U.S. patent application number 17/301118 was filed with the patent office on 2021-07-22 for fusion proteins comprising an anti-cd40 antibody and hiv antigenic peptides.
This patent application is currently assigned to BAYLOR RESEARCH INSTITUTE. The applicant listed for this patent is BAYLOR RESEARCH INSTITUTE. Invention is credited to Jacques F. Banchereau, Anne-Laure FLAMAR, Yves Levy, Monica Montes, Gerard ZURAWSKI.
Application Number | 20210221904 17/301118 |
Document ID | / |
Family ID | 1000005482076 |
Filed Date | 2021-07-22 |
United States Patent
Application |
20210221904 |
Kind Code |
A1 |
ZURAWSKI; Gerard ; et
al. |
July 22, 2021 |
FUSION PROTEINS COMPRISING AN ANTI-CD40 ANTIBODY AND HIV ANTIGENIC
PEPTIDES
Abstract
The present invention includes compositions and methods for the
expression, secretion and use of novel compositions for use as,
e.g., vaccines and antigen delivery vectors, to delivery antigens
to antigen presenting cells. In one embodiment, the vector is an
anti-CD40 antibody, or fragments thereof, and one or more antigenic
peptides linked to the anti-CD40 antibody or fragments thereof,
including humanized antibodies.
Inventors: |
ZURAWSKI; Gerard;
(Midlothian, TX) ; Banchereau; Jacques F.;
(Montclair, NJ) ; FLAMAR; Anne-Laure; (New York,
NY) ; Levy; Yves; (US) ; Montes; Monica;
(Dallas, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BAYLOR RESEARCH INSTITUTE |
Dallas |
TX |
US |
|
|
Assignee: |
BAYLOR RESEARCH INSTITUTE
Dallas
TX
|
Family ID: |
1000005482076 |
Appl. No.: |
17/301118 |
Filed: |
March 25, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15787425 |
Oct 18, 2017 |
10988544 |
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17301118 |
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14610456 |
Jan 30, 2015 |
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15787425 |
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12717789 |
Mar 4, 2010 |
8961991 |
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14610456 |
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61159055 |
Mar 10, 2009 |
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61159059 |
Mar 10, 2009 |
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61159062 |
Mar 10, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2740/16222
20130101; G01N 33/6863 20130101; A61K 39/385 20130101; C07K 14/005
20130101; A61K 2039/6056 20130101; C07K 2317/565 20130101; C07K
2319/40 20130101; Y02A 50/30 20180101; C07K 2319/30 20130101; A61K
2039/64 20130101; C07K 2319/91 20130101; C07K 2317/56 20130101;
A61K 39/00 20130101; A61K 2039/627 20130101; C07K 16/00 20130101;
C07K 2317/74 20130101; C07K 2317/77 20130101; C07K 2317/41
20130101; C12N 2770/24222 20130101; C12N 2770/24234 20130101; C07K
2319/00 20130101; C07K 2317/80 20130101; C12N 2760/16122 20130101;
C12N 2740/16322 20130101; C07K 16/2878 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 14/005 20060101 C07K014/005; A61K 39/385 20060101
A61K039/385; G01N 33/68 20060101 G01N033/68; C07K 16/00 20060101
C07K016/00 |
Goverment Interests
STATEMENT OF FEDERALLY FUNDED RESEARCH
[0002] This invention was made with U.S. Government support under
Contract No. 1U19AI057234-0100003 awarded by the NIH. The
government has certain rights in this invention.
Claims
1-56. (canceled)
57. An antibody-antigen fusion protein comprising: a dendritic cell
(DC)-targeting antibody or antigen binding fragment thereof; one or
more viral antigens; and one or more flexible linkers between the
DC-targeting antibody or antigen binding fragment thereof and the
one or more antigens; wherein the viral antigen is not a tumor
associated antigen.
58. The fusion protein of claim 57, wherein the one or more
flexible linkers comprises a polypeptide with an amino acid
sequence corresponding to one or more of: SEQ ID NO:11-14 and
145.
59. The fusion protein of claim 57, wherein the one or more
antigens comprise one or more of gag, pol, nef, and env HIV
antigens.
60. The fusion protein of claim 59, wherein the one or more
antigens comprise a HIV polypeptide with an amino acid sequence
corresponding to one or more of SEQ ID NOS:1-5.
61. The fusion protein of claim 57, wherein the DC targeting
antibody or antigen binding fragment is selected from an antibody
or antigen binding fragment that specifically binds to MHC class I,
MHC class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16,
CD19, CD20, CD29, CD31, CD40, CD43, CD44, CD45, CD54, CD56, CD57,
CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, CLEC-6, CD40, BDCA-2,
MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2,
IFN-.gamma. receptor and IL-2 receptor, ICAM-1, Fc.gamma. receptor,
LOX-1, or ASGPR.
62. The fusion protein of claim 61, wherein the DC targeting
antibody or antigen binding fragment specifically binds to
CD40.
63. The fusion protein of claim 62, wherein the CD40 antibody
comprises: a heavy chain variable domain (VH) which comprises in
sequence hypervariable regions CDR1H, CDR2H and CDR3H, the CDR1H
having the amino acid sequence GFTFSDYYMY (SEQ ID NO.:45), the
CDR2H having the amino acid sequence YINSGGGSTYYPDTVKG (SEQ ID
NO.:46), and the CDR3H having the amino acid sequence RGLPFHAMDY
(SEQ ID NO.:47); and a light chain variable domain (VL) which
comprises in sequence hypervariable regions CDR1L, CDR2L and CDR3L,
the CDR1L having the amino acid sequence SASQGISNYLN (SEQ ID
NO.:41), the CDR2L having the amino acid sequence YTSILHS (SEQ ID
NO.:42), and the CDR3L having the amino acid sequence QQFNKLPPT
(SEQ ID NO.:43).
64. The fusion protein of claim 62, wherein the CD40 antibody
comprises anti-CD40_12B4.2C10 (ATCC Submission No. HS446, Accession
No. PTA-10653) or a humanized version thereof.
65. The fusion protein of claim 62, wherein the CD40 antibody
comprises anti-CD40_11B6.1C3 (ATCC Submission No. HS440, Accession
No. PTA-10652) or a humanized version thereof.
66. The fusion protein of claim 57, wherein the (DC)-targeting
antibody is a human antibody or a humanized antibody.
67. A vaccine comprising the fusion protein of claim 57.
68. A nucleic acid expression vector encoding the fusion protein of
claim 57.
69. A method for treating or preventing HIV in a patient, the
method comprising administering a vaccine comprising the fusion
protein of claim 59.
70. A method of making HIV peptide-specific T cells comprising:
immunizing a subject with a fusion protein of claim 59.
71. A method of enhancing T cell responses comprising: immunizing a
subject in need of vaccination with an effective amount of a
vaccine of claim 67.
72. The method of claim 69, wherein the vaccine is able to elicit
an HIV-specific T cell immune response.
73. The method of claim 69, wherein the one or more antigens elicit
at least one of a humoral or a cellular immune response in a
host.
74. A method of generating antigen-loaded, activated dendritic
cells comprising: obtaining previously isolated patient dendritic
cells and exposing the dendritic cells to activating amounts of the
fusion protein of claim 57.
75. A method of producing a vaccine of claim 67, the method
comprising introducing a flexible linker between a DC-targeting
antibody or antigen binding fragment thereof and an antigen.
76. A method of making a DC-targeting antibody-antigen fusion
protein, the method comprising expressing the fusion protein of
claim 57 in a host cell and isolating the protein from the host
cell.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 15/787,425, filed Oct. 18, 2017, which is a continuation
of U.S. patent application Ser. No. 14/610,456, filed Jan. 30,
2015, which is a divisional of U.S. patent application Ser. No.
12/717,789 filed Mar. 4, 2010, now issued U.S. Pat. No. 8,961,991,
issued Feb. 24, 2005, which claims priority to U.S. Provisional
Application Ser. No. 61/159,059, filed Mar. 10, 2009, 61/159,055,
filed Mar. 10, 2009, and 61/159,062, filed Mar. 10, 2009, and the
entire contents of each are incorporated herein by reference.
TECHNICAL FIELD OF THE INVENTION
[0003] The present invention relates in general to the field of
immunization, and more particularly, to novel anti-CD40 based
vaccines.
BACKGROUND OF THE INVENTION
[0004] Without limiting the scope of the invention, its background
is described in connection with antigen presentation.
[0005] One example of vaccines and methods for antigen presentation
is taught in U.S. Pat. No. 7,118,751, issued to Ledbetter, et al.,
for DNA vaccines encoding an amino-terminus antigen linked to a
carboxy-terminus domain that binds CD40. Briefly, vaccines are
taught that target one or more antigens to a cell surface receptor
to improve the antigen-specific humoral and cellular immune
response. Antigen(s) linked to a domain that binds to a cell
surface receptor are internalized, carrying antigen(s) into an
intracellular compartment where the antigen(s) are digested into
peptides and loaded onto MHC molecules. T cells specific for the
peptide antigens are activated, leading to an enhanced immune
response. The vaccine may comprise antigen(s) linked to a domain
that binds at least one receptor or a DNA plasmid encoding
antigen(s) linked to a domain that binds at least one receptor. A
preferred embodiment of the invention targets HIV-1 env antigen to
the CD40 receptor, resulting in delivery of antigen to CD40
positive cells, and selective activation of the CD40 receptor on
cells presenting HIV-1 env antigens to T cells.
[0006] Another example is found in United States Patent Application
No. 20080254026, filed by Li, et al., for antagonist anti-CD40
monoclonal antibodies and methods for their use. Briefly,
compositions and methods are disclosed for use in therapy for
treating diseases mediated by stimulation of CD40 signaling on
CD40-expressing cells are provided. The methods comprise
administering a therapeutically effective amount of an antagonist
anti-CD40 antibody or antigen-binding fragment thereof to a patient
in need thereof. The antagonist anti-CD40 antibody or
antigen-binding fragment thereof is free of significant agonist
activity, but exhibits antagonist activity when the antibody binds
a CD40 antigen on a human CD40-expressing cell. Antagonist activity
of the anti-CD40 antibody or antigen-binding fragment thereof
beneficially inhibits proliferation and/or differentiation of human
CD40-expressing cells, such as B cells.
[0007] Yet another example is taught in United States Patent
Application No. 20080241139, filed by Delucia for an adjuvant
combination comprising a microbial TLR agonist, a CD40 or 4-1BB
agonist, and optionally an antigen and the use thereof for inducing
a synergistic enhancement in cellular immunity. Briefly, this
application is said to teach adjuvant combinations comprising at
least one microbial TLR agonist such as a whole virus, bacterium or
yeast or portion thereof such a membrane, spheroplast, cytoplast,
or ghost, a CD40 or 4-1BB agonist and optionally an antigen wherein
all 3 moieties may be separate or comprise the same recombinant
microorganism or virus are disclosed. The use of these immune
adjuvants for treatment of various chronic diseases such as cancers
and HIV infection is also provided.
[0008] United States Patent Application No. 20080199471, filed by
Bernett, et al., is directed to optimized CD40 antibodies and
methods of using the same. Briefly, this application is said to
teach antibodies that target CD40, wherein the antibodies comprise
at least one modification relative to a parent antibody, wherein
the modification alters affinity to an Fc.gamma.R or alters
effector function as compared to the parent antibody. Also
disclosed are methods of using the antibodies of the invention.
[0009] Finally, United States Patent Application No. 20080181915,
file by Tripp, et al., is directed to a CD40 ligand adjuvant for
respiratory syncytial virus. Briefly, this application is said to
teach methods and adjuvants for enhancing an immune response to RSV
in a host, wherein the methods and adjuvants comprise a source of a
CD40 binding protein. Preferably, the CD40 binding protein is CD40L
and the source is a vector comprising a promoter operatively linked
to a CD40L coding region. The enhanced immune response produced by
the adjuvants and methods of the current invention includes both
increased expression of Th1 cytokines and increased production of
antibody.
SUMMARY OF THE INVENTION
[0010] In one embodiment, the present invention is a fusion protein
comprising the formula: Antibody(Ab)-Peptide Linker (PL)-Antigen
(Ag)x, Ab-(PL-Ag)x; Ab-(Ag-PL)x; Ab-(PL-Ag-PL)x; Ab-(Ag-PL-Ag)x;
Ab-(PL-Ag)x-PL; or Ab-(Ag-PL)x-Ag; wherein Ab is an antibody or
fragment thereof; wherein PL is at least one peptide linker
comprising at least one glycosylation site; wherein Ag is at least
one antigen; and wherein x is an integer from 1 to 20, the fusion
protein having more stability in solution than the same fusion
protein without the glycosylation site. In one aspect, Ag is
selected from a viral antigen, a tumor antigen, an infectious
disease antigen, an autoimmune antigen, a toxin or combinations
thereof. In another aspect, the Ag is a peptide concatemer. In
another aspect, the PL is a peptide concatemer. In another aspect,
the -(PL-Ag)x, -(Ag-PL)x, -(PL-Ag-PL)x, or -(Ag-PL-Ag)x are located
at the carboxy terminus of the Ab heavy chain or fragment thereof.
In another aspect, the Ag elicits a humoral immune response and/or
cellular immune response in a host. In one aspect, the Ab comprises
at least the variable region of anti-CD40_12E12.3F3 (ATCC Accession
No. PTA-9854), anti-CD40_12B4.2C10 (Deposit Submission No. HS446,
ATCC Accession No. PTA-10653), and anti-CD40_11B6.1C3 (Deposit
Submission No. HS440, ATCC Accession No. PTA-10652).
[0011] In one aspect, the Ag is selected from autoimmune diseases
or disorders associated with antigens involved in autoimmune
disease selected from glutamic acid decarboxylase 65 (GAD 65),
native DNA, myelin basic protein, myelin proteolipid protein,
acetylcholine receptor components, thyroglobulin, and the thyroid
stimulating hormone (TSH) receptor. In another aspect, the Ag is
selected from infectious disease antigens selected from bacterial,
viral, parasitic, and fungal antigens. In another aspect, x
comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, or 19. In another aspect, the fusion protein comprises two
or more Ags from different antigens separated by at least one PL.
In another aspect, the fusion protein comprises two or more Ags
separated by at least one PL comprising an alanine and a serine. In
another aspect, the Ab is an antibody fragment selected from Fv,
Fab, Fab', F(ab')2, Fc, or a ScFv.
[0012] In one aspect, the Ab binds specifically to an MHC class I,
MHC class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD
19, CD20, CD29, CD31, CD40, CD43, CD44, CD45, CD54, CD56, CD57,
CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40,
BDCA-2, MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1,
B7-2, IFN-.gamma. receptor and IL-2 receptor, ICAM-1, Fc.gamma.
receptor, T cell receptor, or lectin. In another aspect, the Ab is
an IgA, IgD, IgE, IgG or IgM or isotype thereof. In another aspect,
the Ab is a human antibody or a humanized antibody. In another
aspect, the PL comprises an alanine and a serine. In another
aspect, the PL is selected from: SSVSPTTSVHPTPTSVPPTPTKSSP (SEQ ID
NO.: 11); PTSTPADSSTITPTATPTATPTIKG (SEQ ID NO.: 12);
TVTPTATATPSAIVTTITPTATTKP (SEQ ID NO.: 13); or
TNGSITVAATAPTVTPTVNATPSAA (SEQ ID NO.: 14).
[0013] Yet another embodiment of the present invention is a nucleic
acid expression vector encoding a fusion protein comprising: a
first polynucleotide encoding an antibody light chain or fragment
thereof; and a second polynucleotide encoding an antibody heavy
chain or fragment thereof; wherein the fusion protein comprises the
following formula: Ab-(PL-Ag)x or Ab-(Ag-PL)x; wherein Ab is an
antibody or fragment thereof; wherein PL is at least one peptide
linker comprising at least one glycosylation site; wherein Ag is at
least one antigen; and wherein x is an integer from 1 to 20, the
fusion protein having more stability in solution than the same
fusion protein without the glycosylation site. In one aspect, the
(PL-Ag)x or (Ag-PL)x are located at the carboxy terminus of the Ab
heavy chain or fragment thereof. In another aspect, the first and
second polynucleotide are on a single expression vector. In another
aspect, the Ag is selected from infectious disease antigens
selected from bacterial, viral, parasitic, and fungal antigens. In
another aspect, x comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, or 19. In another aspect, the fusion
protein comprises two or more Ags from different antigens separated
by at least one PL. In another aspect, the fusion protein comprises
two or more Ags separated by at least on PL comprising an alanine
and a serine. In another aspect, the Ab is an antibody fragment
selected from Fv, Fab, Fab', F(ab')2, Fc, or a ScFv. In another
aspect, the Ab binds specifically to an MHC class I, MHC class II,
CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD 19, CD20,
CD29, CD31, CD40, CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83,
CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2,
MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2,
IFN-.gamma. receptor and IL-2 receptor, ICAM-1, Fc.gamma. receptor,
T cell receptor, or lectin. In another aspect, the Ab is an IgA,
IgD, IgE, IgG or IgM or isotype thereof. In another aspect, the Ab
is a human antibody or a humanized antibody. In another aspect, the
PL is comprises an alanine and a serine and/or the PL is selected
from: SSVSPTTSVHPTPTSVPPTPTKSSP (SEQ ID NO.: 11);
PTSTPADSSTITPTATPTATPTIKG (SEQ ID NO.: 12);
TVTPTATATPSAIVTTITPTATTKP (SEQ ID NO.: 13); or
TNGSITVAATAPTVTPTVNATPSAA (SEQ ID NO.: 14). In another aspect, the
first and second polynucleotides are downstream from a constitutive
promoter.
[0014] Yet another embodiment of the present invention is a stable,
secretable fusion protein comprising the formula:
NH.sub.2-Ab-(PL-Ag)x-COOH or NH2-Ab-(Ag-PL)x-COOH; wherein Ab is an
antibody or fragment thereof; wherein PL is at least one peptide
linker comprising at least one glycosylation site; wherein Ag is at
least one immunogenic antigen; and wherein x is an integer from 1
to 20, the fusion protein being stable and soluble in solution as
compared to an Ab-Ag protein alone that is not soluble or
stable.
[0015] Another embodiment is a method of stabilizing antigenic
peptides comprising: incorporating one or more antigenic peptides
that are unstable or insoluble into a fusion protein, wherein the
fusion protein has the following structure: Ab-(PL-Ag)x or
Ab-(Ag-PL)x; wherein Ab is an antibody or fragment thereof; wherein
PL is at least one peptide linker comprising at least one
glycosylation site; wherein Ag is at least one antigen; and wherein
x is an integer from 1 to 20, the fusion protein being stable and
soluble in solution wherein the Ab-Ag is not soluble or stable.
[0016] Yet another embodiment of the present invention is a host
cell comprising a nucleic acid expression vector comprising: a
first polynucleotide encoding an antibody light chain; and a second
polynucleotide encoding an antibody heavy chain fusion protein, the
fusion protein comprising the following formula: Ab-(PL-Ag)x or
Ab-(Ag-PL)x; wherein Ab is an antibody or fragment thereof; wherein
PL is at least one peptide linker comprising at least one
glycosylation site; wherein Ag is at least one antigen; and wherein
x is an integer from 1 to 20, the fusion protein having more
stability is solution than the fusion protein without the
glycosylation site. In another embodiment, the host cell comprises
an expression vector that produces a fusion protein comprising the
formula: Ab-(PL-Ag)x; Ab-(Ag-PL)x; Ab-(PL-Ag-PL)x; Ab-(Ag-PL-Ag)x;
Ab-(PL-Ag)x-PL; or Ab-(Ag-PL)x-Ag; wherein Ab is an antibody or
fragment thereof; wherein PL is at least one peptide linker
comprising at least one glycosylation site; wherein Ag is at least
one antigen; and wherein x is an integer from 1 to 20, the fusion
protein having more stability in solution than the same fusion
protein without the glycosylation site.
[0017] The present invention also includes a pharmaceutical
composition comprising the antibody having the formula comprising
the formula: Ab-(PL-Ag)x; Ab-(Ag-PL)x; Ab-(PL-Ag-PL)x;
Ab-(Ag-PL-Ag)x; Ab-(PL-Ag)x-PL; or Ab-(Ag-PL)x-Ag; wherein Ab is an
antibody or fragment thereof; wherein PL is at least one peptide
linker comprising at least one glycosylation site; wherein Ag is at
least one antigen; and wherein x is an integer from 1 to 20, the
fusion protein having more stability in solution than the same
fusion protein without the glycosylation site.
[0018] Yet another embodiment of the present invention is a fusion
protein comprising the formula: Ab-(PL-Ag)x-(PLy-Agz)n; or
Ab-(Ag-PL)x-(PLy-Agz)n; wherein Ab is an antibody or fragment
thereof; wherein PL is at least one peptide linker comprising at
least one glycosylation site; wherein Ag is at least one antigen;
and wherein x is an integer from 1 to 20; wherein n is 0 to 19; and
wherein y or z is 0 to 10, wherein the fusion protein has more
stability in solution than the same fusion protein without the
glycosylation site.
[0019] Another embodiment is an isolated and purified vaccine
comprising: a heavy chain selected from at least one of SEQ ID
NOS.: 6, 7, 8, 9, 10, 16, 17, 18, 19, 20, 36, 37, 96, 97, 98, 99,
110, 111, 112, 118, 119, 134, 136, 138, 146, and 147 that binds
specifically to CD40; and a light chain that binds specifically to
CD40. In one aspect, the antibody is defined further as a humanized
antibody.
[0020] Yet another embodiment of the present invention is a fusion
protein comprising the formula: Ab-(PL-Ag)x; Ab-(Ag-PL)x;
Ab-(PL-Ag-PL)x; Ab-(Ag-PL-Ag)x; Ab-(PL-Ag)x-PL; or Ab-(Ag-PL)x-Ag;
wherein Ab is an antibody or fragment thereof; PL is at least one
peptide linker comprising at least one glycosylation site; Ag is at
least one viral antigen; and x is an integer from 1 to 20. In one
aspect, the fusion protein has more stability is solution than the
PL without the glycosylation site. In another aspect, the Ag
comprises a peptide from an adenovirus, retrovirus, picornavirus,
herpesvirus, rotaviruses, hantaviruses, coronavirus, togavirus,
flavirvirus, rhabdovirus, paramyxovirus, orthomyxovirus,
bunyavirus, arenavirus, reovirus, papilomavirus, parvovirus,
poxvirus, hepadnavirus, or spongiform virus. In another aspect, the
Ag comprises a peptide from at least one of HIV, CMV, hepatitis A,
B, and C, influenza; measles, polio, smallpox, rubella, respiratory
syncytial, herpes simplex, varicella zoster, Epstein-Barr, Japanese
encephalitis, rabies, flu, or cold viruses.
[0021] In another aspect, the Ag is selected from: Nef (66-97):
VGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGL (SEQ ID NO.: 1); Nef (116-145):
HTQGYFPDWQNYTPGPGVRYPLTFGWLYKL (SEQ ID NO.: 2); Gag p17 (17-35):
EKIRLRPGGKKKYKLKHIV (SEQ ID NO.: 3); Gag p17-p24 (253-284):
NPPIPVGEIYKRWIILGLNKIVRMYSPTSILD (SEQ ID NO.: 4); or Pol 325-355
(RT 158-188) is: AIFQSSMTKILEPFRKQNPDIVIYQYMDDLY (SEQ ID NO.: 5).
In another aspect, the Ag is 19 to 32 residues. In another aspect,
the Ag is selected from a cytotoxic T lymphocyte (CTL) epitope
identified in the HIV-1 Nef, Gag and Env proteins presented in the
context of MHC-class I molecules. In another aspect, the Ag is
selected from HIV gp120, gp41, Gag, p17, p24, p2, p7, p1, p6, Tat,
Rev, PR, RT, IN, Vif, Vpr, Vpx, Vpu and Nef. In another aspect, x
comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18 or 19. In another aspect, the Ag comprises virus peptides
from different antigens separated by different peptide linkers. In
another aspect, the Ag is separated by at least one PL comprising
an alanine and a serine. In another aspect, the fusion protein is
selected from SEQ ID NOS.: 21, 22, 23, 24, 25, 26 or 36. In another
aspect, the fusion protein is isolated from a cell that comprises a
polynucleotide vector that encodes the fusion protein, the
polynucleotide vector comprising SEQ ID NOS.: 21, 22, 23, 24, 25,
26 or 36. In another aspect, the Ab comprises SEQ ID NOS.: 37 and
38.
[0022] In another aspect, the fusion protein is isolated from a
cell that comprises a polynucleotide vector that expresses the
fusion protein and the Ab portion comprises SEQ ID NOS.: 39 and 40.
In another aspect, Ag is selected from at least one of SEQ ID NOS.:
52-56, 58-60, 61-69, 70-72, or 73-77. In another aspect, the Ag is
17 to 60 residues. In another aspect, the Ag is 8, 10, 12, 14, 15,
16, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55 to 60 residues long. In
another aspect, the Ag comprises at least one lipopeptide. In
another aspect, the Ag is at the carboxy-terminus and further
comprises a carboxy-terminus (Palm)-NH.sub.2 group. In another
aspect, the PL is selected from: SSVSPTTSVHPTPTSVPPTPTKSSP (SEQ ID
NO.: 11); PTSTPADSSTITPTATPTATPTIKG (SEQ ID NO.: 12);
TVTPTATATPSAIVTTITPTATTKP (SEQ ID NO.: 13); or
TNGSITVAATAPTVTPTVNATPSAA (SEQ ID NO.: 14). In another aspect, the
PL comprises an alanine and a serine.
[0023] Another embodiment is the present invention is a viral
antigen delivery vector comprising: a fusion protein comprising an
anti-CD40 antibody or fragment thereof and one or more viral
peptides at the carboxy-terminus of the anti-CD40 antibody, wherein
when two or more viral peptides are present the viral peptides are
separated by the one or more peptide linkers comprising at least
one potential glycosylation site. In another aspect, an antigen
delivery vector is an anti-CD40 antibody or fragment thereof and
two or more viral peptides at the carboxy-terminus of the light
chain, the heavy chain or both the light and heavy chains of the
anti-CD40 antibody, wherein when two or more viral peptides are
separated by the one or more peptide linkers that comprise at least
one potential glycosylation site.
[0024] Yet another embodiment of the present invention is a method
of stabilizing viral peptides comprising: incorporating one or more
viral peptides that are unstable or insoluble into a fusion protein
with an antibody, wherein the antibody and the viral peptides are
separated by one or more peptide linkers that comprise one or more
glycosylation sites. Yet another embodiment is a method of
enhancing T cell responses comprising: immunizing a subject in need
of vaccination with an effective amount of a vaccine comprising the
formula: Ab-(PL-Ag)x or Ab-(Ag-PL)x; wherein Ab is an antibody or
fragment thereof; PL is at least one peptide linker comprising at
least one glycosylation site; Ag is at least one viral antigen; and
x is an integer from 1 to 20. In one aspect, the fusion protein has
more stability in solution than an identical fusion protein without
the glycosylation site. In another aspect, the at least one viral
antigen comprise peptides from adenovirus, retrovirus,
picornavirus, herpesvirus, rotaviruses, hantaviruses, coronavirus,
togavirus, flavirvirus, rhabdovirus, paramyxovirus, orthomyxovirus,
bunyavirus, arenavirus, reovirus, papilomavirus, parvovirus,
poxvirus, hepadnavirus, or spongiform virus. In another aspect, the
at least one viral antigen comprise peptides from at least one of
HIV, CMV, hepatitis A, B, and C, influenza; measles, polio,
smallpox, rubella; respiratory syncytial, herpes simplex, varicella
zoster, Epstein-Barr, Japanese encephalitis, rabies, flu, or cold
viruses.
[0025] In one aspect, the Ag is selected from: Nef (66-97):
VGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGL (SEQ ID NO.: 1); Nef (116-145):
HTQGYFPDWQNYTPGPGVRYPLTFGWLYKL (SEQ ID NO.: 2); Gag p17 (17-35):
EKIRLRPGGKKKYKLKHIV (SEQ ID NO.: 3); Gag p17-p24 (253-284):
NPPIPVGEIYKRWIILGLNKIVRMYSPTSILD (SEQ ID NO.: 4); and/or Pol
325-355 (RT 158-188) is: AIFQSSMTKILEPFRKQNPDIVIYQYMDDLY (SEQ ID
NO.: 5). In another aspect, the Ag is 19 to 32 residues and is
selected from a cytotoxic T lymphocyte (CTL) epitope identified in
the HIV-1 Nef, Gag and Env proteins presented in the context of
MHC-class I molecules. In another aspect, the Ag is selected from
HIV gp120, gp41, Gag, p17, p24, p2, p7, p1, p6, Tat, Rev, PR, RT,
IN, Vif, Vpr, Vpx, Vpu and Nef. In another aspect, x comprises 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19.
In another aspect, the Ag comprises two or more viral antigens from
different viruses. In another aspect, PL comprises an alanine and a
serine. In another aspect, the vaccine is selected from SEQ ID
NOS.: 21, 22, 23, 24, 25, 26 or 36. In another aspect, the Ab
comprises SEQ ID NOS.: 37 and 38. In another aspect, the Ag is
selected from at least one of SEQ ID NOS.: 52-56, 58-60, 61-69,
70-72, or 73-77. In another aspect, the Ag is 17 to 60 residues. In
another aspect, the Ag is 8, 10, 12, 14, 15, 16, 18, 19, 20, 25,
30, 35, 40, 45, 50, 55 to 60 residues long. In another aspect, the
Ag is 8, 10, 12, 14, 15, 16, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55
to 60 residues long. In another aspect, the Ag comprise a
lipopeptide. In another aspect, the Ag is at the carboxy-terminus
and comprises a carboxy-terminus (Palm)-NH.sub.2 group. In another
aspect, the PL is selected from: SSVSPTTSVHPTPTSVPPTPTKSSP (SEQ ID
NO.: 11); PTSTPADSSTITPTATPTATPTIKG (SEQ ID NO.: 12);
TVTPTATATPSAIVTTITPTATTKP (SEQ ID NO.: 13); or
TNGSITVAATAPTVTPTVNATPSAA (SEQ ID NO.: 14).
[0026] Yet another embodiment of the present invention is a method
of making HIV peptide-specific IFN.gamma. producing T cells
comprising: immunizing a subject with a fusion protein comprising
an anti-CD40 antibody, or fragment thereof, with one or more HIV
peptides at the carboxy-terminus of the antibody; and isolating
peripheral blood mononuclear cells from the subject, wherein the
isolated peripheral mononuclear cells are enriched for anti-HIV
IFN.gamma. producing T cells, wherein the anti-CD40 antibody
comprises SEQ ID NOS.: 37 and 38 or fragments thereof. In one
aspect, the subject is a patient suspected of having an HIV
infection. In another aspect, the fusion protein comprises two or
more HIV peptides and the peptides are separated by one or more
peptide linkers. In another aspect, the fusion protein comprises
two or more HIV peptides and the peptides are separated by the one
or more peptide linkers comprise glycosylation sequences. In
another aspect, the fusion protein comprises two or more HIV
peptides and the peptides are separated by one or more peptide
linkers comprising an alanine and a serine. In another aspect, the
one or more HIV peptides comprise at least one lipopeptide. In
another aspect, the one or more HIV peptides comprise a
carboxy-terminus (Palm)-NH.sub.2 group. In another aspect, the one
or more HIV peptides are 19- to 32-amino-acid long and are selected
from a cytotoxic T lymphocyte (CTL) epitopes identified in the
HIV-1 Nef, Gag and Env proteins in the context of different
MHC-class I molecules. In another aspect, the one or more HIV
peptides are selected from HIV gp120, gp41, Gag, p17, p24, p2, p7,
p1, p6, Tat, Rev, PR, RT, IN, Vif, Vpr, Vpx, Vpu and Nef. In
another aspect, the one or more viral peptides are selected from at
least one of: Nef (66-97): VGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGL (SEQ ID
NO.: 1); Nef (116-145): HTQGYFPDWQNYTPGPGVRYPLTFGWLYKL (SEQ ID NO.:
2); Gag p17 (17-35): EKIRLRPGGKKKYKLKHIV (SEQ ID NO.: 3); Gag
p17-p24 (253-284): NPPIPVGEIYKRWIILGLNKIVRMYSPTSILD (SEQ ID NO.:
4); and/or Pol 325-355 (RT 158-188) is:
AIFQSSMTKILEPFRKQNPDIVIYQYMDDLY (SEQ ID NO.: 5).
[0027] Yet another embodiment of the present invention is a fusion
protein comprising an anti-CD40 antibody, or fragment thereof, with
one or more viral peptides at the carboxy-terminus of the antibody
separated by a PL comprising at least one alanine and one serine.
In one aspect, the one or more viral peptides are HIV peptides. In
another aspect, the one or more viral peptides are selected from at
least one of: Nef (66-97): VGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGL (SEQ ID
NO.: 1); Nef (116-145): HTQGYFPDWQNYTPGPGVRYPLTFGWLYKL (SEQ ID NO.:
2); Gag p17 (17-35): EKIRLRPGGKKKYKLKHIV (SEQ ID NO.: 3); Gag
p17-p24 (253-284): NPPIPVGEIYKRWIILGLNKIVRMYSPTSILD (SEQ ID NO.:
4); and/or Pol 325-355 (RT 158-188) is:
AIFQSSMTKILEPFRKQNPDIVIYQYMDDLY (SEQ ID NO.: 5).
[0028] The present invention also includes a method of making a
fusion protein comprising: inserting into an expression vector a
nucleic acid construct comprising polynucleotides that encode a
protein having the formula: Ab-(PL-Ag)x or Ab-(Ag-PL)x; wherein Ab
is an antibody or fragment thereof; PL is at least one peptide
linker comprising at least one glycosylation site; Ag is at least
one viral antigen; and x is an integer from 1 to 20; and culturing
the vector under conditions sufficient to permit expression of the
fusion protein. In one aspect, the fusion protein has more
stability in solution than an identical fusion protein without the
glycosylation site. In another aspect, the at least one viral
antigen comprise peptides from an adenovirus, retrovirus,
picornavirus, herpesvirus, rotaviruses, hantaviruses, coronavirus,
togavirus, flavirvirus, rhabdovirus, paramyxovirus, orthomyxovirus,
bunyavirus, arenavirus, reovirus, papilomavirus, parvovirus,
poxvirus, hepadnavirus, or spongiform virus. In another aspect, the
at least one viral antigen comprise peptides from at least one of
HIV, CMV, hepatitis A, B, and C, influenza; measles, polio,
smallpox, rubella, respiratory syncytial, herpes simplex, varicella
zoster, Epstein-Barr, Japanese encephalitis, rabies, flu, or cold
viruses. In another aspect, the fusion protein is the Ab's light
chain, the Ab's heavy chain or both the Ab's light and heavy
chains. In another aspect, the Ag is selected from: Nef (66-97):
VGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGL (SEQ ID NO.: 1); Nef (116-145):
HTQGYFPDWQNYTPGPGVRYPLTFGWLYKL (SEQ ID NO.: 2); Gag p17 (17-35):
EKIRLRPGGKKKYKLKHIV (SEQ ID NO.: 3); Gag p17-p24 (253-284):
NPPIPVGEIYKRWIILGLNKIVRMYSPTSILD (SEQ ID NO.: 4); and/or Pol
325-355 (RT 158-188) is: AIFQSSMTKILEPFRKQNPDIVIYQYMDDLY (SEQ ID
NO.: 5).
[0029] Yet another embodiment of the present invention includes a
method of expanding antigen-specific T cells in vitro comprising:
isolating PBMCs from an HIV patient; incubating the isolated PBMCs
with an effective amount of a .alpha.CD40.LIPO5 HIV peptide
vaccine; expanding the PBMCs in the presence of an effective amount
of IL-2; harvesting the cells; and assessing the cytokine
production by the cells to determine the presence of anti-HIV
specific T cells. Another embodiment is an HIV antigen-specific T
cells made by the method comprising: isolating PBMCs from an HIV
patient; incubating the isolated PBMCs with an effective amount of
a .alpha.CD40.LIPO5 HIV peptide vaccine; expanding the PBMCs in the
presence of an effective amount of IL-2; harvesting the cells; and
assessing the cytokine production by the cells to determine the
presence of anti-HIV specific T cells. Another embodiment is a
method of making a therapeutic vaccine comprising: loading a
dendritic cell with .alpha.CD40.LIPO5 HIV peptide vaccine
comprising: isolating HIV patient monocytes; differentiating the
monocytes into dendritic cells with IFN.alpha. and GM-CSF; and
exposing the differentiated dendritic cells to an .alpha.CD40.LIPO5
HIV peptide, wherein the loaded dendritic cells are capable of
stimulating autologous HIV-peptide specific T cells in vitro.
[0030] The present invention also includes a therapeutic vaccine
made by the method comprising: loading a dendritic cell with
.alpha.CD40.LIPO5 HIV peptide vaccine comprising: isolating HIV
patient monocytes; differentiating the monocytes into dendritic
cells with IFN.alpha. and GM-CSF; and exposing the differentiated
dendritic cells to an .alpha.CD40.LIPO5 HIV peptide, wherein the
loaded dendritic cells are capable of stimulating autologous
HIV-peptide specific T cells in vitro. Another embodiment is a
therapeutic vaccine comprising a polypeptide comprising at least
one of SEQ ID NOS.: 21, 22, 23, 24, 25, 26 or 36. Yet another
embodiment is a therapeutic vaccine comprising a fusion protein
comprising the formula: Ab-(PL-Ag)x; Ab-(Ag-PL)x; Ab-(PL-Ag-PL)x;
Ab-(Ag-PL-Ag)x; Ab-(PL-Ag)x-PL; or Ab-(Ag-PL)x-Ag; wherein Ab is an
antibody or fragment thereof; PL is at least one peptide linker
comprising at least one glycosylation site; Ag is at least one
viral antigen; and x is an integer from 1 to 20.
[0031] Yet another embodiment of the present invention includes a
fusion protein comprising the formula: Ab-(PL-Ag)x; Ab-(Ag-PL)x;
Ab-(PL-Ag-PL)x; Ab-(Ag-PL-Ag)x; Ab-(PL-Ag)x-PL; or Ab-(Ag-PL)x-Ag;
wherein Ab is an antibody or fragment thereof; PL is at least one
peptide linker comprising at least one glycosylation site; Ag is at
least one cancer antigen; and x is an integer from 1 to 20. In one
aspect, the fusion protein has more stability in solution than the
same fusion protein without the glycosylation site. In another
aspect, the Ag is selected from tumor associated antigens selected
from CEA, prostate specific antigen (PSA), HER-2/neu, BAGE, GAGE,
MAGE 1-4, 6 and 12, MUC-related protein (Mucin) (MUC-1, MUC-2,
etc.), GM2 and GD2 gangliosides, ras, myc, tyrosinase, MART
(melanoma antigen), MARCO-MART, cyclin B1, cyclin D, Pmel
17(gp100), GnT-V intron V sequence (N-acetylglucoaminyltransferase
V intron V sequence), Prostate Ca psm, prostate serum antigen
(PSA), PRAME (melanoma antigen), .beta.-catenin, MUM-1-B (melanoma
ubiquitous mutated gene product), GAGE (melanoma antigen) 1, BAGE
(melanoma antigen) 2-10, c-ERB2 (Her2/neu), EBNA (Epstein-Barr
Virus nuclear antigen) 1-6, gp75, human papilloma virus (HPV) E6
and E7, p53, lung resistance protein (LRP), Bcl-2, and Ki-67. In
another aspect, the Ag is selected from tumor associated antigens
comprising antigens from leukemias and lymphomas, neurological
tumors such as astrocytomas or glioblastomas, melanoma, breast
cancer, lung cancer, head and neck cancer, gastrointestinal tumors,
gastric cancer, colon cancer, liver cancer, pancreatic cancer,
genitourinary tumors such cervix, uterus, ovarian cancer, vaginal
cancer, testicular cancer, prostate cancer or penile cancer, bone
tumors, vascular tumors, or cancers of the lip, nasopharynx,
pharynx and oral cavity, esophagus, rectum, gall bladder, biliary
tree, larynx, lung and bronchus, bladder, kidney, brain and other
parts of the nervous system, thyroid, Hodgkin's disease,
non-Hodgkin's lymphoma, multiple myeloma and leukemia.
[0032] In another aspect, the Ag is selected from at least one of:
MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWV (SEQ
ID NO.:74);
LTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHD (SEQ
ID NO.:75);
LMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVIS (SEQ
ID NO.:76);
NDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERP (SEQ
ID NO.:77); or SLYTKVVHYRKWIKDTIVANP (SEQ ID NO.:78), and fragments
thereof. In another aspect, the Ag is selected from at least one
of: IMDQVPFSV (SEQ ID NO.:113); ITDQVPFSV (SEQ ID NO.:114);
YLEPGPVTV (SEQ ID NO.:115); YLEPGPVTA (SEQ ID NO.:116); KTWGQYWQV
(SEQ ID NO.:117);
DTTEPATPTTPVTTPTTTKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEAQRLDCWRGGQVSLKV
SNDGPTLIGANASFSIALNFPGSQKVLPDGQVIWVNNTIINGSQVWGGQPVYPQETDDACIFPDGGP
CPSGSWSQKRSFVYVWKTWGQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSQSYVPL
AHSSSAFTITDQVPFSVSVSQLRALDGGNKHFLRNQ (SEQ ID NO.:122);
PLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRAXVVTHTYLEPGPVTAQVVLQAAIPLTSCGS
SPVPAS (SEQ ID NO.:124);
GTTDGHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTSVQVPTTEVISTAPVQMPTAESTG
MTPEKVPVSEVMGTTLAEMSTPEATGMTPAEVSIVVLSGTTAA (SEQ ID NO.:126);
QVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLRLVKRQVPLDCVLYRYGSFS
VTLDIVQ (SEQ ID NO.:128); and
GIESAEILQAVPSGEGDAFELTVSCQGGLPKEACMEISSPGCQPPAQRLCQPVLPSPACQLVLHQILK
GGSGTYCLNVSLADTNSLAVVSTQLIVPGILLTGQEAGLGQ (SEQ ID NO.:130), and
fragments thereof.
[0033] In another aspect, the Ag is selected from at least one of:
MEMKILRALNFGLGRPLPLHFLRRASKIGEVDVEQHTLAKYLMELTMLDY (SEQ ID
NO.:132); and DWLVQVQMKFRLLQETMYMTVSIIDRFMQNNCVPKK (SEQ ID
NO.:133). In another aspect, the Ag is selected from at least one
of: MEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYFKCV (SEQ ID
NO.:141);
QKEVLPSMRKIVATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEPVKKSRLQLLGATCMFVASK
MKETIPLTAEKLCIYTDNSIRPEELLQMELL (SEQ ID NO.:142);
LVNKLKWNLAAMTPHDFIEHFLSKMPEAEENKQIIRKHAQTFVALCATDVKFISNPPSMV (SEQ
ID NO.:143); and
AAGSVVAAVQGLNLRSPNNFLSYYRLTRFLSRVIKCDPDCLRACQEQIEALLESSLRQAQQNMDPK
AAEEEEEEEEEVDLACTPTDVRDVDI (SEQ ID NO.:144), and fragments thereof.
In another aspect, the Ag is 19 to 32 amino acids long. In another
aspect, the Ag is 17 to 60 amino acids long and is selected from a
cytotoxic T lymphocyte (CTL) epitope identified in PSA or cyclin 1.
In another aspect, x comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, or 19. In another aspect, the Ag
comprises two or more cancer peptides from different cancer
antigens separated by the PL. In another aspect, the Ag is
separated by at least one PL comprising an alanine and a serine. In
another aspect, the Ag is selected from SEQ ID NOS.: 74-78, 79-86,
87-92, 93-95, 113-117, 122-130, 132-133, and 141-144. In another
aspect, the Ab comprises SEQ ID NOS.: 38 and 39. In another aspect,
the Ab is expressed by a nucleic acid expression vector comprising
SEQ ID NOS.: 40 and 41. In another aspect, the PL is selected from:
SSVSPTTSVHPTPTSVPPTPTKSSP (SEQ ID NO.: 11);
PTSTPADSSTITPTATPTATPTIKG (SEQ ID NO.: 12);
TVTPTATATPSAIVTTITPTATTKP (SEQ ID NO.: 13); or
TNGSITVAATAPTVTPTVNATPSAA (SEQ ID NO.: 14). In another aspect, the
PL comprises an alanine and a serine.
[0034] Yet another embodiment of the present invention includes a
antigen delivery vector that expresses an anti-CD40 antibody or
fragment thereof and two or more cancer peptides at the
carboxy-terminus of the light chain, the heavy chain or both the
light and heavy chains of the anti-CD40 antibody, wherein when two
or more cancer peptides are present, the cancer peptides are
separated by the one or more peptide linkers that comprise at least
one glycosylation site. In one aspect, the one or more peptide
linkers are selected from: SSVSPTTSVHPTPTSVPPTPTKSSP (SEQ ID NO.:
11); PTSTPADSSTITPTATPTATPTIKG (SEQ ID NO.: 12);
TVTPTATATPSAIVTTITPTATTKP (SEQ ID NO.: 13); or
TNGSITVAATAPTVTPTVNATPSAA (SEQ ID NO.: 14).
[0035] Yet another embodiment of the present invention includes an
anti-CD40 fusion protein comprising an anti-CD40 antibody or
fragment thereof and one or more cancer peptides at the
carboxy-terminus of the anti-CD40 antibody, wherein when two or
more cancer peptides are present the cancer peptides are separated
by the one or more linker peptides that comprise at least one
glycosylation site. In one aspect, the antibody fragment is
selected from an Fv, Fab, Fab', F(ab').sub.2, Fc, or a ScFv
fragment. In another aspect, the Ag is selected from SEQ ID NOS.:
74-78, 79-86, 87-92, 93-95, 113-117, 122-130, 132-133, and
141-144.
[0036] Yet another embodiment of the present invention includes a
method of stabilizing cancer peptides comprising: incorporating one
or more cancer peptides that are unstable or insoluble into a
fusion protein with an antibody, wherein the antibody and the
cancer peptides are separated by one or more peptide linkers that
comprise one or more glycosylation sites. In another aspect, the
fusion protein comprises two or more cancer peptides and the cancer
peptides are separated by the one or more peptide linkers. In
another aspect, the fusion protein comprises two or more cancer
peptides and the peptides are separated by the one or more peptide
linkers. In another aspect, the fusion protein comprises two or
more cancer peptides and the peptides are separated by one or more
linkers comprising an alanine and a serine. In another aspect, the
cancer peptide is selected from tumor associated antigens selected
from CEA, prostate specific antigen (PSA), HER-2/neu, BAGE, GAGE,
MAGE 1-4, 6 and 12, MUC-related protein (Mucin) (MUC-1, MUC-2,
etc.), GM2 and GD2 gangliosides, ras, myc, tyrosinase, MART
(melanoma antigen), MARCO-MART, cyclin B1, cyclin D, Pmel
17(gp100), GnT-V intron V sequence (N-acetylglucoaminyltransferase
V intron V sequence), Prostate Ca psm, prostate serum antigen
(PSA), PRAME (melanoma antigen), .beta.-catenin, MUM-1-B (melanoma
ubiquitous mutated gene product), GAGE (melanoma antigen) 1, BAGE
(melanoma antigen) 2-10, c-ERB2 (Her2/neu), EBNA (Epstein-Barr
Virus nuclear antigen) 1-6, gp75, human papilloma virus (HPV) E6
and E7, p53, lung resistance protein (LRP), Bcl-2, and Ki-67. In
another aspect, the Ag is selected from tumor associated antigens
comprising antigens from leukemias and lymphomas, neurological
tumors such as astrocytomas or glioblastomas, melanoma, breast
cancer, lung cancer, head and neck cancer, gastrointestinal tumors,
gastric cancer, colon cancer, liver cancer, pancreatic cancer,
genitourinary tumors such cervix, uterus, ovarian cancer, vaginal
cancer, testicular cancer, prostate cancer or penile cancer, bone
tumors, vascular tumors, or cancers of the lip, nasopharynx,
pharynx and oral cavity, esophagus, rectum, gall bladder, biliary
tree, larynx, lung and bronchus, bladder, kidney, brain and other
parts of the nervous system, thyroid, Hodgkin's disease,
non-Hodgkin's lymphoma, multiple myeloma and leukemia.
[0037] In another aspect, the Ag is selected from at least one of:
MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWV (SEQ
ID NO.:74);
LTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHD (SEQ
ID NO.:75);
LMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVIS (SEQ
ID NO.:76);
NDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERP (SEQ
ID NO.:77); or SLYTKVVHYRKWIKDTIVANP (SEQ ID NO.:78).
[0038] In another aspect, the Ag is selected from at least one of:
IMDQVPFSV (SEQ ID NO.:113); ITDQVPFSV (SEQ ID NO.:114); YLEPGPVTV
(SEQ ID NO.:115); YLEPGPVTA (SEQ ID NO.:116); KTWGQYWQV (SEQ ID
NO.:117);
DTTEPATPTTPVTTPTTTKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEAQRLDCWRGGQVSLKV
SNDGPTLIGANASFSIALNFPGSQKVLPDGQVIWVNNTIINGSQVWGGQPVYPQETDDACIFPDGGP
CPSGSWSQKRSFVYVWKTWGQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSQSYVPL
AHSSSAFTITDQVPFSVSVSQLRALDGGNKHFLRNQ (SEQ ID NO.:122);
PLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRAXVVTHTYLEPGPVTAQVVLQAAIPLTSCGS
SPVPAS (SEQ ID NO.:124);
GTTDGHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTSVQVPTTEVISTAPVQMPTAESTG
MTPEKVPVSEVMGTTLAEMSTPEATGMTPAEVSIVVLSGTTAA (SEQ ID NO.:126);
QVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLRLVKRQVPLDCVLYRYGSFS
VTLDIVQ (SEQ ID NO.:128); and
GIESAEILQAVPSGEGDAFELTVSCQGGLPKEACMEISSPGCQPPAQRLCQPVLPSPACQLVLHQILK
GGSGTYCLNVSLADTNSLAVVSTQLIVPGILLTGQEAGLGQ (SEQ ID NO.:130), and
fragments thereof.
[0039] In another aspect, the Ag is selected from at least one of:
MEMKILRALNFGLGRPLPLHFLRRASKIGEVDVEQHTLAKYLMELTMLDY (SEQ ID
NO.:132); and DWLVQVQMKFRLLQETMYMTVSIIDRFMQNNCVPKK (SEQ ID
NO.:133).
[0040] In another aspect, the Ag is selected from at least one of:
MEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYFKCV (SEQ ID NO.:141);
QKEVLPSMRKIVATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEPVKKSRLQLLGATCMFVASK
MKETIPLTAEKLCIYTDNSIRPEELLQMELL (SEQ ID NO.:142);
LVNKLKWNLAAMTPHDFIEHFLSKMPEAEENKQIIRKHAQTFVALCATDVKFISNPPSMV (SEQ
ID NO.:143); and
AAGSVVAAVQGLNLRSPNNFLSYYRLTRFLSRVIKCDPDCLRACQEQIEALLESSLRQAQQNMDPK
AAEEEEEEEEEVDLACTPTDVRDVDI (SEQ ID NO.:144), and fragments thereof.
In another aspect, the Ag is 19 to 32 amino acids long. In another
aspect, the Ag is 17 to 60 amino acids long and is selected from a
cytotoxic T lymphocyte (CTL) epitope identified in PSA or cyclin 1.
In another aspect, x comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, or 19. In another aspect, the fusion
protein comprises cancer peptides from different antigens separated
by different peptide linkers. In another aspect, the fusion protein
comprises two or more cancer peptides separated by one or more
peptide linkers comprising an alanine and a serine. In another
aspect, the antibody comprises SEQ ID NOS.: 38 and 39. In another
aspect, the fusion protein is expressed by a nucleic acid
expression vector comprising SEQ ID NOS.: 40 and 41. In another
aspect, the peptide linker is selected from:
SSVSPTTSVHPTPTSVPPTPTKSSP (SEQ ID NO.: 11);
PTSTPADSSTITPTATPTATPTIKG (SEQ ID NO.: 12);
TVTPTATATPSAIVTTITPTATTKP (SEQ ID NO.: 13); or
TNGSITVAATAPTVTPTVNATPSAA (SEQ ID NO.: 14).
[0041] Yet another embodiment of the present invention includes a
method of enhancing T cell responses comprising: immunizing a
subject in need of vaccination with an effective amount of a
vaccine comprising a fusion protein comprising an anti-CD40
antibody or portion thereof and one or more cancer peptides linked
to the carboxy-terminus of the anti-CD40 antibody. In another
aspect, the cancer peptides are selected from tumor associated
antigens selected from CEA, prostate specific antigen (PSA),
HER-2/neu, BAGE, GAGE, MAGE 1-4, 6 and 12, MUC (Mucin) (e.g.,
MUC-1, MUC-2, etc.), GM2 and GD2 gangliosides, ras, myc,
tyrosinase, MART (melanoma antigen), MARCO-MART, cyclin B1, cyclin
D, Pmel 17(gp100), GnT-V intron V sequence
(N-acetylglucoaminyltransferase V intron V sequence), Prostate Ca
psm, prostate serum antigen (PSA), PRAME (melanoma antigen),
.beta.-catenin, MUM-1-B (melanoma ubiquitous mutated gene product),
GAGE (melanoma antigen) 1, BAGE (melanoma antigen) 2-10, c-ERB2
(Her2/neu), EBNA (Epstein-Barr Virus nuclear antigen) 1-6, gp75,
human papilloma virus (HPV) E6 and E7, p53, lung resistance protein
(LRP), Bcl-2, and Ki-67. In another aspect, the cancer peptides is
selected from tumor associated antigens comprising antigens from
leukemias and lymphomas, neurological tumors such as astrocytomas
or glioblastomas, melanoma, breast cancer, lung cancer, head and
neck cancer, gastrointestinal tumors, gastric cancer, colon cancer,
liver cancer, pancreatic cancer, genitourinary tumors such cervix,
uterus, ovarian cancer, vaginal cancer, testicular cancer, prostate
cancer or penile cancer, bone tumors, vascular tumors, or cancers
of the lip, nasopharynx, pharynx and oral cavity, esophagus,
rectum, gall bladder, biliary tree, larynx, lung and bronchus,
bladder, kidney, brain and other parts of the nervous system,
thyroid, Hodgkin's disease, non-Hodgkin's lymphoma, multiple
myeloma and leukemia.
[0042] Yet another embodiment of the present invention includes a
method of making an anti-CD40-antigen fusion protein comprising:
expressing a fusion protein comprising an anti-CD40 antibody or
fragment thereof in a host cell, the fusion protein comprising one
or more cancer peptides at the carboxy-terminus of the anti-CD40
antibody or fragment thereof, wherein when two or more cancer
peptides are separated by one or more linkers, at least one linker
comprising a glycosylation site; and isolating the fusion protein.
In another aspect, the fusion protein expressed in the host is
further isolated and purified. In another aspect, the host is a
eukaryotic cell. In another aspect, the cancer peptides are
selected from tumor associated antigens selected from CEA, prostate
specific antigen (PSA), HER-2/neu, BAGE, GAGE, MAGE 1-4, 6 and 12,
MUC-related protein (Mucin) (MUC-1, MUC-2, etc.), GM2 and GD2
gangliosides, ras, myc, tyrosinase, MART (melanoma antigen),
MARCO-MART, cyclin B1, cyclin D, Pmel 17(gp100), GnT-V intron V
sequence (N-acetylglucoaminyltransferase V intron V sequence),
Prostate Ca psm, prostate serum antigen (PSA), PRAME (melanoma
antigen), .beta.-catenin, MUM-1-B (melanoma ubiquitous mutated gene
product), GAGE (melanoma antigen) 1, BAGE (melanoma antigen) 2-10,
c-ERB2 (Her2/neu), EBNA (Epstein-Barr Virus nuclear antigen) 1-6,
gp75, human papilloma virus (HPV) E6 and E7, p53, lung resistance
protein (LRP), Bcl-2, and Ki-67. In another aspect, the cancer
peptides are selected from tumor associated antigens comprising
antigens from leukemias and lymphomas, neurological tumors such as
astrocytomas or glioblastomas, melanoma, breast cancer, lung
cancer, head and neck cancer, gastrointestinal tumors, gastric
cancer, colon cancer, liver cancer, pancreatic cancer,
genitourinary tumors such cervix, uterus, ovarian cancer, vaginal
cancer, testicular cancer, prostate cancer or penile cancer, bone
tumors, vascular tumors, or cancers of the lip, nasopharynx,
pharynx and oral cavity, esophagus, rectum, gall bladder, biliary
tree, larynx, lung and bronchus, bladder, kidney, brain and other
parts of the nervous system, thyroid, Hodgkin's disease,
non-Hodgkin's lymphoma, multiple myeloma and leukemia. In another
aspect, the cancer peptides are selected from at least one of:
MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWV (SEQ
ID NO.:74);
LTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHD (SEQ
ID NO.:75);
LMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVIS (SEQ
ID NO.:76);
NDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERP (SEQ
ID NO.:77); or SLYTKVVHYRKWIKDTIVANP (SEQ ID NO.:78).
[0043] In another aspect, the cancer peptides are selected from at
least one of: IMDQVPFSV (SEQ ID NO.:113); ITDQVPFSV (SEQ ID
NO.:114); YLEPGPVTV (SEQ ID NO.:115); YLEPGPVTA (SEQ ID NO.:116);
KTWGQYWQV (SEQ ID NO.:117);
DTTEPATPTTPVTTPTTTKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEAQRLDCWRGGQVSLKV
SNDGPTLIGANASFSIALNFPGSQKVLPDGQVIWVNNTIINGSQVWGGQPVYPQETDDACIFPDGGP
CPSGSWSQKRSFVYVWKTWGQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSQSYVPL
AHSSSAFTITDQVPFSVSVSQLRALDGGNKHFLRNQ (SEQ ID NO.:122);
PLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRAXVVTHTYLEPGPVTAQVVLQAAIPLTSCGS
SPVPAS (SEQ ID NO.:124);
GTTDGHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTSVQVPTTEVISTAPVQMPTAESTG
MTPEKVPVSEVMGTTLAEMSTPEATGMTPAEVSIVVLSGTTAA (SEQ ID NO.:126);
QVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLRLVKRQVPLDCVLYRYGSFS
VTLDIVQ (SEQ ID NO.:128); and
GIESAEILQAVPSGEGDAFELTVSCQGGLPKEACMEISSPGCQPPAQRLCQPVLPSPACQLVLHQILK
GGSGTYCLNVSLADTNSLAVVSTQLIVPGILLTGQEAGLGQ (SEQ ID NO.:130), and
fragments thereof.
[0044] In another aspect, the cancer peptides are selected from at
least one of: MEMKILRALNFGLGRPLPLHFLRRASKIGEVDVEQHTLAKYLMELTMLDY
(SEQ ID NO.:132); and DWLVQVQMKFRLLQETMYMTVSIIDRFMQNNCVPKK (SEQ ID
NO.:133).
[0045] In another aspect, the cancer peptides are selected from at
least one of: MEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYFKCV (SEQ
ID NO.:141);
QKEVLPSMRKIVATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEPVKKSRLQLLGATCMFVAS- K
MKETIPLTAEKLCIYTDNSIRPEELLQMELL (SEQ ID NO.:142);
LVNKLKWNLAAMTPHDFIEHFLSKMPEAEENKQIIRKHAQTFVALCATDVKFISNPPSMV (SEQ
ID NO.:143); and
AAGSVVAAVQGLNLRSPNNFLSYYRLTRFLSRVIKCDPDCLRACQEQIEALLESSLRQAQQNMDPK
AAEEEEEEEEEVDLACTPTDVRDVDI (SEQ ID NO.:144), and fragments
thereof.
[0046] Yet another embodiment of the present invention includes a
method of expanding antigen-specific T cells in vitro comprising:
isolating peripheral blood mononuclear cells (PBMCs) from a cancer
patient; incubating the isolated PBMCs with an immunogenic amount
of an .alpha.CD40-(PL-Ag)x or .alpha.CD40-(Ag-PL)x vaccine, wherein
Ag is a tumor associated antigen and x is an integer 1 to 20;
expanding the PBMCs in the presence of an effective amount of IL-2;
harvesting the cells; and assessing the cytokine production by the
cells to determine the presence of anti-cancer specific T
cells.
[0047] Yet another embodiment of the present invention includes a
tumor associated antigen-specific T cells made by the method
comprising: isolating peripheral blood mononuclear cells (PBMCs)
from a cancer patient; incubating the isolated PBMCs with an
immunogenic amount of an .alpha.CD40-(PL-Ag)x or
.alpha.CD40-(Ag-PL)x vaccine, wherein Ag is a tumor associated
antigen and x is an integer 1 to 20; expanding the PBMCs in the
presence of an effective amount of IL-2; harvesting the cells; and
assessing the cytokine production by the cells to determine the
presence of tumor associated antigen-specific T cells.
[0048] Yet another embodiment of the present invention includes a
therapeutic vaccine comprising a fusion protein comprising the
formula: Ab-(PL-Ag)x; Ab-(Ag-PL)x; Ab-(PL-Ag-PL)x; Ab-(Ag-PL-Ag)x;
Ab-(PL-Ag)x-PL; or Ab-(Ag-PL)x-Ag; wherein Ab is an antibody or
fragment thereof; PL is at least one peptide linker comprising at
least one glycosylation site; Ag is at least one cancer antigen;
and x is an integer from 1 to 20.
BRIEF DESCRIPTION OF THE DRAWINGS
[0049] For a more complete understanding of the features and
advantages of the present invention, reference is now made to the
detailed description of the invention along with the accompanying
figures and in which:
[0050] FIG. 1 shows protein A affinity recombinant antibodies fused
to various HIV peptides (lanes 1 to 5) secreted from transfected
293F cells, analyzed by reducing SDS.PAGE and Coomassie Brilliant
Blue staining.
[0051] FIG. 2 shows protein A affinity purified recombinant
antibodies fused to various HIV peptides (Lanes 1 and 2) secreted
from transfected 293F cells, then analyzed by reducing SDS.PAGE and
Coomassie Brilliant Blue staining.
[0052] FIG. 3 shows protein A affinity purified recombinant
antibodies fused to various HIV peptide strings (Lanes 1 to 5)
secreted from transfected 293F cells, then analyzed by reducing
SDS.PAGE and Coomassie Brilliant Blue staining.
[0053] FIG. 4 shows protein A affinity purified recombinant
antibodies fused to various HIV peptide strings (Lanes 1 to 6)
secreted from transfected 293F cells, then analyzed by reducing
SDS.PAGE and Coomassie Brilliant Blue staining.
[0054] FIG. 5 describes the protocol used in vitro to assay the
potency of .alpha.CD40.LIPO5 HIV peptide fusion recombinant
antibody (.alpha.CD40.LIPO5 rAb) to elicit the expansion of
antigen-specific T cells in the context of a PBMC culture.
[0055] FIGS. 6A-6C show HIV peptide-specific IFN.gamma. production
in PBMCs from HIV patients incubated with various concentrations of
anti-CD40.LIPO5 peptide string vaccine. 6C is the control group,
which received no vaccine, and defines the baseline response of the
culture to each peptide.
[0056] FIG. 7 is a summary of .alpha.CD40.LIPO5 peptide vaccine
responses against the 5 peptide regions from 8 HIV patients.
[0057] FIGS. 8A-8B show that the .alpha.CD40.LIPO5 HIV peptide
vaccine elicits expansion of HIV peptide-specific T cells capable
of secreting multiple cytokines--a desirable feature in a vaccine.
FIGS. 8A-8B also show that the .alpha.CD40.LIPO5 HIV peptide
vaccine elicits gag253, nef66, nef116 and pol325 peptide-specific
responses characterized by production of multiple cytokines
(patient A5).
[0058] FIG. 9 shows the protocol for testing .alpha.CD40.LIPO5 HIV
peptide vaccine for its ability to direct the expansion of
antigen-specific T cells resulting from targeted uptake by DCs and
presentation of peptide epitopes on their surface MHC complex.
[0059] FIGS. 10A-10B show the cytokine secretion in response to HIV
peptides from DC-T cell co-cultures treated with various doses of
.alpha.CD40.LIPO5 HIV peptide vaccine (patient A10).
[0060] FIGS. 11A-11B show PBMCs from patient A4 treated with the
.alpha.CD40.LIPO5 HIV peptide vaccine elicit expansion of
antigen-specific T cells with specificity to the gag253 region, but
not to the flexible linker sequences.
[0061] FIG. 12A is the .alpha.CD40.LIPO5 HIV peptide vaccine heavy
chain sequence showing flexible linker regions in bold, joining
sequences underlined and HIV peptide regions shaded in grey. FIG.
12A shows PBMCs from patient A3 treated with the .alpha.CD40.LIPO5
HIV peptide vaccine elicit expansion of antigen-specific T cells
with specificities to the gag253, nef66, and nef116 regions, but
not to the flexible linker sequences. FIGS. 12B and 12C show HIV
antigen-specific T cell responses evoked from HIV patient A17 PBMCs
incubated with 30 nM of three different HIV5 peptide DC targeting
vaccines. FIGS. 12D and 12E show a similar study to that show in
FIGS. 12B and 12C, except that the PBMCs are from a different HIV
patient (A2). FIG. 12F shows 15 different HIV peptide responses [5
peptide regions sampled in 3 patients], it was found that the
anti-CD40.HIV5pep vaccine was superior to anti-DCIR.HIV5pep,
anti-LOX-1.HIV5pep and non-LIPO5 mix for eliciting a broad range of
HIV peptide-specific CD8+ and CD4+ T responses.
[0062] FIG. 13 shows the internalization of anti-CD40 mAb:IL-4DC.
IL-4DCs were treated with 500 ng/nil of anti-CD40-Alexa 568.
[0063] FIG. 14 shows CD4 and CD8 T cell proliferation by DCs
targeted with anti-CD40-HA1. 5.times.10e3 IFNDCs loaded with 2
ug/ml of anti-CD40-HA or control Ig-HA1 were co-cultured with
CFSE-labeled autologous CD4+ or CD8+ T cells (2.times.10e5) for 7
days. Cells were then then stained with anti-CD4 or anti-CD8
antibodies. Cell proliferation was tested by measuring
CFSE-dilution.
[0064] FIG. 15 shows a titration of HA1 fusion protein on CD4+ T
proliferation. IFNDCs (5K) loaded with fusion proteins were
co-cultured with CFSE-labeled CD4+ T cells (200K) for 7 days.
[0065] FIG. 16 shows IFNDCs targeted with anti-CD40-HA1 activate
HA1-specific CD4+ T cells. CD4+ T cells were re-stimulated with DCs
loaded with 5 uM of indicated peptides, and then intracellular
IFN.gamma. was stained.
[0066] FIG. 17 shows IFNDCs targeted with anti-CD40-HA1 activate
HA1-specific CD4+ T cells. CD4+ T cells were re-stimulated with DCs
loaded with indicated peptides for 36h, and then culture
supernatant was analyzed for measuring IFN.gamma..
[0067] FIG. 18 shows that targeting CD40 results in enhanced
cross-priming of MART-1 specific CD8+ T cells. IFNDCs (5K/well)
loaded with fusion proteins were co-cultured with purified CD8+ T
cells for 10 days. Cells were stained with anti-CD8 and tetramer.
Cells are from healthy donors (HLA-A*0201+).
[0068] FIG. 19 shows targeting CD40 results in enhanced
cross-priming of MART-1 specific CD8+ T cells (Summary of
8-repeated experiments using cells from different healthy
donors).
[0069] FIG. 20 shows CD8+ CTL induced with IFNDCs targeted with
anti-CD40-MART-1 are functional. CD8+ T cells co-cultured with
IFNDCs targeted with fusion proteins were mixed with T2 cells
loaded with 10 uM peptide epitope.
[0070] FIG. 21 shows CD8+ CTL induced with IFNDCs targeted with
anti-CD40-Flu M1 are functional. CD8+ T cells co-cultured with
IFNDCs targeted with fusion proteins were mixed with T2 cells
loaded with 1.0 nM peptide epitope.
[0071] FIG. 22 shows an outline of protocol to test the ability a
vaccine composed of anti-CD4012E12 linked to PSA (prostate specific
antigen) to elicit the expansion from a naive T cell population.
PSA-specific CD4+ T cells corresponding to a broad array of PSA
epitopes. Briefly, DCs derived by culture with IFN.alpha. and
GM-CSF of monocytes from a healthy donor are incubated with the
vaccine. The next day, cells are placed in fresh medium and pure
CD4+ T cells from the same donor are added. Several days later, PSA
peptides are added and, after four hours, secreted gamma-IFN levels
in the culture supernatants are determined.
[0072] FIG. 23 shows that many PSA peptides elicit potent
gamma-IFN-production responses indicating that anti-CD4012E12 and
similar anti-CD40 agents can efficiently deliver antigen to DCs,
resulting in the priming of immune responses against multiple
epitopes of the antigen.
[0073] FIG. 24 shows DCs targeted with anti-CD40-PSA induce
PSA-specific CD8+ T cell responses. IFNDCs were targeted with 1 ug
mAb fusion protein with PSA. Purified autologous CD8+ T cells were
co-cultured for 10 days. Cells were stained with anti-CD8 and PSA
(KLQCVDLHV)-tetramer. Cells are from a HLA-A*0201 positive healthy
donor. The results demonstrate that anti-CD40 effectively deliver
PSA to the DCs, which in turn elicit the expansion of PSA-specific
CD8+ T cells.
[0074] FIG. 25 a scheme (left) and the IFN.gamma. production by T
cells of the pools of peptides and control for Donor 2.
5.times.10e3 IFNDCs loaded with 2 ug/ml of anti-CD40-Cyclin D1 were
co-cultured with purified autologous CD4+ T cells (2.times.10e5)
for 8 days. Cells were then re-stimulated with with 5 uM of
individual peptides derived from CyclinD1 for 5h in the presence of
Brefeldin A. Cells were stained for measuring intracellular
IFN.gamma. expression.
[0075] FIG. 26 shows a peptide scan and IFN.gamma. production by T
cells obtained from the pools of peptides shown in FIG. 25 and
control for Donor 2. 5.times.10e3 IFNDCs loaded with 2 ug/ml of
anti-CD40-Cyclin D1 were co-cultured with purified autologous CD4+
T cells (2.times.10e5) for 8 days. Cells were then re-stimulated
with 5 uM of individual peptides derived from CyclinD1 for 5h in
the presence of Brefeldin A. Cells were stained for measuring
intracellular IFN.gamma. expression.
[0076] FIG. 27 shows the expression and construct design for
anti-CD40-MART-1 peptide antibodies.
[0077] FIG. 28 is a summary of the CD4.sup.+ and CD8.sup.+
immunodominant epitopes for MART-1.
[0078] FIG. 29 shows the expression and construct design for
anti-CD40-gp100 peptide antibodies.
[0079] FIG. 30 shows the design for additional anti-CD40-gp100
peptide antibodies.
[0080] FIG. 31 shows the expression and construct design for
additional anti-CD40-gp100 peptide antibodies.
[0081] FIG. 32 is a summary of the CD4.sup.+ and CD8.sup.+
immunodominant epitopes for gp100.
[0082] FIG. 33 shows the expression and construct design for
additional anti-CD40-gp100 peptide antibodies.
[0083] FIG. 34A shows that full-length Cyclin B1 fused to the
C-terminus of either antibody H chain or cohesion fail to be
secreted from mammalian 293F cells. FIG. 34B shows that full-length
Cyclin B1 fused to the C-terminus of either antibody H chain or
cohesion fail to be secreted from mammalian 293F cells.
[0084] FIG. 35 shows Cyclin B1 segmentation strategy based on known
or predicted structural domain regions.
[0085] FIG. 36 shows that Cyclin D1 segments p1, p3, and p4, but
not p2 express well as direct fusions to the H chain
C-terminus.
[0086] FIG. 37 shows the relative expression levels of various
Cyclin D1 segments as direct fusions to the H chain C-terminus in
various combinations with flexible linker sequences.
[0087] FIG. 38 show a summary of various H chain-Cyclin D1 segment
constructs and their relative expressibility as vaccines.
[0088] FIG. 39 shows that full-length Cyclin D1 fused to the
C-terminus of a DC targeting antibody H chain is very poorly
expressed as a secreted recombinant antibody.
DETAILED DESCRIPTION OF THE INVENTION
[0089] While the making and using of various embodiments of the
present invention are discussed in detail below, it should be
appreciated that the present invention provides many applicable
inventive concepts that can be embodied in a wide variety of
specific contexts. The specific embodiments discussed herein are
merely illustrative of specific ways to make and use the invention
and do not delimit the scope of the invention.
[0090] To facilitate the understanding of this invention, a number
of terms are defined below. Terms defined herein have meanings as
commonly understood by a person of ordinary skill in the areas
relevant to the present invention. Terms such as "a", "an" and
"the" are not intended to refer to only a singular entity, but
include the general class of which a specific example may be used
for illustration. The terminology herein is used to describe
specific embodiments of the invention, but their usage does not
delimit the invention, except as outlined in the claims.
[0091] The invention includes also variants and other modification
of an antibody (or "Ab") of fragments thereof, e.g., anti-CD40
fusion protein (antibody is used interchangeably with the term
"immunoglobulin"). As used herein, the term "antibodies or
fragments thereof," includes whole antibodies or fragments of an
antibody, e.g., Fv, Fab, Fab', F(ab').sub.2, Fc, and single chain
Fv fragments (ScFv) or any biologically effective fragments of an
immunoglobulins that binds specifically to, e.g., CD40. Antibodies
from human origin or humanized antibodies have lowered or no
immunogenicity in humans and have a lower number or no immunogenic
epitopes compared to non-human antibodies. Antibodies and their
fragments will generally be selected to have a reduced level or no
antigenicity in humans.
[0092] As used herein, the terms "Ag" or "antigen" refer to a
substance capable of either binding to an antigen binding region of
an immunoglobulin molecule or of eliciting an immune response,
e.g., a T cell-mediated immune response by the presentation of the
antigen on Major Histocompatibility Antigen (MHC) cellular
proteins. As used herein, "antigen" includes, but is not limited
to, antigenic determinants, haptens, and immunogens which may be
peptides, small molecules, carbohydrates, lipids, nucleic acids or
combinations thereof. The skilled immunologist will recognize that
when discussing antigens that are processed for presentation to T
cells, the term "antigen" refers to those portions of the antigen
(e.g., a peptide fragment) that is a T cell epitope presented by
MHC to the T cell receptor. When used in the context of a B cell
mediated immune response in the form of an antibody that is
specific for an "antigen", the portion of the antigen that binds to
the complementarity determining regions of the variable domains of
the antibody (light and heavy) the bound portion may be a linear or
three-dimensional epitope. In the context of the present invention,
the term antigen is used on both contexts, that is, the antibody is
specific for a protein antigen (CD40), but also carries one or more
peptide epitopes for presentation by MHC to T cells. In certain
cases, the antigens delivered by the vaccine or fusion protein of
the present invention are internalized and processed by antigen
presenting cells prior to presentation, e.g., by cleavage of one or
more portions of the antibody or fusion protein.
[0093] As used herein, the term "antigenic peptide" refers to that
portion of a polypeptide antigen that is specifically recognized by
either B-cells or T-cells. B-cells respond to foreign antigenic
determinants via antibody production, whereas T-lymphocytes are the
mediate cellular immunity. Thus, antigenic peptides are those parts
of an antigen that are recognized by antibodies, or in the context
of an MHC, by T-cell receptors.
[0094] As used herein, the term "epitope" refers to any protein
determinant capable of specific binding to an immunoglobulin or of
being presented by a Major Histocompatibility Complex (MHC) protein
(e.g., Class I or Class II) to a T-cell receptor. Epitopic
determinants are generally short peptides 5-30 amino acids long
that fit within the groove of the MHC molecule that presents
certain amino acid side groups toward the T cell receptor and has
certain other residues in the groove, e.g., due to specific charge
characteristics of the groove, the peptide side groups and the T
cell receptor. Generally, an antibody specifically binds to an
antigen when the dissociation constant is 1 mM, 100 nM or even 10
nM.
[0095] As used herein, the term "vector" is used in two different
contexts. When using the term "vector" with reference to a vaccine,
a vector is used to describe a non-antigenic portion that is used
to direct or deliver the antigenic portion of the vaccine. For
example, an antibody or fragments thereof may be bound to or form a
fusion protein with the antigen that elicits the immune response.
For cellular vaccines, the vector for delivery and/or presentation
of the antigen is the antigen presenting cell, which is delivered
by the cell that is loaded with antigen. In certain cases, the
cellular vector itself may also process and present the antigen(s)
to T cells and activate an antigen-specific immune response. When
used in the context of nucleic acids, a "vector" refers a construct
which is capable of delivering, and preferably expressing, one or
more genes or polynucleotide sequences of interest in a host cell.
Examples of vectors include, but are not limited to, viral vectors,
naked DNA or RNA expression vectors, DNA or RNA expression vectors
associated with cationic condensing agents, DNA or RNA expression
vectors encapsulated in liposomes, and certain eukaryotic cells,
such as producer cells.
[0096] The compositions and methods of the present invention can be
used with a wide variety of peptides and/or protein in which the
antibody or fragment thereof and the peptide linker or "PL" create
a protein that is stable and/or soluble.
[0097] As used herein, the compositions and methods use an antigen
delivery vector comprising the formula: Ab-(PL-Ag)x or Ab-(Ag-PL)x;
wherein Ab is an antibody or fragment thereof; PL is at least one
peptide linker comprising at least one glycosylation site; Ag is at
least one viral antigen; and x is an integer from 1 to 20. One
example of an antibody for use with the present invention comprises
at least the variable region of anti-CD40_12E12.3F3 (ATCC Accession
No. PTA-9854), anti-CD40_12B4.2C10 (Deposit No. HS446, ATCC
Accession No. PTA-10653), and anti-CD40_11B6.1C3 (Deposit No.
HS440, ATCC Accession No. PTA-10652).
[0098] As used herein, the terms "stable" and "unstable" when
referring to proteins is used to describe a peptide or protein that
maintains its three-dimensional structure and/or activity (stable)
or that loses immediately or over time its three-dimensional
structure and/or activity (unstable). As used herein, the term
"insoluble" refers to those proteins that when produced in a cell
(e.g., a recombinant protein expressed in a eukaryotic or
prokaryotic cell or in vitro) are not soluble in solution absent
the use of denaturing conditions or agents (e.g., heat or chemical
denaturants, respectively). The antibody or fragment thereof and
the linkers taught herein have been found to convert antibody
fusion proteins with the peptides from insoluble and/or unstable
into proteins that are stable and/or soluble. Another example of
stability versus instability is when the domain of the protein with
a stable conformation has a higher melting temperature (T.sub.m)
than the unstable domain of the protein when measured in the same
solution. A domain is stable compared to another domain when the
difference in the T.sub.m is at least about 2.degree. C., more
preferably about 4.degree. C., still more preferably about
7.degree. C., yet more preferably about 10.degree. C., even more
preferably about 15.degree. C., still more preferably about
20.degree. C., even still more preferably about 25.degree. C., and
most preferably about 30.degree. C., when measured in the same
solution.
[0099] As used herein, "polynucleotide" or "nucleic acid" refers to
a strand of deoxyribonucleotides or ribonucleotides in either a
single- or a double-stranded form (including known analogs of
natural nucleotides). A double-stranded nucleic acid sequence will
include the complementary sequence. The polynucleotide sequence may
encode variable and/or constant region domains of immunoglobulin
that are formed into a fusion protein with one or more linkers. For
use with the present invention, multiple cloning sites (MCS) may be
engineered into the locations at the carboxy-terminal end of the
heavy and/or light chains of the antibodies to allow for in-frame
insertion of peptide for expression between the linkers. As used
herein, the term "isolated polynucleotide" refers to a
polynucleotide of genomic, cDNA, or synthetic origin or some
combination thereof. By virtue of its origin the "isolated
polynucleotide" (1) is not associated with all or a portion of a
polynucleotide in which the "isolated polynucleotides" are found in
nature, (2) is operably linked to a polynucleotide which it is not
linked to in nature, or (3) does not occur in nature as part of a
larger sequence. The skilled artisan will recognize that to design
and implement a vector having the formula Ab-(PL-Ag)x or
Ab-(Ag-PL)x, can be manipulated at the nucleic acid level by using
techniques known in the art, such as those taught in Current
Protocols in Molecular Biology, 2007 by John Wiley and Sons,
relevant portions incorporated herein by reference. Briefly, the
Ab, Ag and PL encoding nucleic acid sequences can be inserted using
polymerase chain reaction, enzymatic insertion of oligonucleotides
or polymerase chain reaction fragments in a vector, which may be an
expression vector. To facilitate the insertion of (PL-Ag)x or
(Ag-PL)x at the carboxy terminus of the antibody light chain, the
heavy chain, or both, a multiple cloning site (MCS) may be
engineered in sequence with the antibody sequences.
[0100] As used herein, the term "polypeptide" refers to a polymer
of amino acids and does not refer to a specific length of the
product; thus, peptides, oligopeptides, and proteins are included
within the definition of polypeptide. This term also does not refer
to or exclude post expression modifications of the polypeptide, for
example, glycosylations, acetylations, phosphorylations and the
like. Included within the definition are, for example, polypeptides
containing one or more analogs of an amino acid (including, for
example, unnatural amino acids, etc.), polypeptides with
substituted linkages, as well as other modifications known in the
art, both naturally occurring and non-naturally occurring. The term
"domain," or "polypeptide domain" refers to that sequence of a
polypeptide that folds into a single globular region in its native
conformation, and that may exhibit discrete binding or functional
properties.
[0101] A polypeptide or amino acid sequence "derived from" a
designated nucleic acid sequence refers to a polypeptide having an
amino acid sequence identical to that of a polypeptide encoded in
the sequence, or a portion thereof wherein the portion consists of
at least 3-5 amino acids, preferably at least 4-7 amino acids, more
preferably at least 8-10 amino acids, and even more preferably at
least 11-15 amino acids, or which is immunologically identifiable
with a polypeptide encoded in the sequence. This terminology also
includes a polypeptide expressed from a designated nucleic acid
sequence.
[0102] As used herein, "pharmaceutically acceptable carrier" refers
to any material that when combined with an immunoglobulin (Ig)
fusion protein of the present invention allows the Ig to retain
biological activity and is generally non-reactive with the
subject's immune system. Examples include, but are not limited to,
standard pharmaceutical carriers such as a phosphate buffered
saline solution, water, emulsions such as an oil/water emulsion,
and various types of wetting agents. Certain diluents may be used
with the present invention, e.g., for aerosol or parenteral
administration, that may be phosphate buffered saline or normal
(0.85%) saline.
[0103] The invention provides an CD40 binding molecule comprising
at least one immunoglobulin light chain variable domain (V.sub.L)
which comprises in sequence hypervariable regions CDR1.sub.L,
CDR2.sub.L and CDR3.sub.L, the CDR1.sub.L having the amino acid
sequence SASQGISNYLN (SEQ ID NO.:41) the CDR2.sub.L having the
amino acid sequence YTSILHS (SEQ ID NO.:42) and the CDR3.sub.L
having the amino acid sequence QQFNKLPPT (SEQ ID NO.:43) the amino
acid sequences of which are shown in SEQ ID NO. 37; and direct
equivalent thereof.
[0104] Accordingly the invention provides an CD40 binding molecule
which comprises an antigen binding site comprising at least one
immunoglobulin heavy chain variable domain (V.sub.H) which
comprises in sequence hypervariable regions CDR1.sub.H, CDR2.sub.H
and CDR3.sub.H, the CDR1.sub.H having the amino acid sequence
GFTFSDYYMY (SEQ ID NO.:44), the CDR2.sub.H having the amino acid
sequence YINSGGGSTYYPDTVKG (SEQ ID NO.:45), and the CDR3.sub.H
having the amino acid sequence RGLPFHAMDY (SEQ ID NO.:46), the
amino acid sequences of which are shown in SEQ ID NO. 38; and
direct equivalents thereof.
[0105] In one aspect the invention provides a single domain CD40
binding molecule comprising an isolated immunoglobulin light chain
comprising a heavy chain variable domain (V.sub.L) as defined
above. In another aspect the invention provides a single domain
CD40 binding molecule comprising an isolated immunoglobulin heavy
chain comprising a heavy chain variable domain (V.sub.H) as defined
above.
[0106] In another aspect the invention also provides an CD40
binding molecule comprising both heavy (V.sub.H) and light chain
(V.sub.L) variable domains in which the CD40 binding molecule
comprises at least one antigen binding site comprising: a) an
immunoglobulin heavy chain variable domain (V.sub.L) which
comprises in sequence hypervariable regions CDR1.sub.L, CDR2.sub.L
and CDR3.sub.L, the CDR1.sub.L having the amino acid sequence
SASQGISNYLN (SEQ ID NO.:41), the CDR2.sub.L having the amino acid
sequence YTSILHS (SEQ ID NO.:42), and the CDR3.sub.L having the
amino acid sequence QQFNKLPPT (SEQ ID NO.:43), the amino acid
sequences of which are shown in SEQ ID. NO. 1, and b) an
immunoglobulin light chain variable domain (V.sub.H) which
comprises in sequence hypervariable regions CDR1.sub.H, CDR2.sub.H
and CDR3.sub.H, the CDR1.sub.H having the amino acid sequence
GFTFSDYYMY (SEQ ID NO.:44), the CDR2' having the amino acid
sequence YINSGGGSTYYPDTVKG (SEQ ID NO.:45), and the CDR3.sub.H
having the amino acid sequence RGLPFHAMDY (SEQ ID NO.:46), the
amino acid sequences of which are shown in SEQ ID NO. 38; and
direct equivalents thereof.
[0107] Unless otherwise indicated, any polypeptide chain is herein
described as having an amino acid sequence starting at the
N-terminal end and ending at the C-terminal end. When the antigen
binding site comprises both the V.sub.H and V.sub.L domains, these
may be located on the same polypeptide molecule or, preferably,
each domain may be on a different chain, the V.sub.H domain being
part of an immunoglobulin heavy chain or fragment thereof and the
V.sub.L being part of an immunoglobulin light chain or fragment
thereof.
[0108] Non-limiting examples for antigens targeted by the
antibodies of the present invention include, but are not limited
to: cell surface marker selected from MHC class I, MHC class II,
CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD 19, CD20,
CD29, CD31, CD40,CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83,
CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2,
MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2,
IFN-.gamma. receptor and IL-2 receptor, ICAM-1, Fc.gamma. receptor,
T cell receptors, lectins, or other immune cell receptors. In one
specific example, the antigens that are targeted by the antibody
portion of the present invention are specifically expressed by
antigen presenting cells, e.g., dendritic cells, Langerhans cells,
macrophages, and B cells.
[0109] As used herein, the term "CD40 binding molecule" refers to
any molecule capable of binding to the CD40 antigen either alone or
associated with other molecules having one or more the V.sub.L and
V.sub.H CDRs taught herein, in some cases 2, 3, 4, 5, or all 6
CDRs. The binding reaction may be shown by standard methods
(qualitative assays) including, for example, a bioassay for
determining by blocking the binding of other molecules to CD40 or
any kind of binding or activity assays (e.g., activation, reduction
or modulation of an immune response), with reference to a negative
control test in which an antibody of unrelated specificity but of
the same isotype, e.g., an anti-CD25 or anti-CD80 antibody, is
used.
[0110] The present invention may also be made into a single chain
antibody having the variable domains of the heavy and light chains
of an antibody covalently bound by a peptide linker usually
including from 10 to 30 amino acids, preferably from 15 to 25 amino
acids. Therefore, such a structure does not include the constant
part of the heavy and light chains and it is believed that the
small peptide spacer should be less antigenic than a whole constant
part.
[0111] As used herein, the term "chimeric antibody" refers to an
antibody in which the constant regions of heavy or light chains or
both are of human origin while the variable domains of both heavy
and light chains are of non-human (e.g., mouse, hamster or rat)
origin or of human origin but derived from a different human
antibody.
[0112] As used herein, the term "CDR-grafted antibody" refers to an
antibody in which the hypervariable complementarity determining
regions (CDRs) are derived from a donor antibody, such as a
non-human (e.g., mouse) antibody or a different human antibody,
while all or substantially all the other parts of the
immunoglobulin (e.g., the conserved regions of the variable
domains, i.e., framework regions), are derived from an acceptor
antibody (in the case of a humanized antibody--an antibody of human
origin). A CDR-grafted antibody may include a few amino acids of
the donor sequence in the framework regions, for instance in the
parts of the framework regions adjacent to the hypervariable
regions.
[0113] As used herein, the term "human antibody" refers to an
antibody in which the constant and variable regions of both the
heavy and light chains are all of human origin, or substantially
identical to sequences of human origin, not necessarily from the
same antibody and includes antibodies produced by mice in which the
mouse, hamster or rat immunoglobulin variable and constant part
genes have been replaced by their human counterparts, e.g. as
described in general terms in EP 0546073 B1, U.S. Pat. Nos.
5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,
EP 0 438474 B1 and EP 0 463151 B1, relevant portions incorporated
herein by reference.
[0114] The CD40 binding molecule of the invention can be a
humanized antibody that comprises the CDRs obtained from the
anti-CD40_12E12.3F3, the anti-CD40_11B6.1C3, or the
anti-CD40_12B4.2C10 antibodies. One example of a chimeric antibody
includes the variable domains of both heavy and light chains are of
human origin, for instance those variable domains of the
anti-CD40_12E12.3F3 antibody that are part of SEQ ID NO.: 148 and
SEQ ID NO.: 149, anti-CD40_12B4.2C10 in SEQ ID NO.: 150 and SEQ ID
NO.: 151 or SEQ ID NO.: 152, and/or anti-CD40_11B6.1C3, SEQ ID NO.:
153 and SEQ ID NO.: 154, or combination thereof. The constant
region domains preferably also comprise suitable human constant
region domains, for instance as described in "Sequences of Proteins
of Immunological Interest", Kabat E. A. et al, US Department of
Health and Human Services, Public Health Service, National
Institute of Health. The nucleic acid sequences can be found in,
e.g., SEQ ID NOS.: 8 and 9.
[0115] Hypervariable regions may be associated with any kind of
framework regions, e.g., of human origin. Suitable framework
regions were described Kabat E. A. One heavy chain framework is a
heavy chain framework, for instance that of anti-CD40_12E12.3F3
antibody that are part of SEQ ID NO.: 149; anti-CD40_12B4.2C10-SEQ
ID NO.: 151 or SEQ ID NO.: 152, and/or anti-CD40_11B6.1C3-SEQ ID
NO.: 154, or combination thereof, e.g., FR1.sub.L, FR2.sub.L,
FR3.sub.L and FR4.sub.L regions. In a similar manner, SEQ ID NO.
148 shows the anti-CD40_12E12.3F3 (or the equivalents for
anti-CD40_12B4.2C10 and anti-CD40_11B6.1C3, SEQ ID NOS.: 150 and
153, respectively) heavy chain framework that includes the sequence
of FR1H, FR2.sub.H, FR3.sub.H and FR4.sub.H regions. The CDRs may
be added to a human antibody framework, such as those described in
U.S. Pat. No. 7,456,260, issued to Rybak, et al., which teach new
human variable chain framework regions and humanized antibodies
comprising the framework regions, relevant portions and framework
sequences incorporated herein by reference. To accomplish the
engraftment at a genetic level, the present invention also includes
the underlying nucleic acid sequences for the V.sub.L AND V.sub.H
regions as well as the complete antibodies and the humanized
versions thereof. The nucleic acid sequences of the present
invention include SEQ ID NOS.: 155 and 156, which are the anti-CD40
antibody light and the heavy chains, respectively, as well as those
nucleic acid sequences that include variable codon usage for the
same amino acid sequences and conservative variations thereof
having 85, 90, 95 or 100% sequence identity at the nucleic or amino
acid level. Likewise, the CDRs may have 85, 90, 95 or 100% sequence
identity at the nucleic or amino acid level, individually, in
groups or 2, 3, 4 or 5 or all together.
[0116] Monoclonal antibodies raised against a protein naturally
found in all humans are typically developed in a non-human system
e.g. in mice, and as such are typically non-human proteins. As a
direct consequence of this, a xenogenic antibody as produced by a
hybridoma, when administered to humans, elicits an undesirable
immune response that is predominantly mediated by the constant part
of the xenogenic immunoglobulin. Xenogeneic antibodies tend to
elicit a host immune response, thereby limiting the use of such
antibodies as they cannot be administered over a prolonged period
of time. Therefore, it is particularly useful to use single chain,
single domain, chimeric, CDR-grafted, or especially human
antibodies that are not likely to elicit a substantial allogenic
response when administered to humans. The present invention
includes antibodies with minor changes in an amino acid sequence
such as deletion, addition or substitution of one, a few or even
several amino acids which are merely allelic forms of the original
protein having substantially identical properties.
[0117] The inhibition of the binding of CD40 to its receptor may be
conveniently tested in various assays including such assays are
described hereinafter in the text. By the term "to the same extent"
is meant that the reference and the equivalent molecules exhibit,
on a statistical basis, essentially identical CD40 binding
inhibition curves in one of the assays referred to above. For
example, the assay used may be an assay of competitive inhibition
of binding of CD40 by the binding molecules of the invention.
[0118] Generally, the human anti-CD40 antibody comprises at least:
(a) one light chain which comprises a variable domain having an
amino acid sequence substantially identical to that shown in SEQ ID
NO. 1 starting with the amino acid at position 1 and ending with
the amino acid at position 107 and the constant part of a human
light chain; and (b) one heavy chain which comprises a variable
domain having an amino acid sequence substantially identical to
that shown in SEQ ID NO. 2 and the constant part of a human heavy
chain. The constant part of a human heavy chain may be of the
.gamma.1, .gamma.2, .gamma.3, .gamma.4, .mu., .beta.2, or .delta.
or .epsilon. type, preferably of the .gamma.-type, whereas the
constant part of a human light chain may be of the .kappa. or
.lamda. type (which includes the .lamda..sub.1, .lamda..sub.2 and
.lamda..sub.3 subtypes) but is preferably of the .kappa. type. The
amino acid sequences of the general locations of the variable and
constant domains are well known in the art and generally follow the
Kabat nomenclature.
[0119] A CD40 binding molecule of the invention may be produced by
recombinant DNA techniques. In view of this, one or more DNA
molecules encoding the binding molecule must be constructed, placed
under appropriate control sequences and transferred into a suitable
host organism for expression.
[0120] In a very general manner, there are accordingly provided:
(i) DNA molecules encoding a single domain CD40 binding molecule of
the invention, a single chain CD40 binding molecule of the
invention, a heavy or light chain or fragments thereof of a CD40
binding molecule of the invention; and (ii) the use of the DNA
molecules of the invention for the production of a CD40 binding
molecule of the invention by recombinant methods.
[0121] The present state of the art is such that the skilled worker
in the art can synthesize the DNA molecules of the invention given
the information provided herein, i.e., the amino acid sequences of
the hypervariable regions and the DNA sequences coding for them. A
method for constructing a variable domain gene is for example
described in EPA 239 400, relevant portions incorporated herein by
reference. Briefly, a gene encoding a variable domain of a MAb is
cloned. The DNA segments encoding the framework and hypervariable
regions are determined and the DNA segments encoding the
hypervariable regions are removed so that the DNA segments encoding
the framework regions are fused together with suitable restriction
sites at the junctions. The restriction sites may be generated at
the appropriate positions by mutagenesis of the DNA molecule by
standard procedures. Double stranded synthetic CDR cassettes are
prepared by DNA synthesis according to the sequences given in SEQ
ID NO. 1 and 3 or 2 and 4 (amino acid and nucleic acid sequences,
respectively). These cassettes are often provided with sticky ends
so that they can be ligated at the junctions of the framework.
[0122] It is not necessary to have access to the mRNA from a
producing hybridoma cell line in order to obtain a DNA construct
coding for the CD40 binding molecules of the invention. For
example, PCT application WO 90/07861 gives full instructions for
the production of an antibody by recombinant DNA techniques given
only written information as to the nucleotide sequence of the gene,
relevant portions incorporated herein by reference. Briefly, the
method comprises the synthesis of a number of oligonucleotides,
their amplification by the PCR method, and their splicing to give
the desired DNA sequence.
[0123] Expression vectors comprising a suitable promoter or genes
encoding heavy and light chain constant parts are publicly
available. Thus, once a DNA molecule of the invention is prepared
it may be conveniently transferred in an appropriate expression
vector. DNA molecules encoding single chain antibodies may also be
prepared by standard methods, for example, as described in WO
88/1649. In view of the foregoing, no hybridoma or cell line
deposit is necessary to comply with the criteria of sufficiency of
description.
[0124] For example, first and second DNA constructs are made that
bind specifically to CD40. Briefly, a first DNA construct encodes a
light chain or fragment thereof and comprises a) a first part which
encodes a variable domain comprising alternatively framework and
hypervariable regions, the hypervariable regions being in sequence
CDR1.sub.L, CDR2.sub.L and CDR3.sub.L the amino acid sequences of
which are shown in SEQ ID NO. 1; this first part starting with a
codon encoding the first amino acid of the variable domain and
ending with a codon encoding the last amino acid of the variable
domain, and b) a second part encoding a light chain constant part
or fragment thereof which starts with a codon encoding the first
amino acid of the constant part of the heavy chain and ends with a
codon encoding the last amino acid of the constant part or fragment
thereof, followed by a stop codon.
[0125] The first part encodes a variable domain having an amino
acid sequence substantially identical to the amino acid sequence as
shown in SEQ ID NO. 1. A second part encodes the constant part of a
human heavy chain, more preferably the constant part of the human
.gamma.1 chain. This second part may be a DNA fragment of genomic
origin (comprising introns) or a cDNA fragment (without
introns).
[0126] The second DNA construct encodes a heavy chain or fragment
thereof and comprises a) a first part which encodes a variable
domain comprising alternatively framework and hypervariable
regions; the hypervariable regions being CDR1.sub.H and optionally
CDR2.sub.H and CDR3.sub.H, the amino acid sequences of which are
shown in SEQ ID NO. 2; this first part starting with a codon
encoding the first amino acid of the variable domain and ending
with a codon encoding the last amino acid of the variable domain,
and b) a second part encoding a heavy chain constant part or
fragment thereof which starts with a codon encoding the first amino
acid of the constant part of the light chain and ends with a codon
encoding the last amino acid of the constant part or fragment
thereof followed by a stop codon.
[0127] The first part encodes a variable domain having an amino
acid sequence substantially identical to the amino acid sequence as
shown in SEQ ID NO. 2. The first part has the nucleotide sequence
as shown in SEQ ID NO. 2 starting with the nucleotide at position 1
and ending with the nucleotide at position 321. Also preferably the
second part encodes the constant part of a human light chain, more
preferably the constant part of the human .kappa. chain.
[0128] The invention also includes CD40 binding molecules in which
one or more of the residues of CDR1.sub.L, CDR2.sub.L, CDR3.sub.L,
CDR1.sub.H, CDR2.sub.H or CDR3.sub.H or the frameworks, typically
only a few (e.g. FR1-4.sub.L or .sub.H), are changed from the
residues shown in SEQ ID NO. 37 and SEQ ID NO. 38; by, e.g., site
directed mutagenesis of the corresponding DNA sequences. The
invention includes the DNA sequences coding for such changed CD40
binding molecules. In particular the invention includes a CD40
binding molecules in which one or more residues of CDR1.sub.L,
CDR2.sub.L and/or CDR3.sub.L have been changed from the residues
shown in SEQ ID NO. 37 and one or more residues of CDR1.sub.H,
CDR2.sub.H and/or CDR3.sub.H have been changed from the residues
shown in SEQ ID NO. 38.
[0129] Each of the DNA constructs are placed under the control of
suitable control sequences, in particular under the control of a
suitable promoter. Any kind of promoter may be used, provided that
it is adapted to the host organism in which the DNA constructs will
be transferred for expression. However, if expression is to take
place in a mammalian cell, an immunoglobulin gene promoter may be
used in B cells. The first and second parts may be separated by an
intron, and, an enhancer may be conveniently located in the intron
between the first and second parts. The presence of such an
enhancer that is transcribed but not translated, may assist in
efficient transcription. In particular embodiments the first and
second DNA constructs comprise the enhancer of, e.g., a heavy chain
human gene.
[0130] The desired antibody may be produced in a cell culture or in
a transgenic animal A suitable transgenic animal may be obtained
according to standard methods that include micro injecting into
eggs the first and second DNA constructs placed under suitable
control sequences transferring the so prepared eggs into
appropriate pseudo-pregnant females and selecting a descendant
expressing the desired antibody.
[0131] The invention also provides an expression vector able to
replicate in a prokaryotic or eukaryotic cell line, which comprises
at least one of the DNA constructs above described. Each expression
vector containing a DNA construct is then transferred into a
suitable host organism. When the DNA constructs are separately
inserted on two expression vectors, they may be transferred
separately, i.e. one type of vector per cell, or co-transferred,
this latter possibility being preferred. A suitable host organism
may be a bacterium, a yeast or a mammalian cell line, this latter
being preferred. More preferably, the mammalian cell line is of
lymphoid origin, e.g., a myeloma, hybridoma or a normal
immortalized B-cell, which conveniently does not express any
endogenous antibody heavy or light chain.
[0132] When the antibody chains are produced in a cell culture, the
DNA constructs must first be inserted into either a single
expression vector or into two separate but compatible expression
vectors, the latter possibility being preferred. For expression in
mammalian cells it is preferred that the coding sequence of the
CD40 binding molecule is integrated into the host cell DNA within a
locus which permits or favors high level expression of the CD40
binding molecule.
[0133] In a further aspect of the invention there is provided a
process for the product of a CD40 binding molecule that comprises:
(i) culturing an organism which is transformed with an expression
vector as defined above; and (ii) recovering the CD40 binding
molecule from the culture.
[0134] In accordance with the present invention it has been found
that the anti-CD40_12E12.3F3, anti-CD40_12B4.2C10 and/or
anti-CD40_11B6.1C3 antibody appears to have binding specificity for
the antigenic epitope of human CD40. It is therefore most
surprising that antibodies to this epitope, e.g. the
anti-CD40_12E12.3F3 antibody, are capable of delivering antigen
efficiently into dendritic cells (DCs). Antibodies, in particular
chimeric and CDR-grafted antibodies and especially human
antibodies, which have binding specificity for the antigenic
epitope of mature human CD40; and use of such antibodies for DC
antigen loading are novel and are included within the scope of the
present invention.
[0135] To use the anti-CD40 antibody of the present invention for
treatment indications, the appropriate dosage will, of course, vary
depending upon, for example, the antibody disclosed herein to be
employed, the host, the mode of administration and the nature and
severity of the condition being treated. However, in prophylactic
use, satisfactory results are generally found at dosages from about
0.05 mg to about 10 mg per kilogram body weight more usually from
about 0.1 mg to about 5 mg per kilogram body weight. The frequency
of dosing for prophylactic uses will normally be in the range from
about once per week up to about once every 3 months, more usually
in the range from about once every 2 weeks up to about once every
10 weeks, e.g., once every 4 to 8 weeks. The anti-CD40 antibody of
the present can be administered parenterally, intravenously, e.g.,
into the antecubital or other peripheral vein, intramuscularly, or
subcutaneously.
[0136] Pharmaceutical compositions of the invention may be
manufactured in conventional manner, e.g., in a lyophilized form.
For immediate administration it is dissolved in a suitable aqueous
carrier, for example sterile water for injection or sterile
buffered physiological saline. If it is considered desirable to
make up a solution of larger volume for administration by infusion
rather as a bolus injection, it is advantageous to incorporate
human serum albumin or the patient's own heparinized blood into the
saline at the time of formulation. The presence of an excess of
such physiologically inert protein prevents loss of antibody by
adsorption onto the walls of the container and tubing used with the
infusion solution. If albumin is used, a suitable concentration is
from 0.5 to 4.5% by weight of the saline solution.
[0137] One embodiment of the present invention provides an
immunoconjugate comprising a humanized antibody of the invention,
e.g., a humanized anti-CD40 antibody, linked to one or more
effector molecules, antigen(s) and/or a detectable label(s).
Preferably, the effector molecule is a therapeutic molecule such
as, for example, one or more peptides that comprise one or more T
cell epitopes, a toxin, a small molecule, a cytokine or a
chemokine, an enzyme, or a radiolabel.
[0138] Exemplary toxins include, but are not limited to,
Pseudomonas exotoxin or diphtheria toxin. Examples of small
molecules include, but are not limited to, chemotherapeutic
compounds such as taxol, doxorubicin, etoposide, and bleiomycin.
Exemplary cytokines include, but are not limited to, IL-1, IL-2,
IL-4, IL-5, IL-6, and IL-12, IL-17, and IL-25. Exemplary enzymes
include, but are not limited to, RNAses, DNAses, proteases,
kinases, and caspases. Exemplary radioisotopes include, but are not
limited to, .sup.32P and .sup.125I.
[0139] As used herein, the term "epitope" refers to a molecule or
substance capable of stimulating an immune response. In one
example, epitopes include but are not limited to a polypeptide and
a nucleic acid encoding a polypeptide, wherein expression of the
nucleic acid into a polypeptide is capable of stimulating an immune
response when the polypeptide is processed and presented on a Major
Histocompatibility Complex (MHC) molecule. Generally, epitopes
include peptides presented on the surface of cells non-covalently
bound to the binding groove of Class I or Class II MHC, such that
they can interact with T cell receptors and the respective T cell
accessory molecules.
[0140] Proteolytic Processing of Antigens. Epitopes that are
displayed by MHC on antigen presenting cells are cleavage peptides
or products of larger peptide or protein antigen precursors. For
MHC I epitopes, protein antigens are often digested by proteasomes
resident in the cell. Intracellular proteasomal digestion produces
peptide fragments of about 3 to 23 amino acids in length that are
then loaded onto the MHC protein. Additional proteolytic activities
within the cell, or in the extracellular milieu, can trim and
process these fragments further. Processing of MHC Class II
epitopes generally occurs via intracellular proteases from the
lysosomal/endosomal compartment. The present invention includes, in
one embodiment, pre-processed peptides that are attached to the
anti-CD40 antibody (or fragment thereof) that directs the peptides
against which an enhanced immune response is sought directly to
antigen presenting cells.
[0141] To identify epitopes potentially effective as immunogenic
compounds, predictions of MHC binding alone are useful but often
insufficient. The present invention includes methods for
specifically identifying the epitopes within antigens most likely
to lead to the immune response sought for the specific sources of
antigen presenting cells and responder T cells.
[0142] The present invention allows for a rapid and easy assay for
the identification of those epitopes that are most likely to
produce the desired immune response using the patient's own antigen
presenting cells and T cell repertoire. The compositions and
methods of the present invention are applicable to any protein
sequence, allowing the user to identify the epitopes that are
capable of binding to MHC and are properly presented to T cells
that will respond to the antigen. Accordingly, the invention is not
limited to any particular target or medical condition, but instead
encompasses and MHC epitope(s) from any useful source.
[0143] As used herein, the term "veneered" refers to a humanized
antibody framework onto which antigen-binding sites or CDRs
obtained from non-human antibodies (e.g., mouse, rat or hamster),
are placed into human heavy and light chain conserved structural
framework regions (FRs), for example, in a light chain or heavy
chain polynucleotide to "graft" the specificity of the non-human
antibody into a human framework. The polynucleotide expression
vector or vectors that express the veneered antibodies can be
transfected mammalian cells for the expression of recombinant human
antibodies which exhibit the antigen specificity of the non-human
antibody and will undergo posttranslational modifications that will
enhance their expression, stability, solubility, or combinations
thereof.
[0144] Antigens.
[0145] Examples of viral antigens for use with the present
invention include, but are not limited to, e.g., HIV, HCV, CMV,
adenoviruses, retroviruses, picornaviruses, etc. Non-limiting
example of retroviral antigens such as retroviral antigens from the
human immunodeficiency virus (HIV) antigens such as gene products
of the gag, pol, and env genes, the Nef protein, reverse
transcriptase, and other HIV components; hepatitis viral antigens
such as the S, M, and L proteins of hepatitis B virus, the pre-S
antigen of hepatitis B virus, and other hepatitis, e.g., hepatitis
A, B, and C, viral components such as hepatitis C viral RNA;
influenza viral antigens such as hemagglutinin and neuraminidase
and other influenza viral components; measles viral antigens such
as the measles virus fusion protein and other measles virus
components; rubella viral antigens such as proteins E1 and E2 and
other rubella virus components; rotaviral antigens such as VP7sc
and other rotaviral components; cytomegaloviral antigens such as
envelope glycoprotein B and other cytomegaloviral antigen
components; respiratory syncytial viral antigens such as the RSV
fusion protein, the M2 protein and other respiratory syncytial
viral antigen components; herpes simplex viral antigens such as
immediate early proteins, glycoprotein D, and other herpes simplex
viral antigen components; varicella zoster viral antigens such as
gpI, gpII, and other varicella zoster viral antigen components;
Japanese encephalitis viral antigens such as proteins E, M-E,
M-E-NS1, NS1, NS1-NS2A, 80% E, and other Japanese encephalitis
viral antigen components; rabies viral antigens such as rabies
glycoprotein, rabies nucleoprotein and other rabies viral antigen
components. See Fundamental Virology, Second Edition, eds. Fields,
B. N. and Knipe, D. M. (Raven Press, New York, 1991) for additional
examples of viral antigens. The at least one viral antigen may be
peptides from an adenovirus, retrovirus, picornavirus, herpesvirus,
rotaviruses, hantaviruses, coronavirus, togavirus, flavirvirus,
rhabdovirus, paramyxovirus, orthomyxovirus, bunyavirus, arenavirus,
reovirus, papilomavirus, parvovirus, poxvirus, hepadnavirus, or
spongiform virus. In certain specific, non-limiting examples, the
at least one viral antigen are peptides obtained from at least one
of HIV, CMV, hepatitis A, B, and C, influenza, measles, polio,
smallpox, rubella; respiratory syncytial, herpes simplex, varicella
zoster, Epstein-Barr, Japanese encephalitis, rabies, flu, and/or
cold viruses.
[0146] In one aspect, the one or more of the antigenic peptides are
selected from at least one of: Nef (66-97):
VGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGL (SEQ ID NO.: 1); Nef (116-145):
HTQGYFPDWQNYTPGPGVRYPLTFGWLYKL (SEQ ID NO.: 2); Gag p17 (17-35):
EKIRLRPGGKKKYKLKHIV (SEQ ID NO.: 3); Gag p17-p24 (253-284):
NPPIPVGEIYKRWIILGLNKIVRMYSPTSILD (SEQ ID NO.: 4); or Pol 325-355
(RT 158-188) is: AIFQSSMTKILEPFRKQNPDIVIYQYMDDLY (SEQ ID NO.: 5).
In one aspect, the fusion protein peptides are separated by one or
more linkers selected from: SSVSPTTSVHPTPTSVPPTPTKSSP (SEQ ID NO.:
11); PTSTPADSSTITPTATPTATPTIKG (SEQ ID NO.: 12);
TVTPTATATPSAIVTTITPTATTKP (SEQ ID NO.: 13); or
TNGSITVAATAPTVTPTVNATPSAA (SEQ ID NO.: 14).
[0147] Antigenic targets that may be delivered using the
anti-CD40-antigen vaccines of the present invention include genes
encoding antigens such as viral antigens, bacterial antigens,
fungal antigens or parasitic antigens. Pathogens include
trypanosomes, tapeworms, roundworms, helminthes, malaria. Tumor
markers, such as fetal antigen or prostate specific antigen, may be
targeted in this manner Other examples include: HIV env proteins
and hepatitis B surface antigen. Administration of a vector
according to the present invention for vaccination purposes would
require that the vector-associated antigens be sufficiently
non-immunogenic to enable long-term expression of the transgene,
for which a strong immune response would be desired. In some cases,
vaccination of an individual may only be required infrequently,
such as yearly or biennially, and provide long-term immunologic
protection against the infectious agent. Specific examples of
organisms, allergens and nucleic and amino sequences for use in
vectors and ultimately as antigens with the present invention may
be found in U.S. Pat. No. 6,541,011, relevant portions incorporated
herein by reference, in particular, the tables that match organisms
and specific sequences that may be used with the present
invention.
[0148] Bacterial antigens for use with the anti-CD40-antigen
vaccines disclosed herein include, but are not limited to, e.g.,
bacterial antigens such as pertussis toxin, filamentous
hemagglutinin, pertactin, FIM2, FIM3, adenylate cyclase and other
pertussis bacterial antigen components; diptheria bacterial
antigens such as diptheria toxin or toxoid and other diptheria
bacterial antigen components; tetanus bacterial antigens such as
tetanus toxin or toxoid and other tetanus bacterial antigen
components; streptococcal bacterial antigens such as M proteins and
other streptococcal bacterial antigen components; gram-negative
bacilli bacterial antigens such as lipopolysaccharides and other
gram-negative bacterial antigen components, Mycobacterium
tuberculosis bacterial antigens such as mycolic acid, heat shock
protein 65 (HSP65), the 30 kDa major secreted protein, antigen 85A
and other mycobacterial antigen components; Helicobacter pylori
bacterial antigen components; pneumococcal bacterial antigens such
as pneumolysin, pneumococcal capsular polysaccharides and other
pneumococcal bacterial antigen components; haemophilus influenza
bacterial antigens such as capsular polysaccharides and other
haemophilus influenza bacterial antigen components; anthrax
bacterial antigens such as anthrax protective antigen and other
anthrax bacterial antigen components; rickettsiae bacterial
antigens such as rompA and other rickettsiae bacterial antigen
component. Also included with the bacterial antigens described
herein are any other bacterial, mycobacterial, mycoplasmal,
rickettsial, or chlamydial antigens. Partial or whole pathogens may
also be: haemophilus influenza; Plasmodium falciparum; Neisseria
meningitidis; Streptococcus pneumoniae; Neisseria gonorrhoeae;
salmonella serotype typhi; shigella; Vibrio cholerae; Dengue Fever;
Encephalitides; Japanese Encephalitis; lyme disease; Yersinia
pestis; west nile virus; yellow fever; tularemia; hepatitis (viral;
bacterial); RSV (respiratory syncytial virus); HPIV 1 and HPIV 3;
adenovirus; small pox; allergies and cancers.
[0149] Fungal antigens for use with compositions and methods of the
invention include, but are not limited to, e.g., candida fungal
antigen components; histoplasma fungal antigens such as heat shock
protein 60 (HSP60) and other histoplasma fungal antigen components;
cryptococcal fungal antigens such as capsular polysaccharides and
other cryptococcal fungal antigen components; coccidiodes fungal
antigens such as spherule antigens and other coccidiodes fungal
antigen components; and tinea fungal antigens such as trichophytin
and other coccidiodes fungal antigen components.
[0150] Examples of protozoal and other parasitic antigens include,
but are not limited to, e.g., Plasmodium falciparum antigens such
as merozoite surface antigens, sporozoite surface antigens,
circumsporozoite antigens, gametocyte/gamete surface antigens,
blood-stage antigen pf 155/RESA and other plasmodial antigen
components; toxoplasma antigens such as SAG-1, p30 and other
toxoplasmal antigen components; schistosomae antigens such as
glutathione-S-transferase, paramyosin, and other schistosomal
antigen components; Leishmania major and other leishmaniae antigens
such as gp63, lipophosphoglycan and its associated protein and
other leishmanial antigen components; and Trypanosoma cruzi
antigens such as the 75-77 kDa antigen, the 56 kDa antigen and
other trypanosomal antigen components.
[0151] Antigen that can be targeted using the anti-CD40-antigen
vaccines of the present invention will generally be selected based
on a number of factors, including: likelihood of internalization,
level of immune cell specificity, type of immune cell targeted,
level of immune cell maturity and/or activation and the like. In
this embodiment, the antibodies may be mono- or bi-specific
antibodies that include one anti-CD40 binding domain and one
binding domain against a second antigen, e.g., cell surface markers
for dendritic cells such as, MHC class I, MHC Class II, B7-2, CD18,
CD29, CD31, CD43, CD44, CD45, CD54, CD58, CD83, CD86, CMRF-44,
CMRF-56, DCIR and/or Dectin-1 and the like; while in some cases
also having the absence of CD2, CD3, CD4, CD8, CD14, CD15, CD16, CD
19, CD20, CD56, and/or CD57. Examples of cell surface markers for
antigen presenting cells include, but are not limited to, MHC class
I, MHC Class II, CD45, B7-1, B7-2, IFN-.gamma. receptor and IL-2
receptor, ICAM-1 and/or Fc.gamma. receptor. Examples of cell
surface markers for T cells include, but are not limited to, CD3,
CD4, CD8, CD 14, CD20, CD11b, CD16, CD45 and HLA-DR.
[0152] Target antigens on cell surfaces for delivery include those
characteristic of tumor antigens typically will be derived from the
cell surface, cytoplasm, nucleus, organelles and the like of cells
of tumor tissue. Examples of tumor targets for the antibody portion
of the present invention include, without limitation, hematological
cancers such as leukemias and lymphomas, neurological tumors such
as astrocytomas or glioblastomas, melanoma, breast cancer, lung
cancer, head and neck cancer, gastrointestinal tumors such as
gastric or colon cancer, liver cancer, pancreatic cancer,
genitourinary tumors such cervix, uterus, ovarian cancer, vaginal
cancer, testicular cancer, prostate cancer or penile cancer, bone
tumors, vascular tumors, or cancers of the lip, nasopharynx,
pharynx and oral cavity, esophagus, rectum, gall bladder, biliary
tree, larynx, lung and bronchus, bladder, kidney, brain and other
parts of the nervous system, thyroid, Hodgkin's disease,
non-Hodgkin's lymphoma, multiple myeloma and leukemia.
[0153] Examples of antigens that may be delivered alone or in
combination to immune cells for antigen presentation using the
present invention includes tumor proteins, e.g., mutated oncogenes;
viral proteins associated with tumors; and tumor mucins and
glycolipids. The antigens may be viral proteins associated with
tumors would be those from the classes of viruses noted above.
Certain antigens may be characteristic of tumors (one subset being
proteins not usually expressed by a tumor precursor cell), or may
be a protein that is normally expressed in a tumor precursor cell,
but having a mutation characteristic of a tumor. Other antigens
include mutant variant(s) of the normal protein having an altered
activity or subcellular distribution, e.g., mutations of genes
giving rise to tumor antigens.
[0154] Specific non-limiting examples of tumor antigens for use in
an anti-CD40-fusion protein vaccine include, e.g., CEA, prostate
specific antigen (PSA), HER-2/neu, BAGE, GAGE, MAGE 1-4, 6 and 12,
MUC (Mucin) (e.g., MUC-1, MUC-2, etc.), GM2 and GD2 gangliosides,
ras, myc, tyrosinase, MART (melanoma antigen), Pmel 17(gp100),
GnT-V intron V sequence (N-acetylglucoaminyltransferase V intron V
sequence), Prostate Ca psm, PRAME (melanoma antigen),
.beta.-catenin, MUM-1-B (melanoma ubiquitous mutated gene product),
GAGE (melanoma antigen) 1, MAGE, BAGE (melanoma antigen) 2-10,
c-ERB2 (Her2/neu), DAGE, EBNA (Epstein-Barr Virus nuclear antigen)
1-6, gp75, human papilloma virus (HPV) E6 and E7, p53, lung
resistance protein (LRP), Bcl-2, Ki-67, Cyclin B1, gp100, Survivin,
and NYESO-1
[0155] In addition, the immunogenic molecule can be an autoantigen
involved in the initiation and/or propagation of an autoimmune
disease, the pathology of which is largely due to the activity of
antibodies specific for a molecule expressed by the relevant target
organ, tissue, or cells, e.g., SLE or MG. In such diseases, it can
be desirable to direct an ongoing antibody-mediated (i.e., a
Th2-type) immune response to the relevant autoantigen towards a
cellular (i.e., a Th1-type) immune response. Alternatively, it can
be desirable to prevent onset of or decrease the level of a Th2
response to the autoantigen in a subject not having, but who is
suspected of being susceptible to, the relevant autoimmune disease
by prophylactically inducing a Th1 response to the appropriate
autoantigen. Autoantigens of interest include, without limitation:
(a) with respect to SLE, the Smith protein, RNP ribonucleoprotein,
and the SS-A and SS-B proteins; and (b) with respect to MG, the
acetylcholine receptor. Examples of other miscellaneous antigens
involved in one or more types of autoimmune response include, e.g.,
endogenous hormones such as luteinizing hormone, follicular
stimulating hormone, testosterone, growth hormone, prolactin, and
other hormones.
[0156] Antigens involved in autoimmune diseases, allergy, and graft
rejection can be used in the compositions and methods of the
invention. For example, an antigen involved in any one or more of
the following autoimmune diseases or disorders can be used in the
present invention: diabetes, diabetes mellitus, arthritis
(including rheumatoid arthritis, juvenile rheumatoid arthritis,
osteoarthritis, psoriatic arthritis), multiple sclerosis,
myasthenia gravis, systemic lupus erythematosis, autoimmune
thyroiditis, dermatitis (including atopic dermatitis and eczematous
dermatitis), psoriasis, Sjogren's Syndrome, including
keratoconjunctivitis sicca secondary to Sjogren's Syndrome,
alopecia areata, allergic responses due to arthropod bite
reactions, Crohn's disease, aphthous ulcer, iritis, conjunctivitis,
keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma,
cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis,
drug eruptions, leprosy reversal reactions, erythema nodosum
leprosum, autoimmune uveitis, allergic encephalomyelitis, acute
necrotizing hemorrhagic encephalopathy, idiopathic bilateral
progressive sensorineural hearing loss, aplastic anemia, pure red
cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's
granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome,
idiopathic sprue, lichen planus, Crohn's disease, Graves
ophthalmopathy, sarcoidosis, primary biliary cirrhosis, uveitis
posterior, and interstitial lung fibrosis. Examples of antigens
involved in autoimmune disease include glutamic acid decarboxylase
65 (GAD 65), native DNA, myelin basic protein, myelin proteolipid
protein, acetylcholine receptor components, thyroglobulin, and the
thyroid stimulating hormone (TSH) receptor.
[0157] Examples of antigens involved in allergy include pollen
antigens such as Japanese cedar pollen antigens, ragweed pollen
antigens, rye grass pollen antigens, animal derived antigens such
as dust mite antigens and feline antigens, histocompatiblity
antigens, and penicillin and other therapeutic drugs. Examples of
antigens involved in graft rejection include antigenic components
of the graft to be transplanted into the graft recipient such as
heart, lung, liver, pancreas, kidney, and neural graft components.
The antigen may be an altered peptide ligand useful in treating an
autoimmune disease.
[0158] It will be appreciated by those of skill in the art that the
sequence of any protein effector molecule may be altered in a
manner that does not substantially affect the functional advantages
of the effector protein. For example, glycine and alanine are
typically considered to be interchangeable as are aspartic acid and
glutamic acid and asparagine and glutamine. One of skill in the art
will recognize that many different variations of effector sequences
will encode effectors with roughly the same activity as the native
effector. The effector molecule and the antibody may be conjugated
by chemical or by recombinant means as described above. Chemical
modifications include, for example, derivitization for the purpose
of linking the effector molecule and the antibody to each other,
either directly or through a linking compound, by methods that are
well known in the art of protein chemistry. Both covalent and
noncovalent attachment means may be used with the humanized
antibodies of the present invention.
[0159] The procedure for attaching an effector molecule to an
antibody will vary according to the chemical structure of the
moiety to be attached to the antibody. Polypeptides typically
contain a variety of functional groups; e.g., carboxylic acid
(COOH), free amine (--NH.sub.2) or sulfhydryl (--SH) groups, which
are available for reaction with a suitable functional group on an
antibody to result in the binding of the effector molecule.
Alternatively, the antibody can be derivatized to expose or to
attach additional reactive functional groups, e.g., by attachment
of any of a number of linker molecules such as those available from
Pierce Chemical Company, Rockford Ill.
[0160] The linker is capable of forming covalent bonds to both the
antibody and to the effector molecule. Suitable linkers are well
known to those of skill in the art and include, but are not limited
to, straight or branched-chain carbon linkers, heterocyclic carbon
linkers, or peptide linkers. Where the antibody and the effector
molecule are polypeptides, the linkers may be joined to the
constituent amino acids through their side groups (e.g., through a
disulfide linkage to cysteine). However, in a preferred embodiment,
the linkers will be joined to the alpha carbon amino and carboxyl
groups of the terminal amino acids.
[0161] In some circumstances, it is desirable to free the effector
molecule from the antibody when the immunoconjugate has reached its
target site. Therefore, in these circumstances, immunoconjugates
will comprise linkages that are cleavable in the vicinity of the
target site. Cleavage of the linker to release the effector
molecule from the antibody may be prompted by enzymatic activity or
conditions to which the immunoconjugate is subjected either inside
the target cell or in the vicinity of the target site. When the
target site is a tumor, a linker that is cleavable under conditions
present at the tumor site (e.g. when exposed to tumor-associated
enzymes or acidic pH) may be used.
[0162] Exemplary chemical modifications of the effector molecule
and the antibody of the present invention also include
derivitization with polyethylene glycol (PEG) to extend time of
residence in the circulatory system and reduce immunogenicity,
according to well known methods (See for example, Lisi, et al.,
Applied Biochem. 4:19 (1982); Beauchamp, et al., Anal Biochem.
131:25 (1982); and Goodson, et al., Bio/Technology 8:343
(1990)).
[0163] The present invention contemplates vaccines for use in both
active and passive immunization embodiments Immunogenic
compositions, proposed to be suitable for use as a vaccine, may be
prepared most readily directly from immunogenic T-cell stimulating
peptides prepared in a manner disclosed herein. The final
vaccination material is dialyzed extensively to remove undesired
small molecular weight molecules and/or lyophilized for more ready
formulation into a desired vehicle. In certain embodiment of the
present invention, the compositions and methods of the present
invention are used to manufacture a cellular vaccine, e.g., the
antigen-delivering anti-CD40 binding portion of the antibody is
used to direct the antigen(s) to an antigen presenting cell, which
then "loads" the antigen onto MHC proteins for presentation. The
cellular vaccine is, therefore, the antigen presenting cell that
has been loaded using the compositions of the present invention to
generate antigen-loaded antigen presenting cells.
[0164] When the vaccine is the anti-CD40 binding protein itself,
e.g., a complete antibody or fragments thereof, then these "active
ingredients" can be made into vaccines using methods understood in
the art, e.g., U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231;
4,599,230; and 4,578,770, relevant portions incorporated herein by
reference. Typically, such vaccines are prepared as injectables,
e.g., as liquid solutions or suspensions or solid forms suitable
for re-suspension in liquid prior to injection. The preparation may
also be emulsified. The active immunogenic ingredient is often
mixed with excipients that are pharmaceutically acceptable and
compatible with the active ingredient. Suitable excipients are, for
example, water, saline, dextrose, glycerol, ethanol, or the like
and combinations thereof. In addition, if desired, the vaccine may
contain minor amounts of auxiliary substances such as wetting or
emulsifying agents, pH buffering agents, or adjuvants that enhance
the effectiveness of the vaccines.
[0165] The vaccines are administered in a manner compatible with
the dosage formulation, and in such amount as will be
therapeutically effective and immunogenic. The quantity to be
administered depends on the subject to be treated, including, e.g.,
the capacity of the individual's immune system to generate an
immune response. Precise amounts of cells or active ingredient
required to be administered depend on the judgment of the
practitioner. However, suitable dosage ranges are of the order of a
few thousand cells (to millions of cells) for cellular vaccines.
For standard epitope or epitope delivery vaccines then the vaccine
may be several hundred micrograms active ingredient per
vaccination. Suitable regimes for initial administration and
booster shots are also variable, but are typified by an initial
administration followed by subsequent inoculations or other
administrations.
[0166] The manner of application may vary widely, however, certain
embodiments herein will most likely be delivered intravenously or
at the site of a tumor or infection directly. Regardless, any of
the conventional methods for administration of a vaccine are
applicable. The dosage of the vaccine will depend on the route of
administration and will vary according to the size of the host.
[0167] In many instances, it will be desirable to have multiple
administrations of the vaccine, e.g., four to six vaccinations
provided weekly or every other week. A normal vaccination regimen
will often occur in two to twelve week intervals or from three to
six week intervals. Periodic boosters at intervals of 1-5 years,
usually three years, may be desirable to maintain protective levels
of the immune response or upon a likelihood of a remission or
re-infection. The course of the immunization may be followed by
assays for, e.g., T cell activation, cytokine secretion or even
antibody production, most commonly conducted in vitro. These immune
response assays are well known and may be found in a wide variety
of patents and as taught herein.
[0168] The vaccine of the present invention may be provided in one
or more "unit doses" depending on whether the nucleic acid vectors
are used, the final purified proteins, or the final vaccine form is
used. Unit dose is defined as containing a predetermined-quantity
of the therapeutic composition calculated to produce the desired
responses in association with its administration, i.e., the
appropriate route and treatment regimen. The quantity to be
administered, and the particular route and formulation, are within
the skill of those in the clinical arts. The subject to be treated
may also be evaluated, in particular, the state of the subject's
immune system and the protection desired. A unit dose need not be
administered as a single injection but may include continuous
infusion over a set period of time. Unit dose of the present
invention may conveniently be described in terms of DNA/kg (or
protein/Kg) body weight, with ranges between about 0.05, 0.10,
0.15, 0.20, 0.25, 0.5, 1, 10, 50, 100, 1,000 or more mg/DNA or
protein/kg body weight are administered.
[0169] Likewise, the amount of anti-CD40-antigen vaccine delivered
can vary from about 0.2 to about 8.0 mg/kg body weight. Thus, in
particular embodiments, 0.4 mg, 0.5 mg, 0.8 mg, 1.0 mg, 1.5 mg, 2.0
mg, 2.5 mg, 3.0 mg, 4.0 mg, 5.0 mg, 5.5 mg, 6.0 mg, 6.5 mg, 7.0 mg
and 7.5 mg of the vaccine may be delivered to an individual in
vivo. The dosage of vaccine to be administered depends to a great
extent on the weight and physical condition of the subject being
treated as well as the route of administration and the frequency of
treatment. A pharmaceutical composition that includes a naked
polynucleotide prebound to a liposomal or viral delivery vector may
be administered in amounts ranging from 1 .mu.g to 1 mg
polynucleotide to 1 .mu.g to 100 mg protein. Thus, particular
compositions may include between about 1 .mu.g, 5 .mu.g, 10 .mu.g,
20 .mu.g, 30 .mu.g, 40 .mu.g, 50 .mu.g, 60 .mu.g, 70 .mu.g, 80
.mu.g, 100 .mu.g, 150 .mu.g, 200 .mu.g, 250 .mu.g, 500 .mu.g, 600
.mu.g, 700 .mu.g, 800 .mu.g, 900 .mu.g or 1,000 .mu.g
polynucleotide or protein that is bound independently to 1 .mu.g, 5
.mu.g, 10 .mu.g, 20 .mu.g, 3.0 .mu.g, 40 .mu.g 50 .mu.g, 60 .mu.g,
70 .mu.g, 80 .mu.g, 100 .mu.g, 150 .mu.g, 200 .mu.g, 250 .mu.g, 500
.mu.g, 600 .mu.g, 700 .mu.g, 800 .mu.g, 900 .mu.g, 1 mg, 1.5 mg, 5
mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg
or 100 mg vector.
[0170] Antibodies of the present invention may optionally be
covalently or non-covalently linked to a detectable label.
Detectable labels suitable for such use include any composition
detectable by spectroscopic, photochemical, biochemical,
immunochemical, electrical, optical or chemical methods. Useful
labels in the present invention include magnetic beads (e.g.,
DYNABEADS.RTM.), fluorescent dyes (e.g., fluorescein
isothiocyanate, Texas red, rhodamine, green fluorescent protein,
and the like), radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S, or
.sup.32P), enzymes (e.g., horse radish peroxidase, alkaline
phosphatase and others commonly used in an ELISA), and colorimetric
labels such as colloidal gold or colored glass or plastic (e.g.
polystyrene, polypropylene, latex, etc.) beads.
[0171] Methods of detecting such labels are well known to those of
skill in the art. Thus, for example, radiolabels may be detected
using photographic film or scintillation counters, fluorescent
markers may be detected using a photodetector to detect emitted
illumination. Enzymatic labels are typically detected by providing
the enzyme with a substrate and detecting the reaction product
produced by the action of the enzyme on the substrate, and
colorimetric labels are detected by simply visualizing the colored
label.
[0172] The antibody and/or immunoconjugate compositions of this
invention are particularly useful for parenteral administration,
such as intravenous administration or administration into a body
cavity. The compositions for administration will commonly comprise
a solution of the antibody and/or immunoconjugate dissolved in a
pharmaceutically acceptable carrier, preferably an aqueous carrier.
A variety of aqueous carriers can be used, e.g., buffered saline
and the like. These solutions are sterile and generally free of
undesirable matter. These compositions may be sterilized by
conventional, well-known sterilization techniques. The compositions
may contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions such as pH
adjusting and buffering agents, toxicity adjusting agents and the
like, for example, sodium acetate, sodium chloride, potassium
chloride, calcium chloride, sodium lactate and the like. The
concentration of fusion protein in these formulations can vary
widely, and will be selected primarily based on fluid volumes,
viscosities, body weight and the like in accordance with the
particular mode of administration selected and the patient's
needs.
[0173] Thus, a typical pharmaceutical immunoconjugate composition
of the present invention for intravenous administration would be
about 0.1 to 10 mg per patient per day. Dosages from 0.1 up to
about 100 mg per patient per day may be used. Actual methods for
preparing administrable compositions will be known or apparent to
those skilled in the art and are described in more detail in such
publications as REMINGTON'S PHARMACEUTICAL SCIENCE, 19TH ED., Mack
Publishing Company, Easton, Pa. (1995).
[0174] The compositions of the present invention can be
administered for therapeutic treatments. In therapeutic
applications, compositions are administered to a patient suffering
from a disease, in an amount sufficient to cure or at least
partially arrest the disease and its complications. An amount
adequate to accomplish this is defined as a "therapeutically
effective dose." Amounts effective for this use will depend upon
the severity of the disease and the general state of the patient's
health. An effective amount of the compound is that which provides
either subjective relief of a symptom(s) or an objectively
identifiable improvement as noted by the clinician or other
qualified observer.
[0175] Single or multiple administrations of the compositions are
administered depending on the dosage and frequency as required and
tolerated by the patient. In any event, the composition should
provide a sufficient quantity of the proteins of this invention to
effectively treat the patient. Preferably, the dosage is
administered once but may be applied periodically until either a
therapeutic result is achieved or until side effects warrant
discontinuation of therapy. Generally, the dose is sufficient to
treat or ameliorate symptoms or signs of disease without producing
unacceptable toxicity to the patient.
[0176] Controlled release parenteral formulations of the
immunoconjugate compositions of the present invention can be made
as implants, oily injections, or as particulate systems. For a
broad overview of protein delivery systems see, Banga, A. J.,
THERAPEUTIC PEPTIDES AND PROTEINS: FORMULATION, PROCESSING, AND
DELIVERY SYSTEMS, Technomic Publishing Company, Inc., Lancaster,
Pa., (1995) incorporated herein by reference. Particulate systems
include microspheres, microparticles, microcapsules, nanocapsules,
nanospheres, and nanoparticles. Microcapsules contain the
therapeutic protein as a central core. In microspheres the
therapeutic is dispersed throughout the particle. Particles,
microspheres, and microcapsules smaller than about 1 .mu.m are
generally referred to as nanoparticles, nanospheres, and
nanocapsules, respectively. Capillaries have a diameter of
approximately 5 .mu.m so that only nanoparticles are administered
intravenously. Microparticles are typically around 100 .mu.m in
diameter and are administered subcutaneously or
intramuscularly.
[0177] Polymers can be used for ion-controlled release of
immunoconjugate compositions of the present invention. Various
degradable and non-degradable polymeric matrices for use in
controlled drug delivery are known in the art (Langer, R., Accounts
Chem. Res. 26:537-542 (1993)). For example, the block copolymer,
poloxamer 407.RTM. exists as a viscous yet mobile liquid at low
temperatures but forms a semisolid gel at body temperature,
hydroxyapatite has been used as a microcarrier for controlled
release of proteins, and/or liposomes may be used for controlled
release as well as drug targeting of the lipid-capsulated drug.
Numerous additional systems for controlled delivery of therapeutic
proteins are known. See, e.g., U.S. Pat. Nos. 5,055,303, 5,188,837,
4,235,871, 4,501,728, 4,837,028 4,957,735 and 5,019,369, 5,055,303;
5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206,
5,271,961; 5,254,342 and 5,534,496, relevant portions of each of
which are incorporated herein by reference.
[0178] Among various uses of the immunoconjugates of the invention
are included a variety of disease conditions caused by specific
human cells that may be eliminated by the toxic action of the
fusion protein. For example, for the humanized anti-CD40_12E12.3F3
(ATCC Accession No. PTA-9854), anti-CD40_12B4.2C10 (ATCC Accession
No. PTA-10653, Submission No. AB13-22.12B4.2C10 (HS446)), and
anti-CD40_11B6.1C3 (ATCC Accession No. PTA-10652, Submission No.
AB13-22.11B6.1C3 (HS440)), antibodies disclosed herein, one
preferred application for immunoconjugates is the treatment of
malignant cells expressing CD40.
[0179] In another embodiment, this invention provides kits for the
delivery of antigens, e.g., CD40 or an immunoreactive fragment
thereof, conjugated or in the form of a fusion protein with one or
more T cell or B cell epitopes. A "biological sample" as used
herein is a sample of biological tissue or fluid that contains the
antigen. Such samples include, but are not limited to, tissue from
biopsy, blood, and blood cells (e.g., white cells). Preferably, the
cells are lymphocytes, e.g., dendritic cells. Biological samples
also include sections of tissues, such as frozen sections taken for
histological purposes. A biological sample is typically obtained
from a multicellular eukaryote, preferably a mammal such as rat,
mouse, cow, dog, guinea pig, or rabbit, and more preferably a
primate, such as a macaque, chimpanzee, or human Most preferably,
the sample is from a human. The antibodies of the invention may
also be used in vivo, for example, as a diagnostic tool for in vivo
imaging.
[0180] Kits will typically comprise a nucleic acid sequence that
encodes an antibody of the present invention (or fragment thereof)
with one or more framework portions or multiple cloning sites at
the carboxy-terminal end into which the coding sequences for one or
more antigens may be inserted. In some embodiments, the antibody
will be a humanized anti-CD40 Fv fragment, such as an scFv or dsFv
fragment. In addition the kits will typically include instructional
materials disclosing methods of use of an antibody of the present
invention (e.g. for loading into dendritic cells prior to
immunization with the dendritic cells, which can be autologous
dendritic cells). The kits may also include additional components
to facilitate the particular application for which the kit is
designed. Thus, for example, the kit may additionally contain
methods of detecting the label (e.g. enzyme substrates for
enzymatic labels, filter sets to detect fluorescent labels,
appropriate secondary labels such as a sheep anti-mouse-HRP, or the
like). The kits may additionally include buffers and other reagents
routinely used for the practice of a particular method. Such kits
and appropriate contents are well known to those of skill in the
art.
[0181] In another set of uses for the invention, immunoconjugates
targeted by antibodies of the invention can be used to purge
targeted cells from a population of cells in a culture. For
example, if a specific population of T cells is preferred, the
immunoconjugates of the present invention may be used to enrich a
population of T cells having the opposite effect of the on-going
immune response. Thus, for example, cells cultured from a patient
having a cancer can be purged of cancer cells by providing the
patient with dendritic cells that were antigen loaded using the
antibodies of the invention as a targeting moiety for the antigens
that will trigger an immune response against the cancer, virus or
other pathogen. Likewise, the immunoconjugates can be used to
increase the population of regulatory T cells or drive the immune
response toward or away from a cytotoxic T cell response or even
drive a B cell response.
Example 1: Anti-CD40-HIV Peptides Vaccine
[0182] Five 19- to 32-amino-acid long sequences were selected from
a multiplicity of cytotoxic T lymphocyte (CTL) epitopes identified
in the HIV-1 Nef, Gag and Env proteins in the context of different
MHC-class I molecules. It has been reported that CTL responses can
be induced efficiently by lipopeptide vaccines in mice, in
primates, and in humans. The five HIV peptides were then modified
in C-terminal position by a (Palm)-NH2 group and the five HIV
peptide sequences have been well described in the scientific
literature [e.g., Characterization of a multi-lipopeptides mixture
used as an HIV-1 vaccine candidate (1999) Klinguer et al., Vaccine,
Volume 18, 259-267] and in a patent application [Cytotoxic T
lymphocyte-inducing lipopeptides and use as vaccines. Gras-Masse H.
et al., Patent No. EP0491628 (1992-06-24); U.S. Pat. No. 5,871,746
(1999-02-16)].
[0183] A very desirable HIV vaccine would be composed of
recombinant anti-dendritic cell receptor antibody fused to the
above HIV peptides. The present invention includes compositions and
methods to efficiently produce proteins and HIV vaccines.
[0184] The sequences shown below are the amino-acid sequences of
the five selected HIV peptides and the amino-acid positions within
each HIV protein are in brackets.
TABLE-US-00001 Nef (66-97) is: (SEQ ID NO.: 1)
VGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGL Ncf (116-145) is: (SEQ ID NO.: 2)
HTQGYFPDWQNYTPGPGVRYPLTFGWLYKL Gag p17 (17-35) is: (SEQ ID NO.: 3)
EKIRLRPGGKKKYKLKHIV Gag p17-p24 (253-284) is: (SEQ ID NO.: 4)
NPPIPVGEIYKRWIILGLNKIVRMYSPTSILD Pol 325-355 (RT 158-188) is: (SEQ
ID NO.: 5) AIFQSSMTKILEPFRKQNPDIVIYQYMDDLY
[0185] The sequence below is a hIgG4 heavy chain (H)-HIV gag17
fusion protein where the Gag p17 (17-35) region is shown in bold.
The underlined AS residues are joining sequences.
TABLE-US-00002 [mAnti-DCIR_9E8_H-LV-hIgG4H-C-Pep-gag17] C655 is:
(SEQ ID NO.: 6) QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWL
AHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARS
SHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL
GKASEKIRLRPGGKKKYKLKHIVAS.
[0186] The sequence below is an H chain-HIV gag253 fusion protein
where the Gag p17-p24 (253-284) region is shown in bold. The
underlined AS residues are joining sequences.
TABLE-US-00003 [mAnti-DCIR_9E8_H-LV-hIgG4H-C-Pep-gag253] C656 is:
(SEQ ID NO.: 7) QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWL
AHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARS
SHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL
GKASNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDAS.
[0187] The sequence below is an H chain-HIV nef116 fusion protein
where the Nef (116-145) region is shown in bold. The underlined AS
residues are joining sequences.
TABLE-US-00004 [mAnti-DCIR_9E8_H-LV-hIgG4H-C-Pep-nef116] C680 is:
(SEQ ID NO.: 8) QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWL
AHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARS
SHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL
GKASHTQGYFPDWQNYTPGPGVRYPLTFGWLYKLAS.
[0188] The sequence below is a H chain-HIV nef66 fusion protein
where the Nef (66-97) region is shown shaded in bold. The
underlined AS residues are joining sequences.
TABLE-US-00005 [mAnti-DCIR_9E8_H-LV-hIgG4H-C-Pep-nef66] C679 is:
(SEQ ID NO.: 9) QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWL
AHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARS
SHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL
GKASVGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLAS.
[0189] The sequence below is a H chain-HIV pol158 fusion protein
where the Pol 325-355 (RT 158-188) region is shown in bold. The
underlined AS residues are joining sequences.
TABLE-US-00006 [mAnti-DCIR_9E8_H-LV-hIgG4H-C-Pep-pol158] C667 is:
(SEQ ID NO.: 10) QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWL
AHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARS
SHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL
GKASAIFQSSMTKILEPFRKQNPDIVIYQYMDDLYAS.
[0190] FIG. 1 shows protein A affinity purified recombinant
antibodies fused to various HIV peptides (lanes 1 to 5) secreted
from transfected 293F cells, analyzed by reducing SDS.PAGE and
Coomassie Brilliant Blue staining. Expression vectors for the H
chains fused to various C-terminal HIV peptides coding regions were
co-transfected with the matching light chain (L) plasmid into
transient 293F cells for three days before harvesting the
supernatant for subsequent purification. Cell number and DNA amount
were constant between transfections. Since the protein A affinity
matrix was used in excess, the SDS.PAGE analysis defines both the
production yield and the H chain integrity of the various vaccine
constructs. Lanes 1, 4 and 5 (upper bands) show that the H chains
fused directly to HIV gag17, nef66 and pol158 peptides can be
well-secreted. Lane 2 shows that the H chain fused directly to HIV
gag253 peptide expresses poorly. Lane 3 shows that the H chain
fused directly to HIV nef116 peptide is not expressed at all.
[0191] Surprisingly, it was found that the use of flexible
potentially glycosylated inter-peptide coding region linker
sequences improves the secretion of intact recombinant antibody-HIV
peptides fusion proteins.
[0192] The flexible linker sequences used are derived from
cellulosomal anchoring scaffoldin B precursor [Bacteroides
cellulosolvens] and have been described by the present inventors in
co-pending U.S. Patent Application Ser. No. 61/081,234, relevant
portions incorporated herein by reference.
[0193] The sequences shown below are the 25-amino-acid long
sequences of the four selected peptide linker sequences. The
underlined sequences are predicted N-linked glycosylation
sites.
TABLE-US-00007 Flex1 is: (SEQ ID NO.: 11) SSVSPTTSVHPTPTSVPPTPTKSSP
Flex2 is: (SEQ ID NO.: 12) PTSTPADSSTITPTATPTATPTIKG Flex3 is: (SEQ
ID NO.: 13) TVTPTATATPSAIVTTITPTATTKP Flex4 is: (SEQ ID NO.: 14)
TNGSITVAATAPTVTPTVNATPSAA
[0194] These sequences [the linkers shows in bold and underlined
regions obtained from cohesion] are derived from the inter-cohesin
domain spacers of the bacterial protein
>gi|50656899|gb|AAT79550.1| cellulosomal anchoring scaffoldin B
precursor [Bacteroides cellulosolvens]:
TABLE-US-00008 (SEQ ID NO.: 15)
MQSPRLKRKILSVILAVCYIISSFSIQFAATPQVNIIIGSAQGIPGSTVK
VPINLQNVPEIGINNCDFTIKFDSDILDFNSVEAGDIVPLPVASFSSNNS
KDIIKFLFSDATQGNMPINENGLFAVISFKIKDNAQKGISNIKVSSYGSF
SGMSGKEMQSLSPTFFSGSIDVSDVSTSKLDVKVGNVEGIAGTEVNVPIT
FENVPDNGINNCNFTLSYDSNALEFLTTEAGNIIPLAIADYSSYRSMEGK
IKFLFSDSSQGTRSIKNDGVFANIKFKIKGNAIRDTYRIDLSELGSFSSK
QNNNLKSIATQFLSGSVNVKDIESSVSPTTSVHPTPTSVPPTPTKSSPGN
KMKIQIGDVKANQGDTVIVPITFNEVPVMGVNNCNFTLAYDKNIMEFISA
DAGDIVTLPMANYSYNMPSDGLVKFLYNDQAQGAMSIKEDGTFANVKFKI
KQSAAFGKYSVGIKAIGSISALSNSKLIPIESIFKDGSITVTNKPIVNIE
IGKVKVKAGDKIKVPVEIKDIPSIGINNCNFTLKYNSNVLKYVSNEAGTI
VPAPLANLSINKPDEGIIKLLFSDASQGGMPIKDNGIFVNLEFQAVNDAN
IGVYGLELDTIGAFSGISSAKMTSIEPQFNNGSIEIFNSAQTPVPSNTEV
QTPTNTISVTPTNNSTPTNNSTPKPNPLYNLNVNIGEISGEAGGVIEVPI
EFKNVPDFGINNCDFSVKYDKSIFEYVTYEAGSIVKDSIVNLACMENSGI
INLLFNDATQSSSPIKNNGVFAKLKFKINSNAASGTYQINAEGYGKFSGN
LNGKLTSINPIFENGIINIGNVTVKPTSTPADSSTITPTATPTATPTIKG
TPTVTPIYWMNVLIGNMNAAIGEEVVVPIEFKNVPPFGINNCDFKLVYDS
NALELKKVEAGDIVPEPLANLSSNKSEGKIQFLFNDASQGSMQIENGGVF
AKITFKVKSTAASGIYNIRKDSVGSFSGLIDNKMTSIGPKFTDGSIVVGT
VTPTATATPSAIVTTITPTATTKPIATPTIKGTPTATPMYWMNVVIGKMN
AEVGGEVVVPIEFNNVPSFGINNCDFKLVYDATALELKNVEAGDIIKTPL
ANFSNNKSEEGKISFLFNDASQGSMQIENGGVFAKITFKVKSTTATGVYD
LRKDLVGSFSGLKDNKMTSIGAEFTNGSITVAATAPTVTPTVNATPSAAT
PTVTPTATATPSVTIPTVTPTATATPSVTIPTVTPTATATPSAATPTVTP
TATATPSVTIPTVTPTVTATPSDTIPTVTPTATATPSAIVTTITPTATAK
PIATPTIKGTPTATPMYWMNVVIGKMNAEVGGEVVVPIEFKNVPSFGINN
CDFKLVYDATALELKNVEAGDIIKTPLANFSNNKSEEGKISFLFNDASQG
SMQIENGGVSAKITFKVKSTTAIGVYDIRKDLIGSFSGLKDSKMTSIGAE
FTNGSITVATTAPTVTPTATATPSVTIPTVTPTATATPGTATPGTATPTA
TATPGAATPTETATPSVMIPTVTPTATATPTATATPTVKGTPTIKPVYKM
NVVIGRVNVVAGEEVVVPVEFKNIPAIGVNNCNFVLEYDANVLEVKKVDA
GEIVPDALINFGSNNSDEGKVYFLFNDALQGRMQIANDGIFANITFKVKS
SAAAGIYNIRKDSVGAFSGLVDKLVPISAEFTDGSISVESAKSTPTATAT
GTNVTPTVAATVTPTATPASTTPTATPTATSTVKGTPTATPLYSMNVIIG
KVNAEASGEVVVPVEFKDVPSIGINNCNFILEYDASALELDSAEAGEIVP
VPLGNFSSNNKDEGKIYFLFSDGTQGRMQIVNDGIFAKIKFKVKSTASDG
TYYIRKDSVGAFSGLIEKKIIKIGAEFTDGSITVRSLTPTPTVTPNVASP
TPTKVVAEPTSNQPAGPGPITGTIPTATTTATATPTKASVATATPTATPI
VVVEPTIVRPGYNKDADLAVFISSDKSRYEESSIITYSIEYKNIGKVNAT
NVKIAAQIPKFTKVYDAAKGAVKGSEIVWMIGNLAVGESYTKEYKVKVDS
LTKSEEYTDNTVTISSDQTVDIPENITTGNDDKSTIRVMLYSNRFTPGSH
SSYILGYKDKTFKPKQNVTRAEVAAMFARIMGLTVKDGAKSSYKDVSNKH
WALKYIEAVTKSGIFKGYKDSTFHPNAPITRAELSTVIFNYLHLNNIAPS
KVHFTDINKHWAKNYIEEIYRFKLIQGYSDGSFKPNNNITRAEVVTMINR
MLYRGPLKVKVGSFPDVSPKYWAYGDIEEASRNHKYTRDEKDGSEILIE.
[0195] The sequence below is a heavy chain (H)-HIV
gag17-nef66-nef116 peptides fusion protein where the HIV gag17,
nef66, nef116 peptide sequences are bold. The underlined AS
residues are joining sequences.
TABLE-US-00009 [mAnti-DCIR_9E8_H-LV-hIgG4H-C-gag17-nef66-nef116]
C694 is: (SEQ ID NO.: 16)
QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWL
AHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARS
SHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL
GKASEKIRLRPGGKKKYKLKHIVASVGFPVTPQVPLRPMTYKAAVDLSHF
LKEKGGLASHTQGYFPDWQNYTPGPGVRYPLTFGWLYKLAS.
[0196] The sequence below is an H chain-HIV gag17-nef116 peptides
fusion protein where the HIV gag17 and nef116 peptide sequences
[italics] are linked via a spacer f1 [shown in bold]. The
underlined AS residues are joining sequences.
TABLE-US-00010 [mAnti-DCIR_9E8_H-LV-hIgG4H-C-gag17-f1-nef116] C692
is: (SEQ ID NO.: 17)
QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWL
AHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARS
SHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL
GKASEKIRLRPGGKKKYKLKHIVASSSVSPTTSVHPTPTSVPPTPTKSSP
ASHTQGYFPDWQNYTPGPGVRYPLTFGWLYKLAS.
[0197] FIG. 2 shows protein A affinity purified recombinant
antibodies fused to various HIV peptides (Lanes 1 and 2) secreted
from transfected 293F cells, then analyzed by reducing SDS.PAGE and
Coomassie Brilliant Blue staining. Expression vectors for the H
chains fused to various C-terminal HIV peptides coding regions were
co-transfected with the matching L chain plasmid into transient
293F cells for three days before harvesting the supernatant for
subsequent purification. Lanes 1 and 2 (upper bands) show that the
H chains fused directly to a HIV peptide string of
gag17-nef66-nef116 can be well-secreted. Also the H chain product
containing a HIV peptide string of gag17 and nef116 separated by
the flexible spacer f1 (Lane 2) is also well expressed. Thus HIV
nef116 peptide, which is not expressed as a secreted product when
directly fused to the H chain alone, can be well-expressed when
appended in certain other peptide and flexible string contexts.
Note that the H chain fused directly to gag17-f1-nef116 [82
residues] migrates slower than H chain with gag17-nef66-nef116 [89
residues] this suggests that the flexible linker f1 is
glycosylated, possibly also enhancing the production of the
secreted gag17-f1-nef116 fusion antibody versus gag17-nef66-nef116
fusion antibody.
[0198] The sequence below is an H chain-HIV peptides string of
gag17-gag253-nef66 fusion protein where each HIV peptide sequence
[shaded in italics] is separated by a inter-peptide spacer f [shown
in bold]. In this case, a 27-amino-acid long linker flex-v1(v1)
[shown in bold italics] derived from cellulosomal anchoring
scaffoldin B precursor [Bacteroides cellulosolvens regions in
bold-italics-underlined] was inserted between the H chain
C-terminus and the HIV peptides-flexible spacers string. The
underlined AS residues are joining sequences.
TABLE-US-00011 [mAnti-DCIR_9E8_H-LV-hIgG4H-C-Flex-v1-Pep-gag17-
f1-gag253-f2-nef66] C711 is: (SEQ ID NO.: 18)
QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWL
AHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARS
SHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GKAS
ASEKIRLRPGGKKKYKL
KHIVASSSVSPTTSVHPTPTSVPPTPTKSSPASNPPIPVGEIYKRWHLGL
NKIVRMYSPTSILDASPTSTPADSSTITPTATPTATPTIKGASVGFPVTP
QVPLRPMTYKAAVDLSHFLKEKGGLAS.
[0199] The sequence below is an H chain-HIV peptides string of
pol158-gag17-nef66-nef116-gag253 fusion protein where peptide
sequences are shaded in grey. The underlined AS residues are
joining sequences.
TABLE-US-00012 [mAnti-DCIR_9E8_H-LV-hIgG4H-C-Pep-pol158-gag17-
nef66-nef116-gag253] C713 is: (SEQ ID NO.: 19)
QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWL
AHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARS
SHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL
GKASAIFQSSMTKILEPFRKQNPDIVIYQYMDDLYASEKIRLRPGGKKKY
KLKHIVASVGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLASHTQGYFPD
WQNYTPGPGVRYPLTFGWLYKLASNPPIPVGEIYKRWIILGLNKIVRMYS PTSILDAS.
[0200] FIG. 3 shows protein A affinity purified recombinant
antibodies fused to various HIV peptide strings (Lanes 1 to 5)
secreted from transfected 293F cells, then analyzed by reducing
SDS.PAGE and Coomassie Brilliant Blue staining. Expression vectors
for the H chains fused to various C-terminal HIV peptides coding
regions were co-transfected with the matching L chain plasmid into
transient 293F cells for three days before harvesting the
supernatant for subsequent purification. Lanes 1, 2 and 3 (upper
bands) show that the 4 HIV peptides-flexible spacers fused to H
chain via the flexible linker flex-v1 can be well-secreted.
However, a string of 4 HIV peptides fused directly to H chain is
not expressed at all (Lane 4, upper band). Also, lane 5 (upper
band) shows that a string of 5 HIV peptides fused directly to H
chain is not expressed at all. This result suggests that certain
combinations and contexts of flexible linkers and HIV peptide
coding sequences can enhance secretion of recombinant antibody-HIV
peptide fusion proteins (Lanes 1, 2 and 3).
[0201] The sequence below is for an H chain-HIV peptides string of
gag17-gag253-nef66-nef116-pol158 fusion protein where each HIV
peptide sequence [shaded in italics] is separated by an
inter-peptide spacer f [shown in bold]. The flexible linker flex-v1
(v1) [shown in bold-italics] was inserted between the H chain
C-terminus and the HIV peptides-flexible spacers string. The
underlined AS residues are joining sequences.
TABLE-US-00013 [mAnti-DCIR_9E8_H-LV-hIgG4H-C-hIgG4H-Flex-v1-Pep-
gag17-f1-gag253-f2-nef116-f3-nef66-f4-po1158] C825 is: (SEQ ID NO.:
20) QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWL
AHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARS
SHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GKAS
ASEKIRLRPGGKKKYKLKH
IVASSSVSPTTSVHPTPTSVPPTPTKSSPASNPPIPVGEIYKRWHLGLNK
IVRMYSPTSILDASPTSTPADSSTITPTATPTATPTIKGASHTQGYFPDW
QNYTPGPGVRYPLTFGWLYKLASTVTPTATATPSAIVTTITPTATTKPAS
VGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLASTNGSITVAATAPTVTP
TVNATPSAAASAIFQSSMTKILEPFRKQNPDIVIYQYMDDLYAS.
[0202] FIG. 4 shows protein A affinity purified recombinant
antibodies fused to various HIV peptide strings (Lanes 1 to 6)
secreted from transfected 293F cells, then analyzed by reducing
SDS.PAGE and Coomassie Brilliant Blue staining. Expression vectors
for the H chains fused to various C-terminal HIV peptides coding
regions were co-transfected with the matching L chain plasmid into
transient 293F cells for three days before harvesting the
supernatant for subsequent purification. Lanes 1, 3 and 5 (upper
bands) show that the string of 4 HIV peptides-flexible spacers
fused to H chain via the flexible linker flex-v1 can be
well-secreted. Lanes 2 and 6 (upper bands) show that the string of
5 HIV peptides-flexible spacers fused to H chain via the flexible
linker flex-v1 expresses poorly. However certain combinations and
contexts of HIV peptide coding sequences enhance secretion of
recombinant antibody-HIV peptide fusion proteins (Lanes 3 and 4).
Thus H chain fused to a string of 5 HIV peptides-flexible spacers
via the flexible linker flex-v1 can be well-expressed when appended
in certain other peptide and flexible string contexts (Lane 4).
[0203] The present invention includes compositions and methods for
flexible potentially glycosylated inter-peptide coding region
linker sequences and combinations of such HIV peptide coding
regions that are particularly favorable to efficient secretion of
recombinant anti-DC receptor antibody-HIV peptide fusion
vaccines.
[0204] The use of inter-structural domain linker sequences derived
from cellulose-degrading bacteria as preferred inter-domain linker
sequences in protein engineering--particularly those with highly
predicted glycosylation sites. Desirable properties of these
sequences are i) inherent flexibility, thereby facilitating
separation of linked domains which should greatly help their
correct folding and maintaining B cell receptor access to
conformationally-dependent antigen epitopes; ii) glycosylation,
thereby helping secretion and solubility of the intact produced
fusion protein, and also protecting of the linker sequences from
culture medium proteases.
[0205] Certain combinations of HIV peptide coding regions favor
secretion and that particular flexible linker sequences inserted
between the HIV peptide coding sequences can also help secretion of
intact HIV peptide string vaccines--principles that can also be
applied to solve similar issues for other preferred peptide
antigens.
[0206] DNA sequences of preferred linker and antigen coding
sequences. Joining sequence codons and stop codons are in bold:
TABLE-US-00014 [mAnti-DCIR_9E8_H-LV-hIgG4H-C-Pep-gag17] C655 is:
(SEQ ID NO.: 21) GCTAGTGAGAAGATCCGGCTGCGGCCCGGCGGCAAGAAGAAGTACAAGCT
GAAGCACATCGTGGCTAGCTGA [mAnti-DCIR_9E8_H-LV-hIgG4H-C-Pep-nef66]
C679 is: (SEQ ID NO.: 22)
GCTAGTGTGGGCTTCCCCGTGACCCCCCAGGTGCCCCTGCGGCCCATGAC
CTACAAGGCCGCCGTGGACCTGAGCCACTTCCTGAAGGAGAAGGGCGGCC TGGCTAGCTGA
[mAnti-DCIR_9E8_H-LV-hIgG4H-C-Pep-pol158] C667 is: (SEQ ID NO.: 23)
GCTAGTGCCATCTTCCAGAGCAGCATGACCAAGATCCTGGAGCCCTTCCG
GAAGCAGAACCCCGACATCGTGATCTACCAGTACATGGACGACCTGTACG CTAGCTGA
[mAnti-DCIR_9E8_H-LV-hIgG4H-C-Flex-v1-Pep-gag253] C681 is: (SEQ ID
NO.: 24) GCTAGTCAGACCCCCACCAACACCATCAGCGTGACCCCCACCAACAACAG
CACCCCCACCAACAACAGCAACCCCAAGCCCAACCCCGCTAGTAACCCCC
CCATCCCCGTGGGCGAGATCTACAAGCGGTGGATCATCCTGGGCCTGAAC
AAGATCGTGCGGATGTACAGCCCCACCAGCATCCTGGACGCTAGCTGA
[mAnti-DCIR_9E8_H-LV-hIgG4H-Flex-v1-Pep-gag17- nef116] C686 is:
(SEQ ID NO.: 25) GCTAGTCAGACCCCCACCAACACCATCAGCGTGACCCCCACCAACAACAG
CACCCCCACCAACAACAGCAACCCCAAGCCCAACCCCGCTAGTGAGAAGA
TCCGGCTGCGGCCCGGCGGCAAGAAGAAGTACAAGCTGAAGCACATCGTG
GCTAGTCACACCCAGGGCTACTTCCCCGACTGGCAGAACTACACCCCCGG
CCCCGGCGTGCGGTACCCCCTGACCTTCGGCTGGCTGTACAAGCTGGCTA GCTGA
[mAnti-DCIR_9E8_H-LV-hIgG4H-C-hIgG4H-Flex-v1-Pep-
gag17-f1-gag253-f2-nef116-f3-nef66-f4-pol158] C825 is: (SEQ ID NO.:
26) GCTAGTCAGACCCCCACCAACACCATCAGCGTGACCCCCACCAACAACAG
CACCCCCACCAACAACAGCAACCCCAAGCCCAACCCCGCTAGTGAGAAGA
TCCGGCTGCGGCCCGGCGGCAAGAAGAAGTACAAGCTGAAGCACATCGTG
GCTAGTAGCAGCGTGAGCCCCACCACCAGCGTGCACCCCACCCCCACCAG
CGTGCCCCCCACCCCCACCAAGAGCAGCCCCGCTAGTAACCCCCCCATCC
CCGTGGGCGAGATCTACAAGCGGTGGATCATCCTGGGCCTGAACAAGATC
GTGCGGATGTACAGCCCCACCAGCATCCTGGACGCTAGTCCCACCAGCAC
CCCCGCCGACAGCAGCACCATCACCCCCACCGCCACCCCCACCGCCACCC
CCACCATCAAGGGCGCTAGTCACACCCAGGGCTACTTCCCCGACTGGCAG
AACTACACCCCCGGCCCCGGCGTGCGGTACCCCCTGACCTTCGGCTGGCT
GTACAAGCTGGCTAGTACCGTGACCCCCACCGCCACCGCCACCCCCAGCG
CCATCGTGACCACCATCACCCCCACCGCCACCACCAAGCCCGCTAGTGTG
GGCTTCCCCGTGACCCCCCAGGTGCCCCTGCGGCCCATGACCTACAAGGC
CGCCGTGGACCTGAGCCACTTCCTGAAGGAGAAGGGCGGCCTGGCTAGTA
CCAACGGCAGCATCACCGTGGCCGCCACCGCCCCCACCGTGACCCCCACC
GTGAACGCCACCCCCAGCGCCGCCGCTAGTGCCATCTTCCAGAGCAGCAT
GACCAAGATCCTGGAGCCCTTCCGGAAGCAGAACCCCGACATCGTGATCT
ACCAGTACATGGACGACCTGTACGCTAGCTGA.
[0207] DNA sequences of preferred linker and antigen coding
sequences. Joining sequence codons are in bold:
TABLE-US-00015 Nef (66-97) is: (SEQ ID NO.: 27)
GCTAGTGTGGGCTTCCCCGTGACCCCCCAGGTGCCCCTGCGGCCCATGAC
CTACAAGGCCGCCGTGGACCTGAGCCACTTCCTGAAGGAGAAGGGCGGCC TGGCTAGC Nef
(116-145) is: (SEQ ID NO.: 28)
GCTAGTCACACCCAGGGCTACTTCCCCGACTGGCAGAACTACACCCCCGG
CCCCGGCGTGCGGTACCCCCTGACCTTCGGCTGGCTGTACAAGCTGGCTA GC Gag p17
(17-35) is: (SEQ ID NO.: 29)
GCTAGTGAGAAGATCCGGCTGCGGCCCGGCGGCAAGAAGAAGTACAAGCT
GAAGCACATCGTGGCTAGC Gag p17-p24 (253-284) is: (SEQ ID NO.: 30)
GCTAGTAACCCCCCCATCCCCGTGGGCGAGATCTACAAGCGGTGGATCAT
CCTGGGCCTGAACAAGATCGTGCGGATGTACAGCCCCACCAGCATCCTGG ACGCTAGC Pol
325-355 (RT 158-188) is: (SEQ ID NO.: 31)
GCTAGTGCCATCTTCCAGAGCAGCATGACCAAGATCCTGGAGCCCTTCCG
GAAGCAGAACCCCGACATCGTGATCTACCAGTACATGGACGACCTGTACG CTAGC Flex1 is:
(SEQ ID NO.: 32) GCTAGTAGCAGCGTGAGCCCCACCACCAGCGTGCACCCCACCCCCACCAG
CGTGCCCCCCACCCCCACCAAGAGCAGCCCCGCTAGC Flex2 is: (SEQ ID NO.: 33)
GCTAGTCCCACCAGCACCCCCGCCGACAGCAGCACCATCACCCCCACCGC
CACCCCCACCGCCACCCCCACCATCAAGGGCGCTAGC Flex3 is: (SEQ ID NO.: 34)
GCTAGTACCGTGACCCCCACCGCCACCGCCACCCCCAGCGCCATCGTGAC
CACCATCACCCCCACCGCCACCACCAAGCCCGCTAGC Flex4 is: (SEQ ID NO.: 35)
GCTAGTACCAACGGCAGCATCACCGTGGCCGCCACCGCCCCCACCGTGAC
CCCCACCGTGAACGCCACCCCCAGCGCCGCCGCTAGC
[0208] The present invention includes compositions and methods for
assembling constructs encoding HIV peptides and Flexible linker
sequences. The H chain expression vectors typically have a Nhe I
site [g|ctagc] appended to the H chain C-terminal residue codon, or
[for flex-v1 vectors] to the C-terminal codon of the flex-v1
sequence. Flexible linker sequences or HIV peptide sequences have
an Spe I site [a|ctagt] preceding the N-terminal flexible linker or
HIV peptide codon, a Nhe I site appended to the C-terminal flexible
linker or HIV peptide codon, followed by a TGA stop codon, followed
by a Eco RI site, followed by a Not I site. Such flexible linker or
HIV peptide Spe I-Not I fragments are inserted into the H chain
vector prepared with Nhe I-Not I digestion. Nhe I and Spe I are
compatible sites, but when ligated [g|ctagt] is no longer either a
Nhe I or Spe I site. Thus additional Spe I-Not I flexible linker or
HIV peptide fragments can be inserted into the new Nhe I-Not I
interval distal to the initial flexible linker or HIV peptide. In
this way, strings of HIV peptide and/or flexible linker coding
regions can be appended to the expression vector H chain coding
region.
Example 2. HIV Peptides Vaccine--In Vitro Antigen-Targeting
Biology
[0209] Anti-CD40.LIPO5 HIV peptides vaccine tests on HIV patients
in vitro. To study the ability of .alpha.CD40.LIPO5 HIV peptide
fusion recombinant antibody (.alpha.CD40.LIPO5 rAb) to mediate
antigen presentation, the fusion rAb was added to blood cells from
HIV-infected individuals and measured cytokine production form
peripheral blood mononuclear cells (PBMCs).
[0210] FIG. 5 describes the protocol used in vitro to assay the
potency of .alpha.CD40.LIPO5 HIV peptide fusion recombinant
antibody (.alpha.CD40.LIPO5 rAb) to elicit the expansion of
antigen-specific T cells in the context of a PBMC culture. Briefly,
PBMCs (2.times.10.sup.6 cells/ml) from apheresis of HIV patients
are incubated with a dose range of .alpha.CD40.LIPO5 HIV peptide
vaccine. On day 2, 100 U/ml IL-2 are added to the culture and then,
the media is refreshed every 2 days with 100 U/ml IL-2. On day 10,
the expanded cells are challenged for 48 h with the individual long
peptides corresponding to the 5 HIV peptide sequences incorporated
in the .alpha.CD40.LIPO5 HIV peptide fusion rAb. Then, culture
supernatants are harvested and assessed for cytokine production (by
the T cells with T cell receptor [TCR] specificities for peptide
sequences) using multiplex beads assay (Luminex). Antigen-specific
cytokine production detected in such an assay, if it depends on the
presence of the anti-CD40.LIPO5 HIV peptide vaccine, reflects
vaccine uptake by antigen presenting cells [APC] in the culture,
and processing [proteolytic degradation] and presentation of
peptides on MHC. The antigen-MHC complexes are recognized by T
cells with TCR that recognize only the particular HIV antigen-MHC
complex. In a HIV patient, such cells are likely to be memory T
cells that expanded in the patient in response to the HIV
infection.
[0211] Epitopes from all 5 HIV peptide regions of the vaccine can
be presented by APCs. The scheme in FIG. 5 was used to assay the in
vitro expansion of HIV peptide-specific T cells in response to
anti-CD40.LIPO5 peptide vaccine. Results from 7 individuals are
shown in FIGS. 6A-C and indicate that the .alpha.CD40.LIPO5 HIV
peptide fusion rAb elicited HIV peptide-specific IFN.gamma.
responses in all of the patients studied. Thus, the a-CD40.LIPO5
HIV peptide fusion rAb allows DCs to cross-present at least 1 or 2
different peptides out of the 5 peptides within the vaccine to the
T cells of each individual. However, the set of HIV peptides that
stimulated IFN.gamma. production was different for each
patient--most likely reflecting different pools of memory T cells
for HIV specificity.
[0212] FIGS. 6A-C show the HIV peptide-specific IFN.gamma.
production in PBMCs from HIV patients incubated with various
concentrations of anti-CD40.LIPO5 peptide string vaccine. C is the
control group, which received no vaccine, and defines the baseline
response of the culture to each peptide.
[0213] FIG. 7 is a summary of .alpha.CD40.LIPO5 peptide vaccine
responses against the 5 peptide regions from 8 HIV patients. The
data are based on peptide-specific IFN.gamma. production. FIG. 7
shows that the antigen-specific responses observed in 8 HIV
patients. The data demonstrate that all HIV peptide regions on the
vaccine have the capacity to be processed and presented to T
cells--assuming the likely situation that responses to these
peptides will only be observed if the appropriate TCR-bearing cells
are present. Thus, each patient has a characteristic spectrum of
such cells.
[0214] The .alpha.CD40.LIPO5 peptide vaccine can evoke the
proliferation of antigen-specific T cells capable of secreting a
wide spectrum of cytokines
[0215] FIGS. 8A-B show that .alpha.CD40.LIPO5 HIV peptide vaccine
elicits expansion of HIV peptide-specific T cells capable of
secreting multiple cytokines--a desirable feature in a vaccine. In
FIGS. 8A-B .alpha.CD40.LIPO5 HIV peptide vaccine elicits gag253,
nef66, nef116 and pol325 peptide-specific responses characterized
by production of multiple cytokines. This is patient A5.
[0216] Anti-CD40.LIPO5 HIV peptide vaccination of ex vivo DCs.
[0217] FIG. 9 shows the protocol for testing .alpha.CD40.LIPO5 HIV
peptide vaccine for its ability to direct the expansion of
antigen-specific T cells resulting from targeted uptake by DCs and
presentation of peptide epitopes on their surface MHC complex.
Briefly, HIV patient monocytes are differentiated into DCs by
culture for 2 days with IFN.alpha. and GM-CSF. Different doses
.alpha.CD40.LIPO5 HIV peptide vaccine or a mix of the 5 peptides
are then added for 18 h. Autologous T cells were added to the
co-culture (at a ratio of 1:20) on day 3. On day 5, 100 U/ml IL-2
are added to the culture and then, the media is refreshed every 2
days with 100 U/ml IL-2. On day 10, the expanded cells are
rechallenged for 48 h with the individual long peptides
corresponding to the 5 HIV peptide sequences incorporated in the
.alpha.CD40.LIPO5 HIV peptide fusion rAb. Then, culture
supernatants are harvested and assessed for cytokine production
using Luminex.
[0218] FIGS. 10A-B show the cytokine secretion in response to HIV
peptides from DC-T cell co-cultures treated with various doses of
.alpha.CD40.LIPO5 HIV peptide vaccine. This is patient A10. The
results in the patient A10 shown in FIGS. 10A-B demonstrate
expansion of antigen-specific T cells corresponding to epitopes
within the gag17, gag253, and pol325 HIV peptide regions. In most
instances, there is concordance of responses between
.alpha.CD40.LIPO5 HIV peptide vaccine and non-LIPO5 vaccine
[mixture of 5 non-lipidated HIV peptides with sequences
corresponding to those in the .alpha.CD40.LIPO5 HIV peptide
vaccine]. Thus, the .alpha.CD40.LIPO5 HIV peptide vaccine functions
well in this in vitro setting where cultured DCs effectively
process and present the HIV antigens to T cells. This exemplifies
use of the .alpha.CD40.LIPO5 HIV peptide vaccine for ex vivo
vaccination, whereby the `vaccinated DCs` would be cryopreserved
for future re-injection into the same patient.
[0219] .alpha.CD40.LIPO5 HIV peptide vaccine--possible immune
effect of the flexible linker regions. It is possible that the
flexible linker sequences interspersing the HIV peptide sequences
within the .alpha.CD40.LIPO5 HIV peptide vaccine themselves contain
T cell epitopes. FIGS. 11A-B show that patient A4 does not appear
to have a significant pool of memory T cells with specificities to
the five flexible linker sequences within .alpha.CD40.LIPO5 HIV
peptide vaccine. In FIGS. 11A-B, PBMCs from patient A4 treated with
the .alpha.CD40.LIPO5 HIV peptide vaccine elicit expansion of
antigen-specific T cells with specificity to the gag253 region, but
not to the flexible linker sequences. The protocol describe in FIG.
9 was used, with the flexible linker long peptides corresponding in
sequence to the bold areas, the HIV peptides are in bold-italics,
shown in the sequence below.
[0220] .alpha.CD40.LIPO5 HIV peptide vaccine heavy chain sequence
showing flexible linker regions in bold, joining sequences
underlined and HIV peptide regions shaded in bold italics.
TABLE-US-00016 (SEQ ID NO.: 36)
QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKS
RLTISKDTSSNQVFLKITIVDTADAATYYCARSSHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPS
VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS
SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTIS
VTPTNNSTPTNNSNPKPNPAS ASSSVSPTTSVHPTPTSVPPTPTKSSP AS
ASPTSTPADSSTITPTATPTATPTIKGAS ASTVTPTATATPSAIVTTITPTATTKPAS
ASTNGSITVAATAPTVTPTVNATPSAAAS AS..
[0221] In FIG. 12A, the PBMCs from patient A3 treated with the
.alpha.CD40.LIPO5 HIV peptide vaccine elicit expansion of
antigen-specific T cells with specificities to the gag253, nef66,
and nef116 regions, but not to the flexible linker sequences. The
protocol described in FIG. 1 was used, with the flexible linker
long peptides corresponding in sequence to the bold areas shown in
FIGS. 8A-B.
[0222] FIGS. 12B and 12C show HIV antigen-specific T cell responses
evoked from HIV patient A17 PBMCs incubated with 30 nM of three
different HIV5 peptide DC targeting vaccines. Cells were cultured
for 10 days with IL-2 and then stimulated with individual long
peptides corresponding to the 5 HIV peptide sequences encompassed
within the DC-targeting vaccines. After 1 hr brefeldin A was added
and incubation continued for a further 5 hrs before staining for
FACS analysis. The FACS plots show IFN.gamma. and CD8 staining on
CD3+ T cells. Circles indicate significant vaccine-evoked expansion
of IFN.gamma.+ cells compared to cells from PBMCs cultured without
vaccine. CD8- cells are CD4+ T cells. The data show that that
anti-CD40.HIV5pep vaccine evokes a strong expansion of nef66
(N66)-specific CD8+ T cells which is not seen with the other DC
targeting vehicles.
[0223] These are data based on the LIPO5 HIV peptide string. For
example the anti-CD40 H chain is
anti-CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-v1-Pep-gag17-f1-gag253-f2-nef116-f-
3-nef66-f4-pol158] with sequence:
TABLE-US-00017 (SEQ ID NO.: 37)
EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAY
INSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRG
LPFHAMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTY
TCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL
PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQT
PTNTISVTPTNNSTPTNNSNPKPNPASEKIRLRPGGKKKYKLKHIVASSS
VSPTTSVHPTPTSVPPTPTKSSPASNPPIPVGEIYKRWIILGLNKIVRMY
SPTSILDASPTSTPADSSTITPTATPTATPTIKGASHTQGYFPDWQNYTP
GPGVRYPLTFGWLYKLASTVTPTATATPSAIVTTITPTATTKPASVGFPV
TPQVPLRPMTYKAAVDLSHFLKEKGGLASTNGSITVAATAPTVTPTVNAT
PSAAASAIFQSSMTKILEPFRKQNPDIVIYQYMDDLYAS.
[0224] FIGS. 12D and 12E is a similar study to that show in FIGS.
12B and 12C, except that the PBMCs are from a different HIV patient
(A2). The data show antigen-specific CD4+ and CD8+ T cell responses
evoked by anti-CD40.HIV5pep but not the other DC-targeting
vaccines, or by a mixture of the peptides themselves.
[0225] FIG. 12F shows that, based on analysis of 15 different HIV
peptide responses [5 peptide regions sampled in 3 patients],
anti-CD40.HIV5pep vaccine is clearly superior to anti-DCIR.HIV5pep,
anti-LOX-1.HIV5pep and non-LIPO5 mix for eliciting a broad range of
HIV peptide-specific CD8+ and CD4+ T responses.
[0226] The immunogenicity of the flexible linker sequences is of
concern for the .alpha.CD40.LIPO5 HIV peptide vaccine design. The
limited datasets shown above, testing recall of T cells with
specificities for epitopes within the flexible linker sequences,
suggest that the human repertoire against these sequences is
variable. Also, the ability of these sequences to prime responses
de novo is untested. Responses to the .alpha.CD40.LIPO5 HIV peptide
vaccine in monkeys can be tested using the present invention. If
necessary, certain less desirable epitopes within these regions can
be identified by a combination of predictive computational means
and peptide stimulation scans, and then eliminated by introducing
mutational changes that abrogate the TCR interaction.
[0227] The anti-CD40 binding molecule includes a light chain having
the following amino acid sequence (SEQ ID NO. 38). The variable
region of the antibody light chain is underlined and the CDRs are
bolded (SEQ ID NOS.: 42, 43 and 44, respectively).
TABLE-US-00018 (SEQ ID NO.: 38)
MMSSAQFLGLLLLCFQGTRCDIQMTQTTSSLSASLGDRVTISCSASQGIS
NYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTIGNLEP
EDIATYYCQQFNKLPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA
SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.
[0228] The anti-CD40 binding molecule includes a heavy chain having
the following sequence. The variable region of the antibody light
chain is underlined and the CDRs are bolded (SEQ ID NOS.: 45, 46
and 47, respectively).
TABLE-US-00019 (SEQ ID NO.: 39)
MNLGLSLIFLVLVLKGVQCEVKLVESGGGLVQPGGSLKLSCATSGFTFSD
YYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYL
QMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKGPSVFPLA
PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP
EFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGV
EVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI
EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL
HNHYTQKSLSLSLGKAS.
[0229] In one aspect the nucleic acid that encodes the light chain
comprises the SEQ ID NO. The variable region of the antibody light
chain nucleic acid sequence is underlined and the CDRs are
bolded.
TABLE-US-00020 (SEQ ID NO.: 40)
ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGG
TACCAGATGTGATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCT
CTCTAGGAGACAGAGTCACCATCAGTTGCAGTGCAAGTCAGGGCATTAGC
AATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCT
GATCTATTACACATCAATTTTACACTCAGGAGTCCCATCAAGGTTCAGTG
GCAGTGGGTCTGGGACAGATTATTCTCTCACCATCGGCAACCTGGAACCT
GAAGATATTGCCACTTACTATTGTCAGCAGTTTAATAAGCTTCCTCCGAC
GTTCGGTGGAGGCACCAAACTCGAGATCAAACGAACTGTGGCTGCACCAT
CTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCC
TCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACA
GTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCA
CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACG
CTGAGCAAAGCAGACTACGAGAAACACAAAGTCTATGCCTGCGAAGTCAC
CCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GTTAG.
[0230] In one aspect the nucleic acid that encodes the heavy chain
comprises the SEQ ID NO.:40. The variable region of the antibody
heavy chain nucleic acid sequence is underlined and the CDRs are
bolded.
TABLE-US-00021 (SEQ ID NO.: 41)
ATGAACTTGGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGT
CCAGTGTGAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTG
GAGGGTCCCTGAAACTCTCCTGTGCAACCTCTGGATTCACTTTCAGTGAC
TATTACATGTATTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGT
CGCATACATTAATTCTGGTGGTGGTAGCACCTATTATCCAGACACTGTAA
AGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTG
CAAATGAGCCGGCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAG
ACGGGGGTTACCGTTCCATGCTATGGACTATTGGGGTCAAGGAACCTCAG
TCACCGTCTCCTCAGCCAAAACGAAGGGCCCATCCGTCTTCCCCCTGGCG
CCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGT
CAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCC
TGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTC
TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAA
GACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACA
AGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCT
GAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGA
CACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACG
TGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTG
GAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCAC
GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACG
GCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATC
GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTA
CACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGA
CCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAG
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA
CTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCA
GGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTG
CACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAG CTGA.
[0231] A humanized antibody includes the heavy chain variable
region (V.sub.H) and a light chain variable region (V.sub.L),
wherein the framework regions of the heavy chain and light chain
variable regions are from a donor human antibody, and wherein the
light chain complementarity determining regions (CDRs) have at
least 80%, 90%, 95% or higher identity to CDR1.sub.L having the
amino acid sequence SASQGISNYLN (SEQ ID NO.:41), the CDR2.sub.L
having the amino acid sequence YTSILHS (SEQ ID NO.:42) and the
CDR3.sub.L having the amino acid sequence QQFNKLPPT (SEQ ID
NO.:43); and wherein the heavy chain complementarity determining
regions comprise at least 80%, 90%, 95% or higher identity to the
CDR1.sub.H, CDR2.sub.H and CDR3.sub.H, the CDR1.sub.H having the
amino acid sequence GFTFSDYYMY (SEQ ID NO.:45), the CDR2.sub.H
having the amino acid sequence YINSGGGSTYYPDTVKG (SEQ ID NO.:46),
and the CDR3.sub.H having the amino acid sequence RGLPFHAMDY (SEQ
ID NO.:47). For example, the humanized antibody may comprise a VL
framework having at least 95% identity to the framework of SEQ ID
NO.:38 and a VH framework that has at least 95% identity to the
framework of SEQ ID NO.:39. In another aspect, the donor CDR
sequences are from ANTI-CD40_12E12.3F3 and further, wherein the
antibody or fragment thereof specifically binds to CD40.
Example 3. Prostate-Specific Antigen (PSA), Cycline D1, MART-1,
Influenza Viral Nucleoprotein (NP) and HA1 Subunit of Influenza
Viral Hemagglutinin (H1N1, PR8) and Peptide Screen
[0232] Internalization of anti-CD40 mAb. 1.times.10.sup.6 IL-4DCs
were incubated for 1 h in ice with 3 mg/ml human gamma globulin in
PBS containing 3% BSA to block non-specific binding. Cells were
pulsed for 30 minutes on ice with Alexa 568 labeled anti-CD40 mAb
(all at 20 ng/ml final concentration in non-specific block). Cells
were then washed and allowed to internalize surface bound
antibodies for different times, between 0 and 90 minutes, at
37.degree. C. Following internalization, cells were washed twice
with ice-cold PBS containing 1% BSA and 0.05% sodium azide (PBA)
and fixed in ice-cold 1% methanol-free formaldehyde (MFF) in PBS
overnight at 4.degree. C. Cells were permeablized in PBS 3% BSA
containing 0.5% saponin (PBAS) for 20 minutes at 4.degree. C., and
transferred to a 96-well round bottom polypropylene microtiter
plate. After washing twice with ice-cold PBAS, cells were incubated
for 1 h on ice with 3 mg/ml human gamma globulin in PBAS.
BODIPY-phalloidin diluted in PBAS and incubated with cells for 1
hour in ice. Cells were further stained with TOPRO-II, as a nuclear
counterstain. Slides were imaged on a Leica SP1 confocal
microscope.
[0233] Cells. Monoclonal antibodies for cell surface staining were
purchased from BD Biosciences (CA). Monocytes (1.times.10.sup.6/ml)
from healthy donors were cultured in Cellgenics media (France)
containing GM-CSF (100 ng/ml) and IL-4 (50 ng/ml) or GM-CSF (100
ng/ml) and IFN.alpha. (500 Units/ml) (R&D, CA). For IFNDCs,
cells were fed on day 1 with IFN.alpha. and GM-CSF. For IL-4DCs,
the same amounts of cytokines were supplemented into the media on
day one and day three. PBMCs were isolated from Buffy coats using
Percoll.TM. gradients (GE Healthcare, Buckinghamshire, UK) by
density gradient centrifugation. Total CD4+ and CD8+ T cells were
purified by using StemCell kits (CA).
[0234] Peptides. 15-mers (11 amino acid overlapping) for
prostate-specific antigen (PSA), Cycline D1, MART-1, influenza
viral nucleoprotein (NP) and HA1 subunit of influenza viral
hemagglutinin (H1N1, PR8), were synthesized (Mimotopes).
[0235] DCs and T cell co-culture and cytokine expressions.
5.times.10.sup.3 DCs loaded with recombinant fusion proteins
(anti-CD40-HA1, Control Ig-HA1, anti-CD40-PSA, anti-CD40-Cyclin D1,
anti-CD40-MART-1, anti-MARCO-MART-1, and control Ig-MART-1) were
co-cultured with 2.times.10.sup.5 CFSE-labeled CD4+ T cells for 8
days. Proliferation was tested by measuring CFSE dilution after
staining cells with anti-CD4 antibody labeled with APC.
[0236] For measuring the expression of intracellular IFN.gamma.,
CD4+ T cells were restimulated with 1-5 uM of indicated peptides
for 5h in the presence of Brefeldin A. In separate experiments,
CD4+ T cells were restimulated with peptides indicated for 36h, and
then cytokines secreted by CD4+ T cells were measured by the
Luminex.
[0237] CD8+ T cells were co-cultured with DCs for 10 days in the
presence of 20 units/ml IL-2 and 20 units/ml IL-7. On day 10 of the
culture, CD8+ T cells were stained with anti-CD8 and tetramers
indicated.
[0238] CTL assay. On day 10 of the culture, a 5-h .sup.51Cr release
assay was performed. T2 cells pulsed with .sup.51Cr first and then
labeled with 10 uM HLA-A2 epitope of MART-1 or 1 nM epitope of
influenza viral M1. T2 cells without peptide were used as control.
The mean of triplicate samples was calculated, and the percentage
of specific lysis was determined using the following formula:
percentage of specific lysis=100.times.(experimental .sup.51Cr
release-control .sup.51Cr release)/(maximum .sup.51Cr
release-control .sup.51Cr release). The maximum release refers to
counts from targets in 2.5% Triton X-100.
[0239] Preparation of mAbs specific for human CD40. Receptor
ectodomain.hIgG (human IgG1Fc) and AP (human placental alkaline
phosphatase) fusion proteins were produced for immunizing mice and
screening mAbs, respectively. A mammalian vector for human IgFc
fusion proteins was engineered as described [J. Immunol. 163:
1973-1983 (1999)]. The mammalian expression vector for receptor
ectodomain.AP proteins was generated using PCR to amplify cDNA for
AP resides 133-1581 (gb|BC0096471) while adding a proximal in-frame
Xho I site and a distal 6C-terminal His residues followed by a TGA
stop codon and Not I site. This Xho I-Not I fragment replaced the
human IgG Fc coding sequence in the above ectodomain.IgG vector.
Fusion proteins were produced using the FreeStyle.TM. 293
Expression System (Invitrogen, CA) according to the manufacturer's
protocol (1 mg total plasmid DNA with 1.3 ml 293Fectin reagent/L of
transfection). Receptor ectodomain hIgG was purified by 1 ml HiTrap
protein A affinity chromatography (GE Healthcare, CA) eluted with
0.1 M glycine, pH 2.7. Fractions were neutralized with 2M Tris, and
then dialyzed against PBS.
[0240] Mouse mAbs were generated by conventional technology.
Briefly, six-week-old BALB/c mice were immunized i.p. with 20 .mu.g
of receptor ectodomain hIgGFc fusion protein with Ribi adjuvant,
then boosted with 20 .mu.g antigen ten days and fifteen days later.
After three months, the mice were boosted again three days prior to
taking the spleens. Three to four days after a final boosting,
draining lymph nodes (LN) were harvested. B cells from spleen or LN
cells were fused with SP2/O-Ag 14 cells (ATCC). Hybridoma
supernatants were screened to analyze mAbs specific to the receptor
ectodomain fusion protein compared to the fusion partner alone, or
to the receptor ectodomain fused to alkaline phosphatase [J.
Immunol. 163: 1973-1983 (1999)]. Positive wells were then screened
in FACS using 293F cells transiently transfected with expression
plasmids encoding full-length receptor cDNAs. Selected hybridomas
were single cell cloned and expanded in CELLine flasks (Integra,
CA). Hybridoma supernatants were mixed with an equal volume of 1.5
M glycine, 3 M NaCl, 1.times.PBS, pH 7.8 (binding buffer) and
tumbled with MabSelect resin (GE Healthcare, CA) (800 .mu.l/5 ml
supernatant). The resin was washed with binding buffer and eluted
with 0.1 M glycine, pH 2.7. Following neutralization with 2 M Tris,
mAbs were dialyzed against PBS.
[0241] Expression and purification of recombinant mAbs. Total RNA
was prepared from hybridoma cells using RNeasy kit (Qiagen, CA) and
used for cDNA synthesis and PCR (SMART RACE kit, BD Biosciences)
using supplied 5' primers and gene specific 3' primers
(mIgG.kappa., 5' ggatggtgggaagatggatacagttggtgcagcatc3' (SEQ ID
NO.:48); mIgG2a, 5'ccaggcatcctagagtcaccgaggagccagt3') (SEQ ID
NO.:49). PCR products were then cloned (pCR2.1 TA kit, Invitrogen)
and characterized by DNA sequencing (MC Lab, CA). Using the derived
sequences for the mouse heavy (H) and light (L) chain variable
(V)-region cDNAs, specific primers were used to PCR amplify the
signal peptide and V-regions while incorporating flanking
restriction sites for cloning into expression vectors encoding
downstream human IgG.kappa. or IgG4H regions. The vector for
expression of chimeric mV.kappa.-hIg.kappa. was built by amplifying
residues 401-731 (gi|63101937|) flanked by Xho I and Not I sites
and inserting this into the Xho I-Not I interval of pIRES2-DsRed2
(BD Biosciences). PCR was used to amplify the mAb V.kappa. region
from the initiator codon, appending a Nhe I or Spe I site then
CACC, to the region encoding (e.g., residue 126 of gi|76779294|),
appending a distal Xho I site. The PCR fragment was then cloned
into the Nhe I-Not I interval of the above vector. The control
human IgG.kappa. sequence corresponds to gi|49257887| residues
26-85 and gi|21669402| residues 67-709. The control human IgG4H
vector corresponds to residues 12-1473 of gi|19684072| with S229P
and L236E substitutions, which stabilize a disulphide bond and
abrogate residual FcR interaction [J. Immunol. 164: 1925-1933
(2000)], inserted between the Bgl II and Not I sites of
pIRES2-DsRed2 while adding the sequence 5'gctagctgattaattaa 3'
instead of the stop codon. PCR was used to amplify the mAb VH
region from the initiator codon, appending CACC then a Bgl II site,
to the region encoding residue 473 of gi|19684072|. The PCR
fragment was then cloned into the Bgl II-Apa I interval of the
above vector.
[0242] Expression and purification of Flu HA1 fusion protein. The
Flu HA1 antigen coding sequence is a CipA protein [Clostridium.
thermocellum] gi|479126| residues 147-160 preceding hemagglutinin
[Influenza A virus (A/Puerto Rico/8/34(H1N1))] gi|126599271|
residues 18-331 with a P321L change and with 6 C-terminal His
residues was inserted between the H chain vector Nhe I and Not I
sites to encode recombinant antibody-HA1 fusion proteins (rAb.HA1).
Similarly, recombinant antibody-PSA fusion proteins (rAb.PSA) were
encoded by inserting gi|34784812| prostate specific antigen
residues 101-832 with proximal sequence
GCTAGCGATACAACAGAACCTGCAACACCTACAACACCTGTAACAACACCGACAACAACACTT
CTAGCGC (SEQ ID NO.:50) (Nhe I site and CipA spacer) and a distal
Not I site into the same H chain vector. Recombinant antibody
proteins were expressed and purified as described above for hFc
fusion proteins. In some cases the rAb.antigen coding region and
the corresponding L chain coding region were transferred to
separate cetHS-puro UCOE vectors (Millipore, CA). The use of UCOE
vectors in combination with a preadapted serum free, suspension
cell line allowed for rapid production of large quantities of
protein [Cytotechnology 38, 43-46 (2002).] CHO-S cells grown in
CD-CHO with GlutaMAX and HT media supplement (Invitrogen) were
seeded at 5.times.10.sup.5 ml 24h prior to transfection in 500 ml
Corning Ehrlenmyer flasks and incubated in 8% CO.sub.2 at 125 rpm.
On the day of transfection, 1.2.times.10.sup.7 cells with viability
at least 95% were added to a final volume of 30 ml in a 125 ml
flask in CD-CHO with GlutaMAX. 48 .mu.l of FreeStyle Max reagent
(Invitrogen) in 0.6 ml of OptiPRO SFM (Invitrogen) was added with
gentle mixing to 24 .mu.g of Sce I-linearized light chain vector
and 24 .mu.g of Sce I-linearized H chain vector mixed and sterile
filtered in 0.6 ml of OptiPRO SFM. After 20 min, the DNA-lipid
complex was slowly added to the 125 ml CHO-S culture flask with
swirling. Cells were incubated 24h before adding 30 ml of a
combined media solution of CD-CHO with CHO-M5 (Sigma, C0363
component of CHO Kit 1) containing 5 .mu.g/ml of puromycin (A.G.
Scientific, CA), 2.times.GlutaMAX and 0.25.times.Pen/Strep
(Invitrogen). At day 2, another 5 .mu.g/ml of puromycin was added
directly to the culture and selection was allowed to proceed
.about.10-14 days while following cell viability from six days post
transfection. The viable cell count dropped and when the viable
density is .about.2-3.times.10.sup.6/ml, the cells were transferred
to fresh selection medium (CD CHO-S+CHO M5 with 2.times. GlutaMAX,
0.25.times.Pen/Strep, 10 .mu.g/ml Puromycin) at 1 E6/ml. Frozen
cell stocks were prepared when viability reached >90%. Cells
were split in selection medium when cell density exceeded
2.times.10.sup.6/ml until scaled to 4.times.250 ml in 500 ml
flasks. Supernatant was harvested when cell viability dropped below
80% with a maximum final cell density .about.7.times.10.sup.6/ml.
Endotoxin levels were less than 0.2 units/ml.
[0243] Expression and purification of recombinant Flu M1 and MART-1
proteins. PCR was used to amplify the ORF of Influenza A/Puerto
Rico/8/34/Mount Sinai (H1N1) M1 gene while incorporating an Nhe I
site distal to the initiator codon and a Not I site distal to the
stop codon. The digested fragment was cloned into pET-28b(+)
(Novagen), placing the M1 ORF in-frame with a His6 tag, thus
encoding His.Flu M1 protein. A pET28b (+) derivative encoding an
N-terminal 169 residue cohesin domain from C. thermocellum
(unpublished) inserted between the Nco I and Nhe I sites expressed
Coh.His. For expression of Cohesin-Flex-hMART-1-PeptideA-His, the
sequence
GACACCACCGAGGCCCGCCACCCCCACCCCCCCGTGACCACCCCCACCACCACCGACCGGAAG
GGCACCACCGCCGAGGAGCTGGCCGGCATCGGCATCCTGACCGTGATCCTGGGCGGCAAGCG
GACCAACAACAGCACCCCCACCAAGGGCGAATTCTGCAGATATCCATCACACTGGCGGCCG (SEQ
ID NO.:51) (encoding
DTTEARHPHPPVTTPTTDRKGTTAEELAGIGILTVILGGKRTNNSTPTKGEFCRYPSHWRP (SEQ
ID NO.:52)--the italicized residues are the immunodominant
HLA-A2-restricted peptide and the underlined residues surrounding
the peptide are from MART-1) was inserted between the Nhe I and Xho
I sites of the above vector. The proteins were expressed in E. coli
strain BL21 (DE3) (Novagen) or T7 Express (NEB), grown in LB at
37.degree. C. with selection for kanamycin resistance (40 .mu.g/ml)
and shaking at 200 rounds/min to mid log phase growth when 120 mg/L
IPTG was added. After three hours, the cells were harvested by
centrifugation and stored at -80.degree. C. E. coli cells from each
1 L fermentation were resuspended in 30 ml ice-cold 50 mM Tris, 1
mM EDTA pH 8.0 (buffer B) with 0.1 ml of protease inhibitor
Cocktail II (Calbiochem, CA). The cells were sonicated on ice
2.times. 5 min at setting 18 (Fisher Sonic Dismembrator 60) with a
5 min rest period and then spun at 17,000 r.p.m. (Sorvall SA-600)
for 20 min at 4.degree. C. For His.Flu M1 purification the 50 ml
cell lysate supernatant fraction was passed through 5 ml Q
Sepharose beads and 6.25 ml 160 mM Tris, 40 mM imidazole, 4 M NaCl
pH 7.9 was added to the Q Sepharose flow through. This was loaded
at 4 ml/min onto a 5 ml HiTrap chelating HP column charged with
Ni++. The column-bound protein was washed with 20 mM NaPO.sub.4,
300 mM NaCl pH 7.6 (buffer D) followed by another wash with 100 mM
H.sub.3COONa pH 4.0. Bound protein was eluted with 100 mM
H.sub.3COONa pH 4.0. The peak fractions were pooled and loaded at 4
ml/min onto a 5 ml HiTrap S column equilibrated with 100 mM
H.sub.3COONa pH 5.5, and washed with the equilibration buffer
followed by elution with a gradient from 0-1 M NaCl in 50 mM
NaPO.sub.4 pH 5.5. Peak fractions eluting at about 500 mM NaCl were
pooled. For Coh.Flu M1.His purification, cells from 2 L of culture
were lysed as above. After centrifugation, 2.5 ml of Triton X114
was added to the supernatant with incubation on ice for 5 min.
After further incubation at 25.degree. C. for 5 min, the
supernatant was separated from the Triton X114 following
centrifugation at 25.degree. C. The extraction was repeated and the
supernatant was passed through 5 ml of Q Sepharose beads and 6.25
ml 160 mM Tris, 40 mM imidazole, 4 M NaCl pH 7.9 was added to the Q
Sepharose flow through. The protein was then purified by Ni.sup.++
chelating chromatography as described above and eluted with 0-500
mM imidazole in buffer D.
[0244] FIG. 13 shows the internalization of anti-CD40 mAb:IL-4DC.
IL-4DCs were treated with 500 ng/ml of anti-CD40-Alexa 568.
[0245] FIG. 14 shows CD4 and CD8 T cell proliferation by DCs
targeted with anti-CD40-HA1. 5.times.10e3 IFNDCs loaded with 2
ug/ml of anti-CD40-HA or control Ig-HA1 were co-cultured with
CFSE-labeled autologous CD4+ or CD8+ T cells (2.times.10e5) for 7
days. Cells were then then stained with anti-CD4 or anti-CD8
antibodies. Cell proliferation was tested by measuring
CFSE-dilution.
[0246] FIG. 15 shows a titration of HA1 fusion protein on CD4+ T
proliferation. IFNDCs (5K) loaded with fusion proteins were
co-cultured with CFSE-labeled CD4+ T cells (200K) for 7 days.
[0247] FIG. 16 shows IFNDCs targeted with anti-CD40-HA1 activate
HA1-specific CD4+ T cells. CD4+ T cells were restimulated with DCs
loaded with 5 uM of indicated peptides, and then intracellular
IFN.gamma. was stained.
[0248] FIG. 17 shows IFNDCs targeted with anti-CD40-HA1 activate
HA1-specific CD4+ T cells. CD4+ T cells were restimulated with DCs
loaded with indicated peptides for 36h, and then culture
supernatant was analyzed for measuring IFN.gamma..
[0249] FIG. 18 shows that targeting CD40 results in enhanced
cross-priming of MART-1 specific CD8+ T cells. IFNDCs (5K/well)
loaded with fusion proteins were co-cultured with purified CD8+ T
cells for 10 days. Cells were stained with anti-CD8 and tetramer.
Cells are from healthy donors (HLA-A*0201+).
[0250] FIG. 19 shows targeting CD40 results in enhanced
cross-priming of MART-1 specific CD8+ T cells (Summary of
8-repeated experiments using cells from different healthy
donors).
[0251] FIG. 20 shows CD8+ CTL induced with IFNDCs targeted with
anti-CD40-MART-1 are functional. CD8+ T cells co-cultured with
IFNDCs targeted with fusion proteins were mixed with T2 cells
loaded with 10 uM peptide epitope.
[0252] FIG. 21 shows CD8+ CTL induced with IFNDCs targeted with
anti-CD40-Flu M1 are functional. CD8+ T cells co-cultured with
IFNDCs targeted with fusion proteins were mixed with T2 cells
loaded with 1.0 nM peptide epitope.
[0253] FIG. 22 shows an outline of protocol to test the ability a
vaccine composed of anti-CD4012E12 linked to PSA (prostate specific
antigen) to elicit the expansion from a naive T cell population.
PSA-specific CD4+ T cells corresponding to a broad array of PSA
epitopes. Briefly, DCs derived by culture with IFN.alpha. and
GM-CSF of monocytes from a healthy donor are incubated with the
vaccine. The next day, cells are placed in fresh medium and pure
CD4+ T cells from the same donor are added. Several days later, PSA
peptides are added and, after four hours, secreted gamma-IFN levels
in the culture supernatants are determined.
[0254] FIG. 23 shows that many PSA peptides elicit potent
gamma-IFN-production responses indicating that anti-CD4012E12 and
similar anti-CD40 agents can efficiently deliver antigen to DCs,
resulting in the priming of immune responses against multiple
epitopes of the antigen. The peptide mapping of PSA antigens.
5.times.10e3 IFNDCs loaded with 2 ug/ml of anti-CD40-PSA were
co-cultured with purified autologous CD4+ T cells (2.times.10e5)
for 8 days. Cells were then restimulated with 5 uM of individual
peptides derived from PSA for 36h. The amount of IFN.gamma. was
measured by Luminex. Cells are from healthy donors.
[0255] FIG. 24 shows DCs targeted with anti-CD40-PSA induce
PSA-specific CD8+ T cell responses. IFNDCs were targeted with 1 ug
mAb fusion protein with PSA. Purified autologous CD8+ T cells were
co-cultured for 10 days. Cells were stained with anti-CD8 and PSA
(KLQCVDLHV)-tetramer. Cells are from a HLA-A*0201 positive healthy
donor. The results demonstrate that anti-CD40 effectively deliver
PSA to the DCs, which in turn elicit the expansion of PSA-specific
CD8+ T cells. Briefly, 5.times.10e3 IFNDCs loaded with 2 ug/ml of
anti-CD40-PSA were co-cultured with purified autologous CD8+ T
cells (2.times.10e5) for 10 days. Cells were then stained with
tetramer. Cells are from HLA-0*201 positive healthy donor.
[0256] FIG. 25 a scheme (left) and the IFN.gamma. production by T
cells of the pools of peptides and control for Donor 2.
5.times.10e3 IFNDCs loaded with 2 ug/ml of anti-CD40-Cyclin D1 were
co-cultured with purified autologous CD4+ T cells (2.times.10e5)
for 8 days. Cells were then restimulated with with 5 uM of
individual peptides derived from CyclinD1 for 5h in the presence of
Brefeldin A. Cells were stained for measuring intracellular
IFN.gamma. expression.
[0257] FIG. 26 shows a peptide scan and IFN.gamma. production by T
cells obtained from the pools of peptides shown in FIG. 25 and
control for Donor 2. 5.times.10e3 IFNDCs loaded with 2 ug/ml of
anti-CD40-Cyclin D1 were co-cultured with purified autologous CD4+
T cells (2.times.10e5) for 8 days. Cells were then restimulated
with with 5 uM of individual peptides derived from CyclinD1 for 5h
in the presence of Brefeldin A. Cells were stained for measuring
intracellular IFN.gamma. expression.
[0258] In conclusion, delivering antigens to DCs, the most potent
antigen presenting cells, via CD40 is an efficient way to induce
and activate antigen specific both CD4+ and CD8+ T cell-mediated
immunity. Thus, vaccines made of anti-CD40 mAb will induce potent
immunity against cancer and infections.
[0259] Peptide Information:
[0260] HA1 Sequences:
TABLE-US-00022 (SEQ ID NO.: 53)
MKANLLVLLCALAAADADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLL EDSHNGKLCR (SEQ
ID NO.: 54) LKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPG
DFIDYEELRE (SEQ ID NO.: 55)
QLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEK EGSYPKLKNS (SEQ
ID NO.: 56) YVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEI
AERPKVRDQA (SEQ ID NO.: 57)
GRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHE CNTKCQTPLG (SEQ
ID NO.: 58) AINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNIPSI
[0261] Sequences of peptides in FIG. 17
TABLE-US-00023 Peptide 22: (SEQ ID NO.: 59) SSFERFEIFPKESSWPN
Peptide 45: (SEQ ID NO.: 60) GNLIAPWYAFALSRGFG Peptide 46: (SEQ ID
NO.: 61) WYAFALSRGFGSGIITS
[0262] NP Sequences:
TABLE-US-00024 (SEQ ID NO.: 62) MASQGTKRSYEQMETDGERQNATEIRASVG
KMIGGIGRFYIQMCTELKLSDYEGRLIQNS (SEQ ID NO.: 63)
LTIERMVLSAFDERRNKYLEEHPSAGKDPK KTGGPIYRRVNGKWMRELILYDKEEIRRIW (SEQ
ID NO.: 64) RQANNGDDATAGLTHMMIWHSNLNDATYQR
TRALVRTGMDPRMCSLMQGSTLPRRSGA AG (SEQ ID NO.: 65)
AAVKGVGTMVMELVRMIKRGINDRNFWRGE NGRKTRIAYERMCNILKGKFQTAAQKAM MD (SEQ
ID NO.: 66) QVRESRNPGNAEFEDLTFLARSALILRGSV
AHKSCLPACVYGPAVASGYDFEREGYSLVG (SEQ ID NO.: 67)
IDPFRLLQNSQVYSLIRPNENPAHKSQLVW MACHSAAFEDLRVLSFIKGTKVLPRGKLST (SEQ
ID NO.: 68) RGVQIASNENMETMESSTLELRSRYWAIRT
RSGGNTNQQRASAGQISIQPTFSVQRNLPF (SEQ ID NO.: 69)
DRTTIMAAFNGNTEGRTSDMRTEIIRMMES ARPEDVSFQGRGVFELSDEKAASPIVPSFD (SEQ
ID NO.: 70) MSNEGSYFFGDNAEEYDN
[0263] Sequences of peptides in FIG. 23
TABLE-US-00025 Peptide 22: (SEQ ID NO.: 71) GKWVRELVLYDKEEIRR
Peptide 33: (SEQ ID NO.: 72) RTGMDPRMCSLMQGSTL Peptide 46: (SEQ ID
NO.: 73) MCNILKGKFQTAAQKAM
[0264] Prostate Specific Antigen (PSA) Sequence
TABLE-US-00026 (SEQ ID NO.: 74) MWVPVVFLTLSVTWIGAAPLILSRIVGGW
ECEKHSQPWQVLVASRGRAVCGGVLVHPQ WV (SEQ ID NO.: 75)
LTAAHCIRNKSVILLGRHSLFHPEDTGQVF QVSHSFPHPLYDMSLLKNRFLRPGDDSSHD (SEQ
ID NO.: 76) LMLLRLSEPAELTDAVKVMDLPTQEPALGT
TCYASGWGSIEPEEFLTPKKLQCVDLHVIS (SEQ ID NO.: 77)
NDVCAQVHPQKVTKFMLCAGRWTGGKSTCS GDSGGPLVCNGVLQGITSWGSEPCALPERP (SEQ
ID NO.: 78) SLYTKVVHYRKWIKDTIVANP
[0265] Sequences of peptides in FIG. 23
TABLE-US-00027 Peptide 1: (SEQ ID NO.: 79) APLILSRIVGGWECE Peptide
4: (SEQ ID NO.: 80) ECEKHSQPWQVLVAS Peptide 25: (SEQ ID NO.: 81)
GDDSSHDLMLLRLSE Peptide 26: (SEQ ID NO.: 82) SHDLMLLRLSEPAEL
Peptide 49: (SEQ ID NO.: 83) SGDSGGPLVCNGVLQ Peptide 54: (SEQ ID
NO.: 84) GSEPCALPERPSLYT Peptide 56: (SEQ ID NO.: 85)
ERPSLYTKVVHYRKW Peptide 58: (SEQ ID NO.: 86) VVHYRKWIKDTIVAN
[0266] Cyclin D1 Sequence
TABLE-US-00028 (SEQ ID NO.: 87)
MRSYRFSDYLIIMSVSFSNDMDLFCGEDSGVFSGESTVDFSSSEVDSWPG DSIACFIEDER (SEQ
ID NO.: 88) HFVPGHDYLSRFQTRSLDASAREDSVAWILKVQAYYNFQPLTAYLAVNYM
DRFLYARRLP (SEQ ID NO.: 89)
ETSGWPMQLLAVACLSLAAKMEEILVPSLFDFQVAGVKYLFEAKTIKRME LLVLSVLDWR (SEQ
ID NO.: 90) LRSVTPFDFISFFAYKIDPSGTFLGFFISHATEIILSNIKEASFLEYWPS
SIAAAAILCV (SEQ ID NO.: 91)
ANELPSLSSVVNPHESPETWCDGLSKEKIVRCYRLMKAMAIENNRLNTPK VIAKLRVSVR (SEQ
ID NO.: 92) ASSTLTRPSDESSFSSSSPCKRRKLSGYSWVGDETSTSN
[0267] Sequences of peptides in FIG. 26.
TABLE-US-00029 Peptide 7: (SEQ ID NO.: 93) DRVLRAMLKAEETCA Peptide
8: (SEQ ID NO.: 94) RAMLKAEETCAPSVS Peptide 10: (SEQ ID NO.: 95)
TCAPSVSYFKCVQKE
[0268] MART-1 Antigen. MART-1 is a tumor-associated melanocytic
differentiation antigen. Vaccination with MART-1 antigen may
stimulate a host cytotoxic T-cell response against tumor cells
expressing the melanocytic differentiation antigen, resulting in
tumor cell lysis.
[0269] FIG. 27 shows the expression and construct design for
anti-CD40-MART-1 peptide antibodies. FIG. 28 is a summary of the
CD4.sup.+ and CD8.sup.+ immunodominant epitopes for MART-1. FIGS.
27 and 28 show the use of the flexible linker technology to permit
the successful expression of recombinant anti-DC receptor targeting
antibodies fused to significant (.about.2/3) parts of human MART-1.
Recombinant antibody fused at the H chain C-terminus to the entire
MART-1 coding region is not at all secreted from production
mammalian cells [not shown]. The Flex-v1-hMART-1-Pep-3-f4-Pep-1
adduct is particularly well expressed and is one preferred
embodiment of a MART-1-targeting vaccine, as is the Flex-v
1-hMART-1-Pep-3-f4-Pep-1-f3-Pep-2 adduct which bears a maximum load
of MART-1 epitopes. Slide 2 of the MART-1 powerpoint presentation
shows that these adducts can be successfully appended to multiple
anti-DC receptor vehicles.
[0270] The sequence below is a H chain-hMART-1 peptides string of
pep3-pep1-pep2 fusion protein where each hMART1 peptide sequence
[bold-italics] is separated by a inter-peptide spacer f [shown in
bold]. In this case, a 27-amino-acid long linker flex-v1(v1)
[italics] derived from cellulosomal anchoring scaffoldin B
precursor [Bacteroides cellulosolvens--described in the gag-nef
vaccine invention disclosure] was inserted between the H chain
C-terminus and the hMART1 peptides-flexible spaces string. The
underlined AS residues are joining sequences.
TABLE-US-00030
[manti-CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-v1-hMART-1-Pep-3-f4-Pep-1]
C981 is: (SEQ ID NO.: 96)
EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYINSGGGSTYY
PDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKG
PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKV
SNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTIS
VTPTNNSTPTNNSNPKPNPAS ASTNGS ITVAATAPTVTPTVNATPSAAAS
[manti-CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-v1-hMART-1-Pep-3-f4-Pep-1-f3-
Pep-2] C978 is: (SEQ ID NO.: 97)
EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYINSGGGSTYY
PDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKG
PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTKTYTCNVDHKPS
NTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKV
SNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTIS
VTPTNNSTPTNNSNPKPNPAS ASTNGS ITVAATAPTVTPTVNATPSAAAS ASTVT
PTATATPSAIVTTITPTATTKPAS AS
[mAnti-DCIR_9E8_H-LV-hIgG4H-C-Flex-v1-hMART-1-Pep-3-f4-Pep-1] C1012
is: (SEQ ID NO.: 98)
QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWLAHIYWDDDKRY
NPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARSSHYYGYGYGGYFDVWGAGTTVTVSSAK
TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTP
EVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPT
NTISVTPTNNSTPTNNSNPKPNPAS AST NGSITVAATAPTVTPTVNATPSAAAS AS
[mAnti-DCIR_9E8_H-LV-hIgG4H-C-Flex-v1-hMART-1-Pep-3-f4-Pep-1-f3-Pep-2]
C1013 is: (SEQ ID NO.: 99)
QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLEWLAHIYWDDDKRY
NPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARSSHYYGYGYGGYFDVWGAGTTVTVSSAK
TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTP
EVTCVVVDVSQPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNT
ISVTPTNNSTPTNNSNPKPNPAS ASTN GSITVAATAPTVTPTVNATPSAAAS AST
VTPTATATPSAIVTTITPTATTKPAS AS
[0271] MART-1 DNA Sequence:
MART-1 constructs with 3 peptides, Start/stop sites are underlined,
peptide 1 is bold, peptide 2 is bold-italics and peptide 3 is
bold-underlined:
TABLE-US-00031 (SEQ ID NO.: 100)
AACACCGACAACAACAGATGATCTGGATGCAGCTAGTGGGTTTGATCATCGGGACAGCAAA
GTGTCTCTTCAAGAGAAAAACTGTGAACCTGTGGTTCCCAATGCTCCACCTGCTTATGAG
AAACTCTCTGCAGAACAGTCACCACCACCTTATTCACCTGCTAGTACCAACGGCAGCATCA
CCGTGGCCGCCACCGCCCCCACCGTGACCCCCACCGTGAACGCCACCCCCAGCGCCGCCGCTA GT
GCTAGTACCGTGA
CCCCCACCGCCACCGCCACCCCCAGCGCCATCGTGACCACCATCACCCCCACCGCCACCACCA
AGCCCGCTAGTGTCTTACTGCTCATCGGCTGTTGGTATTGTAGAAGACGAAATGGATACA
GAGCCTTGATGGATAAAAGTCTTCATGTTGGCACTCAATGTGCCTTAACAAGAAGATGCC
CACAAGAAGGGtgaGCGGCCGCATCGAAGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCT
GCAGGCATGC
Peptide 3 is bold followed by the Flex-4 amino acid
sequence--underlined.
TABLE-US-00032 (SEQ ID NO.: 101)
GFDHRDSKVSLQEKNCEPVVPNAPPAYEKLSAEQSPPPYSPASTNGSITV AATAPTVTPT
Peptide 1 is bold followed by the Flex-3 amino acid
sequence--underlined.
TABLE-US-00033 (SEQ ID NO.: 102)
VNATPSAAASMPREDAHFIYGYPKKGHGHSYTTAEEAAGIGILTVILGAS TVTPTATATP
Peptide 3 is bold.
TABLE-US-00034 (SEQ ID NO.: 103)
SAIVTTITPTATTKPASVLLLIGCWYCRRRNGYRALMDKSLHVGTQCALT RRCPQEG
[0272] MART1-Peptide 3, the italicized portion is the CD4+
immunodominant epitope.
TABLE-US-00035 (SEQ ID NO.: 104)
GFDHRDSKVSLQEKNCEPVVPNAPPAYEKLSAEQSPPPYSP Flex-4 (SEQ ID NO.: 105)
ASTNGSITVAATAPTVTPTVNATPSAAAS
[0273] MART1-Peptide 1 the italicized portion is the CD4+
immunodominant epitope and the underlined-italicized portion is the
CD8+ immunodominant epitope
TABLE-US-00036 (SEQ ID NO.: 106)
MPREDAHFIYGYPKKGHGHSYTTAEEAAGIGILTVILG Flex-3: (SEQ ID NO.: 107)
ASTVTPTATATPSAIVTTITPTATTKPAS
[0274] MART 1--Peptide 2 the italicized portion is the CD4+
immunodominant epitope.
TABLE-US-00037 (SEQ ID NO.: 108)
VLLLIGCWYCRRRNGYRALMDKSLHVGTQCALTRRCPQEG
[0275] MART1 constructs with two peptides:
[0276] Peptide 3 is bold-italics-underlined, flex-4 is bold and
Peptide 1 is bold-italics-underlined:
TABLE-US-00038 (SEQ ID NO.: 109) ASTNGSITVAATAPTVTPTVNATPSAAAS
[0277] Protein Sequence: C978.
rAB-cetHS-puro[manti-CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-v1-hMART-1-Pep-3
(bold-italics-underlined)-f4 (bold)-Pep-1 (bold-italics)-f3
(italics)-Pep-2 (bold-underlined)]
TABLE-US-00039 (SEQ ID NO.: 110)
MNLGLSLIFLVLVLKGVQCEVKLVESGGGLVQPGGSLKLSCATSGFTFSD
YYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYL
QMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKGPSVFPLA
PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP
EFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGV
EVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI
EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL
HNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNPAS
ASTNGSITVAATAPTVTPTVNATPSAAAS ASTVTPTATATPSAIYTTITPTATT
KPASVLLLIGCWYCRRRNGYRALMDKSLHVGTQCALTRRCPQEGAS
[0278] Protein Sequence: C981.
rAB-cetHS-puro[manti-CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-v1-hMART-1-Pep-3
(bold-italics-underlined)-f4-(bold)-Pep-1](bold-underlined)
TABLE-US-00040 (SEQ ID NO.: 111)
MNLGLSLIFLVLVLKGVQCEVKLVESGGGLVQPGGSLKLSCATSGFTFSD
YYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYL
QMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKGPSVFPLA
PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP
EFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGV
EVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI
EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL
HNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNPAS AST
NGSITVAATAPTVTPTVNATPSAAASMPREDAHFIYGYPKKGHGHSYTT
AEEAAGIGILTVILGAS
[0279] GP100 Antigen. GP100 antigen is a melanoma-associated
antigen. When administered in a vaccine formulation, gp100 antigen
may stimulate a cytotoxic T cell HLA-A2.1-restricted immune
response against tumors that express this antigen, which may result
in a reduction in tumor size.
[0280] GP100 ectodomain coding region fused to recombinant antibody
H chain coding region is not at all secreted by production
mammalian cells [not shown]. The total sequence is shown
below--italics residues are the leader sequence and the
transmembrane domain, the peptides are in bold-italics and the
transmembrane domain is italics-underlined.
TABLE-US-00041 (SEQ ID NO.: 112)
MDLVLKRCLLHLAVIGALLAVGATKVPRNQDWLGVSRQLRTKAWNRQLYP
EWTEAQRLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDG
QVIWVNNTIINGSQVWGGQPVYPQETDDACIFPDGGPCPSGSWSQKRSFV YVW
LGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSRSYVPL AHSSSAFT
SVSQLRALDGGNKHFLRNQPLTFALQLHDPSGY LAEADLSYTWDFGDSSGTLISRALVVTHT
QVVLQAAIPLTS CGSSPVPGTTDGHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTS
VQVPTTEVISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGM
TPAEVSIVVLSGTTAAQVTTTEWVETTARELPIPEPEGPDASSIMSTESI
TGSLGPLLDGTATLRLVKRQVPLDCVLYRYGSFSVTLDIVQGIESAEILQ
AVPSGEGDAFELTVSCQGGLPKEACMEISSPGCQPPAQRLCQPVLPSPAC
QLVLHQILKGGSGTYCLNVSLADTNSLAVVSTQLIMPGQEAGLGQVPLIV
GILLVLMAVVLASLIYRRRLMKQDFSVPQLPHSSSHWLRLPRIFCSCPIG ENSPLLSGQQV
[0281] Known HLA-A0201 restricted peptides sequences are: GP100 M:
209-217 (2M): IMDQVPFSV (SEQ ID NO.:113); 209-217 WT: ITDQVPFSV
(SEQ ID NO.:114) GP100 M: 280-288 (9V): YLEPGPVTV (SEQ ID NO.:115)
280-288 WT: YLEPGPVTA (SEQ ID NO.:116) GP100 WT: 154-162: KTWGQYWQV
(SEQ ID NO.:117)
[0282] FIG. 29-33 show the gp100 adducts which were successfully
expressed as secreted anti-DC receptor targeting vaccines. These
employed the use of the flexible linker sequences and fragmentation
and shuffling of the gp100 ectodomain coding region. Preferred
embodiments of gp100 vaccine adducts are described.
[0283] FIG. 29 shows the expression and construct design for
anti-CD40-gp100 peptide antibodies. FIG. 30 shows the design for
additional anti-CD40-gp100 peptide antibodies. FIG. 31 shows the
expression and construct design for additional anti-CD40-gp100
peptide antibodies. FIG. 32 is a summary of the CD4.sup.+ and
CD8.sup.+ immunodominant epitopes for gp100. FIG. 33 shows the
expression and construct design for additional anti-CD40-gp100
peptide antibodies.
[0284]
rAB-cetHS-puro[manti-CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-hgp100-Pep-1-
-f4-Pep-3-f3-Pep-4-f4-Pep-5-f3-Pep-2] C1285, the peptides are
bold-italics, flexible linkers are bold and the underlined AS
residues are joining sequences:
TABLE-US-00042 (SEQ ID NO.: 118)
EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYINSGGGSTYY
PDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTVSSAKTKG
PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKV
SNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS
ASTNGSITVAATAPTVTPTVNATPSAAAS ASTVTPTATATPSAIVTTITPTATTKPAS
ASTNGSITVAATAPTVTPTVNATPSAAAS ASTVTPTATATPSAIVTTITPTATTKPAS AS
rAB-cetHS-puro[hIgG4H-C-Flex-hgp100-Pep-1-f4-Pep-3-f3-Pep-4-f4-Pep-
5-f3-Pep-2] C1286: (SEQ ID NO.: 119)
RLQLQESGPGLLKPSVTLSLTCTVSGDSVASSSYYWGWVRQPPGKGLEWIGTINFSGNMYY
SPSLRSRVTMSADMSENSFYLKLDSVTAADTAVYYCAAGHLVMGFGAHWGQGKLVSVSPASTKG
PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKV
SNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS
ASTNGSITVAATAPTVTPTVNATPSAAAS ASTVTPTATATPSAIVTTITPTATTKPAS
ASTNGSITVAATAPTVTPTVNATPSAAAS ASTVTPTATATPSAIVTTITPTATTKPAS AS
[0285] gp100:--Nucleic Acid Sequence. Peptide 1--underlined,
Peptide 2--italics, Peptide 3--bold, Peptide 4--bold-underlined,
Peptide 5 bold-italics.
TABLE-US-00043 (SEQ ID NO.: 120)
GATACAACAGAACCTGCAACACCTACAACACCTGTAACAACACCGACAAC
AACAAAAGTACCCAGAAACCAGGACTGGCTTGGTGTCTCAAGGCAACTCA
GAACCAAAGCCTGGAACAGGCAGCTGTATCCAGAGTGGACAGAAGCCCAG
AGACTTGACTGCTGGAGAGGTGGTCAAGTGTCCCTCAAGGTCAGTAATGA
TGGGCCTACACTGATTGGTGCAAATGCCTCCTTCTCTATTGCCTTGAACT
TCCCTGGAAGCCAAAAGGTATTGCCAGATGGGCAGGTTATCTGGGTCAAC
AATACCATCATCAATGGGAGCCAGGTGTGGGGAGGACAGCCAGTGTATCC
CCAGGAAACTGACGATGCCTGCATCTTCCCTGATGGTGGACCTTGCCCAT
CTGGCTCTTGGTCTCAGAAGAGAAGCTTTGTTTATGTCTGGAAGACCTGG
GGCCAATACTGGCAAGTTCTAGGGGGCCCAGTGTCTGGGCTGAGCATTGG
GACAGGCAGGGCAATGCTGGGCACACACACCATGGAAGTGACTGTCTACC
ATCGCCGGGGATCCCAGAGCTATGTGCCTCTTGCTCATTCCAGCTCAGCC
TTCACCATTACTGACCAGGTGCCTTTCTCCGTGAGCGTGTCCCAGTTGCG
GGCCTTGGATGGAGGGAACAAGCACTTCCTGAGAAATCAGGCTAGTACCA
ACGGCAGCATCACCGTGGCCGCCACCGCCCCCACCGTGACCCCCACCGTG
AACGCCACCCCCAGCGCCGCCGCTAGTGGCACCACAGATGGGCACAGGCC
AACTGCAGAGGCCCCTAACACCACAGCTGGCCAAGTGCCTACTACAGAAG
TTGTGGGTACTACACCTGGTCAGGCGCCAACTGCAGAGCCCTCTGGAACC
ACATCTGTGCAGGTGCCAACCACTGAAGTCATAAGCACTGCACCTGTGCA
GATGCCAACTGCAGAGAGCACAGGTATGACACCTGAGAAGGTGCCAGTTT
CAGAGGTCATGGGTACCACACTGGCAGAGATGTCAACTCCAGAGGCTACA
GGTATGACACCTGCAGAGGTATCAATTGTGGTGCTTTCTGGAACCACAGC
TGCAGCTAGTACCGTGACCCCCACCGCCACCGCCACCCCCAGCGCCATCG
TGACCACCATCACCCCCACCGCCACCACCAAGCCCGCTAGTCAGGTAACA
ACTACAGAGTGGGTGGAGACCACAGCTAGAGAGCTACCTATCCCTGAGCC
TGAAGGTCCAGATGCCAGCTCAATCATGTCTACGGAAAGTATTACAGGTT
CCCTGGGCCCCCTGCTGGATGGTACAGCCACCTTAAGGCTGGTGAAGAGA
CAAGTCCCCCTGGATTGTGTTCTGTATCGATATGGTTCCTTTTCCGTCAC
CCTGGACATTGTCCAGGCTAGTACCAACGGCAGCATCACCGTGGCCGCCA
CCGCCCCCACCGTGACCCCCACCGTGAACGCCACCCCCAGCGCCGCCGCT
AGTGGTATTGAAAGTGCCGAGATCCTGCAGGCTGTGCCGTCCGGTGAGGG
GGATGCATTTGAGCTGACTGTGTCCTGCCAAGGCGGGCTGCCCAAGGAAG
CCTGCATGGAGATCTCATCGCCAGGGTGCCAGCCCCCTGCCCAGCGGCTG
TGCCAGCCTGTGCTACCCAGCCCAGCCTGCCAGCTGGTTCTGCACCAGAT
ACTGAAGGGTGGCTCGGGGACATACTGCCTCAATGTGTCTCTGGCTGATA
CCAACAGCCTGGCAGTGGTCAGCACCCAGCTTATCGTGCCTGGGATTCTT
CTCACAGGTCAAGAAGCAGGCCTTGGGCAGTAAGCTAGTACCGTGACCCC
CACCGCCACCGCCACCCCCAGCGCCATCGTGACCACCATCACCCCCACCG
CCACCACCAAGCCCGCTAGT GCTAGC TGA
[0286] GP100-Peptide 1--Nucleic Acid Sequence.
TABLE-US-00044 (SEQ ID NO.: 121)
GATACAACAGAACCTGCAACACCTACAACACCTGTAACAACACCGACAAC
AACAAAAGTACCCAGAAACCAGGACTGGCTTGGTGTCTCAAGGCAACTCA
GAACCAAAGCCTGGAACAGGCAGCTGTATCCAGAGTGGACAGAAGCCCAG
AGACTTGACTGCTGGAGAGGTGGTCAAGTGTCCCTCAAGGTCAGTAATGA
TGGGCCTACACTGATTGGTGCAAATGCCTCCTTCTCTATTGCCTTGAACT
TCCCTGGAAGCCAAAAGGTATTGCCAGATGGGCAGGTTATCTGGGTCAAC
AATACCATCATCAATGGGAGCCAGGTGTGGGGAGGACAGCCAGTGTATCC
CCAGGAAACTGACGATGCCTGCATCTTCCCTGATGGTGGACCTTGCCCAT
CTGGCTCTTGGTCTCAGAAGAGAAGCTTTGTTTATGTCTGGAAGACCTGG
GGCCAATACTGGCAAGTTCTAGGGGGCCCAGTGTCTGGGCTGAGCATTGG
GACAGGCAGGGCAATGCTGGGCACACACACCATGGAAGTGACTGTCTACC
ATCGCCGGGGATCCCAGAGCTATGTGCCTCTTGCTCATTCCAGCTCAGCC
TTCACCATTACTGACCAGGTGCCTTTCTCCGTGAGCGTGTCCCAGTTGCG
GGCCTTGGATGGAGGGAACAAGCACTTCCTGAGAAATCAG
[0287] Protein Sequence:
TABLE-US-00045 (SEQ ID NO.: 122)
DTTEPATPTTPVTTPTTTKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEAQ
RLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIWVN
NTIINGSQVWGGQPVYPQETDDACIFPDGGPCPSGSWSQKRSFVYVWKTW
GQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSQSYVPLAHSSSA
FTITDQVPFSVSVSQLRALDGGNKHFLRNQ GP100-Peptide 3 (SEQ ID NO.: 123)
GGCACCACAGATGGGCACAGGCCAACTGCAGAGGCCCCTAACACCACAGC
TGGCCAAGTGCCTACTACAGAAGTTGTGGGTACTACACCTGGTCAGGCGC
CAACTGCAGAGCCCTCTGGAACCACATCTGTGCAGGTGCCAACCACTGAA
GTCATAAGCACTGCACCTGTGCAGATGCCAACTGCAGAGAGCACAGGTAT
GACACCTGAGAAGGTGCCAGTTTCAGAGGTCATGGGTACCACACTGGCAG
AGATGTCAACTCCAGAGGCTACAGGTATGACACCTGCAGAGGTATCAATT
GTGGTGCTTTCTGGAACCACAGCTGCA
[0288] Protein Sequence:
TABLE-US-00046 (SEQ ID NO.: 124)
GTTDGHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTSVQVPTTE
VISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGMTPAEVSI VVLSGTTAA
GP100-Peptide 4: (SEQ ID NO.: 125)
CAGGTAACAACTACAGAGTGGGTGGAGACCACAGCTAGAGAGCTACCTAT
CCCTGAGCCTGAAGGTCCAGATGCCAGCTCAATCATGTCTACGGAAAGTA
TTACAGGTTCCCTGGGCCCCCTGCTGGATGGTACAGCCACCTTAAGGCTG
GTGAAGAGACAAGTCCCCCTGGATTGTGTTCTGTATCGATATGGTTCCTT
TTCCGTCACCCTGGACATTGTCCAG
[0289] Protein Sequence:
TABLE-US-00047 (SEQ ID NO.: 126)
QVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATL
RLVKRQVPLDCVLYRYGSFSVTLDIVQ GP100-Peptide 5 (SEQ ID NO.: 127)
GGTATTGAAAGTGCCGAGATCCTGCAGGCTGTGCCGTCCGGTGAGGGG
GATGCATTTGAGCTGACTGTGTCCTGCCAAGGCGGGCTGCCCAAGGAA
GCCTGCATGGAGATCTCATCGCCAGGGTGCCAGCCCCCTGCCCAGCGG
CTGTGCCAGCCTGTGCTACCCAGCCCAGCCTGCCAGCTGGTTCTGCAC
CAGATACTGAAGGGTGGCTCGGGGACATACTGCCTCAATGTGTCTCTG
GCTGATACCAACAGCCTGGCAGTGGTCAGCACCCAGCTTATCGTGCCT
GGGATTCTTCTCACAGGTCAAGAAGCAGGCCTTGGGCAG
[0290] Protein Sequence:
TABLE-US-00048 (SEQ ID NO.: 128)
GIESAEILQAVPSGEGDAFELTVSCQGGLPKEACMEISSPGCQPPAQR
LCQPVLPSPACQLVLHQILKGGSGTYCLNVSLADTNSLAVVSTQLIVP GILLTGQEAGLGQ
GP100-Peptide 2 (SEQ ID NO.: 129)
CCTCTGACCTTTGCCCTCCAGCTCCATGACCCTAGTGGCTATCTGGCT
GAAGCTGACCTCTCCTACACCTGGGACTTTGGAGACAGTAGTGGAACC
CTGATCTCTCGGGCACYTGTGGTCACTCATACTTACCTGGAGCCTGGC
CCAGTCACTGCCCAGGTGGTCCTGCAGGCTGCCATTCCTCTCACCTCC
TGTGGCTCCTCCCCAGTTCCAGCTAGC
[0291] Protein Sequence:
TABLE-US-00049 (SEQ ID NO.: 130)
PLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRAXVVTHTYLEPG
PVTAQVVLQAAIPLTSCGSSPVPAS
[0292] Cyclin B1 Antigen. Cyclin B1, also known as CCNB1, is a
human gene that encodes a regulatory protein involved in mitosis.
Cyclin B1 complexes with p34(cdc2) to form the maturation-promoting
factor (MPF). Two alternative transcripts are known that are the
result of alternative transcription initiation sites. A first
transcript encodes a constitutively expressed transcript. The
second transcript is a cell cycle-regulated transcript expressed
predominantly during G2/M phase.
[0293] FIGS. 34A and 34B shows that full-length Cyclin B1 fused to
the C-terminus of either antibody H chain or cohesion fail to be
secreted from mammalian 293F cells. The data are anti-human Fc and
anti-cohesin ELISA on serial dilutions of transfection
supernatants. rAb.Cyclin B1 and Coh.Cyclin B1 proteins are poorly
expressed as products secreted from mammalian cells.
[0294] The following amino acid sequence is human cyclin B1. Two
peptide regions known to contain T cell epitopes are highlighted in
bold-underlined and italics-underlined.
TABLE-US-00050 (SEQ ID NO.: 131)
MALRVTRNSKINAENKAKINMAGAKRVPTAPAATSKPGLRPRTALGDI
GNKVSEQLQAKMPMKKEAKPSATGKVIDKKLPKPLEKVPMLVPVPVSE
PVPEPEPEPEPEPVKEEKLSPEPILVDTASPSPMETSGCAPAEEDLCQ
AFSDVILAVNDVDAEDGADPNLCSEYVKDIYAYLRQLEEEQAVRPKYL
LGREVTGNMRAILIDWINQVQMKFRLLQETMYMTVSIIDRFMQNNCVP
KKMLQLVGVTAMFIASKYEEMYPPEIGDFAFVTDNTYTKHQIRQ
DMVHFPPSQIAAGAFCLALKILDNGEWTPTLQHYLS
YTEESLLPVMQHLAKNVVMVNQGLTKHMTVKNKYATSKHAKISTLPQL
NSALVQDLAKAVAKVHHHHHH Peptide-1 (SEQ ID NO.: 132)
MEMKILRALNFGLGRPLPLHFLRRASKIGEVDVEQHTLAKYLMELTML DY Peptide-2 (SEQ
ID NO.: 133) DWLVQVQMKFRLLQETMYMTVSIIDRFMQNNCVPKK
[0295] FIG. 35 shows a summary of relative expression levels of
prototype Cyclin B1 vaccines secreted from transfected mammalian
293F cells. The flexible linker sequences facilitate secretion.
[0296] C1189
rAB-cetHS-puro[manti-CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-v1
(bold)-hCyclinB 1-Peptide-2(italics)-Peptide-1 (bold-italics)-f4
(bold)] [AS linkers--underlined]
TABLE-US-00051 (SEQ ID NO.: 134)
EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWV
AYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYC
ARRGLPFHAMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS
SSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT
KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS
KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH
NHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNPASDWL
VQVQMKFRLLQETMYMTVSIIDRFMQNNCVPKKASMEMKILRALNFGL GRPLPLHFLRRAS
ASTNDSITVA ATAPTVTPTVNATPSAAAS
[0297] Above is the sequence of the mature secreted H chain for one
form of anti-CD4012E12-cyclin B1 vaccine. The AS residues are from
joining restriction sites. The DNA coding sequence is shown below,
and this includes the signal peptide.
TABLE-US-00052 (SEQ ID NO.: 135)
ATGAACTTGGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGT
GTCCAGTGTGAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAG
CCCGGAGGGTCCCTGAAACTCTCCTGTGCAACCTCTGGATTCACTTTC
AGTGACTATTACATGTATTGGGTTCGCCAGACTCCAGAGAAGAGGCTG
GAGTGGGTCGCATACATTAATTCTGGTGGTGGTAGCACCTATTATCCA
GACACTGTAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAAC
ACCCTGTACCTGCAAATGAGCCGGCTGAAGTCTGAGGACACAGCCATG
TATTACTGTGCAAGACGGGGGTTACCGTTCCATGCTATGGACTATTGG
GGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGAAGGGCCCA
TCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACA
GCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG
GTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCG
GCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACC
GTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGAT
CACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATAT
GGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGAAGGGGGACCA
TCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCC
CGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGAC
CCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAAT
GCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTG
GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAG
TACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAA
ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACC
CTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACC
TGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAG
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTG
GACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAG
AGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAG
GCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGT
AAAGCTAGTCAGACCCCCACCAACACCATCAGCGTGACCCCCACCAAC
AACAGCACCCCCACCAACAACAGCAACCCCAAGCCCAACCCCGCTAGT
GACTGGCTAGTACAGGTTCAAATGAAATTCAGGTTGTTGCAGGAGACC
ATGTACATGACTGTCTCCATTATTGATCGGTTCATGCAGAATAATTGT
GTGCCCAAGAAGGCTAGTATGGAAATGAAGATTCTAAGAGCTTTAAAC
TTTGGTCTGGGTCGGCCTCTACCTTTGCACTTCCTTCGGAGAGCATCT
AAGATTGGAGAGGTTGATGTCGAGCAACATACTTTGGCCAAATACCTG
ATGGAACTAACTATGTTGGACTATGCTAGTACCAACGACAGCATCACC
GTGGCCGCCACCGCCCCCACCGTGACCCCCACCGTGAACGCCACCCCC
AGCGCCGCCGCTAGCTGA
[0298] C1143
rAB-cetHS-puro[manti-CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-v1
(bold)-hCyclinB1-Peptide-2(italics)-f3 (bold)] [AS
linkers--underlined].
TABLE-US-00053 (SEQ ID NO.: 136)
EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWV
AYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYC
ARRGLPFHAMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS
SSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT
KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS
KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH
NHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNPASDWL
VQVQMKFRLLQETMYMTVSIIDRFMQNNCVPKKASTVTPTATATPSAI VTTITPTATTKPAS
[0299] Above is the sequence of the mature secreted H chain for one
form of anti-CD4012E12-cyclin B1 vaccine. The AS residues are from
joining restriction sites. The DNA coding sequence is shown below,
and this includes the signal peptide.
TABLE-US-00054 (SEQ ID NO.: 137)
ATGAACTTGGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGT
GTCCAGTGTGAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAG
CCCGGAGGGTCCCTGAAACTCTCCTGTGCAACCTCTGGATTCACTTTC
AGTGACTATTACATGTATTGGGTTCGCCAGACTCCAGAGAAGAGGCTG
GAGTGGGTCGCATACATTAATTCTGGTGGTGGTAGCACCTATTATCCA
GACACTGTAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAAC
ACCCTGTACCTGCAAATGAGCCGGCTGAAGTCTGAGGACACAGCCATG
TATTACTGTGCAAGACGGGGGTTACCGTTCCATGCTATGGACTATTGG
GGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGAAGGGCCCA
TCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACA
GCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG
GTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCG
GCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACC
GTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGAT
CACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATAT
GGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGAAGGGGGACCA
TCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCC
CGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGAC
CCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAAT
GCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTG
GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAG
TACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAA
ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACC
CTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACC
TGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAG
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTG
GACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAG
AGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAG
GCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGT
AAAGCTAGTCAGACCCCCACCAACACCATCAGCGTGACCCCCACCAAC
AACAGCACCCCCACCAACAACAGCAACCCCAAGCCCAACCCCGCTAGT
GACTGGCTAGTACAGGTTCAAATGAAATTCAGGTTGTTGCAGGAGACC
ATGTACATGACTGTCTCCATTATTGATCGGTTCATGCAGAATAATTGT
GTGCCCAAGAAGGCTAGTACCGTGACCCCCACCGCCACCGCCACCCCC
AGCGCCATCGTGACCACCATCACCCCCACCGCCACCACCAAGCCCGCT AGCTGA
[0300] C911
rAB-cetHS-puro[manti-CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-v1
(bold)-hCyclinB1-Peptide-1 (italics)-f4 (bold)]
TABLE-US-00055 (SEQ ID NO.: 138)
EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWV
AYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYC
ARRGLPFHAMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS
SSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT
KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS
KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH
NHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNPASMEM
KILRALNFGLGRPLPLHFLRRASKIGEVDVEQHTLAKYLMELTMLDYA
STNGSITVAATAPTVTPTVNATPSAAAS
[0301] C911
rAB-cetHS-puro[manti-CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-v1
(bold)-hCyclinB1-Peptide-1 (italics)-f4 (bold)] nucleic acid
sequence.
TABLE-US-00056 (SEQ ID NO.: 139)
ATGAACTTGGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGT
GTCCAGTGTGAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAG
CCCGGAGGGTCCCTGAAACTCTCCTGTGCAACCTCTGGATTCACTTTC
AGTGACTATTACATGTATTGGGTTCGCCAGACTCCAGAGAAGAGGCTG
GAGTGGGTCGCATACATTAATTCTGGTGGTGGTAGCACCTATTATCCA
GACACTGTAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAAC
ACCCTGTACCTGCAAATGAGCCGGCTGAAGTCTGAGGACACAGCCATG
TATTACTGTGCAAGACGGGGGTTACCGTTCCATGCTATGGACTATTGG
GGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGAAGGGCCCA
TCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACA
GCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG
GTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCG
GCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACC
GTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGAT
CACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATAT
GGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGAAGGGGGACCA
TCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCC
CGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGAC
CCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAAT
GCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTG
GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAG
TACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAA
ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACC
CTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACC
TGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAG
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTG
GACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAG
AGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAG
GCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGT
AAAGCTAGTCAGACCCCCACCAACACCATCAGCGTGACCCCCACCAAC
AACAGCACCCCCACCAACAACAGCAACCCCAAGCCCAACCCCGCTAGT
ATGGAAATGAAGATTCTAAGAGCTTTAAACTTTGGTCTGGGTCGGCCT
CTACCTTTGCACTTCCTTCGGAGAGCATCTAAGATTGGAGAGGTTGAT
GTCGAGCAACATACTTTGGCCAAATACCTGATGGAACTAACTATGTTG
GACTATGCTAGTACCAACGGCAGCATCACCGTGGCCGCCACCGCCCCC
ACCGTGACCCCCACCGTGAACGCCACCCCCAGCGCCGCCGCTAGCTGA
[0302] D-type Cyclin Antigen. D-type cyclins are predominantly
expressed in the G1 phase of the cell cycle. The expression pattern
of cyclin D1 has been extensively studied in certain cancer types
including lymphoma and non-small cell lung cancer. Approximately 30
percent of breast carcinomas are Cyclin D1 positive. Over
expression of Cyclin D1 is now a well established criterion for the
diagnosis of Mantle Cell Lymphoma, a malignant, non-Hodgkin's
lymphoma which is characterized by a unique chromosomal
translocation t(11;14).
[0303] Cyclin D1--Peptide 1--bold, Peptide 2--bold-underlined,
Peptide-3 italics, Peptide 4--underlined.
TABLE-US-00057 (SEQ ID NO.: 140)
MEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYFKCV
QKEVLPSMRKIVATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEPVKK
SRLQLLGATCMFVASKMKETIPLTAEKLCIYTDNSIRPEELLQMELLL
VNKLKWNLAAMTPHDFIEHFLSKMPEAEENKQIIRKHAQTFVALCATD
VKFISNPPSMVAAGSVVAAVQGLNLRSPNNFLSYYRLTRFLSRVIKCD
PDCLRACQEQIEALLESSLRQAQQNMDPKAAEEEEEEEEEVDLACTPT DVRDVDI Pep-1:
(SEQ ID NO.: 141) MEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYFKCV
Pep-2 (SEQ ID NO.: 142)
QKEVLPSMRKIVATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEPVKK
SRLQLLGATCMFVASKMKETIPLTAEKLCIYTDNSIRPEELLQMELL Pep-3 (SEQ ID NO.:
143) LVNKLKWNLAAMTPHDFIEHFLSKMPEAEENKQIIRKHAQTFVALCAT DVKFISNPPSMV
Pep-4 (SEQ ID NO.: 144)
AAGSVVAAVQGLNLRSPNNFLSYYRLTRFLSRVIKCDPDCLRACQEQI
EALLESSLRQAQQNMDPKAAEEEEEEEEEVDLACTPTDVRDVDI Flex-4 sequence (SEQ
ID NO.: 14) TNGSITVAATAPTVTPTVNATPSAA Flex-3 sequence (SEQ ID NO.:
13) TVTPTATATPSAIVTTITPTATTKP Flex-var1 (SEQ ID NO.: 145)
QTPTNTISVTPTNNSTPTNNSNPKPNP
[0304] FIG. 35 shows Cyclin B1 segmentation strategy based on known
or predicted structural domain regions.
[0305] FIG. 36 shows that Cyclin D1 segments p1, p3, and p4, but
not p2 express well as direct fusions to the H chain C-terminus.
These are transient transfections of the H chain vectors
co-transfected with the L chain expression vector into 293F cells
and the supernatants harvested after 48-72 hours of expression. The
Cyclin D1 p3+p4 segments joined together at the H chain C-terminus
also express well, but various other combinations, with and without
interspersed flex segments do not express, or express very
poorly.
[0306] FIG. 37 shows the relative expression levels of various
Cyclin D1 segments as direct fusions to the H chain C-terminus in
various combinations with flexible linker sequences. These are
transient transfections of the H chain vectors co-transfected with
the L chain expression vector into 293F cells and the supernatants
harvested after 48-72 hours of expression. The Cyclin D1
p2+p3+p4+f4 segments joined together at the H chain C-terminus also
express well enough for vaccine production.
[0307] Sequences of useful anti-DCIR 9E8--cyclin D1H chain fusion
proteins are below.
[0308] 1082 is rAB-pIRES2[mAnti-DCIR_9E8_H-LV-hIgG4H-C-Flex-v1
(bold)-hCyclinD1-Pep-1 (italics)-f4 (bold)-]
TABLE-US-00058 (SEQ ID NO.: 146)
QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLE
WLAHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYY
CARSSHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTS
ESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFE
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE
VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS
IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV
MHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPN
PASMEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYF
KCVASTNGSITVAATAPTVTPTVNATPSAAAS
[0309] C1086 is rAB-pIRES2[mAnti-DCIR_9 E8_H-LV-hIgG4H-C-Flex-v1
(bold)-hCyclinD1-Pep-2-(bold)-Pep-3(bold-underlined)-Pep-4
(italics)-f4)(bold)]
TABLE-US-00059 (SEQ ID NO.: 147)
QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLSWIRQPSGKGLE
WLAHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYY
CARSSHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTS
ESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFE
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE
VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS
IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV
MHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPN
PASQKEVLPSMRKIVATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEP
VKKSRLQLLGATCMFVASKMKETIPLTAEKLCIYTDNSIRPEELLQME
LLLVNKLKWNLAAMTPHDFIEHFLSKMPEAEENKQIIRKHAQTFVALC
ATDVKFISNPPSMVAAGSVVAAVQGLNLRSPNNFLSYYRLTRFLSRVI
KCDPDCLRACQEQIEALLESSLRQAQQNMDPKAAEEEEEEEEEVDLAC
TPTDVRDVDIASTNGSITVAATAPTVTPTVNATPSAAAS
[0310] FIG. 38 show a summary of various H chain-Cyclin D1 segment
constructs and their relative expressibility as vaccines.
[0311] FIG. 39 above shows that full-length Cyclin D1 fused to the
C-terminus of a DC targeting antibody H chain is very poorly
expressed as a secreted recombinant antibody.
[0312] anti-CD40_12E12.3F3
[0313] anti-CD40_12E12.3F3_H-V-hIgG4H-C--underlined region shows
the Heavy chain V region amino acid sequence:
TABLE-US-00060 (SEQ ID NO.: 148)
MNLGLSLIFLVLVLKGVQCEVKLVESGGGLVQPGGSLKLSCATSGFTF
SDYYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKN
TLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQGTSVTFVSSAKTKG
PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK
YGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE
DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD
KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS
[0314] anti-CD40_12E12.3F3_K-V-hIgGK-C--underlined region shows the
Light chain V region amino acid sequence
TABLE-US-00061 (SEQ ID NO.: 149)
MMSSAQFLGLLLLCFQGTRCDIQMTQTTSSLSASLGDRVTISCSASQG
ISNYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTIG
NLEPEDIATYYCQQFNKLPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST
YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC-
[0315] anti-CD40_12B4.2C10
[0316] anti-CD40_12B4.2C10 Heavy Chain:
TABLE-US-00062 (SEQ ID No.: 150)
MEWSWIFLFLLSGTAGVHSEVQLQQSGPELVKPGASVKMSCKAS
GYTFTDYVLHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKAT
LTSDKSSSTAYMELSSLTSEDSAVYYCARGYPAYSGYAMDYWGQ
GTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEP
VTVTWNSGSLSSGVHTFPAVLQKGEFV
[0317] anti-CD40_12B4.2C10 Light Chain:
TABLE-US-00063 (SEQ ID No.: 151)
MMSSAQFLGLLLLCFQGTRCDIQMTQTTSSLSASLGDRVTISCR
ASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSG
TDYSLTISNLEQEDIATYFCHHGNTLPWTFGGGTKLEIKRADAA
PTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQ
NGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKT STSPIVKSFNRNEC
[0318] anti-CD40_12B4.2C10 Light Chain--alternative clone
(17K6)
TABLE-US-00064 (SEQ ID No.: 152)
MDFQVQIFSFLLISASVIMSRGQIVLTQSPAILSASPGEKVTM
TCSASSSVSYMYRYQQKPGSSPKPWIYGTSNLASGVPARFSGS
GSGTSYSLTISSMEAEDAATYYCQQYHSYPLTFGAGTKLELKR
ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKID
GSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTC
EATHKTSTSPIVKSFNRNEC
[0319] anti-CD40_11B6.1C3
[0320] anti-CD40_11B6.1C3 Heavy Chain:
TABLE-US-00065 (SEQ ID No.: 153)
MGWSWIFLFLLSGTAGVLSEVQLQQSGPELVKPGASVKISCK
ASGYSFTGYYMHWVKQSHVKSLEWIGRINPYNGATSYNQNFKD
KASLTVDKSSSTAYMELHSLTSEDSAVYYCAREDYVYWGQGTT
LTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVT
VTWNSGSLSSGVHTFPAVLQKGEFV
[0321] anti-CD40_11B6.1C3 Light Chain:
TABLE-US-00066 (SEQ ID No.: 154)
MKLPVRLLVLMFWIPASSSDVVMTQTPLSLPVSLGDQASISC
RSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDR
FSGSGSGTDFALKISRVEAEDLGVYFCSQSTHVPWTFGGGTKL
EIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVK
WKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHN
SYTCEATHKTSTSPIVKSFNRNEC
[0322] [anti-CD40_12E12.3F3_K-V-hIgGK-C]--underlined region shows
the Light chain V region sequence
TABLE-US-00067 (SEQ ID NO.: 155)
ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCA
AGGTACCAGATGTGATATCCAGATGACACAGACTACATCCTCCCTGT
CTGCCTCTCTAGGAGACAGAGTCACCATCAGTTGCAGTGCAAGTCAG
GGCATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAAC
TGTTAAACTCCTGATCTATTACACATCAATTTTACACTCAGGAGTCC
CATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTATTCTCTCACC
ATCGGCAACCTGGAACCTGAAGATATTGCCACTTACTATTGTCAGCA
GTTTAATAAGCTTCCTCCGACGTTCGGTGGAGGCACCAAACTCGAGA
TCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCT
GATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAA
TAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACG
CCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGC
AAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGC
AGACTACGAGAAACACAAAGTCTATGCCTGCGAAGTCACCCATCAGG
GCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
[0323] [anti-CD40_12E12.3F3_H-V-hIgG4H-C]--underlined region shows
the Heavy chain V region sequence:
TABLE-US-00068 (SEQ ID NO.: 156)
ATGAACTTGGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGG
TGTCCAGTGTGAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGC
AGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAACCTCTGGATTCACT
TTCAGTGACTATTACATGTATTGGGTTCGCCAGACTCCAGAGAAGAG
GCTGGAGTGGGTCGCATACATTAATTCTGGTGGTGGTAGCACCTATT
ATCCAGACACTGTAAAGGGCCGATTCACCATCTCCAGAGACAATGCC
AAGAACACCCTGTACCTGCAAATGAGCCGGCTGAAGTCTGAGGACAC
AGCCATGTATTACTGTGCAAGACGGGGGTTACCGTTCCATGCTATGG
ACTATTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACG
AAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTC
CGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCG
AACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTG
CACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAG
CAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACA
CCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGA
GTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGA
GTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGG
ACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTG
GACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGA
TGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGT
TCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAG
GACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGG
CCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGC
CCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATG
ACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCC
CAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACA
ACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC
CTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAA
TGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA
CACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGCTGA
[0324] anti-CD40_12B4.2C10_H-V-hIgG4H-C heavy chain
TABLE-US-00069 (SEQ ID NO.: 157)
ATGGAATGGAGTTGGATATTTCTCTTTCTTCTGTCAGGAACTG
CAGGTGTCCACTCTGAGGTCCAGCTGCAGCAGTCTGGACCTGAG
CTGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTC
TGGATACACATTCACTGACTATGTTTTGCACTGGGTGAAACAGA
AGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTAC
AATGATGGTACTAAGTACAATGAGAAGTTCAAAGGCAAGGCCAC
ACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCA
GCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAGG
GGCTATCCGGCCTACTCTGGGTATGCTATGGACTACTGGGGTCA
AGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGAAGGGCCCAT
CCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGC
ACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACC
GGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGC
ACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTC
AGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGAC
CTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGG
ACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGC
CCAGCACCTGAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCC
CCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGG
TCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTC
CAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAA
GACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGG
TCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAG
GAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCAT
CGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCAC
AGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAAC
CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGA
CATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT
ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC
CTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGG
GAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC
ACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGC TGA
[0325] anti-CD40_12B4.2C10_K-V-hIgGK-C (variant 1) light chain
TABLE-US-00070 (SEQ ID NO.: 158)
ATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGC
CTCAGTCATAATGTCCAGGGGACAAATTGTTCTCACCCAGTCTC
CAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACCATGACC
TGCAGTGCCAGCTCAAGTGTAAGTTACATGTACAGGTACCAGCA
GAAGCCAGGATCCTCACCCAAACCCTGGATTTATGGCACATCCA
ACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGATCT
GGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGA
TGCTGCCACTTATTACTGCCAGCAATATCATAGTTACCCGCTCA
CGTTCGGTGCTGGGACCAAGCTCGAGATCAAACGAACTGTGGCT
GCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA
ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATC
CCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAA
TCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGA
CAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAG
ACTACGAGAAACACAAAGTCTATGCCTGCGAAGTCACCCATCAG
GGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG TTAG
[0326] anti-CD40_12B4.2C10_K-V-hIgGK-C (Variant 2) light chain
TABLE-US-00071 (SEQ ID NO.: 159)
ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTT
TCAAGGTACCAGATGTGATATCCAGATGACACAGACTACATCCTC
CCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGC
AAGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAGAAACC
AGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACA
CTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGA
TTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCAC
TTACTTTTGCCATCATGGTAATACGCTTCCGTGGACGTTCGGTGG
AGGCACCAAGCTCGAGATCAAACGAACTGTGGCTGCACCATCTGT
CTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGC
CTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCA
GGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCT
CAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAA
AGTCTATGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGT
CACAAAGAGCTTCAACAGGGGAGAGTGTTAG
[0327] anti-CD40_11B6.1C3_H-V-hIgG4H-C heavy chain
TABLE-US-00072 (SEQ ID NO.: 160)
ATGGGATGGAGCTGGATCTTTCTCTTTCTCCTGTCAGGAACTGCAG
GTGTCCTCTCTGAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGT
GAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTAC
TCATTCACTGGCTACTACATGCACTGGGTGAAGCAAAGCCATGTAA
AGAGCCTTGAGTGGATTGGACGTATTAATCCTTACAATGGTGCTAC
TAGCTACAACCAGAATTTCAAGGACAAGGCCAGCTTGACTGTAGAT
AAGTCCTCCAGCACAGCCTACATGGAGCTCCACAGCCTGACATCTG
AGGACTCTGCAGTCTATTACTGTGCAAGAGAGGACTACGTCTACTG
GGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACGAAGGGC
CCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGA
GCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACC
GGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC
ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCA
GCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACAC
CTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGA
GTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTG
AGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAA
GGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTG
GTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACG
TGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGA
GCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTG
CACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCA
ACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAA
AGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAG
GAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAG
GCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA
GCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC
GGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGT
GGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCT
GCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA GCTAGCTGA
[0328] anti-CD40_11B6.1C3_K-V-hIgGK-C light chain
TABLE-US-00073 (SEQ ID NO: 161)
ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCT
GCTTCCAGCAGTGATGTTGTGATGACCCAAACTCCACTCTCCCTG
CCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGT
CAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTAC
CTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTT
TCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGA
TCAGGGACAGATTTCGCACTCAAGATCAGTAGAGTGGAGGCTGAG
GATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGTGG
ACGTTCGGTGGAGGCACCAAGCTCGAGATCAAACGAACTGTGGCT
GCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAA
TCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCC
AGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCG
GGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTAC
GAGAAACACAAAGTCTATGCCTGCGAAGTCACCCATCAGGGCCTG
AGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
[0329] It is contemplated that any embodiment discussed in this
specification can be implemented with respect to any method, kit,
reagent, or composition of the invention, and vice versa.
Furthermore, compositions of the invention can be used to achieve
methods of the invention.
[0330] It will be understood that particular embodiments described
herein are shown by way of illustration and not as limitations of
the invention. The principal features of this invention can be
employed in various embodiments without departing from the scope of
the invention. Those skilled in the art will recognize, or be able
to ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures described herein. Such
equivalents are considered to be within the scope of this invention
and are covered by the claims.
[0331] All publications and patent applications mentioned in the
specification are indicative of the level of skill of those skilled
in the art to which this invention pertains. All publications and
patent applications are herein incorporated by reference to the
same extent as if each individual publication or patent application
was specifically and individually indicated to be incorporated by
reference.
[0332] The use of the word "a" or "an" when used in conjunction
with the term "comprising" in the claims and/or the specification
may mean "one," but it is also consistent with the meaning of "one
or more," "at least one," and "one or more than one." The use of
the term "or" in the claims is used to mean "and/or" unless
explicitly indicated to refer to alternatives only or the
alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or." Throughout this application, the term "about" is used to
indicate that a value includes the inherent variation of error for
the device, the method being employed to determine the value, or
the variation that exists among the study subjects.
[0333] As used in this specification and claim(s), the words
"comprising" (and any form of comprising, such as "comprise" and
"comprises"), "having" (and any form of having, such as "have" and
"has"), "including" (and any form of including, such as "includes"
and "include") or "containing" (and any form of containing, such as
"contains" and "contain") are inclusive or open-ended and do not
exclude additional, unrecited elements or method steps.
[0334] The term "or combinations thereof" as used herein refers to
all permutations and combinations of the listed items preceding the
term. For example, "A, B, C, or combinations thereof" is intended
to include at least one of: A, B, C, AB, AC, BC, or ABC, and if
order is important in a particular context, also BA, CA, CB, CBA,
BCA, ACB, BAC, or CAB. Continuing with this example, expressly
included are combinations that contain repeats of one or more item
or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so
forth. The skilled artisan will understand that typically there is
no limit on the number of items or terms in any combination, unless
otherwise apparent from the context.
[0335] All of the compositions and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and/or methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. All such similar substitutes and modifications apparent
to those skilled in the art are deemed to be within the spirit,
scope and concept of the invention as defined by the appended
claims.
Sequence CWU 1
1
161132PRTArtificial SequenceSynthetic HIV peptide. 1Val Gly Phe Pro
Val Thr Pro Gln Val Pro Leu Arg Pro Met Thr Tyr1 5 10 15Lys Ala Ala
Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu 20 25
30230PRTArtificial SequenceSynthetic HIV peptide. 2His Thr Gln Gly
Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro1 5 10 15Gly Val Arg
Tyr Pro Leu Thr Phe Gly Trp Leu Tyr Lys Leu 20 25
30319PRTArtificial SequenceSynthetic HIV peptide. 3Glu Lys Ile Arg
Leu Arg Pro Gly Gly Lys Lys Lys Tyr Lys Leu Lys1 5 10 15His Ile
Val432PRTArtificial SequenceSynthetic HIV peptide. 4Asn Pro Pro Ile
Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu1 5 10 15Gly Leu Asn
Lys Ile Val Arg Met Tyr Ser Pro Thr Ser Ile Leu Asp 20 25
30531PRTArtificial SequenceSynthetic HIV peptide. 5Ala Ile Phe Gln
Ser Ser Met Thr Lys Ile Leu Glu Pro Phe Arg Lys1 5 10 15Gln Asn Pro
Asp Ile Val Ile Tyr Gln Tyr Met Asp Asp Leu Tyr 20 25
306475PRTArtificial SequencehIgG4 heavy chain (H) - HIV gag17
fusion protein. 6Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu
Gln Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe
Ser Leu Ser Thr Ser 20 25 30Gly Met Gly Leu Ser Trp Ile Arg Gln Pro
Ser Gly Lys Gly Leu Glu 35 40 45Trp Leu Ala His Ile Tyr Trp Asp Asp
Asp Lys Arg Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg Leu Thr Ile Ser
Lys Asp Thr Ser Ser Asn Gln Val65 70 75 80Phe Leu Lys Ile Thr Ile
Val Asp Thr Ala Asp Ala Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Ser Ser
His Tyr Tyr Gly Tyr Gly Tyr Gly Gly Tyr Phe 100 105 110Asp Val Trp
Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr 115 120 125Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135
140Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu145 150 155 160Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His 165 170 175Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser 180 185 190Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Lys Thr Tyr Thr Cys 195 200 205Asn Val Asp His Lys Pro
Ser Asn Thr Lys Val Asp Lys Arg Val Glu 210 215 220Ser Lys Tyr Gly
Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu225 230 235 240Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250
255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ser Ser 325 330 335Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 355 360 365Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375
380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Arg Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Leu Gly Lys Ala Ser
Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys 450 455 460Lys Lys Tyr Lys
Leu Lys His Ile Val Ala Ser465 470 4757488PRTArtificial SequenceH
chain - HIV gag253 fusion protein. 7Gln Val Thr Leu Lys Glu Ser Gly
Pro Gly Ile Leu Gln Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ser
Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Gly Leu Ser Trp
Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45Trp Leu Ala His Ile
Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg
Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn Gln Val65 70 75 80Phe Leu
Lys Ile Thr Ile Val Asp Thr Ala Asp Ala Ala Thr Tyr Tyr 85 90 95Cys
Ala Arg Ser Ser His Tyr Tyr Gly Tyr Gly Tyr Gly Gly Tyr Phe 100 105
110Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr
115 120 125Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser
Thr Ser 130 135 140Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu145 150 155 160Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His 165 170 175Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys 195 200 205Asn Val Asp
His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 210 215 220Ser
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu225 230
235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser 260 265 270Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Phe Asn Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 325 330 335Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345
350Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Arg Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Leu Gly
Lys Ala Ser Asn Pro Pro Ile Pro Val Gly Glu Ile Tyr 450 455 460Lys
Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg Met Tyr Ser465 470
475 480Pro Thr Ser Ile Leu Asp Ala Ser 4858486PRTArtificial
SequenceH chain - HIV nef116 fusion protein. 8Gln Val Thr Leu Lys
Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln1 5 10 15Thr Leu Ser Leu
Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Gly
Leu Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45Trp Leu
Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser 50 55 60Leu
Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn Gln Val65 70 75
80Phe Leu Lys Ile Thr Ile Val Asp Thr Ala Asp Ala Ala Thr Tyr Tyr
85 90 95Cys Ala Arg Ser Ser His Tyr Tyr Gly Tyr Gly Tyr Gly Gly Tyr
Phe 100 105 110Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
Ala Lys Thr 115 120 125Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys
Ser Arg Ser Thr Ser 130 135 140Glu Ser Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu145 150 155 160Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys 195 200
205Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu
210 215 220Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
Phe Glu225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser 260 265 270Gln Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 405 410 415Val Asp Lys
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440
445Ser Leu Gly Lys Ala Ser His Thr Gln Gly Tyr Phe Pro Asp Trp Gln
450 455 460Asn Tyr Thr Pro Gly Pro Gly Val Arg Tyr Pro Leu Thr Phe
Gly Trp465 470 475 480Leu Tyr Lys Leu Ala Ser 4859488PRTArtificial
SequenceH chain - HIV nef66 fusion protein. 9Gln Val Thr Leu Lys
Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln1 5 10 15Thr Leu Ser Leu
Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Gly
Leu Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45Trp Leu
Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser 50 55 60Leu
Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn Gln Val65 70 75
80Phe Leu Lys Ile Thr Ile Val Asp Thr Ala Asp Ala Ala Thr Tyr Tyr
85 90 95Cys Ala Arg Ser Ser His Tyr Tyr Gly Tyr Gly Tyr Gly Gly Tyr
Phe 100 105 110Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
Ala Lys Thr 115 120 125Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys
Ser Arg Ser Thr Ser 130 135 140Glu Ser Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu145 150 155 160Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys 195 200
205Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu
210 215 220Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
Phe Glu225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser 260 265 270Gln Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 405 410 415Val Asp Lys
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440
445Ser Leu Gly Lys Ala Ser Val Gly Phe Pro Val Thr Pro Gln Val Pro
450 455 460Leu Arg Pro Met Thr Tyr Lys Ala Ala Val Asp Leu Ser His
Phe Leu465 470 475 480Lys Glu Lys Gly Gly Leu Ala Ser
48510487PRTArtificial SequenceH chain - HIV pol158 fusion protein.
10Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr
Ser 20 25 30Gly Met Gly Leu Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly
Leu Glu 35 40 45Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr
Asn Pro Ser 50 55 60Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser
Ser Asn Gln Val65 70 75 80Phe Leu Lys Ile Thr Ile Val Asp Thr Ala
Asp Ala Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Ser Ser His Tyr Tyr Gly
Tyr Gly Tyr Gly Gly Tyr Phe 100 105 110Asp Val Trp Gly Ala Gly Thr
Thr Val Thr Val Ser Ser Ala Lys Thr 115 120 125Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140Glu Ser Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu145 150 155
160Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
165 170 175Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser 180 185 190Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys
Thr Tyr Thr Cys 195 200 205Asn Val Asp His Lys Pro Ser Asn Thr Lys
Val Asp Lys Arg Val Glu 210 215 220Ser Lys Tyr Gly Pro Pro Cys Pro
Pro Cys Pro Ala Pro Glu Phe Glu225 230 235 240Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270Gln
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280
285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
290 295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn305
310 315 320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
Ser Ser 325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
Met Thr Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 405 410 415Val
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 420 425
430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445Ser Leu Gly Lys Ala Ser Ala Ile Phe Gln Ser Ser Met Thr
Lys Ile 450 455 460Leu Glu Pro Phe Arg Lys Gln Asn Pro Asp Ile Val
Ile Tyr Gln Tyr465 470 475 480Met Asp Asp Leu Tyr Ala Ser
4851125PRTArtificial SequenceSynthetic linker sequence. 11Ser Ser
Val Ser Pro Thr Thr Ser Val His Pro Thr Pro Thr Ser Val1 5 10 15Pro
Pro Thr Pro Thr Lys Ser Ser Pro 20 251225PRTArtificial
SequenceSynthetic linker peptide. 12Pro Thr Ser Thr Pro Ala Asp Ser
Ser Thr Ile Thr Pro Thr Ala Thr1 5 10 15Pro Thr Ala Thr Pro Thr Ile
Lys Gly 20 251325PRTArtificial SequenceSynthetic linker peptide.
13Thr Val Thr Pro Thr Ala Thr Ala Thr Pro Ser Ala Ile Val Thr Thr1
5 10 15Ile Thr Pro Thr Ala Thr Thr Lys Pro 20 251425PRTArtificial
SequenceSynthetic linker peptide. 14Thr Asn Gly Ser Ile Thr Val Ala
Ala Thr Ala Pro Thr Val Thr Pro1 5 10 15Thr Val Asn Ala Thr Pro Ser
Ala Ala 20 25152299PRTBacteroides cellulosolvens 15Met Gln Ser Pro
Arg Leu Lys Arg Lys Ile Leu Ser Val Ile Leu Ala1 5 10 15Val Cys Tyr
Ile Ile Ser Ser Phe Ser Ile Gln Phe Ala Ala Thr Pro 20 25 30Gln Val
Asn Ile Ile Ile Gly Ser Ala Gln Gly Ile Pro Gly Ser Thr 35 40 45Val
Lys Val Pro Ile Asn Leu Gln Asn Val Pro Glu Ile Gly Ile Asn 50 55
60Asn Cys Asp Phe Thr Ile Lys Phe Asp Ser Asp Ile Leu Asp Phe Asn65
70 75 80Ser Val Glu Ala Gly Asp Ile Val Pro Leu Pro Val Ala Ser Phe
Ser 85 90 95Ser Asn Asn Ser Lys Asp Ile Ile Lys Phe Leu Phe Ser Asp
Ala Thr 100 105 110Gln Gly Asn Met Pro Ile Asn Glu Asn Gly Leu Phe
Ala Val Ile Ser 115 120 125Phe Lys Ile Lys Asp Asn Ala Gln Lys Gly
Ile Ser Asn Ile Lys Val 130 135 140Ser Ser Tyr Gly Ser Phe Ser Gly
Met Ser Gly Lys Glu Met Gln Ser145 150 155 160Leu Ser Pro Thr Phe
Phe Ser Gly Ser Ile Asp Val Ser Asp Val Ser 165 170 175Thr Ser Lys
Leu Asp Val Lys Val Gly Asn Val Glu Gly Ile Ala Gly 180 185 190Thr
Glu Val Asn Val Pro Ile Thr Phe Glu Asn Val Pro Asp Asn Gly 195 200
205Ile Asn Asn Cys Asn Phe Thr Leu Ser Tyr Asp Ser Asn Ala Leu Glu
210 215 220Phe Leu Thr Thr Glu Ala Gly Asn Ile Ile Pro Leu Ala Ile
Ala Asp225 230 235 240Tyr Ser Ser Tyr Arg Ser Met Glu Gly Lys Ile
Lys Phe Leu Phe Ser 245 250 255Asp Ser Ser Gln Gly Thr Arg Ser Ile
Lys Asn Asp Gly Val Phe Ala 260 265 270Asn Ile Lys Phe Lys Ile Lys
Gly Asn Ala Ile Arg Asp Thr Tyr Arg 275 280 285Ile Asp Leu Ser Glu
Leu Gly Ser Phe Ser Ser Lys Gln Asn Asn Asn 290 295 300Leu Lys Ser
Ile Ala Thr Gln Phe Leu Ser Gly Ser Val Asn Val Lys305 310 315
320Asp Ile Glu Ser Ser Val Ser Pro Thr Thr Ser Val His Pro Thr Pro
325 330 335Thr Ser Val Pro Pro Thr Pro Thr Lys Ser Ser Pro Gly Asn
Lys Met 340 345 350Lys Ile Gln Ile Gly Asp Val Lys Ala Asn Gln Gly
Asp Thr Val Ile 355 360 365Val Pro Ile Thr Phe Asn Glu Val Pro Val
Met Gly Val Asn Asn Cys 370 375 380Asn Phe Thr Leu Ala Tyr Asp Lys
Asn Ile Met Glu Phe Ile Ser Ala385 390 395 400Asp Ala Gly Asp Ile
Val Thr Leu Pro Met Ala Asn Tyr Ser Tyr Asn 405 410 415Met Pro Ser
Asp Gly Leu Val Lys Phe Leu Tyr Asn Asp Gln Ala Gln 420 425 430Gly
Ala Met Ser Ile Lys Glu Asp Gly Thr Phe Ala Asn Val Lys Phe 435 440
445Lys Ile Lys Gln Ser Ala Ala Phe Gly Lys Tyr Ser Val Gly Ile Lys
450 455 460Ala Ile Gly Ser Ile Ser Ala Leu Ser Asn Ser Lys Leu Ile
Pro Ile465 470 475 480Glu Ser Ile Phe Lys Asp Gly Ser Ile Thr Val
Thr Asn Lys Pro Ile 485 490 495Val Asn Ile Glu Ile Gly Lys Val Lys
Val Lys Ala Gly Asp Lys Ile 500 505 510Lys Val Pro Val Glu Ile Lys
Asp Ile Pro Ser Ile Gly Ile Asn Asn 515 520 525Cys Asn Phe Thr Leu
Lys Tyr Asn Ser Asn Val Leu Lys Tyr Val Ser 530 535 540Asn Glu Ala
Gly Thr Ile Val Pro Ala Pro Leu Ala Asn Leu Ser Ile545 550 555
560Asn Lys Pro Asp Glu Gly Ile Ile Lys Leu Leu Phe Ser Asp Ala Ser
565 570 575Gln Gly Gly Met Pro Ile Lys Asp Asn Gly Ile Phe Val Asn
Leu Glu 580 585 590Phe Gln Ala Val Asn Asp Ala Asn Ile Gly Val Tyr
Gly Leu Glu Leu 595 600 605Asp Thr Ile Gly Ala Phe Ser Gly Ile Ser
Ser Ala Lys Met Thr Ser 610 615 620Ile Glu Pro Gln Phe Asn Asn Gly
Ser Ile Glu Ile Phe Asn Ser Ala625 630 635 640Gln Thr Pro Val Pro
Ser Asn Thr Glu Val Gln Thr Pro Thr Asn Thr 645 650 655Ile Ser Val
Thr Pro Thr Asn Asn Ser Thr Pro Thr Asn Asn Ser Thr 660 665 670Pro
Lys Pro Asn Pro Leu Tyr Asn Leu Asn Val Asn Ile Gly Glu Ile 675 680
685Ser Gly Glu Ala Gly Gly Val Ile Glu Val Pro Ile Glu Phe Lys Asn
690 695 700Val Pro Asp Phe Gly Ile Asn Asn Cys Asp Phe Ser Val Lys
Tyr Asp705 710 715 720Lys Ser Ile Phe Glu Tyr Val Thr Tyr Glu Ala
Gly Ser Ile Val Lys 725 730 735Asp Ser Ile Val Asn Leu Ala Cys Met
Glu Asn Ser Gly Ile Ile Asn 740 745 750Leu Leu Phe Asn Asp Ala Thr
Gln Ser Ser Ser Pro Ile Lys Asn Asn 755 760 765Gly Val Phe Ala Lys
Leu Lys Phe Lys Ile Asn Ser Asn Ala Ala Ser 770 775 780Gly Thr Tyr
Gln Ile Asn Ala Glu Gly Tyr Gly Lys Phe Ser Gly Asn785 790 795
800Leu Asn Gly Lys Leu Thr Ser Ile Asn Pro Ile Phe Glu Asn Gly Ile
805 810 815Ile Asn Ile Gly Asn Val Thr Val Lys Pro Thr Ser Thr Pro
Ala Asp 820 825 830Ser Ser Thr Ile Thr Pro Thr Ala Thr Pro Thr Ala
Thr Pro Thr Ile 835 840 845Lys Gly Thr Pro Thr Val Thr Pro Ile Tyr
Trp Met Asn Val Leu Ile 850 855 860Gly Asn Met Asn Ala Ala Ile Gly
Glu Glu Val Val Val Pro Ile Glu865 870 875 880Phe Lys Asn Val Pro
Pro Phe Gly Ile Asn Asn Cys Asp Phe Lys Leu 885 890 895Val Tyr Asp
Ser Asn Ala Leu Glu Leu Lys Lys Val Glu Ala Gly Asp 900 905 910Ile
Val Pro Glu Pro Leu Ala Asn Leu Ser Ser Asn Lys Ser Glu Gly 915 920
925Lys Ile Gln Phe Leu Phe Asn Asp Ala Ser Gln Gly Ser Met Gln Ile
930 935 940Glu Asn Gly Gly Val Phe Ala Lys Ile Thr Phe Lys Val Lys
Ser Thr945 950 955 960Ala Ala Ser Gly Ile Tyr Asn Ile Arg Lys Asp
Ser Val Gly Ser Phe 965 970 975Ser Gly Leu Ile Asp Asn Lys Met Thr
Ser Ile Gly Pro Lys Phe Thr 980 985 990Asp Gly Ser Ile Val Val Gly
Thr Val Thr Pro Thr Ala Thr Ala Thr 995 1000 1005Pro Ser Ala Ile
Val Thr Thr Ile Thr Pro Thr Ala Thr Thr Lys 1010 1015 1020Pro Ile
Ala Thr Pro Thr Ile Lys Gly Thr Pro Thr Ala Thr Pro 1025 1030
1035Met Tyr Trp Met Asn Val Val Ile Gly Lys Met Asn Ala Glu Val
1040 1045 1050Gly Gly Glu Val Val Val Pro Ile Glu Phe Asn Asn Val
Pro Ser 1055 1060 1065Phe Gly Ile Asn Asn Cys Asp Phe Lys Leu Val
Tyr Asp Ala Thr 1070 1075 1080Ala Leu Glu Leu Lys Asn Val Glu Ala
Gly Asp Ile Ile Lys Thr 1085 1090 1095Pro Leu Ala Asn Phe Ser Asn
Asn Lys Ser Glu Glu Gly Lys Ile 1100 1105 1110Ser Phe Leu Phe Asn
Asp Ala Ser Gln Gly Ser Met Gln Ile Glu 1115 1120 1125Asn Gly Gly
Val Phe Ala Lys Ile Thr Phe Lys Val Lys Ser Thr 1130 1135 1140Thr
Ala Thr Gly Val Tyr Asp Leu Arg Lys Asp Leu Val Gly Ser 1145 1150
1155Phe Ser Gly Leu Lys Asp Asn Lys Met Thr Ser Ile Gly Ala Glu
1160 1165 1170Phe Thr Asn Gly Ser Ile Thr Val Ala Ala Thr Ala Pro
Thr Val 1175 1180 1185Thr Pro Thr Val Asn Ala Thr Pro Ser Ala Ala
Thr Pro Thr Val 1190 1195 1200Thr Pro Thr Ala Thr Ala Thr Pro Ser
Val Thr Ile Pro Thr Val 1205 1210 1215Thr Pro Thr Ala Thr Ala Thr
Pro Ser Val Thr Ile Pro Thr Val 1220 1225 1230Thr Pro Thr Ala Thr
Ala Thr Pro Ser Ala Ala Thr Pro Thr Val 1235 1240 1245Thr Pro Thr
Ala Thr Ala Thr Pro Ser Val Thr Ile Pro Thr Val 1250 1255 1260Thr
Pro Thr Val Thr Ala Thr Pro Ser Asp Thr Ile Pro Thr Val 1265 1270
1275Thr Pro Thr Ala Thr Ala Thr Pro Ser Ala Ile Val Thr Thr Ile
1280 1285 1290Thr Pro Thr Ala Thr Ala Lys Pro Ile Ala Thr Pro Thr
Ile Lys 1295 1300 1305Gly Thr Pro Thr Ala Thr Pro Met Tyr Trp Met
Asn Val Val Ile 1310 1315 1320Gly Lys Met Asn Ala Glu Val Gly Gly
Glu Val Val Val Pro Ile 1325 1330 1335Glu Phe Lys Asn Val Pro Ser
Phe Gly Ile Asn Asn Cys Asp Phe 1340 1345 1350Lys Leu Val Tyr Asp
Ala Thr Ala Leu Glu Leu Lys Asn Val Glu 1355 1360 1365Ala Gly Asp
Ile Ile Lys Thr Pro Leu Ala Asn Phe Ser Asn Asn 1370 1375 1380Lys
Ser Glu Glu Gly Lys Ile Ser Phe Leu Phe Asn Asp Ala Ser 1385 1390
1395Gln Gly Ser Met Gln Ile Glu Asn Gly Gly Val Ser Ala Lys Ile
1400 1405 1410Thr Phe Lys Val Lys Ser Thr Thr Ala Ile Gly Val Tyr
Asp Ile 1415 1420 1425Arg Lys Asp Leu Ile Gly Ser Phe Ser Gly Leu
Lys Asp Ser Lys 1430 1435 1440Met Thr Ser Ile Gly Ala Glu Phe Thr
Asn Gly Ser Ile Thr Val 1445 1450 1455Ala Thr Thr Ala Pro Thr Val
Thr Pro Thr Ala Thr Ala Thr Pro 1460 1465 1470Ser Val Thr Ile Pro
Thr Val Thr Pro Thr Ala Thr Ala Thr Pro 1475 1480 1485Gly Thr Ala
Thr Pro Gly Thr Ala Thr Pro Thr Ala Thr Ala Thr 1490 1495 1500Pro
Gly Ala Ala Thr Pro Thr Glu Thr Ala Thr Pro Ser Val Met 1505 1510
1515Ile Pro Thr Val Thr Pro Thr Ala Thr Ala Thr Pro Thr Ala Thr
1520 1525 1530Ala Thr Pro Thr Val Lys Gly Thr Pro Thr Ile Lys Pro
Val Tyr 1535 1540 1545Lys Met Asn Val Val Ile Gly Arg Val Asn Val
Val Ala Gly Glu 1550 1555 1560Glu Val Val Val Pro Val Glu Phe Lys
Asn Ile Pro Ala Ile Gly 1565 1570 1575Val Asn Asn Cys Asn Phe Val
Leu Glu Tyr Asp Ala Asn Val Leu 1580 1585 1590Glu Val Lys Lys Val
Asp Ala Gly Glu Ile Val Pro Asp Ala Leu 1595 1600 1605Ile Asn Phe
Gly Ser Asn Asn Ser Asp Glu Gly Lys Val Tyr Phe 1610 1615 1620Leu
Phe Asn Asp Ala Leu Gln Gly Arg Met Gln Ile Ala Asn Asp 1625 1630
1635Gly Ile Phe Ala Asn Ile Thr Phe Lys Val Lys Ser Ser Ala Ala
1640 1645 1650Ala Gly Ile Tyr Asn Ile Arg Lys Asp Ser Val Gly Ala
Phe Ser 1655 1660 1665Gly Leu Val Asp Lys Leu Val Pro Ile Ser Ala
Glu Phe Thr Asp 1670 1675 1680Gly Ser Ile Ser Val Glu Ser Ala Lys
Ser Thr Pro Thr Ala Thr 1685 1690 1695Ala Thr Gly Thr Asn Val Thr
Pro Thr Val Ala Ala Thr Val Thr 1700 1705 1710Pro Thr Ala Thr Pro
Ala Ser Thr Thr Pro Thr Ala Thr Pro Thr 1715 1720 1725Ala Thr Ser
Thr Val Lys Gly Thr Pro Thr Ala Thr Pro Leu Tyr 1730 1735 1740Ser
Met Asn Val Ile Ile Gly Lys Val Asn Ala Glu Ala Ser Gly 1745 1750
1755Glu Val Val Val Pro Val Glu Phe Lys Asp Val Pro Ser Ile Gly
1760 1765 1770Ile Asn Asn Cys Asn Phe Ile Leu Glu Tyr Asp Ala Ser
Ala Leu 1775 1780 1785Glu Leu Asp Ser Ala Glu Ala Gly Glu Ile Val
Pro Val Pro Leu 1790 1795 1800Gly Asn Phe Ser Ser Asn Asn Lys Asp
Glu Gly Lys Ile Tyr Phe 1805 1810 1815Leu Phe Ser Asp Gly Thr Gln
Gly Arg Met Gln Ile Val Asn Asp 1820 1825 1830Gly Ile Phe Ala Lys
Ile Lys Phe Lys Val Lys Ser Thr Ala Ser 1835 1840 1845Asp Gly Thr
Tyr Tyr Ile Arg Lys Asp Ser Val Gly Ala Phe Ser 1850 1855 1860Gly
Leu Ile Glu Lys Lys Ile Ile Lys Ile Gly Ala Glu Phe Thr 1865 1870
1875Asp Gly Ser Ile Thr Val Arg Ser Leu Thr Pro Thr Pro Thr Val
1880 1885 1890Thr Pro Asn Val Ala Ser Pro Thr Pro Thr Lys Val Val
Ala Glu 1895 1900 1905Pro Thr Ser Asn Gln Pro Ala Gly Pro Gly Pro
Ile Thr Gly Thr 1910 1915 1920Ile Pro Thr Ala Thr Thr Thr Ala Thr
Ala Thr Pro Thr Lys Ala 1925 1930 1935Ser Val Ala Thr Ala Thr Pro
Thr Ala Thr Pro Ile Val Val Val 1940 1945 1950Glu Pro Thr Ile Val
Arg Pro Gly Tyr Asn Lys Asp Ala Asp Leu 1955 1960 1965Ala Val Phe
Ile Ser Ser Asp Lys Ser Arg Tyr Glu Glu Ser Ser 1970 1975 1980Ile
Ile Thr Tyr Ser Ile Glu Tyr Lys Asn Ile Gly Lys Val Asn 1985 1990
1995Ala Thr Asn Val Lys Ile Ala Ala Gln Ile Pro Lys Phe Thr Lys
2000 2005 2010Val Tyr Asp Ala Ala Lys Gly Ala Val Lys Gly Ser Glu
Ile Val 2015 2020 2025Trp Met Ile Gly Asn Leu Ala Val Gly Glu Ser
Tyr Thr Lys Glu 2030 2035 2040Tyr Lys Val Lys Val Asp Ser Leu Thr
Lys Ser Glu Glu Tyr Thr 2045 2050 2055Asp Asn Thr Val Thr Ile Ser
Ser Asp Gln Thr Val Asp Ile Pro 2060 2065 2070Glu Asn Ile Thr Thr
Gly Asn Asp Asp Lys Ser Thr Ile Arg Val 2075 2080 2085Met Leu Tyr
Ser Asn Arg Phe Thr Pro Gly Ser His Ser Ser Tyr 2090 2095 2100Ile
Leu Gly Tyr Lys Asp Lys Thr Phe Lys Pro Lys Gln Asn Val 2105 2110
2115Thr Arg Ala Glu Val Ala Ala Met Phe Ala Arg Ile Met Gly Leu
2120 2125 2130Thr Val Lys Asp Gly Ala Lys Ser Ser Tyr Lys Asp Val
Ser Asn 2135
2140 2145Lys His Trp Ala Leu Lys Tyr Ile Glu Ala Val Thr Lys Ser
Gly 2150 2155 2160Ile Phe Lys Gly Tyr Lys Asp Ser Thr Phe His Pro
Asn Ala Pro 2165 2170 2175Ile Thr Arg Ala Glu Leu Ser Thr Val Ile
Phe Asn Tyr Leu His 2180 2185 2190Leu Asn Asn Ile Ala Pro Ser Lys
Val His Phe Thr Asp Ile Asn 2195 2200 2205Lys His Trp Ala Lys Asn
Tyr Ile Glu Glu Ile Tyr Arg Phe Lys 2210 2215 2220Leu Ile Gln Gly
Tyr Ser Asp Gly Ser Phe Lys Pro Asn Asn Asn 2225 2230 2235Ile Thr
Arg Ala Glu Val Val Thr Met Ile Asn Arg Met Leu Tyr 2240 2245
2250Arg Gly Pro Leu Lys Val Lys Val Gly Ser Phe Pro Asp Val Ser
2255 2260 2265Pro Lys Tyr Trp Ala Tyr Gly Asp Ile Glu Glu Ala Ser
Arg Asn 2270 2275 2280His Lys Tyr Thr Arg Asp Glu Lys Asp Gly Ser
Glu Ile Leu Ile 2285 2290 2295Glu16541PRTArtificial Sequenceheavy
chain (H) - HIV gag17-nef66-nef116 peptides fusion protein. 16Gln
Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln1 5 10
15Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser
20 25 30Gly Met Gly Leu Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu
Glu 35 40 45Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn
Pro Ser 50 55 60Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser
Asn Gln Val65 70 75 80Phe Leu Lys Ile Thr Ile Val Asp Thr Ala Asp
Ala Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Ser Ser His Tyr Tyr Gly Tyr
Gly Tyr Gly Gly Tyr Phe 100 105 110Asp Val Trp Gly Ala Gly Thr Thr
Val Thr Val Ser Ser Ala Lys Thr 115 120 125Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140Glu Ser Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu145 150 155 160Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170
175Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
180 185 190Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr
Thr Cys 195 200 205Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys Arg Val Glu 210 215 220Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys
Pro Ala Pro Glu Phe Glu225 230 235 240Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270Gln Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 290 295
300Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ser Ser 325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Gln
Glu Glu Met Thr Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 405 410
415Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
420 425 430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu 435 440 445Ser Leu Gly Lys Ala Ser Glu Lys Ile Arg Leu Arg
Pro Gly Gly Lys 450 455 460Lys Lys Tyr Lys Leu Lys His Ile Val Ala
Ser Val Gly Phe Pro Val465 470 475 480Thr Pro Gln Val Pro Leu Arg
Pro Met Thr Tyr Lys Ala Ala Val Asp 485 490 495Leu Ser His Phe Leu
Lys Glu Lys Gly Gly Leu Ala Ser His Thr Gln 500 505 510Gly Tyr Phe
Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro Gly Val Arg 515 520 525Tyr
Pro Leu Thr Phe Gly Trp Leu Tyr Lys Leu Ala Ser 530 535
54017534PRTArtificial SequenceH chain - HIV gag17-nef116 peptides
fusion protein. 17Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu
Gln Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe
Ser Leu Ser Thr Ser 20 25 30Gly Met Gly Leu Ser Trp Ile Arg Gln Pro
Ser Gly Lys Gly Leu Glu 35 40 45Trp Leu Ala His Ile Tyr Trp Asp Asp
Asp Lys Arg Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg Leu Thr Ile Ser
Lys Asp Thr Ser Ser Asn Gln Val65 70 75 80Phe Leu Lys Ile Thr Ile
Val Asp Thr Ala Asp Ala Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Ser Ser
His Tyr Tyr Gly Tyr Gly Tyr Gly Gly Tyr Phe 100 105 110Asp Val Trp
Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr 115 120 125Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135
140Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu145 150 155 160Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His 165 170 175Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser 180 185 190Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Lys Thr Tyr Thr Cys 195 200 205Asn Val Asp His Lys Pro
Ser Asn Thr Lys Val Asp Lys Arg Val Glu 210 215 220Ser Lys Tyr Gly
Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu225 230 235 240Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250
255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ser Ser 325 330 335Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 355 360 365Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375
380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Arg Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Leu Gly Lys Ala Ser
Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys 450 455 460Lys Lys Tyr Lys
Leu Lys His Ile Val Ala Ser Ser Ser Val Ser Pro465 470 475 480Thr
Thr Ser Val His Pro Thr Pro Thr Ser Val Pro Pro Thr Pro Thr 485 490
495Lys Ser Ser Pro Ala Ser His Thr Gln Gly Tyr Phe Pro Asp Trp Gln
500 505 510Asn Tyr Thr Pro Gly Pro Gly Val Arg Tyr Pro Leu Thr Phe
Gly Trp 515 520 525Leu Tyr Lys Leu Ala Ser 53018626PRTArtificial
SequenceH chain - HIV peptides string of gag17-gag253- nef66 fusion
protein. 18Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro
Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu
Ser Thr Ser 20 25 30Gly Met Gly Leu Ser Trp Ile Arg Gln Pro Ser Gly
Lys Gly Leu Glu 35 40 45Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys
Arg Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp
Thr Ser Ser Asn Gln Val65 70 75 80Phe Leu Lys Ile Thr Ile Val Asp
Thr Ala Asp Ala Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Ser Ser His Tyr
Tyr Gly Tyr Gly Tyr Gly Gly Tyr Phe 100 105 110Asp Val Trp Gly Ala
Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr 115 120 125Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140Glu
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu145 150
155 160Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His 165 170 175Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser 180 185 190Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Lys Thr Tyr Thr Cys 195 200 205Asn Val Asp His Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg Val Glu 210 215 220Ser Lys Tyr Gly Pro Pro Cys
Pro Pro Cys Pro Ala Pro Glu Phe Glu225 230 235 240Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265
270Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro Ser Ser 325 330 335Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr Leu Pro
Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 355 360 365Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro385 390
395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu 435 440 445Ser Leu Gly Lys Ala Ser Gln Thr Pro
Thr Asn Thr Ile Ser Val Thr 450 455 460Pro Thr Asn Asn Ser Thr Pro
Thr Asn Asn Ser Asn Pro Lys Pro Asn465 470 475 480Pro Ala Ser Glu
Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr 485 490 495Lys Leu
Lys His Ile Val Ala Ser Ser Ser Val Ser Pro Thr Thr Ser 500 505
510Val His Pro Thr Pro Thr Ser Val Pro Pro Thr Pro Thr Lys Ser Ser
515 520 525Pro Ala Ser Asn Pro Pro Ile Pro Val Gly Glu Ile Tyr Lys
Arg Trp 530 535 540Ile Ile Leu Gly Leu Asn Lys Ile Val Arg Met Tyr
Ser Pro Thr Ser545 550 555 560Ile Leu Asp Ala Ser Pro Thr Ser Thr
Pro Ala Asp Ser Ser Thr Ile 565 570 575Thr Pro Thr Ala Thr Pro Thr
Ala Thr Pro Thr Ile Lys Gly Ala Ser 580 585 590Val Gly Phe Pro Val
Thr Pro Gln Val Pro Leu Arg Pro Met Thr Tyr 595 600 605Lys Ala Ala
Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu 610 615 620Ala
Ser62519608PRTArtificial SequenceH chain - HIV peptides string of
pol158-gag17- nef66-nef116-gag253 fusion protein. 19Gln Val Thr Leu
Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln1 5 10 15Thr Leu Ser
Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met
Gly Leu Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45Trp
Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser 50 55
60Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn Gln Val65
70 75 80Phe Leu Lys Ile Thr Ile Val Asp Thr Ala Asp Ala Ala Thr Tyr
Tyr 85 90 95Cys Ala Arg Ser Ser His Tyr Tyr Gly Tyr Gly Tyr Gly Gly
Tyr Phe 100 105 110Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser
Ser Ala Lys Thr 115 120 125Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Cys Ser Arg Ser Thr Ser 130 135 140Glu Ser Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu145 150 155 160Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys 195 200
205Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu
210 215 220Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
Phe Glu225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser 260 265 270Gln Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 405 410 415Val Asp Lys
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440
445Ser Leu Gly Lys Ala Ser Ala Ile Phe Gln Ser Ser Met Thr Lys Ile
450 455 460Leu Glu Pro Phe Arg Lys Gln Asn Pro Asp Ile Val Ile Tyr
Gln Tyr465 470 475 480Met Asp Asp Leu Tyr Ala Ser Glu Lys Ile Arg
Leu Arg Pro Gly Gly 485 490 495Lys Lys Lys Tyr Lys Leu Lys His Ile
Val Ala Ser Val Gly Phe Pro 500 505 510Val Thr Pro Gln Val Pro Leu
Arg Pro Met Thr Tyr Lys Ala Ala Val 515 520 525Asp Leu Ser His Phe
Leu Lys Glu Lys Gly Gly Leu Ala Ser His Thr 530 535 540Gln Gly Tyr
Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro Gly Val545 550 555
560Arg Tyr Pro Leu Thr Phe Gly Trp Leu Tyr Lys Leu Ala Ser Asn Pro
565 570 575Pro Ile Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu
Gly Leu 580 585 590Asn Lys Ile Val Arg Met Tyr Ser Pro Thr Ser Ile
Leu Asp Ala Ser 595 600 60520745PRTArtificial SequenceH chain - HIV
peptides string of gag17-gag253- nef66-nef116-pol158 fusion
protein. 20Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro
Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu
Ser Thr Ser 20 25 30Gly Met Gly Leu Ser Trp Ile Arg Gln Pro Ser Gly
Lys Gly Leu Glu 35 40 45Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys
Arg Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp
Thr Ser Ser Asn Gln Val65 70 75 80Phe Leu Lys Ile Thr Ile Val Asp
Thr Ala Asp Ala Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Ser Ser His Tyr
Tyr Gly Tyr Gly Tyr Gly Gly Tyr Phe 100 105 110Asp Val Trp Gly Ala
Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr 115 120 125Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140Glu
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu145 150
155 160Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His 165 170 175Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser 180 185 190Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Lys Thr Tyr Thr Cys 195 200 205Asn Val Asp His Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg Val Glu 210 215 220Ser Lys Tyr Gly Pro Pro Cys
Pro Pro Cys Pro Ala Pro Glu Phe Glu225 230 235 240Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265
270Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro Ser Ser 325 330 335Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr Leu Pro
Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 355 360 365Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro385 390
395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu 435 440 445Ser Leu Gly Lys Ala Ser Gln Thr Pro
Thr Asn Thr Ile Ser Val Thr 450 455 460Pro Thr Asn Asn Ser Thr Pro
Thr Asn Asn Ser Asn Pro Lys Pro Asn465 470 475 480Pro Ala Ser Glu
Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr 485 490 495Lys Leu
Lys His Ile Val Ala Ser Ser Ser Val Ser Pro Thr Thr Ser 500 505
510Val His Pro Thr Pro Thr Ser Val Pro Pro Thr Pro Thr Lys Ser Ser
515 520 525Pro Ala Ser Asn Pro Pro Ile Pro Val Gly Glu Ile Tyr Lys
Arg Trp 530 535 540Ile Ile Leu Gly Leu Asn Lys Ile Val Arg Met Tyr
Ser Pro Thr Ser545 550 555 560Ile Leu Asp Ala Ser Pro Thr Ser Thr
Pro Ala Asp Ser Ser Thr Ile 565 570 575Thr Pro Thr Ala Thr Pro Thr
Ala Thr Pro Thr Ile Lys Gly Ala Ser 580 585 590His Thr Gln Gly Tyr
Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro 595 600 605Gly Val Arg
Tyr Pro Leu Thr Phe Gly Trp Leu Tyr Lys Leu Ala Ser 610 615 620Thr
Val Thr Pro Thr Ala Thr Ala Thr Pro Ser Ala Ile Val Thr Thr625 630
635 640Ile Thr Pro Thr Ala Thr Thr Lys Pro Ala Ser Val Gly Phe Pro
Val 645 650 655Thr Pro Gln Val Pro Leu Arg Pro Met Thr Tyr Lys Ala
Ala Val Asp 660 665 670Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu
Ala Ser Thr Asn Gly 675 680 685Ser Ile Thr Val Ala Ala Thr Ala Pro
Thr Val Thr Pro Thr Val Asn 690 695 700Ala Thr Pro Ser Ala Ala Ala
Ser Ala Ile Phe Gln Ser Ser Met Thr705 710 715 720Lys Ile Leu Glu
Pro Phe Arg Lys Gln Asn Pro Asp Ile Val Ile Tyr 725 730 735Gln Tyr
Met Asp Asp Leu Tyr Ala Ser 740 7452172DNAArtificial SequenceLinker
and antigen coding sequences. 21gctagtgaga agatccggct gcggcccggc
ggcaagaaga agtacaagct gaagcacatc 60gtggctagct ga
7222111DNAArtificial SequenceAntigen and linker coding sequence.
22gctagtgtgg gcttccccgt gaccccccag gtgcccctgc ggcccatgac ctacaaggcc
60gccgtggacc tgagccactt cctgaaggag aagggcggcc tggctagctg a
11123108DNAArtificial SequenceAntigen and linker coding sequence.
23gctagtgcca tcttccagag cagcatgacc aagatcctgg agcccttccg gaagcagaac
60cccgacatcg tgatctacca gtacatggac gacctgtacg ctagctga
10824198DNAArtificial SequenceAntigen and linker coding sequence.
24gctagtcaga cccccaccaa caccatcagc gtgaccccca ccaacaacag cacccccacc
60aacaacagca accccaagcc caaccccgct agtaaccccc ccatccccgt gggcgagatc
120tacaagcggt ggatcatcct gggcctgaac aagatcgtgc ggatgtacag
ccccaccagc 180atcctggacg ctagctga 19825255DNAArtificial
SequenceAntigen and linker coding sequence. 25gctagtcaga cccccaccaa
caccatcagc gtgaccccca ccaacaacag cacccccacc 60aacaacagca accccaagcc
caaccccgct agtgagaaga tccggctgcg gcccggcggc 120aagaagaagt
acaagctgaa gcacatcgtg gctagtcaca cccagggcta cttccccgac
180tggcagaact acacccccgg ccccggcgtg cggtaccccc tgaccttcgg
ctggctgtac 240aagctggcta gctga 25526882DNAArtificial
SequenceANtigen and linker coding sequence. 26gctagtcaga cccccaccaa
caccatcagc gtgaccccca ccaacaacag cacccccacc 60aacaacagca accccaagcc
caaccccgct agtgagaaga tccggctgcg gcccggcggc 120aagaagaagt
acaagctgaa gcacatcgtg gctagtagca gcgtgagccc caccaccagc
180gtgcacccca cccccaccag cgtgcccccc acccccacca agagcagccc
cgctagtaac 240ccccccatcc ccgtgggcga gatctacaag cggtggatca
tcctgggcct gaacaagatc 300gtgcggatgt acagccccac cagcatcctg
gacgctagtc ccaccagcac ccccgccgac 360agcagcacca tcacccccac
cgccaccccc accgccaccc ccaccatcaa gggcgctagt 420cacacccagg
gctacttccc cgactggcag aactacaccc ccggccccgg cgtgcggtac
480cccctgacct tcggctggct gtacaagctg gctagtaccg tgacccccac
cgccaccgcc 540acccccagcg ccatcgtgac caccatcacc cccaccgcca
ccaccaagcc cgctagtgtg 600ggcttccccg tgacccccca ggtgcccctg
cggcccatga cctacaaggc cgccgtggac 660ctgagccact tcctgaagga
gaagggcggc ctggctagta ccaacggcag catcaccgtg 720gccgccaccg
cccccaccgt gacccccacc gtgaacgcca cccccagcgc cgccgctagt
780gccatcttcc agagcagcat gaccaagatc ctggagccct tccggaagca
gaaccccgac 840atcgtgatct accagtacat ggacgacctg tacgctagct ga
88227108DNAArtificial SequenceAntigen and linker coding sequence.
27gctagtgtgg gcttccccgt gaccccccag gtgcccctgc ggcccatgac ctacaaggcc
60gccgtggacc tgagccactt cctgaaggag aagggcggcc tggctagc
10828102DNAArtificial SequenceAntigen and linker coding sequence.
28gctagtcaca cccagggcta cttccccgac tggcagaact acacccccgg ccccggcgtg
60cggtaccccc tgaccttcgg ctggctgtac aagctggcta gc
1022969DNAArtificial SequenceAntigen and linker coding sequence.
29gctagtgaga agatccggct gcggcccggc ggcaagaaga agtacaagct gaagcacatc
60gtggctagc 6930108DNAArtificial SequenceAntigen and linker coding
sequence. 30gctagtaacc cccccatccc cgtgggcgag atctacaagc ggtggatcat
cctgggcctg 60aacaagatcg tgcggatgta cagccccacc agcatcctgg acgctagc
10831105DNAArtificial SequenceAntigen and linker coding sequence.
31gctagtgcca tcttccagag cagcatgacc aagatcctgg agcccttccg gaagcagaac
60cccgacatcg tgatctacca gtacatggac gacctgtacg ctagc
1053287DNAArtificial SequenceAntigen and linker coding sequence.
32gctagtagca gcgtgagccc caccaccagc gtgcacccca cccccaccag cgtgcccccc
60acccccacca agagcagccc cgctagc 873387DNAArtificial SequenceAntigen
and linker coding sequence. 33gctagtccca ccagcacccc cgccgacagc
agcaccatca cccccaccgc cacccccacc 60gccaccccca ccatcaaggg cgctagc
873487DNAArtificial SequenceANtigen and linker coding sequence.
34gctagtaccg tgacccccac cgccaccgcc acccccagcg ccatcgtgac caccatcacc
60cccaccgcca ccaccaagcc cgctagc 873587DNAArtificial SequenceAntigen
and linker coding sequence. 35gctagtacca acggcagcat caccgtggcc
gccaccgccc ccaccgtgac ccccaccgtg 60aacgccaccc ccagcgccgc cgctagc
8736745PRTArtificial SequenceSynthesized HIV peptide vaccine. 36Gln
Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln1 5 10
15Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser
20 25 30Gly Met Gly Leu Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu
Glu 35 40 45Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn
Pro Ser 50 55 60Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser
Asn Gln Val65 70 75 80Phe Leu Lys Ile Thr Ile Val Asp Thr Ala Asp
Ala Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Ser Ser His Tyr Tyr Gly Tyr
Gly Tyr Gly Gly Tyr Phe 100 105 110Asp Val Trp Gly Ala Gly Thr Thr
Val Thr Val Ser Ser Ala Lys Thr 115 120 125Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140Glu Ser Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu145 150 155 160Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170
175Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
180 185 190Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr
Thr Cys 195 200 205Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys Arg Val Glu 210 215 220Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys
Pro Ala Pro Glu Phe Glu225 230 235 240Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270Gln Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 290 295
300Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ser Ser 325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Gln
Glu Glu Met Thr Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 405 410
415Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
420 425 430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu 435 440 445Ser Leu Gly Lys Ala Ser Gln Thr Pro Thr Asn Thr
Ile Ser Val Thr 450 455 460Pro Thr Asn Asn Ser Thr Pro Thr Asn Asn
Ser Asn Pro Lys Pro Asn465 470 475 480Pro Ala Ser Glu Lys Ile Arg
Leu Arg Pro Gly Gly Lys Lys Lys Tyr 485 490 495Lys Leu Lys His Ile
Val Ala Ser Ser Ser Val Ser Pro Thr Thr Ser 500 505 510Val His Pro
Thr Pro Thr Ser Val Pro Pro Thr Pro Thr Lys Ser Ser 515 520 525Pro
Ala Ser Asn Pro Pro Ile Pro Val Gly Glu Ile Tyr Lys Arg Trp 530 535
540Ile Ile Leu Gly Leu Asn Lys Ile Val Arg Met Tyr Ser Pro Thr
Ser545 550 555 560Ile Leu Asp Ala Ser Pro Thr Ser Thr Pro Ala Asp
Ser Ser Thr Ile 565 570 575Thr Pro Thr Ala Thr Pro Thr Ala Thr Pro
Thr Ile Lys Gly Ala Ser 580 585 590His Thr Gln Gly Tyr Phe Pro Asp
Trp Gln Asn Tyr Thr Pro Gly Pro 595 600 605Gly Val Arg Tyr Pro Leu
Thr Phe Gly Trp Leu Tyr Lys Leu Ala Ser 610 615 620Thr Val Thr Pro
Thr Ala Thr Ala Thr Pro Ser Ala Ile Val Thr Thr625 630 635 640Ile
Thr Pro Thr Ala Thr Thr Lys Pro Ala Ser Val Gly Phe Pro Val 645 650
655Thr Pro Gln Val Pro Leu Arg Pro Met Thr Tyr Lys Ala Ala Val Asp
660 665 670Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu Ala Ser Thr
Asn Gly 675 680 685Ser Ile Thr Val Ala Ala Thr Ala Pro Thr Val Thr
Pro Thr Val Asn 690 695 700Ala Thr Pro Ser Ala Ala Ala Ser Ala Ile
Phe Gln Ser Ser Met Thr705 710 715 720Lys Ile Leu Glu Pro Phe Arg
Lys Gln Asn Pro Asp Ile Val Ile Tyr 725 730 735Gln Tyr Met Asp Asp
Leu Tyr Ala Ser 740 74537739PRTArtificial SequenceSynthetic HIV
peptide. 37Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe
Ser Asp Tyr 20 25 30Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg
Leu Glu Trp Val 35 40 45Ala Tyr Ile Asn Ser Gly Gly Gly Ser Thr Tyr
Tyr Pro Asp Thr Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Ser Arg Leu Lys Ser
Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Arg Gly Leu Pro Phe
His Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Ser Val Thr Val
Ser Ser Ala Lys Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala
Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150
155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
Val Asp His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Ser Lys Tyr Gly Pro Pro 210 215 220Cys Pro Pro Cys Pro Ala Pro
Glu Phe Glu Gly Gly Pro Ser Val Phe225 230 235 240Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255Glu Val
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265
270Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn Lys
Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360
365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp385 390 395 400Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
Asp Lys Ser Arg Trp 405 410 415Gln Glu Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His 420 425 430Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Leu Gly Lys Ala Ser 435 440 445Gln Thr Pro Thr Asn
Thr Ile Ser Val Thr Pro Thr Asn Asn Ser Thr 450 455 460Pro Thr Asn
Asn Ser Asn Pro Lys Pro Asn Pro Ala Ser Glu Lys Ile465 470 475
480Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Lys Leu Lys His Ile Val
485 490 495Ala Ser Ser Ser Val Ser Pro Thr Thr Ser Val His Pro Thr
Pro Thr 500 505 510Ser Val Pro Pro Thr Pro Thr Lys Ser Ser Pro Ala
Ser Asn Pro Pro 515 520 525Ile Pro Val Gly Glu Ile Tyr Lys Arg Trp
Ile Ile Leu Gly Leu Asn 530 535 540Lys Ile Val Arg Met Tyr Ser Pro
Thr Ser Ile Leu Asp Ala Ser Pro545 550 555 560Thr Ser Thr Pro Ala
Asp Ser Ser Thr Ile Thr Pro Thr Ala Thr Pro 565 570 575Thr Ala Thr
Pro Thr Ile Lys Gly Ala Ser His Thr Gln Gly Tyr Phe 580 585 590Pro
Asp Trp Gln Asn Tyr Thr Pro Gly Pro Gly Val Arg Tyr Pro Leu 595 600
605Thr Phe Gly Trp Leu Tyr Lys Leu Ala Ser Thr Val Thr Pro Thr Ala
610 615 620Thr Ala Thr Pro Ser Ala Ile Val Thr Thr Ile Thr Pro Thr
Ala Thr625 630 635 640Thr Lys Pro Ala Ser Val Gly Phe Pro Val Thr
Pro Gln Val Pro Leu 645 650 655Arg Pro Met Thr Tyr Lys Ala Ala Val
Asp Leu Ser His Phe Leu Lys 660 665 670Glu Lys Gly Gly Leu Ala Ser
Thr Asn Gly Ser Ile Thr Val Ala Ala 675 680 685Thr Ala Pro Thr Val
Thr Pro Thr Val Asn Ala Thr Pro Ser Ala Ala 690 695 700Ala Ser Ala
Ile Phe Gln Ser Ser Met Thr Lys Ile Leu Glu Pro Phe705 710 715
720Arg Lys Gln Asn Pro Asp Ile Val Ile Tyr Gln Tyr Met Asp Asp Leu
725 730 735Tyr Ala Ser38234PRTArtificial SequenceSynthetic
anti-CD40 binding molecule sequence (light chain). 38Met Met Ser
Ser Ala Gln Phe Leu Gly Leu Leu Leu Leu Cys Phe Gln1 5 10 15Gly Thr
Arg Cys Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser 20 25 30Ala
Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly 35 40
45Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val
50 55 60Lys Leu Leu Ile Tyr Tyr Thr Ser Ile Leu His Ser Gly Val Pro
Ser65 70 75 80Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu
Thr Ile Gly 85 90 95Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr Cys
Gln Gln Phe Asn 100 105 110Lys Leu Pro Pro Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys Arg 115 120 125Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr145 150 155 160Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185
190Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
195 200 205His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro 210 215 220Val Thr Lys Ser Phe Asn Arg Gly Glu Cys225
23039467PRTArtificial SequenceSynthetic anti-CD 40 binding molecule
sequence (heavy chain). 39Met Asn Leu Gly Leu Ser Leu Ile Phe Leu
Val Leu Val Leu Lys Gly1 5 10 15Val Gln Cys Glu Val Lys Leu Val Glu
Ser Gly Gly Gly Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Lys Leu Ser
Cys Ala Thr Ser Gly Phe Thr Phe 35 40 45Ser Asp Tyr Tyr Met Tyr Trp
Val Arg Gln Thr Pro Glu Lys Arg Leu 50 55 60Glu Trp Val Ala Tyr Ile
Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Pro65 70 75 80Asp Thr Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95Thr Leu Tyr
Leu Gln Met Ser Arg Leu Lys Ser Glu Asp Thr Ala Met 100 105 110Tyr
Tyr Cys Ala Arg Arg Gly Leu Pro Phe His Ala Met Asp Tyr Trp 115 120
125Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Lys Gly Pro
130 135 140Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr145 150 155 160Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr 165 170 175Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro 180 185 190Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205Val Pro Ser Ser Ser
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 210 215 220His Lys Pro
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr225 230 235
240Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro
245 250 255Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser 260 265 270Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser Gln Glu Asp 275 280 285Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn 290 295 300Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Tyr Arg Val305 310 315 320Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 325 330 335Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 340 345 350Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 355 360
365Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
370 375 380Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu385 390 395 400Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu 405 410 415Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Arg Leu Thr Val Asp Lys 420 425 430Ser Arg Trp Gln Glu Gly Asn
Val Phe Ser Cys Ser Val Met His Glu 435 440 445Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 450 455 460Lys Ala
Ser46540705DNAArtificial SequenceSynthetic anti-CD40 binding
molecule nucleic acid sequence (light chain). 40atgatgtcct
ctgctcagtt ccttggtctc ctgttgctct gttttcaagg taccagatgt 60gatatccaga
tgacacagac tacatcctcc ctgtctgcct ctctaggaga cagagtcacc
120atcagttgca gtgcaagtca gggcattagc aattatttaa actggtatca
gcagaaacca 180gatggaactg ttaaactcct gatctattac acatcaattt
tacactcagg agtcccatca 240aggttcagtg gcagtgggtc tgggacagat
tattctctca ccatcggcaa cctggaacct 300gaagatattg ccacttacta
ttgtcagcag tttaataagc ttcctccgac gttcggtgga 360ggcaccaaac
tcgagatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca
420tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa
taacttctat 480cccagagagg ccaaagtaca gtggaaggtg gataacgccc
tccaatcggg taactcccag 540gagagtgtca cagagcagga cagcaaggac
agcacctaca gcctcagcag caccctgacg 600ctgagcaaag cagactacga
gaaacacaaa gtctatgcct gcgaagtcac ccatcagggc 660ctgagctcgc
ccgtcacaaa gagcttcaac aggggagagt gttag 705411404DNAArtificial
SequenceSynthetic anti-CD40 binding molecule nucleic acid sequence
(heavy chain). 41atgaacttgg ggctcagctt gattttcctt gtccttgttt
taaaaggtgt ccagtgtgaa 60gtgaagctgg tggagtctgg gggaggctta gtgcagcctg
gagggtccct gaaactctcc 120tgtgcaacct ctggattcac tttcagtgac
tattacatgt attgggttcg ccagactcca 180gagaagaggc tggagtgggt
cgcatacatt aattctggtg gtggtagcac ctattatcca 240gacactgtaa
agggccgatt caccatctcc agagacaatg ccaagaacac cctgtacctg
300caaatgagcc ggctgaagtc tgaggacaca gccatgtatt actgtgcaag
acgggggtta 360ccgttccatg ctatggacta ttggggtcaa ggaacctcag
tcaccgtctc ctcagccaaa 420acgaagggcc catccgtctt ccccctggcg
ccctgctcca ggagcacctc cgagagcaca 480gccgccctgg gctgcctggt
caaggactac ttccccgaac cggtgacggt gtcgtggaac 540tcaggcgccc
tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc
600tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcacgaa
gacctacacc 660tgcaacgtag atcacaagcc cagcaacacc aaggtggaca
agagagttga gtccaaatat 720ggtcccccat gcccaccctg cccagcacct
gagttcgaag ggggaccatc agtcttcctg 780ttccccccaa aacccaagga
cactctcatg atctcccgga cccctgaggt cacgtgcgtg 840gtggtggacg
tgagccagga agaccccgag gtccagttca actggtacgt ggatggcgtg
900gaggtgcata atgccaagac aaagccgcgg gaggagcagt tcaacagcac
gtaccgtgtg 960gtcagcgtcc tcaccgtcct gcaccaggac tggctgaacg
gcaaggagta caagtgcaag 1020gtctccaaca aaggcctccc gtcctccatc
gagaaaacca tctccaaagc caaagggcag 1080ccccgagagc cacaggtgta
caccctgccc ccatcccagg aggagatgac caagaaccag 1140gtcagcctga
cctgcctggt caaaggcttc taccccagcg acatcgccgt ggagtgggag
1200agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga
ctccgacggc 1260tccttcttcc tctacagcag gctaaccgtg gacaagagca
ggtggcagga ggggaatgtc 1320ttctcatgct ccgtgatgca tgaggctctg
cacaaccact acacacagaa gagcctctcc 1380ctgtctctgg gtaaagctag ctga
1404427PRTArtificial SequenceHeavy chain variable domain region of
the antigen binding site of a synthetic CD 40 binding molecule.
42Tyr Thr Ser Ile Leu His Ser1 5439PRTArtificial SequenceHeavy
chain variable domain region of the antigen binding site of a
synthetic CD 40 binding molecule. 43Gln Gln Phe Asn Lys Leu Pro Pro
Thr1 54410PRTArtificial SequenceLight chain variable domain region
of the antigen binding site of a synthetic CD 40 binding molecule.
44Gly Phe Thr Phe Ser Asp Tyr Tyr Met Tyr1 5 104517PRTArtificial
SequenceLight chain variable domain region of the antigen binding
site of a synthetic CD 40 binding molecule. 45Tyr Ile Asn Ser Gly
Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val Lys1 5 10
15Gly4610PRTArtificial SequenceLight chain variable domain region
of the antigen binding site of a synthetic CD 40 binding molecule.
46Arg Gly Leu Pro Phe His Ala Met Asp Tyr1 5 104710PRTArtificial
SequenceLight chain variable domain region of the antigen binding
site of a synthetic CD 40 binding molecule. 47Arg Gly Leu Pro Phe
His Ala Met Asp Tyr1 5 104836DNAArtificial SequenceSynthetic
primers for PCR. 48ggatggtggg aagatggata cagttggtgc agcatc
364931DNAArtificial SequenceSynthetic primers for PCR. 49ccaggcatcc
tagagtcacc gaggagccag t 315070DNAArtificial SequenceAntibody-PSA
fusion protein sequence. 50gctagcgata caacagaacc tgcaacacct
acaacacctg taacaacacc gacaacaaca 60cttctagcgc 7051186DNAArtificial
SequenceCohesin-Flex-hMART-1-PeptideA-His nucleic acid sequence.
51gacaccaccg aggcccgcca cccccacccc cccgtgacca cccccaccac caccgaccgg
60aagggcacca ccgccgagga gctggccggc atcggcatcc tgaccgtgat cctgggcggc
120aagcggacca acaacagcac ccccaccaag ggcgaattct gcagatatcc
atcacactgg 180cggccg 1865261PRTArtificial
SequenceCohesin-Flex-hMART-1-PeptideA-His amino-acid sequence.
52Asp Thr Thr Glu Ala Arg His Pro His Pro Pro Val Thr Thr Pro Thr1
5 10 15Thr Asp Arg Lys Gly Thr Thr Ala Glu Glu Leu Ala Gly Ile Gly
Ile 20 25 30Leu Thr Val Ile Leu Gly Gly Lys Arg Thr Asn Asn Ser Thr
Pro Thr 35 40 45Lys Gly Glu Phe Cys Arg Tyr Pro Ser His Trp Arg Pro
50 55 605360PRTArtificial SequenceHA1 peptide sequence. 53Met Lys
Ala Asn Leu Leu Val Leu Leu Cys Ala Leu Ala Ala Ala Asp1 5 10 15Ala
Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25
30Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn
35 40 45Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Arg 50 55
605460PRTArtificial SequenceHA1 peptide sequence. 54Leu Lys Gly Ile
Ala Pro Leu Gln Leu Gly Lys Cys Asn Ile Ala Gly1 5 10 15Trp Leu Leu
Gly Asn Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser 20 25 30Trp Ser
Tyr Ile Val Glu Thr Pro Asn Ser Glu Asn Gly Ile Cys Tyr 35 40 45Pro
Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu 50 55
605560PRTArtificial SequenceHA1 peptide sequence. 55Gln Leu Ser Ser
Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys1 5 10 15Glu Ser Ser
Trp Pro Asn His Asn Thr Asn Gly Val Thr Ala Ala Cys 20 25 30Ser His
Glu Gly Lys Ser Ser Phe Tyr Arg Asn Leu Leu Trp Leu Thr 35 40 45Glu
Lys Glu Gly Ser Tyr Pro Lys Leu Lys Asn Ser 50 55
605660PRTArtificial SequenceHA1 peptide sequence. 56Tyr Val Asn Lys
Lys Gly Lys Glu Val Leu Val Leu Trp Gly Ile His1 5 10 15His Pro Pro
Asn Ser Lys Glu Gln Gln Asn Leu Tyr Gln Asn Glu Asn 20 25 30Ala Tyr
Val Ser Val Val Thr Ser Asn Tyr Asn Arg Arg Phe Thr Pro 35 40 45Glu
Ile Ala Glu Arg Pro Lys Val Arg Asp Gln Ala 50 55
605760PRTArtificial SequenceHA1 peptide sequence. 57Gly Arg Met Asn
Tyr Tyr Trp Thr Leu Leu Lys Pro Gly Asp Thr Ile1 5 10 15Ile Phe Glu
Ala Asn Gly Asn Leu Ile Ala Pro Met Tyr Ala Phe Ala 20 25 30Leu Ser
Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser Asn Ala Ser Met 35 40 45His
Glu Cys Asn Thr Lys Cys Gln Thr Pro Leu Gly 50 55
605840PRTArtificial SequenceHA1 peptide sequence. 58Ala Ile Asn Ser
Ser Leu Pro Tyr Gln Asn Ile His Pro Val Thr Ile1 5 10 15Gly Glu Cys
Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr 20 25 30Gly Leu
Arg Asn Ile Pro Ser Ile 35 405917PRTArtificial SequenceSynthetic
peptide. 59Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser
Trp Pro1 5 10 15Asn6017PRTArtificial SequenceSynthetic peptide.
60Gly Asn Leu Ile Ala Pro Trp Tyr Ala Phe Ala Leu Ser Arg Gly Phe1
5 10 15Gly6117PRTArtificial SequenceSynthetic peptide. 61Trp Tyr
Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr1 5 10
15Ser6260PRTArtificial SequenceNP sequence. 62Met Ala Ser Gln Gly
Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Asp1 5 10 15Gly Glu Arg Gln
Asn Ala Thr Glu Ile Arg Ala Ser Val Gly Lys Met 20 25 30Ile Gly Gly
Ile Gly Arg Phe Tyr Ile Gln Met Cys Thr Glu Leu Lys 35 40 45Leu Ser
Asp Tyr Glu Gly Arg Leu Ile Gln Asn Ser 50 55 606360PRTArtificial
SequenceNP sequence. 63Leu Thr Ile Glu Arg Met Val
Leu Ser Ala Phe Asp Glu Arg Arg Asn1 5 10 15Lys Tyr Leu Glu Glu His
Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr 20 25 30Gly Gly Pro Ile Tyr
Arg Arg Val Asn Gly Lys Trp Met Arg Glu Leu 35 40 45Ile Leu Tyr Asp
Lys Glu Glu Ile Arg Arg Ile Trp 50 55 606458PRTArtificial
SequenceNP sequence. 64Arg Gln Ala Asn Asn Gly Asp Asp Ala Thr Ala
Gly Leu Thr His Met1 5 10 15Met Ile Trp His Ser Asn Leu Asn Asp Ala
Thr Tyr Gln Arg Thr Arg 20 25 30Ala Leu Val Arg Thr Gly Met Asp Pro
Arg Met Cys Ser Leu Met Gln 35 40 45Gly Ser Thr Leu Pro Arg Arg Ser
Gly Ala 50 556560PRTArtificial SequenceNP sequence. 65Ala Ala Val
Lys Gly Val Gly Thr Met Val Met Glu Leu Val Arg Met1 5 10 15Ile Lys
Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg Gly Glu Asn Gly 20 25 30Arg
Lys Thr Arg Ile Ala Tyr Glu Arg Met Cys Asn Ile Leu Lys Gly 35 40
45Lys Phe Gln Thr Ala Ala Gln Lys Ala Met Met Asp 50 55
606660PRTArtificial SequenceNP sequence. 66Gln Val Arg Glu Ser Arg
Asn Pro Gly Asn Ala Glu Phe Glu Asp Leu1 5 10 15Thr Phe Leu Ala Arg
Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His 20 25 30Lys Ser Cys Leu
Pro Ala Cys Val Tyr Gly Pro Ala Val Ala Ser Gly 35 40 45Tyr Asp Phe
Glu Arg Glu Gly Tyr Ser Leu Val Gly 50 55 606760PRTArtificial
SequenceNP sequence. 67Ile Asp Pro Phe Arg Leu Leu Gln Asn Ser Gln
Val Tyr Ser Leu Ile1 5 10 15Arg Pro Asn Glu Asn Pro Ala His Lys Ser
Gln Leu Val Trp Met Ala 20 25 30Cys His Ser Ala Ala Phe Glu Asp Leu
Arg Val Leu Ser Phe Ile Lys 35 40 45Gly Thr Lys Val Leu Pro Arg Gly
Lys Leu Ser Thr 50 55 606860PRTArtificial SequenceNP sequence.
68Arg Gly Val Gln Ile Ala Ser Asn Glu Asn Met Glu Thr Met Glu Ser1
5 10 15Ser Thr Leu Glu Leu Arg Ser Arg Tyr Trp Ala Ile Arg Thr Arg
Ser 20 25 30Gly Gly Asn Thr Asn Gln Gln Arg Ala Ser Ala Gly Gln Ile
Ser Ile 35 40 45Gln Pro Thr Phe Ser Val Gln Arg Asn Leu Pro Phe 50
55 606960PRTArtificial SequenceNP sequence. 69Asp Arg Thr Thr Ile
Met Ala Ala Phe Asn Gly Asn Thr Glu Gly Arg1 5 10 15Thr Ser Asp Met
Arg Thr Glu Ile Ile Arg Met Met Glu Ser Ala Arg 20 25 30Pro Glu Asp
Val Ser Phe Gln Gly Arg Gly Val Phe Glu Leu Ser Asp 35 40 45Glu Lys
Ala Ala Ser Pro Ile Val Pro Ser Phe Asp 50 55 607018PRTArtificial
SequenceNP sequence. 70Met Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp
Asn Ala Glu Glu Tyr1 5 10 15Asp Asn7117PRTArtificial
SequenceSynthetic peptide. 71Gly Lys Trp Val Arg Glu Leu Val Leu
Tyr Asp Lys Glu Glu Ile Arg1 5 10 15Arg7217PRTArtificial
SequenceSynthetic peptide. 72Arg Thr Gly Met Asp Pro Arg Met Cys
Ser Leu Met Gln Gly Ser Thr1 5 10 15Leu7317PRTArtificial
SequenceSynthetic peptide. 73Met Cys Asn Ile Leu Lys Gly Lys Phe
Gln Thr Ala Ala Gln Lys Ala1 5 10 15Met7460PRTArtificial
SequencePSA sequence. 74Met Trp Val Pro Val Val Phe Leu Thr Leu Ser
Val Thr Trp Ile Gly1 5 10 15Ala Ala Pro Leu Ile Leu Ser Arg Ile Val
Gly Gly Trp Glu Cys Glu 20 25 30Lys His Ser Gln Pro Trp Gln Val Leu
Val Ala Ser Arg Gly Arg Ala 35 40 45Val Cys Gly Gly Val Leu Val His
Pro Gln Trp Val 50 55 607560PRTArtificial SequencePSA sequence.
75Leu Thr Ala Ala His Cys Ile Arg Asn Lys Ser Val Ile Leu Leu Gly1
5 10 15Arg His Ser Leu Phe His Pro Glu Asp Thr Gly Gln Val Phe Gln
Val 20 25 30Ser His Ser Phe Pro His Pro Leu Tyr Asp Met Ser Leu Leu
Lys Asn 35 40 45Arg Phe Leu Arg Pro Gly Asp Asp Ser Ser His Asp 50
55 607660PRTArtificial SequencePSA sequence. 76Leu Met Leu Leu Arg
Leu Ser Glu Pro Ala Glu Leu Thr Asp Ala Val1 5 10 15Lys Val Met Asp
Leu Pro Thr Gln Glu Pro Ala Leu Gly Thr Thr Cys 20 25 30Tyr Ala Ser
Gly Trp Gly Ser Ile Glu Pro Glu Glu Phe Leu Thr Pro 35 40 45Lys Lys
Leu Gln Cys Val Asp Leu His Val Ile Ser 50 55 607760PRTArtificial
SequencePSA sequence. 77Asn Asp Val Cys Ala Gln Val His Pro Gln Lys
Val Thr Lys Phe Met1 5 10 15Leu Cys Ala Gly Arg Trp Thr Gly Gly Lys
Ser Thr Cys Ser Gly Asp 20 25 30Ser Gly Gly Pro Leu Val Cys Asn Gly
Val Leu Gln Gly Ile Thr Ser 35 40 45Trp Gly Ser Glu Pro Cys Ala Leu
Pro Glu Arg Pro 50 55 607821PRTArtificial SequencePSA sequence.
78Ser Leu Tyr Thr Lys Val Val His Tyr Arg Lys Trp Ile Lys Asp Thr1
5 10 15Ile Val Ala Asn Pro 207915PRTArtificial SequenceSynthetic
peptide. 79Ala Pro Leu Ile Leu Ser Arg Ile Val Gly Gly Trp Glu Cys
Glu1 5 10 158015PRTArtificial SequenceSynthetic peptide. 80Glu Cys
Glu Lys His Ser Gln Pro Trp Gln Val Leu Val Ala Ser1 5 10
158115PRTArtificial sequenceSynthetic peptide. 81Gly Asp Asp Ser
Ser His Asp Leu Met Leu Leu Arg Leu Ser Glu1 5 10
158215PRTArtificial SequenceSynthetic peptide. 82Ser His Asp Leu
Met Leu Leu Arg Leu Ser Glu Pro Ala Glu Leu1 5 10
158315PRTArtificial SequenceSynthetic peptide. 83Ser Gly Asp Ser
Gly Gly Pro Leu Val Cys Asn Gly Val Leu Gln1 5 10
158415PRTArtificial SequenceSynthetic peptide. 84Gly Ser Glu Pro
Cys Ala Leu Pro Glu Arg Pro Ser Leu Tyr Thr1 5 10
158515PRTArtificial SequenceSynthetic peptide. 85Glu Arg Pro Ser
Leu Tyr Thr Lys Val Val His Tyr Arg Lys Trp1 5 10
158615PRTArtificial SequenceSynthetic peptide. 86Val Val His Tyr
Arg Lys Trp Ile Lys Asp Thr Ile Val Ala Asn1 5 10
158760PRTArtificial SequenceCyclin D1 sequence. 87Met Arg Ser Tyr
Arg Phe Ser Asp Tyr Leu His Met Ser Val Ser Phe1 5 10 15Ser Asn Asp
Met Asp Leu Phe Cys Gly Glu Asp Ser Gly Val Phe Ser 20 25 30Gly Glu
Ser Thr Val Asp Phe Ser Ser Ser Glu Val Asp Ser Trp Pro 35 40 45Gly
Asp Ser Ile Ala Cys Phe Ile Glu Asp Glu Arg 50 55
608860PRTArtificial SequenceCyclin D1 sequence. 88His Phe Val Pro
Gly His Asp Tyr Leu Ser Arg Phe Gln Thr Arg Ser1 5 10 15Leu Asp Ala
Ser Ala Arg Glu Asp Ser Val Ala Trp Ile Leu Lys Val 20 25 30Gln Ala
Tyr Tyr Asn Phe Gln Pro Leu Thr Ala Tyr Leu Ala Val Asn 35 40 45Tyr
Met Asp Arg Phe Leu Tyr Ala Arg Arg Leu Pro 50 55
608960PRTArtificial SequenceCyclin D1 sequence. 89Glu Thr Ser Gly
Trp Pro Met Gln Leu Leu Ala Val Ala Cys Leu Ser1 5 10 15Leu Ala Ala
Lys Met Glu Glu Ile Leu Val Pro Ser Leu Phe Asp Phe 20 25 30Gln Val
Ala Gly Val Lys Tyr Leu Phe Glu Ala Lys Thr Ile Lys Arg 35 40 45Met
Glu Leu Leu Val Leu Ser Val Leu Asp Trp Arg 50 55
609060PRTArtificial SequenceCyclin D1 sequence. 90Leu Arg Ser Val
Thr Pro Phe Asp Phe Ile Ser Phe Phe Ala Tyr Lys1 5 10 15Ile Asp Pro
Ser Gly Thr Phe Leu Gly Phe Phe Ile Ser His Ala Thr 20 25 30Glu Ile
Ile Leu Ser Asn Ile Lys Glu Ala Ser Phe Leu Glu Tyr Trp 35 40 45Pro
Ser Ser Ile Ala Ala Ala Ala Ile Leu Cys Val 50 55
609160PRTArtificial SequenceCyclin D1 sequence. 91Ala Asn Glu Leu
Pro Ser Leu Ser Ser Val Val Asn Pro His Glu Ser1 5 10 15Pro Glu Thr
Trp Cys Asp Gly Leu Ser Lys Glu Lys Ile Val Arg Cys 20 25 30Tyr Arg
Leu Met Lys Ala Met Ala Ile Glu Asn Asn Arg Leu Asn Thr 35 40 45Pro
Lys Val Ile Ala Lys Leu Arg Val Ser Val Arg 50 55
609239PRTArtificial SequenceCyclin D1 sequence. 92Ala Ser Ser Thr
Leu Thr Arg Pro Ser Asp Glu Ser Ser Phe Ser Ser1 5 10 15Ser Ser Pro
Cys Lys Arg Arg Lys Leu Ser Gly Tyr Ser Trp Val Gly 20 25 30Asp Glu
Thr Ser Thr Ser Asn 359315PRTArtificial SequenceSynthetic peptide.
93Asp Arg Val Leu Arg Ala Met Leu Lys Ala Glu Glu Thr Cys Ala1 5 10
159415PRTArtificial SequenceSynthetic peptide. 94Arg Ala Met Leu
Lys Ala Glu Glu Thr Cys Ala Pro Ser Val Ser1 5 10
159515PRTArtificial SequenceSynthetic peptide. 95Thr Cys Ala Pro
Ser Val Ser Tyr Phe Lys Cys Val Gln Lys Glu1 5 10
1596587PRTArtificial SequenceH chain - hMART-1 peptide. 96Glu Val
Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Tyr 20 25
30Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
35 40 45Ala Tyr Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Ser Arg Leu Lys Ser Glu Asp Thr Ala
Met Tyr Tyr Cys 85 90 95Ala Arg Arg Gly Leu Pro Phe His Ala Met Asp
Tyr Trp Gly Gln Gly 100 105 110Thr Ser Val Thr Val Ser Ser Ala Lys
Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His
Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys
Tyr Gly Pro Pro 210 215 220Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu
Gly Gly Pro Ser Val Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255Glu Val Thr Cys Val
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270Gln Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285Lys
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295
300Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys305 310 315 320Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro 340 345 350Ser Gln Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val 355 360 365Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp385 390 395 400Gly
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410
415Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
Ala Ser 435 440 445Gln Thr Pro Thr Asn Thr Ile Ser Val Thr Pro Thr
Asn Asn Ser Thr 450 455 460Pro Thr Asn Asn Ser Asn Pro Lys Pro Asn
Pro Ala Ser Gly Phe Asp465 470 475 480His Arg Asp Ser Lys Val Ser
Leu Gln Glu Lys Asn Cys Glu Pro Val 485 490 495Val Pro Asn Ala Pro
Pro Ala Tyr Glu Lys Leu Ser Ala Glu Gln Ser 500 505 510Pro Pro Pro
Tyr Ser Pro Ala Ser Thr Asn Gly Ser Ile Thr Val Ala 515 520 525Ala
Thr Ala Pro Thr Val Thr Pro Thr Val Asn Ala Thr Pro Ser Ala 530 535
540Ala Ala Ser Met Pro Arg Glu Asp Ala His Phe Ile Tyr Gly Tyr
Pro545 550 555 560Lys Lys Gly His Gly His Ser Tyr Thr Thr Ala Glu
Glu Ala Ala Gly 565 570 575Ile Gly Ile Leu Thr Val Ile Leu Gly Ala
Ser 580 58597656PRTArtificial SequenceH chain - hMART-1 peptide.
97Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp
Tyr 20 25 30Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu
Trp Val 35 40 45Ala Tyr Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Pro
Asp Thr Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Ser Arg Leu Lys Ser Glu Asp
Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Arg Gly Leu Pro Phe His Ala
Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Ser Val Thr Val Ser Ser
Ala Lys Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Cys
Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155
160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val
Asp His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Arg Val Glu
Ser Lys Tyr Gly Pro Pro 210 215 220Cys Pro Pro Cys Pro Ala Pro Glu
Phe Glu Gly Gly Pro Ser Val Phe225 230 235 240Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255Glu Val Thr
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280
285Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys305 310 315 320Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser Gln Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp385 390 395
400Gly Ser Phe Phe
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415Gln Glu
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425
430Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala Ser
435 440 445Gln Thr Pro Thr Asn Thr Ile Ser Val Thr Pro Thr Asn Asn
Ser Thr 450 455 460Pro Thr Asn Asn Ser Asn Pro Lys Pro Asn Pro Ala
Ser Gly Phe Asp465 470 475 480His Arg Asp Ser Lys Val Ser Leu Gln
Glu Lys Asn Cys Glu Pro Val 485 490 495Val Pro Asn Ala Pro Pro Ala
Tyr Glu Lys Leu Ser Ala Glu Gln Ser 500 505 510Pro Pro Pro Tyr Ser
Pro Ala Ser Thr Asn Gly Ser Ile Thr Val Ala 515 520 525Ala Thr Ala
Pro Thr Val Thr Pro Thr Val Asn Ala Thr Pro Ser Ala 530 535 540Ala
Ala Ser Met Pro Arg Glu Asp Ala His Phe Ile Tyr Gly Tyr Pro545 550
555 560Lys Lys Gly His Gly His Ser Tyr Thr Thr Ala Glu Glu Ala Ala
Gly 565 570 575Ile Gly Ile Leu Thr Val Ile Leu Gly Ala Ser Thr Val
Thr Pro Thr 580 585 590Ala Thr Ala Thr Pro Ser Ala Ile Val Thr Thr
Ile Thr Pro Thr Ala 595 600 605Thr Thr Lys Pro Ala Ser Val Leu Leu
Leu Ile Gly Cys Trp Tyr Cys 610 615 620Arg Arg Arg Asn Gly Tyr Arg
Ala Leu Met Asp Lys Ser Leu His Val625 630 635 640Gly Thr Gln Cys
Ala Leu Thr Arg Arg Cys Pro Gln Glu Gly Ala Ser 645 650
65598593PRTArtificial SequenceH chain - hMART-1 peptide. 98Gln Val
Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln1 5 10 15Thr
Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25
30Gly Met Gly Leu Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu
35 40 45Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro
Ser 50 55 60Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn
Gln Val65 70 75 80Phe Leu Lys Ile Thr Ile Val Asp Thr Ala Asp Ala
Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Ser Ser His Tyr Tyr Gly Tyr Gly
Tyr Gly Gly Tyr Phe 100 105 110Asp Val Trp Gly Ala Gly Thr Thr Val
Thr Val Ser Ser Ala Lys Thr 115 120 125Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140Glu Ser Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu145 150 155 160Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170
175Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
180 185 190Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr
Thr Cys 195 200 205Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys Arg Val Glu 210 215 220Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys
Pro Ala Pro Glu Phe Glu225 230 235 240Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270Gln Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 290 295
300Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ser Ser 325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Gln
Glu Glu Met Thr Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 405 410
415Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
420 425 430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu 435 440 445Ser Leu Gly Lys Ala Ser Gln Thr Pro Thr Asn Thr
Ile Ser Val Thr 450 455 460Pro Thr Asn Asn Ser Thr Pro Thr Asn Asn
Ser Asn Pro Lys Pro Asn465 470 475 480Pro Ala Ser Gly Phe Asp His
Arg Asp Ser Lys Val Ser Leu Gln Glu 485 490 495Lys Asn Cys Glu Pro
Val Val Pro Asn Ala Pro Pro Ala Tyr Glu Lys 500 505 510Leu Ser Ala
Glu Gln Ser Pro Pro Pro Tyr Ser Pro Ala Ser Thr Asn 515 520 525Gly
Ser Ile Thr Val Ala Ala Thr Ala Pro Thr Val Thr Pro Thr Val 530 535
540Asn Ala Thr Pro Ser Ala Ala Ala Ser Met Pro Arg Glu Asp Ala
His545 550 555 560Phe Ile Tyr Gly Tyr Pro Lys Lys Gly His Gly His
Ser Tyr Thr Thr 565 570 575Ala Glu Glu Ala Ala Gly Ile Gly Ile Leu
Thr Val Ile Leu Gly Ala 580 585 590Ser99660PRTArtificial SequenceH
chain - hMART-1 peptide. 99Gln Val Thr Leu Lys Glu Ser Gly Pro Gly
Ile Leu Gln Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ser Phe Ser
Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Gly Leu Ser Trp Ile Arg
Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45Trp Leu Ala His Ile Tyr Trp
Asp Asp Asp Lys Arg Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg Leu Thr
Ile Ser Lys Asp Thr Ser Ser Asn Gln Val65 70 75 80Phe Leu Lys Ile
Thr Ile Val Asp Thr Ala Asp Ala Ala Thr Tyr Tyr 85 90 95Cys Ala Arg
Ser Ser His Tyr Tyr Gly Tyr Gly Tyr Gly Gly Tyr Phe 100 105 110Asp
Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr 115 120
125Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
130 135 140Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu145 150 155 160Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His 165 170 175Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser 180 185 190Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Lys Thr Tyr Thr Cys 195 200 205Asn Val Asp His Lys
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 210 215 220Ser Lys Tyr
Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu225 230 235
240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser 260 265 270Gln Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 325 330 335Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360
365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Arg Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440 445Gly Lys Ala Ser Gln
Thr Pro Thr Asn Thr Ile Ser Val Thr Pro Thr 450 455 460Asn Asn Ser
Thr Pro Thr Asn Asn Ser Asn Pro Lys Pro Asn Pro Ala465 470 475
480Ser Gly Phe Asp His Arg Asp Ser Lys Val Ser Leu Gln Glu Lys Asn
485 490 495Cys Glu Pro Val Val Pro Asn Ala Pro Pro Ala Tyr Glu Lys
Leu Ser 500 505 510Ala Glu Gln Ser Pro Pro Pro Tyr Ser Pro Ala Ser
Thr Asn Gly Ser 515 520 525Ile Thr Val Ala Ala Thr Ala Pro Thr Val
Thr Pro Thr Val Asn Ala 530 535 540Thr Pro Ser Ala Ala Ala Ser Met
Pro Arg Glu Asp Ala His Phe Ile545 550 555 560Tyr Gly Tyr Pro Lys
Lys Gly His Gly His Ser Tyr Thr Thr Ala Glu 565 570 575Glu Ala Ala
Gly Ile Gly Ile Leu Thr Val Ile Leu Gly Ala Ser Thr 580 585 590Val
Thr Pro Thr Ala Thr Ala Thr Pro Ser Ala Ile Val Thr Thr Ile 595 600
605Thr Pro Thr Ala Thr Thr Lys Pro Ala Ser Val Leu Leu Leu Ile Gly
610 615 620Cys Trp Tyr Cys Arg Arg Arg Asn Gly Tyr Arg Ala Leu Met
Asp Lys625 630 635 640Ser Leu His Val Gly Thr Gln Cys Ala Leu Thr
Arg Arg Cys Pro Gln 645 650 655Glu Gly Ala Ser
660100630DNAArtificial SequenceMART-1 DNA synthetic construct.
100aacaccgaca acaacagatg atctggatgc agctagtggg tttgatcatc
gggacagcaa 60agtgtctctt caagagaaaa actgtgaacc tgtggttccc aatgctccac
ctgcttatga 120gaaactctct gcagaacagt caccaccacc ttattcacct
gctagtacca acggcagcat 180caccgtggcc gccaccgccc ccaccgtgac
ccccaccgtg aacgccaccc ccagcgccgc 240cgctagtatg ccaagagaag
atgctcactt catctatggt taccccaaga aggggcacgg 300ccactcttac
accacggctg aagaggccgc tgggatcggc atcctgacag tgatcctggg
360agctagtacc gtgaccccca ccgccaccgc cacccccagc gccatcgtga
ccaccatcac 420ccccaccgcc accaccaagc ccgctagtgt cttactgctc
atcggctgtt ggtattgtag 480aagacgaaat ggatacagag ccttgatgga
taaaagtctt catgttggca ctcaatgtgc 540cttaacaaga agatgcccac
aagaagggtg agcggccgca tcgaagagct cggtacccgg 600ggatcctcta
gagtcgacct gcaggcatgc 63010160PRTArtificial SequenceSynthetic
peptide. 101Gly Phe Asp His Arg Asp Ser Lys Val Ser Leu Gln Glu Lys
Asn Cys1 5 10 15Glu Pro Val Val Pro Asn Ala Pro Pro Ala Tyr Glu Lys
Leu Ser Ala 20 25 30Glu Gln Ser Pro Pro Pro Tyr Ser Pro Ala Ser Thr
Asn Gly Ser Ile 35 40 45Thr Val Ala Ala Thr Ala Pro Thr Val Thr Pro
Thr 50 55 6010260PRTArtificial SequenceSynthetic peptide. 102Val
Asn Ala Thr Pro Ser Ala Ala Ala Ser Met Pro Arg Glu Asp Ala1 5 10
15His Phe Ile Tyr Gly Tyr Pro Lys Lys Gly His Gly His Ser Tyr Thr
20 25 30Thr Ala Glu Glu Ala Ala Gly Ile Gly Ile Leu Thr Val Ile Leu
Gly 35 40 45Ala Ser Thr Val Thr Pro Thr Ala Thr Ala Thr Pro 50 55
6010357PRTArtificial SequenceSynthetic peptide. 103Ser Ala Ile Val
Thr Thr Ile Thr Pro Thr Ala Thr Thr Lys Pro Ala1 5 10 15Ser Val Leu
Leu Leu Ile Gly Cys Trp Tyr Cys Arg Arg Arg Asn Gly 20 25 30Tyr Arg
Ala Leu Met Asp Lys Ser Leu His Val Gly Thr Gln Cys Ala 35 40 45Leu
Thr Arg Arg Cys Pro Gln Glu Gly 50 5510441PRTArtificial
SequenceSynthetic MART1 peptide. 104Gly Phe Asp His Arg Asp Ser Lys
Val Ser Leu Gln Glu Lys Asn Cys1 5 10 15Glu Pro Val Val Pro Asn Ala
Pro Pro Ala Tyr Glu Lys Leu Ser Ala 20 25 30Glu Gln Ser Pro Pro Pro
Tyr Ser Pro 35 4010529PRTArtificial SequenceLinker peptide
sequence. 105Ala Ser Thr Asn Gly Ser Ile Thr Val Ala Ala Thr Ala
Pro Thr Val1 5 10 15Thr Pro Thr Val Asn Ala Thr Pro Ser Ala Ala Ala
Ser 20 2510638PRTArtificial SequenceMART1 peptide sequence with
CD4+ and CD8+ epitopes. 106Met Pro Arg Glu Asp Ala His Phe Ile Tyr
Gly Tyr Pro Lys Lys Gly1 5 10 15His Gly His Ser Tyr Thr Thr Ala Glu
Glu Ala Ala Gly Ile Gly Ile 20 25 30Leu Thr Val Ile Leu Gly
3510729PRTArtificial SequenceLinker peptide sequence. 107Ala Ser
Thr Val Thr Pro Thr Ala Thr Ala Thr Pro Ser Ala Ile Val1 5 10 15Thr
Thr Ile Thr Pro Thr Ala Thr Thr Lys Pro Ala Ser 20
2510840PRTArtificial SequenceMART1 peptide sequence with CD4+
epitope. 108Val Leu Leu Leu Ile Gly Cys Trp Tyr Cys Arg Arg Arg Asn
Gly Tyr1 5 10 15Arg Ala Leu Met Asp Lys Ser Leu His Val Gly Thr Gln
Cys Ala Leu 20 25 30Thr Arg Arg Cys Pro Gln Glu Gly 35
40109110PRTArtificial SequenceSynthetic peptide. 109Gly Phe Asp His
Arg Asp Ser Lys Val Ser Leu Gln Glu Lys Asn Cys1 5 10 15Glu Pro Val
Val Pro Asn Ala Pro Pro Ala Tyr Glu Lys Leu Ser Ala 20 25 30Glu Gln
Ser Pro Pro Pro Tyr Ser Pro Ala Ser Thr Asn Gly Ser Ile 35 40 45Thr
Val Ala Ala Thr Ala Pro Thr Val Thr Pro Thr Val Asn Ala Thr 50 55
60Pro Ser Ala Ala Ala Ser Met Pro Arg Glu Asp Ala His Phe Ile Tyr65
70 75 80Gly Tyr Pro Lys Lys Gly His Gly His Ser Tyr Thr Thr Ala Glu
Glu 85 90 95Ala Ala Gly Ile Gly Ile Leu Thr Val Ile Leu Gly Ala Ser
100 105 110110675PRTArtificial SequenceSynthetic MART1 peptide.
110Met Asn Leu Gly Leu Ser Leu Ile Phe Leu Val Leu Val Leu Lys Gly1
5 10 15Val Gln Cys Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val
Gln 20 25 30Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe
Thr Phe 35 40 45Ser Asp Tyr Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu
Lys Arg Leu 50 55 60Glu Trp Val Ala Tyr Ile Asn Ser Gly Gly Gly Ser
Thr Tyr Tyr Pro65 70 75 80Asp Thr Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn 85 90 95Thr Leu Tyr Leu Gln Met Ser Arg Leu
Lys Ser Glu Asp Thr Ala Met 100 105 110Tyr Tyr Cys Ala Arg Arg Gly
Leu Pro Phe His Ala Met Asp Tyr Trp 115 120 125Gly Gln Gly Thr Ser
Val Thr Val Ser Ser Ala Lys Thr Lys Gly Pro 130 135 140Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr145 150 155
160Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
165 170 175Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro 180 185 190Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr 195 200 205Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
Tyr Thr Cys Asn Val Asp 210 215 220His Lys Pro Ser Asn Thr Lys Val
Asp Lys Arg Val Glu Ser Lys Tyr225 230 235 240Gly Pro Pro Cys Pro
Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro 245 250 255Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 260 265 270Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 275 280
285Pro Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 290 295
300Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg
Val305 310 315 320Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu 325 330 335Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ser Ser Ile Glu Lys 340 345 350Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr 355 360 365Leu Pro Pro Ser Gln Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr 370 375 380Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu385 390 395 400Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 405 410
415Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
420 425 430Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
His Glu 435 440 445Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Leu Gly 450 455 460Lys Ala Ser Gln Thr Pro Thr Asn Thr Ile
Ser Val Thr Pro Thr Asn465 470 475 480Asn Ser Thr Pro Thr Asn Asn
Ser Asn Pro Lys Pro Asn Pro Ala Ser 485 490 495Gly Phe Asp His Arg
Asp Ser Lys Val Ser Leu Gln Glu Lys Asn Cys 500 505 510Glu Pro Val
Val Pro Asn Ala Pro Pro Ala Tyr Glu Lys Leu Ser Ala 515 520 525Glu
Gln Ser Pro Pro Pro Tyr Ser Pro Ala Ser Thr Asn Gly Ser Ile 530 535
540Thr Val Ala Ala Thr Ala Pro Thr Val Thr Pro Thr Val Asn Ala
Thr545 550 555 560Pro Ser Ala Ala Ala Ser Met Pro Arg Glu Asp Ala
His Phe Ile Tyr 565 570 575Gly Tyr Pro Lys Lys Gly His Gly His Ser
Tyr Thr Thr Ala Glu Glu 580 585 590Ala Ala Gly Ile Gly Ile Leu Thr
Val Ile Leu Gly Ala Ser Thr Val 595 600 605Thr Pro Thr Ala Thr Ala
Thr Pro Ser Ala Ile Val Thr Thr Ile Thr 610 615 620Pro Thr Ala Thr
Thr Lys Pro Ala Ser Val Leu Leu Leu Ile Gly Cys625 630 635 640Trp
Tyr Cys Arg Arg Arg Asn Gly Tyr Arg Ala Leu Met Asp Lys Ser 645 650
655Leu His Val Gly Thr Gln Cys Ala Leu Thr Arg Arg Cys Pro Gln Glu
660 665 670Gly Ala Ser 675111606PRTArtificial SequenceSynthetic
MART1 peptide. 111Met Asn Leu Gly Leu Ser Leu Ile Phe Leu Val Leu
Val Leu Lys Gly1 5 10 15Val Gln Cys Glu Val Lys Leu Val Glu Ser Gly
Gly Gly Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala
Thr Ser Gly Phe Thr Phe 35 40 45Ser Asp Tyr Tyr Met Tyr Trp Val Arg
Gln Thr Pro Glu Lys Arg Leu 50 55 60Glu Trp Val Ala Tyr Ile Asn Ser
Gly Gly Gly Ser Thr Tyr Tyr Pro65 70 75 80Asp Thr Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95Thr Leu Tyr Leu Gln
Met Ser Arg Leu Lys Ser Glu Asp Thr Ala Met 100 105 110Tyr Tyr Cys
Ala Arg Arg Gly Leu Pro Phe His Ala Met Asp Tyr Trp 115 120 125Gly
Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Lys Gly Pro 130 135
140Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser
Thr145 150 155 160Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr 165 170 175Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro 180 185 190Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr 195 200 205Val Pro Ser Ser Ser Leu
Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 210 215 220His Lys Pro Ser
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr225 230 235 240Gly
Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro 245 250
255Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
260 265 270Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
Glu Asp 275 280 285Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 290 295 300Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
Asn Ser Thr Tyr Arg Val305 310 315 320Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu 325 330 335Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 340 345 350Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 355 360 365Leu
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 370 375
380Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu385 390 395 400Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu 405 410 415Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Arg Leu Thr Val Asp Lys 420 425 430Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser Cys Ser Val Met His Glu 435 440 445Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 450 455 460Lys Ala Ser Gln
Thr Pro Thr Asn Thr Ile Ser Val Thr Pro Thr Asn465 470 475 480Asn
Ser Thr Pro Thr Asn Asn Ser Asn Pro Lys Pro Asn Pro Ala Ser 485 490
495Gly Phe Asp His Arg Asp Ser Lys Val Ser Leu Gln Glu Lys Asn Cys
500 505 510Glu Pro Val Val Pro Asn Ala Pro Pro Ala Tyr Glu Lys Leu
Ser Ala 515 520 525Glu Gln Ser Pro Pro Pro Tyr Ser Pro Ala Ser Thr
Asn Gly Ser Ile 530 535 540Thr Val Ala Ala Thr Ala Pro Thr Val Thr
Pro Thr Val Asn Ala Thr545 550 555 560Pro Ser Ala Ala Ala Ser Met
Pro Arg Glu Asp Ala His Phe Ile Tyr 565 570 575Gly Tyr Pro Lys Lys
Gly His Gly His Ser Tyr Thr Thr Ala Glu Glu 580 585 590Ala Ala Gly
Ile Gly Ile Leu Thr Val Ile Leu Gly Ala Ser 595 600
605112661PRTArtificial SequenceGP100 antigen sequence. 112Met Asp
Leu Val Leu Lys Arg Cys Leu Leu His Leu Ala Val Ile Gly1 5 10 15Ala
Leu Leu Ala Val Gly Ala Thr Lys Val Pro Arg Asn Gln Asp Trp 20 25
30Leu Gly Val Ser Arg Gln Leu Arg Thr Lys Ala Trp Asn Arg Gln Leu
35 40 45Tyr Pro Glu Trp Thr Glu Ala Gln Arg Leu Asp Cys Trp Arg Gly
Gly 50 55 60Gln Val Ser Leu Lys Val Ser Asn Asp Gly Pro Thr Leu Ile
Gly Ala65 70 75 80Asn Ala Ser Phe Ser Ile Ala Leu Asn Phe Pro Gly
Ser Gln Lys Val 85 90 95Leu Pro Asp Gly Gln Val Ile Trp Val Asn Asn
Thr Ile Ile Asn Gly 100 105 110Ser Gln Val Trp Gly Gly Gln Pro Val
Tyr Pro Gln Glu Thr Asp Asp 115 120 125Ala Cys Ile Phe Pro Asp Gly
Gly Pro Cys Pro Ser Gly Ser Trp Ser 130 135 140Gln Lys Arg Ser Phe
Val Tyr Val Trp Lys Thr Trp Gly Gln Tyr Trp145 150 155 160Gln Val
Leu Gly Gly Pro Val Ser Gly Leu Ser Ile Gly Thr Gly Arg 165 170
175Ala Met Leu Gly Thr His Thr Met Glu Val Thr Val Tyr His Arg Arg
180 185 190Gly Ser Arg Ser Tyr Val Pro Leu Ala His Ser Ser Ser Ala
Phe Thr 195 200 205Ile Thr Asp Gln Val Pro Phe Ser Val Ser Val Ser
Gln Leu Arg Ala 210 215 220Leu Asp Gly Gly Asn Lys His Phe Leu Arg
Asn Gln Pro Leu Thr Phe225 230 235 240Ala Leu Gln Leu His Asp Pro
Ser Gly Tyr Leu Ala Glu Ala Asp Leu 245 250 255Ser Tyr Thr Trp Asp
Phe Gly Asp Ser Ser Gly Thr Leu Ile Ser Arg 260 265 270Ala Leu Val
Val Thr His Thr Tyr Leu Glu Pro Gly Pro Val Thr Ala 275 280 285Gln
Val Val Leu Gln Ala Ala Ile Pro Leu Thr Ser Cys Gly Ser Ser 290 295
300Pro Val Pro Gly Thr Thr Asp Gly His Arg Pro Thr Ala Glu Ala
Pro305 310 315 320Asn Thr Thr Ala Gly Gln Val Pro Thr Thr Glu Val
Val Gly Thr Thr 325 330 335Pro Gly Gln Ala Pro Thr Ala Glu Pro Ser
Gly Thr Thr Ser Val Gln 340 345 350Val Pro Thr Thr Glu Val Ile Ser
Thr Ala Pro Val Gln Met Pro Thr 355 360 365Ala Glu Ser Thr Gly Met
Thr Pro Glu Lys Val Pro Val Ser Glu Val 370 375 380Met Gly Thr Thr
Leu Ala Glu Met Ser Thr Pro Glu Ala Thr Gly Met385 390 395 400Thr
Pro Ala Glu Val Ser Ile Val Val Leu Ser Gly Thr Thr Ala Ala 405 410
415Gln Val Thr Thr Thr Glu Trp Val Glu Thr Thr Ala Arg Glu Leu Pro
420 425 430Ile Pro Glu Pro Glu Gly Pro Asp Ala Ser Ser Ile Met Ser
Thr Glu 435 440 445Ser Ile Thr Gly Ser Leu Gly Pro Leu Leu Asp Gly
Thr Ala Thr Leu 450 455 460Arg Leu Val Lys Arg Gln Val Pro Leu Asp
Cys Val Leu Tyr Arg Tyr465 470 475 480Gly Ser Phe Ser Val Thr Leu
Asp Ile Val Gln Gly Ile Glu Ser Ala 485 490 495Glu Ile Leu Gln Ala
Val Pro Ser Gly Glu Gly Asp Ala Phe Glu Leu 500 505 510Thr Val Ser
Cys Gln Gly Gly Leu Pro Lys Glu Ala Cys Met Glu Ile 515 520 525Ser
Ser Pro Gly Cys Gln Pro Pro Ala Gln Arg Leu Cys Gln Pro Val 530 535
540Leu Pro Ser Pro Ala Cys Gln Leu Val Leu His Gln Ile Leu Lys
Gly545 550 555 560Gly Ser Gly Thr Tyr Cys Leu Asn Val Ser Leu Ala
Asp Thr Asn Ser 565 570 575Leu Ala Val Val Ser Thr Gln Leu Ile Met
Pro Gly Gln Glu Ala Gly 580 585 590Leu Gly Gln Val Pro Leu Ile Val
Gly Ile Leu Leu Val Leu Met Ala 595 600 605Val Val Leu Ala Ser Leu
Ile Tyr Arg Arg Arg Leu Met Lys Gln Asp 610 615 620Phe Ser Val Pro
Gln Leu Pro His Ser Ser Ser His Trp Leu Arg Leu625 630 635 640Pro
Arg Ile Phe Cys Ser Cys Pro Ile Gly Glu Asn Ser Pro Leu Leu 645 650
655Ser Gly Gln Gln Val 6601139PRTArtificial SequenceSynthetic
peptide. 113Ile Met Asp Gln Val Pro Phe Ser Val1 51149PRTArtificial
SequenceSynthetic peptide. 114Ile Thr Asp Gln Val Pro Phe Ser Val1
51159PRTArtificial SequenceSynthetic peptide. 115Tyr Leu Glu Pro
Gly Pro Val Thr Val1 51169PRTArtificial SequenceSynthetic peptide.
116Tyr Leu Glu Pro Gly Pro Val Thr Ala1 51179PRTArtificial
SequenceSynthetic peptide. 117Lys Thr Trp Gly Gln Tyr Trp Gln Val1
51181160PRTArtificial SequenceSynthetic peptide. 118Glu Val Lys Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys
Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30Tyr Met
Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45Ala
Tyr Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Ser Arg Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Arg Arg Gly Leu Pro Phe His Ala Met Asp Tyr Trp Gly
Gln Gly 100 105 110Thr Ser Val Thr Val Ser Ser Ala Lys Thr Lys Gly
Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
Glu Ser Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser
Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200
205Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro
210 215 220Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser
Val Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro 245 250 255Glu Val Thr Cys Val Val Val Asp Val
Ser Gln Glu Asp Pro Glu Val 260 265 270Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315
320Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser
325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro 340 345 350Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val 355 360 365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp385 390 395 400Gly Ser Phe Phe Leu
Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415Gln Glu Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala Ser 435 440
445Asp Thr Thr Glu Pro Ala Thr Pro Thr Thr Pro Val Thr Thr Pro Thr
450 455 460Thr Thr Lys Val Pro Arg Asn Gln Asp Trp Leu Gly Val Ser
Arg Gln465 470 475 480Leu Arg Thr Lys Ala Trp Asn Arg Gln Leu Tyr
Pro Glu Trp Thr Glu 485 490 495Ala Gln Arg Leu Asp Cys Trp Arg Gly
Gly Gln Val Ser Leu Lys Val 500 505 510Ser Asn Asp Gly Pro Thr Leu
Ile Gly Ala Asn Ala Ser Phe Ser Ile 515 520 525Ala Leu Asn Phe Pro
Gly Ser Gln Lys Val Leu Pro Asp Gly Gln Val 530 535 540Ile Trp Val
Asn Asn Thr Ile Ile Asn Gly Ser Gln Val Trp Gly Gly545 550 555
560Gln Pro Val Tyr Pro Gln Glu Thr Asp Asp Ala Cys Ile Phe Pro Asp
565 570 575Gly Gly Pro Cys Pro Ser Gly Ser Trp Ser Gln Lys Arg Ser
Phe Val 580 585 590Tyr Val Trp Lys Thr Trp Gly Gln Tyr Trp Gln Val
Leu Gly Gly Pro 595 600 605Val Ser Gly Leu Ser Ile Gly Thr Gly Arg
Ala Met Leu Gly Thr His 610 615 620Thr Met Glu Val Thr Val Tyr His
Arg Arg Gly Ser Gln Ser Tyr Val625 630 635 640Pro Leu Ala His Ser
Ser Ser Ala Phe Thr Ile Thr Asp Gln Val Pro 645 650 655Phe Ser Val
Ser Val Ser Gln Leu Arg Ala Leu Asp Gly Gly Asn Lys 660 665 670His
Phe Leu Arg Asn Gln Ala Ser Thr Asn Gly Ser Ile Thr Val Ala 675 680
685Ala Thr Ala Pro Thr Val Thr Pro Thr Val Asn Ala Thr Pro Ser Ala
690 695 700Ala Ala Ser Gly Thr Thr Asp Gly His Arg Pro Thr Thr Glu
Ala Pro705 710 715 720Asn Thr Thr Ala Gly Gln Val Pro Thr Thr Glu
Val Val Gly Thr
Thr 725 730 735Pro Gly Gln Ala Pro Thr Ala Glu Pro Ser Gly Thr Thr
Ser Val Gln 740 745 750Val Pro Thr Thr Glu Val Ile Ser Thr Ala Pro
Val Gln Met Pro Thr 755 760 765Ala Glu Ser Thr Gly Met Thr Pro Glu
Lys Val Pro Val Ser Glu Val 770 775 780Met Gly Thr Thr Leu Ala Glu
Met Ser Thr Pro Glu Ala Thr Gly Met785 790 795 800Thr Pro Ala Glu
Val Ser Ile Val Val Leu Ser Gly Thr Thr Ala Ala 805 810 815Ala Ser
Thr Val Thr Pro Thr Ala Thr Ala Thr Pro Ser Ala Ile Val 820 825
830Thr Thr Ile Thr Pro Thr Ala Thr Thr Lys Pro Ala Ser Gln Val Thr
835 840 845Thr Thr Glu Trp Val Glu Thr Thr Ala Arg Glu Leu Pro Ile
Pro Glu 850 855 860Pro Glu Gly Pro Asp Ala Ser Ser Ile Met Ser Thr
Glu Ser Ile Thr865 870 875 880Gly Ser Leu Gly Pro Leu Leu Asp Gly
Thr Ala Thr Leu Arg Leu Val 885 890 895Lys Arg Gln Val Pro Leu Asp
Cys Val Leu Tyr Arg Tyr Gly Ser Phe 900 905 910Ser Val Thr Leu Asp
Ile Val Gln Ala Ser Thr Asn Gly Ser Ile Thr 915 920 925Val Ala Ala
Thr Ala Pro Thr Val Thr Pro Thr Val Asn Ala Thr Pro 930 935 940Ser
Ala Ala Ala Ser Gly Ile Glu Ser Ala Glu Ile Leu Gln Ala Val945 950
955 960Pro Ser Gly Glu Gly Asp Ala Phe Glu Leu Thr Val Ser Cys Gln
Gly 965 970 975Gly Leu Pro Lys Glu Ala Cys Met Glu Ile Ser Ser Pro
Gly Cys Gln 980 985 990Pro Pro Ala Gln Arg Leu Cys Gln Pro Val Leu
Pro Ser Pro Ala Cys 995 1000 1005Gln Leu Val Leu His Gln Ile Leu
Lys Gly Gly Ser Gly Thr Tyr 1010 1015 1020Cys Leu Asn Val Ser Leu
Ala Asp Thr Asn Ser Leu Ala Val Val 1025 1030 1035Ser Thr Gln Leu
Ile Val Pro Gly Ile Leu Leu Thr Gly Gln Glu 1040 1045 1050Ala Gly
Leu Gly Gln Ala Ser Thr Val Thr Pro Thr Ala Thr Ala 1055 1060
1065Thr Pro Ser Ala Ile Val Thr Thr Ile Thr Pro Thr Ala Thr Thr
1070 1075 1080Lys Pro Ala Ser Pro Leu Thr Phe Ala Leu Gln Leu His
Asp Pro 1085 1090 1095Ser Gly Tyr Leu Ala Glu Ala Asp Leu Ser Tyr
Thr Trp Asp Phe 1100 1105 1110Gly Asp Ser Ser Gly Thr Leu Ile Ser
Arg Ala Leu Val Val Thr 1115 1120 1125His Thr Tyr Leu Glu Pro Gly
Pro Val Thr Ala Gln Val Val Leu 1130 1135 1140Gln Ala Ala Ile Pro
Leu Thr Ser Cys Gly Ser Ser Pro Val Pro 1145 1150 1155Ala Ser
11601191161PRTArtificial SequenceSynthetic peptide. 119Arg Leu Gln
Leu Gln Glu Ser Gly Pro Gly Leu Leu Lys Pro Ser Val1 5 10 15Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Val Ala Ser Ser 20 25 30Ser
Tyr Tyr Trp Gly Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40
45Trp Ile Gly Thr Ile Asn Phe Ser Gly Asn Met Tyr Tyr Ser Pro Ser
50 55 60Leu Arg Ser Arg Val Thr Met Ser Ala Asp Met Ser Glu Asn Ser
Phe65 70 75 80Tyr Leu Lys Leu Asp Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr 85 90 95Cys Ala Ala Gly His Leu Val Met Gly Phe Gly Ala
His Trp Gly Gln 100 105 110Gly Lys Leu Val Ser Val Ser Pro Ala Ser
Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185
190Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys
195 200 205Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr
Gly Pro 210 215 220Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly
Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val
Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270Val Gln Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310
315 320Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro 340 345 350Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425
430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala
435 440 445Ser Asp Thr Thr Glu Pro Ala Thr Pro Thr Thr Pro Val Thr
Thr Pro 450 455 460Thr Thr Thr Lys Val Pro Arg Asn Gln Asp Trp Leu
Gly Val Ser Arg465 470 475 480Gln Leu Arg Thr Lys Ala Trp Asn Arg
Gln Leu Tyr Pro Glu Trp Thr 485 490 495Glu Ala Gln Arg Leu Asp Cys
Trp Arg Gly Gly Gln Val Ser Leu Lys 500 505 510Val Ser Asn Asp Gly
Pro Thr Leu Ile Gly Ala Asn Ala Ser Phe Ser 515 520 525Ile Ala Leu
Asn Phe Pro Gly Ser Gln Lys Val Leu Pro Asp Gly Gln 530 535 540Val
Ile Trp Val Asn Asn Thr Ile Ile Asn Gly Ser Gln Val Trp Gly545 550
555 560Gly Gln Pro Val Tyr Pro Gln Glu Thr Asp Asp Ala Cys Ile Phe
Pro 565 570 575Asp Gly Gly Pro Cys Pro Ser Gly Ser Trp Ser Gln Lys
Arg Ser Phe 580 585 590Val Tyr Val Trp Lys Thr Trp Gly Gln Tyr Trp
Gln Val Leu Gly Gly 595 600 605Pro Val Ser Gly Leu Ser Ile Gly Thr
Gly Arg Ala Met Leu Gly Thr 610 615 620His Thr Met Glu Val Thr Val
Tyr His Arg Arg Gly Ser Gln Ser Tyr625 630 635 640Val Pro Leu Ala
His Ser Ser Ser Ala Phe Thr Ile Thr Asp Gln Val 645 650 655Pro Phe
Ser Val Ser Val Ser Gln Leu Arg Ala Leu Asp Gly Gly Asn 660 665
670Lys His Phe Leu Arg Asn Gln Ala Ser Thr Asn Gly Ser Ile Thr Val
675 680 685Ala Ala Thr Ala Pro Thr Val Thr Pro Thr Val Asn Ala Thr
Pro Ser 690 695 700Ala Ala Ala Ser Gly Thr Thr Asp Gly His Arg Pro
Thr Thr Glu Ala705 710 715 720Pro Asn Thr Thr Ala Gly Gln Val Pro
Thr Thr Glu Val Val Gly Thr 725 730 735Thr Pro Gly Gln Ala Pro Thr
Ala Glu Pro Ser Gly Thr Thr Ser Val 740 745 750Gln Val Pro Thr Thr
Glu Val Ile Ser Thr Ala Pro Val Gln Met Pro 755 760 765Thr Ala Glu
Ser Thr Gly Met Thr Pro Glu Lys Val Pro Val Ser Glu 770 775 780Val
Met Gly Thr Thr Leu Ala Glu Met Ser Thr Pro Glu Ala Thr Gly785 790
795 800Met Thr Pro Ala Glu Val Ser Ile Val Val Leu Ser Gly Thr Thr
Ala 805 810 815Ala Ala Ser Thr Val Thr Pro Thr Ala Thr Ala Thr Pro
Ser Ala Ile 820 825 830Val Thr Thr Ile Thr Pro Thr Ala Thr Thr Lys
Pro Ala Ser Gln Val 835 840 845Thr Thr Thr Glu Trp Val Glu Thr Thr
Ala Arg Glu Leu Pro Ile Pro 850 855 860Glu Pro Glu Gly Pro Asp Ala
Ser Ser Ile Met Ser Thr Glu Ser Ile865 870 875 880Thr Gly Ser Leu
Gly Pro Leu Leu Asp Gly Thr Ala Thr Leu Arg Leu 885 890 895Val Lys
Arg Gln Val Pro Leu Asp Cys Val Leu Tyr Arg Tyr Gly Ser 900 905
910Phe Ser Val Thr Leu Asp Ile Val Gln Ala Ser Thr Asn Gly Ser Ile
915 920 925Thr Val Ala Ala Thr Ala Pro Thr Val Thr Pro Thr Val Asn
Ala Thr 930 935 940Pro Ser Ala Ala Ala Ser Gly Ile Glu Ser Ala Glu
Ile Leu Gln Ala945 950 955 960Val Pro Ser Gly Glu Gly Asp Ala Phe
Glu Leu Thr Val Ser Cys Gln 965 970 975Gly Gly Leu Pro Lys Glu Ala
Cys Met Glu Ile Ser Ser Pro Gly Cys 980 985 990Gln Pro Pro Ala Gln
Arg Leu Cys Gln Pro Val Leu Pro Ser Pro Ala 995 1000 1005Cys Gln
Leu Val Leu His Gln Ile Leu Lys Gly Gly Ser Gly Thr 1010 1015
1020Tyr Cys Leu Asn Val Ser Leu Ala Asp Thr Asn Ser Leu Ala Val
1025 1030 1035Val Ser Thr Gln Leu Ile Val Pro Gly Ile Leu Leu Thr
Gly Gln 1040 1045 1050Glu Ala Gly Leu Gly Gln Ala Ser Thr Val Thr
Pro Thr Ala Thr 1055 1060 1065Ala Thr Pro Ser Ala Ile Val Thr Thr
Ile Thr Pro Thr Ala Thr 1070 1075 1080Thr Lys Pro Ala Ser Pro Leu
Thr Phe Ala Leu Gln Leu His Asp 1085 1090 1095Pro Ser Gly Tyr Leu
Ala Glu Ala Asp Leu Ser Tyr Thr Trp Asp 1100 1105 1110Phe Gly Asp
Ser Ser Gly Thr Leu Ile Ser Arg Ala Leu Val Val 1115 1120 1125Thr
His Thr Tyr Leu Glu Pro Gly Pro Val Thr Ala Gln Val Val 1130 1135
1140Leu Gln Ala Ala Ile Pro Leu Thr Ser Cys Gly Ser Ser Pro Val
1145 1150 1155Pro Ala Ser 11601202142DNAArtificial SequenceGP100
nucleic acid sequence. 120gatacaacag aacctgcaac acctacaaca
cctgtaacaa caccgacaac aacaaaagta 60cccagaaacc aggactggct tggtgtctca
aggcaactca gaaccaaagc ctggaacagg 120cagctgtatc cagagtggac
agaagcccag agacttgact gctggagagg tggtcaagtg 180tccctcaagg
tcagtaatga tgggcctaca ctgattggtg caaatgcctc cttctctatt
240gccttgaact tccctggaag ccaaaaggta ttgccagatg ggcaggttat
ctgggtcaac 300aataccatca tcaatgggag ccaggtgtgg ggaggacagc
cagtgtatcc ccaggaaact 360gacgatgcct gcatcttccc tgatggtgga
ccttgcccat ctggctcttg gtctcagaag 420agaagctttg tttatgtctg
gaagacctgg ggccaatact ggcaagttct agggggccca 480gtgtctgggc
tgagcattgg gacaggcagg gcaatgctgg gcacacacac catggaagtg
540actgtctacc atcgccgggg atcccagagc tatgtgcctc ttgctcattc
cagctcagcc 600ttcaccatta ctgaccaggt gcctttctcc gtgagcgtgt
cccagttgcg ggccttggat 660ggagggaaca agcacttcct gagaaatcag
gctagtacca acggcagcat caccgtggcc 720gccaccgccc ccaccgtgac
ccccaccgtg aacgccaccc ccagcgccgc cgctagtggc 780accacagatg
ggcacaggcc aactgcagag gcccctaaca ccacagctgg ccaagtgcct
840actacagaag ttgtgggtac tacacctggt caggcgccaa ctgcagagcc
ctctggaacc 900acatctgtgc aggtgccaac cactgaagtc ataagcactg
cacctgtgca gatgccaact 960gcagagagca caggtatgac acctgagaag
gtgccagttt cagaggtcat gggtaccaca 1020ctggcagaga tgtcaactcc
agaggctaca ggtatgacac ctgcagaggt atcaattgtg 1080gtgctttctg
gaaccacagc tgcagctagt accgtgaccc ccaccgccac cgccaccccc
1140agcgccatcg tgaccaccat cacccccacc gccaccacca agcccgctag
tcaggtaaca 1200actacagagt gggtggagac cacagctaga gagctaccta
tccctgagcc tgaaggtcca 1260gatgccagct caatcatgtc tacggaaagt
attacaggtt ccctgggccc cctgctggat 1320ggtacagcca ccttaaggct
ggtgaagaga caagtccccc tggattgtgt tctgtatcga 1380tatggttcct
tttccgtcac cctggacatt gtccaggcta gtaccaacgg cagcatcacc
1440gtggccgcca ccgcccccac cgtgaccccc accgtgaacg ccacccccag
cgccgccgct 1500agtggtattg aaagtgccga gatcctgcag gctgtgccgt
ccggtgaggg ggatgcattt 1560gagctgactg tgtcctgcca aggcgggctg
cccaaggaag cctgcatgga gatctcatcg 1620ccagggtgcc agccccctgc
ccagcggctg tgccagcctg tgctacccag cccagcctgc 1680cagctggttc
tgcaccagat actgaagggt ggctcgggga catactgcct caatgtgtct
1740ctggctgata ccaacagcct ggcagtggtc agcacccagc ttatcgtgcc
tgggattctt 1800ctcacaggtc aagaagcagg ccttgggcag taagctagta
ccgtgacccc caccgccacc 1860gccaccccca gcgccatcgt gaccaccatc
acccccaccg ccaccaccaa gcccgctagt 1920cctctgacct ttgccctcca
gctccatgac cctagtggct atctggctga agctgacctc 1980tcctacacct
gggactttgg agacagtagt ggaaccctga tctctcgggc acytgtggtc
2040actcatactt acctggagcc tggcccagtc actgcccagg tggtcctgca
ggctgccatt 2100cctctcacct cctgtggctc ctccccagtt ccagctagct ga
2142121690DNAArtificial SequenceGP100 peptide nucleic acid
sequence. 121gatacaacag aacctgcaac acctacaaca cctgtaacaa caccgacaac
aacaaaagta 60cccagaaacc aggactggct tggtgtctca aggcaactca gaaccaaagc
ctggaacagg 120cagctgtatc cagagtggac agaagcccag agacttgact
gctggagagg tggtcaagtg 180tccctcaagg tcagtaatga tgggcctaca
ctgattggtg caaatgcctc cttctctatt 240gccttgaact tccctggaag
ccaaaaggta ttgccagatg ggcaggttat ctgggtcaac 300aataccatca
tcaatgggag ccaggtgtgg ggaggacagc cagtgtatcc ccaggaaact
360gacgatgcct gcatcttccc tgatggtgga ccttgcccat ctggctcttg
gtctcagaag 420agaagctttg tttatgtctg gaagacctgg ggccaatact
ggcaagttct agggggccca 480gtgtctgggc tgagcattgg gacaggcagg
gcaatgctgg gcacacacac catggaagtg 540actgtctacc atcgccgggg
atcccagagc tatgtgcctc ttgctcattc cagctcagcc 600ttcaccatta
ctgaccaggt gcctttctcc gtgagcgtgt cccagttgcg ggccttggat
660ggagggaaca agcacttcct gagaaatcag 690122230PRTArtificial
SequenceAmino acid sequence. 122Asp Thr Thr Glu Pro Ala Thr Pro Thr
Thr Pro Val Thr Thr Pro Thr1 5 10 15Thr Thr Lys Val Pro Arg Asn Gln
Asp Trp Leu Gly Val Ser Arg Gln 20 25 30Leu Arg Thr Lys Ala Trp Asn
Arg Gln Leu Tyr Pro Glu Trp Thr Glu 35 40 45Ala Gln Arg Leu Asp Cys
Trp Arg Gly Gly Gln Val Ser Leu Lys Val 50 55 60Ser Asn Asp Gly Pro
Thr Leu Ile Gly Ala Asn Ala Ser Phe Ser Ile65 70 75 80Ala Leu Asn
Phe Pro Gly Ser Gln Lys Val Leu Pro Asp Gly Gln Val 85 90 95Ile Trp
Val Asn Asn Thr Ile Ile Asn Gly Ser Gln Val Trp Gly Gly 100 105
110Gln Pro Val Tyr Pro Gln Glu Thr Asp Asp Ala Cys Ile Phe Pro Asp
115 120 125Gly Gly Pro Cys Pro Ser Gly Ser Trp Ser Gln Lys Arg Ser
Phe Val 130 135 140Tyr Val Trp Lys Thr Trp Gly Gln Tyr Trp Gln Val
Leu Gly Gly Pro145 150 155 160Val Ser Gly Leu Ser Ile Gly Thr Gly
Arg Ala Met Leu Gly Thr His 165 170 175Thr Met Glu Val Thr Val Tyr
His Arg Arg Gly Ser Gln Ser Tyr Val 180 185 190Pro Leu Ala His Ser
Ser Ser Ala Phe Thr Ile Thr Asp Gln Val Pro 195 200 205Phe Ser Val
Ser Val Ser Gln Leu Arg Ala Leu Asp Gly Gly Asn Lys 210 215 220His
Phe Leu Arg Asn Gln225 230123327DNAArtificial SequenceSynthetic
oligonucleotide. 123ggcaccacag atgggcacag gccaactgca gaggccccta
acaccacagc tggccaagtg 60cctactacag aagttgtggg tactacacct ggtcaggcgc
caactgcaga gccctctgga 120accacatctg tgcaggtgcc aaccactgaa
gtcataagca ctgcacctgt gcagatgcca 180actgcagaga gcacaggtat
gacacctgag aaggtgccag tttcagaggt catgggtacc 240acactggcag
agatgtcaac tccagaggct acaggtatga cacctgcaga ggtatcaatt
300gtggtgcttt ctggaaccac agctgca 327124109PRTArtificial
SequenceSynthetic GP-100 peptide. 124Gly Thr Thr Asp Gly His Arg
Pro Thr Ala Glu Ala Pro Asn Thr Thr1 5 10 15Ala Gly Gln Val Pro Thr
Thr Glu Val Val Gly Thr Thr Pro Gly Gln 20 25 30Ala Pro Thr Ala Glu
Pro Ser Gly Thr Thr Ser Val Gln Val Pro Thr 35 40 45Thr Glu Val Ile
Ser Thr Ala Pro Val Gln Met Pro Thr Ala Glu Ser 50 55
60Thr Gly Met Thr Pro Glu Lys Val Pro Val Ser Glu Val Met Gly Thr65
70 75 80Thr Leu Ala Glu Met Ser Thr Pro Glu Ala Thr Gly Met Thr Pro
Ala 85 90 95Glu Val Ser Ile Val Val Leu Ser Gly Thr Thr Ala Ala 100
105125225DNAArtificial SequenceSynthetic GP100 oligonucleotide.
125caggtaacaa ctacagagtg ggtggagacc acagctagag agctacctat
ccctgagcct 60gaaggtccag atgccagctc aatcatgtct acggaaagta ttacaggttc
cctgggcccc 120ctgctggatg gtacagccac cttaaggctg gtgaagagac
aagtccccct ggattgtgtt 180ctgtatcgat atggttcctt ttccgtcacc
ctggacattg tccag 22512675PRTArtificial SequenceSynthetic GP100
oligonucleotide. 126Gln Val Thr Thr Thr Glu Trp Val Glu Thr Thr Ala
Arg Glu Leu Pro1 5 10 15Ile Pro Glu Pro Glu Gly Pro Asp Ala Ser Ser
Ile Met Ser Thr Glu 20 25 30Ser Ile Thr Gly Ser Leu Gly Pro Leu Leu
Asp Gly Thr Ala Thr Leu 35 40 45Arg Leu Val Lys Arg Gln Val Pro Leu
Asp Cys Val Leu Tyr Arg Tyr 50 55 60Gly Ser Phe Ser Val Thr Leu Asp
Ile Val Gln65 70 75127327DNAArtificial SequenceSynthetic GP100
oligonucleotide. 127ggtattgaaa gtgccgagat cctgcaggct gtgccgtccg
gtgaggggga tgcatttgag 60ctgactgtgt cctgccaagg cgggctgccc aaggaagcct
gcatggagat ctcatcgcca 120gggtgccagc cccctgccca gcggctgtgc
cagcctgtgc tacccagccc agcctgccag 180ctggttctgc accagatact
gaagggtggc tcggggacat actgcctcaa tgtgtctctg 240gctgatacca
acagcctggc agtggtcagc acccagctta tcgtgcctgg gattcttctc
300acaggtcaag aagcaggcct tgggcag 327128109PRTArtificial
SequenceSynthetic GP100 amino-acid sequence. 128Gly Ile Glu Ser Ala
Glu Ile Leu Gln Ala Val Pro Ser Gly Glu Gly1 5 10 15Asp Ala Phe Glu
Leu Thr Val Ser Cys Gln Gly Gly Leu Pro Lys Glu 20 25 30Ala Cys Met
Glu Ile Ser Ser Pro Gly Cys Gln Pro Pro Ala Gln Arg 35 40 45Leu Cys
Gln Pro Val Leu Pro Ser Pro Ala Cys Gln Leu Val Leu His 50 55 60Gln
Ile Leu Lys Gly Gly Ser Gly Thr Tyr Cys Leu Asn Val Ser Leu65 70 75
80Ala Asp Thr Asn Ser Leu Ala Val Val Ser Thr Gln Leu Ile Val Pro
85 90 95Gly Ile Leu Leu Thr Gly Gln Glu Ala Gly Leu Gly Gln 100
105129219DNAArtificial SequenceSynthetic GP100 oligonucleotide.
129cctctgacct ttgccctcca gctccatgac cctagtggct atctggctga
agctgacctc 60tcctacacct gggactttgg agacagtagt ggaaccctga tctctcgggc
acytgtggtc 120actcatactt acctggagcc tggcccagtc actgcccagg
tggtcctgca ggctgccatt 180cctctcacct cctgtggctc ctccccagtt ccagctagc
21913073PRTArtificial SequenceSynthetic GP100 amino acid
sequencemisc_feature(38)..(38)Xaa can be any naturally occurring
amino acid 130Pro Leu Thr Phe Ala Leu Gln Leu His Asp Pro Ser Gly
Tyr Leu Ala1 5 10 15Glu Ala Asp Leu Ser Tyr Thr Trp Asp Phe Gly Asp
Ser Ser Gly Thr 20 25 30Leu Ile Ser Arg Ala Xaa Val Val Thr His Thr
Tyr Leu Glu Pro Gly 35 40 45Pro Val Thr Ala Gln Val Val Leu Gln Ala
Ala Ile Pro Leu Thr Ser 50 55 60Cys Gly Ser Ser Pro Val Pro Ala
Ser65 70131438PRTArtificial SequenceHuman CyclinB1 amino-acid
sequence. 131Met Ala Leu Arg Val Thr Arg Asn Ser Lys Ile Asn Ala
Glu Asn Lys1 5 10 15Ala Lys Ile Asn Met Ala Gly Ala Lys Arg Val Pro
Thr Ala Pro Ala 20 25 30Ala Thr Ser Lys Pro Gly Leu Arg Pro Arg Thr
Ala Leu Gly Asp Ile 35 40 45Gly Asn Lys Val Ser Glu Gln Leu Gln Ala
Lys Met Pro Met Lys Lys 50 55 60Glu Ala Lys Pro Ser Ala Thr Gly Lys
Val Ile Asp Lys Lys Leu Pro65 70 75 80Lys Pro Leu Glu Lys Val Pro
Met Leu Val Pro Val Pro Val Ser Glu 85 90 95Pro Val Pro Glu Pro Glu
Pro Glu Pro Glu Pro Glu Pro Val Lys Glu 100 105 110Glu Lys Leu Ser
Pro Glu Pro Ile Leu Val Asp Thr Ala Ser Pro Ser 115 120 125Pro Met
Glu Thr Ser Gly Cys Ala Pro Ala Glu Glu Asp Leu Cys Gln 130 135
140Ala Phe Ser Asp Val Ile Leu Ala Val Asn Asp Val Asp Ala Glu
Asp145 150 155 160Gly Ala Asp Pro Asn Leu Cys Ser Glu Tyr Val Lys
Asp Ile Tyr Ala 165 170 175Tyr Leu Arg Gln Leu Glu Glu Glu Gln Ala
Val Arg Pro Lys Tyr Leu 180 185 190Leu Gly Arg Glu Val Thr Gly Asn
Met Arg Ala Ile Leu Ile Asp Trp 195 200 205Leu Val Gln Val Gln Met
Lys Phe Arg Leu Leu Gln Glu Thr Met Tyr 210 215 220Met Thr Val Ser
Ile Ile Asp Arg Phe Met Gln Asn Asn Cys Val Pro225 230 235 240Lys
Lys Met Leu Gln Leu Val Gly Val Thr Ala Met Phe Ile Ala Ser 245 250
255Lys Tyr Glu Glu Met Tyr Pro Pro Glu Ile Gly Asp Phe Ala Phe Val
260 265 270Thr Asp Asn Thr Tyr Thr Lys His Gln Ile Arg Gln Met Glu
Met Lys 275 280 285Ile Leu Arg Ala Leu Asn Phe Gly Leu Gly Arg Pro
Leu Pro Leu His 290 295 300Phe Leu Arg Arg Ala Ser Lys Ile Gly Glu
Val Asp Val Glu Gln His305 310 315 320Thr Leu Ala Lys Tyr Leu Met
Glu Thr Met Leu Asp Tyr Asp Met Val 325 330 335His Phe Pro Pro Ser
Gln Ile Ala Ala Gly Ala Phe Cys Leu Ala Leu 340 345 350Lys Ile Leu
Asp Asn Gly Glu Trp Thr Pro Thr Leu Gln His Tyr Leu 355 360 365Ser
Tyr Thr Glu Glu Ser Leu Leu Pro Val Met Gln His Leu Ala Lys 370 375
380Asn Val Val Met Val Asn Gln Gly Leu Thr Lys His Met Thr Val
Lys385 390 395 400Asn Lys Tyr Ala Thr Ser Lys His Ala Lys Ile Ser
Thr Leu Pro Gln 405 410 415Leu Asn Ser Ala Leu Val Gln Asp Leu Ala
Lys Ala Val Ala Lys Val 420 425 430His His His His His His
43513250PRTArtificial SequenceSynthetic peptide. 132Met Glu Met Lys
Ile Leu Arg Ala Leu Asn Phe Gly Leu Gly Arg Pro1 5 10 15Leu Pro Leu
His Phe Leu Arg Arg Ala Ser Lys Ile Gly Glu Val Asp 20 25 30Val Glu
Gln His Thr Leu Ala Lys Tyr Leu Met Glu Leu Thr Met Leu 35 40 45Asp
Tyr 5013336PRTArtificial SequenceSynthetic peptide. 133Asp Trp Leu
Val Gln Val Gln Met Lys Phe Arg Leu Leu Gln Glu Thr1 5 10 15Met Tyr
Met Thr Val Ser Ile Ile Asp Arg Phe Met Gln Asn Asn Cys 20 25 30Val
Pro Lys Lys 35134594PRTArtificial SequenceSynthetic peptide. 134Glu
Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp
Val 35 40 45Ala Tyr Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp
Thr Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Ser Arg Leu Lys Ser Glu Asp Thr
Ala Met Tyr Tyr Cys 85 90 95Ala Arg Arg Gly Leu Pro Phe His Ala Met
Asp Tyr Trp Gly Gln Gly 100 105 110Thr Ser Val Thr Val Ser Ser Ala
Lys Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Cys Ser
Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His
Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys
Tyr Gly Pro Pro 210 215 220Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu
Gly Gly Pro Ser Val Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255Glu Val Thr Cys Val
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270Gln Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285Lys
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295
300Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys305 310 315 320Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro 340 345 350Ser Gln Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val 355 360 365Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp385 390 395 400Gly
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410
415Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
Ala Ser 435 440 445Gln Thr Pro Thr Asn Thr Ile Ser Val Thr Pro Thr
Asn Asn Ser Thr 450 455 460Pro Thr Asn Asn Ser Asn Pro Lys Pro Asn
Pro Ala Ser Asp Trp Leu465 470 475 480Val Gln Val Gln Met Lys Phe
Arg Leu Leu Gln Glu Thr Met Tyr Met 485 490 495Thr Val Ser Ile Ile
Asp Arg Phe Met Gln Asn Asn Cys Val Pro Lys 500 505 510Lys Ala Ser
Met Glu Met Lys Ile Leu Arg Ala Leu Asn Phe Gly Leu 515 520 525Gly
Arg Pro Leu Pro Leu His Phe Leu Arg Arg Ala Ser Lys Ile Gly 530 535
540Glu Val Asp Val Glu Gln His Thr Leu Ala Lys Tyr Leu Met Glu
Leu545 550 555 560Thr Met Leu Asp Tyr Ala Ser Thr Asn Asp Ser Ile
Thr Val Ala Ala 565 570 575Thr Ala Pro Thr Val Thr Pro Thr Val Asn
Ala Thr Pro Ser Ala Ala 580 585 590Ala Ser1351842DNAArtificial
SequenceSynthetic oligonucleotide sequence. 135atgaacttgg
ggctcagctt gattttcctt gtccttgttt taaaaggtgt ccagtgtgaa 60gtgaagctgg
tggagtctgg gggaggctta gtgcagcccg gagggtccct gaaactctcc
120tgtgcaacct ctggattcac tttcagtgac tattacatgt attgggttcg
ccagactcca 180gagaagaggc tggagtgggt cgcatacatt aattctggtg
gtggtagcac ctattatcca 240gacactgtaa agggccgatt caccatctcc
agagacaatg ccaagaacac cctgtacctg 300caaatgagcc ggctgaagtc
tgaggacaca gccatgtatt actgtgcaag acgggggtta 360ccgttccatg
ctatggacta ttggggtcaa ggaacctcag tcaccgtctc ctcagccaaa
420acgaagggcc catccgtctt ccccctggcg ccctgctcca ggagcacctc
cgagagcaca 480gccgccctgg gctgcctggt caaggactac ttccccgaac
cggtgacggt gtcgtggaac 540tcaggcgccc tgaccagcgg cgtgcacacc
ttcccggctg tcctacagtc ctcaggactc 600tactccctca gcagcgtggt
gaccgtgccc tccagcagct tgggcacgaa gacctacacc 660tgcaacgtag
atcacaagcc cagcaacacc aaggtggaca agagagttga gtccaaatat
720ggtcccccat gcccaccctg cccagcacct gagttcgaag ggggaccatc
agtcttcctg 780ttccccccaa aacccaagga cactctcatg atctcccgga
cccctgaggt cacgtgcgtg 840gtggtggacg tgagccagga agaccccgag
gtccagttca actggtacgt ggatggcgtg 900gaggtgcata atgccaagac
aaagccgcgg gaggagcagt tcaacagcac gtaccgtgtg 960gtcagcgtcc
tcaccgtcct gcaccaggac tggctgaacg gcaaggagta caagtgcaag
1020gtctccaaca aaggcctccc gtcctccatc gagaaaacca tctccaaagc
caaagggcag 1080ccccgagagc cacaggtgta caccctgccc ccatcccagg
aggagatgac caagaaccag 1140gtcagcctga cctgcctggt caaaggcttc
taccccagcg acatcgccgt ggagtgggag 1200agcaatgggc agccggagaa
caactacaag accacgcctc ccgtgctgga ctccgacggc 1260tccttcttcc
tctacagcag gctaaccgtg gacaagagca ggtggcagga ggggaatgtc
1320ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacacagaa
gagcctctcc 1380ctgtctctgg gtaaagctag tcagaccccc accaacacca
tcagcgtgac ccccaccaac 1440aacagcaccc ccaccaacaa cagcaacccc
aagcccaacc ccgctagtga ctggctagta 1500caggttcaaa tgaaattcag
gttgttgcag gagaccatgt acatgactgt ctccattatt 1560gatcggttca
tgcagaataa ttgtgtgccc aagaaggcta gtatggaaat gaagattcta
1620agagctttaa actttggtct gggtcggcct ctacctttgc acttccttcg
gagagcatct 1680aagattggag aggttgatgt cgagcaacat actttggcca
aatacctgat ggaactaact 1740atgttggact atgctagtac caacgacagc
atcaccgtgg ccgccaccgc ccccaccgtg 1800acccccaccg tgaacgccac
ccccagcgcc gccgctagct ga 1842136542PRTArtificial SequenceSynthetic
peptide. 136Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe
Ser Asp Tyr 20 25 30Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg
Leu Glu Trp Val 35 40 45Ala Tyr Ile Asn Ser Gly Gly Gly Ser Thr Tyr
Tyr Pro Asp Thr Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Ser Arg Leu Lys Ser
Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Arg Gly Leu Pro Phe
His Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Ser Val Thr Val
Ser Ser Ala Lys Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala
Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150
155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
Val Asp His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Ser Lys Tyr Gly Pro Pro 210 215 220Cys Pro Pro Cys Pro Ala Pro
Glu Phe Glu Gly Gly Pro Ser Val Phe225 230 235 240Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255Glu Val
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265
270Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
Ser Val 290 295 300Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn Lys Gly Leu Pro Ser
Ser Ile Glu Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser Gln Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp385 390
395 400Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
Trp 405 410 415Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His 420 425 430Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Leu Gly Lys Ala Ser 435 440 445Gln Thr Pro Thr Asn Thr Ile Ser Val
Thr Pro Thr Asn Asn Ser Thr 450 455 460Pro Thr Asn Asn Ser Asn Pro
Lys Pro Asn Pro Ala Ser Asp Trp Leu465 470 475 480Val Gln Val Gln
Met Lys Phe Arg Leu Leu Gln Glu Thr Met Tyr Met 485 490 495Thr Val
Ser Ile Ile Asp Arg
Phe Met Gln Asn Asn Cys Val Pro Lys 500 505 510Lys Ala Ser Thr Val
Thr Pro Thr Ala Thr Ala Thr Pro Ser Ala Ile 515 520 525Val Thr Thr
Ile Thr Pro Thr Ala Thr Thr Lys Pro Ala Ser 530 535
5401371686DNAArtificial SequenceSynthetic oligonucleotide.
137atgaacttgg ggctcagctt gattttcctt gtccttgttt taaaaggtgt
ccagtgtgaa 60gtgaagctgg tggagtctgg gggaggctta gtgcagcccg gagggtccct
gaaactctcc 120tgtgcaacct ctggattcac tttcagtgac tattacatgt
attgggttcg ccagactcca 180gagaagaggc tggagtgggt cgcatacatt
aattctggtg gtggtagcac ctattatcca 240gacactgtaa agggccgatt
caccatctcc agagacaatg ccaagaacac cctgtacctg 300caaatgagcc
ggctgaagtc tgaggacaca gccatgtatt actgtgcaag acgggggtta
360ccgttccatg ctatggacta ttggggtcaa ggaacctcag tcaccgtctc
ctcagccaaa 420acgaagggcc catccgtctt ccccctggcg ccctgctcca
ggagcacctc cgagagcaca 480gccgccctgg gctgcctggt caaggactac
ttccccgaac cggtgacggt gtcgtggaac 540tcaggcgccc tgaccagcgg
cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 600tactccctca
gcagcgtggt gaccgtgccc tccagcagct tgggcacgaa gacctacacc
660tgcaacgtag atcacaagcc cagcaacacc aaggtggaca agagagttga
gtccaaatat 720ggtcccccat gcccaccctg cccagcacct gagttcgaag
ggggaccatc agtcttcctg 780ttccccccaa aacccaagga cactctcatg
atctcccgga cccctgaggt cacgtgcgtg 840gtggtggacg tgagccagga
agaccccgag gtccagttca actggtacgt ggatggcgtg 900gaggtgcata
atgccaagac aaagccgcgg gaggagcagt tcaacagcac gtaccgtgtg
960gtcagcgtcc tcaccgtcct gcaccaggac tggctgaacg gcaaggagta
caagtgcaag 1020gtctccaaca aaggcctccc gtcctccatc gagaaaacca
tctccaaagc caaagggcag 1080ccccgagagc cacaggtgta caccctgccc
ccatcccagg aggagatgac caagaaccag 1140gtcagcctga cctgcctggt
caaaggcttc taccccagcg acatcgccgt ggagtgggag 1200agcaatgggc
agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc
1260tccttcttcc tctacagcag gctaaccgtg gacaagagca ggtggcagga
ggggaatgtc 1320ttctcatgct ccgtgatgca tgaggctctg cacaaccact
acacacagaa gagcctctcc 1380ctgtctctgg gtaaagctag tcagaccccc
accaacacca tcagcgtgac ccccaccaac 1440aacagcaccc ccaccaacaa
cagcaacccc aagcccaacc ccgctagtga ctggctagta 1500caggttcaaa
tgaaattcag gttgttgcag gagaccatgt acatgactgt ctccattatt
1560gatcggttca tgcagaataa ttgtgtgccc aagaaggcta gtaccgtgac
ccccaccgcc 1620accgccaccc ccagcgccat cgtgaccacc atcaccccca
ccgccaccac caagcccgct 1680agctga 1686138556PRTArtificial
SequenceSynthetic peptide. 138Glu Val Lys Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Thr
Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30Tyr Met Tyr Trp Val Arg Gln
Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45Ala Tyr Ile Asn Ser Gly
Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met
Ser Arg Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg
Arg Gly Leu Pro Phe His Ala Met Asp Tyr Trp Gly Gln Gly 100 105
110Thr Ser Val Thr Val Ser Ser Ala Lys Thr Lys Gly Pro Ser Val Phe
115 120 125Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr
Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205Ser Asn Thr
Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220Cys
Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe225 230
235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro 245 250 255Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
Pro Glu Val 260 265 270Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu Gln Phe Asn Ser
Thr Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn
Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345
350Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly 370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp385 390 395 400Gly Ser Phe Phe Leu Tyr Ser Arg Leu
Thr Val Asp Lys Ser Arg Trp 405 410 415Gln Glu Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His 420 425 430Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala Ser 435 440 445Gln Thr Pro
Thr Asn Thr Ile Ser Val Thr Pro Thr Asn Asn Ser Thr 450 455 460Pro
Thr Asn Asn Ser Asn Pro Lys Pro Asn Pro Ala Ser Met Glu Met465 470
475 480Lys Ile Leu Arg Ala Leu Asn Phe Gly Leu Gly Arg Pro Leu Pro
Leu 485 490 495His Phe Leu Arg Arg Ala Ser Lys Ile Gly Glu Val Asp
Val Glu Gln 500 505 510His Thr Leu Ala Lys Tyr Leu Met Glu Leu Thr
Met Leu Asp Tyr Ala 515 520 525Ser Thr Asn Gly Ser Ile Thr Val Ala
Ala Thr Ala Pro Thr Val Thr 530 535 540Pro Thr Val Asn Ala Thr Pro
Ser Ala Ala Ala Ser545 550 5551391728DNAArtificial
SequenceSynthetic oligonucleotide. 139atgaacttgg ggctcagctt
gattttcctt gtccttgttt taaaaggtgt ccagtgtgaa 60gtgaagctgg tggagtctgg
gggaggctta gtgcagcccg gagggtccct gaaactctcc 120tgtgcaacct
ctggattcac tttcagtgac tattacatgt attgggttcg ccagactcca
180gagaagaggc tggagtgggt cgcatacatt aattctggtg gtggtagcac
ctattatcca 240gacactgtaa agggccgatt caccatctcc agagacaatg
ccaagaacac cctgtacctg 300caaatgagcc ggctgaagtc tgaggacaca
gccatgtatt actgtgcaag acgggggtta 360ccgttccatg ctatggacta
ttggggtcaa ggaacctcag tcaccgtctc ctcagccaaa 420acgaagggcc
catccgtctt ccccctggcg ccctgctcca ggagcacctc cgagagcaca
480gccgccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt
gtcgtggaac 540tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg
tcctacagtc ctcaggactc 600tactccctca gcagcgtggt gaccgtgccc
tccagcagct tgggcacgaa gacctacacc 660tgcaacgtag atcacaagcc
cagcaacacc aaggtggaca agagagttga gtccaaatat 720ggtcccccat
gcccaccctg cccagcacct gagttcgaag ggggaccatc agtcttcctg
780ttccccccaa aacccaagga cactctcatg atctcccgga cccctgaggt
cacgtgcgtg 840gtggtggacg tgagccagga agaccccgag gtccagttca
actggtacgt ggatggcgtg 900gaggtgcata atgccaagac aaagccgcgg
gaggagcagt tcaacagcac gtaccgtgtg 960gtcagcgtcc tcaccgtcct
gcaccaggac tggctgaacg gcaaggagta caagtgcaag 1020gtctccaaca
aaggcctccc gtcctccatc gagaaaacca tctccaaagc caaagggcag
1080ccccgagagc cacaggtgta caccctgccc ccatcccagg aggagatgac
caagaaccag 1140gtcagcctga cctgcctggt caaaggcttc taccccagcg
acatcgccgt ggagtgggag 1200agcaatgggc agccggagaa caactacaag
accacgcctc ccgtgctgga ctccgacggc 1260tccttcttcc tctacagcag
gctaaccgtg gacaagagca ggtggcagga ggggaatgtc 1320ttctcatgct
ccgtgatgca tgaggctctg cacaaccact acacacagaa gagcctctcc
1380ctgtctctgg gtaaagctag tcagaccccc accaacacca tcagcgtgac
ccccaccaac 1440aacagcaccc ccaccaacaa cagcaacccc aagcccaacc
ccgctagtat ggaaatgaag 1500attctaagag ctttaaactt tggtctgggt
cggcctctac ctttgcactt ccttcggaga 1560gcatctaaga ttggagaggt
tgatgtcgag caacatactt tggccaaata cctgatggaa 1620ctaactatgt
tggactatgc tagtaccaac ggcagcatca ccgtggccgc caccgccccc
1680accgtgaccc ccaccgtgaa cgccaccccc agcgccgccg ctagctga
1728140294PRTArtificial SequenceSynthetic Cyclin D1 peptide. 140Glu
His Gln Leu Leu Cys Cys Glu Val Glu Thr Ile Arg Arg Ala Tyr1 5 10
15Pro Asp Ala Asn Leu Leu Asn Asp Arg Val Leu Arg Ala Met Leu Lys
20 25 30Ala Glu Glu Thr Cys Ala Pro Ser Val Ser Tyr Phe Lys Cys Val
Gln 35 40 45Lys Glu Val Leu Pro Ser Met Arg Lys Ile Val Ala Thr Trp
Met Leu 50 55 60Glu Val Cys Glu Glu Gln Lys Cys Glu Glu Glu Val Phe
Pro Leu Ala65 70 75 80Met Asn Tyr Leu Asp Arg Phe Leu Ser Leu Glu
Pro Val Lys Lys Ser 85 90 95Arg Leu Gln Leu Leu Gly Ala Thr Cys Met
Phe Val Ala Ser Lys Met 100 105 110Lys Glu Thr Ile Pro Leu Thr Ala
Glu Lys Leu Cys Ile Tyr Thr Asp 115 120 125Asn Ser Ile Arg Pro Glu
Glu Leu Leu Gln Met Glu Leu Leu Leu Val 130 135 140Asn Lys Leu Lys
Trp Asn Leu Ala Ala Met Thr Pro His Asp Phe Ile145 150 155 160Glu
His Phe Leu Ser Lys Met Pro Glu Ala Glu Glu Asn Lys Gln Ile 165 170
175Ile Arg Lys His Ala Gln Thr Phe Val Ala Leu Cys Ala Thr Asp Val
180 185 190Lys Phe Ile Ser Asn Pro Pro Ser Met Val Ala Ala Gly Ser
Val Val 195 200 205Ala Ala Val Gln Gly Leu Asn Leu Arg Ser Pro Asn
Asn Phe Leu Ser 210 215 220Tyr Tyr Arg Leu Thr Arg Phe Leu Ser Arg
Val Ile Lys Cys Asp Pro225 230 235 240Asp Cys Leu Arg Ala Cys Gln
Glu Gln Ile Glu Ala Leu Leu Glu Ser 245 250 255Ser Leu Arg Gln Ala
Gln Gln Asn Met Asp Pro Lys Ala Ala Glu Glu 260 265 270Glu Glu Glu
Glu Glu Glu Glu Val Asp Leu Ala Cys Thr Pro Thr Asp 275 280 285Val
Arg Asp Val Asp Ile 29014148PRTArtificial SequenceSynthetic peptide
sequence. 141Met Glu His Gln Leu Leu Cys Cys Glu Val Glu Thr Ile
Arg Arg Ala1 5 10 15Tyr Pro Asp Ala Asn Leu Leu Asn Asp Arg Val Leu
Arg Ala Met Leu 20 25 30Lys Ala Glu Glu Thr Cys Ala Pro Ser Val Ser
Tyr Phe Lys Cys Val 35 40 4514295PRTArtificial SequenceSynthetic
peptide sequence. 142Gln Lys Glu Val Leu Pro Ser Met Arg Lys Ile
Val Ala Thr Trp Met1 5 10 15Leu Glu Val Cys Glu Glu Gln Lys Cys Glu
Glu Glu Val Phe Pro Leu 20 25 30Ala Met Asn Tyr Leu Asp Arg Phe Leu
Ser Leu Glu Pro Val Lys Lys 35 40 45Ser Arg Leu Gln Leu Leu Gly Ala
Thr Cys Met Phe Val Ala Ser Lys 50 55 60Met Lys Glu Thr Ile Pro Leu
Thr Ala Glu Lys Leu Cys Ile Tyr Thr65 70 75 80Asp Asn Ser Ile Arg
Pro Glu Glu Leu Leu Gln Met Glu Leu Leu 85 90 9514360PRTArtificial
SequenceSynthetic peptide sequence. 143Leu Val Asn Lys Leu Lys Trp
Asn Leu Ala Ala Met Thr Pro His Asp1 5 10 15Phe Ile Glu His Phe Leu
Ser Lys Met Pro Glu Ala Glu Glu Asn Lys 20 25 30Gln Ile Ile Arg Lys
His Ala Gln Thr Phe Val Ala Leu Cys Ala Thr 35 40 45Asp Val Lys Phe
Ile Ser Asn Pro Pro Ser Met Val 50 55 6014492PRTArtificial
SequenceSynthetic peptide sequence. 144Ala Ala Gly Ser Val Val Ala
Ala Val Gln Gly Leu Asn Leu Arg Ser1 5 10 15Pro Asn Asn Phe Leu Ser
Tyr Tyr Arg Leu Thr Arg Phe Leu Ser Arg 20 25 30Val Ile Lys Cys Asp
Pro Asp Cys Leu Arg Ala Cys Gln Glu Gln Ile 35 40 45Glu Ala Leu Leu
Glu Ser Ser Leu Arg Gln Ala Gln Gln Asn Met Asp 50 55 60Pro Lys Ala
Ala Glu Glu Glu Glu Glu Glu Glu Glu Glu Val Asp Leu65 70 75 80Ala
Cys Thr Pro Thr Asp Val Arg Asp Val Asp Ile 85 9014527PRTArtificial
SequenceSynthetic peptide sequence. 145Gln Thr Pro Thr Asn Thr Ile
Ser Val Thr Pro Thr Asn Asn Ser Thr1 5 10 15Pro Thr Asn Asn Ser Asn
Pro Lys Pro Asn Pro 20 25146560PRTArtificial SequenceSynthetic
cyclin D1H chain fusion protein. 146Gln Val Thr Leu Lys Glu Ser Gly
Pro Gly Ile Leu Gln Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ser
Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Gly Leu Ser Trp
Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45Trp Leu Ala His Ile
Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg
Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn Gln Val65 70 75 80Phe Leu
Lys Ile Thr Ile Val Asp Thr Ala Asp Ala Ala Thr Tyr Tyr 85 90 95Cys
Ala Arg Ser Ser His Tyr Tyr Gly Tyr Gly Tyr Gly Gly Tyr Phe 100 105
110Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr
115 120 125Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser
Thr Ser 130 135 140Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu145 150 155 160Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His 165 170 175Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys 195 200 205Asn Val Asp
His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 210 215 220Ser
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu225 230
235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser 260 265 270Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Phe Asn Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 325 330 335Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345
350Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Arg Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Leu Gly
Lys Ala Ser Gln Thr Pro Thr Asn Thr Ile Ser Val Thr 450 455 460Pro
Thr Asn Asn Ser Thr Pro Thr Asn Asn Ser Asn Pro Lys Pro Asn465 470
475 480Pro Ala Ser Met Glu His Gln Leu Leu Cys Cys Glu Val Glu Thr
Ile 485 490 495Arg Arg Ala Tyr Pro Asp Ala Asn Leu Leu Asn Asp Arg
Val Leu Arg 500 505 510Ala Met Leu Lys Ala Glu Glu Thr Cys Ala Pro
Ser Val Ser Tyr Phe 515 520 525Lys Cys Val Ala Ser Thr Asn Gly Ser
Ile Thr Val Ala Ala Thr Ala 530 535 540Pro Thr Val Thr Pro Thr Val
Asn Ala Thr Pro Ser Ala Ala Ala Ser545 550 555
560147759PRTArtificial SequenceSynthetic peptide. 147Gln Val Thr
Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln1 5 10 15Thr Leu
Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly
Met Gly Leu Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu
35 40 45Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro
Ser 50 55 60Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn
Gln Val65 70 75 80Phe Leu Lys Ile Thr Ile Val Asp Thr Ala Asp Ala
Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Ser Ser His Tyr Tyr Gly Tyr Gly
Tyr Gly Gly Tyr Phe 100 105 110Asp Val Trp Gly Ala Gly Thr Thr Val
Thr Val Ser Ser Ala Lys Thr 115 120 125Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140Glu Ser Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu145 150 155 160Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170
175Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
180 185 190Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr
Thr Cys 195 200 205Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys Arg Val Glu 210 215 220Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys
Pro Ala Pro Glu Phe Glu225 230 235 240Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270Gln Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 290 295
300Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ser Ser 325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Gln
Glu Glu Met Thr Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 405 410
415Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
420 425 430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu 435 440 445Ser Leu Gly Lys Ala Ser Gln Thr Pro Thr Asn Thr
Ile Ser Val Thr 450 455 460Pro Thr Asn Asn Ser Thr Pro Thr Asn Asn
Ser Asn Pro Lys Pro Asn465 470 475 480Pro Ala Ser Gln Lys Glu Val
Leu Pro Ser Met Arg Lys Ile Val Ala 485 490 495Thr Trp Met Leu Glu
Val Cys Glu Glu Gln Lys Cys Glu Glu Glu Val 500 505 510Phe Pro Leu
Ala Met Asn Tyr Leu Asp Arg Phe Leu Ser Leu Glu Pro 515 520 525Val
Lys Lys Ser Arg Leu Gln Leu Leu Gly Ala Thr Cys Met Phe Val 530 535
540Ala Ser Lys Met Lys Glu Thr Ile Pro Leu Thr Ala Glu Lys Leu
Cys545 550 555 560Ile Tyr Thr Asp Asn Ser Ile Arg Pro Glu Glu Leu
Leu Gln Met Glu 565 570 575Leu Leu Leu Val Asn Lys Leu Lys Trp Asn
Leu Ala Ala Met Thr Pro 580 585 590His Asp Phe Ile Glu His Phe Leu
Ser Lys Met Pro Glu Ala Glu Glu 595 600 605Asn Lys Gln Ile Ile Arg
Lys His Ala Gln Thr Phe Val Ala Leu Cys 610 615 620Ala Thr Asp Val
Lys Phe Ile Ser Asn Pro Pro Ser Met Val Ala Ala625 630 635 640Gly
Ser Val Val Ala Ala Val Gln Gly Leu Asn Leu Arg Ser Pro Asn 645 650
655Asn Phe Leu Ser Tyr Tyr Arg Leu Thr Arg Phe Leu Ser Arg Val Ile
660 665 670Lys Cys Asp Pro Asp Cys Leu Arg Ala Cys Gln Glu Gln Ile
Glu Ala 675 680 685Leu Leu Glu Ser Ser Leu Arg Gln Ala Gln Gln Asn
Met Asp Pro Lys 690 695 700Ala Ala Glu Glu Glu Glu Glu Glu Glu Glu
Glu Val Asp Leu Ala Cys705 710 715 720Thr Pro Thr Asp Val Arg Asp
Val Asp Ile Ala Ser Thr Asn Gly Ser 725 730 735Ile Thr Val Ala Ala
Thr Ala Pro Thr Val Thr Pro Thr Val Asn Ala 740 745 750Thr Pro Ser
Ala Ala Ala Ser 755148468PRTArtificial SequenceSynthetic fusion
protein. 148Met Asn Leu Gly Leu Ser Leu Ile Phe Leu Val Leu Val Leu
Lys Gly1 5 10 15Val Gln Cys Glu Val Lys Leu Val Glu Ser Gly Gly Gly
Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Thr Ser
Gly Phe Thr Phe 35 40 45Ser Asp Tyr Tyr Met Tyr Trp Val Arg Gln Thr
Pro Glu Lys Arg Leu 50 55 60Glu Trp Val Ala Tyr Ile Asn Ser Gly Gly
Gly Ser Thr Tyr Tyr Pro65 70 75 80Asp Thr Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95Thr Leu Tyr Leu Gln Met Ser
Arg Leu Lys Ser Glu Asp Thr Ala Met 100 105 110Tyr Tyr Cys Ala Arg
Arg Gly Leu Pro Phe His Ala Met Asp Tyr Trp 115 120 125Gly Gln Gly
Thr Ser Val Thr Phe Val Ser Ser Ala Lys Thr Lys Gly 130 135 140Pro
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser145 150
155 160Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val 165 170 175Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe 180 185 190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val 195 200 205Thr Val Pro Ser Ser Ser Leu Gly Thr
Lys Thr Tyr Thr Cys Asn Val 210 215 220Asp His Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg Val Glu Ser Lys225 230 235 240Tyr Gly Pro Pro
Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly 245 250 255Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265
270Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
275 280 285Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His 290 295 300Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
Ser Thr Tyr Arg305 310 315 320Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys 325 330 335Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro Ser Ser Ile Glu 340 345 350Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365Thr Leu Pro
Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 370 375 380Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp385 390
395 400Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val 405 410 415Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
Thr Val Asp 420 425 430Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
Cys Ser Val Met His 435 440 445Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Leu 450 455 460Gly Lys Ala
Ser465149234PRTArtificial SequenceSynthetic fusion protein. 149Met
Met Ser Ser Ala Gln Phe Leu Gly Leu Leu Leu Leu Cys Phe Gln1 5 10
15Gly Thr Arg Cys Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser
20 25 30Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln
Gly 35 40 45Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
Thr Val 50 55 60Lys Leu Leu Ile Tyr Tyr Thr Ser Ile Leu His Ser Gly
Val Pro Ser65 70 75 80Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr
Ser Leu Thr Ile Gly 85 90 95Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr
Tyr Cys Gln Gln Phe Asn 100 105 110Lys Leu Pro Pro Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys Arg 115 120 125Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr145 150 155 160Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170
175Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
180 185 190Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys 195 200 205His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro 210 215 220Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys225 230150203PRTArtificial SequenceSynthetic anti-CD40 heavy
chain sequence. 150Met Glu Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser
Gly Thr Ala Gly1 5 10 15Val His Ser Glu Val Gln Leu Gln Gln Ser Gly
Pro Glu Leu Val Lys 20 25 30Pro Gly Ala Ser Val Lys Met Ser Cys Lys
Ala Ser Gly Tyr Thr Phe 35 40 45Thr Asp Tyr Val Leu His Trp Val Lys
Gln Lys Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly Tyr Ile Asn Pro
Tyr Asn Asp Gly Thr Lys Tyr Asn65 70 75 80Glu Lys Phe Lys Gly Lys
Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser 85 90 95Thr Ala Tyr Met Glu
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110Tyr Tyr Cys
Ala Arg Gly Tyr Pro Ala Tyr Ser Gly Tyr Ala Met Asp 115 120 125Tyr
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr 130 135
140Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr
Asn145 150 155 160Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr
Phe Pro Glu Pro 165 170 175Val Thr Val Thr Trp Asn Ser Gly Ser Leu
Ser Ser Gly Val His Thr 180 185 190Phe Pro Ala Val Leu Gln Lys Gly
Glu Phe Val 195 200151234PRTArtificial SequenceSynthetic anti-CD40
light chain sequence. 151Met Met Ser Ser Ala Gln Phe Leu Gly Leu
Leu Leu Leu Cys Phe Gln1 5 10 15Gly Thr Arg Cys Asp Ile Gln Met Thr
Gln Thr Thr Ser Ser Leu Ser 20 25 30Ala Ser Leu Gly Asp Arg Val Thr
Ile Ser Cys Arg Ala Ser Gln Asp 35 40 45Ile Ser Asn Tyr Leu Asn Trp
Tyr Gln Gln Lys Pro Asp Gly Thr Val 50 55 60Lys Leu Leu Ile Tyr Tyr
Thr Ser Arg Leu His Ser Gly Val Pro Ser65 70 75 80Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser 85 90 95Asn Leu Glu
Gln Glu Asp Ile Ala Thr Tyr Phe Cys His His Gly Asn 100 105 110Thr
Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 115 120
125Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln
130 135 140Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn
Phe Tyr145 150 155 160Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp
Gly Ser Glu Arg Gln 165 170 175Asn Gly Val Leu Asn Ser Trp Thr Asp
Gln Asp Ser Lys Asp Ser Thr 180 185 190Tyr Ser Met Ser Ser Thr Leu
Thr Leu Thr Lys Asp Glu Tyr Glu Arg 195 200 205His Asn Ser Tyr Thr
Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro 210 215 220Ile Val Lys
Ser Phe Asn Arg Asn Glu Cys225 230152235PRTArtificial
SequenceSynthetic protein. 152Met Asp Phe Gln Val Gln Ile Phe Ser
Phe Leu Leu Ile Ser Ala Ser1 5 10 15Val Ile Met Ser Arg Gly Gln Ile
Val Leu Thr Gln Ser Pro Ala Ile 20 25 30Leu Ser Ala Ser Pro Gly Glu
Lys Val Thr Met Thr Cys Ser Ala Ser 35 40 45Ser Ser Val Ser Tyr Met
Tyr Arg Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60Pro Lys Pro Trp Ile
Tyr Gly Thr Ser Asn Leu Ala Ser Gly Val Pro65 70 75 80Ala Arg Phe
Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95Ser Ser
Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr 100 105
110His Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
115 120 125Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser
Ser Glu 130 135 140Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe
Leu Asn Asn Phe145 150 155 160Tyr Pro Lys Asp Ile Asn Val Lys Trp
Lys Ile Asp Gly Ser Glu Arg 165 170 175Gln Asn Gly Val Leu Asn Ser
Trp Thr Asp Gln Asp Ser Lys Asp Ser 180 185 190Thr Tyr Ser Met Ser
Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 195 200 205Arg His Asn
Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 210 215 220Pro
Ile Val Lys Ser Phe Asn Arg Asn Glu Cys225 230
235153196PRTArtificial SequenceSynthetic protein. 153Met Gly Trp
Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly1 5 10 15Val Leu
Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys 20 25 30Pro
Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe 35 40
45Thr Gly Tyr Tyr Met His Trp Val Lys Gln Ser His Val Lys Ser Leu
50 55 60Glu Trp Ile Gly Arg Ile Asn Pro Tyr Asn Gly Ala Thr Ser Tyr
Asn65 70 75 80Gln Asn Phe Lys Asp Lys Ala Ser Leu Thr Val Asp Lys
Ser Ser Ser 85 90 95Thr Ala Tyr Met Glu Leu His Ser Leu Thr Ser Glu
Asp Ser Ala Val 100 105 110Tyr Tyr Cys Ala Arg Glu Asp Tyr Val Tyr
Trp Gly Gln Gly Thr Thr 115 120 125Leu Thr Val Ser Ser Ala Lys Thr
Thr Pro Pro Ser Val Tyr Pro Leu 130 135 140Ala Pro Gly Ser Ala Ala
Gln Thr Asn Ser Met Val Thr Leu Gly Cys145 150 155 160Leu Val Lys
Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser 165 170 175Gly
Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Lys 180 185
190Gly Glu Phe Val 195154238PRTArtificial SequenceSynthetic
protein. 154Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile
Pro Ala1 5 10 15Ser Ser Ser Asp Val Val Met Thr Gln Thr Pro Leu Ser
Leu Pro Val 20 25 30Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu 35 40 45Val His Ser Asn Gly Asn Thr Tyr Leu His Trp
Tyr Leu Gln Lys Pro 50 55 60Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys
Val Ser Asn Arg Phe Ser65 70 75 80Gly Val Pro Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Ala 85 90 95Leu Lys Ile Ser Arg Val Glu
Ala Glu Asp Leu Gly Val Tyr Phe Cys 100 105 110Ser Gln Ser Thr His
Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu 115 120 125Glu Ile Lys
Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro 130 135
140Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe
Leu145 150 155 160Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp
Lys Ile Asp Gly 165 170 175Ser Glu Arg Gln Asn Gly Val Leu Asn Ser
Trp Thr Asp Gln Asp Ser 180 185 190Lys Asp Ser Thr Tyr Ser Met Ser
Ser Thr Leu Thr Leu Thr Lys Asp 195 200 205Glu Tyr Glu Arg His Asn
Ser Tyr Thr Cys Glu Ala Thr His Lys Thr 210 215 220Ser Thr Ser Pro
Ile Val Lys Ser Phe Asn Arg Asn Glu Cys225 230
235155705DNAArtificial Sequenceanti-CD40 light chain synthetic
olignucleotide sequence. 155atgatgtcct ctgctcagtt ccttggtctc
ctgttgctct gttttcaagg taccagatgt 60gatatccaga tgacacagac tacatcctcc
ctgtctgcct ctctaggaga cagagtcacc 120atcagttgca gtgcaagtca
gggcattagc aattatttaa actggtatca gcagaaacca 180gatggaactg
ttaaactcct gatctattac acatcaattt tacactcagg agtcccatca
240aggttcagtg gcagtgggtc tgggacagat tattctctca ccatcggcaa
cctggaacct 300gaagatattg ccacttacta ttgtcagcag tttaataagc
ttcctccgac gttcggtgga 360ggcaccaaac tcgagatcaa acgaactgtg
gctgcaccat ctgtcttcat cttcccgcca 420tctgatgagc agttgaaatc
tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480cccagagagg
ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag
540gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag
caccctgacg 600ctgagcaaag cagactacga gaaacacaaa gtctatgcct
gcgaagtcac ccatcagggc 660ctgagctcgc ccgtcacaaa gagcttcaac
aggggagagt gttag 7051561404DNAArtificial Sequenceanti-CD40
synthetic oligonucleotide heavy chain sequence. 156atgaacttgg
ggctcagctt gattttcctt gtccttgttt taaaaggtgt ccagtgtgaa 60gtgaagctgg
tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaactctcc
120tgtgcaacct ctggattcac tttcagtgac tattacatgt attgggttcg
ccagactcca 180gagaagaggc tggagtgggt cgcatacatt aattctggtg
gtggtagcac ctattatcca 240gacactgtaa agggccgatt caccatctcc
agagacaatg ccaagaacac cctgtacctg 300caaatgagcc ggctgaagtc
tgaggacaca gccatgtatt actgtgcaag acgggggtta 360ccgttccatg
ctatggacta ttggggtcaa ggaacctcag tcaccgtctc ctcagccaaa
420acgaagggcc catccgtctt ccccctggcg ccctgctcca ggagcacctc
cgagagcaca 480gccgccctgg gctgcctggt caaggactac ttccccgaac
cggtgacggt gtcgtggaac 540tcaggcgccc tgaccagcgg cgtgcacacc
ttcccggctg tcctacagtc ctcaggactc 600tactccctca gcagcgtggt
gaccgtgccc tccagcagct tgggcacgaa gacctacacc 660tgcaacgtag
atcacaagcc cagcaacacc aaggtggaca agagagttga gtccaaatat
720ggtcccccat gcccaccctg cccagcacct gagttcgaag ggggaccatc
agtcttcctg 780ttccccccaa aacccaagga cactctcatg atctcccgga
cccctgaggt cacgtgcgtg 840gtggtggacg tgagccagga agaccccgag
gtccagttca actggtacgt ggatggcgtg 900gaggtgcata atgccaagac
aaagccgcgg gaggagcagt tcaacagcac gtaccgtgtg 960gtcagcgtcc
tcaccgtcct gcaccaggac tggctgaacg gcaaggagta caagtgcaag
1020gtctccaaca aaggcctccc gtcctccatc gagaaaacca tctccaaagc
caaagggcag 1080ccccgagagc cacaggtgta caccctgccc ccatcccagg
aggagatgac caagaaccag 1140gtcagcctga cctgcctggt caaaggcttc
taccccagcg acatcgccgt ggagtgggag 1200agcaatgggc agccggagaa
caactacaag accacgcctc ccgtgctgga ctccgacggc 1260tccttcttcc
tctacagcag gctaaccgtg gacaagagca ggtggcagga ggggaatgtc
1320ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacacagaa
gagcctctcc 1380ctgtctctgg gtaaagctag ctga 14041571410DNAArtificial
SequenceSynthetic oligonucleotide sequence. 157atggaatgga
gttggatatt tctctttctt ctgtcaggaa ctgcaggtgt ccactctgag 60gtccagctgc
agcagtctgg acctgagctg gtaaagcctg gggcttcagt gaagatgtcc
120tgcaaggctt ctggatacac attcactgac tatgttttgc actgggtgaa
acagaagcct 180gggcagggcc ttgagtggat tggatatatt aatccttaca
atgatggtac taagtacaat 240gagaagttca aaggcaaggc cacactgact
tcagacaaat cctccagcac agcctacatg 300gagctcagca gcctgacctc
tgaggactct gcggtctatt actgtgcaag gggctatccg 360gcctactctg
ggtatgctat ggactactgg ggtcaaggaa cctcagtcac cgtctcctca
420gccaaaacga agggcccatc cgtcttcccc ctggcgccct gctccaggag
cacctccgag 480agcacagccg ccctgggctg cctggtcaag gactacttcc
ccgaaccggt gacggtgtcg 540tggaactcag gcgccctgac cagcggcgtg
cacaccttcc cggctgtcct acagtcctca 600ggactctact ccctcagcag
cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 660tacacctgca
acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc
720aaatatggtc ccccatgccc accctgccca gcacctgagt tcgaaggggg
accatcagtc 780ttcctgttcc ccccaaaacc caaggacact ctcatgatct
cccggacccc tgaggtcacg 840tgcgtggtgg tggacgtgag ccaggaagac
cccgaggtcc agttcaactg gtacgtggat 900ggcgtggagg tgcataatgc
caagacaaag ccgcgggagg agcagttcaa cagcacgtac 960cgtgtggtca
gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag
1020tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc
caaagccaaa 1080gggcagcccc gagagccaca ggtgtacacc ctgcccccat
cccaggagga gatgaccaag 1140aaccaggtca gcctgacctg cctggtcaaa
ggcttctacc ccagcgacat cgccgtggag 1200tgggagagca atgggcagcc
ggagaacaac tacaagacca cgcctcccgt gctggactcc 1260gacggctcct
tcttcctcta cagcaggcta accgtggaca agagcaggtg gcaggagggg
1320aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac
acagaagagc 1380ctctccctgt ctctgggtaa agctagctga
1410158708DNAArtificial SequenceSynthetic oligonucleotide sequence.
158atggattttc aagtgcagat tttcagcttc ctgctaatca gtgcctcagt
cataatgtcc 60aggggacaaa ttgttctcac ccagtctcca gcaatcctgt ctgcatctcc
aggggagaag 120gtcaccatga cctgcagtgc cagctcaagt gtaagttaca
tgtacaggta ccagcagaag 180ccaggatcct cacccaaacc ctggatttat
ggcacatcca acctggcttc tggagtccct 240gctcgcttca gtggcagtgg
atctgggacc tcttattctc tcacaatcag cagcatggag 300gctgaagatg
ctgccactta ttactgccag caatatcata gttacccgct cacgttcggt
360gctgggacca agctcgagat caaacgaact gtggctgcac catctgtctt
catcttcccg 420ccatctgatg agcagttgaa atctggaact gcctctgttg
tgtgcctgct gaataacttc 480tatcccagag aggccaaagt acagtggaag
gtggataacg ccctccaatc gggtaactcc 540caggagagtg tcacagagca
ggacagcaag gacagcacct acagcctcag cagcaccctg 600acgctgagca
aagcagacta cgagaaacac aaagtctatg cctgcgaagt cacccatcag
660ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgttag
708159705DNAArtificial SequenceSynthetic oligonucleotide sequence.
159atgatgtcct ctgctcagtt ccttggtctc ctgttgctct gttttcaagg
taccagatgt 60gatatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga
cagagtcacc 120atcagttgca gggcaagtca ggacattagc aattatttaa
actggtatca gcagaaacca 180gatggaactg ttaaactcct gatctactac
acatcaagat tacactcagg agtcccatca 240aggttcagtg gcagtgggtc
tggaacagat tattctctca ccattagcaa cctggagcaa 300gaagatattg
ccacttactt ttgccatcat ggtaatacgc ttccgtggac gttcggtgga
360ggcaccaagc tcgagatcaa acgaactgtg gctgcaccat ctgtcttcat
cttcccgcca 420tctgatgagc agttgaaatc tggaactgcc tctgttgtgt
gcctgctgaa taacttctat 480cccagagagg ccaaagtaca gtggaaggtg
gataacgccc tccaatcggg taactcccag 540gagagtgtca cagagcagga
cagcaaggac agcacctaca gcctcagcag caccctgacg 600ctgagcaaag
cagactacga gaaacacaaa gtctatgcct gcgaagtcac ccatcagggc
660ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag
7051601389DNAArtificial SequenceSynthetic oligonucleotide sequence.
160atgggatgga gctggatctt tctctttctc ctgtcaggaa ctgcaggtgt
cctctctgag 60gtccagctgc aacagtctgg acctgagctg gtgaagcctg gggcttcagt
gaagatatcc 120tgcaaggctt ctggttactc attcactggc tactacatgc
actgggtgaa gcaaagccat 180gtaaagagcc ttgagtggat tggacgtatt
aatccttaca atggtgctac tagctacaac 240cagaatttca aggacaaggc
cagcttgact gtagataagt cctccagcac agcctacatg 300gagctccaca
gcctgacatc tgaggactct gcagtctatt actgtgcaag agaggactac
360gtctactggg gccaaggcac cactctcaca gtctcctcag ccaaaacgaa
gggcccatcc 420gtcttccccc tggcgccctg ctccaggagc acctccgaga
gcacagccgc cctgggctgc 480ctggtcaagg actacttccc cgaaccggtg
acggtgtcgt ggaactcagg cgccctgacc 540agcggcgtgc acaccttccc
ggctgtccta cagtcctcag gactctactc cctcagcagc 600gtggtgaccg
tgccctccag cagcttgggc acgaagacct acacctgcaa cgtagatcac
660aagcccagca acaccaaggt ggacaagaga gttgagtcca aatatggtcc
cccatgccca 720ccctgcccag cacctgagtt cgaaggggga ccatcagtct
tcctgttccc cccaaaaccc 780aaggacactc tcatgatctc ccggacccct
gaggtcacgt gcgtggtggt ggacgtgagc 840caggaagacc ccgaggtcca
gttcaactgg tacgtggatg gcgtggaggt gcataatgcc 900aagacaaagc
cgcgggagga gcagttcaac agcacgtacc gtgtggtcag cgtcctcacc
960gtcctgcacc aggactggct gaacggcaag gagtacaagt gcaaggtctc
caacaaaggc 1020ctcccgtcct ccatcgagaa aaccatctcc aaagccaaag
ggcagccccg agagccacag 1080gtgtacaccc tgcccccatc ccaggaggag
atgaccaaga accaggtcag cctgacctgc 1140ctggtcaaag gcttctaccc
cagcgacatc gccgtggagt gggagagcaa tgggcagccg 1200gagaacaact
acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac
1260agcaggctaa ccgtggacaa gagcaggtgg caggagggga atgtcttctc
atgctccgtg 1320atgcatgagg ctctgcacaa ccactacaca cagaagagcc
tctccctgtc tctgggtaaa 1380gctagctga 1389161717DNAArtificial
SequenceSynthetic Ooigonucleotide sequence. 161atgaagttgc
ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagcagtgat 60gttgtgatga
cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc
120tcttgcagat ctagtcagag ccttgtacac agtaatggaa acacctattt
acattggtac 180ctgcagaagc caggccagtc tccaaagctc ctgatctaca
aagtttccaa ccgattttct 240ggggtcccag acaggttcag tggcagtgga
tcagggacag atttcgcact caagatcagt 300agagtggagg ctgaggatct
gggagtttat ttctgctctc aaagtacaca tgttccgtgg 360acgttcggtg
gaggcaccaa gctcgagatc aaacgaactg tggctgcacc atctgtcttc
420atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt
gtgcctgctg 480aataacttct atcccagaga ggccaaagta cagtggaagg
tggataacgc cctccaatcg 540ggtaactccc aggagagtgt cacagagcag
gacagcaagg acagcaccta cagcctcagc 600agcaccctga cgctgagcaa
agcagactac gagaaacaca aagtctatgc ctgcgaagtc 660acccatcagg
gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgttag 717
* * * * *