U.S. patent application number 15/126990 was filed with the patent office on 2017-05-25 for immunogenic glycopeptides, composition comprising the glycopeptides and use thereof.
This patent application is currently assigned to MacKay Medical Foundation The Presbyterian Church In Taiwan MacKay Memorial Hospital. The applicant listed for this patent is MacKay Medical Foundation The Presbyterian Church In Taiwan MacKay Memorial Hospital. Invention is credited to Chih-long CHANG, Chao-Chih WU.
Application Number | 20170143810 15/126990 |
Document ID | / |
Family ID | 54145306 |
Filed Date | 2017-05-25 |
United States Patent
Application |
20170143810 |
Kind Code |
A1 |
CHANG; Chih-long ; et
al. |
May 25, 2017 |
IMMUNOGENIC GLYCOPEPTIDES, COMPOSITION COMPRISING THE GLYCOPEPTIDES
AND USE THEREOF
Abstract
Disclosed herein are an immunogenic glycopeptide for inducing
immune response to treat cancer. Other aspects of the present
disclosure are pharmaceutical composition comprising the
immunogenic glycopeptide; and method for preventing and/or treating
a cancer.
Inventors: |
CHANG; Chih-long; (Taipei
City, TW) ; WU; Chao-Chih; (Taipei City, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MacKay Medical Foundation The Presbyterian Church In Taiwan MacKay
Memorial Hospital |
Taipei City, OT |
|
TW |
|
|
Assignee: |
MacKay Medical Foundation The
Presbyterian Church In Taiwan MacKay Memorial Hospital
Taipei City, OT
TW
|
Family ID: |
54145306 |
Appl. No.: |
15/126990 |
Filed: |
March 19, 2015 |
PCT Filed: |
March 19, 2015 |
PCT NO: |
PCT/US15/21421 |
371 Date: |
September 16, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61955216 |
Mar 19, 2014 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/24 20130101;
C07K 16/3076 20130101; A61K 39/001173 20180801; C07K 2317/565
20130101; A61P 35/00 20180101; C07K 7/08 20130101; A61K 39/0011
20130101; A61K 2039/70 20130101; A61K 2039/6012 20130101; C07K
2317/73 20130101; C07K 2317/732 20130101; A61K 2039/55577 20130101;
C07K 2317/734 20130101; A61K 39/39558 20130101; A61P 35/04
20180101; C07K 16/3069 20130101; A61K 2039/55566 20130101; A61K
2039/505 20130101; A61K 45/06 20130101; A61K 2039/6081 20130101;
C07K 2317/92 20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07K 7/08 20060101 C07K007/08 |
Claims
1. An immunogenic glycopeptide or a derivative thereof, wherein the
immunogenic glycopeptide has the structure of formula I:
##STR00003## wherein the PADRE is a pan-DR epitope and has at least
10 consecutive amino acid residues that is at least 90% identical
to the amino acid sequence of SEQ ID No. 1.
2. The immunogenic glycopeptide or a derivative thereof of claim 1,
wherein the amino acid sequence of the PADRE is identical to the
amino acid sequence of SEQ ID No. 1.
3. The immunogenic glycopeptide or a derivative thereof of claim 1,
wherein the derivative has the structure of formula II,
##STR00004##
4. The immunogenic glycopeptide or a derivative thereof of claim 1,
wherein the derivative has the structure of formula III,
##STR00005##
5. A pharmaceutical composition for treating Globo H-positive
cancer in a subject in need thereof, comprising a therapeutically
effective amount of the immunogenic glycopeptide of claim 1 and a
pharmaceutically acceptable carrier.
6. The pharmaceutical composition of claim 5, wherein the PADRE has
the amino acid sequence that is identical to the amino acid
sequence of SEQ ID No. 1.
7. The pharmaceutical composition of claim 5, wherein the Globo
H-positive cancer is breast cancer, ovarian cancer, pancreatic
cancer, prostate cancer, colorectal cancer or lung cancer.
8. The pharmaceutical composition of claim 7, wherein the ovarian
cancer is primary ovarian cancer.
9. An antibody which specifically binds to at least one epitope
defined by the immunogenic glycopeptide of claim 1.
10. The antibody of claim 9, wherein the PADRE has the amino acid
sequence that is identical to the amino acid sequence of SEQ ID No.
1.
11. The antibody of claim 9, wherein the antibody is a humanized
antibody and has a heavy chain and a light chain respectively
having, in a variable region, an amino acid sequence at least 90%
identical to SEQ ID No. 2 and SEQ ID No. 3.
12. The antibody of claim 11, wherein the amino acid sequence of
the variable region of the heavy chain is identical to SEQ ID No.
2.
13. The antibody of claim 11, wherein the amino acid sequence of
the variable region of the light chain is identical to SEQ ID No. 3
or SEQ ID No. 4.
14. A pharmaceutical composition for treating Globo H-positive
cancer in a subject in need thereof, comprising a therapeutically
effective amount of the antibody of claim 9 and a pharmaceutically
acceptable carrier.
15. The pharmaceutical composition of claim 14, wherein the PADRE
has the amino acid sequence that is identical to the amino acid
sequence of SEQ ID No. 1.
16. The pharmaceutical composition of claim 14, wherein the
antibody is a humanized antibody and has a heavy chain and a light
chain respectively having an amino acid sequence at least 90%
identical to SEQ ID No. 2 and SEQ ID No. 3.
17. The pharmaceutical composition of claim 16, wherein the amino
acid sequence of the heavy chain is identical to the amino acid
sequence of SEQ ID No. 2.
18. The pharmaceutical composition of claim 16, wherein the amino
acid sequence of the light chain is identical to the amino acid
sequence of SEQ ID No. 3 or SEQ ID No. 4.
19. The pharmaceutical composition of claim 14, wherein the Globo
H-positive cancer is breast cancer, ovarian cancer, pancreatic
cancer, prostate cancer, colorectal cancer or lung cancer.
20. The pharmaceutical composition of claim 19, wherein the ovarian
cancer is primary ovarian cancer.
21. A method of treating Globo H-positive cancer in a subject in
need thereof, comprising the step of administering to the subject
the pharmaceutical composition of claim 12.
22. The method of claim 21, wherein the Globo H-positive cancer is
breast cancer, ovarian cancer, pancreatic cancer, prostate cancer,
colorectal cancer or lung cancer.
23. The method of claim 22, wherein the ovarian cancer is primary
ovarian cancer.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the field of immunotherapy
of cancer. In particular, the disclosed invention relates to an
immunogenic glycopeptide, a pharmaceutical composition comprising
the glycopeptide and to the use thereof for enhancing the immune
response and notably in cancer therapy.
BACKGROUND OF THE INVENTION
[0002] Globo H
(Fuc.alpha.1.fwdarw.2Gal.beta.1.fwdarw.3GalNAc.beta.1.fwdarw.3Gal.alpha.1-
.fwdarw.4Gal.beta.1.fwdarw.4Glc.beta.1.fwdarw.O-cer) is a
hexasaccharide and belongs to a large number of tumor-associated
carbohydrate antigens that are overexpressed on the surface of
various epithelial cancer cells, including breast, colon, ovarian,
pancreatic, lung, and prostate cancer cells. The aberrant
expression of Globo H renders it an attractive candidate for
immunotherapy and the development of cancer vaccines for Globo
H-expressing cancers. In addition to Globo H, other known
carbohydrate antigens including GM2, GD2, GD3, fucosyl-GM1,
Globo-H, Lewis.sup.y (Le.sup.y,
Fuc.alpha.1.fwdarw.2Gal.beta.1.fwdarw.4[Fuc.alpha.1-3]GlcNAc.beta.1.fwdar-
w.3Gal.beta.1.fwdarw.O-cer, Tn (GalNAc.alpha.-O-Ser/Thr), TF
(Gal.beta.1.fwdarw.3GalNAc.alpha.-O-Ser/Thr and STn
(NeuAc.alpha.2.fwdarw.6GalNAc.alpha.-O-Ser/Thr are also used as
target antigens for cancer immunotherapy (Susan F Slovin et al.,
Carbohydrate Vaccines as Immunotherapy for Cancer, Immunology and
Cell Biology (2005) 83, 418-428; Zhongwu Guo and Qianli Wang,
Recent Development in Carbohydrate-Based Cancer Vaccines, Curr Opin
Chem Biol. 2009 December; 13(5-6): 608-617; Therese Buskas et al.,
Immunotherapy for Cancer: Synthetic Carbohydrate-based Vaccines,
Chem Commun (Camb). 2009 Sep. 28; (36): 5335-5349).
[0003] However, most carbohydrate antigens are often tolerated by
the immune system, and consequently, the immunogenicity induced by
them is limited. Further, the production of antibody against a
specific immunogen typically involves the cooperative interaction
of two . types of lymphocytes, B-cells and helper T-cells. For
example, Globo H alone cannot activate helper T-cells, which also
attributes to the poor immunogenicity of Globo H. Accordingly, the
immunization with Globo H is often typified by low titer of
immunoglobulin M (IgM) and failure to class switch to
immunoglobulin G (IgG), as well as ineffective antibody affinity
maturation.
[0004] Various approaches have been developed to address the
above-mentioned deficiencies. In certain researches, foreign
carrier proteins or peptides having T-epitopes (such as keyhole
limpet hemocyanin (KLH) or detoxified tetanus toxoid (TT)) have
been conjugated with carbohydrate antigens hoping to enhance the
immunogenicity of the carbohydrate antigens. U.S. 20010048929
provided a multivalent immunogenic molecule, comprising a carrier
molecule containing at least one functional T-cell epitope, and
multiple different carbohydrate fragments each linked to the
carrier molecule and each containing at least one functional B-cell
epitope, wherein said carrier molecule imparts enhanced
immunogenicity to said multiple carbohydrate fragments and wherein
the carbohydrate fragment is Globo H, Le.sub.Y or STn. U.S.
20120328646 provides a carbohydrate based vaccine containing Globo
H (B cell epitope) chemically conjugated to the immunogenic carrier
diphtheria toxin cross-reacting material 197 (DT-CRM 197) (Th
epitope) via a p-nitrophenyl linker, which provides immunogenicity
in breast cancer models, showing delayed tumorigenesis in xenograft
studies. U.S. 20120263749 relates to a polyvalent vaccine for
treating cancer comprising at least two conjugated antigens
selected from a group containing glycolipid antigen such as Globo
H, a Lewis antigen and a ganglioside, polysaccharide antigen, mucin
antigen, glycosylated mucin antigen and an appropriate
adjuvant.
[0005] Nonetheless, conjugation of carbohydrates to a carrier
protein poses several new problems. According to Ingale et al., the
foreign carrier protein and the linker for attaching the carrier
protein and the carbohydrate may elicit strong B-cell responses,
thereby leading to the suppression of an antibody response against
the carbohydrate epitope (Ingale S. et al. Robust immune responses
elicited by a fully synthetic three-component vaccine. Nat Chem
Biol. 2007 October;3(10):663-7. Epub 2007 Sep. 2). Furthermore,
Ingale et al. also indicated that the conjugation chemistry is
difficult to control, resulting in conjugates with ambiguities in
composition and structure, which may affect the reproducibility of
an immune response. Considering the above-mentioned factors, Ingale
et al. concluded that it is not surprising that preclinical and
clinical studies using carbohydrate-protein conjugates have led to
mixed results. For example, Kuduk et al. taught that the
immunization with a trimeric cluster of Tn-antigens conjugated to
KLH in the presence of the adjuvant QS-21 elicited modest titers of
IgG antibodies in mice (Kuduk S D, et al. Synthetic and
immunological studies on clustered modes of mucin-related Tn and TF
O-linked antigens: the preparation of a glycopeptide-based vaccine
for clinical trials against prostate cancer. J Am Chem Soc. 1998;
120:12474-12485); while Slovin et al. taught that the same vaccine
gave low median IgG and IgM antibody titers in a clinical trial of
relapsed prostate cancer patients (Slovin SF, et al. Fully
synthetic carbohydrate-based vaccines in biochemically relapsed
prostate cancer: clinical trial results with
alpha-N-acetylgalactosamine-O-serine/threonine conjugate vaccine. J
Clin Oncol. 2003;21:4292-4298).
[0006] Moreover, for cancer patients with hypoimmune status;
particular in patients receiving chemotherapy or radiation therapy,
as well as late-stage cancer patients, the efficacy of active
immune intervention is often limited, for these patients may not be
able to produce sufficient antibodies to elicit the anti-tumor
effect.
[0007] In view of the foregoing, there exists a need in the art for
developing alternative strategies for improving the immunization
and/or therapeutic efficacy of carbohydrate-based vaccines.
SUMMARY OF THE INVENTION
[0008] The following presents a simplified summary of the
disclosure in order to provide a basic understanding to the reader.
This summary is not an extensive overview of the disclosure and it
does not identify key/critical elements of the present invention or
delineate the scope of the present invention. Its sole purpose is
to present some concepts disclosed herein in a simplified form as a
prelude to the more detailed description that is presented
later.
[0009] In one aspect, the present disclosure is directed to an
immunogenic glycopeptide or a derivative thereof, wherein the
immunogenic glycopeptide or a derivative thereof may elicit high
titers of immunoglobulin G (IgG) and immunoglobulin M (IgM)
antibodies against Globo H in vivo.
[0010] According to one embodiment of the present disclosure, the
immunogenic glycopeptide has the structure of:
##STR00001##
wherein the PADRE is a pan-DR epitope and has at least 10
consecutive amino acid residues that is at least 90% identical to
the amino acid sequence of AKXVAAWTLKAAA (SEQ ID NO: 1), where X is
a cyclohexylalanine residue; and wherein P is Globo H, GD2, GM2,
SSEA 4, Lewis Y or STn.
[0011] According to another embodiment, the amino acid sequence of
the PADRE is identical to the amino acid sequence of SEQ ID NO:
1.
[0012] In another aspect, the present disclosure is directed to a
pharmaceutical composition for treating a cancer in a subject in
need thereof.
[0013] According to one embodiment of the present disclosure, the
pharmaceutical composition comprises, (1) a therapeutically
effective amount of the immunogenic glycopeptide according to any
of the above-mentioned aspect/embodiments of the present disclosure
and (2) a pharmaceutically acceptable carrier.
[0014] According to certain embodiments of the present disclosure,
the cancer is any of tumor-associated carbohydrate-expressing
cancers; preferably, the cancer is breast cancer, ovarian cancer,
pancreatic cancer, prostate cancer, colorectal cancer or lung
cancer.
[0015] In yet another aspect, the present disclosure is directed to
a method of treating a tumor-associated carbohydrate-expressing
cancer in a subject in need thereof.
[0016] According to embodiments of the present disclosure, the
method includes administering to the subject the immunogenic
glycopeptide or pharmaceutical composition according to any of the
aspects/embodiments described in the present disclosure.
[0017] Many of the attendant features and advantages of the present
disclosure will becomes better understood with reference to the
following detailed description considered in connection with the
accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWING
[0018] FIGS. 1 (A) and (B) provide bar graphs illustrating the IgM
titers of mice immunized with the Globo H-PADRE glycopeptide
according to one working example of the present disclosure (FIG.
1(A) for diluted serum IgM 1:100 and FIG. 1(B) for diluted serum
IgM 1:1000).
[0019] FIGS. 2 (A) and (B) provide bar graphs illustrating the IgG
titers of mice immunized with the Globo H-PADRE glycopeptide
according to one working example of the present disclosure (FIG.
2(A) for diluted serum IgG 1:100 and FIG. 2(B) for diluted serum
IgG 1:1000).
[0020] FIGS. 3 (A) and (B) show the binding affinity of the
anti-Globo H IgG and IgM antibodies with Globo H (FIG. 3(A) for
binding affinity of anti-Globo H IgG antibodies with Globo H and
FIG. 3(B) for binding affinity of anti-Globo H IgM antibodies with
Globo H).
[0021] FIGS. 4 (A) and (B) show that mMouse immunized with 2 .mu.g
of glycopeptide by direct conjugation of PADRE to Globo and QS21
adjuvant (2 .mu.g) exhibits high-titer of anti-Globo H IgG and IgM
with immune boost effect. MZ-11-Globo H: Globo H-PADRE,
MZ-11-4KA-Globo H: PADRE-branched Globo H.
[0022] FIG. 5 shows that antibodies in serum from mice vaccinated
with Globo H-PADRE (+adjuvant QS21) bind to Globo H-expressing
MCF-7 cells. MZ-11-Globo H: Globo H-PADRE.
[0023] FIGS. 6 (A) and (B) show that glycopeptide Globo H-PADRE (M)
induces higher titer of anti-Globo H IgG antibody than general
carrier protein-Globo H conjugation (C) does. C: control; Q:
adjuvant QS21. FIG. 6(A) refers to the results of mouse-anti-Globo
IgG ELISA and FIG. 6(B) refers to the results of mouse-anti-Globo
IgM ELISA.
[0024] FIGS. 7(A) and 7(B) shows that antibody titers in individual
mouse receiving glycopeptide Globo H-PADRE are constantly high,
whereas antibody titers in mouse receiving carrier protein-Globo H
conjugation are variable and most are low. FIG. 7(A) refers to the
results of G vaccine Mouse anti-GloboH IgG ELISA and FIG. 7(B)
refers to the results of MZ-11-Globo vaccine Mouse anti-Globo IgG
ELISA. G1-G10 represent mouse No.1-No.10 receiving carrier
protein-Globo H vaccine. M1-M10 represent mouse No.1-N.10 receiving
glycopeptide Globo H-PADRE vaccine.
[0025] FIGS. 8 (A) and (B) show that glycopeptide Globo H-PADRE (M)
induces long-term anti-Globo H IgG, whereas general carrier
protein-Globo H conjugation (G) does not. FIG. 8(A) refers to the
results of mouse serum anti-GloboH IgG and FIG. 8(B) refers to the
results of mouse serum anti-GloboH IgM.
[0026] FIGS. 9(A) and (B) show that dissection of individual mouse
receiving glycopeptide Globo H-PADRE shows constantly long-lived
high-titer anti-Globo H IgG antibody (FIG. 9(A) for D109 mouse
serum anti-GliboH IgG and FIG. 9(B) for D109 mouse serum
anti-GliboH IgM). G1-G10 represent mouse No.1-No.10 receiving
carrier protein-Globo H vaccine. Ml-M10 represent mouse No.1-N.10
receiving glycopeptide Globo H-PADRE vaccine.
[0027] FIGS. 10(A) and (B) show that GM2-PADRE glycopeptide induces
high-titer anti-carbohydrate IgG antibody (FIG. 10(A) for the
induction of IgG and FIG. 10(B) for the induction of IgM).
[0028] FIG. 11 shows that mouse treated by Globo H-PADRE vaccine
demonstrated slower tumor growth.
[0029] FIG. 12 shows that mouse treated by adoptive transfer of
serum from mice immunized by Globo H-PADRE vaccine showed small
tumor burden.
[0030] FIGS. 13(A) to (H) shows polyvalent vaccines composed of
Globo H-, GM2-, Lewis Y-PADRE conjugation mixtures or SSEA4, GM2,
Lewis Y-PADRE conjugation mixtures can induce high-titer of IgG
against each of respective carbohydrate antigen (FIG. 13(A) for
Globo IgG; FIG. 13(B) for Globo IgM; FIG. 13(C) for GM 2IgG; FIG.
13(D) for GM2 IgM; FIG. 13 (E) for LewisY IgG; FIG. 13(F) for
LewisY IgM; FIG. 13(G) for SSEA4 IgG and FIG. 13(H) for SSEA4
IgM).
DETAILED DESCRIPTION OF THE INVENTION
[0031] The present invention is based, at least, on the finding
that the glycopeptide conjugate of a tumor-associated carbohydrate
antigen and the PADRE sequence is capable of eliciting an immune
response in a mammal. The glycopeptide facilitates the activation
of both B cells and T cells, thereby resulting in the production of
IgM and IgG that specifically bind to the carbohydrate antigen.
Particularly, the glycopeptide conjugate can be used as a vaccine
capable of inducing high-titer anti-carbohydrate IgG antibody for
treating cancer expression tumor-associated carbohydrate antigens.
More particularly, polyvalent glycopeptide conjugate vaccine
elicites high-titer polyvalent anti-carbohydrate IgG antibodies for
treating cancer expressing tumor-associated carbohydrate
antigens.
[0032] Therefore, in one aspect, the present disclosure is directed
to an immunogenic glycopeptide. Moreover, the immunogenic
glycopeptide according to the present disclosure can be provided
for use in treating (including preventing) cancer; for example, it
shall be manufactured as a medicament, e.g., comprised in a
pharmaceutical composition. The present immunogenic glycopeptide
and the pharmaceutical composition comprising the same can also be
applied in a method for treating and/or preventing cancer.
Accordingly, the present disclosure also contemplates a method for
treating cancer in a subject suffering therefrom comprising
administering to said subject a therapeutically effective amount of
the glycopeptide or pharmaceutical composition as defined
herein.
Definition
[0033] Unless otherwise defined herein, scientific and technical
terminologies employed in the present disclosure shall have the
meanings that are commonly understood and used by one of ordinary
skill in the art. Unless otherwise required by context, it will be
understood that singular terms shall include plural forms of the
same and plural terms shall include the singular. Specifically, as
used herein and in the claims, the singular forms "a" and "an"
include the plural reference unless the context clearly indicates
otherwise. Also, as used herein and in the claims, the terms "at
least one" and "one or more" have the same meaning and include one,
two, three, or more.
[0034] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical value, however,
inherently contains certain errors necessarily resulting from the
standard deviation found in the respective testing measurements.
Also, as used herein, the term "about" generally means within 10%,
5%, 1%, or 0.5% of a given value or range. Alternatively, the term
"about" means within an acceptable standard error of the mean when
considered by one of ordinary skill in the art. Other than in the
operating/working examples, or unless otherwise expressly
specified, all of the numerical ranges, amounts, values and
percentages such as those for quantities of materials, durations of
times, temperatures, operating conditions, ratios of amounts, and
the likes thereof disclosed herein should be understood as modified
in all instances by the term "about." Accordingly, unless indicated
to the contrary, the numerical parameters set forth in the present
disclosure and attached claims are approximations that can vary as
desired. At the very least, each numerical parameter should at
least be construed in light of the number of reported significant
digits and by applying ordinary rounding techniques.
[0035] The term "antigen" as used herein is defined as a substance
capable of eliciting an immune response. Said immune response may
involve either antibody production, or the activation of specific
immunologically-competent cells, or both. As used herein, the term
"immunogen" refers to an antigen capable of inducing the production
of an antibody. Also, the term "immunogenicity" generally refers to
the ability of an immunogen or antigen to stimulate an immune
response.
[0036] The term "epitope" refers to a unit of structure
conventionally bound by an immunoglobulin V.sub.H/V.sub.Lpair. An
epitope defines the minimum binding site for an antibody, and thus
represent the target of specificity of an antibody.
[0037] As used herein, the term "glycopeptide" refers to a compound
in which carbohydrate is covalently attached to a peptide or
oligopeptide.
[0038] Unless specified otherwise, in the peptide notation used
herein, the left-hand direction is the amino-terminal (N-terminal)
direction and the right-hand direction is the carboxy-terminal
(C-terminal) direction, in accordance with standard usage and
convention.
[0039] "Percentage (%) amino acid sequence identity" with respect
to the amino acid sequences identified herein is defined as the
percentage of amino acid residues in a candidate sequence that are
identical with the amino acid residues in the specific polypeptide
sequence, after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity, and
not considering any conservative substitutions as part of the
sequence identity. Alignment for purposes of determining percentage
sequence identity can be achieved in various ways that are within
the skill in the art, for instance, using publicly available
computer software such as BLAST, BLAST-2, ALIGN or Megalign
(DNASTAR) software. Those skilled in the art can determine
appropriate parameters for measuring alignment, including any
algorithms needed to achieve maximal alignment over the full length
of the sequences being compared. For purposes herein, sequence
comparison between two amino acid sequences was carried out by
computer program Blastp (protein-protein BLAST) provided online by
Nation Center for Biotechnology Information (NCBI). Specifically,
the percentage amino acid sequence identity of a given amino acid
sequence A to a given amino acid sequence B (which can
alternatively be phrased as a given amino acid sequence A that has
a certain % amino acid sequence identity to a given amino acid
sequence B) is calculated by the formula as follows:
X Y 100 % ##EQU00001##
where X is the number of amino acid residues scored as identical
matches by the sequence alignment program BLAST in that program's
alignment of A and B, and where Y is the total number of amino acid
residues in A or B, whichever is shorter.
[0040] As discussed herein, minor variations in the amino acid
sequences of proteins/polypeptides are contemplated as being
encompassed by the presently disclosed and claimed inventive
concept(s), providing that the variations in the amino acid
sequence maintain at least 90%, such as at least 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% and 99%. In particular, conservative amino
acid replacements are contemplated. Conservative replacements are
those that take place within a family of amino acids that are
related in their side chains. Genetically encoded amino acids are
generally divided into families: (1) acidic=aspartate, glutamate;
(2) basic lysine, arginine, histidine; (3) nonpolar alanine,
valine, leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine,
cysteine, serine, threonine, tyrosine. More preferred families are:
serine and threonine are aliphatic-hydroxy family; asparagine and
glutamine are an amide-containing family; alanine, valine, leucine
and isoleucine are an aliphatic family; and phenylalanine,
tryptophan, and tyrosine are an aromatic family. For example, it is
reasonable to expect that an isolated replacement of a leucine with
an isoleucine or valine, an aspartate with a glutamate, a threonine
with a serine, or a similar replacement of an amino acid with a
structurally related amino acid will not have a major effect on the
binding or properties of the resulting molecule, especially if the
replacement does not involve an amino acid within a framework site.
Whether an amino acid change results in a functional peptide can
readily be determined by assaying the specific activity of the
polypeptide derivative. Fragments or analogs of
proteins/polypeptides can be readily prepared by those of ordinary
skill in the art. Preferred amino-and carboxy-termini of fragments
or analogs occur near boundaries of functional domains.
[0041] Unless contrary to the context, the term "treatment" are
used herein broadly to include a preventative (e.g., prophylactic),
curative, or palliative measure that results in a desired
pharmaceutical and/or physiological effect. Preferably, the effect
is therapeutic in terms of partially or completely curing or
preventing cancer. Also, the terms "treatment" and "treating" as
used herein refer to application or administration of the present
immunogenic glycopeptide, antibody, or pharmaceutical composition
comprising any of the above to a subject, who has cancer, a symptom
of cancer, a disease or disorder secondary to cancer, or a
predisposition toward cancer, with the purpose to partially or
completely alleviate, ameliorate, relieve, delay onset of, inhibit
progression of, reduce severity of, and/or reduce incidence of one
or more symptoms or features of cancer. Generally, a "treatment"
includes not just the improvement of symptoms or decrease of
markers of the disease, but also a cessation or slowing of progress
or worsening of a symptom that would be expected in absence of
treatment. The term "treating" can also be used herein in a
narrower sense which refers only to curative or palliative measures
intended to ameliorate and/or cure an already present disease state
or condition in a patient or subject.
[0042] The term "preventing" as used herein refers to a
preventative or prophylactic measure that stops a disease state or
condition from occurring in a patient or subject. Prevention can
also include reducing the likelihood of a disease state or
condition from occurring in a patient or subject and impeding or
arresting the onset of said disease state or condition.
[0043] As used herein, the term "therapeutically effective amount"
refers to the quantity of an active component which is sufficient
to yield a desired therapeutic response. A therapeutically
effective amount is also one in which any toxic or detrimental
effects of the compound or composition are outweighed by the
therapeutically beneficial effects.
[0044] As used herein, a "pharmaceutically acceptable carrier" is
one that is suitable for use with the subjects without undue
adverse side effects (such as toxicity, irritation, and allergic
response) commensurate with a reasonable benefit/risk ratio. Also,
each carrier must be "acceptable" in the sense of being compatible
with the other ingredients of the pharmaceutical composition. The
carrier can be in the form of a solid, semi-solid, or liquid
diluent, cream or a capsule. The carrier must be "acceptable" in
the sense of being compatible with the other ingredients of the
formulation, and is selected to minimize any degradation of the
active agent and to minimize any adverse side effects in the
subject.
[0045] As used herein, the term "adjuvant" refers to an
immunological agent that modifies the effect of an immunogen, while
having few if any direct effects when administered by itself It is
often included in vaccines to enhance the recipient's immune
response to a supplied antigen, while keeping the injected foreign
material to a minimum. Adjuvants are added to vaccines to stimulate
the immune system's response to the target antigen, but do not in
themselves confer immunity.
[0046] As used herein, the term "subject" refers to a mammal
including the human species that is treatable with antibody. The
term "subject" is intended to refer to both the male and female
gender unless one gender is specifically indicated.
Immunogenic Glycopeptide of the Invention
[0047] In one aspect, the present invention provides an immunogenic
glycopeptide having the following structure:
##STR00002##
wherein the PADRE is a pan-DR epitope and has at least 10
consecutive amino acid residues that is at least 80% identical to
the amino acid sequence of AKXVAAWTLKAAA (SEQ ID No. 1), where X is
a cyclohexylalanine residue; and wherein P is Globo H, GD2, GM2,
SSEA 4, Lewis, LewisY or STn. Preferably, the sequence identity as
mentioned above is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
[0048] The PADRE sequence is a non-natural sequence engineered to
introduce anchor residues for different known DR-binding motifs.
For example, X (cyclohexylalanine) in position 3 is an aliphatic
residue corresponding to the position 1 of DR-binding motif, T in
position 8 is a non-charged hydroxylated residue corresponding to
position 6 of DR-binding; while A in position 11 is a small
hydrophobic residue corresponding to position 9 of the DR-binding
motif Generally, substituting one residue with another residue of
substantially the same chemical and/or structural property, e.g.,
substituting X (cyclohexylalanine) with aromatic F (phenylalanine),
will not significantly affect the binding affinity of the PADRE
sequence.
[0049] According to another embodiment, the amino acid sequence of
the PADRE is identical to the amino acid sequence of SEQ ID No.
1.
[0050] According to various embodiments of the present disclosure,
the PADRE sequence has 10 to 20 amino acid residues. In one
embodiment, the last residue (K) can be omitted. In certain
embodiments, the first residue (A) or the first two residues (A and
K) are omitted. In one embodiment, the PADRE sequence is lack of
the first two residues and the last residue.
Compositions and Applications of Immunogenic Glycopeptide of
Antibodiy of the Invention
[0051] To prevent a subject from contracting cancer, the present
immunogenic glycopeptide or a pharmaceutical composition comprising
the same is administered to the subject in a therapeutically (or
immunogenically) effective amount. Accordingly, the pharmaceutical
composition and treating method also fall within the scope of the
present invention.
[0052] In one aspect, the invention provides a pharmaceuticala
composition comprising one or more immunogenic glycopeptide of the
present invention as described herein. In one embodiment, the
composition is a vaccine. In one embodiment, the composition is a
vaccine. In a further embodiment, the composition is a polyvalent
vaccine comprising one or more Globo H- , GM2-, Lewis Y, or
SSEA4-PADRE glycopeptide as described herein. In another further
embodiment, the composition is a polyvalent vaccine comprises Globo
H- , GM2- and Lewis Y-PADRE glycopeptides or SSEA4- , GM2 and Lewis
Y-PADRE glycopeptides, as described herein.
[0053] In addition to the immunogenic glycopeptide, said
pharmaceutical composition can further comprises a pharmaceutically
acceptable carrier. The pharmaceutical composition may further
comprises one or more pharmaceutically acceptable additives,
including binders, flavorings, buffering agents, thickening agents,
coloring agents, anti-oxidants, diluents, stabilizers, buffers,
emulsifiers, dispersing agents, suspending agents, antiseptics and
the like.
[0054] In addition to the immunogenic glycopeptide, said
pharmaceutical composition further comprises a pharmaceutically
acceptable carrier. The pharmaceutical composition may further
comprises one or more pharmaceutically acceptable additives,
including binders, flavorings, buffering agents, thickening agents,
coloring agents, anti-oxidants, diluents, stabilizers, buffers,
emulsifiers, dispersing agents, suspending agents, antiseptics and
the like.
[0055] The choice of a pharmaceutically-acceptable carrier to be
used in conjunction with the present glycopeptide is basically
determined by the way the composition is to be administered. The
pharmaceutical composition of the present invention may be
administered orally or subcutaneous, intravenous, intrathecal or
intramuscular injection.
[0056] Injectables for administration can be prepared in sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents include, but are not limited to,
propylene glycol, polyethylene glycol, vegetable oils such as olive
oil, and injectable organic esters such as ethyl oleate.
Illustrative examples of aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Common parenteral vehicles include
sodium chloride solution, Ringer's dextrose, dextrose and sodium
chloride, lactated Ringer's, or fixed oils; whereas intravenous
vehicles often include fluid and nutrient replenishers, electrolyte
replenishers (such as those based on Ringer's dextrose), and the
like.
[0057] Optionally, the pharmaceutically acceptable carrier may be
an immunogenic adjuvant. Alternatively, the present pharmaceutical
composition may optionally comprise an immunogenic adjuvant. An
immunogenic adjuvant is a compound that, when combined with an
antigen, increases the immune response to the antigen as compared
to the response induced by the antigen alone. For example, an
adjuvant may augment humoral immune responses, cell-mediated immune
responses, or both. Exemplary immunogenic adjuvants include, but
are not limited to mineral salts, polynucleotides, polyarginines,
ISCOMs, saponins, monophosphoryl lipid A, imiquimod, CCR-5
inhibitors, toxins, polyphosphazenes, cytokines, immunoregulatory
proteins, immunostimulatory fusion proteins, co-stimulatory
molecules, and combinations thereof. Mineral salts include, but are
not limited to, AIK(SO.sub.4).sub.2, AINa(SO.sub.4).sub.2,
AlNH(SO.sub.4).sub.2, silica, alum, Al(OH).sub.3,
Ca.sub.3(PO4).sub.2, kaolin, or carbon. Useful immunostimulatory
polynucleotides include, but are not limited to, CpG
oligonucleotides with or without immune stimulating complexes
(ISCOMs), CpG oligonucleotides with or without polyarginine, poly
IC or poly AU acids. Toxins include cholera toxin. Saponins
include, but are not limited to, QS21, QS17 or QS7. Also, examples
of are muramyl dipeptides,
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP),
N-acetyl-nornuramyl-L-alanyl-D-isoglutamine,
N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1
'2'-dipalmitoyl-sn-glycero-3-hydroxphosphoryloxy)-ethylamine RIBI
(MPL+TDM+CWS) in a 2 percent squalene/TWEEN 80 emulsion,
lipopolysaccharides and its various derivatives, including lipid A,
Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvants,
Merck Adjuvant 65, polynucleotides (e.g. poly IC and poly AU
acids), wax D from Mycobacterium tuberculosis, substances found in
Corynebacterium parvum, Bordetella pertussis, and members of the
genus Brucella, Titermax, Quil A, ALUN, Lipid A derivatives,
choleratoxin derivatives, HSP derivatives, LPS derivatives,
synthetic peptide matrixes or GMDP, Montanide ISA-51 and QS-21, CpG
oligonucleotide, poly I:C, and GMCSF. Combinations of adjuvants can
also be used. Preferably, the adjuvant is aluminum salts (such as
aluminum phosphate and aluminum hydroxide), calcium phosphate,
polyinosinic-polycytidylic acid (poly I:C), CpG motif, and saponins
(such as Quil A or QS21). Preferably, the adjuvant is aluminum
hydroxide or QS21.
[0058] In another aspect, the present invention provides a method
for preventing and/or treating a cancer, comprises administering an
effective amount of the immunogenic glycopeptide described herein
or a derivative thereof to a subject.
[0059] According to various working examples presented below, adult
C57BL/6 mice (weight 20-25 grams) immunized with about 2 .mu.g to
54 .mu.g of the immunogenic glycopeptide elicited desired immune
response. Hence, in certain embodiments of the present disclosure,
the therapeutically effective amount of the immunogenic
glycopeptide for mice could be expressed as 0.08-27 mg/kg body
weight.
[0060] The therapeutically Effective amount for a human subject can
be estimated from the animal doses according to various
well-established standards or conversion means. For example, the
"Guidance for Industry Estimating the Maximum Safe Starting Dose in
Initial Clinical Trials for Therapeutics in Adult Healthy
Volunteers" by Food and Drug Administration of U.S. Department of
Health and Human Services provides several conversion factors for
converting animal doses to human equivalent doses (HEDs). For mice
weighted between 11 to 34 grams, to convert the mice dose (in
mg/kg) to HED (in mg/kg) for a 60 kg adult human, the mice dose is
multiplied by 0.081. In the instant case, the therapeutically
effective amount of the present immunogenic glycopeptide for an
adult human subject is 0.06-2.2 mg/kg body weight. According to
various embodiments of the present disclosure, when the subject is
human, the therapeutically effective amount of the immunogenic
glycopeptide can be at least 1 mg/kg.
[0061] According to various embodiments of the present disclosure,
the cancer treatable by the immunogenic glycopeptide, the
pharmaceutical composition comprising the same or the treating
method described herein is tumor-associated carbohydrate-expressing
cancers; preferably, the cancer is breast cancer, ovarian cancer,
pancreatic cancer, prostate cancer, colorectal cancer or lung
cancer.
[0062] The following Examples are provided to elucidate certain
aspects of the present invention and to aid those of skilled in the
art in practicing this invention. These Examples are in no way to
be considered to limit the scope of the invention in any manner.
Without further elaboration, it is believed that one skilled in the
art can, based on the description herein, utilize the present
invention to its fullest extent. All publications cited herein are
hereby incorporated by reference in their entirety.
EXAMPLE
Example 1 Preparation of Immunogenic Globo H-PADRE Glycopeptide
[0063] 5.5 mg of customly synthesized PADRE-azide was dissolved in
110 .mu.l of DMSO, and 5 mg of Globo H-b-N-acetyl propargyl
(Carbosynth) was dissolved in 1 ml of distilled water, wherein
PADRE has the sequence as shown in SEQ ID NO: 1. For click
reaction, 1 .mu.mole of both PADRE-azide and Globo H-b-acetyl
propargyl were first mixed and added with distilled water to a
final volume of 500 .mu.l, and then 500 .mu.l of t-butanol (Sigma),
200 .mu.l of 100 mM CuSO.sub.4.5H.sub.2O (Sigma) and 160 .mu.l of
500 mM fresh prepared Na-L-ascorbate (Sigma) were sequentially
added under magnetic stirring. The mixture was incubated overnight
under stir at room temperature, followed by addition of 50 .mu.l of
27% ammonium hydroxide (Sigma). The product, the Globo H-PADRE
glycopeptide, was further diluted with one volume of distilled
water and stored at 4.degree. C.
Example 2 Production of Anti-Globo H IgG and IgM antibodies
[0064] Adult female C57BL/6 mice (5 in each group at 5 weeks old,
average weight 16-20 gm; Biolasco, Taiwan, R.O.C.) were injected
subcutaneously to abdomen region with the Globo H-PADRE
glycopeptide of Example 1, above, together with the complete
Freund's adjuvant (CFA; from Sigma) as the adjuvant. Three
immunizations were given at a 2-week interval; each vaccination
contained 2, 6 or 18 .mu.g Globo H-PADRE glycopeptide with 50 .mu.l
adjuvant. Serum was collected one week after the last immunization,
and then subjected to enzyme-linked immunosorbent assay (ELISA) to
measure the production of the anti-Globo H antibody. Serum from
naive mice injected with PBS and serum from mice immunized with the
adjuvant only were used as negative controls. Sera raised against
the MBr1 antibodies (Enzo Life Science; 0.5 .mu.g/ml) or MZ-2
antibodies (produced in Example 3 below; 1 .mu.g/ml) were used as
positive controls.
[0065] For ELISA, diluted serum (1:100 or 1:1000) from mice
immunized with Globo H-PADRE was added into designated wells of a
96-well ELISA plate and incubated at room temperature for one hour.
Wells were then washed six times with 0.1% Tween-20 in 1XPBS.
Thereafter, 1:2500 diluted anti-mouse IgG-HRP or anti-mouse IgM-HRP
(Jackson Immuno Research) was added to the wells and incubated at
room temperature for another one hour, and washed six times with
0.1% Tween-20 in 1XPBS. Color development was performed by
incubation of the washed wells with DMT ELISA kit, and stopped by
adding 2N H.sub.2SO.sub.4. Signals were read and recorded by ELISA
reader at O.D. 450 nm (reference: 540 nm). Elisa results are
depicted in FIG. 1 (FIG. (A) for diluted serum IgM 1:100 and FIG.
(B) for diluted serum igM 1:1000) and FIG. 2 (FIG. (A) for diluted
serum IgG 1:100 and FIG. (B) for diluted serum IgG 1:1000).
[0066] The data in FIG. 1 indicate that Globo H-PADRE glycopeptide
induced the production of anti-Globo H IgM. For mice immunized with
2 .mu.g Globo H-PADRE glycopeptide, the anti-Globo H IgM titers
increased as immunization proceeded.
[0067] A cell binding assay was performed to elucidate the binding
affinity of the anti-Globo H IgG and IgM antibodies with Globo H.
Briefly, 100 .mu.l of 1:10 diluted serum or 10 .mu.g/ml of
monoclonal antibodies in 1.times. PBS were incubated with
2.times.10.sup.5 of cells at room temperature for 20 minutes. The
cells were washed once with 2 ml of 1.times.PBS. After
centrifugation, the wash buffer was discarded and cells were
resuspended in 100.mu.l of 1:100 diluted PE anti-mouse IgG-Fc
(Jackson immunoresearch) or 100 .mu.l of 1:100 diluted PE
anti-mouse IgM (eBioscience) and incubated again at room
temperature for 20 minutes. The cells were washed with PBS and
resuspended in 200 .mu.l of 1.times. PBS after centrifugation. The
binding of antibodies with cells were detected by flow cytometry.
Results of cell binding assay are summarized in FIG. 3 (FIG. 3(A)
for binding affinity of anti-Globo H IgG antibodies with Globo H
and FIG. 3(B) for binding affinity of anti-Globo H IgM antibodies
with Globo H). As can be seen in FIG. 3, anti-Globo H IgG
antibodies obtained from immunizations with the present Globo
H-PADRE glycopeptide displayed excellent recognition of MCF-7 cells
which express the Globo H antigen.
Example 3 Direct Conjugation of PADRE with Globo H Induces
High-titer of Anti-Globo H IgG with Boost effect
[0068] C57BL/6 mice were immunized 3 times with 2 ug or 8 ug of
single Globo H conjugated vaccine (MZ-11-Globo H) or 8 ug of 4
Globo H conjugated vaccine (MZ-11-4KA-Globo H) plus QS-21 as
adjuvant at a 2-week interval. Serum was harvested before and 7
days after each immunization. For ELISA assay, 1 ug of streptavidin
(21135, Thermo) was dissolved in 100 uL of 1.times. PBS and coated
on 96-well Costar assay plate (9018, Coming) before loading of
biotin-Globo H (0.1 ug/well). The wells were then blocked with 1%
BSA in 1.times. PBS, and incubated with serum 1:1000 diluted in the
same blocking solution, followed by washing with 1.times. PBS-0.1%
Tween 20. The bound mouse IgG and IgM were detected using
HRP-conjugated goat anti-mouse IgG-Fc (1:5000; 115-035-071, Jackson
Immunoresearch) and HRP-conjugated goat-anti-mouse IgM .mu. chain
(1:5000; AP128P, MILLIPORE). The color development was performed by
adding 100 uL of NeA-Blue solution (010116-1, Clinical Science
Products) and stopped with 50 uL of 2N sulfuric acid. The O.D. was
read at 450 nm subtracted 540 nm as reference. FIG. 4 shows that
mMouse immunized with 2 .mu.g of glycopeptide by direct conjugation
of PADRE to Globo and QS21 adjuvant (2 .mu.g) exhibits high-titer
of anti-Globo H IgG and IgM with immune boost effect. MZ-11-Globo
H: Globo H-PADRE, MZ-11-4KA-Globo H: PADRE-branched Globo H.
Example 4 IgG in Sera from Mice Immunized with Globo H-PADRE
Efficiently Bind to Globo H-expression Breast Cancer Cell Line
(MCF-7)
[0069] C57BL/6 mice were immunized 3 times with adjuvant alone or
2, 6, or 18 ug of Globo H-PADRE (MZ-11-Globo H) at a 2-week
interval. Anti-serum were harvested 7 days after last immunization.
Serum from mice without immunization was collected as control. For
FACS, 5.times.10.sup.5 of MCF-7 cells were stained with 100 uL of
1:10 diluted serum in flow tube followed by 100 uL of 1:100 diluted
PE-conjugated goat anti-mouse IgG-Fc antibody (115-116-071, Jackson
immunoresearch) and 1:100 diluted APC-conjugated rat anti-mouse IgM
(17-5790-82, eBioscience). The stained cells were analyzed using BD
FACSCalibur. FIG. 5 shows that antibodies in serum from mice
vaccinated with Globo H-PADRE (+adjuvant QS21) bind to Globo
H-expressing MCF-7 cells. MZ-11-Globo H: Globo H-PADRE.
Example 5 Globo H-PADRE (M) Induces Much Higher Titer Anti-Globo H
IgG Than Carrier Protein-Globo H (G) with Class Switch
[0070] C57BL/6 mice were immunized with adjuvant (QS21 20 ug/mice),
2 ug of general carrier protein-Globo H conjugation (G) vaccine, or
Globo H-PADRE conjugation (MZ11-GloboH) vaccine at a 2-week
interval. Anti-Globo H serum was harvested before and 7 days after
each vaccination. The titer of anti-Globo H serum in pooled serum
or each mice were detected by ELISA assay with appropriated
secondary antibody. FIG. 6 shows that glycopeptide Globo H-PADRE
(M) induces higher titer of anti-Globo H IgG antibody than general
carrier protein-Globo H conjugation (C) does. C: control; Q:
adjuvant QS21. FIG. 7 shows that antibody titers in individual
mouse receiving glycopeptide Globo H-PADRE are constantly high,
whereas antibody titers in mouse receiving carrier protein-Globo H
conjugation are variable and most are low and it represents that
Globo H-PADRE (M) stably induces high titer of anti-Globo H IgG in
individual mouse.
Example 8 Globo H-PADRE (M) Induces Long-Lived Anti-Globo H IgG
Antibody and B cell Memory Responses
[0071] Anti-Globo H serum was harvested on 36 and 81 days after
last Vaccination (D64 and D109). The titer of anti-Globo H
antibodies The titers of anti-Globo H serum in pooled serum or each
mouse were detected by ELISA assay with appropriated secondary
antibody with 1/10000 dilution. FIG. 8 shows that glycopeptide
Globo H-PADRE (M) induces long-term anti-Globo H IgG, whereas
general carrier protein-Globo H conjugation (G) does not and FIG. 9
shows that dissection of individual mouse receiving glycopeptide
Globo H-PADRE shows constantly long-lived high-titer anti-Globo H
IgG antibody. Very low level of anti-Globo H IgG antibody is noted
in mouse receiving carrier protein-Globo H conjugation.
Example 9 Carbohydrate-PADRE Glycopeptide Induces High-titer
Anti-Carbohydrate IgG Antibody (GM2 as example)
[0072] C57BL/6 mice were immunized with adjuvant (QS21 20 ug/mice)
or GM2-PADRE conjugation vaccine with adjuvant (QS-21 20 ug/mice)
at a 2-week interval. Anti-GM2 serum was harvested before and 7
days after each vaccination. The titer of anti-GM2 serum in pooled
serum or each mice were detected by ELISA assay with appropriated
secondary antibody. FIG. 10 shows that GM2-PADRE glycopeptide
induces high-titer anti-carbohydrate IgG antibody.
Example 10 Anti-tumor Effect of Globo H-PADRE Glycopeptide Vaccine
in Immunocompetent Mouse Model
[0073] Mice were divided into 3 groups and subcutaneously (s.c.)
administered with 1.times. PBS (control), 20 ug of QS-21 alone or 6
ug of Globo H-PADRE (MZ11-Globo H) plus 20 ug of QS-21 at a 2-week
interval. Seven days after third vaccination, mice were s.c.
implanted 1.times.10.sup.5 of LLC1 cells and were concomitantly
vaccinated again. The vaccination interval was changed to 7 days
after tumor inoculation. Tumor size was measured by caliper at day
7, 10, 14 and 18 after tumor implantation and calculated at
length.times.width.times.height. FIG. 11 shows that mouse treated
by Globo H-PADRE vaccine demonstrated slower tumor growth (LLC1
cells subcutaneous tumor model in immuno-competent mice).
Example 11 Adoptive Transfer of Immunized Serum to Intra-Peritoneal
Ovarian Tumor Model Showed Obvious Anti-tumor Efficacy
[0074] Mice were divided into 2 groups. Serum was collected from
group 1 mice without immunization as control. Serum was also
collected from group 2 mice vaccinated with Globo H-PADRE
(MZ11-Globo H) 3 times at a 2-week interval as anti-Globo H serum.
One million TOV21G cells were intra-peritoneal (i.p.) implanted
into 5-week-old female NU/NU mice (BioLASCO Taiwan). After 4 days,
mice were administered with 200 .mu.L of control serum or
anti-GloboH serum 3 times a week through i.p. route. Untreated mice
were set as control. For monitoring tumor growth, tumor bearing
mice were i.p. injected 2004 of luciferin (3.9 mg/ml). The
chemoluminescent intensity of each mouse was detected by a
non-invasive IVIS system (Xenogen) with fixed exposure condition
per batch of experiment.
Sequence CWU 1
1
1113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Ala Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala Ala
Ala 1 5 10
* * * * *