U.S. patent application number 16/433415 was filed with the patent office on 2020-05-21 for controlled modulation of amino acid side chain length of peptide antigens.
This patent application is currently assigned to Board of Regents, The University of Texas System. The applicant listed for this patent is Board of Regents, The University of Texas System Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc.. Invention is credited to Martin L. Campbell, Constantin G. Ioannides, Catherine A. O'Brian, George E. Peoples.
Application Number | 20200157146 16/433415 |
Document ID | / |
Family ID | 27807976 |
Filed Date | 2020-05-21 |
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United States Patent
Application |
20200157146 |
Kind Code |
A1 |
Ioannides; Constantin G. ;
et al. |
May 21, 2020 |
CONTROLLED MODULATION OF AMINO ACID SIDE CHAIN LENGTH OF PEPTIDE
ANTIGENS
Abstract
The invention provides a method for the creation of peptide
antigens comprising epitopes with at least a first modification
comprising a shortened or lengthened amino acid side chain. By
extension or shortening of the side chain with CH3/CH2 groups, for
example, made by computer assisted modeling of the tumor antigen
(peptide) bound in the MHC-I-groove, immunogenicity can be improved
with minimal modification of adjacent tertiary structure, thereby
avoiding cross-reactivity. Provided by the invention are methods of
creating such antigens, as well as methods for therapeutic or
prophylactic treatment of various conditions comprising
administration of the antigens.
Inventors: |
Ioannides; Constantin G.;
(Houston, TX) ; Campbell; Martin L.; (Colorado
Springs, CO) ; O'Brian; Catherine A.; (Chicago,
IL) ; Peoples; George E.; (San Antonio, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Board of Regents, The University of Texas System
Henry M. Jackson Foundation for the Advancement of Military
Medicine, Inc. |
Austin
Rockville |
TX
MD |
US
US |
|
|
Assignee: |
Board of Regents, The University of
Texas System
Austin
TX
Henry M. Jackson Foundation for the Advancement of Military
Medicine, Inc.
Rockville
MD
|
Family ID: |
27807976 |
Appl. No.: |
16/433415 |
Filed: |
June 6, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16169280 |
Oct 24, 2018 |
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16433415 |
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14445776 |
Jul 29, 2014 |
10239916 |
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16169280 |
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10507009 |
Mar 28, 2005 |
8802618 |
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PCT/US2003/006952 |
Mar 6, 2003 |
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14445776 |
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60412441 |
Sep 20, 2002 |
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60362778 |
Mar 8, 2002 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/4705 20130101;
C07K 7/06 20130101 |
International
Class: |
C07K 7/06 20060101
C07K007/06; C07K 14/47 20060101 C07K014/47 |
Goverment Interests
[0002] This invention was made with Government support under grant
numbers 17-97-I 7098 and I-01-299 awarded by the Department of
Defense. The Government has certain rights in the invention.
Claims
1. A method for preparing a peptide antigen with modulated
immunogenicity comprising substituting at least a first amino acid
located in a CTL epitope with a first substitute amino acid having
an extended or shortened side chain as compared to the first amino
acid.
2. The method of claim 1, wherein the first substitute amino acid:
has the same base residue as the first amino acid; b) is a
non-natural amino acid; c) extends the side chain of the first
amino acid; d) adds a --CH.sub.2/CH.sub.3 group to the side chain
of the first amino acid; e) adds two --CH.sub.2/CH.sub.3 groups to
the side chain of the first amino acid; f) shortens the side chain
of the first amino acid; g) reduces one --CH.sub.2/CH.sub.3 group
on the side chain of the first amino acid; h) acid reduces two
--CH.sub.2/CH.sub.3 groups on the side chain of the first amino
acid; i) eliminates an --OH group from the side chain of the first
amino acid; j) eliminates an --NH.sub.2 group from the side chain
of the first amino acid; or k) adds an --NH.sub.2 group to the side
chain of the first amino acid.
3. (canceled)
4. The method of claim 1, wherein the side chain of the first
substituted amino acid is an aliphatic side chain.
5.-13. (canceled)
14. The method of claim 1, further comprising determining the CTL
epitope of the antigen.
15. The method of claim 1, further comprising modeling the CTL
epitope while bound in the MHC-1 groove or the MHCII groove.
16. (canceled)
17. The method of claim 1, further comprising a) substituting a
second amino acid located in the CTL epitope with a second
substitute amino acid having an extended or shortened side chain as
compared to the second amino acid in the CTL epitope; b)
substituting a second and third amino acid located in the CTL
epitope with a second and third substitute amino acid each having
an extended or shortened side chain as compared to the second and
third amino acid of the CTL epitope; or c) substituting a second,
third and fourth amino acid located in the CTL epitope with a
second, third and fourth substitute amino acid each having an
extended or shortened side chain as compared to the second, third
and fourth amino acid of the CTL epitope.
18.-19. (canceled)
20. The method of claim 1, wherein the antigen is a tumor
antigen.
21. The method of claim 20, wherein the tumor antigen is derived
from breast cancer, ovarian cancer, prostate cancer, blood cancer,
skin cancer, uterine cancer, cervical cancer, liver cancer, colon
cancer, lung cancer brain cancer, head & neck cancer, stomach
cancer, esophageal cancer, pancreatic cancer, or testicular
cancer.
22. The method of claim 21, wherein the tumor antigen is HER-2.
23. The method of claim 1, wherein the antigen is a viral antigen
bacterial antigen or a parasitic antigen.
24.-25. (canceled)
26. The method of claim 1, wherein modulated immunogenicity of the
peptide antigen comprises an increase in the antigen's ability to
selectively activate high-avidity or low-avidity CTL
precursors.
27. (canceled)
28. The method of claim 1, wherein the modulated immunogenicity of
the peptide antigen comprises a) an increase in the antigen's
ability to protect CTLs from activation induced cell death, b) an
increase in the antigen's ability to selectively activate cytokine
production, c) an increase in the antigen's ability to induce CTL
proliferation, d) increases the affinity of the antigen for a T
cell receptor or e) reduces interactions that interference with T
cell receptor binding.
29.-32. (canceled)
33. A method of inducing immunity in a subject comprising
administering to said subject a modified peptide antigen comprising
a CTL epitope, wherein said modified peptide antigen has at least
one amino acid with a length-modified side chain, as compared to
the amino acid in same position, within the naturally occurring CTL
epitope.
34. The method of claim 33, wherein the subject is a human.
35. The method of claim 33, wherein said modified peptide antigen
is a modified tumor peptide antigen.
36. The method of claim 33, wherein the length-modified side chain
of the modified peptide antigen a) is extended as compared to the
amino acid in the same position in the natural CTL epitope, or b)
is shortened as compared to the amino acid in the same position in
the natural CTL epitope.
37.-40. (canceled)
41. The method of treating a HER-2 related cancer comprising
administering to said subject a modified E75 peptide, wherein said
modified E75 peptide has at least one amino acid with a
length-modified side chain, as compared to the amino acid in the
same position in the natural E75 peptide.
42. The method of claim 41, wherein the HER-2 related cancer is
breast or ovarian cancer.
43. A peptide antigen with modulated immunogenicity comprising at
least a first substituted amino acid having an extended or
shortened side chain as compared to the amino acid amino acid in
same position in the natural CTL epitope.
44. The method of claim 33, wherein the modified peptide antigen
further comprises a) a second amino acid with a length-modified
side chain, b) a second and third amino acid with a length-modified
side chain, or c) a second, third and fourth amino acid with a
length-modified side chain.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 16/169,280, filed Oct. 24, 2018, which is a continuation of
U.S. application Ser. No. 14/445,776, filed Jul. 29, 2014, now U.S.
Pat. No. 10,239,916, which is divisional of U.S. application Ser.
No. 10/507,009, filed Mar. 28, 2005, now U.S. Pat. No. 8,802,618,
which is a national stage application under 35 U.S.C. .sctn. 371 of
International Application No. PCT/US03/06952, filed Mar. 6, 2003,
which claims priority to U.S. Provisional Patent Application Ser.
Nos. 60/362,778, filed Mar. 8, 2002, and 60/412,441, filed Sep. 20,
2002, each of which is incorporated in its entirety by
reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0003] The present invention relates generally to the fields of
immunology and cancer biology. More particularly, it concerns
modified peptide antigen compositions and methods of use
therefor.
2. Description of Related Art
[0004] Immunotherapy refers to the technique of using a patient's
immune system against tumor cells or infectious organisms. With
respect to cancer, the objective is to direct the patient's immune
system against tumor cells by targeting antigens (Ag) that are
specific to or preferentially expressed by tumor cells. These
antigens thus represent a potential target for methods and
compositions of immunotherapy. However, some antigens are present
either in low levels in normal cells or in fetal development. For
example, oncofetal antigen is a carcinoembryonic antigen (CEA)
which is expressed in fetal development and in most adenocarcinomas
of entodermally-derived digestive system epithelia, as well as in
breast tumor cells and non-small-cell lung cancer cells (Thomas et
al., 1990).
[0005] As tumor antigen are self-antigen, they are recognized with
low-affinity by both cytotoxic T lymphocytes-tumor infiltrating
lymphocytes (CTL-TIL) and vaccination-induced CTL, because high
avidity (hi-av) CTL are silenced. In addition to being weak
immunogens, the effectors induced by antigen variants are often
cross-reactive rather than specific for the tumor antigen. A second
limitation of the antigen of the type used above is that the tumor
antigen is presented in small amounts, in part due to the decreased
levels of MHC-I expressed by the tumor compared with healthy
tissue. Thus, although a number of approaches have been developed
recently for tumor vaccination, these approaches have failed to
show significant effects both on cure-rate, and immunological
responses to vaccine treatment in patients. This poor
immunogenicity requires novel methods to improve the immunogenicity
of the tumor antigen.
[0006] Typically, the induction of tumor immunity by functional CTL
requires: (1) expansion of "naive" or "stand-in" precursors of
effector CTL (eCTL) to increase the pool of responders to tumor.
This is because disease progression may expand tumor cells to very
high numbers, thus only a large pool of CTL precursors can assure
expansion of eCTL to similarly high numbers, without exhaustion due
to end-stage proliferation and differentiation (2) generation of
hi-av eCTL which recognize even small amounts of antigen on tumor;
(3) protection of hi-av eCTL from deletion (elimination) at re
stimulation with antigen and cytokines; and (4) induction of hi-av
memory CTL (mCTL), from eCTL or activated CTL.
[0007] Recent advances provided partial answers to the first and
second requirements by: (1) expanding precursors of CTL for model
antigen using weak and null agonists; (2) identifying hi-av CTL in
melanoma, although in small numbers. The other requirements, hi-av
CTL protection from elimination and induction of mCTL, are still
poorly understood. However, novel approaches are needed to induce,
to protect from apoptosis, and to direct hi-av CTL to the memory
pool, as shifting the response to low-affinity CTL or non-specific
effectors occurs when enhancer antigen generated by sequence
changes induce cross-reactive CTL.
[0008] Developing successful immunotherapies, including cancer
therapies, thus imposes significant constraints for CTL induction,
because of (a) the tolerance and anergy induced by inappropriate
antigen stimulation plus type II cytokines; (b) the predominance of
low-affinity CTL in the periphery: either escaped from tolerance,
or induced by antigen and their agonists (an increase in the number
of eCTL may not compensate for their low affinity for tumors); (c)
the limited understanding of the relationship between the
activation of TCR signaling, cytokine signaling and activation of
survival pathways in mCTL; (d) costimulatory molecules, cytokine
receptors and death receptors are not clone specific; (e) induction
of memory cells requires either weaker costimulation and/or a
slower rate of proliferation of activated CTL than that of effector
CTL; and (f) survival effects are mediated by CD95 and Bcl-2 family
pathways. Therefore, there is a need for novel methods and
compositions for modulating a CTL response and for improved methods
of immunotherapy.
SUMMARY OF THE INVENTION
[0009] In one aspect, the invention provides a method for preparing
a peptide antigen with modulated immunogenicity comprising
substituting at least a first amino acid located in a CTL epitope
with a first substitute amino acid having an extended or shortened
side chain as compared to the first amino acid. The first
substituted amino acid may have the same base (i.e. be a derivative
or modification of the amino acid being substituted, such as having
a derivatized or modified side chain) or a different residue as the
first amino acid. The substituted amino acid may be a natural or
non-natural amino acid. In certain embodiments of the invention, a
modified side chain may be an aliphatic side chain. The first
substitute amino acid may extend or shorten the side chain. In one
embodiment of the invention, the first substitute amino acid adds
1, 2, 3, 4, 5 or more --CH.sub.2/CH.sub.3 groups to the side chain.
In another embodiment of the invention, the first substitute amino
acid shortens the side chain by 1, 2, 3 or more --CH.sub.2/CH.sub.3
groups on the side chain. A substitute amino acid may also
eliminate an --OH group from the side chain. In still further
embodiments of the invention, the first substitute amino acid
eliminates or adds an --NH.sub.2 group of a side chain. In certain
aspects of the invention, the amino acid substitution increases the
affinity of the antigen for a T cell receptor. In other embodiments
of the invention, the substitution reduces interactions that
interfer with T cell receptor binding.
[0010] In another aspect of the invention, the method for preparing
a peptide antigen with modulated immunogenicity further comprises
determining the CTL epitope of the antigen. In one embodiment of
the invention, the method for preparing a peptide antigen comprises
modeling a CTL epitope, including a CTL epitope bound in the MHC-I
or MHC-II groove.
[0011] In still another aspect of the invention, the method for
preparing a peptide antigen with modulated immunogenicity may
comprise substituting at least a second amino acid located in the
CTL epitope with a second substitute amino acid having an extended
or shortened side chain as compared to the second amino acid. The
method may also still further comprise substituting a third amino
acid located in the CTL epitope with a third substitute amino acid
having an extended or shortened side chain as compared to the third
amino acid. In still further embodiments of the invention, the
method may further comprise substituting a fourth amino acid
located in the CTL epitope with a fourth substitute amino acid
having an extended or shortened side chain as compared to the
fourth amino acid.
[0012] The antigen may, in one embodiment of the invention, be a
tumor antigen, including, for example, an antigen derived from
breast cancer, ovarian cancer, prostate cancer, blood cancer, skin
cancer, uterine cancer, cervical cancer, liver cancer, colon
cancer, lung cancer brain cancer, head & neck cancer, stomach
cancer, esophageal cancer, pancreatic cancer, or testicular cancer.
In one embodiment of the invention, the tumor antigen is HER-2. In
another embodiment of the invention, the antigen is a viral,
bacterial or parasitic antigen.
[0013] In the method of preparing a peptide antigen with modulated
immunogenicity, modulation of immunogenicity may comprise an
increase in the antigen's ability to selectively activate
high-avidity CTL precursors and/or low-avidity CTLs. Modulation of
immunogenicity may still further comprise an increase in the
antigen's ability to protect CTLs from activation induced cell
death. Modulation may also comprise an increase in the antigen's
ability to selectively activate cytokine production. In yet another
embodiment of the invention, modulation of immunogenicity may
comprise an increase in the antigen's ability to induce CTL
proliferation.
[0014] In still yet another aspect, the invention provides a method
of inducing immunity in a subject comprising administering to said
subject a modified peptide antigen comprising a CTL epitope,
wherein said antigen has at least one amino acid with a
length-modified side chain, as compared to the same position in the
natural molecule, within the CTL epitope. In the method, the
subject may be an animal, including a human. The modified peptide
antigen may be a modified tumor peptide antigen, and may also be a
viral, bacterial or parasite antigen. The length-modified side
chain may be extended or shortened as compared to the same position
in the natural molecule. The modified peptide may further comprise
a second amino acid with a length-modified side chain, as well as a
third or fourth amino acid with a length-modified side chain. Each
of these modified side chains may be shortened or lengthened as
compared to the same position in the natural amino acid.
[0015] In still yet another aspect, the invention provides a method
of treating a HER-2 related cancer comprising administering to said
subject a modified E75 peptide, wherein said peptide has at least
one amino acid with a length-modified side chain, as compared to
the same position in the natural molecule. In the method, the HER-2
related cancer may be breast or ovarian cancer.
[0016] In still yet another aspect, the invention provides a
peptide antigen with modulated immunogenicity prepared by
substituting at least a first amino acid located in a CTL epitope
with a first substitute amino acid having an extended or shortened
side chain as compared to the first amino acid. Still further
provided are vaccine compositions comprising the antigen as well as
methods for therapeutically or prophylactically treating a patient
for a tumor, viral, bacterial or parasitic disease comprising
administering the vaccine to the patient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0018] FIGS. 1A-1C. Induction or IFN-.gamma. by A7.2, A7.3 and G4.3
in three healthy donors of PBMC (FIGS. 1A, 1B and 1C) Autologous
MO-derived autologous DC were used as APC. Peptides were pulsed at
25 .mu.g/ml exogenous concentration. IFN-.gamma. was determined
from the supernatant using specific ELISA.
[0019] FIGS. 2A-2B. (FIG. 2A) 7.2-primed T cells from Donor 1
respond to E7S with higher induction of IFN-.gamma. "production
rate". The x-axis intercept, tentatively indicate, the amount of
A7.2 required to induce the same account of IFN-.gamma. as E7S.
(FIG. 2B) IL-2 production of CH2-E7S primed T cells at
restimulation with the same peptide.
[0020] FIGS. 3A-3C. (FIGS. 3A and 3B) Induction of higher CTL
activity in CTL-TIL-1 at priming and restimulation with A7.3.
S5.1+A7.2=specificity control immunogen made from A7.2 with Ser 5
replaced by homoserine (S5.1). (FIG. 3C) CTL-4 (E75 responding)
after three stimulations with A7.0, A7.2, and A7.3, A7.3-induced
CTL recognize E75 at 25 nM.
[0021] FIGS. 4A-4C. (FIGS. 4A and 4B) Priming with A7.2 followed by
restimulation with A7.3 increase the numbers of hi-av E7S-specific
CTL. (FIG. 4C) LU (E7s-specific) were determined from LU against
T2-E7s minus LU against T2-NP. Rested "post effector" A7.3 induced
CTL recognize E7s after restimulatlon with peptide.
[0022] FIGS. 5A-5D. CH2-E75 induced CTL recognize endogenously
presented epitope. (FIG. 5A) CTL-3 was primed with E75 and
restimulated with A7.2 and A7.3, respectively. (insert-IFN-7 and
IL-2 responses to A7.0, A7.2 and A7.3 at priming). (FIG. 5B)
Cold-target inhibition of lysis of SKOV3-A2 cells by CTL-3. A
subpopulation of E75-specific cells recognize endogenously
presented E75, with higher affinity based on 27% inhibition of
lysis by T2-E75 (at 100 nM). (FIGS. 5C and 5D) CTL-TIL-HI
recognition of SKOV3.A2 but not of SKOV3 is inhibited by T2-E75,
confirming the specificity of these CTL.
[0023] FIGS. 6A-6C. Two color-FACS analysis of F8-1 (FIG. 6B), E75
(FIG. 6A), and positive control (FIG. 6C) influenza
matrix-stimulated CD8.sup.+ cells--Donor 1 after culture in IL-1.
for 20 days. CD61L.sup.+ CFSE (upper left quadrant, 1). CD6L.sup.+
CFSE. cells (upper right quadrant, 2). F8-1 primed cells secreted
higher levels of IFN-.gamma. at restimulation with E75 (<200
pg/ml) E75 primed cells within 16 h (<75 pg/ml). In F8-1 cells,
IFN-.gamma. was also detected at 6 h.
[0024] FIGS. 7A-7B. (FIG. 7A) F42SK-CTL line were stimulated with
agonistic .alpha.Fas mAb (CH11) in the absence or presence of F42
or E75. Cell cycle analysis was performed 24 h and 96 h later in
CD8.sup.+ cells stained with propidium iodide (PI). Results
indicate % apoptotic cells; i.e. cells in the sub Go phase.
Exogenously pulsed F42 and E75 inhibited the residual Fas-apoptosis
on day 1, but only E75 inhibited on day 4. (FIG. 7B) F42SK-CTL were
restimulated with the indicated agonists pulsed on T2 cells. The
sensitivity of F42-stimulated cells to aFas was paralleled by Bad
up-regulation by F42 and lower Bcl-XL/Bad ratios than E75. (0 and
NP) indicate either nonstimulated cells or cells stimulated with T2
with peptide. Equal numbers of cells were lysed, separated by
SDS-PAGE, expression of Bcl-2, Bcl-XL and Bad determined with
specific antibodies followed by Scanning Densitometry. Numbers
indicate band intensity.
[0025] FIGS. 8A-8F Induction of effector functions in donor 1
(FIGS. 8A and 8B) and donor 2 (FIGS. 8C, 8D and 8E) at priming with
the wild-type CTL epitope E75 and its variants. FIGS. 8A and 8C,
IFN-.gamma.; FIGS. 8B, 8D and 8E, Cytolysis. FIGS. 8A and 8C,
IFN-.gamma. was determined from supernatants collected from the
same cultures which were used on day 8 for CTL assays. FIGS. 8B,
8D, and 8E, Equal numbers of effectors from each culture were
tested in the same study. Results indicate the percentage of
E75-specific lysis obtained by subtracting the specific lysis of T2
cells not pulsed with peptide, from the specific lysis of T2 cells
pulsed with 25 .mu.g/ml E75 in the same study. The E:T was 20:1.
Stimulators were autologous DCs pulsed with 25 .mu.g/ml peptide.
NPs indicate control effectors that were stimulated only with
autologous DCs which were not pulsed with peptide. FIG. 8E,
Effectors E75-CTL, S5K-CTL, and S5A-CTL lysed the indicator ovarian
tumor SKOV3.A2. Specific cold target inhibition indicated the
percentage of inhibition of lysis of SKOV3.2 cells by cold
(unlabeled) T2-E75 cells minus inhibition of lysis in the presence
of T2-NP cells. S5G-CTL were not used here because their numbers
declined rapidly after restimulation. E:T ratio was 30:1, cold:hot
ratio was 10:1. FIG. 8F, Percentage of live cells in donor 2
cultures primed and restimulated with each variant 30 days after
priming. Note the decrease in live cells in cultures stimulated
with S5A or S5G. *, p<0.05.
[0026] FIGS. 9A-9C. FIG. 9A, Kinetics of IFN-.gamma. production;
FIG. 9B, E75-specific CTL induction; and FIG. 9C, survival of donor
3 CTL stimulated by E75 and S5K. Study details as described in
Examples and the legend to the FIG. 8A, IFN-.gamma. was determined
on day 3 after stimulation with each peptide. The numbers 1, 2, and
3 indicate the number of stimulations. Equal numbers of live cells
from E75- and S5K-stimulated cultures were stimulated with
autologous DC pulsed with the corresponding peptide. FIG. 9C, The
number of live cells recovered was determined 1 wk after the third
and the fifth stimulations.
[0027] FIGS. 10A-10C. antigen specificity of S5A-CTL, S5K-CTL, and
E75-CTL. FIG. 10A, Donor 1 S5A-CTL recognized S5K less efficiently
than S5A. Donor 3 S5K-CTL recognized E75 with lower affinity than
S5K. T2 cells were pulsed with E75 and S5K at 10 .mu.g/ml. FIG.
10B, Donor 3 E75-CTL recognized S5K with lower affinity than
E75.
[0028] FIG. 10C, Donor 3 S5K-CTL recognized E75 with lower affinity
than S5K-CTL. Concentration dependent recognition of E75 and S5K in
the same study. Targets were T2 cells pulsed with the indicated
concentrations of peptide. FIGS. 10B and 10C, Results of a 6-h CTL
assay. E:T ratio was 10:1. *, p<0.05.
[0029] FIGS. 11A-11C. S5K-CTL recognized endogenous E75 presented
by ovarian tumor cells. FIGS. 11A and 11B, Cold target inhibition
of cytolysis of OVA-16 (HLA-A2, HER-2.sup.high). Cold targets were
T2 pulsed with E75, using as specificity control T2 which were not
pulsed with peptide (T2-NP). Numbers in the parentheses indicate
the percentage of inhibition of lysis of S5K-CTL by T2-E75 compared
with lysis of tumor in the presence of T2-NP. *, p<0.05. E:T
ratio was 10:1; the ratio of cold to hot targets was 1:1. C, IFN
.gamma. induction. IL-12 was used at 3 IU (300 pg/ml); the
responders to SKOV3. A2 stimulator ratio was 40:1.
[0030] FIGS. 12A-12D. Expansion of CD8.sup.+ cells from S5K-CTL
after stimulation with E75 (FIG. 12A) or S5K (FIG. 12B) in the
absence (o) or presence ( ) of CH11 mAb. Equal numbers of S5K-CTL
were stimulated with DCs pulsed with 0, 25, and 50 .mu.g/ml of each
peptide. The number of CD8.sup.+ cells was determined by
flow-cytometry using anti-CD8 mAb-FITC conjugated. FIG. 12C,
antigen-induced resistance to CD95-mediated apoptosis. S5K-CTL were
stimulated with autologous DCs pulsed with E75 or S5K at 5 and 25
.mu.g/ml or control no peptide (0). CH11 mAb was added 1 h later.
The number of apoptotic cells was determined 1 and 4 days later.
FIG. 12D, Restimulation with E75 and S5K-induced resistance to
CD95-mediated apoptosis in S5K-CTL stimulated 1 wk before with S5K.
Apoptotic cells are shown in the panel subG1. Results are from one
study representative of three independently performed studies. Bars
indicate unstimulated (.box-solid.), E75 stimulated (),
E75+anti-Fas stimulated (), S5K-stimulated (), and S5K.sup.+
anti-Fas stimulated ().
[0031] FIGS. 13A-13D. FIG. 13A, Expression levels of Bcl-family
members by S5K-CTL stimulated with the indicated peptides; or FIG.
13B, with PHA for 96 h. The same blot was used for probing with all
Abs. 1 indicates unstimulated; 2 indicates PHA-stimulated cells.
The numbers below the bands indicate the densitometric values
(pixel total.times.10.sup.-3) FIGS. 13C and 13D, Expansion of
E75.sup.+TCR cells in S5K-CTL stimulated in parallel with T2-E75
(E75), T2-S5K (S5K), or with T2-NP (NP) as control for 1 wk. The
presence of E75.sup.+TCR cells was determined using dE75 (y-axis).
Forward scatter (FW) is shown on x-axis. FIG. 13C, E75.sup.+TCR
cells expression in large lymphocytes (FW: 640-1000); FIG. 13D,
E75.sup.+TCR expression on small lymphocytes (FW: 380-600). The
percentage of dNP.sup..+-. cells ranged from 0.1-0.5% in both
populations.
[0032] FIGS. 14A-14D. Stimulation of S5K-CTL with E75 significantly
increased the number of E75.sup.+TCR cells. FIG. 14A, Percentage of
E75.sup.+TCR cells in the large () and small () lymphocytes was
determined immediately after staining and 50 min after washing and
incubation of cells in PBS to dissociate low-affinity
(t.sub.1/2<50 min) TCR-dE75 complexes. Most small lymphocytes
recognized E75 with t.sub.1/2 of <50 min, while .about.50% of
large lymphocytes had a t.sub.1/2 of 50 min for E75. FIG. 14B,
Increase in the numbers of E75.sup.+TCR cells of S5K-CTL after
stimulation with E75 and S5K large () and small () lymphocytes. The
numbers of live cells recovered after stimulation with T2-NP,
T2-E75, and T2-S5K, and expansion in IL-2 were 2.7, 3.2, and
2.9.times.10.sup.6 cells, respectively. FIG. 14C, Increased levels
of expression of E75.sup.+TCR in large lymphocytes stimulated with
E75 compared with S5K. The differences in MCF in small lymphocytes
were minimal: 202 for E75, 180 for S5K. FIG. 14D, Increased levels
of expression of Bcl-2 in E75.sup.+TCR large lymphocytes but not in
small lymphocytes at stimulation with E75 or S5K. All
determinations were performed in the same study. Results are from
one determination representative of two with similar results.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0033] The invention overcomes the limitations of the prior art by
providing methods for the modulation of TCR signaling using
modified antigens, thereby providing each of the required steps for
developing successful immunological therapies, including cancer
therapies. For example, this can be accomplished in accordance with
the invention by introducing discrete changes in the aliphatic side
chain length of the same residue, at the same position in the
stimulating antigen (Ag). This can comprise the addition or removal
of CH.sub.2 (methylene) groups from the side chain. CH.sub.2 groups
are smaller than OH and NH.sub.2 groups and do not form
electrostatic or H-bonds, but do form weaker hydrophobic van der
Waals bonds which increase in proportion to the number of CH.sub.2
added. These bonds should also modulate the avidity (or half-life)
of peptide for TCR, which is a requirement for transformation of an
antigen into a stronger or a weaker agonist. HAB antigen can also
modulate survival and avoid inducing apoptosis by over-stimulation
by decreasing the number of CH.sub.2 groups. T cell development
studies have shown that TCR modifies its response to antigen side
chain changes even at the level of one CH.sub.2 group.
[0034] By extension or shortening of the side chain with
CH.sub.3/CH.sub.2 groups, for example, done by computer assisted
modeling of the tumor antigen (peptide) bound in the MHC-I groove,
immunogenicity can be improved with minimal modification of
adjacent tertiary structure, thereby avoiding cross-reactivity.
Detection of T cell activation by this novel method allows
modification of the stimulating intensity of the tumor antigen by
CH.sub.2 deletion or addition, allowing circumvention of induction
of apoptosis by under/over-stimulation of lymphocytes.
[0035] The invention thus, in one aspect, provides methods of
activation of immunity to a tumor or pathogen as follows: (1) the
methods may be used for selective activation of high-avidity
precursors of tumor/pathogen reactive CTL using an attenuated
antigen from the tumor or pathogen comprising shortened CH.sub.2
side chains (type 1 agonists); (2) activation of low-avidity CTL
against the tumor/pathogen using CH.sub.2 side chain extended
antigen ("amplifying agonists") (type 2 agonists); (3) protection
from activation induced cell death (AICD) by re-stimulation of
cells previously activated with type 2 agonists above with even
more attenuated agonists (type 3) than the ones listed in type 1;
(4) selective activation of cytokine production or of both cytokine
production and proliferation by the manipulation of the CH.sub.2
side chain length at two distinct positions; (5) ability to target
the positions where the changes will be made using molecular
modeling of the tumor/pathogen antigen MHC-I, as well as
tumor/pathogen antigen-MHC-II complex; (6) applicability to most if
not all tumor or pathogen antigen. The poor immunogenicity of tumor
and some other antigen may not be due only to the low affinity of
the antigen for the TCR but also to interference of side chains
with interactions by other side chains with TCR. Therefore, the
invention may be used to overcome these limitations.
[0036] Vaccine therapies raise the need for hi-av CTL for the
peptide-MHC complex presented by the target antigen. Induction of
hi-av CTL using "heteroclitic antigen" requires replacement of core
residues in the peptide. While this approach was found to enhance
responses to some antigen, for others the resulting CTL were of
lower affinity for a target tumor than wild-type CTL. Excess
signaling by heteroclitic antigen and partial signaling by wild
type antigen could specifically eliminate both antigen-specific and
cross-reactive CTL in vivo, and this elimination extends to
bystander T-cells.
[0037] There were previously no approaches for modulation of TCR
signaling of hi-av CTL for protection from apoptosis, survival, and
progression to memory. This is important because: (a) TCR signals
at re-stimulation with the initiating antigen enhance the
susceptibility to Fas-mediated apoptosis, while IL-2 amplifies the
death inducing effects of Fas; (b) cessation of antigen stimulation
and withdrawal of growth factors also lead to death of effectors;
(c) extensive proliferation rapidly leads to generation of
end-stage of differentiated CTL, which die via apoptosis even
before disease recurrence. This shortens the life-span of mCTL; (d)
whether optimal generation of mCTL requires them to revert to a
resting Go/G.sub.1 phenotype and to proliferate slowly and
intermittently in response to antigen or continuously in response
to cytokines has not yet been elucidated; (e) the dependence of
cell survival on MAPK (ERK) controlled pathways raise the need for
intermittent antigen signaling (as an alternative) when cytokines
(IL-15) are absent or are below the levels that can activate
survival pathways.
[0038] Thus, the concept for vaccination by changing TCR signaling
in a subtle manner to direct the progression of CTL through the
desired steps entails the use of three immunogens targeting the
same CTL, but each acting at a defined step and endowed with the
ability to expand precursors, activate and expand eCTL, and protect
mCTL, respectively. HAB antigen maintain the core residues of the
wild type antigen with their charged and --OH groups, thus the
position and orientation in the binding pocket is unchanged.
I. MODIFIED ANTIGENS
[0039] Identification and modification of tumor or pathogen
antigens which are the target of CTL allows vaccination for
therapeutic or prophylactic benefit. A number of antigen, and in
particular, the majority of tumor antigen, are weak partial
agonists, which induce low levels of cytolysis by low-avidity CTL.
Little is known about the strategies that may render success in
using tumor or pathogen antigens for vaccination of a subject. The
outcome of TCR activation is dependent on the affinity of TCR for
the peptide-MHC. Extended TCR stimulation may activate TCR-negative
feedback pathways. The current invention can be used to avoid such
problems.
[0040] In accordance with the invention, however, induction of
high-avidity CTL can be carried out by modulation of TCR signaling
using modified antigen of focused specificity and increased
capability for van derWaals forces. Initial studies to demonstrate
the effectiveness of the approach were carried out using molecular
modeling of the HER-2 peptide E75-HLA-A2 complex and identification
of CH.sub.2 side chains pointing upwards and sideways. E75 is
recognized frequently by tumor reacting ovarian and breast CTL-TIL,
as well as by CTL from transgenic models. Phase I clinical studies,
show that E75 lacks toxicity, but induce immune responses being
presented by the tumor.
[0041] Modifications of the length of side-chains were made in
Gly4, Ala7, and Phe8 by replacement with NVal, NLeu and
HomoPhe(--CH.sub.2), respectively and Ser5 by replacement with Gly.
The corresponding immunogens were designated as G4.3, A7.2, A7.3
8.-1, and S5.-1, respectively. Of these, A7.2 induced higher levels
of IFN-.gamma. than A7.3 and G4.3 in cells from donors and ovarian
cancer patients which did not respond to E75, indicating increased
signaling by a CH.sub.2 extension. Further, A7.2-primed cells
responded to E75 faster with higher levels of IFN-.gamma., than
E75-primed cells. E75-specific CTL were induced by A7.2 and
A7.3.
[0042] A. Design of CH.sub.2-Modified Immunogens
[0043] The HER-2 peptide E75 (369-377) has been identified as an
immunodominant epitope recognized by ovarian tumor reactive CTL
(Fisk et al., 1995; Rongcun et al., 1999; zum Buschenfelde et al.,
2000). This raises the possibility of using E75 or of fragments of
HER-2 containing this epitope for cancer vaccination. E75 induced
IFN-.gamma. even in unfractionated PBMC in the majority of healthy
donors or E75 vaccinated patients (Lee et al., 2000; Zaks and
Rosenberg, 1998; Anderson et al., 2000). E75-induced CTL also
recognized HLA-A2.sup.+ HER-2.sup.+ tumors by secretion of
IFN-.gamma.. These effects could be augmented by addition of low
levels (100 pg/ml) of IL-12 (Anderson et al., 2000).
[0044] E75 is a weak inducer of cytolytic activity against tumor
cells (Zaks and Rosenberg, 1998; Anderson et al., 2000). The
cytolytic activity of E75 (peptide)-induced CTL was significantly
weaker against tumor cells expressing HER-2. Endogenously, E75 is
presented by tumor cells, indicating that E75 is an important
immunogen for induction of anti-tumor activity (zum Buschenfelde et
al., 2000; Fisk et al., 1997). E75 is a weak partial agonist of
which the ability to induce lytic effector does not improve by the
use of DC as APC, IL-2, IL-12, TNF-.alpha. or of various
pretreatments (IL-2, RANTES) of responders (Lee et al., 2000;
Anderson et al., 2000). This demonstrated the need for optimization
of immunogenicity of E75, to induce eCTL, and to enhance survival
of mCTL.
[0045] B. Modeling of CTL Epitope-MHC Complexes
[0046] One aspect of the invention comprises identifying a CTL
epitope and discerning the secondary structure of the complex
between CTL epitopes and class I and/or class II MHC molecules.
With this information, side chains involved in the interaction with
the T-cell receptor can be modified as described herein. Numerous
scientific publications have been devoted to the prediction of
secondary structure of a given epitope or molecule and may be used
in accordance with the invention (see, e.g., Chou and Fasman,
1974a,b; 1978a,b, 1979). Moreover, computer programs are currently
available to assist with predicting an antigenic portion and an
epitopic core region of one or more proteins, polypeptides or
peptides. Examples include those programs based upon the
Jameson-Wolf analysis (Jameson and Wolf, 1988; Wolf et al., 1988),
the program PepPlot.RTM. (Brutlag et al., 1990; Weinberger et al.,
1985), and other programs for protein tertiary structure prediction
(Fetrow and Bryant, 1993). Another commercially available software
program capable of carrying out such analyses is MacVector (IBI,
New Haven, Conn.). In addition to the computer programs
commercially available for analysis of protein-protein
interactions, Simon et al. (2002), for example, described a program
optimized for analysis of MHC class II molecules complexed with
various peptides fitting into the MHC class II groove.
[0047] To determine whether a modification to an epitope will
affect the interaction with a TCR, the putative location of the
modified amino acid(s) could be determined by comparison of the
mutated sequence to that of the unmutated polypeptide's secondary
and tertiary structure, as determined by such methods known to
those of ordinary skill in the art including, but not limited to,
X-ray crystallography, NMR or computer modeling. X-ray
crystallography in particular has proved useful for the
determination of the structure of antigen-MHC complexes. For
example, the elucidation of the structure of different peptide
complexes between an antigen and MHC molecules by X-ray
crystallography was described by, e.g., Madden (1995) and Stern and
Wily (1994). X-ray crystallography has also been used to elucidate
the structure of a viral peptide-HLA-A2 complex bound in the human
TCR (Garboci et al., 1996).
[0048] In further embodiments of the invention, major CTL epitopes
of a polypeptide antigen may be identified by an empirical approach
in which portions of the gene encoding the polypeptide are
expressed in a recombinant host, and/or the resulting proteins
tested for their ability to elicit an immune response. For example,
PCR.TM. can be used to prepare a range of peptides lacking
successively longer fragments of the C-terminus of the protein. The
immunoactivity of each of these peptides is determined to identify
those fragments and/or domains of the polypeptide that are
immunodominant. Further studies in which only a small number of
amino acids are removed at each iteration then allows the location
of the antigenic determinants of the polypeptide to be more
precisely determined.
[0049] Once one and/or more such analyses are completed, epitopes
may be modified as is described herein. The peptides may then be
employed in the methods of the invention to modulate an immune
response as is desired by administration of an antigen bearing the
epitope to a mammal, preferably a human.
[0050] C. Cytotoxic T Lymphocytes
[0051] T lymphocytes arise from hematopoietic stem cells in the
bone marrow, and migrate to the thymus gland to mature. T cells
express a unique antigen binding receptor on their membrane (T-cell
receptor), which can only recognize antigen in association with
major histocompatibility complex (MHC) molecules on the surface of
other cells. There are at least two populations of T cells, known
as T helper cells and T cytotoxic cells. T helper cells and T
cytotoxic cells are primarily distinguished by their display of the
membrane bound glycoproteins CD4 and CD8, respectively. T helper
cells secret various lymphokines, that are crucial for the
activation of B cells, T cytotoxic cells, macrophages and other
cells of the immune system. In contrast, a T cytotoxic cells that
recognizes an antigen-MHC complex proliferates and differentiates
into an effector cell called a cytotoxic T lymphocyte (CTL). CTLs
eliminate cells of the body displaying antigen, such as virus
infected cells and tumor cells, by producing substances that result
in cell lysis.
[0052] The major histocompatibility complex (MHC) is a large
genetic complex with multiple loci. The MHC loci encode two major
classes of MHC membrane molecules, referred to as class I and class
II MHCs. T helper lymphocytes generally recognize antigen
associated with MHC class II molecules, and T cytotoxic lymphocytes
recognize antigen associated with MHC class I molecules. In humans
the MHC is refereed to as the HLA complex and in mice the H-2
complex.
[0053] In certain embodiments of the invention, T-lymphocytes are
specifically activated by contact with an antigenic composition
comprising a modified CTL epitope. In one embodiment of the
invention, this could comprise activating T-lymphocytes by contact
with an antigen presenting cell that is in contact with an antigen
of the invention. T cells express a unique antigen binding receptor
on their membrane, a T-cell receptor (TCR), which can only
recognize antigen in association with major histocompatibility
complex (MHC) molecules on the surface of other cells. There are
several populations of T cells, such as T helper cells and T
cytotoxic cells. T helper cells and T cytotoxic cells are primarily
distinguished by their display of the membrane bound glycoproteins
CD4 and CD8, respectively. T helper cells secret various
lymphokines, that are crucial for the activation of B cells, T
cytotoxic cells, macrophages and other cells of the immune system.
In contrast, a T cytotoxic cell that recognizes an antigen-MHC
complex proliferates and differentiates into an effector cell
called a cytotoxic T lymphocyte (CTL). CTLs eliminate cells of the
body displaying antigen, such as virus infected cells and tumor
cells, by producing substances that result in cell lysis.
II. MODIFIED CTL EPITOPES
[0054] Optimization of immunogenicity requires approaches for
controlled modulation of TCR (T-cell antigen receptor) signaling by
antigen (antigen). Studies on positive selection, survival, as well
as induction of memory CTL (mCTL), indicate the requirement for
modulation of TCR signaling to allow progression of T cells from
naive to effector CTL (eCTL) and memory CTL (mCTL) (Williams et
al., 1999; Roy and Nicholson, 2000; Krammer, 2000). Attenuation of
the strength of TCR signaling should thus be able to avoid
AICD-mediated death by overstimulation, as well as induce
homeostatic proliferation of precursors of hi-av T cells which
should be more sensitive to low affinity ligands.
[0055] The approach of the inventors thus provides methods for
modulation of TCR signaling. By addition of CH.sub.2-groups in the
side chains of the amino acids of a target antigen in positions
pointing upwards and sideways, this will allow increased affinity
of the peptide for the TCR because of increased availability of
CH.sub.2-- groups to form van der Waals interactions with TCR.
Because the maintenance of the peptide core, and of charged polar,
or phenol rings in place, these antigen will be less cross-reactive
with other TCR than CTL induced by enhancer agonists. Thus,
modulation of TCR signaling will not require amino acid
substitution in the core with unpredicted effects due to
modification of positions of core residues in the groove, and
modification of the surfaces presented to TCR. The
increase/decrease in the available CH.sub.2 groups should modify
the half-life and the affinity (Kd) of the TCR for the peptide.
Thus, modification of CH.sub.2 side chain length at defined
positions allows modulation of TCR signaling, according to the
requirements for overt or attenuated stimulation of cells in
various stages of differentiation.
[0056] It is thus indicated that if new interactions created by
CH.sub.2 addition are functional, they will either increase the
affinity for TCR or will disrupt existent nonproductive
interactions. Thus, corresponding analogs should be more
immunogenic than the Wild type agonists, for activation of same
effector function. If CH.sub.2 extension is done in residues with
short or absent side chains (Ala, Gly) which do not point upward,
the interference with existent interactions by other side chains
will be minimized. Thus, the objectives of the studies described
below were to determine the immunogenicity of CH.sub.2-E75
analogs.
[0057] Since it is the interactive capacity and nature of an
antigen that defines its biological (e.g., immunological)
functional activity, certain amino acid sequence substitutions
should be made with consideration to the structure of the amino
acid substituted. As used herein, an "amino molecule" refers to any
amino acid, whether natural or non-natural, including amino acid
derivatives or amino acid mimics as would be known to one of
ordinary skill in the art. In certain embodiments, the residues of
the antigenic composition comprise amino molecules that are
sequential, without any non-amino molecule interrupting the
sequence of amino molecule residues. In other embodiments, the
sequence may comprise one or more non-amino molecule moieties. In
particular embodiments, the sequence of residues of the antigenic
composition may be interrupted by one or more non-amino molecule
moieties. In certain other embodiment of the invention, non-natural
amino acids are used to replace natural amino acids in a native CTL
epitope. Accordingly, antigenic compositions prepared in accordance
with the invention may encompass an amino molecule sequence
comprising at least one of the 20 common amino acids in naturally
synthesized proteins, as well as at least one modified or unusual
amino acid, including but not limited to those shown in Tables 1
and 2 below.
[0058] In substituting amino acids, it may also be desired to
consider the relative hydrophobicity, hydrophilicity, charge, size,
and/or the like of the amino acids. An analysis of the size, shape
and/or type of the amino acid side-chain substituents reveals that
arginine, lysine and/or histidine are all positively charged
residues; that alanine, glycine and/or serine are all a similar
size; and/or that phenylalanine, tryptophan and/or tyrosine all
have a generally similar shape.
[0059] Antigenic epitope-bearing peptides and polypeptides of the
invention designed according the guidelines described herein
generally will preferably contain a sequence of at least seven to
about 15 to about 30 amino acids contained within the amino acid
sequence of a polypeptide of the invention. Preferably, the amino
acid sequence of the epitope-bearing peptide is selected to provide
substantial solubility in aqueous solvents (i.e., the sequence
includes relatively hydrophilic residues and highly hydrophobic
sequences are preferably avoided); and sequences containing proline
residues are particularly preferred.
[0060] In terms of immunologically functional equivalents, it is
well understood by the skilled artisan that there is a limit to the
number of changes that may be made within a defined portion of a
molecule and still result in a molecule with an acceptable level of
equivalent immunological activity. An immunologically functional
equivalent peptide or polypeptide are thus defined herein as those
peptide(s) or polypeptide(s) in which certain, typically not most
or all, of the amino acid(s) may be substituted. In particular,
where a shorter length peptide is concerned, it is contemplated
that fewer amino acid substitutions should be made within the given
peptide. A longer polypeptide may have an intermediate number of
changes. The full length protein will have the most tolerance for a
larger number of changes. Of course, a plurality of distinct
polypeptides/peptides with different substitutions may easily be
made and used in accordance with the invention.
[0061] Further still, U.S. Pat. No. 5,194,392 to Geysen (1990)
describes a general method of detecting or determining the sequence
of monomers (amino acids or other compounds) which is a topological
equivalent of the epitope (i.e., a "mimotope") which is
complementary to a particular paratope (antigen binding site) of an
antibody of interest. More generally, U.S. Pat. No. 4,833,092 to
Geysen (1989) describes a method of detecting or determining a
sequence of monomers which is a topographical equivalent of a
ligand which is complementary to the ligand binding site of a
particular receptor of interest.
[0062] Similarly, U.S. Pat. No. 5,480,971 to Houghten, et al.
(1996) on Peralkylated Oligopeptide Mixtures discloses linear
C.sub.1-C.sub.7-alkyl peralkylated oligopeptides and sets and
libraries of such peptides, as well as methods for using such
oligopeptide sets and libraries for determining the sequence of a
peralkylated oligopeptide that preferentially binds to an acceptor
molecule of interest. Thus, non-peptide analogs of the
epitope-bearing peptides of the invention also can be made
routinely by these methods.
[0063] It also is well understood that where certain residues are
shown to be particularly important to the immunological or
structural properties of a protein or peptide, e.g., residues in
binding regions or active sites, such residues may not generally be
exchanged absent the side-chain changes described herein. In this
manner, functional equivalents are defined herein as those peptides
or polypeptides which maintain a substantial amount of their native
immunological activity or possess increased immunological
activity.
[0064] To effect more quantitative changes, the hydropathic index
of amino acids may be considered. Each amino acid has been assigned
a hydropathic index on the basis of their hydrophobicity and charge
characteristics, these are: isoleucine (+4.5); valine (+4.2);
leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine
(-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline
(-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);
aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine
(-4.5).
[0065] The importance of the hydropathic amino acid index in
conferring interactive biological function on a protein,
polypeptide or peptide is generally understood in the art (Kyte and
Doolittle, 1982, incorporated herein by reference). It is known
that certain amino acids may be substituted for other amino acids
having a similar hydropathic index or score and still retain a
similar biological activity. In making changes based upon the
hydropathic index, the substitution of amino acids whose
hydropathic indices are within .+-.2 is preferred, those which are
within .+-.1 are particularly preferred, and those within .+-.0.5
are even more particularly preferred.
[0066] It also is understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity, particularly where the immunological functional
equivalent polypeptide or peptide thereby created is intended for
use in immunological embodiments, as in certain embodiments of the
present invention. U.S. Pat. No. 4,554,101, incorporated herein by
reference, states that the greatest local average hydrophilicity of
a protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with its immunogenicity and antigenicity, i.e.,
with a immunological property of the protein.
[0067] As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate
(+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4). In making changes based
upon similar hydrophilicity values, the substitution of amino acids
whose hydrophilicity values are within .+-.2 is preferred, those
which are within .+-.1 are particularly preferred, and those within
.+-.0.5 are even more particularly preferred.
[0068] While discussion has focused on functionally equivalent
polypeptides arising from amino acid changes, it will be
appreciated that these changes may be effected by alteration of the
encoding DNA; taking into consideration also that the genetic code
is degenerate and that two or more codons may code for the same
amino acid. Nucleic acids encoding these antigenic compositions
also can be constructed and inserted into one or more expression
vectors by standard methods (Sambrook et al., 2001), for example,
using PCR.TM. cloning methodology.
[0069] Certain aspects of the instant invention comprise synthesis
of peptide and polypeptide epitopes in cyto, via transcription and
translation of appropriate polynucleotides. These peptides and
polypeptides will include the twenty "natural" amino acids, and
post-translational modifications thereof. However, in vitro peptide
synthesis permits the use of modified and/or unusual amino acids.
As described herein, these amino acids may in particular find use
in the creation of modified CTL epitopes. A table of exemplary, but
not limiting, modified and/or unusual amino acids is provided
herein below in Table 1.
TABLE-US-00001 TABLE 1 Modified and/or Unusual Amino Acids Abbr.
Amino Acid Aad 2-Aminoadipic acid BAad 3- Aminoadipic acid BAla
Beta-alanine, beta-Amino-propionic acid Abu 2-Aminobutyric acid
4Abu 4- Aminobutyric acid, piperidinic acid Acp 6-Aminocaproic acid
Ahe 2-Aminoheptanoic acid Aib 2-Aminoisobutyric acid BAib
3-Aminoisobutyric acid Apm 2-Aminopimelic acid Dbu
2,4-Diaminobutyric acid Des Desmosine Dpm 2,2'-Diaminopimelic acid
Dpr 2,3-Diaminopropionic acid EtGly N-Ethylglycine EtAsn
N-Ethylasparagine Hyl Hydroxylysine AHyl allo-Hydroxylysine 3Hyp
3-Hydroxyproline 4Hyp 4-Hydroxyproline Ide Isodesmosine Aile
allo-Isoleucine MeGly N-Methylglycine, sarcosine MeIle
N-Methylisoleucine MeLys 6-N-Methyllysine MeVal N-Methylvaline Nva
Norvaline Nle Norleucine Orn Ornithine
[0070] In making amino acid substitutions in accordance with the
invention, it will be desired to particularly consider side-chain
modifications. Non-limiting examples of specific side-chain
modifications contemplated for use with the current invention,
including specific side chain lengthening or shortening
modifications, are set forth below in Table 2:
TABLE-US-00002 TABLE 2 Exemplary Amino Acid Substitutions for
Linear Lengthening or Shortening of Side Chains with Un-Natural
Amino Acids Analogs of Ala7: (Ala7: R chain = CH.sub.3) Reagent
Used with side chain amino Compound acid longer than Natural
Glycine CH.sub.2.dbd.O R chain = CH.sub.2CH.sub.3 -aminobutyric
acid (+1 CH.sub.2) Fmoc-Abu--OH
N-.alpha.-fmoc-L-.alpha.-aminobutyric acid Fmac-2-aminobutanaic
acid C.sub.19H.sub.19NO.sub.4: M.W.: 325-4 R chain =
Ch.sub.2CH.sub.2CH.sub.3 Norvaline (+2 CH.sub.2) Fmoc-Nle--OH
N-.alpha.-fmac-L-norvaline C.sub.20H.sub.21NO.sub.4; M.W.: 339.4 R
chain = CH.sub.2CH.sub.2CH.sub.2CH.sub.3 Norleucine (+3 CH.sub.2)
Fmoc-Nle--OH N-.alpha.-fmac-L-norleucine CAS No. 77284 32-3;
C.sub.21H.sub.23NO.sub.4; M.W.: 353.4 Analogs of Phe8 (Phe8: R
chain = Ch.sub.2(C.sub.6H.sub.5)) Compound Reagent Used R chain =
C.sub.6H.sub.5 Phenyl Glycine (-1CH.sub.2) Fmac-Phg--OH
N-.alpha.-fmac-L-phenylglycine C.sub.23H.sub.19NO.sub.4; M.W.:
373.4 Analogs of Lys1 (R chain =
CH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2) Compound Reagent R chain
= Ch.sub.2CH.sub.2CH.sub.2NH.sub.2 Ornithine (--CH.sub.2)
Fmac-Orn(Bac)--OH N-.alpha.-Fmac-N-.delta.-Bac-L-ornithine CAS No.:
109425-55-0; C.sub.25H.sub.30N.sub.2O.sub.6; M.W.: 454.5 R chain =
CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2 Homolysine (+1
CH.sub.2) ##STR00001## Analogs of the Ile2: (R chain =
(CH(CH.sub.3)CH.sub.2CH.sub.3)* Compound Reagent R chain:
CH.sub.2CH(CH.sub.3)CH.sub.2CH.sub.3 .gamma.-Methyl_l-leucine(+1
CH.sub.2) H-Leu(.gamma.Me)--OH .gamma.-Methyl-L-leucine
C.sub.7H.sub.15NO.sub.2; M.W.: 145.2 R chain:
CH.sub.2CH.sub.2(C.sub.6H.sub.5) Homophenylalaine (+1 CH.sub.2)
##STR00002## Gly4: (R chain = H)** CH.sub.2-Analogs of Ser 5 (R
chain = CH.sub.2OH) Compound Reagent R chain: OH 2-amino-2-hydroxy
Acetic Acid (-1 CH.sub.2) (unstable under peptide synthesis
conditions) R chain: CH.sub.2CH.sub.2OH Homoserine (+1 CH.sub.2)
Fmoc-Hse(Trt)--OH N-.alpha.-fmac-O-trityl-L-homoserine
C.sub.38H.sub.33NO.sub.5; M.W.: 583.7 Analogs of Leu6 (R Chain =
CH.sub.2CH(CH.sub.3).sub.2*** Compound Reagent R chain:
Ch.sub.2(CH.sub.2CH(CH.sub.3).sub.2 Homoleucine (+1 CH.sub.2) Ala7:
R Chain--CH.sub.3 Previously teted See Phe3 See Leu6 *Since the
first carbon of the R Chain is branched, eliminating this carbon to
form a (-1 CH) structure would radically affect the makeup of this
amino acid and may cause unwarranted side reactions. **Any
alterations in the side chain of this amino acid results in a
non-homologous amino acid. ***Removing the first methylene group to
make a (-1 CH.sub.2) compound results in the formation of the
natural amino acid Valine.
[0071] A. Epitopic Core Sequences
[0072] One aspect of the current invention provides for the
modification of peptide epitope-bearing portions of an antigen in
order to modulate TCR signaling and to achieve a therapeutic
benefit therefrom. The epitope of this antigen can be termed an
immunogenic or antigenic epitope. An "immunogenic epitope" is
defined as a part of a antigen that interacts with MHC class I
and/or class II molecules and/or the TCR, eliciting a TCR-mediated
response to the antigen. These immunogenic epitopes are believed to
be confined to a few loci on the molecule. On the other hand, a
region of a protein molecule to which an antibody can bind is
defined as an "antigenic epitope." The number of immunogenic
epitopes of a protein generally is less than the number of
antigenic epitopes. See, for instance, Geysen, (1984).
[0073] Peptides capable of eliciting protein-reactive sera are
frequently represented in the primary sequence of a protein, can be
characterized by a set of simple chemical rules, and are confined
neither to immunodominant regions of intact proteins (i.e.,
immunogenic epitopes) nor to the amino or carboxyl terminals.
Peptides that are extremely hydrophobic and those of six or fewer
residues generally are ineffective at inducing antibodies that bind
to the mimicked protein; longer, soluble peptides, especially those
containing proline residues, usually are effective. Sutcliffe et
al., 1984. For instance, 18 of 20 peptides designed according to
these guidelines, containing 8-39 residues covering 75% of the
sequence of the influenza virus hemagglutinin HA1 polypeptide
chain, induced antibodies that reacted with the HA1 protein or
intact virus; and 12/12 peptides from the MuLV polymerase and 18/18
from the rabies glycoprotein induced antibodies that precipitated
the respective proteins.
[0074] B. Identifying CTL Epitopes
[0075] Numerous techniques for the identification of CTL epitopes
are known to those of skill in the art and may be employed in
connection with the instant invention. For example, various
computer-based prediction algorithms have been described and are
publicly available to identify tumor-reactive CTL epitopes (see,
e.g., Lu and Celis, 2000). For example, Falk et al. (1991) describe
a method for prediction of HLA A2.1 haplotypes by computer
software. A common strategy in the search for epitope containing
antigens is to first isolate T-cells specific for the antigen and
attempt to identify the antigen(s) recognized by the T-cells. For
example, in patients with cancer, specific CTLs have been often
derived from lymphocytic infiltrates present at the tumor site
(Weidmann et al., 1994). Tumor-specific CTLs have also been found
in peripheral blood or malignant ascites of patients with cancer,
indicating that a systemic response to the tumor may be present or
that redistribution of CTLs from the tumor to the periphery might
occur (Wallace et al., 1993).
[0076] Common protocols for CTL epitope identification involve
isolating and assaying extremely pure MHC molecules from
antigen-presenting cells. Prior to peptide extraction, all
contaminating proteinaceous material must be removed (Chicz and
Urban, 1994). Using immunoaffinity purification, bound HLA
molecules are obtained. From these, smaller amounts of bound
peptide can be isolated and further purified, such as by HPLC.
These fractions can be assayed for reactivity with cloned CTLs and
can be sequenced or otherwise characterized.
[0077] In another technique, developed by Van der Zee et al. (1989)
and referred to as the "pepscan" technique, dozens of peptides are
simultaneously synthesized on polyethylene rods arrayed in a
96-well microtiter plate pattern. Peptides are then chemically
cleaved from the solid support and supplied to irradiated syngeneic
thymocytes for antigen presentation. A cloned CTL line is then
tested for reactivity in a proliferation assay monitored by
.sup.3H-thymidine incorporation. This type of analysis particularly
suits a CTL stimulation assay since it can be automated using a
microtiter plate reader and employs relatively low levels of
radiation. The technique is highly specific.
[0078] Yet another method for identification of CTL epitopes is
described in U.S. Pat. No. 6,338,945, the entire disclosure of
which is specifically incorporated herein by reference. In this
technique, CTL epitopes are identified by screening solid phase
combinatorial libraries of molecules in a cytotoxic T cell assay.
In this way, CTLs activated by the molecules in the library are
identified.
[0079] T cell epitopes may also be predicted utilizing the HLA A2.1
motif described by Falk et al. (1991). From this analysis, peptides
may be synthesized and used as targets in an in vitro cytotoxic
assay. Still another method that may also be utilized to predict
immunogenic portions is to determine which portion has the property
of CTL induction in mice utilizing retroviral vectors (see, Warner
et al., 1991). As noted in Warner et al., CTL induction in mice may
be utilized to predict cellular immunogenicity in humans. Preferred
immunogenic portions may also be deduced by determining which
fragments of an antigen are capable of inducing lysis by autologous
patient lymphocytes of target cells (e.g., autologous
EBV-transformed lymphocytes) expressing the fragments after vector
transduction of the corresponding genes.
[0080] U.S. Pat. No. 4,554,101, (Hopp) incorporated herein by
reference, teaches the identification and/or preparation of
epitopes from primary amino acid sequences on the basis of
hydrophilicity. Through the methods disclosed in Hopp, one of skill
in the art would be able to identify epitopes from within an amino
acid sequence.
[0081] C. Production of Modified Antigens
[0082] The epitope-bearing peptides and polypeptides of the
invention may be produced by any conventional means for making
peptides or polypeptides including recombinant means using nucleic
acid molecules of the invention. For instance, a short
epitope-bearing amino acid sequence may be fused to a larger
polypeptide which acts as a carrier during recombinant production
and purification, as well as during immunization to produce
anti-peptide antibodies. Epitope-bearing peptides also may be
synthesized using known methods of chemical synthesis. For
instance, Houghten has described a simple method for synthesis of
large numbers of peptides, such as 10-20 mg of 248 different 13
residue peptides representing single amino acid variants of a
segment of the HA1 polypeptide which were prepared and
characterized (by ELISA-type binding studies) in less than four
weeks, Houghten, (1985). This "Simultaneous Multiple Peptide
Synthesis (SMPS)" process is further described in U.S. Pat. No.
4,631,211 to Houghten et al. (1986). In this procedure the
individual resins for the solid-phase synthesis of various peptides
are contained in separate solvent-permeable packets, enabling the
optimal use of the many identical repetitive steps involved in
solid-phase methods. A completely manual procedure allows 500-1000
or more syntheses to be conducted simultaneously. Houghten et al.,
supra, at 5134.
[0083] Immunogenic TCL epitope-bearing peptides of the invention,
i.e., those parts of a antigen that interact with MHC molecules
and/or TCR, may be prepared according to methods known in the art.
For instance, Geysen et al., 1984, supra, discloses a procedure for
rapid concurrent synthesis on solid supports of hundreds of
peptides of sufficient purity to react in an enzyme-linked
immunosorbent assay. Modulation of immunogenicity can then be
easily assayed as described herein below. In this manner a peptide
bearing a modified immunomodulatory CTL epitope may be identified
routinely by one of ordinary skill in the art.
[0084] For instance, combining synthetic preparation of the
immunologically important epitope in the coat protein of
foot-and-mouth disease virus combined with assays for immunologic
activity, Geysen et al. identified the epitope with a resolution of
seven amino acids by synthesis of an overlapping set of all 208
possible hexapeptides covering the entire 213 amino acid sequence
of the protein. Then, a complete replacement set of peptides in
which all 20 amino acids were substituted in turn at every position
within the epitope were synthesized, and the particular amino acids
conferring specificity for the reaction with antibody were
determined. Thus, peptide analogs of the epitope-bearing peptides
of the invention can be made by this method. U.S. Pat. No.
4,708,871 to Geysen (1987) further describes this method of
identifying a peptide bearing an immunogenic epitope of a desired
protein.
III. TREATMENT OR PREVENTION OF DISEASE WITH THE INVENTION
[0085] The disclosures presented herein have significant relevance
to immunotherapy of human diseases and disorders including, but not
limited to, cancer. In using the immunotherapeutic compositions and
methods of the present invention in treatment methods, other
standard treatments also may be employed, such as radiotherapy or
chemotherapy. However, in certain instances it may be preferred to
use the immunotherapy alone initially so that its effectiveness can
be readily assessed. Certain aspects of the invention thus concern
methods for the prevention or treatment of disease. Such disease
may be external in origin, for example, in the case of infection by
bacterial, viral, parasitic or other types of causative agents. The
disease may also be internal in origin, for example, in the case of
spontaneous carcinogenesis.
[0086] A. Vaccine Preparations
[0087] A modified antigenic composition of the present invention
may be mixed with one or more additional components (e.g.,
excipients, salts, etc.) which are pharmaceutically acceptable and
compatible with at least one active ingredient (e.g., antigen) to
form a composition suitable for administration to an animal, for
example, a human. Such a composition may be termed a "vaccine". As
used herein, the term "vaccine" refers to any composition
formulated for administration to an animal, including a human, and
which includes one or more antigen(s) prepared in accordance with
the invention, whether used or intended to be used for prophylactic
administration to prevent development of disease and/or for
therapeutic administration for mitigation or elimination of an
existing disease state. The preparation of such vaccines is
generally well understood in the art, as exemplified by U.S. Pat.
Nos. 4,608,251, 4,601,903, 4,599,231, 4,599,230, and 4,596,792, all
incorporated herein by reference. These methods may therefore be
used to prepare a vaccine comprising an antigenic composition
comprising one or more epitopes modified as described herein as an
active ingredient. In preferred embodiments, the compositions of
the present invention are prepared to be pharmacologically
acceptable vaccines.
[0088] Pharmaceutical vaccine compositions of the present invention
comprise an effective amount of one or more modified antigens and
any desired additional agents dissolved or dispersed in a
pharmaceutically acceptable carrier. The phrases "pharmaceutical or
pharmacologically acceptable" refers to molecular entities and
compositions that do not produce an adverse, allergic or other
untoward reaction when administered to an animal, such as, for
example, a human, as appropriate. The preparation of an
pharmaceutical composition that contains at least one antigen
prepared in accordance with the invention or additional active
ingredient will be known to those of skill in the art in light of
the present disclosure, as exemplified by Remington's
Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990,
incorporated herein by reference. Moreover, for animal (e.g.,
human) administration, it will be understood that preparations
should meet sterility, pyrogenicity, general safety and purity
standards as required by FDA Office of Biological Standards.
[0089] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
surfactants, antioxidants, preservatives (e.g., antibacterial
agents, antifungal agents), isotonic agents, absorption delaying
agents, salts, preservatives, drugs, drug stabilizers, binders,
excipients, disintegration agents, lubricants, sweetening agents,
flavoring agents, dyes, such like materials and combinations
thereof, as would be known to one of ordinary skill in the art
(see, for example, Remington's Pharmaceutical Sciences, 18th Ed.
Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by
reference). The composition may comprise different types of
carriers depending on whether it is to be administered in solid,
liquid or aerosol form, and whether it need to be sterile for such
routes of administration as injection. Except insofar as any
conventional carrier is incompatible with the active ingredient,
its use in the therapeutic or pharmaceutical compositions is
contemplated.
[0090] In any case, the composition may comprise various
antioxidants to retard oxidation of one or more component.
Additionally, the prevention of the action of microorganisms can be
brought about by preservatives such as various antibacterial and
antifungal agents, including but not limited to parabens (e.g.,
methylparabens, propylparabens), chlorobutanol, phenol, sorbic
acid, thimerosal or combinations thereof.
[0091] The compositions may be formulated in a free base, neutral
or salt form. Pharmaceutically acceptable salts, include the acid
addition salts, e.g., those formed with the free amino groups of a
proteinaceous composition, or which are formed with inorganic acids
such as for example, hydrochloric or phosphoric acids, or such
organic acids as acetic, oxalic, tartaric or mandelic acid. Salts
formed with the free carboxyl groups can also be derived from
inorganic bases such as for example, sodium, potassium, ammonium,
calcium or ferric hydroxides; or such organic bases as
isopropylamine, trimethylamine, histidine or procaine.
[0092] In embodiments where the composition is in a liquid form, a
carrier can be a solvent or dispersion medium comprising but not
limited to, water, ethanol, polyol (e.g., glycerol, propylene
glycol, liquid polyethylene glycol, etc., lipids (e.g.,
triglycerides, vegetable oils, liposomes) and combinations thereof.
The proper fluidity can be maintained, for example, by the use of a
coating, such as lecithin; by the maintenance of the required
particle size by dispersion in carriers such as, for example liquid
polyol or lipids; by the use of surfactants such as, for example
hydroxypropylcellulose; or combinations thereof such methods. In
many cases, it will be preferable to include isotonic agents, such
as, for example, sugars, sodium chloride or combinations
thereof.
[0093] In other embodiments, one may use eye drops, nasal solutions
or sprays, aerosols or inhalants in the present invention. Such
compositions are generally designed to be compatible with the
target tissue type. In a non-limiting example, nasal solutions are
usually aqueous solutions designed to be administered to the nasal
passages in drops or sprays. Nasal solutions are prepared so that
they are similar in many respects to nasal secretions, so that
normal ciliary action is maintained. Thus, in preferred embodiments
the aqueous nasal solutions usually are isotonic or slightly
buffered to maintain a pH of about 5.5 to about 6.5. In addition,
antimicrobial preservatives, similar to those used in ophthalmic
preparations, drugs, or appropriate drug stabilizers, if required,
may be included in the formulation. For example, various commercial
nasal preparations are known and include drugs such as antibiotics
or antihistamines.
[0094] In certain embodiments of the invention, the antigen may be
prepared for administration by such routes as oral ingestion. In
these embodiments, the solid composition may comprise, for example,
solutions, suspensions, emulsions, tablets, pills, capsules (e.g.,
hard or soft shelled gelatin capsules), sustained release
formulations, buccal compositions, troches, elixirs, suspensions,
syrups, wafers, or combinations thereof. Oral compositions may be
incorporated directly with the food of the diet. Preferred carriers
for oral administration comprise inert diluents, assimilable edible
carriers or combinations thereof. In other aspects of the
invention, the oral composition may be prepared as a syrup or
elixir. A syrup or elixir, and may comprise, for example, at least
one active agent, a sweetening agent, a preservative, a flavoring
agent, a dye, a preservative, or combinations thereof.
[0095] In certain preferred embodiments an oral composition may
comprise one or more binders, excipients, disintegration agents,
lubricants, flavoring agents, and combinations thereof. In certain
embodiments, a composition may comprise one or more of the
following: a binder, such as, for example, gum tragacanth, acacia,
cornstarch, gelatin or combinations thereof; an excipient, such as,
for example, dicalcium phosphate, mannitol, lactose, starch,
magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate or combinations thereof; a disintegrating agent, such as,
for example, corn starch, potato starch, alginic acid or
combinations thereof; a lubricant, such as, for example, magnesium
stearate; a sweetening agent, such as, for example, sucrose,
lactose, saccharin or combinations thereof; a flavoring agent, such
as, for example peppermint, oil of wintergreen, cherry flavoring,
orange flavoring, etc.; or combinations thereof the foregoing. When
the dosage unit form is a capsule, it may contain, in addition to
materials of the above type, carriers such as a liquid carrier.
Various other materials may be present as coatings or to otherwise
modify the physical form of the dosage unit. For instance, tablets,
pills, or capsules may be coated with shellac, sugar or both.
[0096] Additional formulations which are suitable for other modes
of administration include suppositories. Suppositories are solid
dosage forms of various weights and shapes, usually medicated, for
insertion into the rectum, vagina or urethra. After insertion,
suppositories soften, melt or dissolve in the cavity fluids. In
general, for suppositories, traditional carriers may include, for
example, polyalkylene glycols, triglycerides or combinations
thereof. In certain embodiments, suppositories may be formed from
mixtures containing, for example, the active ingredient in the
range of about 0.5% to about 10%, and preferably about 1% to about
2%.
[0097] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and/or the other ingredients. In the case of
sterile powders for the preparation of sterile injectable
solutions, suspensions or emulsion, the preferred methods of
preparation are vacuum-drying or freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered liquid medium
thereof. The liquid medium should be suitably buffered if necessary
and the liquid diluent first rendered isotonic prior to injection
with sufficient saline or glucose. The preparation of highly
concentrated compositions for direct injection is also
contemplated, where the use of DMSO as solvent is envisioned to
result in extremely rapid penetration, delivering high
concentrations of the active agents to a small area.
[0098] The composition must be stable under the conditions of
manufacture and storage, and preserved against the contaminating
action of microorganisms, such as bacteria and fungi. It will be
appreciated that endotoxin contamination should be kept minimally
at a safe level, for example, less that 0.5 ng/mg protein.
[0099] In particular embodiments, prolonged absorption of an
injectable composition can be brought about by the use in the
compositions of agents delaying absorption, such as, for example,
aluminum monostearate, gelatin or combinations thereof.
[0100] For an antigenic composition to be useful as a vaccine, an
antigenic composition must induce an immune response to the antigen
in a cell, tissue or animal (e.g., a human). As used herein, an
"antigenic composition" refers to a composition comprising one or
more antigens comprising at least a first epitope modified as
described herein. In other embodiments, the antigenic composition
is in a mixture that comprises an additional immunostimulatory
agent or nucleic acids encoding such an agent. Immunostimulatory
agents include but are not limited to an additional antigen, an
immunomodulator and an antigen presenting cell or an adjuvant. In
other embodiments, one or more of the additional agent(s) is
covalently bonded to the antigen or an immunostimulatory agent, in
any combination. In certain embodiments, the antigenic composition
is conjugated to or comprises an HLA anchor motif amino acids.
[0101] In certain embodiments of the invention, an antigenic
composition or immunologically functional equivalent, may be used
as an effective vaccine in modulating a humoral and/or
cell-mediated immune response in an animal. Such modulation may,
for example, be used for the treatment or prevention of cancer or
of a disease caused by an infective agent as described herein. One
or more antigenic compositions or vaccines may be used in both
active and passive immunization embodiments. In a non-limiting
example, a nucleic acid encoding an antigen might also be
formulated with a proteinaceous adjuvant. Of course, it will be
understood that various compositions described herein may further
comprise additional components. For example, one or more vaccine
components may be comprised in a lipid or liposome. In another
non-limiting example, a vaccine may comprise one or more adjuvants.
A vaccine of the present invention, and its various components, may
be prepared and/or administered by any method disclosed herein or
as would be known to one of ordinary skill in the art, in light of
the present disclosure. One of skill in the art may wish to add one
or more components to such a vaccine in addition to an antigen of
the invention, including, but not limited to the agents discussed
below.
[0102] B. Additional Vaccine Components
[0103] 1. Immunomodulators
[0104] It is contemplated that immunomodulators can be included in
a vaccine to augment a cell's or a patient's (e.g., an animal's)
response. Immunomodulators can be included as purified proteins,
nucleic acids encoding immunomodulators, and/or cells that express
immunomodulators in the vaccine composition. The following sections
list non-limiting examples of immunomodulators that are of
interest, and it is contemplated that various combinations of
immunomodulators may be used in certain embodiments (e.g., a
cytokine and a chemokine).
[0105] a. Cytokines
[0106] Interleukins, cytokines, nucleic acids encoding interleukins
or cytokines, and/or cells expressing such compounds are
contemplated as possible vaccine components. Interleukins and
cytokines, include but are not limited to interleukin 1 (IL-1),
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,
IL-12, IL-13, IL-14, IL-15, IL-18, .beta.-interferon,
.alpha.-interferon, .gamma.-interferon, angiostatin,
thrombospondin, endostatin, GM-CSF, G-CSF, M-CSF, METH-1, METH-2,
tumor necrosis factor, TGF.beta., LT and combinations thereof.
[0107] b. Chemokines
[0108] Chemokines, nucleic acids that encode for chemokines, and/or
cells that express such also may be used as vaccine components.
Chemokines generally act as chemoattractants to recruit immune
effector cells to the site of chemokine expression. It may be
advantageous to express a particular chemokine coding sequence in
combination with, for example, a cytokine coding sequence, to
enhance the recruitment of other immune system components to the
site of treatment. Such chemokines include, for example, RANTES,
MCAF, MIP1-alpha, MIP1-Beta, IP-10 and combinations thereof. The
skilled artisan will recognize that certain cytokines are also
known to have chemoattractant effects and could also be classified
under the term chemokines.
[0109] c. Immunogenic Carrier Proteins
[0110] In certain embodiments, an antigenic composition of the
invention may be chemically coupled to a carrier or recombinantly
expressed with a immunogenic carrier peptide or polypeptide (e.g.,
a antigen-carrier fusion peptide or polypeptide) to enhance an
immune reaction. Exemplary immunogenic carrier amino acid sequences
include hepatitis B surface antigen, keyhole limpet hemocyanin
(KLH) and bovine serum albumin (BSA). Other albumins such as
ovalbumin, mouse serum albumin or rabbit serum albumin also can be
used as immunogenic carrier proteins. Means for conjugating a
polypeptide or peptide to a immunogenic carrier protein are well
known in the art and include, for example, glutaraldehyde,
m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide and
bis-biazotized benzidine.
[0111] d. Biological Response Modifiers
[0112] It may be desirable to coadminister biologic response
modifiers (BRM), which have been shown to upregulate T cell
immunity or downregulate suppressor cell activity. Such BRMs
include, but are not limited to, cimetidine (CIM; 1200 mg/d)
(Smith/Kline, PA); low-dose cyclophosphamide (CYP; 300 mg/m.sup.2)
(Johnson/Mead, NJ), or a gene encoding a protein involved in one or
more immune helper functions, such as B-7.
[0113] 2. Adjuvants
[0114] Immunization protocols have used adjuvants to stimulate
responses for many years, and as such adjuvants are well known to
one of ordinary skill in the art. Some adjuvants affect the way in
which antigens are presented. For example, the immune response is
increased when protein antigens are precipitated by alum.
Emulsification of antigens also prolongs the duration of antigen
presentation.
[0115] In one aspect, an adjuvant effect is achieved by use of an
agent, such as alum, used in about 0.05 to about 0.1% solution in
phosphate buffered saline. Alternatively, the antigen is made as an
admixture with synthetic polymers of sugars (Carbopol.RTM.) used as
an about 0.25% solution. Adjuvant effect may also be made by
aggregation of the antigen in the vaccine by heat treatment with
temperatures ranging between about 70.degree. to about 101.degree.
C. for a 30-second to 2-minute period, respectively. Aggregation by
reactivating with pepsin treated (Fab) antibodies to albumin,
mixture with bacterial cell(s) such as C. parvum, an endotoxin or a
lipopolysaccharide component of Gram-negative bacteria, emulsion in
physiologically acceptable oil vehicles, such as mannide
mono-oleate (Aracel A), or emulsion with a 20% solution of a
perfluorocarbon (Fluosol-DA.RTM.) used as a block substitute, also
may be employed.
[0116] Some adjuvants, for example, certain organic molecules
obtained from bacteria, act on the host rather than on the antigen.
An example is muramyl dipeptide
(N-acetylmuramyl-L-alanyl-D-isoglutamine [MDP]), a bacterial
peptidoglycan. The effects of MDP, as with most adjuvants, are not
fully understood. MDP stimulates macrophages but also appears to
stimulate B cells directly. The effects of adjuvants, therefore,
are not antigen-specific. If they are administered together with a
purified antigen, however, they can be used to selectively promote
the response to the antigen.
[0117] Adjuvants have been used experimentally to promote a
generalized increase in immunity against unknown antigens (e.g.,
U.S. Pat. No. 4,877,611). This has been attempted particularly in
the treatment of cancer. For many cancers, there is compelling
evidence that the immune system participates in host defense
against the tumor cells. The current invention provides for such
treatments by providing improved antigen compounds.
[0118] Various polysaccharide adjuvants may also be used. For
example, the use of various pneumococcal polysaccharide adjuvants
on the antibody responses of mice has been described (Yin et al.,
1989). The doses that produce optimal responses, or that otherwise
do not produce suppression, should be employed as indicated (Yin et
al., 1989). Polyamine varieties of polysaccharides are particularly
preferred, such as chitin and chitosan, including deacetylated
chitin. Another group of adjuvants are the muramyl dipeptide (MDP,
N-acetylmuramyl-L-alanyl-D-isoglutamine) group of bacterial
peptidoglycans. Derivatives of muramyl dipeptide, such as the amino
acid derivative threonyl-MDP, and the fatty acid derivative MTPPE,
are also contemplated.
[0119] U.S. Pat. No. 4,950,645 describes a lipophilic
disaccharide-tripeptide derivative of muramyl dipeptide which is
described for use in artificial liposomes formed from phosphatidyl
choline and phosphatidyl glycerol. It is the to be effective in
activating human monocytes and destroying tumor cells, but is
non-toxic in generally high doses. The compounds of U.S. Pat. No.
4,950,645 and PCT Patent Application WO 91/16347, are contemplated
for use with cellular carriers and other embodiments of the present
invention.
[0120] Another adjuvant contemplated for use in the present
invention is BCG. BCG (bacillus Calmette-Guerin, an attenuated
strain of Mycobacterium) and BCG-cell wall skeleton (CWS) may also
be used as adjuvants in the invention, with or without trehalose
dimycolate. Trehalose dimycolate may be used itself. Trehalose
dimycolate administration has been shown to correlate with
augmented resistance to influenza virus infection in mice (Azuma et
al., 1988). Trehalose dimycolate may be prepared as described in
U.S. Pat. No. 4,579,945.
[0121] Amphipathic and surface active agents, e.g., saponin and
derivatives such as QS21 (Cambridge Biotech), form yet another
group of adjuvants for use with the immunogens of the present
invention. Nonionic block copolymer surfactants (Rabinovich et al.,
1994) may also be employed. Oligonucleotides are another useful
group of adjuvants (Yamamoto et al., 1988). Quil A and lentinen are
other adjuvants that may be used in certain embodiments of the
present invention.
[0122] One group of adjuvants preferred for use in the invention
are the detoxified endotoxins, such as the refined detoxified
endotoxin of U.S. Pat. No. 4,866,034. These refined detoxified
endotoxins are effective in producing adjuvant responses in
mammals. Of course, the detoxified endotoxins may be combined with
other adjuvants to prepare multi-adjuvant-incorporated cells. For
example, combination of detoxified endotoxins with trehalose
dimycolate is particularly contemplated, as described in U.S. Pat.
No. 4,435,386. Combinations of detoxified endotoxins with trehalose
dimycolate and endotoxic glycolipids is also contemplated (U.S.
Pat. No. 4,505,899), as is combination of detoxified endotoxins
with cell wall skeleton (CWS) or CWS and trehalose dimycolate, as
described in U.S. Pat. Nos. 4,436,727, 4,436,728 and 4,505,900.
Combinations of just CWS and trehalose dimycolate, without
detoxified endotoxins, is also envisioned to be useful, as
described in U.S. Pat. No. 4,520,019.
[0123] In other embodiments, the present invention contemplates
that a variety of adjuvants may be employed in the membranes of
cells, resulting in an improved immunogenic composition. The only
requirement is, generally, that the adjuvant be capable of
incorporation into, physical association with, or conjugation to,
the cell membrane of the cell in question. Those of skill in the
art will know the different kinds of adjuvants that can be
conjugated to cellular vaccines in accordance with this invention
and these include alkyl lysophosphilipids (ALP); BCG; and biotin
(including biotinylated derivatives) among others. Certain
adjuvants particularly contemplated for use are the teichoic acids
from Gram negative cells. These include the lipoteichoic acids
(LTA), ribitol teichoic acids (RTA) and glycerol teichoic acid
(GTA). Active forms of their synthetic counterparts may also be
employed in connection with the invention (Takada et al.,
1995).
[0124] Various adjuvants, even those that are not commonly used in
humans, may still be employed in animals, where, for example, one
desires to raise antibodies or to subsequently obtain activated T
cells. The toxicity or other adverse effects that may result from
either the adjuvant or the cells, e.g., as may occur using
non-irradiated tumor cells, is irrelevant in such
circumstances.
[0125] One group of adjuvants preferred for use in some embodiments
of the present invention are those that can be encoded by a nucleic
acid (e.g., DNA or RNA). It is contemplated that such adjuvants may
be encoded in a nucleic acid (e.g., an expression vector) encoding
the antigen, or in a separate vector or other construct. These
nucleic acids encoding the adjuvants can be delivered directly,
such as for example with lipids or liposomes.
[0126] C. Preparation of Proteinaceous Antigens
[0127] It is understood that an antigenic composition of the
present invention may be made by a method that is well known in the
art, including but not limited to chemical synthesis by solid phase
synthesis and purification away from the other products of the
chemical reactions by HPLC, or production by the expression of a
nucleic acid sequence (e.g., a DNA sequence) encoding a peptide or
polypeptide comprising an antigen of the present invention in an in
vitro translation system or in a living cell. Preferably the
antigenic composition is isolated and extensively dialyzed to
remove one or more undesired small molecular weight molecules
and/or lyophilized for more ready formulation into a desired
vehicle. It is further understood that additional amino acids,
mutations, chemical modification and such like, if any, that are
made in a vaccine component will preferably not substantially
interfere with recognition of the epitopic sequence.
[0128] A peptide antigen modified in accordance with the invention
may be synthesized by methods known to those of ordinary skill in
the art, such as, for example, peptide synthesis using automated
peptide synthesis machines, such as those available from Applied
Biosystems (Foster City, Calif.). Longer peptides or polypeptides
also may be prepared, e.g., by recombinant means. Polypeptides
produced by these or other techniques may be modified by
substitution or modification of one or more side chains, in
addition to replacement or deletion of one or more amino acids.
[0129] D. Genetic Vaccine Antigens
[0130] In certain embodiments, an immune response may be promoted
by transfecting or inoculating an animal with a nucleic acid
encoding an antigen comprising a CTL epitope modified in accordance
with the invention. Such a nucleic acid can be designed using
codons known to those of skill in the art, based on the chemical
structure of the respective amino acids. One or more cells
comprised within a target animal can then expresses the sequences
encoded by the nucleic acid after administration of the nucleic
acid to the animal. Thus, the vaccine may comprise a "genetic
vaccine" useful for immunization protocols. A vaccine may also be
in the form, for example, of a nucleic acid (e.g., a cDNA or an
RNA) encoding all or part of the peptide or polypeptide sequence of
an antigen. Expression in vivo by the nucleic acid may be, for
example, by a plasmid type vector, a viral vector, or a
viral/plasmid construct vector.
[0131] The nucleotide and protein, polypeptide and peptide encoding
sequences for various antigens have been previously disclosed, and
may be found at computerized databases known to those of ordinary
skill in the art. One such database is the National Center for
Biotechnology Information's Genbank and GenPept databases (found on
the world wide web at ncbi.nlm.nih.gov). The coding regions for
these known antigens may be amplified and/or expressed using the
techniques disclosed herein or by any technique that would be know
to those of ordinary skill in the art (e.g., Sambrook et al.,
2001). Though a nucleic acid may be expressed in an in vitro
expression system, in certain embodiments of the invention the
nucleic acid comprises a vector for in vivo replication and/or
expression.
[0132] E. Cellular Vaccine Antigens
[0133] In another embodiment, a cell expressing the antigen may be
included in the vaccine. The cell may be isolated from a culture,
tissue, organ or organism and administered to an animal as a
cellular vaccine. Thus, the present invention contemplates a
"cellular vaccine." The cell may be transfected with a nucleic acid
encoding an antigen to enhance its expression of the antigen. Of
course, the cell may also express one or more additional vaccine
components, such as immunomodulators or adjuvants. A vaccine may
comprise all or part of the cell.
[0134] In particular embodiments, it is contemplated that nucleic
acids encoding antigens of the present invention may be transfected
into plants, particularly edible plants, and all or part of the
plant material used to prepare a vaccine, such as for example, an
oral vaccine. Such methods are described in U.S. Pat. Nos.
5,484,719, 5,612,487, 5,914,123, 5,977,438 and 6,034,298, each
incorporated herein by reference.
[0135] F. Vaccine Component Purification
[0136] A vaccine component, including an antigenic peptide in
accordance with the invention, may be isolated and/or purified from
chemical synthesis reagents, cell or cellular components. In a
method of producing the vaccine component, purification may be
accomplished by any appropriate technique that is described herein
or well-known to those of skill in the art (e.g., Sambrook et al.,
2001). Although preferred for use in certain embodiments, there is
no general requirement that an antigenic composition of the present
invention or other vaccine component always be provided in their
most purified state. Indeed, it is contemplated that less
substantially purified vaccine component, which is nonetheless
enriched in the desired compound, relative to the natural state,
will have utility in certain embodiments.
[0137] The present invention also provides purified, and in
preferred embodiments, substantially purified vaccines or vaccine
components. The term "purified vaccine component" as used herein,
is intended to refer to at least one vaccine component (e.g., a
modified peptide antigen), wherein the component is purified to any
degree relative to its naturally-obtainable state, e.g., relative
to its purity within a cellular extract or reagents of chemical
synthesis. In certain aspects wherein the vaccine component is a
wild-type or mutant protein, polypeptide, or peptide free from the
environment in which it naturally occurs.
[0138] Where the term "substantially purified" is used, this will
refer to a composition in which the specific compound forms the
major component of the composition, such as constituting about 50%
of the compounds in the composition or more. In certain
embodiments, a substantially purified vaccine component will
constitute more than about 60%, about 70%, about 80%, about 90%,
about 95%, about 99% or even more of the compounds in the
composition.
[0139] In further embodiments, a vaccine component may be purified
to homogeneity. As applied to the present invention, "purified to
homogeneity," means that the vaccine component has a level of
purity where the compound is substantially free from other
chemicals, biomolecules or cells. For example, a purified peptide,
polypeptide or protein will often be sufficiently free of other
protein components so that degradative sequencing may be performed
successfully. Various methods for quantifying the degree of
purification of a vaccine component will be known to those of skill
in the art in light of the present disclosure. These include, for
example, determining the specific protein activity of a fraction
(e.g., antigenicity), or assessing the number of polypeptides
within a fraction by gel electrophoresis.
[0140] Various techniques suitable for use in chemical, biomolecule
or biological purification, well known to those of skill in the
art, may be applicable to preparation of a vaccine component of the
present invention. These include, for example, precipitation with
ammonium sulfate, PEG, antibodies and the like or by heat
denaturation, followed by centrifugation; fractionation,
chromatographic procedures, including but not limited to, partition
chromatograph (e.g., paper chromatograph, thin-layer chromatograph
(TLC), gas-liquid chromatography and gel chromatography) gas
chromatography, high performance liquid chromatography, affinity
chromatography, supercritical flow chromatography ion exchange, gel
filtration, reverse phase, hydroxylapatite, lectin affinity;
isoelectric focusing and gel electrophoresis (see for example,
Sambrook et al. 2001; and Freifelder, Physical Biochemistry, Second
1982, incorporated herein by reference).
[0141] Given that many DNA and proteins are known (see for example,
the National Center for Biotechnology Information's Genbank and
GenPept databases (found on the world wide web at
ncbi.nlm.nih.gov)), or may be identified and amplified using the
methods described herein, any purification method for recombinantly
expressed nucleic acid or proteinaceous sequences known to those of
skill in the art can now be employed. In certain aspects, a nucleic
acid may be purified on polyacrylamide gels, and/or cesium chloride
centrifugation gradients, or by any other means known to one of
ordinary skill in the art (see for example, Sambrook et al. 2001
incorporated herein by reference). In further aspects, a
purification of a proteinaceous sequence may be conducted by
recombinantly expressing the sequence as a fusion protein. Such
purification methods are routine in the art. This is exemplified by
the generation of an specific protein-glutathione S-transferase
fusion protein, expression in E. coli, and isolation to homogeneity
using affinity chromatography on glutathione-agarose or the
generation of a polyhistidine tag on the N- or C-terminus of the
protein, and subsequent purification using Ni-affinity
chromatography.
[0142] In particular aspects, cells or other components of a
vaccine may be purified by flow cytometry. Flow cytometry involves
the separation of cells or other particles in a liquid sample, and
is well known in the art (see, for example, U.S. Pat. Nos.
3,826,364, 4,284,412, 4,989,977, 4,498,766, 5,478,722, 4,857,451,
4,774,189, 4,767,206, 4,714,682, 5,160,974 and 4,661,913). Any of
these techniques described herein, and combinations of these and
any other techniques known to skilled artisans, may be used to
purify and/or assay the purity of the various chemicals,
proteinaceous compounds, nucleic acids, cellular materials and/or
cells that may comprise a vaccine of the present invention. As is
generally known in the art, it is believed that the order of
conducting the various purification steps may be changed, or that
certain steps may be omitted, and still result in a suitable method
for the preparation of a substantially purified antigen or other
vaccine component.
[0143] G. Enhancement of Immune Response
[0144] The present invention includes a method of enhancing the
immune response in a subject comprising the steps of contacting one
or more lymphocytes with an antigen modified as is described
herein. In certain embodiments, a modified antigen may be
conjugated to or comprises an HLA anchor motif amino acids. In
other embodiments, a composition comprising an antigen as described
herein is contained in a mixture that comprises an additional
immunostimulatory agent. Immunostimulatory agents include but are
not limited to an additional antigen, an immunomodulator, an
antigen presenting cell or an adjuvant. In other embodiments, one
or more of the additional agent(s) is covalently bonded to an
antigen or an agent, in any combination.
[0145] In certain embodiments, a lymphocyte contacted with a
modified CTL epitope is comprised in an animal, such as a human. In
certain embodiments, the animal is a human cancer patient, for
example, a human breast cancer patient or a human prostate cancer
patient. In a preferred aspect, the one or more lymphocytes
comprise a T-lymphocyte. In a particularly preferred facet, the
T-lymphocyte is a cytotoxic T-lymphocyte.
[0146] The enhanced immune response may be an active or a passive
immune response. Alternatively, the response may be part of an
adoptive immunotherapy approach in which lymphocyte(s) are obtained
with from an animal (e.g., a patient), then pulsed with a
composition comprising a modified antigenic composition. In this
embodiment, the antigenic composition may comprise an additional
immunostimulatory agent. The lymphocyte(s) may be obtained from the
blood of the subject, or alternatively from tumor tissue to obtain
tumor infiltrating lymphocyte(s) as disclosed in Rosenberg et al.,
1986, incorporated herein by reference. In certain preferred
embodiments, the lymphocyte(s) are peripheral blood lymphocyte(s).
In a one embodiment, the lymphocyte(s) are administered to the same
or a different animal (e.g., same or different donors). In another
embodiment, the animal (e.g., a patient) has or is suspected of
having a cancer, such as for example, a breast or prostate cancer.
One type of such therapy is active immunotherapy.
[0147] In active immunotherapy, the antigen, for example,
comprising a CTL epitope modified as described herein, is
administered, generally with a distinct bacterial adjuvant
(Ravindranath & Morton, 1991; Mitchell et al., 1990; Mitchell
et al., 1993). For example, even with prior techniques, in melanoma
immunotherapy, those patients who elicit high IgM response often
survive better than those who elicit no or low IgM antibodies
(Morton et al., 1992). IgM antibodies are often transient
antibodies and the exception to the rule appears to be
anti-ganglioside or anticarbohydrate antibodies.
[0148] H. Vaccine Administration
[0149] The manner of administration of a vaccine comprising an
antigen prepared in accordance with the invention may be varied
widely. Any of the conventional methods for administration of a
vaccine are applicable. For example, a vaccine may be
conventionally administered intravenously, intradermally,
intraarterially, intraperitoneally, intralesionally,
intracranially, intraarticularly, intraprostaticaly,
intrapleurally, intratracheally, intranasally, intravitreally,
intravaginally, intratumorally, intramuscularly, intraperitoneally,
subcutaneously, intravesicularlly, mucosally, intrapericardially,
orally, rectally, nasally, topically, in eye drops, locally, using
aerosol, injection, infusion, continuous infusion, localized
perfusion bathing target cells directly, via a catheter, via a
lavage, in cremes, in lipid compositions (e.g., liposomes), or by
other method or any combination of the forgoing as would be known
to one of ordinary skill in the art (see, for example, Remington's
Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990,
incorporated herein by reference).
[0150] A vaccination schedule and dosages may be varied on a
patient by patient basis, taking into account, for example, factors
such as the weight and age of the patient, the type of disease
being treated, the severity of the disease condition, previous or
concurrent therapeutic interventions, the manner of administration
and the like, which can be readily determined by one of ordinary
skill in the art.
[0151] A vaccine is administered in a manner compatible with the
dosage formulation, and in such amount as will be therapeutically
effective and immunogenic. For example, the intramuscular route may
be preferred in the case of antigens with short half lives in vivo.
The quantity to be administered depends on the subject to be
treated, including, e.g., the capacity of the individual's immune
system to synthesize antibodies, and the degree of protection
desired. The dosage of the vaccine will depend on the route of
administration and will vary according to the size of the host.
[0152] Precise amounts of an active ingredient required to be
administered depend on the judgment of the practitioner. In certain
embodiments, pharmaceutical compositions may comprise, for example,
at least about 0.1% of an active compound. In other embodiments,
the an active compound may comprise between about 2% to about 75%
of the weight of the unit, or between about 25% to about 60%, for
example, and any range derivable therein However, a suitable dosage
range may be, for example, of the order of several hundred
micrograms of active ingredient per vaccination. In other
non-limiting examples, a dose may also comprise from about 1
microgram/kg/body weight, about 5 microgram/kg/body weight, about
10 microgram/kg/body weight, about 50 microgram/kg/body weight,
about 100 microgram/kg/body weight, about 200 microgram/kg/body
weight, about 350 microgram/kg/body weight, about 500
microgram/kg/body weight, about 1 milligram/kg/body weight, about 5
milligram/kg/body weight, about 10 milligram/kg/body weight, about
50 milligram/kg/body weight, about 100 milligram/kg/body weight,
about 200 milligram/kg/body weight, about 350 milligram/kg/body
weight, about 500 milligram/kg/body weight, to about 1000
mg/kg/body weight or more per vaccination, and any range derivable
therein. In non-limiting examples of a derivable range from the
numbers listed herein, a range of about 5 mg/kg/body weight to
about 100 mg/kg/body weight, about 5 microgram/kg/body weight to
about 500 milligram/kg/body weight, etc., can be administered,
based on the numbers described above. A suitable regime for initial
administration and booster administrations (e.g., inoculations) are
also variable, but are typified by an initial administration
followed by subsequent inoculation(s) or other
administration(s).
[0153] In many instances, it will be desirable to have multiple
administrations of the vaccine, usually not exceeding six
vaccinations, more usually not exceeding four vaccinations and
preferably one or more, usually at least about three vaccinations.
In prophylactic embodiments, the vaccinations will normally be at
from two to twelve week intervals, more usually from three to five
week intervals. Periodic boosters at intervals of 1-5 years,
usually three years, will be desirable to maintain protective
levels of the antibodies.
[0154] The course of the immunization may be followed by assays for
antibodies for the modified antigens. The assays may be performed
by labeling with conventional labels, such as radionuclides,
enzymes, fluorescents, and the like. These techniques are well
known and may be found in a wide variety of patents, such as U.S.
Pat. Nos. 3,791,932; 4,174,384 and 3,949,064, as illustrative of
these types of assays.
[0155] I. Infectious Disease States
[0156] In addition to the treatment of cancer, the current
invention is applicable to the treatment or prevention of diseases
mediated by an infectious agent, for example, a bacteria, virus or
parasite. In particular, by modulation of an immunologic response
to a CTL epitope of an infectious agent, an immune reaction to the
antigen bearing the given CTL epitope may be modulated for clinical
benefit. Such CTL epitopes may be found and modified from viral and
bacterial pathogens, as well as various parasitic organisms.
Non-limiting examples of such causative agents which may be treated
with the invention are presented below.
[0157] 1. Viral Infections
[0158] Certain aspects of the current invention concern treatment
or prevention of viral diseases by modulation of an immunologic
response to viral infection. In particular, by identification and
modification of a viral CTL epitope, as is described herein,
certain therapeutic or prophylactic benefits may be obtained. Such
viruses may enter or exit the body through the mucosal surfaces
such as the following pathogenic viruses which are mentioned by way
of example, influenza A, B and C, parainfluenza, paramyxoviruses,
Newcastle disease virus, respiratory syncytial virus, measles,
mumps, adenoviruses, adenoassociated viruses, parvoviruses,
Epstein-Barr virus, rhinoviruses, coxsackievirus es, echoviruses,
reoviruses, rhabdoviruses, lymphocytic choriomeningitis,
coronavirus, polioviruses, herpes simplex, human immunodeficiency
viruses, cytomegaloviruses, papillomaviruses, virus B,
varicella-zoster, poxviruses, rubella, rabies, picornaviruses,
rotavirus and Kaposi associated herpes virus.
[0159] 2. Bacterial Infections
[0160] The invention may also find use in the treatment or
prevention of a disease mediated by bacterial infection. As
indicated, this may be carried out by identifying and modifying a
bacterial CTL epitope and administering this to an individual in
need thereof. Again, this may be done either in response to an
ongoing bacterial disease and/or for the prevention of such a
disease. Examples of such bacterial infections that could be
treated or prevented with the invention, include, but are not
limited to, the 83 or more distinct serotypes of pneumococci,
streptococci such as S. pyogenes, S. agalactiae, S. equi, S. canis,
S. bovis, S. equinus, S. anginosus, S. sanguis, S. salivarius, S.
mitis, S. mutans, other viridans streptococci, peptostreptococci,
other related species of streptococci, enterococci such as
Enterococcus faecalis, Enterococcus faecium, Staphylococci, such as
Staphylococcus epidermidis, Staphylococcus aureus, particularly in
the nasopharynx, Hemophilus influenzae, pseudomonas species such as
Pseudomonas aeruginosa, Pseudomonas pseudomallei, Pseudomonas
mallei, brucellas such as Brucella melitensis, Brucella suis,
Brucella abortus, Bordetella pertussis, Neisseria meningitidis,
Neisseria gonorrhoeae, Moraxella catarrhalis, Corynebacterium
diphtheriae, Corynebacterium ulcerans, Corynebacterium
pseudotuberculosis, Corynebacterium pseudodiphtheriticum,
Corynebacterium urealyticum, Corynebacterium hemolyticum,
Corynebacterium equi, etc. Listeria monocytogenes, Nocordia
asteroides, Bacteroides species, Actinomycetes species, Treponema
pallidum, Leptospirosa species and related organisms. The invention
may also be useful against gram negative bacteria such as
Klebsiella pneumoniae, Escherichia coli, Proteus, Serratia species,
Acinetobacter, Yersinia pestis, Francisella tularensis,
Enterobacter species, Bacteroides and Legionella species and the
like.
[0161] 3. Parasitic Infections
[0162] In addition, the invention may prove useful in controlling
protozoan or macroscopic infections by organisms such as
Cryptosporidium, Isospora belli, Toxoplasma gondii, Trichomonas
vaginalis, Cyclospora species, for example, and for Chlamydia
trachomatis and other Chlamydia infections such as Chlamydia
psittaci, or Chlamydia pneumoniae, for example. Of course it is
understood that the invention may be used on any pathogen for which
a CTL epitope can be identified and modified in accordance with the
invention.
[0163] J. Vectors
[0164] In certain embodiments of the invention, a modified CTL
epitope-containing antigen may be administered to a patient in need
thereof in the form of a transformation vector. For example, such
vectors may be administered to a patient to achieve expression of
the epitope or may be administered as cells which have been
transformed with the vector. The transfection of cells may thus be
used, in certain embodiments, to recombinantly produce one or more
vaccine components for subsequent purification and preparation into
a pharmaceutical vaccine. In other embodiments, the nucleic acid is
transfected into a cell and the cell administered to an animal as a
cellular vaccine component.
[0165] The term "vector" is used to refer to a carrier nucleic acid
molecule into which a nucleic acid sequence can be inserted for
introduction into a cell where it can be replicated. A nucleic acid
sequence can be "exogenous," which means that it is foreign to the
cell into which the vector is being introduced or that the sequence
is homologous to a sequence in the cell but in a position within
the host cell nucleic acid in which the sequence is ordinarily not
found. Vectors include plasmids, cosmids, viruses (bacteriophage,
animal viruses, and plant viruses), and artificial chromosomes
(e.g., YACs). One of skill in the art would be well equipped to
construct a vector through standard recombinant techniques (see,
for example, Maniatis et al., 1988 and Ausubel et al., 1994, both
incorporated herein by reference).
[0166] The nucleic acid encoding the antigenic composition or other
vaccine component may be stably integrated into the genome of the
cell, or may be stably maintained in the cell as a separate,
episomal segment of DNA. Such nucleic acid segments or "episomes"
encode sequences sufficient to permit maintenance and replication
independent of or in synchronization with the host cell cycle.
Vectors and expression vectors may contain nucleic acid sequences
that serve other functions as well and are described infra. How the
expression construct is delivered to a cell and where in the cell
the nucleic acid remains is dependent on the type of expression
construct employed.
[0167] A "promoter" is a control sequence that is a region of a
nucleic acid sequence at which initiation and rate of transcription
are controlled. It may contain genetic elements at which regulatory
proteins and molecules may bind, such as RNA polymerase and other
transcription factors, to initiate the specific transcription a
nucleic acid sequence. Promoter for use in different cell types,
including mammalian and human cells, are well known to those of
skill in the art.
[0168] Numerous different types of vectors for transformation of
cells are known. The ability of certain viruses to infect cells or
enter cells via receptor-mediated endocytosis, and to integrate
into host cell genome and express viral genes stably and
efficiently have made them attractive candidates for the transfer
of foreign nucleic acids into cells (e.g., mammalian cells). For
example, a modified CTL epitope could be encoded by a nucleic acid
or other components such as, for example, an immunomodulator or
adjuvant, could be encoded by the vector. Many types of viral
vectors are known and could be used with the invention, including
adenoviral vectors, adeno-associated viruses (AAV), retroviral
vectors or other types of viral vectors.
[0169] Suitable methods for nucleic acid delivery for
transformation of cells are also well known to those of skill in
the art. Examples of such methods known to those of skill in the
art include, but are by no means limited to: calcium phosphate
precipitation, use of DEAE-dextran followed by polyethylene glycol,
direct sonic loading and liposome-mediated transfection. Any such
of these methods or other methods may thus be used with the
invention.
IV. SCREENING FOR MODULATION OF IMMUNOGENICITY
[0170] In certain aspects of the invention, assays for modulation
of immunogenicity may be used for the assessment of particular
modified antigen epitopes. In this manner, modifications may be
optimized for the desired immunologic effect. For example, assays
of CTL activity may be used following administration of modified
antigens. CTL activity can be assessed by methods described herein
or as would be known to one of skill in the art. Such assays may
find use in accordance with the invention for the assessment of
modified CTL epitopes for the ability to modulate immunogenicity.
For example, CTLs may be assessed in freshly isolated peripheral
blood mononuclear cells (PBMC), in a phytohaemaglutinin-stimulated
IL-2 expanded cell line established from PBMC (Bernard et al.,
1998) or by T cells isolated from a previously immunized subject
and restimulated for 6 days with antigen using standard 4 h
.sup.51Cr release microtoxicity assays. One type of assay uses
cloned T-cells.
[0171] Cloned T-cells have been tested for their ability to mediate
both perforin and Fas ligand-dependent killing in redirected
cytotoxicity assays (Simpson et al., 1998). The cloned cytotoxic T
lymphocytes displayed both Fas- and perforin-dependent killing.
Recently, an in vitro dehydrogenase release assay has been
developed that takes advantage of a new fluorescent amplification
system (Page et al., 1998). This approach is sensitive, rapid,
reproducible and may be used advantageously for mixed lymphocyte
reaction (MLR). It may easily be further automated for large scale
cytotoxicity testing using cell membrane integrity, and is thus
could be used in the present invention. In another fluorometric
assay developed for detecting cell-mediated cytotoxicity, the
fluorophore used is the non-toxic molecule alamarBlue (Nociari et
al., 1998). The alamarBlue is fluorescently quenched (i.e., low
quantum yield) until mitochondrial reduction occurs, which then
results in a dramatic increase in the alamarBlue fluorescence
intensity (i.e., increase in the quantum yield). This assay is
reported to be extremely sensitive, specific and requires a
significantly lower number of effector cells than the standard
.sup.51Cr release assay.
[0172] In certain aspects, T helper cell responses can be measured
by in vitro or in vivo assay with peptides, polypeptides or
proteins. In vitro assays include measurement of a specific
cytokine release by enzyme, radioisotope, chromaphore or
fluorescent assays. In vivo assays include delayed type
hypersensitivity responses called skin tests, as would be known to
one of ordinary skill in the art.
V. EXAMPLES
[0173] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Modified Epitopes
[0174] A modified epitope was created based on the CTL epitope from
the HER-2 proto-oncogene protein product. The sequence of the
native peptide (SEQ ID NO:2) is as follows:
[0175] A preliminary analysis of the possible orientation of the
amino acids in this peptide when bound to HLA-A2 indicated that
Gly4 and Alal were good candidates for CH.sub.2-extension because
Gly4 lacks a side chain, and Ala7 has one CH.sub.3 group as a side
chain. Since Ala7 is preceded by Leu6 and followed by Phe8 and
Leu9, it was hypothesized that the CH.sub.3 side chain in Ala7
points either sideways or upwards (Leu9, down, Phe8 side or up and
Ala7, side or up). Based on this, it was decided to replace the
Ala7 with the unnatural aminoacids: .gamma.-aminobutyric (Abu)
which has 1 CH.sub.2 group extension compared with Ala7 (designated
herein A7.1), norvaline (NVal) which has 2 CH.sub.2 groups
extending linearly from Ala7 (designated herein A7.2) and
norleucine (Nleu) which has 3 CH.sub.2 groups extending from Ala to
the side chain of Ala7 (designated herein A7.3). The same approach
was used for extending the Gly4 with 4 CH.sub.2 groups (+1, +2, +3,
+4) by replacing successively Gly4 with Ala, ABu, NVal and
Nleu.
[0176] A second approach to this CH.sub.2 modification is to
shorten the side chain in Phe8 ("attenuation") by replacement of
Phe8 with IsoPhe8. IsoPhe lacks the CH.sub.2 group between the
phenol ring and the peptide bond.
[0177] Molecular modeling showed that indeed CH.sub.2 extension at
Ala7 lead to a C-chain which is oriented upward (i.e., toward the
TCR). The structures of E75 (Ala), A7.1 (Abu), A7.2 (Nval), A7.3
(Nleu) in the HLA-A2 were modeled by downloading the coordinates of
the HLA-A2 native structure (Saper, 1999) from the Brookhaven
protein database. This file was used as a template for
manipulations with the Swiss Model program (Peitsch,. 1997),
available through the Expasy web site. A bound Tax peptide was
mutated manually to yield the bound E75 peptide. The new structure
was optimized and energy minimized with the GROMOS96 implementation
or the Swiss-Pdb Viewer. The van der Waals radii or the equivalent
atoms were depicted as spheres. The CH.sub.2-extended Ala-side
chains were presented in yellow (structure not shown). The
HLA-A2-peptide structure was presented for each peptide in a
corresponding box.
[0178] The possibility of inducing side chain changes is
controllable. For example Lysl can be replaced with ornithine (-1
CH.sub.2). Arg can be replaced by citrulline, etc. Thus in E75
successive attenuation could be obtained by removal of 5 CH.sub.2
groups in Lysl, Phe3, Leu6, Ala7, and Phe8. Gradual attenuation can
be achieved by successful removal of these groups. This approach
can be used for other tumor peptides which bind not only HLA-A2,
but also other MHC-(class I and class II) molecules.
Example 2
Modeling of the E75-HLA-A2 Complex
[0179] E75-HLA-A2, models were generated by replacement of the
HTLV-1 (Tax) peptide with E75 (Garboczi et al., 1996; Madden et
al., 1993; Baker et al., 2000; Gillogly et al., 2000). Tax shows
the highest structural similarity of the models available in public
databases. The Tax peptide: L L F G Y P V Y V (SEQ ID NO:1) is
similar to E75: KIEG SL AFL, (SEQ ID NO:2) with respect aliphatic
side chain extension in the first 4 and the last 3 amino acids,
with only Lysl and Phe8 differing by NH.sub.3 and OH group
extensions. The central area of Tax is currently under intense
scrutiny, with the analog P6A showing even more similarity in the
core with E75 (Leu6) (Baker et al., 2000).
[0180] Here, the inventors replaced Ala7 with the unnatural amino
acids .gamma.ABU, NVal, and NLeu, because the side chains of these
amino acids linearly extend the CH.sub.3 group of Ala7, with 1, 2,
and 3 CH.sub.2 groups, respectively. This was deemed preferred over
the replacements with Val and ILe, because their branched chains
are less flexible. The HLA-A2-E75 structure was modeled using pdb
entry 1BD2, an HLA-A2 crystal structure with bound Tax peptide and
was analyzed for accessible surface area using the program GETAREA
1.1. The results indicate no significant overall changes in
accessible surface area comparing the initial structure Tax HLA-A2
from structures of pdb versus the model structures and no
significant change between model structures. This is compatible
with the hypothesis that large conformational changes do not occur
upon binding of any of Ala7 variant peptides. Thus it was indicated
that it was likely that this would allow for TCR specific for E75
to bind the peptide. The surface areas calculated for each
structure were: (in .ANG.): Starting structure: (Tax) 18723.12
.ANG.; (HER-2): Ala: (E75)=18707.55 .ANG., .gamma.Abu: (A7.1),
18737.80 .ANG., NVal: (A7.2), 18748.92 .ANG., NLeu: (A7.3),
18775.04 .ANG.. Therefore there is a very small change in the
surface (0.036%) between E75 and A7.3.
Example 3
Confirmation of CTL Epitope Modification Effect
[0181] The CH.sub.2 side chains of the Ala7(E75=A7.0) and
.gamma.ABU7 (A7.1) point sideways while the side chains of NVal
(A7.2) and of NLeu point upwards. Since Gly4 lacks side chains, it
is likely that addition of CH.sub.2 side chains in Gly4 by
replacement with Ala, .gamma.Abu, NVal, and Nleu will lead to
peptide with CH.sub.2 side chains pointing upwards and/or sideways,
creating new contacts for TCR. The fact that the substitution Gly
to NVal is immunogenic was demonstrated by the ability of peptide
G4.3 to induce both IFN-.gamma. and IL-2 at stimulation of PBMC.
A7.1, A7.2, A7.3 were of similar although slightly lower HLA-A2
stabilizing ability, as determined by on- and off-kinetics.
Example 4
Priming with CH.sub.2 Extended E75 Analogs Induced High Levels or
IFN-7 and IL-2 in Weak E75-Responder PBMC
[0182] To establish the ability of CH2-E75 to activate T cells, the
ability of A7.2, A7.3 and G4.3 to activate induction of IFN-.gamma.
at priming was determined. Two donors were selected based on their
weak ability to respond to E75 priming even in the presence of
IL-12. FIGS. 1A and 1B shows that each of the A7.2, A7.3, G4.3 at
25 .mu.M on autologous DC induced higher levels of IFN-.gamma. than
E75 in both donors tested. These results were confirmed with Donor
4, known to respond to E75 by rapid IFN-.gamma. induction. FIG. 1C
shows that peptide F8-1 induced lower levels of IFN-.gamma. than
E75. IsoPhe lacks the intermediate CH.sub.2 group of Phe between
the benzene ring and the peptide chain, thus is 1 CH.sub.2
"shorter" than Phe8.
[0183] To address whether CH2-E75-activated T cells recognized E75,
E75- primed and A7.2-primed T cells were cultured in low
concentrations of IL-2 (40-60 IU/ml) for one week, rested, and
tested for their ability to respond to E75 within 16 h at a lower
exogenous pulsed concentration 2 .mu.g/ml. FIG. 2A shows that at 2
.mu.M, A7.2-primed T cells responded to E75 with 3-fold higher
levels of IFN-.gamma. than E75-primed T cells. This suggested that
A7.2-primed T cells recognized E75 with higher affinity than
E75-primed T cells. To address whether CH2-E75 induce higher levels
of IL-2 than E75, all analogs were tested again in Donor 1 in the
same study. FIG. 2B show that G4.3 induced high levels of IL-2 in
this donor, compared with E75, A7.2, and A7.3.
Example 5
Priming with CH.sub.2 Extended Analogs Induced-E75-Specific CTL of
Higher Avidity for E75 than Priming with E75
[0184] To address whether A 7.1, A 7.2 and A7.3 induced lytic
effectors, their ability to activate lytic function in CD8+ cells
isolated from TIL of an HLA-A2.sup.+ ovarian patient was tested. T2
were used to present peptides to minimize the cross-reactivity of
TIL with allo-DC. FIG. 3A show that the affinity for E75 of CTL
primed with CH2-E75 decreased in the order
A7.3>A7.2>A7.1=A7.0. A7.0 and A7.1 stimulated CTL-TIL did not
recognize E75. Restimulation of A7.3-induced CTL with A7.3 enhanced
their affinity for E75 to the 200 nM level (FIG. 3B) while an
additional stimulation with A7.2 increased their sensitivity for
E75 at 50 nM level. This sensitivity is at least 100-fold higher
than the optimal sensitivity of E75-induced CTL (5000-25000 nM)
(zum Buschenfelde et al., 2000; Anderson et al., 2000). To address
whether A7.2 and A7.3 activate lytic function of peripheral T
cells, the ability of A7.3, A7.2 and A7.0 (E75) to activate
E75-specific cytolysis was tested. Similar results were obtained
with Donor 4 (FIG. 3C). FIG. 3C shows that A 7.3-induced
CTL-recognized E75 at 25 nM exogenous pulsed concentration with
higher affinity that E75-primed CTL.
Example 6
Attenuation of Signaling by CH.sub.2 Deletion
[0185] As A 7.3 activated CTL decreased in numbers at subsequent
restimulations with A7.3, it was investigated whether attenuation
of signaling by CH.sub.2 deletion can increase their numbers. Two
times stimulated (2.times.A7.3) cells were restimulated two more
times, in parallel, with A7.2 or A7.3. A7.2 stimulated cells
increased in numbers compared with A7.3-stimulated cells.
2.times.A7.3 to 2.times.A7.2 stimulated cells contained a higher
number of E75-specific lytic effectors compared with 4.times.A7.3
cells, as indicated by lytic units, LU (FIG. 4B). These results
demonstrated that attenuation of TCR signaling using less
CH.sub.2-extended E75 enhanced the overall yields of high affinity
CTL. Since the numbers of CD8.sup.+ cells induced by the schedule
2.times.A7.3 to 2.times.A7.2 were 4 times higher than the numbers
of CD8.sup.+ cells induced by the schedule 4.times.A7.3 and the
number of E75-specific LU induced by the first schedule was two
times higher, this suggests an 8-fold (4.times.2) increase in the
number of E75-lytic specific effectors by alternation of stronger
and weaker signaling.
Example 7
Rested (Post-Effector) A7.3-Induced CTL Required Restimulation for
Activation of Lytic Function
[0186] To elucidate whether A7.3-induced CTL express lytic function
without stimulation, A 7.3 induced CTL were rested (posteffectors)
and restimulated with A7.3, A7.2 and A7.0, pulsed on autologous DC,
or with autologous DC which were not pulsed with peptide group (0)
in the absence of IL-2. A7.3-CTL were tested 30 h later for
recognition of E75 pulsed on .sup.51Cr-labelled T2 cells. A7.3-CTL
required antigen-stimulation for expression of lytic function
because they responded to either E75 or to A7.2 and A7.3 analogs by
expression of lytic function. Although somewhat higher levels of
lytic activity were induced in A7.3 CTL by restimulation with A7.2,
confirming the results in FIG. 4B, the fact that A7.3-induced CTL
activated a lytic function in response to E75 suggested that such
CTL may be activated in response to tumor antigen.
Example 8
A7.3-Induced CTL Recognized Endogenously Presented E75
[0187] E75-induced CTL, in some instances, failed to recognize
tumor cells presenting E75 because of their low affinity for the
antigen. To verify that A7.3-induced CTL recognize E75 with high
avidity, Donor 3 CTL-3.sup.hi were induced after priming with E75
and re stimulation with A7.3 from a Donor 3 which responded weakly
to E75)(CTL-3.sup.lo, (Zaks and Rosenberg, 1998). In this donor
A7.2 was a stronger inducer of IFN-.gamma. while A7.3 a stronger
inducer of IL-2 than A7.2 (FIG. 5). CTL-3 recognized E75 with high
avidity. To verify that CTL-3.sup.hi recognized endogenous E75 with
high avidity T2 were pulsed with 100 nM E75 and used to inhibit
lysis. To address whether A7.3-induced CTL recognize ovarian tumor
SKOV3.A2, the inventors performed cold-target inhibition
experiments (FIG. 5B, 6C, 6D). CTL-TIL-lysed SKOV3.A2
(HLA-A2.sup.+), but not SKOV3 cells in the presence of unlabelled
T2 cells which were not pulsed with peptide. When T2 cells were
pulsed with E75, SKOV3.A2 lysis was inhibited by 60% in a 5 h CTL
assay. T2-E75 continued to inhibit SKOV3.A2 lysis at the same or
even higher levels when the assay was continued for 16 h,
suggesting that diversion of E75-specificity was a stable
effect.
Example 9
Stimulation of T-Cells with "Attenuated E75" (F8-1) Increased
Expansion of CD62L+ Cells Compared with E75
[0188] To address whether "attenuated E75" analogs activate
T-cells, the inventors used F8-1. As control "attenuated E75," the
entire CH.sub.2-0H group in the position 5 was deleted by replacing
Ser with Gly (analog S5.0). Isolated CD8.sup.+ cells from Donor 1
were labeled with CFSE, then stimulated with E75, S5.0, F8.1 and as
positive control with the influenza matrix CTL epitope M1: 58-66,
pulsed on autologous DC. IL-2 was added at 100 IU/ml (16 Cetus U)
two days later. Cells were maintained in culture for 20 additional
days, then stained with PE-conjugated mAb to CD62L and examined by
two color fluorescence analysis. FIG. 6 shows that F8-1 induced a
significant increase in the CD62L.sup.+ cells, representing 10.8%
of the resulting population, while in E75 and S.5.0-stimulated
cells, they represented only 3.5% and 4.8%, respectively. CD62L is
down regulated during the first 2-3 divisions, then re-expressed at
higher levels after 6 divisions (Baker et al., 2000). Lack of CFSE
fluorescence in live CD62L.sup.+ cells suggested that these cells
underwent at least 6-7 divisions (Baker et al., 2000). In positive
control, MI stimulated cells CD62L.sup.+ CFSE cells were 21%. This
suggested that under identical conditions F8-1 enhanced
proliferation of a CD62L.sup.+ sub-population compared with
E75.
Example 10
Stimulation of E75-Specific CTL Line (F42SK) with "Attenuated E75"
F8-1 Induced Significantly Higher Levels of BcL-2 and Bcl-XL than
Wild-Type E75, and CH.sub.2-Extended A7.3
[0189] To address whether "attenuated E75" enhanced survival
proteins expression, the E75-specific CTL line F42SK (Gillogly et
al., 2000) was used as a target developed by stimulation of T cells
from a healthy donor which responded weakly to E75, with an
"enhancer agonist" designated F42, developed by replacement of Ser5
with Lys5. Replacement of a OH group with a charged residue
enhanced the affinity of the analog for TCR illustrated by higher
IFN-.gamma. induction by F42 than E75. F42SK-CTL recognized
exogenous pulsed E75 although with lower affinity (5000 ng/ml);
they also recognized SKOV3.A2 cells in the context of HLA-A2, as
demonstrated by cold-target inhibition and antibody-inhibition
assays (Gillogly et al., 2000).
[0190] F42SK-CTL were subjected to multiple rounds of F42
stimulation. The responders never encountered A7.3 or F8-1.
F42SK-CTL showed residual Fas-mediated apoptosis (.gtoreq.30%). E75
induced more protection than the inducer F42 from residual
apoptosis induced by a Fas mAb (FIG. 7A). Since apoptosis
resistance in day 4 stimulated T cells is mainly due to the
intrinsic pathway (Roy and Nicholson, 2000; Krammer, 2000) and
resistance to Fas induced apoptosis was suggestive of TCR induced
protection, the inventors investigated the effects of E75, and F42
in upregulation of Bcl-2 and Bcl-XL and Bad. The effects of A7.3
and F8-1 tested in parallel. FIG. 7B show that F42 and E75 had
similar effects in upregulating Bcl-XL and Bcl-2. F42 was a
slightly stronger up-regulator of Bcl-2 than E75. Their effects on
Bcl-XL were similar. E75 was a stronger inhibitor of Bad than F42.
Both A7.3 and F8-1 were significantly stronger stimulators for
up-regulation of Bcl-2 and Bcl-XL than F42. F8-1 was the strongest
inducer of Bcl-2 and Bcl-XL. Since A7.3 differs from E75 by
addition of 3 CH.sub.2 groups, F8-1 differs from E75 by deletion of
1 CH.sub.2 group, these results demonstrate that E75-specific CTL
are highly sensitive to modulation of CH.sub.2 length by
upregulation of pro survival molecules.
Example 11
Materials and Methods
Cells, Abs, and Cytokines
[0191] HLA-A2.sup.+ and PBMC were obtained from completely
HLA-typed healthy volunteers. T2 cells, ovarian SKOV3, SKOV3.A2
cells, and indicator tumors from ovarian ascites were as described
(Lee et al., 2000; Anderson et al., 2000; Fisk et al., 1995). mAb
to CD3, CD4, CD8 (Ortho Diagnostics, Rantory, N.J.), CD13 and CD14
(Caltag Laboratories, San Francisco, Calif.), and HLA-A2 (clone
BB7.2; American Type Culture Collection, Manassas, Va.) were either
unconjugated or conjugated with FITC or
[0192] PE. antigen expression by dendritic cells (DCs) and T cells
was determined by FACS analysis using a flow cytometer
(EPICS-Profile Analyzer; Coulter Electronics, Hialeah, Fla.).
GM-CSF of specific activity (1.25.times.10.sup.7 CFU/250 mg) was
from Immunex, Seattle, Wash.; TNF-.alpha. of specific activity
(2.5.times.10.sup.7 U/mg) was from Cetus (Emeryville, Calif.); IL-4
of specific activity (5.times.10.sup.6 IU/mg) was from Biosource
International (Camarillo, Calif.); IL-2 of specific activity
(18.times.10.sup.6 IU/mg) was from Cetus; IL-12 of specific
activity (5.times.10.sup.6 U/mg) was a kind gift from Dr. S. Wolf
(Department of Immunology, Genetics Institute, Cambridge, Mass.).
The anti-human-Fas mAb CH11 was purchased from Upstate
Biotechnology (Lake Placid, N.Y.). mAb to actin, Bcl-2,
Bcl-x.sub.L, and Bad were purchased from Santa Cruz Biotechnology
(Santa Cruz, Calif.). All other specific mAb and isotype controls
were obtained from BD PharMingen (San Diego, Calif.).
Synthetic Peptides
[0193] Peptides E75 (HER-2: 369-377) and its mutated analogs were
used and are given in Table II. To facilitate presentation, E75
variants mutated at Ser5 are abbreviated based on the position and
the substitution. For example, the variant in which serine (S) was
replaced by alanine (A) is S5A and the variant in which serine was
replaced with glycine (G) is S5G. A7.3, in which the alanine side
chain was extended with two methylene groups, was obtained by
replacement of Ala with Norleucine (linear side chain). F8-1 was
obtained by replacing of Phe8 with isophenylalanine (IsoPhe) (1
CH.sub.2) deletion. All peptides were prepared by the Synthetic
Antigen Laboratory of M. D. Anderson Cancer Center (Houston, Tex.)
and purified by HPLC. The purity of the peptides ranged from
95-97%. Peptides were dissolved in PBS and stored frozen at
-20.degree. C. in aliquots of 2 mg/ml.
Molecular Modeling of the Peptide: HLA-A2 Complex
[0194] The coordinates of the native HLA-A2 structure (Garboczi et
al., 1996; Saper et al., 1999; Berman et al., 2000) were downloaded
from the Brookhaven protein database (ID number: 3HLA). This file
was used as a template for manipulations with the Swiss Model
(Peitsch et al., 1997) program available through the Expasy web
site. The Tax peptide bound to the HLA-A2 (Hausman et al., 1999)
was mutated manually to yield the bound E75 peptide and the AlaS,
Gly5, and Lys5 variants. Each new structure was submitted for
energy minimization with the GROMOS96 implementation of the
Swiss-PdbViewer. Solvent-accessible surface area was calculated
with the GETAREA1.1 online program with the default probe radius,
set at 1.4 .ANG..
T Cell Stimulation by Peptide-Pulsed DC
[0195] DCs generated from peripheral blood were plated at
1.2.times.10.sup.5 cell/well in 24-well culture plates and pulsed
with peptides at 50 .mu.g/ml in serum-free medium for 2 h before
the addition of responders, as described (Lee et al., 2000;
Anderson et al., 2000). E75-induced and S5K-induced CTL lines were
maintained by periodic stimulation with peptide pulsed on DCs,
followed by expansion in the presence of irradiated feeder cells
and PHA. The number of cells expressing a TCR that was specific for
HLA-A2 bound to the E75 peptide (E75-TCR cells) was performed using
E75 dimers (dE75) prepared as described in the manufacturer's
instructions. Empty HLA-A2:IgG dimers were obtained from BD
Pharmingen. Control without peptide dimers not pulsed with peptide
(NP) were prepared in parallel and tested in the same study.
Positive control influenza matrix peptide M1 (58-66) dimers (dM1)
were prepared simultaneously and used in the same study. For
analysis, cells were incubated in parallel with dNP, and dE75
followed by PE-conjugated anti-mouse IgG1. Intracellular expression
of Bcl-2 was determined, following manufacturer's instructions
using FITC-conjugated Bcl-2, Ab, and a matched FITC-conjugated
isotype control.
CTL and Cytokine Assays
[0196] Recognition by CTL of peptides used as immunogens was
performed as described (Fisk et al., 1995). Recognition of E75 and
of its variants was considered specific when the percent specific
lysis of T2 cells pulsed with E75 minus the SD was higher by at
least 5% than the percentage of specific lysis of T2 cells that had
been pulsed with peptide plus the SD, as described (Knutson et al.,
2001). A significant increase/decrease in CTL activity was defined
as an increase/decrease of >20% in the lysis of T2 cells pulsed
with peptide by variant induced CTL compared with wild-type
E75-induced CTL. Similarly, a significant increase in IFN-.gamma.
induction was defined as an increase of >20% in IFN-.gamma.
levels after stimulation with the variant versus after stimulation
with the wild-type E75. The 20% value was chosen as a cut-off for
significant increase based on the assumption that if a 2-fold
increase of the minimum 5% increase (defined above) is 10%, then an
increase >10% should be significant if it equals at least 20%.
Equal numbers of viable effectors were used in all assays. IL-2,
IL-4, and IFN-.gamma. were detected using cytokine ELISA kits
(Biosource International or R&D Systems, Minneapolis, Minn.)
with a sensitivity of 4-7 pg/ml (Lee et al., 2000).
Apoptosis Assays
[0197] E75- and S5K-CTL lines were activated by autologous DCs
pulsed with various concentrations of E75 or S5K in the presence or
absence of 100 .mu.g/ml of CH11. For anti-CD3-mediated apoptosis,
OKT3 mAb was absorbed on wells of 96-well plates overnight before
addition of lymphocytes (DiSomma et al., 1999). For day 1 apoptosis
assays, IL-2 was not added to the cultures. For day 4 apoptosis
assays, IL-2 (300 IU/ml) was added to the cultures at 24 and 72 h
after stimulation with DC-pulsed peptides. Detection of
Fas-mediated apoptosis was performed in the presence or absence of
the agonistic mAb CH11 (anti-Fas mAb) as described (DiSomma et al.,
1999). Cells were labeled by incubation in PBS containing 0.1%
Triton X-100 and 50 .mu.g/ml propidium iodide, and the DNA content
was determined by using flow cytometry.
Western Analysis
[0198] A total of 2.times.10.sup.6 S5K-CD8.sup.+ cells were
stimulated for 96 h with E75, S5K, A7.3, or F8-1 peptides pulsed on
DCs at a final concentration of 25 .mu.g/ml. Additional controls
included cells that were stimulated with T2 that had not been
pulsed with peptide, or S5K cells that were not stimulated or cells
that were stimulated with PHA. A total of 20 .mu.g of protein from
supernatants from 10,000 g of postnuclear detergent lysates were
separated on a 12% SDS-PAGE gel and immunoblotted as described
(Ward et al., 2000). Membranes were probed with monoclonal
anti-actin, anti-Bcl-2 (1:500), anti-Bad (1:500), or
anti-Bcl-x.sub.L (1:500) in 1% BSA-TBS containing 0.1% Tween 20 for
2 h at 25.degree. C., and probed with peroxidase-linked sheep
antimouse Ig (1:1000) in 1% BSA-TBS containing 0.1% Tween 20.
Immunoreactive bands were detected by ECL as described (Ward et
al., 2000).
Example 12
Molecular Modeling
[0199] To address deficiencies in the art, binding of the HER-2/neu
protooncogene (HER-2), CTL epitope E75 (369-377) to HLA-A2 was
examined at the atomic level. Molecular models of the E75-HLA-A2
complex indicated that the side chain of the central Ser5 (S373)
points upward. Thus, the OH group can either enhance binding at the
TCR via a hydrogen bond, or sterically hinder the interaction with
the TCR by decreasing the affinity of the TCR for the pMHC-I. If
the first hypothesis is true, then removal of the OH group should
decrease the affinity of binding by the TCR and decrease signaling,
hence variants in which the central Ser is replaced by Ala or Gly
should be less immunogenic than wild-type E75. If the second
hypothesis is true, then Ala/Gly variants should be more
immunogenic than the wild-type E75. To address the requirement that
variant-induced CTLs survive their encounter with the wild-type
antigen, another variant was created to demonstrate that
stimulation with that variant should protect responding cells from
death by over-stimulation. This variant should stimulate some of
the effector functions weaker than E75, and E75 should activate the
variant-induced effectors. The only alternatives that would not
disturb the peptide bond were positively and negatively charged
side chains. Because the negatively charged amino acids Glu and Asp
have bulky carboxyl groups, Ser5 was replaced with the positively
charged Lys5 (variant S5K). The aminopropyl group of Lys extends
farther and has a greater flexibility than the acetyl group of the
Glu.
[0200] Priming with variants S5A and S5G enhanced the induction of
IFN-.gamma. and E75-specific cytolysis of CTL from two donors known
to respond to E75, but the responders died faster than did the
cells that had been stimulated by E75. In contrast, variant S5K
induced higher levels of IFN-.gamma., but not of CTL activity
against E75 than the E75-induced CTL (E75-CTL). In a "weak
responder" to E75, S5K-induced CTL (S5K-CTL) recognized E75 with
lower affinity than did E75-induced CTL. S5K-CTL survived longer
than the E75-CTL, which became apoptotic at restimulation with E75.
Of interest, restimulation with E75 resulted in better protection
from apoptosis in the S5K-CTL than did restimulation with S5K. This
protection was paralleled by higher Bcl-x.sub.L to Bad ratios and
higher Bcl-2 levels than the ones induced by S5K. Thus, the side
chain variants that were less activating than the wild-type antigen
induced specific CTL for the E75 expressed on tumors. Such CTL were
then expanded by E75, indicating that the nominal antigen or
stronger agonistic variants can use priming with weak agonists to
bypass induction of apoptosis.
Example 13
Generation of E75 Variants Directed by Molecular Modeling
[0201] This approach was designed to identify amino acids in E75
permissive to replacement that would be substituted without
abolishing the objects of the variant peptide to induce CTL
responses. Substitutions in side chains that maintain the overall
conformation of the peptide backbone in the HLA-2 were deemed more
likely to lead to cross-reactive antigen for wild-type
antigen-specific CTL than substitutions that change the peptide
backbone conformation. The E75-HLA-A2 complex was modeled by
replacing the human T cell leukemia virus-1 peptide Tax with E75.
The Tax peptide (Ding et al., 1999; Baker et al., 2000) shows the
highest structural similarity with E75 of the models available in
the databases. The Tax sequence LLFGYPVYV (SEQ ID NO. 1) is similar
to that of E75:KIFGSLAFL with respect to the position of aromatic
residues in P3 and P8 and the aliphatic side chain extensions in
the first four and the last three amino acids (only K1 and F8
differ by an NH.sub.3 and an OH group extension). The major
differences rest in the central area P5 P6:YP versus SL. One Tax
analog, P6A, showed even more similarity with E75 YA versus SL,
with Ala and Leu differing only in the propyl side chain. This
comparison allowed identification of the side chains that point
upwards or sideways and are thus more likely to contact TCR.
[0202] The results show that the side chains of Lys1, Ser5, and
Phe8 point out of the binding pocket of the MHC. The side chains of
Phe3, Leu6, and Ala7 point toward the helical "walls" of the
pocket. The models of the TCR-pMHC-I (HLA-A2) interaction predicted
that of the side chains pointing away from the MHC, Ser5, Leu6, and
Ala7 were most likely to contact the CDR3 (Va+V(3) region. Ser5 was
focused on because the change induced by the removal of the
hydroxyl group was likely to have the strongest effects. Ser was
substituted with Ala, Gly, and Lys. These substitutions removed an
HO-group (Ala), a HO--CH.sub.2-group (Gly), or replaced the OH
group with the aminopropyl (CH.sub.2--CH.sub.2--CH.sub.2--NH.sub.3)
group. The position of the OH suggested that it was less involved
in interactions with the HLA-A2. No significant changes of the MHC
molecule were necessary to accommodate these modifications. Ser5
was preceded by Gly4, which because it does not have a side chain,
is very flexible and may allow small accommodations in the model.
The positions of Phe3 and Lys1 that precede the Ser5 seem to be
unchanged among the four models. These results indicated that Ser5
is in a good structural position to allow side chain replacements
in the antigenic peptide that can modify its interactions with TCR.
S5A, S5G, and S5K bound to HLA-A2 with similar affinity as did E75
(Table II). In T2-stabilization assays, S5A, S5G, and S5K showed
similar stabilizing ability for HLA-A2 as determined with mAb MA2.1
(Table II, legend), and similar scores for times of dissociation
and ligation strengths (Table II) with those of E75 as determined
using the HLA-peptide binding prediction (Parker et al., 1994) and
SYFPEITHI programs (Rammensee et al., 1999).
Example 14
Increased IFN-.gamma.-Inducing and E75-Specific CTL-Inducing
Ability of the E75-Variants S5A and S5G
[0203] To demonstrate modification of the E75 side chain by
deletion or extension to increase or decrease the ability of the
modified antigen to stimulate CTL induction and survival, several
healthy donors known from previous studies were tested to produce
E75-specific CTL at priming ("strong responders", donors 1 and 2)
or exhibit weak CTL activity after several repeated stimulations
(weak responders, donor 3). PBMC were stimulated in parallel with
autologous DCs pulsed with E75 variants. Donor 1 responded with
higher levels of IFN-.gamma. at priming with variants S5K, S5G, and
S5A, and lower levels of IFN-.gamma. at priming with control
variants F8Y and F8K than at priming with E75 (FIG. 8A and FIG.
8B). CTL induced by priming with E75 recognized E75 better than a
CTL induced by S5K, F8Y, or F8K, whereas CTL induced by S5G and S5A
recognized E75 better than CTL induced by E75. S5A and S5G induced
both higher levels of IFN-.gamma. and higher cytolytic activity
than did E75. Thus, removal of the OH group correlated with higher
IFN-.gamma. induction and higher lytic activity against E75.
[0204] CTL induced by S5K secreted higher levels of IFN-.gamma.,
but their recognition of E75 was weaker. Thus, replacement of OH
group with aminopropyl group had more selective effect than removal
of the OH group. Extension of these results with cells from donor 2
revealed that all the E75 variants induced higher levels of
IFN-.gamma. at priming than did E75: S5K by 36%, S5A by 100%, and
S5G by 64% (FIG. 8C). Significantly higher levels of IFN-.gamma.
were detected 96 h after stimulation with each variant in response
to the highest dose (25 .mu.g) of exogenously pulsed peptide in the
presence of IL-2 for 2 days. Significant differences in IFN-.gamma.
induction were not observed when E75 or its variants were used at
1.0 or 5.0 .mu.g/ml at 48 or 72 h. The E75-specific lytic activity
of CTL induced by S5A was significantly higher than the lytic
activity of CTL induced by E75 (FIG. 8D). The increase in lytic
activity by S5A paralleled the increase in IFN-.gamma. in response
to S5A. Recognition of E75 by S5KCTL was lower than the recognition
by E75-CTL. CTL induced by the E75, S5K-CTL, and S5A-CTL all
recognized the indicator SKOV3.A2 tumor. To determine whether
E75-specific tumor-lytic CTLs were present in the variant-induced
CTL, the inventors performed cold-target inhibition of tumor lysis.
Tumor lysis by S5K-CTL was inhibited less by T2-E75 than lysis by
E75-CTL (FIG. 8E). This confirmation that S5A can induce both
higher IFN-.gamma. and higher lytic activity against E75 suggested
that the OH group of Ser5 hindered the TCR interaction with
peptide-HLA-A2 and that removal of the OH group allowed a stronger
TCR activation. However, at restimulation, the number of cells
stimulated by S5A and S5G dropped faster than the number of cells
that had been stimulated by E75. Cells stimulated by S5K survived
longer than E75-stimulated cells (FIG. 8F), suggesting that the
stimulus from the (CH.sub.2).sub.3--NH.sub.3 was more effective
than stimuli from the CH.sub.3 or the CH.sub.2--OH in maintaining
the survival of responders.
TABLE-US-00003 TABLE II HLA-A2 Binding Stability by E75 and its
Variants.sup.a Binding Liga- Stabil- tion.sup.b Code Sequence ity
Strength Change E75 KIFGSLAFL 482 28 Wild type SEQ ID NO. 2 K1G
GIFGSLAFL 138 28 Positive SEQ ID NO. charge.fwdarw.neutral 3 S5A
KIFGALAFL 482 28 OH.fwdarw.nonpolar SEQ ID NO. aliphatic 4 S5G
KIFGGLAFL 483 30 OH.fwdarw.neutral SEQ ID NO. 5 S5K KIFGKLAFL 482
29 OH.fwdarw.positive SEQ ID NO. charge 6 F8K KIFSGSLAKL 88 30
Aromatic to SEQ ID NO. (+) charged 7 F8Y KIFGSLAYL 482 28 OH in
aromatic SEQ ID NO. residue 8 F8D KIFGSLADL 236 28 Aromatic to SEQ
ID NO. (-) charged 9 A7.3 K1FGSL Nd.sup.c Nd 2 CH.sub.2 exten-
(NLeu)FL sion of Ala.sup.7 SEQ ID NO. 10 F8-1 K1FGSLAL Nd Nd 1
CH.sub.2 deletion (Iso-Phe)L of Phe.sup.8 SEQ ID NO. 11 .sup.aThe
binding stability is an estimate of half time of dissociation (in
minutes) from HLA-A2 of peptides of the sequence listed above. The
theoretical half-life of dissociation was calculated using Parker's
algorithm (Parker et al., 1994) available on the world wide web at
bimas.dcrt.ig. gov/molbiol/hla-bind. .sup.bThe ligation strength
was calculated using the SYFPEITHI program (Rammensee et al.,
1999). The experimentally determ