though the P6 Abu
analog was close at 44%.
Example 41
Cross-Reactivity and Functional Avidity of Analogs Substituted at
Two Positions
[0457] Double substitution analogs were created at P1 and P2, P2
and P.OMEGA., and P1 and P.OMEGA. using various combinations of the
single substitutions above (FIGS. 27A and 27B). None of the P1-P2
double substitutions examined had radical changes to binding
affinity or stability, but none of the exhibited significant
cross-reactivity in the IFN-.gamma. assay. A similar pattern is
seen with the P2-P.OMEGA. double substitution analogs, however, the
L426Nva L433Nle analog shows a significant level of
cross-recativity with the native peptide in the IFN-.gamma. assay
along with its similar, somewhat improved binding characteristics.
Finally for the P1-P.OMEGA. double substitutions, the examined
analogs also conformed to the general pattern of having at least
similar binding characteristics, but eliciting negligible
IFN-.gamma. in the cross-reactivity assay. The exception in this
grouping were the S425T L433Nle analog, which exhibited somewhat
improved binding stability and significant cross-reactivity, and
the S425F L433Nle analog, which had a more that fourfold reduced
ED.sub.50, a nearly doubled halftime of dissociation, and elicited
more IFN-.gamma. than the native peptide.
Example 42
Cross-Reactivity and Functional Avidity of Analogs Substituted at
Three Positions
[0458] Four triple substitution analogs were investigated, having
Phe or Thr at P1, Nva or Met at P2, and NMe at P.OMEGA.. The S425T
L426M L433Nle analog had similar affinity whereas the affinity was
improved for the other three analogs. Both analogs with P2 Nva
substitutions displayed increased stability of binding and
significant levels of cross-reactivity. See FIG. 28.
Example 43
Cross-Reactive Immunogenicity of the L426NVA L433NLE Analog
[0459] Two groups of HHD transgenic mice (n=8) were immunized with
a plasmid, pCTLR2 described in example 49 below, expressing
PRAME.sub.425-433 by direct inoculation into the inguinal lymph
nodes of 25 .mu.g in 25 .mu.l of PBS to each lymph node at day 0,
3, 14 and 17. This was followed by two peptide boosts (2.5 .mu.g)
at day 28 and 31, of native peptide or the PRAME epitope analog
L426Nva L433Nle.
[0460] Mice were sacrificed at 10 days after the last boost,
splenocytes prepared and assessed for IFN-.gamma. production after
in vitro stimulation at 0.5.times.10.sup.6 cells/well, with 10
ug/ml of native peptide. At 48 hours after incubation, the
supernatant was harvested and the concentration of IFN-.gamma.
produced in response to the PRAME.sub.425-433 peptide was measured
by ELISA. The data were represented in FIG. 29 and show a
significant enhancement of IFN-.gamma. production in mice boosted
with the PRAME.sub.425-433 L426Nva L433Nle analog.
Example 44
In Vivo Cytotoxicity Induced by the L426NVA L433NLE Analog
[0461] Two groups of HHD transgenic mice (n=8) were immunized as
described in Example 43 above.
[0462] To evaluate the in vivo responses obtained against the
native epitope, splenocytes were isolated from littermate control
HHD mice and incubated with 20 .mu.g/mL or 1 .mu.g/ml of native
peptide for 2 hours. These cells were then stained with CFSE.sup.hi
and CFSE.sup.med fluorescence (4.0 .mu.M or 1 .mu.M, respectively,
for 15 minutes) and intravenously co-injected into immunized mice
with an equal number of control splenocytes stained with
CFSE.sup.lo fluorescence (0.4 .mu.M). Eighteen hours later the
specific elimination of target cells was measured by removing the
spleens and PBMC from challenged animals and measuring CFSE
fluorescence by flow cytometry. The relative depletion of the
populations corresponding to peptide loaded splenocytes was
calculated relative to the control (unloaded) population and
expressed as % specific lysis. The results in FIG. 30 showed
preserved induction of cytotoxicity when the analog replaced the
natural peptide as a booster agent. The trend indicates that the
analog can improve on induction of cytotoxic immunity.
Example 45
In Vivo Cytotoxicity and Tetramer Staining
[0463] Seven groups of HHD transgenic mice (n=4) were immunized
with a plasmid, pCTLP2, expressing PRAME.sub.425-433 by direct
inoculation into the inguinal lymph nodes of 25 .mu.g in 25 .mu.l
of PBS to each lymph node at day 0, 3, 14 and 17. This was followed
by two peptide boosts (2.5 .mu.g) at day 28 and 31, of native
peptide, negative control (EAAGIGILTV peptide), or
PRAME.sub.425-433 epitope analogs bearing mutations at the primary
and/or secondary anchor residues--S425F, L426Nva L433Nle, S425T
L433NIe, and S425T L426Nva L433Nle.
[0464] To evaluate the in vivo response against native peptide,
splenocytes were isolated from littermate control HHD mice and
incubated with 0.2 ug/ml or 20 ug/ml of native peptide for 2 hours.
These cells were then stained with CFSE fluorescence (1 and 2.5
.mu.M respectively, for 15 minutes) and intravenously co-injected
into immunized mice with an equal number of control splenocytes
stained with CFSE.sup.lo fluorescence (0.4 .mu.M). Eighteen hours
later the specific elimination of target cells was measured by
removing the spleen from challenged animals and measuring CFSE
fluorescence in the resulting cell suspensions, by flow cytometry.
The relative depletion of the populations corresponding to
peptide-loaded splenocytes was calculated relative to the control
(unloaded) population and expressed as % specific lysis. In
addition, the frequency of PRAME.sub.425-433-specific T cells, was
evaluated by tetramer/CD8 co-staining. The boost with analogs
encompassing mutations at primary or secondary anchor residues
showed comparable immune activity as compared to the native
peptide, based on in vivo cytotoxicity and tetramer staining. The
analogs were capable of amplifying the immune response as shown by
comparison with the "EAA" group, boosted with an irrelevant
peptide. In that regard, analogs comprising S425F, L33Nle, L426Nva
L433Nle, S425T L433Nle, or S425T L426Nva L433Nle were all capable
of expanding the immunity against the native epitope, as assessed
by in vivo cytotoxicity. However, only the L433Nle, L426Nva
L433Nle, and S425T L426Nva L433NIe analogs expanded the subset of T
cells specific against the native epitope to a level significantly
higher that in mice primed with plasmid and boosted with the
negative control peptide (FIG. 31).
Example 46
Ex Vivo Cytokine Production
[0465] Three groups of HHD transgenic mice (n=4) were immunized
with a plasmid, pCTLR2, expressing PRAME.sub.425-433 by direct
inoculation into the inguinal lymph nodes of 25 .mu.g in 25 .mu.l
of PBS to each lymph node at day 0, 3, 14 and 17. This was followed
by two peptide boosts (2.5 .mu.g) at day 28 and 31, of the PRAME
epitope analogs L426Nva L433Nle and S425T L426Nva L433Nle or the
negative control peptide Melan A (EAAGIGILTV).
[0466] Mice were sacrificed at 10 days after the last boost,
splenocytes prepared and assessed for IFN-.gamma. production by
ELISA at 48 hours after incubation with 10 .mu.g/ml of native
peptide. The data were represented in FIG. 32 as cytokine
concentration in pg/ml (mean of triplicates.+-.SD). The data showed
ex vivo cytokine production by splenocytes from mice boosted with
both analogs, and greater response to L426Nva L433Nle than to S425T
1426Nva L433Nle.
Example 47
Ex Vivo Cytotoxicity Against a Human Tumor Cell Line After Peptide
Boost With Analog
[0467] HHD transgenic mice (n=4) were immunized with a plasmid,
pCTLR2, expressing PRAME.sub.425-433 by direct inoculation into the
inguinal lymph nodes of 25 .mu.g in 25 .mu.l of PBS to each lymph
node at day 0, 3, 14 and 17. This was followed by two peptide
boosts (2.5 .mu.g) at day 28 and 31, with the analog L426Nva
L433Nle. One week after the boost, splenocytes were stimulated ex
vivo with the native peptide and tested against .sup.51Cr-labeled
human tumor cells (PRAME.sup.+ 624.38 melanoma cells pretreated or
not with IFN-.gamma.; or negative control 624.38 cells, deficient
in HLA-A2 expression) at various E:T ratios. The analog L426Nva
L433Nle elicited immune responses that mediated significant
cytotoxicity against human tumor cells expressing A2 (624.38),
slightly elevated upon their pre-treatment with IFN.gamma.. In
contrast, no significant activity was measured against A2-624.28
control cells. See FIG. 33.
Example 48
In Vitro Immunization to PRAME.sub.425-433
[0468] In vitro immunization was carried out according to the
general scheme presented in FIG. 34. Peripheral blood mononuclear
cells (PBMCs) were obtained from healthy donors (HLA-A*0201.sup.+)
by Ficoll-separation. Fresh PBMCs (2.5.times.10.sup.6), together
with 5 ng/ml PRAME.sub.425-433 or peptide analog were plated in
T-cell culture medium. Subsequently 20 IU/ml of interleukin 2 was
added to each well after 72 and 96 hours and addition peptide (5
ng/ml) was added at day 7. Cultures were maintained for an
additional 10 days before effector cells were harvested and used in
tetramer staining. IVS PBMCs were labeled with PRAME.sub.425-433
tetramer and analyzed on the FACSCalibur (BD, San Jose, Calif.).
Quadrants were set based on negative controls, stained with
irrelevant HBV tetramer and SSX2 tetramer, and a minimum of 10,000
gated events were captured. Tetramer-positive cells are expressed
as a percentage of the lymphocyte population.
PRAME.sub.425-433-specific tetramers was significantly enhanced
following IVS with peptide analog as compared with native peptide.
See FIG. 35. This demonstrates that the analog can be a preferable
immunogen.
Example 49
pCTLR2, A Plasmid Expressing the PRAME.sub.425-433 EPITOPE
[0469] pCTLR2 is a recombinant DNA plasmid vaccine which encodes
one polypeptide with an HLA A2-specific CTL epitope, SLLQHLIGL,
from PRAME amino acid residues 425-433, and an epitope cluster
region of PRAME, amino acids 422-509. The cDNA sequence for the
polypeptide in the plasmid is under the control of
promoter/enhancer sequence from cytomegalovirus (CMVp) which allows
efficient transcription of messenger for the polypeptide upon
uptake by antigen presenting cells. The bovine growth hormone
polyadenylation signal (BGH polyA) at the 3' end of the encoding
sequence provides signal for polyadenylation of the messenger to
increase its stability as well as translocation out of nucleus into
the cytoplasm. To facilitate plasmid transport into the nucleus, a
nuclear import sequence (NIS) from Simian virus 40 has been
inserted in the plasmid backbone. One copy of CpG immunostimulatory
motif is engineered into the plasmid to further boost immune
responses. Lastly, two prokaryotic genetic elements in the plasmid
are responsible for amplification in E. coli, the kanamycin
resistance gene (Kan R) and the pMB bacterial origin of
replication. (See FIG. 36).
Immunogen Translation Product Sequence
[0470] The amino acid sequence of the encoded polypeptide (150
amino acid residues in length) is given below.
[0471]
malqsllqhliglsnlthvlypvplesyedihgtlhlerlaylharlrellcelgrpsmvwlsanp-
cp
hcgdrtfydpepilcpcfmpnkrsllqhliglgdaaysllqhliglispekeeqyiasllqhligtkrpsi-
krsllqhligl (SEQ ID NO:100).
[0472] The first 89 amino acid residues are an epitope cluster
region representing PRAME 422-509. Within this epitope cluster
region, a number of potential HLA A2-specific CTL epitopes have
been found using a variety of epitope prediction algorithms. Amino
acid residues 90-150 are an epitope liberation (SYNCHROTOPE.TM.
sequence) sequence with four copies of PRAME 425-433 CTL epitope
(boldface) embedded. Flanking the defined PRAME CTL epitope are
short amino acid sequences that have been shown to play an
important role in the processing of the PRAME CTL epitope. In
addition, the amino acid sequence ispekeeqyia (corresponding to
PRAME amino acid 276-286, in italics) is engineered into the
sting-of-beads region to facilitate the detection of expression of
encoded polypeptide.
[0473] Using a variety of immunological assays including tetramer,
ELISPOT, ELISA, and cytotoxicity, strong CTL responses specific for
epitope PRAME.sub.425-433 have been detected from HLA-A2 transgenic
mice immunized with the pCTLR2 plasmid, suggesting immunogenic
potency for pCTLR2. These data indicated that the plasmid has been
taken up by antigen presenting cells, the encoded polypeptide has
been synthesized and proteolytically processed to produce the
nonamer epitope peptide, and become HLA-A2 bound for
presentation.
Plasmid Construction
[0474] Stepwise ligation of sets of long complementary
oligonucleotides resulted in generation of cDNA encoding amino acid
residues in the "String-of-Beads" epitope liberation sequence
(amino acids 90-150). These cDNA bore appropriate cohesive ends for
restriction enzymes that can be used for further ligation with cDNA
encoding the PRAME epitope cluster region (amino acid 1-89), which
were amplified by performing PCR on cDNA encoding PRAME as
template. The entire insert was then ligated into vector backbone
between Afl II and EcoR I sites. The entire coding sequence was
verified by DNA sequencing.
Example 50
Generation of Antigen Specific T Cell Responses
[0475] H-2 class I-negative, HLA-A2.1-transgenic HHD mice were
housed under pathogen-free conditions and used for evaluation of
the immunogenicity of HLA-A2.1-restricted human tumor-associated
cytotoxic T lymphocyte (CTL) epitopes. Female mice 8-12 weeks of
age were used for intralymphatic immunization and for isolation of
splenocytes for in vivo cytotoxicity studies. The mice were
immunized via bilateral inguinal lymph node injection. Mice were
anesthetized by inhalation of isofluorane and surgeries were
conducted under aseptic conditions. Following preparation for
surgery, an incision 0.5 cm in length was made in the inguinal fold
and the inguinal lymph node was exposed. A maximum volume of 25
.mu.l (25 .mu.g) of plasmid DNA vaccine or peptide was injected
directly into the lymph node using a 0.5 mL insulin syringe. The
wound was closed with sterile 6-0 nylon skin sutures.
Example 51
Ex Vivo Cytotoxicity Against Human Tumor Cells
[0476] HHD transgenic mice (n=4/group) were immunized with the
plasmid pSEM (described more fully in U.S. patent application Ser.
No. 10/292,413 (Pub. No. 20030228634 A1)) incorporated by reference
above in its entirety) expressing melan-A.sub.26-35 A27L epitope
analog, by direct inoculation into the inguinal lymph nodes with 25
ug in 25 ul of PBS/each lymph node at day 0, 3, 14 and 17. This was
followed by two additional peptide boosts (same amount) at day 28
and 31, with the analogs A27L, A27Nva, or A27L V35Nva. One week
after the boost, splenocytes were stimulated ex vivo with the
native melan-A.sub.26-35 peptide and tested against
.sup.51Cr-labeled human tumor cells (624.38 cells) at various E:T
ratios. The resulting immunity after boosting with the A27L or
A27Nva analogs was comprable and more effective than the native
peptide EAAGIGILTV (FIG. 37). Since the priming plasmid expresses
the A27L analog the experiment had a potential bias in favor that
peptide, so that the substantial cytotoxicity obtained with the
A27Nva analog may be an underestimate of it potency if priming made
use of that same sequence.
Example 52
Tetramer Analysis
[0477] Enumeration of CD8+ antigen-specific T cells requires
cognate recognition of the T cell receptor (TCR) by a Class I
MHC/peptide complex. This can be done using Class I MHC tetramers
which are composed of a complex of four HLA MHC Class I molecules
each bound to the specific peptide and conjugated with a
fluorescent protein. Thus tetramer assays allow quantitation of the
total T cell population specific for a given peptide complexed in a
particular MHC molecule. Furthermore, since binding does not depend
on functional pathways, this population includes all specific CD8+
T cells regardless of functional status. The CTL response in
immunized animals was measured by co-staining mononuclear cells
isolated from peripheral blood after density centrifugation
(Lympholyte Mammal, Cedarlane Labs) with HLA-A*0201 MART1
(ELAGIGILTV)-PE MHC tetramer (Beckman Coulter, T01008) or a
Tyrosinase.sub.369-377 (YMDGTMSQV) specific tetramer reagent
(HLA-A*0201 Tyrosinase-PE, Beckman Coulter) and FITC conjugated rat
anti-mouse CD8a (Ly-2) monoclonal antibody (BD Biosciences). Data
was collected using a BD FACS Calibur flow cytometer and analysed
using cellquest software by gating on the lymphocyte population and
calculating the percent of tetramer.sup.+ cells within the
CD8.sup.+ CTL population.
Example 53
Tetramer Staining (Plasmid Priming, Peptide Boost--Native Versus
Analog)
[0478] Two groups of HHD transgenic mice (n=8) were immunized with
plasmid expressing Tyrosinase 369-377, by direct inoculation into
the inguinal lymph nodes with 25 ug in 25 ul of PBS/each lymph node
at day 0, 3, 14 and 17. This was followed by two additional peptide
boosts (similar amount) at day 28 and 31, of natural peptide or the
377Nva analog. Ten days later, the immune response was monitored
using a Tyrosinase 369-377 specific tetramer reagent (HLA-A*0201
Tyrosinase-PE, Beckman Coulter). Individual mice were bled via the
retro-orbital sinus vein and PBMC were isolated using density
centrifugation (Lympholyte Mammal, Cedarlane Labs) at 2000 rpm for
25 minutes. PBMC were co-stained with a mouse specific antibody to
CD8 (BD Biosciences) and the Tyrosinase tetramer reagent and
specific percentages were determined by flow cytometery using a
FACS caliber flow cytometer (BD). The percentages of Tyrosinase
specific CD8.sup.+ cells, show that replacement of the native
peptide with the analog, preserved the expansion of
Tyrosinase-specific subset. The trend indicates that the analog can
improve on the expansion of Tyrosinase specific T cells (FIG.
38).
Example 54
In Vivo Cytotoxicity and Tetramer Staining (Head to Head Comparison
Between Native Peptide and a Panel of Analog Candidates)
[0479] Four groups of HHD transgenic mice (n=6) were immunized with
plasmid (pSEM) expressing Tyrosinase.sub.369-377 and
Melan-A.sub.26-35 A27L epitopes, by direct inoculation into the
inguinal lymph nodes of 25 ug of plasmid in 25 ul of PBS per lymph
node at day 0, 3, 14 and 17. This was followed by two peptide
boosts (similar amount) at days 28 and 31, of Melan-A.sub.26-35
A27L into the left inguinal lymph node and Tyrosinase.sub.369-377
analogs, bearing substitutions at the primary and/or secondary
anchor residues, into the right lymph node. As controls, mice
immunized with plasmid only or naive mice were used.
[0480] To evaluate the in vivo response against natural Tyrosinase
and Melan A epitopes, splenocytes were isolated from littermate
control HHD mice and incubated separately, with 20 ug/ml of natural
peptide (Melan-A.sub.26-35 or Tyrosinase.sub.369-377) for 2 hours
in HL-1 serum free medium (Cambrex) at a concentration of
20.times.10.sup.6 cells/mL. These cells were then stained with CFSE
(Vybrant CFDA SE cell tracer kit, Molecular Probes) (1 and 2.5
.mu.M respectively, for 15 minutes) and intravenously co-injected
into immunized or naive control HHD mice with an equal number of
control non-peptide coated splenocytes stained with CFSE.sup.lo
fluorescence (0.4 .mu.M). Eighteen hours later the specific
elimination of target cells was measured by removing the spleen
from challenged animals and measuring CFSE fluorescence in the
resulting cell suspensions, by flow cytometry. The relative
depletion of the populations corresponding to peptide-loaded
splenocytes was calculated relative to the control (unloaded)
population and expressed as % specific lysis. In addition, the
frequency of Tyrosinase.sub.369-377- and Melan-A.sub.26-35-specific
T cells, was evaluated by tetramer/CD8 co-staining
(HLA-A*0201-tetramers, Beckman Coulter).
[0481] The tyrosinase analog V377Nva was capable of expanding the
population of tyrosinase-specific T cells and amplifying cytotoxic
immunity, similarly to the native peptide and greater than the
Tyrosinase analog M370V V377Nva (FIG. 39).
Example 55
Ex Vivo Cytotoxicity Against Human Tumor Cells
[0482] HHD transgenic mice (n=4/group) were immunized (according to
the general protocol in FIG. 40) with plasmid (pSEM) expressing the
Tyrosinase.sub.369-377 epitope, by direct inoculation into the
inguinal lymph nodes of 25 ug of plasmid in 25 ul of PBS per lymph
node at day 0, 3, 14 and 17. This was followed by two peptide
boosts (same amount) at day 28 and 31, with the native peptide or
analogs bearing substitutions at primary anchor residues P2 and
P.OMEGA. (370 and 377). One week after the boost, splenocytes were
stimulated ex vivo with the native Tyrosinase.sub.369-377 peptide
and assayed against .sup.51Cr-labeled human tumor cells (624.38
cells) at various E:T ratios. Both the native peptide and the M370V
V377Nva analog generated robust cytotoxicity against 624.38 cells
(FIG. 41). Whereas there was some dilution of cytolytic activity
with the native peptide there was none with the analog reinforcing
the indication of greater immunogenicity gained from the tetramer
results in Example 52. Together with the preceding example, this
observation illustrates the usefulness of complementing more
stringent assays (in vivo cytotoxicity and tetramer staining) with
more sensitive assays (ex vivo cytotoxicity after in vitro
stimulation), to outline potentially useful analogs.
[0483] The various methods and techniques described above provide a
number of ways to carry out the invention. Of course, it is to be
understood that not necessarily all objectives or advantages
described may be achieved in accordance with any particular
embodiment described herein. Thus, for example, those skilled in
the art will recognize that the methods may be performed in a
manner that achieves or optimizes one advantage or group of
advantages as taught herein without necessarily achieving other
objectives or advantages as may be taught or suggested herein.
[0484] Furthermore, the skilled artisan will recognize the
interchangeability of various features from different embodiments.
Similarly, the various elements, features and steps discussed
above, as well as other known equivalents for each such element,
feature or step, can be mixed and matched by one of ordinary skill
in this art to perform methods in accordance with principles
described herein. Among the various elements, features, and steps
some will be specifically included and others specifically excluded
in diverse embodiments.
[0485] Although the invention has been disclosed in the context of
certain embodiments and examples, it will be understood by those
skilled in the art that the invention extends beyond the
specifically disclosed embodiments to other alternative embodiments
and/or uses and obvious modifications and equivalents thereof.
* * * * *