U.S. patent application number 16/502844 was filed with the patent office on 2020-03-12 for novel glycan conjugates and methods of use thereof.
The applicant listed for this patent is ACADEMIA SINICA. Invention is credited to Chi-Huey WONG, Chung-Yi WU.
Application Number | 20200078452 16/502844 |
Document ID | / |
Family ID | 56417553 |
Filed Date | 2020-03-12 |
![](/patent/app/20200078452/US20200078452A1-20200312-C00001.png)
![](/patent/app/20200078452/US20200078452A1-20200312-C00002.png)
![](/patent/app/20200078452/US20200078452A1-20200312-C00003.png)
![](/patent/app/20200078452/US20200078452A1-20200312-C00004.png)
![](/patent/app/20200078452/US20200078452A1-20200312-C00005.png)
![](/patent/app/20200078452/US20200078452A1-20200312-C00006.png)
![](/patent/app/20200078452/US20200078452A1-20200312-C00007.png)
![](/patent/app/20200078452/US20200078452A1-20200312-C00008.png)
![](/patent/app/20200078452/US20200078452A1-20200312-C00009.png)
![](/patent/app/20200078452/US20200078452A1-20200312-C00010.png)
![](/patent/app/20200078452/US20200078452A1-20200312-C00011.png)
View All Diagrams
United States Patent
Application |
20200078452 |
Kind Code |
A1 |
WONG; Chi-Huey ; et
al. |
March 12, 2020 |
NOVEL GLYCAN CONJUGATES AND METHODS OF USE THEREOF
Abstract
The present disclosure is directed to vaccines, antibodies,
and/or immunogenic conjugate compositions targeting the
SSEA3/SSEA4/GloboH associated epitopes (natural and modified) which
elicit antibodies and/or binding fragment production useful for
modulating the globo-series glycosphingolipid synthesis. The
present disclosure relates to methods and compositions which can
modulate the globo-series glycosphingolipid synthesis.
Particularly, the present disclosure is directed to glycoenzyme
inhibitor compound and compositions and methods of use thereof that
can modulate the synthesis of globo-series glycosphingolipid
SSEA3/SSEA4/GloboH in the biosynthetic pathway; particularly, the
glycoenzyme inhibitors target the alpha-4GalT; beta-4GalNAcT-I; or
beta-3GalT-V enzymes in the globo-series synthetic pathway.
Moreover, the present disclosure is also directed to the method of
using the compositions described herein for the treatment or
detection of hyperproliferative diseases and/or conditions.
Inventors: |
WONG; Chi-Huey; (La Jolla,
CA) ; WU; Chung-Yi; (TAIPEI, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ACADEMIA SINICA |
Taipei |
|
TW |
|
|
Family ID: |
56417553 |
Appl. No.: |
16/502844 |
Filed: |
July 3, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14832993 |
Aug 21, 2015 |
10342858 |
|
|
16502844 |
|
|
|
|
62107378 |
Jan 24, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07H 15/04 20130101;
A61K 31/715 20130101; A61K 2039/55511 20130101; C07H 15/26
20130101; C07H 5/06 20130101; A61K 39/0011 20130101; C07K 16/3076
20130101; A61K 2039/6037 20130101; C07H 5/02 20130101; A61K
39/001169 20180801; A61K 31/7028 20130101; C08B 37/006 20130101;
C07K 16/44 20130101; C07H 5/04 20130101; A61K 2039/627 20130101;
A61K 39/001173 20180801 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07K 16/30 20060101 C07K016/30; C07H 15/04 20060101
C07H015/04; C08B 37/00 20060101 C08B037/00; C07H 5/02 20060101
C07H005/02; C07H 5/06 20060101 C07H005/06; A61K 31/715 20060101
A61K031/715; C07H 5/04 20060101 C07H005/04; A61K 31/7028 20060101
A61K031/7028; C07H 15/26 20060101 C07H015/26; C07K 16/44 20060101
C07K016/44 |
Claims
1. An immunogenic composition comprising: (a) a glycan conjugate
including a carrier and one or more glycans, and optionally (b) an
adjuvant; wherein each of the one or more glycans is conjugated
with the carrier through a linker having the formula (III):
##STR00026## wherein: X.sub.1 is --OR or --SR, wherein R is an
oxygen or sulfur protecting group, optionally substituted
C.sub.1-10 alkyl, optionally substituted aryl, optionally
substituted acyl, or optionally substituted imidoyl; each instance
of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6 and L is
independently selected from hydrogen, halogen, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted heterocyclyl,
optionally substituted aryl, --N.sub.3, --NO.sub.2,
--N(R.sup.B).sub.2, --N(R.sup.A)C(O)R.sup.A, --OR.sup.A,
--OC(O)R.sup.A, --CR.sup.A, --C(O)N(R.sup.B).sub.2, --CN,
--C(O)R.sup.A, --C(O)OR.sup.A, --S(O)R.sup.A, --SO.sub.2R.sup.A,
--SO.sub.2N(R.sup.B).sub.2, and --NHSO.sub.2R.sup.B; each instance
of R.sup.A is independently selected from hydrogen, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted heterocyclyl, and
optionally substituted aryl; each instance of R.sup.B is
independently selected from hydrogen, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted heterocyclyl, and optionally substituted
aryl; and provided the glycan conjugate is not of the formula
(III-a) and (III-b): ##STR00027##
2. The immunogenic composition of claim 1, wherein L is --OH.
3. The immunogenic composition of claim 2, wherein at least one
instance of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6
is --N.sub.3.
4. The immunogenic composition of claim 2, wherein at least one
instance of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6
is --F.
5. The immunogenic composition of claim 1, wherein L is of the
formula: ##STR00028## wherein: each instance of R.sup.8, R.sup.9,
R.sup.10 and R.sup.11 is independently selected from hydrogen,
halogen, optionally substituted alkyl, optionally substituted
alkenyl, optionally substituted alkynyl, optionally substituted
heterocyclyl, optionally substituted aryl, --N.sub.3, --NO.sub.2,
--N(R.sup.B).sub.2, --N(R.sup.A)C(O)R.sup.A, --OR.sup.A,
--OC(O)R.sup.A, --CR.sup.A, --C(O)N(R.sup.B).sub.2, --CN,
--C(O)R.sup.A, --C(O)OR.sup.A, --S(O)R.sup.A, --SO.sub.2R.sup.A,
--SO.sub.2N(R.sup.B).sub.2, and --NHSO.sub.2R.sup.B; wherein
R.sup.12 is H, OH, or halogen; R.sub.N is selected from --N.sub.3,
--NO.sub.2, --N(R.sup.B).sub.2, --N(R.sup.A)C(O)R.sup.A,
--OR.sup.A, --OC(O)R.sup.A, --SR.sup.A, --C(O)N(R.sup.B).sub.2,
--CN, --C(O)R.sup.A, --C(O)OR.sup.A, --S(O)R.sup.A,
--SO.sub.2R.sup.A, --SO.sub.2N(R.sup.B).sub.2, and
--NHSO.sub.2R.sup.B; each instance of R.sup.A is independently
selected from hydrogen, optionally substituted alkyl, optionally
substituted alkenyl, optionally substituted alkynyl, optionally
substituted heterocyclyl, and optionally substituted aryl; each
instance of R.sup.B is independently selected from hydrogen,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted
heterocyclyl, and optionally substituted aryl.
6. The immunogenic composition of claim 5, wherein the glycan
conjugate is of formula (IV): ##STR00029##
7. The immunogenic composition of claim 6, wherein at least one
instance of R.sup.1, R.sup.2, R.sup.3, R.sup.8, R.sup.9, R.sup.10,
and R.sup.11 is --N.sub.3.
8. The immunogenic composition of claim 6, wherein at least one
instance of R.sup.2, R.sup.3, R.sup.8, R.sup.9, R.sup.10, and
R.sup.11 is --F.
9. The immunogenic composition of claim 1, wherein the carrier is a
protein, a lipid, a lipolized protein, a virus, a peptide, or a
dendrimer of glycopeptides.
10. The immunogenic composition of claim 9, wherein the carrier is
a protein selected from the group consisting of tetanus toxoid
(TT), diphtheria toxoid (DT), diphtheria toxin cross-reacting
material .sub.197 (CRM.sub.197), fragment C of TT, Keyhole limpet
hemocyanin (KLH), bovine serum albumin (BSA), protein D,
outer-membrane protein (OMP) and pneumolysin.
11. The immunogenic composition of claim 10, wherein the carrier
protein is selected from the group consisting of TT, DT and
CRM.sub.197.
12. The immunogenic composition of claim 11, wherein the glycan
conjugate is of the formula (IV-a) or (IV-b): ##STR00030## wherein
m is an integer of 1 to 40, inclusive.
13. The immunogenic composition of claim 1, wherein the linker is a
hetero- or homo-bifunctional linker.
14. The immunogenic composition of claim 1, wherein the adjuvant is
a glycolipid capable of binding a CD1d molecule on a dendritic
cell.
15. The immunogenic composition of claim 1, wherein the adjuvant is
C34, 7DW8-5, C17, C23, Gluco-C34, Aluminum salt, Squalene, MF59, or
QS-21.
16. The immunogenic composition of claim 1, wherein the immunogenic
composition is capable of eliciting an immune response against a
cancer cell.
17. The immunogenic composition of claim 16, wherein the cancer
cell is selected from the group consisting of a brain cancer cell,
a lung cancer cell, a breast cancer cell, an oral cancer cell, an
esophagus cancer cell, a stomach cancer cell, a liver cancer cell,
a bile duct cancer cell, a pancreatic cancer cell, a colon cancer
cell, a kidney cancer cell, a bone cancer cell, a skin cancer cell,
a cervical cancer cell, an ovarian cancer cell, and a prostate
cancer cell.
18. The immunogenic composition of claim 16, wherein the immune
response includes generation of antibodies that specifically bind
to one or more of the antigens selected from the group consisting
of SSEA3 and SSEA4.
19. The immunogenic composition of claim 18, wherein the antibodies
neutralize one or more of SSEA3 and SSEA4 antigen expressed on the
surface of cancer cells or cancer stem cells.
20. The immunogenic composition of claim 18, wherein the antibodies
predominantly includes IgG antibodies.
21. A cancer vaccine, comprising a therapeutically effective amount
of the immunogenic composition of claim 1 and a pharmaceutically
acceptable excipient.
22. The cancer vaccine of claim 21, wherein the cancer vaccine is
able to induce an anti-cancer immune response in a subject.
23. A method of treating cancer in a subject in need thereof
wherein the method comprising administering a therapeutically
effective amount of the immunogenic composition of claim 1.
24. The method of claim 23 wherein the vaccine is co-administered
in combination with another therapeutic agent.
25. The method of claim 23, wherein the cancer is selected from the
group consisting of brain cancer, lung cancer, breast cancer, oral
cancer, esophageal cancer, stomach cancer, liver cancer, bile duct
cancer, pancreatic cancer, colon cancer, kidney cancer, bone
cancer, skin cancer, cervical cancer, ovarian cancer, and prostate
cancer.
26. The method of claim 25, wherein the cancer cell expresses SSEA3
and/or SSEA4 antigen on the surface of the cell.
27. The method of claim 26, wherein the subject is a human.
28. The method of claim 23, wherein the immunogenic composition or
the cancer vaccine is administered subcutaneously.
29. An isolated monoclonal antibody and/or a binding fragment
thereof raised against the immunogenic composition of claim 1.
30. A composition comprising an effective amount of the antibody or
antigen-binding fragment of claim 29 and a pharmaceutically
acceptable carrier.
31. A method for making the immunogenic composition of claim 1.
32. The method of claim 31 wherein the method comprising: providing
a carrier; conjugating one or more glycan to the carrier by
conjugation reaction; wherein each of the one or more glycan is
SSEA3 or SSEA4.
33. An immunogenic composition comprising multivalent construct
targeting one or more of SSEA4 and SSEA3 and their analogs thereof
wherein the glycans are linked to a template and a carrier,
##STR00031## wherein n can be an integer from 1 to 10; wherein
glycan can be selected from the group consisting of Formulas I, II,
III, and IV; wherein if n is 2 or more, each glycan can be the same
as another glycan on the aspartyl peptide or a difference glycan on
the aspartyl peptide.
34. The composition of claim 33 wherein the glycan is selected from
the group consisting of SSEA3 and SSEA4.
35. The composition of claim 33 wherein the multivalent construct
has the structure: ##STR00032## ##STR00033## wherein R.sup.1,
R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, and L on each glycan
moiety can be the same or different.
36. A compound having the formula (I): ##STR00034## or a salt
thereof, wherein: X.sub.1 is --OR or --SR, wherein R is hydrogen, a
oxygen or sulfur protecting group, optionally substituted
C.sub.1-10 alkyl, optionally substituted aryl, optionally
substituted acyl, or optionally substituted imidoyl; each instance
of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6 and L is
independently selected from hydrogen, halogen, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted heterocyclyl,
optionally substituted aryl, --N.sub.3, --NO.sub.2,
--N(R.sup.B).sub.2, --N(R.sup.A)C(O)R.sup.A, --OR.sup.A,
--OC(O)R.sup.A, --SR.sup.A, --C(O)N(R.sup.B).sub.2, --CN,
--C(O)R.sup.A, --C(O)OR.sup.A, --S(O)R.sup.A, --SO.sub.2R.sup.A,
--SO.sub.2N(R.sup.B).sub.2, and --NHSO.sub.2R.sup.B; each instance
of R.sup.A is independently selected from hydrogen, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted heterocyclyl, and
optionally substituted aryl; each instance of R.sup.B is
independently selected from hydrogen, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted heterocyclyl, and optionally substituted
aryl; and provided the compound is not of the formula (I-a) or
(I-b): ##STR00035##
37. The compound of claim 35, wherein L is --OH.
38. The compound of claim 35, wherein L is of the formula:
##STR00036## wherein: each instance of R.sup.8, R.sup.9, R.sup.10
and R.sup.11 is independently selected from hydrogen, halogen,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted
heterocyclyl, optionally substituted aryl, --N.sub.3, --NO.sub.2,
--N(R.sup.B).sub.2, --N(R.sup.A)C(O)R.sup.A, --OR.sup.A,
--OC(O)R.sup.A, --SR.sup.A, --C(O)N(R.sup.B).sub.2, --CN,
--C(O)R.sup.A, --C(O)OR.sup.A, --S(O)R.sup.A, --SO.sub.2R.sup.A,
--SO.sub.2N(R.sup.B).sub.2, and --NHSO.sub.2R.sup.B; wherein
R.sup.12 is H, OH, or halogen; R.sub.N is selected from --N.sub.3,
--NO.sub.2, --N(R.sup.B).sub.2, --N(R.sup.A)C(O)R.sup.A,
--OR.sup.A, --OC(O)R.sup.A, --SR.sup.A, --C(O)N(R.sup.B).sub.2,
--CN, --C(O)R.sup.A, --C(O)OR.sup.A, --S(O)R.sup.A,
--SO.sub.2R.sup.A, --SO.sub.2N(R.sup.B).sub.2, and
--NHSO.sub.2R.sup.B; each instance of R.sup.A is independently
selected from hydrogen, optionally substituted alkyl, optionally
substituted alkenyl, optionally substituted alkynyl, optionally
substituted heterocyclyl, and optionally substituted aryl; and each
instance of R.sup.B is independently selected from hydrogen,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted
heterocyclyl, and optionally substituted aryl.
39. The compound of claim 35, wherein the compound is of Formula
(II): ##STR00037##
40. The compound of claim 39, wherein at least one instance of
R.sup.1, R.sup.2, R.sup.3, R.sup.8, R.sup.9, R.sup.10 and R.sup.11
is --F.
41. The compound of claim 39, wherein at least one instance of
R.sup.1, R.sup.2, R.sup.3, R.sup.8, R.sup.9, R.sup.10 and R.sup.11
is --N.sub.3.
42. The method of treating hyperproliferative disease or condition
comprising the administering to a subject in need thereof a
therapeutically effective amount of the compound of claim 36.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/832,993, filed Aug. 21, 2015, which claims the benefit under
35 U.S.C. 119(e) of U.S. Provisional Patent Application No.
62/107,378, filed on Jan. 24, 2015, entitled NOVEL GLYCAN
CONJUGATES AND METHODS OF USE THEREOF, the contents of which is
hereby incorporated by reference as if set forth in its
entirety.
FIELD
[0002] The present disclosure relates to methods and compositions
which can modulate the globo-series glycosphingolipid synthesis.
Particularly, the present disclosure is directed to glycoenzyme
inhibitor compound and compositions and methods of use thereof that
can modulate the synthesis of globo-series glycosphingolipid
SSEA3/SSEA4/GloboH in the biosynthetic pathway; particularly, the
glycoenzyme inhibitors target the alpha-4GalT; beta-4GalNAcT-I; or
beta-3GalT-V enzymes in the globo-series synthetic pathway.
Additionally, the present disclosure is also directed to vaccines,
antibodies, and/or immunogenic conjugate compositions targeting the
SSEA3/SSEA4/GloboH associated epitopes (natural and modified) which
can elicit antibodies and/or binding fragment production useful for
modulating the globo-series glycosphingolipid synthesis. Moreover,
the present disclosure is also directed to the method of using the
compositions described herein for the treatment or detection of
hyperproliferative diseases and/or conditions.
BACKGROUND OF THE INVENTION
[0003] The carbohydrate antigens GloboH, stage-specific embryonic
antigen-3 (SSEA3), and stage-specific embryonic antigen-4 (SSEA4)
are closely related to one another in either structure or in
function. GloboH, SSEA3 and SSEA4 are globo-series
glycosphingolipids, with SSEA3 being the non-fucosylated
pentasaccharide precursor structure of GloboH, SSEA4 is sialylated
SSEA3 with sialic acid a2-3 links to the non-reducing end of
galactose of SSEA3.
[0004] Stage-specific embryonic antigen-3 (SSEA3) was first
identified and defined by the reactivity of an IgM monoclonal
antibody generated in a rat immunized with 4- to 8-cell stage mouse
embryos. This monoclonal antibody reacted with all mouse
preimplantation embryos from oocytes up to the early blastocyst
stage where its expression became more restricted, in the primitive
endoderm after implantation. The SSEA3 antigenic determinant was
determined to be a carbohydrate present on glycolipids and
glycoproteins; it was also found on human teratocarcinoma cells and
human erythrocytes. In a panel of structures isolated from the
2102Ep human teratocarcinoma cell line, the SSEA3 antibody had the
highest affinity for Gal.beta.(1-3)GalNAc
.beta.(1-3)Gal.alpha.(1-4)Gal.beta.(1-4)Glc.beta.(1)Cer. This
structure is also known as Gb5, galactosyl-globoside, or
globopentaosylceramide.
[0005] Synthesis of SSEA3 occurs when
31,3-.sub.galactosyltransferase V (.beta.3GalT-V) transfers
galactose to the GalNAc of globoside to form Gb5 or
galactosyl-globoside. It was determined that SSEA3 was not
expressed in hematopoietic or mesenchymal stem cells. Based on
immortalized lymph node lymphocytes from primary lung cancer
patients, generated hybridomas, and selected for antibody secreting
clones; monoclonal antibodies were then generated from two of these
clones--J309 and D579, which recognized the SSEA3 antigenic
determinant. The antibodies recognized SSEA3 on several tumor cell
lines including lung and breast cancer cell lines, and a
teratocarcinoma cell line; in an immune adherence assay, rodent
monoclonal SSEA3 antibody, also referred to as MC631, reacted
against the same cell lines as the J309 and D579 antibodies. SSEA3
has also been found on testicular germ cell tumors, as well as in
breast cancer and in BCSCs (breast cancer stem cells).
[0006] Chang et al. looked at SSEA3 expression on normal tissues
using a tissue microarray because its location outside of cancer
and development was largely unknown. The group found SSEA3 to be
expressed on normal epithelium of colon, esophagus, small
intestine, kidney, prostate, rectum, skin, testis, thymus, and
uterine cervix. Expression was located only on the apical surfaces
of epithelial cells or in the cytoplasm, which are considered
immune system restricted or inaccessible sites. In an experiment
using a KLH conjugated GloboH monovalent vaccine in mice, an
antibody response was made to only the GloboH antigen. When
.alpha.-GalCer was added as an adjuvant, the amount of overall
antibody production increased and the mice made polyclonal
antibodies to both the GloboH, the SSEA3 and the SSEA4 antigen
structures, which vaccination was unable to generate in the absence
of the adjuvant. This result showed that SSEA3, GloboH and SSEA4
could make promising targets for cancer vaccines and could be
targeted simultaneously.
[0007] However, most tumor associated carbohydrate antigens have
poor immunogenicity and many approaches have been developed to
increase the immune response of carbohydrate-based vaccines,
including conjugation with a carrier protein,administration with an
immunologic adjuvant using unnatural glycosidic linkage, clustered
antigens, unimolecular polyvalent vaccine or hetero-glycan
multivalent vaccine. Using these strategies, a few
carbohydrate-based vaccines that could elicit significant immune
responses to target glycan structures were designed for cancer
therapy and entered clinical trials. Among them, the clinical
trials of Theratope and GMK with adjuvant QS-21 failed to produce
statistically significant difference between time-to-disease and
overall survival rate. Mot likely these two vaccines could not
elicit robust T cell-dependent immune response in patients.
Specifically, Theratope and GMK induced a higher level of IgM in
patients but could not induce a strong immune IgG response, which
is a major problem in carbohydrate-based vaccine development.
[0008] Previous studies showed that modification of carbohydrate
antigen structures (MCAS) could effectively elicit a higher level
of immune response. For example, in the modification study of the
capsular polysaccharide of group B meningococci, the N-acetyl
groups of .alpha.-(2,8)-linked polysialic acid (PSA) was replaced
with the N-propinoyl group and such a modification elicited a high
antibody response to recognize not only the N-propinoyl PSA, but
also the nature N-acetyl PSA. Similar approaches were applied to
STn and GM3 antigens to produce high antibody titers against
modified and nature forms. The results indicated that
N-phenylacetyl, N-fluoroacetyl or N-difluoroacetyl modifications on
glycan antigens could improve the immunogenicity. Moreover, the
Schultz group reported that incorporation of a p-nitrophenylalanine
into the tumor necrosis factor-.alpha. (TNF-.alpha.) could break
immune tolerance and induce more antibody response to TNF-.alpha..
Using glycans as antigens, although some progress has been
achieved, most cases are the N-modification of disaccharide (STn),
trisaccharide (GM3) and polysialic acid (PSA) and some are based on
fluorinated MUC1 glycopeptide antigens.
SUMMARY OF THE INVENTION
[0009] The present disclosure is based on the discovery that the
modification of the stage-specific embryonic antigens (SSEA3 and
SSEA4) with certain groups disclosed herein elicited robust IgG
antibody response to specifically recognize SSEA3 and SSEA4,
respectively. The antibodies induced by an immunogenic composition
comprising such unnatural glycan moiety are able to mediate the
complement-dependent cell cytotoxicity against tumor cells.
[0010] Accordingly, the present invention features the design of
antibodies against SSEA3 and/or SSEA4 for treating cancers. The
present invention also features novel compounds consisting of the
modified carbohydrate antigens (SSEA3 and SSEA4), glycan conjugates
comprising such, and immunogenic compositions and vaccines
thereof.
[0011] The present disclosure also provides methods of using
synthetic glycan conjugates described herein to treat or reduce
hyperproliferative disease such as cancer.
[0012] Additionally, the present disclosure is also directed to
vaccines and/or immunogenic conjugate compositions targeting the
SSEA3/SSEA4/GloboH associated epitopes (natural and modified) which
can elicit antibodies and/or binding fragment production useful for
modulating the globo-series glycosphingolipid synthesis. Moreover,
the present disclosure is also directed to the method of using the
compositions described herein for the treatment or detection of
hyperproliferative diseases and/or conditions.
[0013] Accordingly, the present invention features the design of
antibodies against SSEA3 for treating cancers. The present
invention also features novel compounds consisting of the modified
carbohydrate antigens (SSEA3, SSEA4), glycan conjugates comprising
such, and immunogenic compositions and vaccines thereof
[0014] In one aspect, the present invention provides a compound of
formula (I):
##STR00001##
or a salt thereof, wherein X.sub.1, R.sup.1, R.sup.2, R.sup.3,
R.sup.4, R.sup.5, R.sup.6 and L are as described herein. In certain
embodiments, a compound of Formula (I) is useful for making an
immunogenic composition for treating cancers.
[0015] In another aspect, the present invention provides a compound
of Formula (II):
##STR00002##
[0016] or a salt thereof, wherein X.sub.1, R.sup.1, R.sup.2,
R.sup.3, R.sup.8, R.sup.9, R.sup.10, R.sup.11, and R.sub.N are as
described herein. In certain embodiments, a compound of Formula
(II) is useful for making an immunogenic composition for treating
cancers.
[0017] In another aspect, the present invention provides an
immunogenic composition, comprising (a) a glycan conjugate
including a carrier and one or more glycans, and optionally (b) an
adjuvant,
[0018] wherein: each of the one or more glycans is conjugated with
the carrier through a linker, having the formula (III) or (IV):
##STR00003##
[0019] wherein X.sub.1, R.sup.1, R.sup.2, R.sup.3, R.sup.4,
R.sup.5, R.sup.6, R.sup.8, R.sup.9, R.sup.10, R.sup.11, and R.sub.N
are as described herein.
[0020] In certain aspects, it is contemplated that any construct of
vaccine containing a combination of any one or more of the three
glycans (SSEA3, SSEA4 and GloboH) and analogs thereof in any ratio
can be linked to a carrier.
##STR00004##
[0021] wherein n can be an integer from 1 to 10;
[0022] wherein Glycan can be selected from the group consisting of
Formulas I, II, III, and IV;
[0023] wherein if n is 2 or more, each Glycan can be the same as
another Glycan on the aspartyl peptide or a difference Glycan on
the aspartyl peptide.
[0024] In some embodiments, Glycan can be selected from the group
consisting of SSEA3, SSEA4, and GloboH.
[0025] In some embodiments, the exemplary multivalent construct can
be:
##STR00005## ##STR00006##
[0026] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, and L on each glycan moiety can be the same or
different.
[0027] In certain embodiments, the immunogenic composition of the
invention comprises an adjuvant. Exemplary adjuvants suitable for
the invention are as described herein.
[0028] In certain embodiments, the immunogenic composition is
capable of eliciting an immune response against a cancer cell in a
subject. In certain embodiments, the cancer cell is selected from
the group consisting of a brain cancer cell, a lung cancer cell, a
breast cancer cell, an oral cancer cell, an esophageal cancer cell,
a stomach cancer cell, a liver cancer cell, a bile duct cancer
cell, a pancreatic cancer cell, a colon cancer cell, a kidney
cancer cell, a bone cancer cell, a skin cancer cell, a cervical
cancer cell, an ovarian cancer cell, and a prostate cancer
cell.
[0029] In certain embodiments, the immune response includes
generation of antibodies that specifically bind to one or more of
the antigens selected from the group consisting of GloboH, SSEA3
and SSEA4. In certain embodiments, the antibodies are developed to
target one or more of GloboH, SSEA3 and SSEA4 expressed on the
surface of cancer cells or cancer stem cells , and trigger CDC
and/or ADCC to kill these cells. In certain embodiments, the
antibodies predominantly include IgG antibodies. In certain
embodiments, the immunogenic compositions provided herein mainly
induce IgG1, IgG2b, IgG2c and IgG3.
[0030] Further, the present disclosure features monoclonal
antibodies and binding fragments raised against the immunogenic
composition described herein.
[0031] In one embodiment, the antibody is a human antibody.
[0032] In one embodiment, the antibody is a humanized antibody.
[0033] In one embodiment, the antibody is specifically targeted
against one or more of SSEA4, SSEA3, or GloboH.
[0034] In one embodiment, the antibody is specifically targeted
against SSEA3.
[0035] In one embodiment, the antibody is specifically targeted
against SSEA4.
[0036] In one embodiment, the antibody is a homogeneous antibody
having the biantennary glycan terminated by two sialic acid in
alpha-2,6-linkage.
[0037] In one aspect, the present disclosure provides a
pharmaceutical composition comprising an effective amount of the
antibody or antigen-binding fragment specifically targeted against
one or more of SSEA4, SSEA3, or GloboH and a pharmaceutically
acceptable carrier
[0038] In one embodiment, the pharmaceutical composition comprises
a combination of antibodies and/or binding fragment thereof each
independently targeting one or more of the SSEA4, SSEA3, and/or
GloboH glycans.
[0039] In one embodiment, the pharmaceutical composition is useful
for the treatment of cancer, infectious diseases, and/or
anti-inflammatory diseases,
[0040] In one embodiment, the pharmaceutical composition comprises
antibodies or binding fragments thereof having universal
biantennary N-glycan terminated with sialic acid in
alpha-2,6-linkage.
[0041] In another aspect, the present invention provides a cancer
vaccine comprising an immunogenic composition described herein and
a pharmaceutically acceptable excipient.
[0042] In another aspect, the present invention provides methods
for treating and/or reducing the risk for cancer in a subject
comprising administering to a subject in need thereof a
therapeutically effective amount of an immunogenic composition or a
cancer vaccine as described herein.
[0043] The treatment results in reduction of tumor size,
elimination of malignant cells, prevention of metastasis,
prevention of relapse, reduction or killing of disseminated cancer,
prolongation of survival and/or prolongation of time to tumor
cancer progression.
[0044] In some embodiments, the treatment further comprises
administering an additional therapy to the subject prior to, during
or subsequent to the administering of the immunogenic composition
or the cancer vaccine described herein. In some embodiments, the
additional therapy is treatment with a chemotherapeutic agent. In
some embodiments, the additional therapy is radiation therapy.
[0045] Another aspect of the present disclosure features a method
of vaccinating a mammal against cancers, comprising administering
to the mammal a pharmacologically effective amount of an
immunogenic composition or a cancer vaccine as described
herein.
[0046] In some embodiments, the mammal is a human. In some
embodiments, the immunogenic composition or the cancer vaccine
described herein is administered subcutaneously.
[0047] Examples of the cancer include, but are not limited to,
brain cancer, lung cancer, breast cancer, oral cancer, esophagus
cancer, stomach cancer, liver cancer, bile duct cancer, pancreas
cancer, colon cancer, kidney cancer, cervix cancer, ovary cancer
and prostate cancer. In some embodiments, the cancer is brain
cancer, lung cancer, breast cancer, ovarian cancer, prostate
cancer, colon cancer, or pancreas cancer.
[0048] In another aspect, the present invention provides methods of
synthesizing the compounds of the invention as described
herein.
[0049] In yet another aspect, the present disclosure features the
process for making an immunogenic composition or a cancer vaccine
as described herein.
[0050] The details of certain embodiments of the invention are set
forth herein. Other features, objects, and advantages of the
invention will be apparent from the Detailed Description, the
Figures, the Examples, and the Claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] FIG. 1: Biosynthetic Pathway of Globo Series of
Glycosphingolipids.
[0052] FIG. 2: The induced GloboH-IgG collected from different
epitope ratios of SSE4-CRM197 or SSEA4-Gc-CRM197 immunization.
[0053] FIG. 3A: Native SSEA4, as well as all eight SSEA4 analogs,
could elicit IgG antibodies against SSEA4 when combining the use of
Gal-C34.
[0054] FIG. 3B: Native SSEA4, as well as all eight SSEA4 analogs,
could elicit IgM antibodies against SSEA4 when combining the use of
Gal-C34.
[0055] FIG. 4A: Native SSEA4, as well as all eight SSEA4 analogs,
could elicit IgG antibodies against SSEA4 when combining the use of
Glc-C34.
[0056] FIG. 4B: Native SSEA4, as well as all eight SSEA4 analogs,
could elicit IgM antibodies against SSEA4 when combining the use of
Glc-C34.
[0057] FIG. 5: The glycan-protein conjugation method affects the
immune response.
DETAILED DESCRIPTIONS
[0058] The present disclosure is based on the surprising discovery
that the modification of the stage-specific embryonic antigens
(SSEA3 and SSEA4) with certain groups elicited robust IgG antibody
response to specifically recognize SSEA3 and SSEA4,
respectively.
[0059] In some examples, the modification of SSEA3 comprises a
fluoro, an azido or an O-phenyl group at the one or more positions
of the glucose of SSEA3. In some examples, the modification of
SSEA3 comprises a fluoro, an azido or an O-phenyl group at the one
or more positions of the non-reducing end galactose. In some
examples, the modification of SSEA4 comprises a fluoro, an azido or
an O-phenyl group at one or more positions of the glucose of SSEA4.
In some examples, the modification of SSEA4 comprises a fluoro, an
azido or an O-phenyl group at one or more positions of the sialic
acid residue.
[0060] Described herein are SSEA3 and SSEA4 analogs having the
modification at the reducing and/or non-reducing end. Such SSEA3
and SSEA4 analogs can elicit a stronger immune response (e.g.,
induction of IgG antibodies against SSEA3 and/or SSEA4) as compared
to the native SSEA3 and SSEA4. The antibodies induced by an
immunogenic composition comprising such unnatural glycan moiety are
able to mediate the complement-dependent cell cytotoxicity against
tumor cells.
Chemical Definitions
[0061] Definitions of specific functional groups and chemical terms
are described in more detail below. The chemical elements are
identified in accordance with the Periodic Table of the Elements,
CAS version, Handbook of Chemistry and Physics, 75th Ed., inside
cover, and specific functional groups are generally defined as
described therein. Additionally, general principles of organic
chemistry, as well as specific functional moieties and reactivity,
are described in Thomas Sorrell, Organic Chemistry, University
Science Books, Sausalito, 1999; Smith and March, March's Advanced
Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New
York, 2001; Larock, Comprehensive Organic Transformations, VCH
Publishers, Inc., New York, 1989; and Carruthers, Some Modern
Methods of Organic Synthesis, 3rd Edition, Cambridge University
Press, Cambridge, 1987. Moreover, exemplary glycan and antibody
methodologies are described in Wong et al, US20100136042,
US20090317837, and US20140051127, the disclosures of each of which
are hereby incorporated by reference.
[0062] Compounds described herein can comprise one or more
asymmetric centers, and thus can exist in various isomeric forms,
e.g., enantiomers and/or diastereomers. For example, the compounds
described herein can be in the form of an individual enantiomer,
diastereomer or geometric isomer, or can be in the form of a
mixture of stereoisomers, including racemic mixtures and mixtures
enriched in one or more stereoisomer. Isomers can be isolated from
mixtures by methods known to those skilled in the art, including
chiral high pressure liquid chromatography (HPLC) and the formation
and crystallization of chiral salts; or preferred isomers can be
prepared by asymmetric syntheses. See, for example, Jacques et al.,
Enantiomers, Racemates and Resolutions (Wiley Interscience, New
York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel,
Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and
Wilen, Tables of Resolving Agents and Optical Resolutions p. 268
(E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind.
1972). The invention additionally encompasses compounds described
herein as individual isomers substantially free of other isomers,
and alternatively, as mixtures of various isomers.
[0063] When a range of values is listed, it is intended to
encompass each value and sub-range within the range. For example
"C1-6" is intended to encompass C1, C2, C3, C4, C5, C6, C1-6, C1-5,
C1-4, C1-3, C1-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6,
C4-5, and C5-6.
[0064] "Alkyl" refers to a radical of a straight-chain or branched
saturated hydrocarbon group having from 1 to 20 carbon atoms
("C1-20 alkyl"). In some embodiments, an alkyl group has 1 to 10
carbon atoms ("C1-10 alkyl"). In some embodiments, an alkyl group
has 1 to 9 carbon atoms ("C1-9 alkyl"). In some embodiments, an
alkyl group has 1 to 8 carbon atoms ("C1-8 alkyl"). In some
embodiments, an alkyl group has 1 to 7 carbon atoms ("C1-7 alkyl").
In some embodiments, an alkyl group has 1 to 6 carbon atoms ("C1-6
alkyl"). In some embodiments, an alkyl group has 1 to 5 carbon
atoms ("C1-5 alkyl"). In some embodiments, an alkyl group has 1 to
4 carbon atoms ("C1-4 alkyl"). In some embodiments, an alkyl group
has 1 to 3 carbon atoms ("C1-3 alkyl"). In some embodiments, an
alkyl group has 1 to 2 carbon atoms ("C1-2 alkyl"). In some
embodiments, an alkyl group has 1 carbon atom ("C1 alkyl"). In some
embodiments, an alkyl group has 2 to 6 carbon atoms ("C2-6 alkyl").
Examples of C1-6 alkyl groups include methyl (C1), ethyl (C2),
n-propyl (C3), iso-propyl (C3), n-butyl (C4), tert-butyl (C4),
sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5),
amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl
(C5), and n-hexyl (C6). Additional examples of alkyl groups include
n-heptyl (C7), n-octyl (C8) and the like.
[0065] Unless otherwise specified, each instance of an alkyl group
is independently optionally substituted, i.e., unsubstituted (an
"unsubstituted alkyl") or substituted (a "substituted alkyl") with
one or more substituents. In certain embodiments, the alkyl group
is unsubstituted C1-10 alkyl (e.g., --CH3). In certain embodiments,
the alkyl group is substituted C1-10 alkyl.
[0066] "Alkenyl" refers to a radical of a straight-chain or
branched hydrocarbon group having from 2 to 20 carbon atoms, one or
more carbon-carbon double bonds, and no triple bonds ("C2-20
alkenyl"). In some embodiments, an alkenyl group has 2 to 10 carbon
atoms ("C2-10 alkenyl"). In some embodiments, an alkenyl group has
2 to 9 carbon atoms ("C2-9 alkenyl"). In some embodiments, an
alkenyl group has 2 to 8 carbon atoms ("C2-8 alkenyl"). In some
embodiments, an alkenyl group has 2 to 7 carbon atoms ("C2-7
alkenyl"). In some embodiments, an alkenyl group has 2 to 6 carbon
atoms ("C2-6 alkenyl"). In some embodiments, an alkenyl group has 2
to 5 carbon atoms ("C2-5 alkenyl"). In some embodiments, an alkenyl
group has 2 to 4 carbon atoms ("C2-4 alkenyl"). In some
embodiments, an alkenyl group has 2 to 3 carbon atoms ("C2-3
alkenyl"). In some embodiments, an alkenyl group has 2 carbon atoms
("C2 alkenyl"). The one or more carbon-carbon double bonds can be
internal (such as in 2-butenyl) or terminal (such as in 1-butenyl).
Examples of C2-4 alkenyl groups include ethenyl (C2), 1-propenyl
(C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl
(C4), and the like. Examples of C2-6 alkenyl groups include the
aforementioned C2-4 alkenyl groups as well as pentenyl (C5),
pentadienyl (C5), hexenyl (C6), and the like. Additional examples
of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8),
and the like. Unless otherwise specified, each instance of an
alkenyl group is independently optionally substituted, i.e.,
unsubstituted (an "unsubstituted alkenyl") or substituted (a
"substituted alkenyl") with one or more substituents. In certain
embodiments, the alkenyl group is unsubstituted C2-10 alkenyl. In
certain embodiments, the alkenyl group is substituted C2-10
alkenyl.
[0067] "Alkynyl" refers to a radical of a straight-chain or
branched hydrocarbon group having from 2 to 20 carbon atoms, one or
more carbon-carbon triple bonds, and optionally one or more double
bonds ("C2-20 alkynyl"). In some embodiments, an alkynyl group has
2 to 10 carbon atoms ("C2-10 alkynyl"). In some embodiments, an
alkynyl group has 2 to 9 carbon atoms ("C2-9 alkynyl"). In some
embodiments, an alkynyl group has 2 to 8 carbon atoms ("C2-8
alkynyl"). In some embodiments, an alkynyl group has 2 to 7 carbon
atoms ("C2-7 alkynyl"). In some embodiments, an alkynyl group has 2
to 6 carbon atoms ("C2-6 alkynyl"). In some embodiments, an alkynyl
group has 2 to 5 carbon atoms ("C2-5 alkynyl"). In some
embodiments, an alkynyl group has 2 to 4 carbon atoms ("C2-4
alkynyl"). In some embodiments, an alkynyl group has 2 to 3 carbon
atoms ("C2-3 alkynyl"). In some embodiments, an alkynyl group has 2
carbon atoms ("C2 alkynyl"). The one or more carbon-carbon triple
bonds can be internal (such as in 2-butynyl) or terminal (such as
in 1-butynyl). Examples of C2-4 alkynyl groups include, without
limitation, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3),
1-butynyl (C4), 2-butynyl (C4), and the like. Examples of C2-6
alkenyl groups include the aforementioned C2-4 alkynyl groups as
well as pentynyl (C5), hexynyl (C6), and the like. Additional
examples of alkynyl include heptynyl (C7), octynyl (C8), and the
like. Unless otherwise specified, each instance of an alkynyl group
is independently optionally substituted, i.e., unsubstituted (an
"unsubstituted alkynyl") or substituted (a "substituted alkynyl")
with one or more substituents. In certain embodiments, the alkynyl
group is unsubstituted C2-10 alkynyl. In certain embodiments, the
alkynyl group is substituted C2-10 alkynyl.
[0068] "Heterocyclyl" or "heterocyclic" refers to a radical of a 3-
to 10-membered non-aromatic ring system having ring carbon atoms
and 1 to 4 ring heteroatoms, wherein each heteroatom is
independently selected from nitrogen, oxygen, sulfur, boron,
phosphorus, and silicon ("3-10 membered heterocyclyl"). In certain
embodiments, the heteroatom is independently selected from
nitrogen, sulfur, and oxygen. In heterocyclyl groups that contain
one or more nitrogen atoms, the point of attachment can be a carbon
or nitrogen atom, as valency permits. A heterocyclyl group can
either be monocyclic ("monocyclic heterocyclyl") or a fused,
bridged or spiro ring system such as a bicyclic system ("bicyclic
heterocyclyl"), and can be saturated or partially unsaturated.
Heterocyclyl bicyclic ring systems can include one or more
heteroatoms in one or both rings. "Heterocyclyl" also includes ring
systems wherein the heterocyclic ring, as defined above, is fused
with one or more carbocyclyl groups wherein the point of attachment
is either on the carbocyclyl or heterocyclic ring, or ring systems
wherein the heterocyclic ring, as defined above, is fused with one
or more aryl or heteroaryl groups, wherein the point of attachment
is on the heterocyclic ring, and in such instances, the number of
ring members continue to designate the number of ring members in
the heterocyclic ring system. Unless otherwise specified, each
instance of heterocyclyl is independently optionally substituted,
i.e., unsubstituted (an "unsubstituted heterocyclyl") or
substituted (a "substituted heterocyclyl") with one or more
substituents. In certain embodiments, the heterocyclyl group is
unsubstituted 3-10 membered heterocyclyl. In certain embodiments,
the heterocyclyl group is substituted 3-10 membered
heterocyclyl.
[0069] "Aryl" refers to a radical of a monocyclic or polycyclic
(e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g.,
having 6, 10, or 14 .pi. electrons shared in a cyclic array) having
6-14 ring carbon atoms and zero heteroatoms in the aromatic ring
system ("C6-14 aryl"). In some embodiments, an aryl group has six
ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments,
an aryl group has ten ring carbon atoms ("C10 aryl"; e.g., naphthyl
such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl
group has fourteen ring carbon atoms ("C14 aryl"; e.g., anthracyl).
"Aryl" also includes ring systems wherein the aryl ring, as defined
above, is fused with one or more carbocyclyl or heterocyclyl groups
wherein the radical or point of attachment is on the aryl ring, and
in such instances, the number of carbon atoms continue to designate
the number of carbon atoms in the aryl ring system. Unless
otherwise specified, each instance of an aryl group is
independently optionally substituted, i.e., unsubstituted (an
"unsubstituted aryl") or substituted (a "substituted aryl") with
one or more substituents. In certain embodiments, the aryl group is
unsubstituted C6-14 aryl. In certain embodiments, the aryl group is
substituted C6-14 aryl.
[0070] Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl,
and heteroaryl groups, as defined herein, which are divalent
bridging groups are further referred to using the suffix -ene,
e.g., alkylene, alkenylene, alkynylene, carbocyclylene,
heterocyclylene, arylene, and heteroarylene.
[0071] The term "alkoxy" or "alkyloxy" refers to an --O-alkyl
radical, wherein alkyl is optionally substituted alkyl as defined
herein. Examples of alkoxy include, but are not limited to,
methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy,
sec-butoxy, and tert-butoxy.
[0072] The term "aryloxy" refers to an --O-aryl, wherein aryl is
optionally substituted aryl as defined herein.
[0073] As used herein, the term "optionally substituted" refers to
a substituted or unsubstituted moiety.
[0074] Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl,
and heteroaryl groups, as defined herein, are optionally
substituted (e.g., "substituted" or "unsubstituted" alkyl,
"substituted" or "unsubstituted" alkenyl, "substituted" or
"unsubstituted" alkynyl, "substituted" or "unsubstituted"
carbocyclyl, "substituted" or "unsubstituted" heterocyclyl,
"substituted" or "unsubstituted" aryl or "substituted" or
"unsubstituted" heteroaryl group). In general, the term
"substituted", whether preceded by the term "optionally" or not,
means that at least one hydrogen present on a group (e.g., a carbon
or nitrogen atom) is replaced with a permissible substituent, e.g.,
a substituent which upon substitution results in a stable compound,
e.g., a compound which does not spontaneously undergo
transformation such as by rearrangement, cyclization, elimination,
or other reaction. Unless otherwise indicated, a "substituted"
group has a substituent at one or more substitutable positions of
the group, and when more than one position in any given structure
is substituted, the substituent is either the same or different at
each position. The term "substituted" is contemplated to include
substitution with all permissible substituents of organic
compounds, any of the substituents described herein that results in
the formation of a stable compound. The present invention
contemplates any and all such combinations in order to arrive at a
stable compound. For purposes of this invention, heteroatoms such
as nitrogen may have hydrogen substituents and/or any suitable
substituent as described herein which satisfy the valencies of the
heteroatoms and results in the formation of a stable moiety.
[0075] "Halo" or "halogen" refers to fluorine (fluoro, --F),
chlorine (chloro, --Cl), bromine (bromo, --Br), or iodine (iodo,
--I).
[0076] "Acyl" as used herein refers to a moiety selected from the
group consisting of --C(.dbd.O)Raa,--CHO, --CO2Raa,
--C(.dbd.O)N(Rbb)2, --C(.dbd.NRbb)Raa, --C(.dbd.NRbb)ORaa,
--C(.dbd.NRbb)N(Rbb)2, --C(.dbd.O)NRbbSO2Raa, --C(.dbd.S)N(Rbb)2,
--C(.dbd.O)SRaa, and --C(.dbd.S)SRaa, wherein Raa and Rbb are as
defined herein.
[0077] Nitrogen atoms can be substituted or unsubstituted as
valency permits, and include primary, secondary, tertiary, and
quarternary nitrogen atoms. Exemplary nitrogen atom substituents
include, but are not limited to, hydrogen, --OH, --ORaa, --N(Rcc)2,
--CN, --C(.dbd.O)Raa, --C(.dbd.O)N(Rcc)2, --CO2Raa, --SO2Raa,
--C(.dbd.NRbb)Raa, --C(.dbd.NRcc)ORaa, --C(.dbd.NRcc)N(Rcc)2,
--SO2N(Rcc)2, --SO2Rcc, --SO2ORcc, --SORaa, --C(.dbd.S)N(Rcc)2,
--C(.dbd.O)SRcc, --C(.dbd.S)SRcc, --P(.dbd.O)2Raa,
--P(.dbd.O)(Raa)2, --P(.dbd.O)2N(Rcc)2, --P(.dbd.O)(NRcc)2, C1-10
alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, C3-10
carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14
membered heteroaryl, or two Rcc groups attached to a nitrogen atom
are joined to form a 3-14 membered heterocyclyl or 5-14 membered
heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,
heterocyclyl, aryl, and heteroaryl is independently substituted
with 0, 1, 2, 3, 4, or 5 Rdd groups, and wherein Raa, Rbb, Rcc, and
Rdd are as defined above.
[0078] In certain embodiments, the substituent present on an oxygen
atom is an oxygen protecting group (also referred to as a hydroxyl
protecting group). Oxygen protecting groups include, but are not
limited to, --Raa, --N(Rbb)2, --C(.dbd.O)SRaa, --C(.dbd.O)Raa,
--CO2Raa, --C(.dbd.O)N(Rbb)2, --C(.dbd.NRbb)Raa,
--C(.dbd.NRbb)ORaa, --C(.dbd.NRbb)N(Rbb)2, --S(.dbd.O)Raa,
--SO2Raa, --Si(Raa)3, --P(Rcc)2, --P(Rcc)3, --P(.dbd.O)2Raa,
--P(.dbd.O)(Raa)2, --P(.dbd.O)(ORcc)2, --P(.dbd.O)2N(Rbb)2, and
--P(.dbd.0)(NRbb)2, wherein Raa, Rbb, and Rcc are as defined
herein. Oxygen protecting groups are well known in the art and
include those described in Protecting Groups in Organic Synthesis,
T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons,
1999, incorporated herein by reference.
[0079] Exemplary oxygen protecting groups include, but are not
limited to, methyl, methoxylmethyl (MOM), methylthiomethyl (MTM),
t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM),
benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM),
(4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM),
t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl,
2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,
bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),
tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,
tetrahydrothiopyranyl, 1-methoxycyclohexyl,
4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl,
4-methoxytetrahydrothiopyranyl S,S-dioxide,
1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP),
1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,
2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,
1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,
1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,
2,2,2-trichloroethyl, 2-trimethylsilylethyl,
2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl,
p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl,
3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl,
2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl,
4-picolyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl,
p,p'-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl,
.alpha.-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl,
di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl,
4-(4'-bromophenacyloxyphenyl)diphenylmethyl,
4,4',4''-tris(4,5-dichlorophthalimidophenyl)methyl,
4,4',4''-tris(levulinoyloxyphenyl)methyl,
4,4',4''-tris(benzoyloxyphenyl)methyl,
3-(imidazol-1-yl)bis(4',4''-dimethoxyphenyl)methyl,
1,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl,
9-(9-phenyl)xanthenyl, 9- (9-phenyl-10-oxo)anthryl,
1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido,
trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl
(TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl
(DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS),
t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl,
triphenylsilyl, diphenylmethylsilyl (DPMS),
t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate,
acetate, chloroacetate, dichloroacetate, trichloroacetate,
trifluoroacetate, methoxyacetate, triphenylmethoxyacetate,
phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate,
4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate
(levulinoyldithioacetal), pivaloate, adamantoate, crotonate,
4-methoxycrotonate, benzoate, p-phenylbenzoate,
2,4,6-trimethylbenzoate (mesitoate), methyl carbonate,
9-fluorenylmethyl carbonate (Fmoc), ethyl carbonate,
2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl
carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),
2-(triphenylphosphonio) ethyl carbonate (Peoc), isobutyl carbonate,
vinyl carbonate, allyl carbonate, t-butyl carbonate (BOC),
p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzyl
carbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate,
p-nitrobenzyl carbonate, S-benzyl thiocarbonate,
4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate,
2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate,
o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate,
2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate,
2-(methylthiomethoxymethyl)benzoate,
2,6-dichloro-4-methylphenoxyacetate,
2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,
2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,
isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,
o-(methoxyacyl)benzoate, .alpha.-naphthoate, nitrate, alkyl
N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,
borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,
sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate
(Ts).
[0080] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
reference unless the context clearly dictates otherwise. As well,
the terms "a" (or "an"), "one or more" and "at least one" can be
used interchangeably herein. It is also to be noted that the terms
"comprising", "including", and "having" can be used
interchangeably.
[0081] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
microbiology, recombinant DNA, and immunology, which are within the
skill of the art. Such techniques are explained fully in the
literature. See, for example, Molecular Cloning A Laboratory
Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring
Harbor Laboratory Press, 1989); DNA Cloning, Volumes I and II (D.
N. Glover ed., 1985); Culture Of Animal Cells (R. I. Freshney, Alan
R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press,
1986); B. Perbal, A Practical Guide To Molecular Cloning (1984);
the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.);
Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P.
Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In
Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical
Methods In Cell And Molecular Biology (Mayer and Walker, eds.,
Academic Press, London, 1987); Antibodies: A Laboratory Manual, by
Harlow and Lane s (Cold Spring Harbor Laboratory Press, 1988); and
Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and
C. C. Blackwell, eds., 1986).
[0082] As used herein, the term "glycan" refers to a
polysaccharide, or oligosaccharide. Glycan is also used herein to
refer to the carbohydrate portion of a glycoconjugate, such as a
glycoprotein, glycolipid, glycopeptide, glycoproteome,
peptidoglycan, lipopolysaccharide or a proteoglycan. Glycans
usually consist solely of O-glycosidic linkages between
monosaccharides. For example, cellulose is a glycan (or more
specifically a glucan) composed of .beta.-1,4-linked D-glucose, and
chitin is a glycan composed of .beta.-1,4-linked
N-acetyl-D-glucosamine. Glycans can be homo or heteropolymers of
monosaccharide residues, and can be linear or branched. Glycans can
be found attached to proteins as in glycoproteins and
proteoglycans. They are generally found on the exterior surface of
cells. O- and N-linked glycans are very common in eukaryotes but
may also be found, although less commonly, in prokaryotes. N-Linked
glycans are found attached to the R-group nitrogen (N) of
asparagine in the sequon. The sequon is a Asn-X-Ser or Asn-X-Thr
sequence, where X is any amino acid except praline.
[0083] As used herein, the term "antigen" is defined as any
substance capable of eliciting an immune response.
[0084] As used herein, the term "immunogenicity" refers to the
ability of an immunogen, antigen, or vaccine to stimulate an immune
response.
[0085] As used herein, the term "CD1d" refers to a member of the
CD1 (cluster of differentiation 1) family of glycoproteins
expressed on the surface of various human antigen-presenting cells.
CD1d presented lipid antigens activate natural killer T cells. CD1d
has a deep antigen-binding groove into which glycolipid antigens
bind. CD1d molecules expressed on dendritic cells can bind and
present glycolipids, including alpha-GalCer analogs such as
C34.
[0086] As used herein, the term "epitope" is defined as the parts
of an antigen molecule which contact the antigen binding site of an
antibody or a T cell receptor.
[0087] As used herein, the term "vaccine" refers to a preparation
that contains an antigen, consisting of whole disease-causing
organisms (killed or weakened) or components of such organisms,
such as proteins, peptides, or polysaccharides, that is used to
confer immunity against the disease that the organisms cause.
Vaccine preparations can be natural, synthetic or derived by
recombinant DNA technology.
[0088] As used herein, the term "antigen specific" refers to a
property of a cell population such that supply of a particular
antigen, or a fragment of the antigen, results in specific cell
proliferation.
[0089] As used herein, the term "specifically binding," refers to
the interaction between binding pairs (e.g., an antibody and an
antigen). In various instances, specifically binding can be
embodied by an affinity constant of about 10-6 moles/liter, about
10-7 moles/liter, or about 10-8 moles/liter, or less.
[0090] As used herein, the terms glycoenzymes refers to at least in
part the enzymes in the globo-series biosynthetic pathway;
exemplary glycoenzymes include alpha-4GalT; beta-4GalNAcT-I; or
beta-3GalT-V enzymes.
[0091] As used herein, the term "globo-series pathway" includes to
a biosynthetic and enzymatic pathways described in FIG. 1.
[0092] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with research, diagnostic or
therapeutic uses for the antibody, and may include enzymes,
hormones, and other proteinaceous or nonproteinaceous solutes. In
one embodiment, the antibody will be purified (1) to greater than
95% by weight of antibody as determined by, for example, the Lowry
method, and in some embodiments more than 99% by weight, (2) to a
degree sufficient to obtain at least 15 residues of N-terminal or
internal amino acid sequence by use of, for example, a spinning cup
sequenator, or (3) to homogeneity by SDS-PAGE under reducing or
nonreducing conditions using, for example, Coomassie blue or silver
stain. Isolated antibody includes the antibody in situ within
recombinant cells since at least one component of the antibody's
natural environment will not be present. Ordinarily, however,
isolated antibody will be prepared by at least one purification
step.
[0093] "Binding affinity" generally refers to the strength of the
sum total of noncovalent interactions between a single binding site
of a molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (Kd).
Affinity can be measured by common methods known in the art,
including those described herein. Low-affinity antibodies generally
bind antigen slowly and tend to dissociate readily, whereas
high-affinity antibodies generally bind antigen faster and tend to
remain bound longer. A variety of methods of measuring binding
affinity are known in the art, any of which can be used for
purposes of the present invention. Specific illustrative
embodiments are described in the following.
[0094] "Antibody fragments" comprise only a portion of an intact
antibody, wherein the portion retains at least one, and as many as
most or all, of the functions normally associated with that portion
when present in an intact antibody. In one embodiment, an antibody
fragment comprises an antigen binding site of the intact antibody
and thus retains the ability to bind antigen. In another
embodiment, an antibody fragment, for example one that comprises
the Fc region, retains at least one of the biological functions
normally associated with the Fc region when present in an intact
antibody, such as FcRn binding, antibody half life modulation, ADCC
function and complement binding. In one embodiment, an antibody
fragment is a monovalent antibody that has an in vivo half life
substantially similar to an intact antibody. For example, such an
antibody fragment may comprise an antigen binding arm linked to an
Fc sequence capable of conferring in vivo stability to the
fragment.
[0095] The monoclonal antibodies herein specifically include
"chimeric" antibodies in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad.
Sci. USA 81:6851-6855 (1984)).
[0096] Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. In one embodiment, a humanized antibody
is a human immunoglobulin (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and/or capacity. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally will also comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the
following review articles and references cited therein: Vaswani and
Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998);
Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and
Gross, Curr. Op. Biotech. 5:428-433 (1994).
[0097] A "blocking" antibody or an "antagonist" antibody is one
which inhibits or reduces biological activity of the antigen it
binds. Certain blocking antibodies or antagonist antibodies
substantially or completely inhibit the biological activity of the
antigen.
[0098] An "agonist antibody", as used herein, is an antibody which
mimics at least one of the functional activities of a polypeptide
of interest.
[0099] A "disorder" is any condition that would benefit from
treatment with an antibody of the invention. This includes chronic
and acute disorders or diseases including those pathological
conditions which predispose the mammal to the disorder in question.
Non-limiting examples of disorders to be treated herein include
cancer.
[0100] The terms "cell proliferative disorder" and "proliferative
disorder" refer to disorders that are associated with some degree
of abnormal cell proliferation. In one embodiment, the cell
proliferative disorder is cancer.
[0101] "Tumor," as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues. The terms "cancer,"
"cancerous," "cell proliferative disorder," "proliferative
disorder" and "tumor" are not mutually exclusive as referred to
herein.
[0102] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth/proliferation. Examples of cancer
include, but are not limited to, carcinoma, lymphoma (e.g.,
Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and
leukemia. More particular examples of such cancers include squamous
cell cancer, small-cell lung cancer, non-small cell lung cancer,
adenocarcinoma of the lung, squamous carcinoma of the lung, cancer
of the peritoneum, hepatocellular cancer, gastrointestinal cancer,
pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,
liver cancer, bladder cancer, hepatoma, breast cancer, colon
cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary gland carcinoma, kidney cancer, liver cancer, prostate
cancer, vulval cancer, thyroid cancer, hepatic carcinoma, leukemia
and other lymphoproliferative disorders, and various types of head
and neck cancer.
[0103] The term "globo-series -related disorder" refers to or
describes a disorder that is typically characterized by or
contributed to by aberrant functioning or presentation of the
pathway. Examples of such disorders include, but are not limited
to, hyperproliferative diseases, including cancer.
[0104] Examples of immunologic deficiency syndromes include, but
are not limited to, ataxia telangiectasia, leukocyte-adhesion
deficiency syndrome, lymphopenia, dysgammaglobulinemia, HIV or
deltaretrovirus infections, common variable immunodeficiency,
severe combined immunodeficiency, phagocyte bactericidal
dysfunction, agammaglobulinemia, DiGeorge syndrome, and
Wiskott-Aldrich syndrome. Examples of hypersensitivity include, but
are not limited to, allergies, asthma, dermatitis, hives,
anaphylaxis, Wissler's syndrome, and thrombocytopenic purpura.
[0105] As used herein, "treatment" refers to clinical intervention
in an attempt to alter the natural course of the individual or cell
being treated, and can be performed either for prophylaxis or
during the course of clinical pathology. Desirable effects of
treatment include preventing occurrence or recurrence of disease,
alleviation of symptoms, diminishment of any direct or indirect
pathological consequences of the disease, preventing or decreasing
inflammation and/or tissue/organ damage, decreasing the rate of
disease progression, amelioration or palliation of the disease
state, and remission or improved prognosis. In some embodiments,
antibodies of the invention are used to delay development of a
disease or disorder.
[0106] An "individual" or a "subject" is a vertebrate. In certain
embodiments, the vertebrate is a mammal. Mammals include, but are
not limited to, farm animals (such as cows), sport animals, pets
(such as cats, dogs, and horses), primates, mice and rats. In
certain embodiments, the vertebrate is a human.
[0107] "Mammal" for purposes of treatment refers to any animal
classified as a mammal, including humans, domestic and farm
animals, and zoo, sports, or pet animals, such as dogs, horses,
cats, cows, etc. In certain embodiments, the mammal is human.
[0108] An "effective amount" refers to an amount effective, at
dosages and for periods of time necessary, to achieve the desired
therapeutic or prophylactic result.
[0109] A "therapeutically effective amount" of a substance/molecule
of the invention may vary according to factors such as the disease
state, age, sex, and weight of the individual, and the ability of
the substance/molecule, to elicit a desired response in the
individual. A therapeutically effective amount is also one in which
any toxic or detrimental effects of the substance/molecule are
outweighed by the therapeutically beneficial effects. A
"prophylactically effective amount" refers to an amount effective,
at dosages and for periods of time necessary, to achieve the
desired prophylactic result. Typically but not necessarily, since a
prophylactic dose is used in subjects prior to or at an earlier
stage of disease, the prophylactically effective amount would be
less than the therapeutically effective amount.
[0110] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g., At211, 1131, 1125, Y90, Rel86, Rel88,
Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu),
chemotherapeutic agents (e.g., methotrexate, adriamicin, vinca
alkaloids (vincristine, vinblastine, etoposide), doxorubicin,
melphalan, mitomycin C, chlorambucil, daunorubicin or other
intercalating agents, enzymes and fragments thereof such as
nucleolyticenzymes, antibiotics, and toxins such as small molecule
toxins or enzymatically active toxins of bacterial, fungal, plant
or animal origin, including fragments and/or variants thereof, and
the various antitumor or anticancer agents disclosed below. Other
cytotoxic agents are described below. A tumoricidal agent causes
destruction of tumor cells.
[0111] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and CYTOXAN.RTM.
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan
and piposulfan; aziridines such as benzodopa, carboquone,
meturedopa, and uredopa; ethylenimines and methylamelamines
including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); delta-9-tetrahydrocannabinol (dronabinol,
MARINOL.RTM.); beta-lapachone; lapachol; colchicines; betulinic
acid; a camptothecin (including the synthetic analogue topotecan
(HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.),
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; callystatin; CC-1065 (including its adozelesin,
carzelesin and bizelesin synthetic analogues); podophyllotoxin;
podophyllinic acid; teniposide; cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlomaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e.g., calicheamicin, especially calicheamicin
gammall and calicheamicin omegaIl (see, e.g., Agnew, Chem. Intl.
Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A;
an esperamicin; as well as neocarzinostatin chromophore and related
chromoprotein enediyne antiobiotic chromophores), aclacinomysins,
actinomycin, authramycin, azaserine, bleomycins, cactinomycin,
carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,
ADRIAMYCIN.RTM. doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elformithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;
procarbazine; PSK.RTM. polysaccharide complex (JHS Natural
Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran;
spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine
(ELDISINE.RTM., FILDESIN.RTM.); dacarbazine; mannomustine;
mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside
("Ara-C"); thiotepa; taxoids, e.g., TAXOL.RTM. paclitaxel
(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE.TM.
Cremophor-free, albumin-engineered nanoparticle formulation of
paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.),
and TAXOTERE.RTM. doxetaxel (Rhone-Poulenc Rorer, Antony, France);
chloranbucil; gemcitabine (GEMZAR.RTM.); 6-thioguanine;
mercaptopurine; methotrexate; platinum analogs such as cisplatin
and carboplatin; vinblastine (VELBAN.RTM.); platinum; etoposide
(VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN.RTM.);
oxaliplatin; leucovovin; vinorelbine (NAVELBINE.RTM.); novantrone;
edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase
inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such
as retinoic acid; capecitabine (XELODA.RTM.); pharmaceutically
acceptable salts, acids or derivatives of any of the above; as well
as combinations of two or more of the above such as CHOP, an
abbreviation for a combined therapy of cyclophosphamide,
doxorubicin, vincristine, and prednisolone, and FOLFOX, an
abbreviation for a treatment regimen with oxaliplatin
(ELOXATIN.TM.) combined with 5-FU and leucovovin.
[0112] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications and patents specifically mentioned herein are
incorporated by reference for all purposes including describing and
disclosing the chemicals, cell lines, vectors, animals,
instruments, statistical analysis and methodologies which are
reported in the publications which might be used in connection with
the invention. All references cited in this specification are to be
taken as indicative of the level of skill in the art. Nothing
herein is to be construed as an admission that the invention is not
entitled to antedate such disclosure by virtue of prior
invention.
[0113] In one aspect, the present disclosure is based on the
surprising discovery that the modification of the stage-specific
embryonic antigens (SSEA3 and SSEA4) with certain groups elicited
robust IgG antibody response to specifically recognize SSEA3 and
SSEA4, respectively.
[0114] In some examples, the modification of SSEA3 comprises a
fluoro, an azido or an O-phenyl group at the one or more positions
of the glucose of SSEA3. In some examples, the modification of
SSEA3 comprises a fluoro, an azido or an O-phenyl group at the one
or more positions of the non-reducing end galactose. In some
examples, the modification of SSEA4 comprises a fluoro, an azido or
an O-phenyl group at one or more positions of the glucose of SSEA4.
In some examples, the modification of SSEA4 comprises a fluoro, an
azido or an O-phenyl group at one or more positions of the sialic
acid residue.
[0115] In certain aspects, the present disclosure provides SSEA3
and SSEA4 analogs having the modification at the reducing and/or
non-reducing end. Such SSEA3 and SSEA4 analogs can elicit a
stronger immune response (e.g., induction of IgG antibodies against
SSEA3 and/or SSEA4) as compared to the native SSEA3 and SSEA4. The
antibodies induced by an immunogenic composition comprising such
unnatural glycan moiety are able to mediate the
complement-dependent cell cytotoxicity against tumor cells.
[0116] Compounds
[0117] Accordingly, the present invention also features novel
compounds consisting of the modified carbohydrate antigens (SSEA3
and SSEA4), glycan conjugates comprising such, and immunogenic
compositions and vaccines thereof
[0118] In one aspect, the present invention provides a compound of
formula (I):
##STR00007##
[0119] or a salt thereof,
[0120] wherein: [0121] X1 is --OR or --SR, wherein R is hydrogen, a
oxygen or sulfur protecting group, optionally substituted C1-10
alkyl, optionally substituted aryl, optionally substituted acyl, or
optionally substituted imidoyl; [0122] each instance of R1, R2, R3,
R4, R5, R6 and L is independently selected from hydrogen, halogen,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted
heterocyclyl, optionally substituted aryl, --N3, --NO2, --N(RB)2,
--N(RA)C(O)RA, --ORA, --OC(O)RA, --SRA, --C(O)N(RB)2, --CN,
--C(O)RA, --C(O)ORA, --S(O)RA, --SO2RA, --SO2N(RB)2, and --NHSO2RB;
[0123] each instance of RA is independently selected from hydrogen,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted
heterocyclyl, and optionally substituted aryl; [0124] each instance
of RB is independently selected from hydrogen, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted heterocyclyl, and
optionally substituted aryl; and [0125] provided the compound is
not of the formula:
##STR00008##
[0126] In certain embodiments, X1 is in the alpha configuration. In
certain embodiments, X1 is in the beta configuration.
[0127] In some embodiments, X1 is --ORA. In some embodiments, X1 is
--OH. In some embodiments, X1 is --O(protecting group). In some
embodiments, X1 is --ORA, wherein RA is unsubstituted C1-10 alkyl.
In some embodiments, X1 is --ORA, wherein RA is substituted C1-10
alkyl. In some embodiments, X1 is --ORA, wherein RA is
unsubstituted aryl. In some embodiments, X1 is --ORA, wherein RA is
substituted aryl. In some embodiments, X1 is --ORA, wherein RA is
unsubstituted acyl. In some embodiments, X1 is --ORA, wherein RA is
substituted acyl. In some embodiments, X1 is --ORA, wherein RA is
unsubstituted imidoyl. In some embodiments, X1 is --ORA, wherein RA
is substituted imidoyl.
[0128] In some embodiments, X1 is --SRA. In some embodiments, X1 is
--SH. In some embodiments, X1 is --S(protecting group). In some
embodiments, X1 is --SRA, wherein RA is unsubstituted C1-10 alkyl.
In some embodiments, X1 is --SRA, wherein RA is substituted C1-10
alkyl. In certain embodiments, X1 is --SCH3. In some embodiments,
X1 is --SRA, wherein RA is unsubstituted aryl. In some embodiments,
X1 is --SRA, wherein RA is substituted aryl. In some embodiments,
X1 is --SRA, wherein RA is unsubstituted acyl. In some embodiments,
X1 is --SRA, wherein RA is substituted acyl. In some embodiments,
X1 is --SRA, wherein RA is unsubstituted imidoyl. In some
embodiments, X1 is --SRA, wherein RA is substituted imidoyl.
[0129] In some embodiments, X1 is C1-10 alkoxy. In some
embodiments, X1 is C1-3 alkoxy.
[0130] In some embodiments, X1 is selected from the group
consisting of alpha-thiomethyl, beta-thiomethyl, alpha-thiocresyl,
beta-thiocresyl, alpha-t-butyldiphenylsilyloxy,
beta-t-butyldiphenylsilyloxy, and alpha-methoxy.
[0131] In some embodiments, R1 is --N3 or --N(RW)2, wherein each RW
is independently hydrogen or a nitrogen protecting group. In
certain embodiments, R1 is --N3. In certain embodiments, R1 is
--N(RW)2, wherein each RW is independently hydrogen or a nitrogen
protecting group. In certain embodiments, R1 is --NH2. In certain
embodiments, R1 is --NHRW, wherein RW is a nitrogen protecting
group. In certain embodiments, R1 is --N(RW)2, wherein each RW is a
nitrogen protecting group. In certain embodiments, R1 is selected
from the group consisting of --N3, --NH(Cbz), --NH(Boc),
--NH(Fmoc), --NHC(O)CCl3, --NHC(O)CH3, and --N(C(O)CH3)2.
[0132] In certain embodiments, R1 is --NH(Cbz). In certain
embodiments, R1 is --NH(Fmoc). In certain embodiments, R1 is
--NHC(O)CCl3. In certain embodiments, R1 is --NHC(O)CH3. In certain
embodiments, R1 is --N(C(O)CH3)2.
[0133] In some embodiments, R2 is --N3 or --N(RW)2, wherein each RW
is independently hydrogen or a nitrogen protecting group. In
certain embodiments, R2 is --N3. In certain embodiments, R2 is --N3
or --N(RW)2, wherein each RW is independently hydrogen or a
nitrogen protecting group. In certain embodiments, R2 is --NH2. In
certain embodiments, R2 is --NHRW, wherein RW is a nitrogen
protecting group. In certain embodiments, R2 is --N(RW)2, wherein
each RW is a nitrogen protecting group. In certain embodiments, R2
is selected from the group consisting of --N3, --NH(Cbz),
--NH(Boc), --NH(Fmoc), --NHC(O)CCl3, --NHC(O)CH3, and
--N(C(O)CH3)2. In certain embodiments, R2 is --NH(Cbz). In certain
embodiments, R2 is --NH(Fmoc). In certain embodiments, R2 is
--NHC(O)CCl3. In certain embodiments, R2 is --NHC(O)CH3. In certain
embodiments, R2 is --N(C(O)CH3)2.
[0134] In some embodiments, R3 is --N3 or --N(RW)2, wherein each RW
is independently hydrogen or a nitrogen protecting group. In
certain embodiments, R3 is --N3. In certain embodiments, R3 is --N3
or --N(RW)2, wherein each RW is independently hydrogen or a
nitrogen protecting group. In certain embodiments, R3 is --NH2. In
certain embodiments, R3 is --NHRW, wherein RW is a nitrogen
protecting group. In certain embodiments, R3 is --N(RW)2, wherein
each RW is a nitrogen protecting group. In certain embodiments, R3
is selected from the group consisting of --N3, --NH(Cbz),
--NH(Boc), --NH(Fmoc), --NHC(O)CCl3, --NHC(O)CH3, and
--N(C(O)CH3)2. In certain embodiments, R3 is --NH(Cbz). In certain
embodiments, R3 is --NH(Fmoc). In certain embodiments, R3 is
--NHC(O)CCl3. In certain embodiments, R3 is --NHC(O)CH3. In certain
embodiments, R3 is --N(C(O)CH3)2.
[0135] In some embodiments, R4 is --N3 or --N(RW)2, wherein each RW
is independently hydrogen or a nitrogen protecting group. In
certain embodiments, R4 is --N3. In certain embodiments, R4 is
--N(RW)2, wherein each RW is independently hydrogen or a nitrogen
protecting group. In certain embodiments, R4 is --NH2. In certain
embodiments, R4 is --NHRW, wherein RW is a nitrogen protecting
group. In certain embodiments, R4 is --N(RW)2, wherein each RW is a
nitrogen protecting group. In certain embodiments, R4 is selected
from the group consisting of --N3, --NH(Cbz), --NH(Boc),
--NH(Fmoc), --NHC(O)CCl3, --NHC(O)CH3, and --N(C(O)CH3)2.
[0136] In certain embodiments, R4 is --NH(Cbz). In certain
embodiments, R4 is --NH(Fmoc). In certain embodiments, R4 is
--NHC(O)CCl3. In certain embodiments, R4 is --NHC(O)CH3. In certain
embodiments, R4 is --N(C(O)CH3)2.
[0137] In some embodiments, R5 is --N3 or --N(RW)2, wherein each RW
is independently hydrogen or a nitrogen protecting group. In
certain embodiments, R5 is --N3. In certain embodiments, R5 is --N3
or --N(RW)2, wherein each RW is independently hydrogen or a
nitrogen protecting group. In certain embodiments, R5 is --NH2. In
certain embodiments, R5 is --NHRW, wherein RW is a nitrogen
protecting group. In certain embodiments, R5 is --N(RW)2, wherein
each RW is a nitrogen protecting group. In certain embodiments, R5
is selected from the group consisting of --N3, --NH(Cbz),
--NH(Boc), --NH(Fmoc), --NHC(O)CCl3, --NHC(O)CH3, and
--N(C(O)CH3)2. In certain embodiments, R5 is --NH(Cbz). In certain
embodiments, R5 is --NH(Fmoc). In certain embodiments, R5 is
--NHC(O)CCl3. In certain embodiments, R5 is --NHC(O)CH3. In certain
embodiments, R5 is --N(C(O)CH3)2.
[0138] In some embodiments, R6 is --N3 or --N(RW)2, wherein each RW
is independently hydrogen or a nitrogen protecting group. In
certain embodiments, R6 is --N3. In certain embodiments, R6 is --N3
or --N(RW)2, wherein each RW is independently hydrogen or a
nitrogen protecting group. In certain embodiments, R6 is --NH2. In
certain embodiments, R6 is --NHRW, wherein RW is a nitrogen
protecting group. In certain embodiments, R6 is --N(RW)2, wherein
each RW is a nitrogen protecting group. In certain embodiments, R6
is selected from the group consisting of --N3, --NH(Cbz),
--NH(Boc), --NH(Fmoc), --NHC(O)CCl3, --NHC(O)CH3, and
--N(C(O)CH3)2. In certain embodiments, R6 is --NH(Cbz). In certain
embodiments, R6 is --NH(Fmoc). In certain embodiments, R6 is
--NHC(O)CCl3. In certain embodiments, R6 is --NHC(O)CH3. In certain
embodiments, R6 is --N(C(O)CH3)2.
[0139] In some embodiments, R1, R2 and R3 are the same. In some
embodiments, R1, R2 and R3 are --OH. In some embodiments, R4, R5
and R6 are the same. In some embodiments, R4, R5 and R6 are
--OH.
[0140] In certain embodiments, L is --OH.
[0141] In certain embodiments, L is --OH and R1 is --N3. In certain
embodiments, L is --OH, R1 is --N3, and each instance of R2, R3,
R4, R5 and R6 is --OH.
[0142] In certain embodiments, L is --OH and R2 is --N3. In certain
embodiments, L is --OH, R2 is --N3, and each instance of R1, R3,
R4, R5 and R6 is --OH.
[0143] In certain embodiments, L is --OH and R3 is --N3. In certain
embodiments, L is --OH, R3 is --N3, and each instance of R1, R2,
R4, R5 and R6 is --OH.
[0144] In certain embodiments, L is --OH and R4 is --N3. In certain
embodiments, L is --OH, R4 is --N3, and each instance of R1, R2,
R3, R5 and R6 is --OH.
[0145] In certain embodiments, L is --OH and R5 is --N3. In certain
embodiments, L is --OH, R5 is --N3, and each instance of R1, R2,
R3, R4 and R6 is --OH.
[0146] In certain embodiments, L is --OH and R6 is --N3. In certain
embodiments, L is --OH, R6 is --N3, and each instance of R1, R2,
R3, R4 and R5 is --OH.
[0147] In certain embodiments, each instance of R1, R2, R3, R4, R5,
R6 and L is --F. In certain embodiments, R1 is --F. In certain
embodiments, R2 is --F. In certain embodiments, R3 is --F. In
certain embodiments, R4 is --F. In certain embodiments, R5 is --F.
In certain embodiments, R6 is --F. In certain embodiments, L is
--F.
[0148] In certain embodiments, L is of the following structure:
##STR00009##
[0149] wherein: [0150] each instance of R8, R9, R10 and R11 is
independently selected from hydrogen, halogen, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted heterocyclyl,
optionally substituted aryl, --N3, --NO2, --N(RB)2, --N(RA)C(O)RA,
--ORA, --OC(O)RA, --SRA, -- C(O)N(RB)2, --CN, --C(O)RA, --C(O)ORA,
--S(O)RA, --SO2RA, --SO2N(RB)2, and --NHSO2RB; [0151] R.sub.N is
selected from --N3, --NO2, --N(RB)2, --N(RA)C(O)RA, --ORA,
--OC(O)RA, --SRA, --C(O)N(RB)2, --CN, --C(O)RA, --C(O)ORA,
--S(O)RA, --SO2RA, --SO2N(RB)2, and --NHSO2RB; [0152] each instance
of RA is independently selected from hydrogen, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted heterocyclyl, and
optionally substituted aryl; and [0153] each instance of RB is
independently selected from hydrogen, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted heterocyclyl, and optionally substituted
aryl.
[0154] In some embodiments, the compound is of Formula (II)
##STR00010##
[0155] wherein: R1, R2, R3, R8, R9, R10, R11 and R.sub.N and X1 are
as described herein, and
[0156] provided the compound is not of the formula:
##STR00011##
[0157] In some embodiments, R8 is --N3 or --N(RW)2, wherein each RW
is independently hydrogen or a nitrogen protecting group. In
certain embodiments, R8 is --N3. In certain embodiments, R8 is
--N(RW)2, wherein each RW is independently hydrogen or a nitrogen
protecting group. In certain embodiments, R8 is --NH2. In certain
embodiments, R8 is --NHRW, wherein RW is a nitrogen protecting
group. In certain embodiments, R8 is --N(RW)2, wherein each RW is a
nitrogen protecting group. In certain embodiments, R8 is selected
from the group consisting of --N3, --NH(Cbz), --NH(Boc),
--NH(Fmoc), --NHC(O)CCl3, --NHC(O)CH3, and --N(C(O)CH3)2. In
certain embodiments, R8 is --NH(Cbz). In certain embodiments, R8 is
--NH(Fmoc). In certain embodiments, R8 is --NHC(O)CCl3. In certain
embodiments, R8 is --NHC(O)CH3. In certain embodiments, R8 is
--N(C(O)CH3)2.
[0158] In some embodiments, R9 is --N3 or --N(RW)2, wherein each RW
is independently hydrogen or a nitrogen protecting group. In
certain embodiments, R9 is --N3. In certain embodiments, R9 is
--N(RW)2, wherein each RW is independently hydrogen or a nitrogen
protecting group. In certain embodiments, R9 is --NH2. In certain
embodiments, R9 is --NHRW, wherein RW is a nitrogen protecting
group. In certain embodiments, R9 is --N(RW)2, wherein each RW is a
nitrogen protecting group. In certain embodiments, R9 is selected
from the group consisting of --N3, --NH(Cbz), --NH(Boc),
--NH(Fmoc), --NHC(O)CCl3, --NHC(O)CH3, and --N(C(O)CH3)2. In
certain embodiments, R9 is --NH(Cbz). In certain embodiments, R9 is
--NH(Fmoc). In certain embodiments, R9 is --NHC(O)CCl3. In certain
embodiments, R9 is --NHC(O)CH3. In certain embodiments, R9 is
--N(C(O)CH3)2.
[0159] In some embodiments, R10 is --N3 or --N(RW)2, wherein each
RW is independently hydrogen or a nitrogen protecting group. In
certain embodiments, R10 is --N3. In certain embodiments, R10 is
--N(RW)2, wherein each RW is independently hydrogen or a nitrogen
protecting group. In certain embodiments, R10 is --NH2. In certain
embodiments, R10 is --NHRW, wherein RW is a nitrogen protecting
group. In certain embodiments, R10 is --N(RW)2, wherein each RW is
a nitrogen protecting group. In certain embodiments, R10 is
selected from the group consisting of --N3, --NH(Cbz), --NH(Boc),
--NH(Fmoc), --NHC(O)CCl3, --NHC(O)CH3, and --N(C(O)CH3)2. In
certain embodiments, R10 is --NH(Cbz). In certain embodiments, R10
is --NH(Fmoc). In certain embodiments, R10 is --NHC(O)CCl3. In
certain embodiments, R10 is --NHC(O)CH3. In certain embodiments,
R10 is --N(C(O)CH3)2.
[0160] In some embodiments, R11 is --N3 or --N(RW)2, wherein each
RW is independently hydrogen or a nitrogen protecting group. In
certain embodiments, R11 is --N3. In certain embodiments, R11 is
--N(RW)2, wherein each RW is independently hydrogen or a nitrogen
protecting group. In certain embodiments, R11 is --NH2. In certain
embodiments, R11 is --NHRW, wherein RW is a nitrogen protecting
group. In certain embodiments, R11 is --N(RW)2, wherein each RW is
a nitrogen protecting group. In certain embodiments, R11 is
selected from the group consisting of --N3, --NH(Cbz), --NH(Boc),
--NH(Fmoc), --NHC(O)CCl3, --NHC(O)CH3, and --N(C(O)CH3)2. In
certain embodiments, R11 is --NH(Cbz). In certain embodiments, R11
is --NH(Fmoc). In certain embodiments, R11 is --NHC(O)CCl3. In
certain embodiments, R11 is --NHC(O)CH3. In certain embodiments,
R11 is --N(C(O)CH3)2.
[0161] In some embodiments, R12 is --N3 or --N(RW)2, wherein each
RW is independently hydrogen or a nitrogen protecting group. In
certain embodiments, R12 is --N3. In certain embodiments, R12 is
--N(RW)2, wherein each RW is independently hydrogen or a nitrogen
protecting group. In certain embodiments, R12 is --NH2. In certain
embodiments, R12 is --NHRW, wherein RW is a nitrogen protecting
group. In certain embodiments, R12 is --N(RW)2, wherein each RW is
a nitrogen protecting group. In certain embodiments, R12 is
selected from the group consisting of --N3, --NH(Cbz), --NH(Boc),
--NH(Fmoc), --NHC(O)CCl3, --NHC(O)CH3, and --N(C(O)CH3)2. In
certain embodiments, R12 is --NH(Cbz). In certain embodiments, R12
is --NH(Fmoc). In certain embodiments, R12 is --NHC(O)CCl3. In
certain embodiments, R12 is --NHC(O)CH3. In certain embodiments,
R12 is --N(C(O)CH3)2.
[0162] In some embodiments, R.sub.N is --N3 or --N(RW)2, wherein
each RW is independently hydrogen or a nitrogen protecting group.
In certain embodiments, RN is --N3. In certain embodiments, R.sub.N
is --N(RW)2, wherein each RW is independently hydrogen or a
nitrogen protecting group. In certain embodiments, R.sub.N is
--NH2. In certain embodiments, R.sub.N is --NHRW, wherein RW is a
nitrogen protecting group. In certain embodiments, R.sub.N is
--N(RW)2, wherein each RW is a nitrogen protecting group. In
certain embodiments, R.sub.N is selected from the group consisting
of --N3, --NH(Cbz), --NH(Boc), --NH(Fmoc), --NHC(O)CCl3,
--NHC(O)CH3, and --N(C(O)CH3)2. In certain embodiments, R.sub.N is
--NH(Cbz). In certain embodiments, R.sub.N is --NH(Fmoc). In
certain embodiments, R.sub.N is --NHC(O)CCl3. In certain
embodiments, R.sub.N is --NHC(O)CH3. In certain embodiments,
R.sub.N is --N(C(O)CH3)2.
[0163] Immunogenic Compositions
[0164] In another aspect, the present invention provides an
immunogenic composition, comprising (a) a glycan conjugate
including a carrier and one or more glycans, and optionally (b) an
adjuvant,
[0165] wherein: each of the one or more glycans is conjugated with
the carrier through a linker, having the formula (III) or (IV):
##STR00012##
[0166] wherein X1, R1, R2, R3, R4, R5, R6, R8, R9, R10, R11, L and
R.sub.N are as described herein.
[0167] In certain embodiments, the linker is a hetero- or
homo-bifunctional linker.
[0168] In certain embodiments, the linker is a homo-bifunctional
p-nitrophenyl linker.
[0169] In certain embodiments, the linker includes at least one
sulfur atom, carboxylate group, amide group, carbamate group,
carbonate group, thiocarbamate group, thiocarbonate group,
thioether group, succinamide group, n-hydroxy succinamide group, or
any combination thereof.
[0170] In certain embodiments, the linker is wherein
--L.sup.1--L.sup.2-- is a bond, O, S , NRL1a--, --C(.dbd.O)--,
--NRL1 aC(.dbd.O)--, --NRL1 aC(.dbd.O)O--, --C(.dbd.O)NRL1a--,
--OC(.dbd.O)NRL1a--, --SC(.dbd.O)--, --C(.dbd.O)S--,
--OC(.dbd.O)--, --C(.dbd.O)O--, --NRL1aC(.dbd.S)--,
--C(.dbd.S)NRL1a--, trans-CRL1b=CRL1b--, cis-CRL1b=CRL1b ,
C.ident.C , OC(RL1b)2-, --C(RL1b)2O--, --NRL1aC(RL1b)2--,
--C(RL1b)2NRL 1 a--, --SC(RL1b)2-, --C(RL1b)2S--, --S(.dbd.O)2O--,
--OS(.dbd.O)2--, --S(.dbd.O)2NRL1a--, --NRL1aS(.dbd.O)2-, or an
optionally substituted C1-20 hydrocarbon chain, optionally wherein
one or more carbon units of the hydrocarbon chain is replaced with
--O--, --S--, --NRL1a--, --C(.dbd.O)--, NRL1 aC(.dbd.O)--, --NRL1
aC(.dbd.O)O--, --C(.dbd.O)NRL 1 a--, --OC(.dbd.O)NRL 1 a--, --S
C(.dbd.O)--, --C(.dbd.O)S--, --OC(.dbd.O)--, --C(.dbd.O)O--,
--NRL1aC(.dbd.S)--, --C(.dbd.S)NRL1a--, trans-CRL1b=CRL1b--,
cis-CRL1b=CRL1b , C.ident.C , S(.dbd.O)20-, --OS(.dbd.O)2--,
--S(.dbd.O)2NRL1a--, or --NRL1aS(.dbd.O)2--, wherein RL1a is
hydrogen, optionally substituted C1-6 alkyl, or a nitrogen
protecting group, or RL1a is joined with the adjacent carbon atom
to form an optionally substituted heterocyclic ring, and wherein
each occurrence of RL1b is independently selected from the group
consisting of hydrogen, halogen, optionally substituted C1-10
alkyl, optionally substituted alkenyl, optionally substituted
alkynyl, optionally substituted carbocyclyl, optionally substituted
heterocyclyl, optionally substituted aryl, and optionally
substituted heteroaryl, or RL1b is joined with the adjacent carbon
or nitrogen or oxygen atom to form an optionally substituted
carbocyclic or heterocyclic ring, or two RL1b groups are joined to
form an optionally substituted carbocyclic or optionally
substituted heterocyclic ring; and L2 is a moiety derived from a
crosslinking reagent capable of crosslinking the carrier and
L1.
[0171] The carrier can be a protein, a lipid, a lipolized protein,
a virus, a peptide, or a dendrimer of glycopeptides. In certain
embodiments, the carrier is a peptide comprising a T cell
epitope.
[0172] Examples of carrier proteins which may be used in the
present invention are tetanus toxoid (TT), diphtheria toxoid (DT),
diphtheria toxin cross-reacting material 197 (CRM197), fragment C
of TT, Keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA),
protein D, outer-membrane protein (OMP) and pneumolysin, diphtheria
toxin cross-reacting material 197 (CRM197) or other DT point
mutants, such as CRM176, CRM228, CRM 45 (Uchida et al J. Biol.
Chem. 218; 3838-3844, 1973); CRM 9, CRM 45, CRM102, CRM 103 and
CRM107 and other mutations described in the art.
[0173] In certain embodiments, the glycan conjugate is of the
formula (IV-a) or (IV-b):
##STR00013##
[0174] wherein m is an integer of 1 to 40, inclusive.
[0175] In certain embodiments, m is an integer of 1 to 30,
inclusive. As generally defined herein, m is an integer of 1 to 20
inclusive. In certain embodiments, m is 1. In certain embodiments,
m is 2. In certain embodiments, m is 4. In certain embodiments, m
is 6. In certain embodiments, m is 8. In certain embodiments, m is
10. In certain embodiments, m is 15. In certain embodiments, m is
20. In certain embodiments, m is 30. In certain embodiments, m is
40.
[0176] In another aspect, the present invention provides a glycan
conjugate mixture comprising at least two of the glycan conjugates
as described herein. In certain embodiments, the average value of w
in the glycan mixture is from about 1.0 to about 40.0. In certain
embodiments, the average value of w in the glycan mixture is from
about 1.0 to 10.0. In certain embodiments, the average value of w
in the glycan mixture is about 5.7, 4.9, 2.9, 2.8, or 3.1. In
certain embodiments, the average value of w in the glycan mixture
is about 4.9, 2.9, 2.8, or 3.1.
[0177] In certain embodiments, the immunogenic compositions
described herein include an immunogenically effective amount of a
glycan conjugate of the invention. In certain embodiments, the
immunogenic composition includes a pharmaceutically effective
amount of the inventive glycan conjugate.
[0178] The compounds of the invention can be synthesized using
procedures described herein and also see US20140051127.
[0179] The immunogenic conjugate of the invention may include one
or more molecules (e.g., 1-40, 1-20, 1-25, 1-30,) of the same or
different SSEA3 and/or SSEA4 analogs and/or related derivatives.
Additional descriptions and related procedures for generating
glycan conjugates are described below. Also see U.S. Pat. No.
8,268,969. The contents of which is hereby incorporated by
reference.
[0180] In certain embodiments, the immunogenic composition of the
invention may include one or more adjuvants. Suitable adjuvants can
include, for example, C34, 7DW8-5, C17, C23, C-30,
alpha-galactoceramide, Gluco-C34, Aluminum salt, Squalene, MF59,
and QS-21).
[0181] As used herein, the term "alum adjuvant" refers to an
aluminum salt with immune adjuvant activity. This agent adsorbs and
precipitates protein antigens in solution; the resulting
precipitate improves vaccine immunogenicity by facilitating the
slow release of antigen from the vaccine depot formed at the site
of inoculation.
[0182] As used herein, the term "immunologic adjuvant" refers to a
substance used in conjunction with an immunogen which enhances or
modifies the immune response to the immunogen. The .alpha.-GalCer
analogs of the present disclosure are used as immunologic adjuvants
to modify or augment the effects of a vaccine by stimulating the
immune system of a patient who is administered the vaccine to
respond to the vaccine more vigorously. In an exemplary
implementation, the analog C34 is used as an adjuvant. The
structures of C34 and other alpha-galactosyl ceramide analogs and
their use as adjuvants are disclosed in detail in U.S. Pat. No.
7,928,077.
[0183] As used herein, the term "glycolipid" refers to a
carbohydrate-attached lipid that serves as a marker for cellular
recognition.
[0184] The glycolipids C34, Gluco-C34, C23 and 7DW8-5 have the
following structures:
##STR00014##
[0185] The immunogenic composition can further include a
pharmaceutically acceptable excipient. In certain embodiments, the
immunogenic compositions described herein include a
pharmaceutically effective amount of a glycan conjugate of the
invention.
[0186] In another aspect, the present invention provides a cancer
vaccine comprising an immunogenic composition described herein and
a pharmaceutically acceptable excipient.
[0187] The cancer vaccines of the invention may include a single
dose or multiple doses of the inventive glycan conjugates, a glycan
conjugate mixture thereof, or immunogenic compositions thereof. The
provided cancer vaccines may be useful for treating or reducing the
risk of cancers. The cancer vaccines may also include packaging
information describing the use or prescribing information for the
subject or a health care professional. Such information may be
required by a regulatory agency such as the U.S. Food and Drug
Administration (FDA). The cancer vaccine may also optionally
include a device for administration of the compound or composition,
for example, a syringe for parenteral administration.
[0188] Pharmaceutical Formulations
[0189] The immune composition is administered in a manner
compatible with the dosage formulation, and in an amount that is
therapeutically effective, protective and immunogenic. The quantity
to be administered depends on the subject to be treated, including,
for example, the capacity of the individual's immune system to
synthesize antibodies, and if needed, to produce a cell-mediated
immune response. Precise amounts of active ingredient required to
be administered depend on the judgment of the practitioner.
However, suitable dosage ranges are readily determinable by one
skilled in the art. Suitable regimes for initial administration and
booster doses are also variable, but may include an initial
administration followed by subsequent administrations. The dosage
of the vaccine may also depend on the route of administration and
varies according to the size of the host.
[0190] The immune composition of this invention can also be used to
generate antibodies in animals for production of antibodies, which
can be used in both cancer treatment and diagnosis. Methods of
making monoclonal and polyclonal antibodies and fragments thereof
in animals (e.g., mouse, rabbit, goat, sheep, or horse) are well
known in the art. See, for example, Harlow and Lane, (1988)
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New
York. The term "antibody" includes intact immunoglobulin molecules
as well as fragments thereof, such as Fab, F(ab')2, Fv, scFv
(single chain antibody), and dAb (domain antibody; Ward, et. al.
(1989) Nature, 341, 544).
[0191] The compositions disclosed herein can be included in a
pharmaceutical composition together with additional active agents,
carriers, vehicles, excipients, or auxiliary agents identifiable by
a person skilled in the art upon reading of the present
disclosure.
[0192] The pharmaceutical compositions preferably comprise at least
one pharmaceutically acceptable carrier. In such pharmaceutical
compositions, the compositions disclosed herein form the "active
compound," also referred to as the "active agent." As used herein
the language "pharmaceutically acceptable carrier" includes
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like,
compatible with pharmaceutical administration. Supplementary active
compounds can also be incorporated into the compositions. A
pharmaceutical composition is formulated to be compatible with its
intended route of administration. Examples of routes of
administration include parenteral, e.g., intravenous, intradermal,
subcutaneous, oral (e.g., inhalation), transdermal (topical),
transmucosal, and rectal administration. Solutions or suspensions
used for parenteral, intradermal, or subcutaneous application can
include the following components: a sterile diluent such as water
for injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol, or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates, or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable
syringes, or multiple dose vials made of glass or plastic.
[0193] Clinical Applications
[0194] The present invention provides glycan conjugates,
immunogenic compositions or vaccines useful for the treatment of a
proliferative disease such as cancer (e.g. lung cancer, large bowel
cancer, pancreas cancer, biliary tract cancer, or endometrial
cancer), benign neoplasm, or angiogenesis in a subject.
[0195] The immunogenic compositions or vaccines described herein
can also be used to generate antibodies in human or animals for
production of antibodies, which can be used in both cancer
treatment and diagnosis. In some embodiments, the immunogenic
compositions or vaccines described herein can also be used to
generate the production of GloboH, SSEA3 and/or SSEA4 antibodies.
Methods of making monoclonal and polyclonal antibodies and
fragments thereof in human and/or animals (e.g., mouse, rabbit,
goat, sheep, or horse) are well known in the art. See, for example,
Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York. The term "antibody" includes
intact immunoglobulin molecules as well as fragments thereof, such
as Fab, F(ab').sub.2, Fv, scFv (single chain antibody), and dAb
(domain antibody; Ward, et. al. (1989) Nature, 341, 544).
[0196] Compositions comprising at least one anti-SSEA3/SSEA4/GloboH
antibody or at least one polynucleotide comprising sequences
encoding an anti-SSEA3/SSEA4/GloboH antibody are provided. In
certain embodiments, a composition may be a pharmaceutical
composition. As used herein, compositions comprise one or more
antibodies that bind to one or more SSEA3/SSEA4/GloboH and/or one
or more polynucleotides comprising sequences encoding one or more
antibodies that bind to one or more SSEA3/SSEA4/GloboH. These
compositions may further comprise suitable carriers, such as
pharmaceutically acceptable excipients including buffers, which are
well known in the art.
[0197] Isolated antibodies and polynucleotides are also provided.
In certain embodiments, the isolated antibodies and polynucleotides
are substantially pure.
[0198] In one embodiment, anti-SSEA3/SSEA4/GloboH antibodies are
monoclonal. In another embodiment, fragments of the
anti-SSEA3/SSEA4/GloboH antibodies (e.g., Fab, Fab'-SH and F(ab!)2
fragments) are provided. These antibody fragments can be created by
traditional means, such as enzymatic digestion, or may be generated
by recombinant techniques. Such antibody fragments may be chimeric,
humanized, or human. These fragments are useful for the diagnostic
and therapeutic purposes set forth below.
[0199] Pharmaceutical Formulations
[0200] Therapeutic formulations comprising an pharmaceutical agents
of the invention are prepared for storage by mixing the antibody
having the desired degree of purity with optional physiologically
acceptable carriers, excipients or stabilizers (Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the
form of aqueous solutions, lyophilized or other dried formulations.
Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations employed, and include
buffers such as phosphate, citrate, histidine and other organic
acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0201] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
including, but not limited to those with complementary activities
that do not adversely affect each other. Such molecules are
suitably present in combination in amounts that are effective for
the purpose intended.
[0202] The active ingredients may also be entrapped in microcapsule
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsule and poly-(methylmethacylate) microcapsule,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
[0203] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0204] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the
immunoglobulin of the invention, which matrices are in the form of
shaped articles, e.g., films, or microcapsule. Examples of
sustained-release matrices include polyesters, hydrogels (for
example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated immunoglobulins remain
in the body for a long time, they may denature or aggregate as a
result of exposure to moisture at 37.degree. C., resulting in a
loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S--S bond formation through
thiol-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
[0205] Pharmaceutical compositions of the invention can be used to
treat, inhibit, delay progression of, prevent/delay recurrence of,
ameliorate, or prevent diseases, disorders or conditions associated
with abnormal expression and/or activity of SSEA3/SSEA4/GloboHs and
SSEA3/SSEA4/GloboH related proteins, including but not limited to
cancer, muscular disorders, ubiquitin-pathway-related genetic
disorders, immune/inflammatory disorders, neurological disorders,
and other ubiquitin pathway-related disorders.
[0206] In one aspect, a blocking antibody of the invention is
specific for a SSEA3/SSEA4/GloboH.
[0207] Pharmaceutical compositions of the invention can be used
either alone or in combination with other compositions in a
therapy. For instance, an antibody of the invention may be
co-administered with another antibody, and/or adjuvant/therapeutic
agents (e.g., steroids). For instance, an antibody of the invention
may be combined with an anti-inflammatory and/or antiseptic in a
treatment scheme, e.g. in treating any of the diseases described
herein, including cancer, muscular disorders,
ubiquitin-pathway-related genetic disorders, immune/inflammatory
disorders, neurological disorders, and other ubiquitin
pathway-related disorders. Such combined therapies noted above
include combined administration (where the two or more agents are
included in the same or separate formulations), and separate
administration, in which case, administration of the antibody of
the invention can occur prior to, and/or following, administration
of the adjunct therapy or therapies.
[0208] Pharmaceutical compositions of the invention (and adjunct
therapeutic agent) can be administered by any suitable means,
including parenteral, subcutaneous, intraperitoneal,
intrapulmonary, and intranasal, and, if desired for local
treatment, intralesional administration. Parenteral infusions
include intramuscular, intravenous, intraarterial, intraperitoneal,
or subcutaneous administration. In addition, the Pharmaceutical
composition can be suitably administered by pulse infusion,
particularly with declining doses of the antibody. Dosing can be by
any suitable route, e.g. by injections, such as intravenous or
subcutaneous injections, depending in part on whether the
administration is brief or chronic.
[0209] The location of the binding target of an antibody of the
invention may be taken into consideration in preparation and
administration of the antibody. When the binding target is an
intracellular molecule, certain embodiments of the invention
provide for the antibody or antigen-binding fragment thereof to be
introduced into the cell where the binding target is located. In
one embodiment, an antibody of the invention can be expressed
intracellularly as an intrabody. The term "intrabody," as used
herein, refers to an antibody or antigen-binding portion thereof
that is expressed intracellularly and that is capable of
selectively binding to a target molecule, as described in Marasco,
Gene Therapy 4: 11-15 (1997); Kontermann, Methods 34: 163-170
(2004); U.S. Pat. Nos. 6,004,940 and 6,329,173; U.S. Patent
Application Publication No. 2003/0104402, and PCT Publication No.
WO2003/077945. Intracellular expression of an intrabody is effected
by introducing a nucleic acid encoding the desired antibody or
antigen-binding portion thereof (lacking the wild-type leader
sequence and secretory signals normally associated with the gene
encoding that antibody or antigen-binding fragment) into a target
cell. Any standard method of introducing nucleic acids into a cell
may be used, including, but not limited to, microinjection,
ballistic injection, electroporation, calcium phosphate
precipitation, liposomes, and transfection with retroviral,
adenoviral, adeno-associated viral and vaccinia vectors carrying
the nucleic acid of interest.
[0210] Pharmaceutical compositions of the invention would be
formulated, dosed, and administered in a fashion consistent with
good medical practice. Factors for consideration in this context
include the particular disorder being treated, the particular
mammal being treated, the clinical condition of the individual
patient, the cause of the disorder, the site of delivery of the
agent, the method of administration, the scheduling of
administration, and other factors known to medical practitioners.
The antibody need not be, but is optionally formulated with one or
more agents currently used to prevent or treat the disorder in
question. The effective amount of such other agents depends on the
amount of antibodies of the invention present in the formulation,
the type of disorder or treatment, and other factors discussed
above. These are generally used in the same dosages and with
administration routes as described herein, or about from 1 to 99%
of the dosages described herein, or in any dosage and by any route
that is empirically/clinically determined to be appropriate.
[0211] For the prevention or treatment of disease, the appropriate
dosage of an Pharmaceutical compositions of the invention (when
used alone or in combination with other agents such as
chemotherapeutic agents) will depend on the type of disease to be
treated, the type of antibody, the severity and course of the
disease, whether the antibody is administered for preventive or
therapeutic purposes, previous therapy, the patient's clinical
history and response to the antibody, and the discretion of the
attending physician. The antibody is suitably administered to the
patient at one time or over a series of treatments. Depending on
the type and severity of the disease, about 1 .mu.g/kg to 15 mg/kg
(e.g. 0.1 mg/kg-10 mg/kg) of antibody can be an initial candidate
dosage for administration to the patient, whether, for example, by
one or more separate administrations, or by continuous infusion.
One typical daily dosage might range from about 1 .mu.g/kg to 100
mg/kg or more, depending on the factors mentioned above. For
repeated administrations over several days or longer, depending on
the condition, the treatment would generally be sustained until a
desired suppression of disease symptoms occurs. One exemplary
dosage of the antibody would be in the range from about 0.05 mg/kg
to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0
mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be
administered to the patient. Such doses may be administered
intermittently, e.g. every week or every three weeks (e.g. such
that the patient receives from about two to about twenty, or e.g.
about six doses of the antibody). An initial higher loading dose,
followed by one or more lower doses may be administered. An
exemplary dosing regimen comprises administering an initial loading
dose of about 4 mg/kg, followed by a weekly maintenance dose of
about 2 mg/kg of the antibody. However, other dosage regimens may
be useful. The progress of this therapy is easily monitored by
conventional techniques and assays.
[0212] Articles of Manufacture
[0213] In another aspect of the invention, an article of
manufacture containing materials useful for the treatment,
prevention and/or diagnosis of the disorders described above is
provided. The article of manufacture comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
etc. The containers may be formed from a variety of materials such
as glass or plastic. The container holds a composition which is by
itself or when combined with another composition effective for
treating, preventing and/or diagnosing the condition and may have a
sterile access port (for example the container may be an
intravenous solution bag or a vial having a stopper pierceable by a
hypodermic injection needle). At least one active agent in the
composition is an antibody of the invention. The label or package
insert indicates that the composition is used for treating the
condition of choice. Moreover, the article of manufacture may
comprise (a) a first container with a composition contained
therein, wherein the composition comprises an antibody of the
invention; and (b) a second container with a composition contained
therein, wherein the composition comprises a further cytotoxic or
otherwise therapeutic agent. The article of manufacture in this
embodiment of the invention may further comprise a package insert
indicating that the compositions can be used to treat a particular
condition. Alternatively, or additionally, the article of
manufacture may further comprise a second (or third) container
comprising a pharmaceutically-acceptable buffer, such as
bacteriostatic water for injection (BWFI), phosphate-buffered
saline, Ringer's solution and dextrose solution. It may further
include other materials desirable from a commercial and user
standpoint, including other buffers, diluents, filters, needles,
and syringes.
[0214] The following are examples of the methods and compositions
of the invention. It is understood that various other embodiments
may be practiced, given the general description provided above.
[0215] In some embodiments, the provided glycan conjugates,
immunogenic compositions or vaccines are useful in treating, or
diagnosing a cancer, including, but are not limited to, acoustic
neuroma, adenocarcinoma, adrenal gland cancer, anal cancer,
angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma,
hemangiosarcoma), appendix cancer, benign monoclonal gammopathy,
biliary cancer (e.g., cholangiocarcinoma), bladder cancer, breast
cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of
the breast, mammary cancer, medullary carcinoma of the breast),
brain cancer (e.g., meningioma; glioma, e.g., astrocytoma,
oligodendroglioma; medulloblastoma), bronchus cancer, carcinoid
tumor, cervical cancer (e.g., cervical adenocarcinoma),
choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer
(e.g., colon cancer, rectal cancer, colorectal adenocarcinoma),
epithelial carcinoma, ependymoma, endotheliosarcoma (e.g., Kaposi's
sarcoma, multiple idiopathic hemorrhagic sarcoma), endometrial
cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer
(e.g., adenocarcinoma of the esophagus, Barrett's adenocarinoma),
Ewing sarcoma, eye cancer (e.g., intraocular melanoma,
retinoblastoma), familiar hypereosinophilia, gall bladder cancer,
gastric cancer (e.g., stomach adenocarcinoma), gastrointestinal
stromal tumor (GIST), head and neck cancer (e.g., head and neck
squamous cell carcinoma, oral cancer (e.g., oral squamous cell
carcinoma (OSCC), throat cancer (e.g., laryngeal cancer, pharyngeal
cancer, nasopharyngeal cancer, oropharyngeal cancer)),
hematopoietic cancers (e.g., leukemia such as acute lymphocytic
leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic
leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic
leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic
lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma
such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and
non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large
cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)),
follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic
lymphoma (CLL/SLL), mantle cell lymphoma (MCL), marginal zone
B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT)
lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal
zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt
lymphoma, lymphoplasmacytic lymphoma (i.e., "Waldenstrom's
macroglobulinemia"), hairy cell leukemia (HCL), immunoblastic large
cell lymphoma, precursor B-lymphoblastic lymphoma and primary
central nervous system (CNS) lymphoma; and T-cell NHL such as
precursor T-lymphoblastic lymphoma/leukemia, peripheral T-cell
lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g.,
mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell
lymphoma, extranodal natural killer T-cell lymphoma, enteropathy
type T-cell lymphoma, subcutaneous panniculitis-like T-cell
lymphoma, anaplastic large cell lymphoma); a mixture of one or more
leukemia/lymphoma as described above; and multiple myeloma (MM)),
heavy chain disease (e.g., alpha chain disease, gamma chain
disease, mu chain disease), hemangioblastoma, inflammatory
myofibroblastic tumors, immunocytic amyloidosis, kidney cancer
(e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma),
liver cancer (e.g., hepatocellular cancer (HCC), malignant
hepatoma), lung cancer (e.g., bronchogenic carcinoma, small cell
lung cancer (SCLC), non-small cell lung cancer (NSCLC),
adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis
(e.g., systemic mastocytosis), myelodysplastic syndrome (MDS),
mesothelioma, myeloproliferative disorder (MPD) (e.g., polycythemia
Vera (PV), essential thrombocytosis (ET), agnogenic myeloid
metaplasia (AMM), a.k.a. myelofibrosis (MF), chronic idiopathic
myelofibrosis, chronic myelocytic leukemia (CML), chronic
neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)),
neuroblastoma, neurofibroma (e.g., neurofibromatosis (NF) type 1 or
type 2, schwannomatosis), neuroendocrine cancer (e.g.,
gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid
tumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma,
ovarian embryonal carcinoma, ovarian adenocarcinoma), papillary
adenocarcinoma, pancreatic cancer (e.g., pancreatic
andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN),
islet cell tumors), penile cancer (e.g., Paget's disease of the
penis and scrotum), pinealoma, primitive neuroectodermal tumor
(PNT), prostate cancer (e.g., prostate adenocarcinoma), rectal
cancer, rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g.,
squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma,
basal cell carcinoma (BCC)), small bowel cancer (e.g., appendix
cancer), soft tissue sarcoma (e.g., malignant fibrous histiocytoma
(MFH), liposarcoma, malignant peripheral nerve sheath tumor
(MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous
gland carcinoma, sweat gland carcinoma, synovioma, testicular
cancer (e.g., seminoma, testicular embryonal carcinoma), thyroid
cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid
carcinoma (PTC), medullary thyroid cancer), urethral cancer,
vaginal cancer and vulvar cancer (e.g., Paget's disease of the
vulva). In certain embodiments, the provided glycan conjugates,
immunogenic compositions or vaccines are useful for treating brain
cancer, lung cancer, breast cancer, oral cancer, esophagus cancer,
stomach cancer, liver cancer, bile duct cancer, pancreas cancer,
colon cancer, kidney cancer, bone cancer, skin cancer, cervix
cancer, ovary cancer, and prostate cancer.
[0216] To perform the treatment methods described herein, an
effective amount of any of the glycan conjugates or immunogenic
compositions or vaccines described herein may be administered to a
subject in need of the treatment via a suitable route, as described
above. The subject, such as a human subject, can be a patient
having cancer, suspected of having cancer, or susceptible to
cancer. The amount of the glycan conjugate or immunogenic
composition administered to the subject may be effective in
eliciting immune responses specific to the glycan moiety in the
conjugate or composition. In some embodiments, the amount of the
glycan conjugate or immunogenic composition is sufficient to elicit
immune responses leading to the inhibition of cancer growth and/or
reduction of tumor mass. In other embodiments, the amount of the
glycan conjugate or immunogenic composition may be effective in
delaying the onset of the target cancer or reducing the risk for
developing the cancer. The exact amount of the provided glycan
conjugates, immunogenic compositions or vaccines required to
achieve an effective amount will vary from subject to subject,
depending, for example, on species, age, and general condition of a
subject, severity of the side effects or disorder, identity of the
particular compound(s), mode of administration, and the like. The
desired dosage can be delivered three times a day, two times a day,
once a day, every other day, every third day, every week, every two
weeks, every three weeks, or every four weeks. In certain
embodiments, the desired dosage can be delivered using multiple
administrations (e.g., two, three, four, five, six, seven, eight,
nine, ten, eleven, twelve, thirteen, fourteen, or more
administrations).
[0217] In certain embodiments, an effective amount of the provided
glycan conjugates, immunogenic compositions or vaccines for
administration one or more times a day to a 70 kg adult human may
comprise about 0.0001 mg to about 3000 mg, about 0.0001 mg to about
2000 mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about
1000 mg, about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000
mg, about 1 mg to about 1000 mg, about 1 mg to about 100 mg, about
10 mg to about 1000 mg, or about 100 mg to about 1000 mg, of a
compound per unit dosage form.
[0218] In certain embodiments, the provided glycan conjugates,
immunogenic compositions or vaccines may be administered orally or
parenterally at dosage levels sufficient to deliver from about
0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50
mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg,
preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01
mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg,
and more preferably from about 1 mg/kg to about 25 mg/kg, of
subject body weight per day, one or more times a day, to obtain the
desired therapeutic effect.
[0219] It will be appreciated that dose ranges as described herein
provide guidance for the administration of the provided glycan
conjugates, immunogenic compositions or vaccines to an adult. The
amount to be administered to, for example, a child or an adolescent
can be determined by a medical practitioner or person skilled in
the art and can be lower or the same as that administered to an
adult.
[0220] It will be also appreciated that the provided glycan
conjugates, immunogenic compositions or vaccines can be
administered in combination with one or more additional
therapeutically active agents. The provided glycan conjugates,
immunogenic compositions or vaccines can be administered in
combination with additional therapeutically active agents that
improve their bioavailability, reduce and/or modify their
metabolism, inhibit their excretion, and/or modify their
distribution within the body. It will also be appreciated that the
therapy employed may achieve a desired effect for the same
disorder, and/or it may achieve different effects.
[0221] The provided glycan conjugates, immunogenic compositions or
vaccines can be administered concurrently with, prior to, or
subsequent to, one or more additional therapeutically active
agents. In general, each agent will be administered at a dose
and/or on a time schedule determined for that agent. In will
further be appreciated that the additional therapeutically active
agent utilized in this combination can be administered together in
a single composition or administered separately in different
compositions. The particular combination to employ in a regimen
will take into account compatibility of the inventive compound with
the additional therapeutically active agent and/or the desired
therapeutic effect to be achieved. In general, it is expected that
additional therapeutically active agents utilized in combination be
utilized at levels that do not exceed the levels at which they are
utilized individually. In some embodiments, the levels utilized in
combination will be lower than those utilized individually.
[0222] In certain embodiments, the provided glycan conjugate,
immunogenic composition or vaccine is administered in combination
with one or more additional pharmaceutical agents described herein.
In certain embodiments, the additional pharmaceutical agent is an
anti-cancer agent. Anti-cancer agents encompass biotherapeutic
anti-cancer agents as well as chemotherapeutic agents.
[0223] Exemplary biotherapeutic anti-cancer agents include, but are
not limited to, interferons, cytokines (e.g., tumor necrosis
factor, interferon a, interferon y), vaccines, hematopoietic growth
factors, monoclonal serotherapy, immunostimulants and/or
immunodulatory agents (e.g., IL-1, 2, 4, 6, or 12), immune cell
growth factors (e.g., GM-CSF) and antibodies (e.g. Herceptin
(trastuzumab), T-DM1, AVASTIN (bevacizumab), ERBITUX (cetuximab),
Vectibix (panitumumab), Rittman (rituximab), Bexxar
(tositumomab)).
[0224] Exemplary chemotherapeutic agents include, but are not
limited to, anti-estrogens (e.g. tamoxifen, raloxifene, and
megestrol), LHRH agonists (e.g. goscrclin and leuprolide),
anti-androgens (e.g. flutamide and bicalutamide), photodynamic
therapies (e.g. vertoporfin (BPD-MA), phthalocyanine,
photosensitizer Pc4, and demethoxy-hypocrellin A (2BA-2-DMHA)),
nitrogen mustards (e.g. cyclophosphamide, ifosfamide, trofosfamide,
chlorambucil, estramustine, and melphalan), nitrosoureas (e.g.
carmustine (BCNU) and lomustine (CCNU)), alkylsulphonates (e.g.
busulfan and treosulfan), triazenes (e.g. dacarbazine,
temozolomide), platinum containing compounds (e.g. cisplatin,
carboplatin, oxaliplatin), vinca alkaloids (e.g. vincristine,
vinblastine, vindesine, and vinorelbine), taxoids (e.g. paclitaxel
or a paclitaxel equivalent such as nanoparticle albumin-bound
paclitaxel (Abraxane), docosahexaenoic acid bound-paclitaxel
(DHA-paclitaxel, Taxoprexin), polyglutamate bound-paclitaxel
(PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX), the
tumor-activated prodrug (TAP) ANG1005 (Angiopep-2 bound to three
molecules of paclitaxel), paclitaxel-EC-1 (paclitaxel bound to the
erbB2-recognizing peptide EC-1), and glucose-conjugated paclitaxel,
e.g., 2'-paclitaxel methyl 2-glucopyranosyl succinate; docetaxel,
taxol), epipodophyllins (e.g. etoposide, etoposide phosphate,
teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan,
irinotecan, crisnatol, mytomycin C), anti-metabolites, DHFR
inhibitors (e.g. methotrexate, dichloromethotrexate, trimetrexate,
edatrexate), IMP dehydrogenase inhibitors (e.g. mycophenolic acid,
tiazofurin, ribavirin, and EICAR), ribonuclotide reductase
inhibitors (e.g. hydroxyurea and deferoxamine), uracil analogs
(e.g. 5-fluorouracil (5-FU), floxuridine, doxifluridine,
ratitrexed, tegafur-uracil, capecitabine), cytosine analogs (e.g.
cytarabine (ara C), cytosine arabinoside, and fludarabine), purine
analogs (e.g. mercaptopurine and Thioguanine), Vitamin D3 analogs
(e.g. EB 1089, CB 1093, and KH 1060), isoprenylation inhibitors
(e.g. lovastatin), dopaminergic neurotoxins (e.g.
1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g.
staurosporine), actinomycin (e.g. actinomycin D, dactinomycin),
bleomycin (e.g. bleomycin A2, bleomycin B2, peplomycin),
anthracycline (e.g. daunorubicin, doxorubicin, pegylated liposomal
doxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin,
mitoxantrone), MDR inhibitors (e.g. verapamil), Ca2+ ATPase
inhibitors (e.g. thapsigargin), imatinib, thalidomide,
lenalidomide, tyrosine kinase inhibitors (e.g., axitinib
(AG013736), bosutinib (SKI-606), cediranib (RECENTIN.TM., AZD2171),
dasatinib (SPRYCEL.RTM., BMS-354825), erlotinib (TARCEVA.RTM.),
gefitinib (IRESSA.RTM.), imatinib (Gleevec.RTM., CGP57148B,
STI-571), lapatinib (TYKERB.RTM., TYVERB.RTM.), lestaurtinib
(CEP-701), neratinib (HKI-272), nilotinib (TASIGNA.RTM.), semaxanib
(semaxinib, SU5416), sunitinib (SUTENT.RTM., SU11248), toceranib
(PALLADIA.RTM.), vandetanib (ZACTIMA.RTM., ZD6474), vatalanib
(PTK787, PTK/ZK), trastuzumab (HERCEPTIN.RTM.), bevacizumab
(AVASTIN.RTM.), rituximab (RITUXAN.RTM.), cetuximab (ERBITUX.RTM.),
panitumumab (VECTIBIX.RTM.), ranibizumab (Lucentis.RTM.), nilotinib
(TASIGNA.RTM.), sorafenib (NEXAVAR.RTM.), everolimus
(AFINITOR.RTM.), alemtuzumab (CAMPATH.RTM.), gemtuzumab ozogamicin
(MYLOTARG.RTM.), temsirolimus (TORISEL.RTM.), ENMD-2076, PCI-32765,
AC220, dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOKTM),
SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869,
MP470, BIBF 1120 (VARGATEF.RTM.), AP24534, JNJ-26483327, MGCD265,
DCC-2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930,
MM-121, XL-184, XL-647, and/or XL228), proteasome inhibitors (e.g.,
bortezomib (Velcade)), mTOR inhibitors (e.g., rapamycin,
temsirolimus (CCI-779), everolimus (RAD-001), ridaforolimus,
AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235 (Novartis), BGT226
(Norvartis), XL765 (Sanofi Aventis), PF-4691502 (Pfizer), GDC0980
(Genetech), SF1126 (Semafoe) and OSI-027 (OSI)), oblimersen,
gemcitabine, carminomycin, leucovorin, pemetrexed,
cyclophosphamide, dacarbazine, procarbizine, prednisolone,
dexamethasone, campathecin, plicamycin, asparaginase, aminopterin,
methopterin, porfiromycin, melphalan, leurosidine, leurosine,
chlorambucil, trabectedin, procarbazine, discodermolide,
carminomycin, aminopterin, and hexamethyl melamine.
[0225] In certain embodiments, the subject being treated is a
mammal. In certain embodiments, the subject is a human. In certain
embodiments, the subject is a domesticated animal, such as a dog,
cat, cow, pig, horse, sheep, or goat. In certain embodiments, the
subject is a companion animal such as a dog or cat. In certain
embodiments, the subject is a livestock animal such as a cow, pig,
horse, sheep, or goat. In certain embodiments, the subject is a zoo
animal. In another embodiment, the subject is a research animal
such as a rodent, dog, or non-human primate. In certain
embodiments, the subject is a non-human transgenic animal such as a
transgenic mouse or transgenic pig.
EXAMPLES
[0226] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Exemplary Syntheses of SSEA3 Analogues
[0227] A: Chemo-enzymatic synthesis of SSEA3 analog-NH2
##STR00015##
[0228] The combined compounds Gb4 analog, ATP, UTP, galactose
analog, phosphoenolpyruvate, MgCl.sub.2 with enzymes galactokinase
(GalK), UDP-sugar pyrophosphorylase (AtUSP),
beta-1,3-galactosyltransferase (LgtD,), pyruvate kinase (PK), and
inorganic pyrophosphatase (PPA) in the solution, and the reaction
was initiated at room temperature with the pH controlled at 7.0,
and the reaction was monitored by TLC until no more product could
be observed. After completion of the reaction, the proteins in the
reaction mixture were removed by heating for 30 min followed by
centrifugation and filtration with 0.22 .quadrature.M filter. The
filtrate was then purified by C-18 gel chromatography. Fractions
were collected and monitored by TLC.
Example 2
Exemplary Syntheses of SSEA4 Analogues
[0229] A : Chemical Synthesis of SSEA4-Gc-NH2
##STR00016## ##STR00017##
[0230] Powdered molecular sieves (4 A, 0.5 g) was added to a
solution of acceptor 3 (93 mg, 0.045 mmol) and imidates 6 (76 mg,
0.068 mmol) in 6 mL of dichloromethane (CH2C12). The mixture was
stirred at room temperature for 2 hrs. After cooled to -10.degree.
C., TMSOTf (5 .mu.L, 0.03 mmol) was added, and the mixture was
stirred at 5.degree. C. (cold room) overnight. The reaction mixture
was quenched by the addition of triethylamine (0.5 mL), diluted
with CH.sub.2Cl.sub.2 and filtered through a pad of celite. The
filtrate was washed with saturated sodium bicarbonate (NaHCO.sub.3)
aqueous solution, dried over sodium sulfate (Na.sub.2SO.sub.4),
filtered, and concentrated. The residue was purified by flash
silica gelchromatography (50-100% EtOAc in Hexane) to afford
hexasaccharide 7 contaminated with impurities from disaccharide
imidates 6. The yield was determined by NMR (90 mg, 68%).
[0231] Zinc dust (1g) was added to a solution of hexasaccharide 7
(90 mg, 0.03 mmol) in glacial acetic acid (5.0 mL) and the mixture
was stirred for 1-2 hrs, until compound 7 was consumed by TLC
analysis. The reaction mixture was diluted with CH2C12, filtered
through a pad of celite, and concentrated under reduced pressure.
The residue was dissolved in pyridine/Ac.sub.2O (1:1, 2.0 mL),
stirred for 1 h, and concentrated. The residue was purified by
flash silica get chromatography. The acylated material was
dissolved in anhydrous CH.sub.2Cl.sub.2 and MeOH (2:8, 10 mL) and
treated with NaOMe (45 mg). After stirring at room temperature for
4 hrs, water (0.2 mL) was added, and the resulting mixture was
stirred for 16 hrs. The reaction mixture was neutralized with
amberlyst IR-120, filtered, and concentrated. The residue was
purified by reverse phase chromatography (RP-18).
[0232] Palladium hydroxide (20% in Charcoal, 50 mg) was added to
the adduct in a mixture of methanol/water/ Acetic acid (10:10:0.5,
6 mL) and the reaction mixture was stirred at room temperature
under a positive pressure of hydrogen for 16 hrs. The reaction
mixture was filtered through a pad of celite and concentrated. The
residue was purified by reverse phase chromatography to afford 8
(17 mg, 43%).
[0233] B: Chemoenzymatic Synthesis of SSEA4 analog-NH2
##STR00018##
[0234] SSEA4 analogs-NH2 were synthesized via enzymatic
regeneration strategy as described in Scheme 3. In this system,
ManNAc derivatives were reacted with pyruvate and transformed into
Neu5Ac analogs by aldolase catalysis, followed by incorporation
with Gb5-NH2 in the regeneration system (J. Am. Chem. Soc. 2013,
135, 14831-14839) to obtain the exemplary SSEA4 analogs-NH2.
[0235] Detail of the reaction condition is described as follows:
Gb5-NH2 (18 .mu.mol), CTP (5 .mu.mol), ManNAc derivative (27
.mu.mol), sodium pyruvate (81 .mu.mol), PEP (55 .mu.mol), and ATP
(5 .mu.mol), were dissolved in 50 mM Tris-HCl buffer (pH 8.0).
Enzymes alpha-(2,3)-sialyltransferase (20 units), sialic acid
aldolase(20 units) CMK (10 units), Pykf (10 units), PPA (10 units),
and Pmcss (10 units) were added to the solution, and the reaction
was incubated at 37.degree. C. for 8 hours and monitored by TLC
plate. At the end of reaction, enzyme was denatured by heating at
100.degree. C. for 5 minutes. The desired SSEA4 analog-NH2 was
purified by G25, DEAE, and SP column (80%).
[0236] .sup.1H NMR of SSEA4analogs-NH2
[0237] B-1. SSEA4-pentylamine (RN=NHAc, R10=OH)
[0238] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 4.94 (d, J=3.8 Hz,
1H), 4.72 (d, J=8.5 Hz, 1H), 4.54-4.50 (m, 3H), 4.40 (t, J=6.4 Hz,
1H), 4.27 (d, J=2.0 Hz, 1H), 4.20 (d, J=2.8 Hz, 1H), 4.10-3.54 (m,
37 H), 3.34-3.31 (m, 1H), 3.02 (t, J=7.6 Hz, 2H), 2.78 (dd, J=12.4,
4.6 Hz, 1H), 2.05 (m, 6H), 1.80 (t, 12.2 Hz, 1H), 1.74-1.67 (m,
4H), 1.51-1.45 (m, 2H)
[0239] B-2. Neu5Gc_SSEA4-pentylamine (RN=NHGc, R10=OH)
[0240] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 4.89 (d, J=3.6 Hz,
1H), 4.66 (d, J=8.2 Hz, 1H), 4.52-4.45 (m, 3H), 4.37 (t, J=6.8 Hz,
1H), 4.23 (d, J=3.2 Hz, 1H), 4.15 (d, J=2.8 Hz, 1H), 4.10-3.48 (m,
35 H), 3.27 (m, 1H), 2.98 (t, J=7.6 Hz, 2H), 2.73 (dd, J=4.8, 12.4
Hz, 1H), 2.00 (s, 3H), 1.77 (t, J=12.0 Hz, 1H), 1.72-1.61 (m, 4H),
1.48-1.39 (m, 2 H).
[0241] B-3. Ac-Alkynyl_SSEA4-pentylamine
(RN=NHCOC.sub.2H.sub.4C.ident.CH, R10=OH)
[0242] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 4.89 (d, J=4.0 Hz,
1H), 4.67 (d, J=8.4 Hz, 1H), 4.52-4.45 (m, 3H), 4.37 (t, J=6.4 Hz,
1H), 4.23 (d, J=2.4 Hz, 1H), 4.08-3.54 (m, 38 H), 3.28 (m, 1H),
2.99 (t, J=7.6 Hz, 2H), 2.53-2.4 (m, 4H), 2.37 (s, 1H), 2.01 (s,
3H), 1.77 (t, J=12.0 Hz, 1H), 1.72-1.62 (m, 4H), 1.49-1.41 (m, 2
H).
[0243] B-4. Ac-Fluoride_SSEA4-pentylamine (RN=NHCOCH.sub.2F,
R10=OH)
[0244] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 4.90 (d, J=46.4 Hz,
2H), 4.90 (d, J=4.0 Hz, 1 H), 4.67 (d, J=8.8 Hz, 1H), 4.53-4.46 (m,
3H), 4.37 (t, J=6.8 Hz, 1 H), 4.24 (d, J=2.8 Hz, 2H), 4.16 (d,
J=3.2 Hz, 1 H), 4.09-3.51 (m, 34H), 3.28 (m, 1H), 2.99 (t, J=7.2
Hz, 1H), 2.75 (dd, J=4.8, 12.4 Hz, 1H), 2.01 (s, 3H), 1.79 (t,
J=12.0 Hz, 1H), 1.72-1.62 (m, 4H), 1.48-1.40 (m, 2 H).
[0245] B-5. Ac-Phenyl _SSEA4-pentylamine (RN=NHCOCH.sub.2Ph,
R10=OH)
[0246] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 7.39-7.30 (m, 5H),
4.90 (d, J=4.0 Hz, 1H), 4.66 (d, J=8.4 Hz, 1H), 4.52-4.46 (m, 3H),
4.37 (t, J=6.8 Hz, 1H), 4.23 (d, J=2.8 Hz, 1H), 4.15 (d, J=3.2 Hz,
1H), 4.08-3.47 (m, 38H), 3.36(dd, J=1.6, 9.2 Hz, 1H), 3.28 (m, 1H),
2.99 (t, J=7.6 Hz, 2H), 2.73 (dd, J=4.8, 12.4 Hz, 1H), 2.00 (s,
3H), 1.76 (t, J=12.0 Hz, 1H), 1.72-1.61 (m, 4H), 1.51-1.40 (m, 2
H).
[0247] B-6. Ac-Azido_SSEA4-pentylamine (RN=NHCOCH.sub.2N.sub.3,
R10=OH)
[0248] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 4.88 (d, J=3.6 Hz,
1H), 4.66 (d, J=8.4 Hz, 1H), 4.52-4.44 (m, 3H), 4.36 (t, J=6.4 Hz,
1H), 4.23 (d, J=2.4 Hz, 1H), 4.08-3.54 (m, 35 H), 3.27 (m, 1H),
2.98 (t, J=7.2 Hz, 2H), 2.73 (dd, J=4.8, 12.4 Hz, 1H), 2.00 (s,
3H), 1.77 (t, J=12.4 Hz, 1H), 1.72-1.60 (m, 4H), 1.48-1.39 (m, 2
H).
[0249] B-7. 5'-Azido_SSEA4-pentylamine (RN=N.sub.3, R10=OH)
[0250] .sup.1H NMR (400 MHz, D.sub.2O): .delta. 4.90 (d, J=3.6 Hz,
1H), 4.67 (d, J=8.4 Hz, 1H), 4.51-4.47 (m, 3H), 4.37 (t, J=6.4 Hz,
1H), 4.23 (d, J=2.8 Hz, 1H), 4.15 (d, J=3.2 Hz, 1H), 4.08-3.44 (m,
35H), 3.31-3.27 (m, 1H), 2.99 (t, J=7.2 Hz, 1H), 2.73 (dd, J=4.8,
12.4 Hz, 1H), 2.01 (s, 3H), 1.76 (t, J=12.0 Hz, 1H), 1.72-1.63 (3,
4H), 1.48-1.41 (m, 2H); HRMS (ESI-TOF, M-H-)
C.sub.46H.sub.78N.sub.5O.sub.33- calcd for 1228.4579, found
1228.4621.
[0251] B-8. 9'-Azido_SSEA4-pentylamine (RN=NHAc, R10=N.sub.3)
[0252] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 4.85 (d, J=3.8 Hz,
1H), 4.67 (d, J=8.4 Hz, 1H), 4.51-4.44 (m, 3H), 4.37 (t, J=6.4 Hz,
1H), 4.23 (d, J=2.8 Hz, 1H), 4.10-3.40 (m, 33 H), 3.27 (m, 1H),
2.98 (t, J=7.6 Hz, 2H), 2.72 (dd, J=4.8, 12.8 Hz, 1H), 2.00 (s,
3H), 2.00 (s, 3H), 1.75 (t, J=12.4 Hz, 1H), 1.72-1.60 (m, 4H),
1.58-1.38 (m, 2 H).
[0253] B-9. NHBz_SSEA4-pentylamine (RN=NHBz, R10=OH)
[0254] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 7.80-7.73 (m, 2H),
7.63 (m, 1H), 7.56-7.51 (m, 2H), 4.92 (d, J=4.0 Hz, 1H), 4.70 (d,
J=8.4 Hz, 1H), 4.58-4.47 (m, 3H), 4.40 (t, J=6.4 Hz, 1H), 4.26 (d,
J=2.8 Hz, 1H), 4.19 (d, J=3.2 Hz, 1H), 4.15-3.53 (m, 36H), 3.31 (m,
1H), 3.01 (t, J=7.6 Hz, 2H), 2.82 (dd, J=4.4, 12.4 Hz, 1H), 2.00
(s, 3H), 1.87 (t, J=12.0 Hz, 1H), 1.72-1.60 (m, 4H), 1.48-1.39 (m,
2 H).
[0255] C: Cross-Linking Reaction for SSEA4 Analog-SH
##STR00019##
[0256] In certain embodiments, DTSSP (2.0 eq) and SSEA4 analog-NH2
(1.0 eq) was mixed in 0.1 M phosphate buffer, pH 7.4 (.about.3
mg/ml). The solution was stirred at room temperature for overnight.
Then the reaction mixture was warmed to 40.degree. C. and added
with DTT (9.0 eq). After stirring for 1.5 hrs at 40.degree. C., the
reaction mixture was concentrated in vacuo, and the residue was
purified by LH-20 column to afford a white solid SSEA4 analogs-SH.
(Scheme 4)
[0257] .sup.1H NMR of SSEA4 analogs-SH
[0258] C-1: SSEA4-SH (RN=NHAc, R10=OH)
[0259] .sup.1H NMR (400 Hz, D.sub.2O) .delta. 4.88 (d, J=4.0 Hz,
1H), 4.65 (d, J=8.5 Hz, 1H), 4.50-4.44 (m, 3H), 4.36 (t, J=6.5 Hz,
1H), 4.22 (d, J=2.9 Hz, 1H), 4.14 (d, J=3.1 Hz, 1H), 4.04-3.55 (m,
35H), 3.26 (t, J=8.5 Hz, 1H), 3.18 (t, J=6.8 Hz, 2H), 2.74-2.70 (m,
3H), 2.49 (t, J=6.8 Hz, 2H), 1.994 (s, 3H), 1.992 (s, 3H), 1.75 (t,
J=12.2 Hz, 1H), 1.61 (tt, J=6.7, 6.7 HZ, 2H), 1.52 (tt, J=7.1, 7.1
Hz, 2H), 1.40-1.36 (m, 2H);
[0260] C-2: Neu5Gc_SSEA4-SH (RN=NHGc, R10=OH)
[0261] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 4.89 (d, J=3.9 Hz,
1H), 4.66 (d, J=8.5 Hz, 1H), 4.53- 4.43 (m, 3H), 4.36 (t, J=6.5 Hz,
1H), 4.22 (d, J=3.0 Hz, 1H), 4.15 (d, J=3.1 Hz, 1H), 4.11-3.48 (m,
38H), 3.27 (t, J=8.4 Hz, 1H), 3.19 (t, J=6.7 Hz, 2H), 2.78-2.71 (m,
3H), 2.51 (t, J=6.7 Hz, 2H), 2.00 (s, 3H), 1.78 (t, J=12.1 Hz, 1H),
1.61 (q, J=7.1 Hz, 2H), 1.52 (q, J=7.1 Hz, 2H), 1.39 (q, J=8.0 Hz,
2H).
[0262] C-3: Ac-Alkynyl_SSEA4-SH (RN=NHCOC.sub.2H.sub.4CCH,
R10=OH)
[0263] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 4.94 (d, J=3.9 Hz,
1H), 4.72 (d, J=8.4 Hz, 1H), 4.58- 4.48 (m, 3H), 4.41 (t, J=6.5 Hz,
1H), 4.30-4.26 (m, 1H), 4.21 (d, J=3.1 Hz, 1H), 4.14 -3.54 (m,
37H), 3.32 (t, J=8.6 Hz, 1H), 3.24 (t, J=6.8 Hz, 2H), 2.83-2.74 (m,
3H), 2.59-2.49 (m, 5H), 2.43 (s, 1H), 2.06 (s, 3H), 1.82 (t, J=12.1
Hz, 1H), 1.67 (p, J=6.9 Hz, 2H), 1.58 (p, J=6.9 Hz, 2H), 1.48-1.38
(m, 2H).
[0264] C-4: Ac-Fluoride_SSEA4-SH (RN=NHCOCH.sub.2F, R10=OH)
[0265] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 4.90 (d, J=46.4 Hz,
2H), 4.95 (d, J=4.0 Hz, 1H), 4.72 (d, J=8.5 Hz, 1H), 4.59-4.48 (m,
3H), 4.41 (t, J=6.6 Hz, 1H), 4.31- 4.26 (m, 1H), 4.23 -4.18 (m,
1H), 4.14-3.54 (m, 36H), 3.36-3.29 (m, 1H), 3.25 (t, J=6.8 Hz, 2H),
2.80 (m, 3H), 2.57 (t, J=6.7 Hz, 2H), 2.06 (s, 3H), 1.84 (t, J=12.2
Hz, 1H), 1.67 (p, J=6.9 Hz, 2H), 1.58 (p, J=7.0 Hz, 2H), 1.43 (q,
J=8.3 Hz, 2H).
[0266] C-5: Ac-Phenyl_SSEA4-SH (RN=NHCOCH.sub.2Ph, R10=OH)
[0267] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 7.48-7.32 (m, 5H),
4.94 (d, J=3.6 Hz, 1H), 4.73-4.68 (d, J=8.4 Hz, 1H), 4.52 (m, 3H),
4.41 (t, J=6.4 Hz, 1H), 4.29-4.26 (m, 1H), 4.20 (d, J=3.0 Hz, 1H),
4.13-3.51 (m, 37H), 3.39 (dd, J=9.0, 1.8 Hz, 1H), 3.32 (t, J=8.6
Hz, 1H), 3.25 (t, J=6.7 Hz, 2H), 2.83-2.74 (m, 3H), 2.56 (t, J=6.7
Hz, 2H), 2.04 (s, 3H), 1.80 (t, J=12.1 Hz, 1H), 1.67 (q, J=7.2 Hz,
2H), 1.57 (q, J=7.1 Hz, 2H), 1.48-1.38 (m, 2H).
[0268] C-6: Ac-Azido_SSEA4-SH (RN=NHCOCH.sub.2N.sub.3, R10=OH)
[0269] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 4.88 (d, J=3.9 Hz,
1H), 4.66 (d, J=8.5 Hz, 1H), 4.52- 4.43 (m, 3H), 4.36 (t, J=6.5 Hz,
1H), 4.22 (d, J=3.1 Hz, 1H), 4.14 (d, J=3.1 Hz, 1H), 4.08-3.47 (m,
38H), 3.26 (t, J=8.4 Hz, 1H), 3.19 (t, J=6.8 Hz, 2H), 2.74 (m, 3H),
2.51 (t, J=6.7 Hz, 2H), 2.00 (s, 3H), 1.76 (t, J=12.1 Hz, 1H), 1.61
(q, J=7.1 Hz, 2H), 1.53 (p, J=7.0 Hz, 2H), 1.38 (q, J=8.3 Hz,
2H).
[0270] C-7: 5'-Azido_SSEA4-SH (RN=N.sub.3, R10=OH)
[0271] .sup.1H NMR (400 Hz, D.sub.2O) .delta. 4.90 (d, J=4.0 Hz,
1H), 4.67 (d, J=8.4 Hz, 1H), 4.51-4.46 (m, 3H), 4.37 (t, J=6.4 Hz,
1H), 4.24 (d, J=2.8 Hz, 1H), 4.15 (d, J=2.8 Hz, 1H), 4.01-3.44 (m,
35H), 3.28 (t, J=8.4 Hz, 1H), 3.21 (t, J=6.8 Hz, 2H), 2.78-2.72 (m,
3H), 2.52 (t, J=7.2 Hz, 2H), 2.02 (s, 3H), 1.77 (t, J=12.0 Hz, 1H),
1.67-1.60 (m, 2H), 1.58-1.50 (m, 2H), 1.43-1.37 (m, 2H)
[0272] C-8: 9'-Azido_SSEA4-SH (RN=NHAc, R10=N3)
[0273] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 4.90 (d, J=3.9 Hz,
1H), 4.68 (d, J=8.5 Hz, 1H), 4.48 (dd, J=13.2, 7.9 Hz, 3H), 4.37
(t, J=6.5 Hz, 1H), 4.26-4.22 (m, 1H), 4.16 (d, J=3.3 Hz, 1H),
4.09-3.44 (m, 36H), 3.31-3.24 (m, 1H), 3.20 (t, J=6.8 Hz, 2H),
2.79-2.70 (m, 3H), 2.52 (t, J=6.7 Hz, 2H), 2.02 (d, J=2.0 Hz, 6H),
1.76 (t, J=12.1 Hz, 1H), 1.63 (p, J=6.9 Hz, 2H), 1.54 (p, J=6.9 Hz,
2H), 1.39 (q, J=8.3 Hz, 2H).
[0274] C-9: NHBz_SSEA4-SH (RN=NHBz, R10=OH)
[0275] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 7.80-7.73 (m, 2H),
7.66-7.58 (m, 1H), 7.52 (dd, J=8.4, 7.0 Hz, 2H), 4.91 (d, J=3.9 Hz,
1H), 4.69 (d, J=8.5 Hz, 1H), 4.57-4.44 (m, 3H), 4.38 (t, J=6.5 Hz,
1H), 4.27-4.22 (m, 1H), 4.18 (d, J=3.1 Hz, 1H), 4.16-3.52 (m, 36H),
3.29 (t, J=8.5 Hz, 1H), 3.20 (t, J=6.8 Hz, 2H), 2.84-2.72 (m, 3H),
2.52 (t, J=6.7 Hz, 2H), 2.03 (s, 3H), 1.89 (t, J=12.2 Hz, 1H), 1.63
(p, J=6.8 Hz, 2H), 1.53 (q, J=7.1 Hz, 2H), 1.40 (q, J=8.2 Hz,
2H).
[0276] D: Chemoenzymatic Synthesis of SSEA4 analog-allyl
##STR00020##
[0277] SSEA4 analogs-allyl were synthesized via enzymatic
regeneration strategy as described in Scheme5. In this system,
ManNAc derivatives were reacted with pyruvate and transformed into
NeuSAc analogs by aldolase catalysis, followed by incorporation
with Gb5-ally in the regeneration system (J. Am. Chem. Soc. 2013,
135, 14831-14839) to obtain the exemplary SSEA4 analogs-allyl.
(Scheme 5)
[0278] Detail of the reaction condition is described as follows:
Gb5-allyl (18 .mu.mol), CTP (5 .mu.mop, ManNAc derivative (27
.mu.mol), sodium pyruvate (81 .mu.mol), PEP (55 .mu.mol), and ATP
(5 .mu.mol), were dissolved in 50 mM Tris-HCl buffer (pH 8.0).
Enzymesalpha-(2,3)-sialyltransferase (20 units), sialic acid
aldolase(20 units) CMK (10 units), Pykf (10 units), PPA (10 units),
and Pmcss (10 units) were added to the solution, and the reaction
was incubated at 37.degree. C. for 8 hours and monitored by TLC
plate. At the end of reaction, enzyme was denatured by heating at
100.degree. C. for 5 minutes. The desired SSEA4-analog-allyl was
purified by G25, DEAE, and SP column (80%).
[0279] .sup.1H NMR of SSEA4 analogs-allyl
[0280] D-1. SSEA4-allyl (R1=OH, RN=NHAc, R10=OH)
[0281] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 6.00 (m, 1H),
5.40-5.37 (d, J=17.3 Hz, 1H), 5.30-5.28 (d, J=10.4 Hz, 1H), 4.92
(d, J=3.9 Hz, 1H), 4.70 (d, J=8.5 Hz, 1H), 4.54-4.51 (m, 3H),
4.40-4.38 (m, 2H), 4.25-4.18 (m, 3H), 4.10-3.52 (m, 34 H),
3.35-3.32 (t, J=8.6 Hz, 1H), 2.77 (dd, J=12.5, 4.6 Hz, 1H), 2.03
(s, 6H), 1.80 (t, J=12.1 Hz, 1H)
[0282] D-2. Neu5Gc_SSEA4-allyl (R1=OH, RN=NHGc, R10=OH)
[0283] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 5.99 (m, 1H), 5.38
(dd, J=1.2, 17.2 Hz, 1H), 5.29 (dd, J=1.2, 10.0 Hz, 1H), 4.93 (d,
J=4.0 Hz, 1H), 4.69 (d, J=8.4 Hz, 1H), 4.58-4.51 (m, 3H), 4.43-4.37
(m, 2H), 4.28-4.17 (m, 3H), 4.14-3.52 (m, 34 H), 3.33 (t, J=8.8 Hz,
1H), 2.77 (dd, J=4.8, 12.4 Hz, 1H), 2.03 (s, 3H), 1.81 (t, J=12.0
Hz, 1H).
[0284] D-3. Ac-Fluoride_SSEA4-allyl (R1=OH, RN=NHCOCH.sub.2F,
R10=OH)
[0285] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 5.96 (m, 1H), 5.36
(dd, J=1.6, 17.2 Hz, 1H), 5.25 (dd, J=1.6, 10.4 Hz, 1H), 4.89 (d,
J=46.4 Hz, 2H), 4.88 (d, J=3.6 Hz, 1 H), 4.65 (d, J=8.4 Hz, 1H),
4.53-4.45 (m, 3H), 4.39-4.32 (m, 2H), 4.22-3.51 (m, 37H), 3.30 (t,
J=8.4 Hz, 1H), 2.73 (dd, J=4.4, 12.4 Hz, 1H), 2.00 (s, 3H), 1.85
(t, J=12.4 Hz, 1H).
[0286] D-4. Ac-Phenyl_SSEA4-allyl (R1=OH, RN=NHCOCH.sub.2Ph,
R10=OH)
[0287] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 7.45-7.34 (m, 5H),
6.02 (m, 1H), 5.42 (dd, J=1.2, 17.2 Hz, 1H), 5.32 (dd, J=1.2, 10.4
Hz, 1H), 4.94 (d, J=4.0 Hz, 1H), 4.72 (d, J=8.4 Hz, 1H), 4.59-4.52
(m, 3H), 4.46-4.38 (m, 2H), 4.30-3.50 (m, 38 H), 3.42-3.32 (m, 4H),
2.77 (dd, J=4.4, 12.8 Hz, 1H), 2.05 (s, 3H), 1.90 (t, J=12.0 Hz,
1H).
[0288] D-5. Ac-Azido_SSEA4-allyl (R1=OH, RN=NHCOCH.sub.2N.sub.3,
R10=OH)
[0289] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 5.95 (m, 1H), 5.35
(dd, J=1.6, 17.2 Hz, 1H), 5.25 (dd, J=1.2, 10.4 Hz, 1H), 4.88 (d,
J=3.6 Hz, 1H), 4.65 (d, J=8.4 Hz, 1H), 4.52-4.46 (m, 3H), 4.40-4.32
(m, 2H), 4.23-4.18 (m, 3H), 4.12-3.50 (m, 36 H), 3.30 (t, J=5.6 Hz,
1H), 2.72 (dd, J=4.8, 12.8 Hz, 1H), 2.00 (s, 3H), 1.84 (t, J=12.4
Hz, 1H).
[0290] D-6. 5'-Azido_SSEA4-allyl (R1=OH, RN=N.sub.3, R10=OH)
[0291] .sup.1H NMR (400 MHz, D.sub.2O): 6 5.99 (m, 1H), 4.40 (dd,
J=1.6, 17.2 Hz, 1H), 5.29 (d, J=10.4 Hz, 1H), 4.92 (d, J=3.6 Hz,
1H), 4.70 (d, J=8.4 Hz, 1H), 4.56-4.51 (m, 3H), 4.43-4.38 (m, 2H),
4.26 (d, J=3.6 Hz, 2H), 4.22 (d, J=6.4 Hz, 1H), 4.10-3.46 (m, 35H),
3.36-3.32 (m, 1H), 2.74 (dd, J=4.8, 12.4 Hz, 1H), 2.04 (s, 3H),
1.79 (t, J=12.4 Hz); HRMS (ESI-TOF, M-H-)
C.sub.44H.sub.71N.sub.4O.sub.33 calcd for 1183.4001, found
1183.4056.
[0292] D-7. 9'-Azido_SSEA4-allyl (R1=OH, RN=NHAc, R10=N.sub.3)
[0293] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 5.96 (m, 1H), 5.36
(dd, J=1.6, 17.3 Hz, 1H), 5.26 (dd, J=1.6, 10.4 Hz, 1H), 4.90 (d,
J=3.6 Hz, 1H), 4.68 (d, J=8.4 Hz, 1H), 4.55-4.47 (m, 3H), 4.41-4.35
(m, 2H), 4.25-4.14 (m, 3H), 4.10-3.41 (m, 34 H), 3.31 (t, J=6.8 Hz,
1H), 2.72 (dd, J=4.8, 12.8 Hz, 1H), 2.02 (s, 3H), 1.79 (t, J=12.0
Hz, 1H).
[0294] D-8. NHBz_SSEA4-allyl (R1=OH, RN=NHBz, R10=OH)
[0295] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 7.76-7.73 (m, 2H),
7.59 (m, 1H), 7.51-7.46 (m, 2H), 5.90 (m, 1H), 5.30 (dd, J=1.6,
17.2 Hz, 1H), 5.25 (dd, J=1.6, 10.8 Hz, 1H), 4.89 (d, J=3.6 Hz,
1H), 4.67 (d, J=8.8 Hz, 1H), 4.55-4.45 (m, 3H), 4.39-4.38 (m, 2H),
4.24-3.50 (m, 34H), 3.30 (t, J=8.0 Hz, 1H), 2.77 (dd, J=4.4, 12.4
Hz, 1H), 2.01 (s, 3H), 1.90 (t, J=12.4 Hz, 1H).
[0296] E: Oxidation reaction for SSEA4 analog-aldehyde
##STR00021##
[0297] In certain exemplary embodiments, a stirred solution of the
SSEA4 analogs-allyl in methanol and H.sub.2O was ozonolysis for 15
minutes under O.sub.3 gas atmosphere at -70.degree. C. The reaction
mixture was quenched by dimethyl sulfide (Me.sub.2S) and then the
solution was evaporated in vacuo. The desired SSEA4
analogs-aldehyde was then purified by G15. (Scheme 6)
[0298] .sup.1HNMR of SSEA4 analogs-aldehyde
[0299] E-1: SSEA4-aldehyde (RN=NHAc, R10=OH)
[0300] 1H NMR (400 MHz, D.sub.2O) .delta. 5.19 (t, J=4.9 Hz, 1H),
4.89 (d, J=3.9 Hz, 1H), 4.66 (d, J=8.4 Hz, 1H), 4.54-4.45 (m, 3H),
4.36 (t, J=6.5 Hz, 1H), 4.25-4.20 (m, 1H), 4.15 (d, J=3.1 Hz, 1H),
4.08-3.47 (m, 32H), 3.37-3.30 (m, 1H), 2.73 (dd, J=12.4, 4.6 Hz,
1H), 2.00 (d, J=0.9 Hz, 6H), 1.76 (t, J=12.1 Hz, 1H).
[0301] E-2: Neu5Gc_SSEA4-aldehyde (RN=NHGc, R10=OH)
[0302] .sup.1HNMR (400 MHz, D.sub.2O) .delta. 5.20 (t, J=4.9 Hz,
1H), 4.91 (d, J=3.9 Hz, 1H), 4.68 (d, J=8.5 Hz, 1H), 4.52 (dt,
J=8.5, 4.5 Hz, 3H), 4.38 (t, J=6.5 Hz, 1H), 4.27-4.22 (m, 1H), 4.17
(d, J=3.1 Hz, 1H), 4.13-3.51 (m, 34H), 3.38-3.32 (m, 1H), 2.76 (dd,
J=12.4, 4.6 Hz, 1H), 2.02 (s, 3H), 1.80 (t, J=12.1 Hz, 1H).
[0303] E-3: Ac-Fluoride_SSEA4-aldehyde (RN=NHCOCH.sub.2F,
R10=OH)
[0304] 1H NMR (400 MHz, D.sub.2O) .delta. 5.21 (t, J=4.9 Hz, 1H),
4.90 (d, J=46.4 Hz, 2H), 4.69 (d, J=8.5 Hz, 1H), 4.52 (t, J=8.0 Hz,
3H), 4.38 (t, J=6.4 Hz, 1H), 4.24 (d, J=3.1 Hz, 1H), 4.17 (d, J=3.2
Hz, 1H), 4.10-3.45 (m, 33H), 3.40-3.32 (m, 1H), 2.78 (dd, J=12.4,
4.6 Hz, 1H), 2.03 (s, 3H), 1.81 (t, J=12.2 Hz, 1H).
[0305] E-4: Ac-Phenyl_SSEA4-aldehyde (RN=NHCOCH.sub.2Ph,
R10=OH)
[0306] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 7.48-7.27 (m, 5H),
5.22 (t, J=4.9 Hz, 1H), 4.92 (d, J=4.0 Hz, 1H), 4.69 (d, J=8.4 Hz,
1H), 4.56-4.49 (m, 3H), 4.39 (t, J=6.5 Hz, 1H), 4.26 (m, 1H), 4.18
(m, 1H), 4.10-3.45 (m, 34H), 3.43-3.34 (m, 1H), 2.76 (dd, J=12.4,
4.6 Hz, 1H), 2.03 (s, 3H), 1.78 (t, J=12.3 Hz, 1H).
[0307] E-5: Ac-Azido_SSEA4-aldehyde (RN=NHCOCH.sub.2N.sub.3,
R10=OH)
[0308] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 5.20 (t, J=4.9 Hz,
1H), 4.90 (d, J=3.9 Hz, 1H), 4.68 (d, J=8.5 Hz, 1H), 4.54-4.48 (m,
3H), 4.38 (t, J=6.4 Hz, 1H), 4.24 (d, J=3.1 Hz, 1H), 4.17 (d, J=3.1
Hz, 1H), 4.13-3.51 (m, 34H), 3.39-3.32 (m, 1H), 2.75 (dd, J=12.4,
4.6 Hz, 1H), 2.02 (s, 3H), 1.79 (t, J=12.2 Hz, 1H).
[0309] E-6: 9'-Azido_SSEA4-aldehyde (RN=NHAc, R10=N.sub.3)
[0310] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 5.20 (t, J=4.9 Hz,
1H), 4.91 (d, J=3.9 Hz, 1H), 4.69 (d, J=8.5 Hz, 1H), 4.52 (t, J=8.0
Hz, 3H), 4.38 (t, J=6.4 Hz, 1H), 4.24 (d, J=3.1 Hz, 1H), 4.17 (d,
J=3.2 Hz, 1H), 4.10-3.45 (m, 32H), 3.39-3.32 (m, 1H), 2.74 (dd,
J=12.5, 4.6 Hz, 1H), 2.03 (d, J=2.1 Hz, 6H), 1.77 (t, J=12.1 Hz,
1H).
[0311] E-7: NHBz_SSEA4-aldehyde (RN=NHBz, R10=OH)
[0312] .sup.1H NMR (400 MHz, D.sub.2O) .delta. 7.83-7.76 (m, 2H),
7.63 (t, J=7.3 Hz, 1H), 7.53 (t, J=7.7 Hz, 2H), 5.21 (t, J=4.9 Hz,
1H), 4.92 (d, J=3.8 Hz, 1H), 4.70 (d, J=8.5 Hz, 1H), 4.57-4.49 (m,
3H), 4.39 (t, J=6.5 Hz, 1H), 4.26 (d, J=3.1 Hz, 1H), 4.19 (d, J=3.3
Hz, 1H), 4.16-3.52 (m, 32H), 3.40-3.34 (m, 1H), 2.82 (dd, J=12.4,
4.6 Hz, 1H), 2.04 (d, J=4.7 Hz, 3H), 1.87 (t, J=12.1 Hz, 1H).
Example 3
Synthesis of SSEA3/SSEA4 Analog CRM197-Conjugates Via Sulfo-EMCS
Crosslink
##STR00022##
[0314] General Methods:
[0315] Step A. To modify SSEA3 analog-NH2 or SSEA4 analog-NH2 into
SSEA3 analog- SH or SSEA4-analog-SH
[0316] To synthesize SSEA3/4 analog CRM197-conjugates, the
amine-terminated SSEA3/4 analogs were reacted with the DTSSP linker
in PBS buffer (pH 7.4) at room temperature. To monitor the pH value
of solution by pH paper, and add some NaOH solution to the solution
when the solution became neuter or acid. After the reaction was
stirred at room temperature for 12 hours, DTT was added to the
solution at room temperature. The solution was kept stirring at
40.degree. C., and then the solvent was removed under reduced
pressure. The residue was purified by LH-20 column chromatography
to give SSEA3/4 analog-SH.
[0317] Step B: To modify CRM197 into CRM197-maleimide.
[0318] After the salt of commercial CRM197 (1.0 mg) was removed via
alternate dissolving in water and dialyzing (Amicon Ultra-0.5, 10
kDa,), the residue was dissolved in PBS buffer (pH 6.5, 1.0 mL) and
transferred into a sample vial. Sulfo-EMCS (1.0 mg,
8.22.times.10.sup.-6 mol) was added to the solution, and then the
reaction was kept stirring at room temperature for 2 hours. The
mixture was purified by Amicon Ultra-0.5 (10 kDa). After using
MALDI-TOF to check the molecular weight and BCA assay to calculate
the amount of protein, the CRM197-maleimid was stored in PBS buffer
(pH 7.2, 1.0 mg/mL) for next step. According to the data of
MALDI-TOF, the amount of maleimid function groups could be
calculated. For example, when the molecular weight of
CRM197-maleimid was 61841, the numbers of maleimide function groups
on CRM197-maleimid were (61841-58326)/193=18.2.
[0319] Step C: The Synthesis of SSEA3/4 analog-CRM197
Conjugates
[0320] The CRM197-maleimids were dissolved in PBS buffer (pH 7.2,
the concentration was 1.0 mg/mL) and then different amount of
SSEA3/4 analog-SH (5.0 mg/mL in PBS buffer, pH 7.2) were added into
the solution. The mixtures were stirred at room temperature for 2
hours. The SSEA3/4 analog-CRM197 conjugates were purified by using
Amicon Ultra-0.5 (10 kDa) to remove the nonreactive SSEA3/4
analog-SH and sodium phosphate salt via dialysis. The obtained
SSEA3/4 analog-CRM197 conjugates could be characterized by
MALDI-TOF analysis to determine the carbohydrate incorporation
rate. The nonreactive SSEA3/4 analog-SH could be recovered after
reacting with DTT and purifying by LH-20 column chromatography.
TABLE-US-00001 TABLE 1 Carbohydrate incorporation rate of SSEA4
analog with CRM-197 via Sulfo-EMCS Molecular weight (y) average
Code Sugar after glycosylation incorporation rate M1 SSEA4 75465
8.84 M2 Neu5Gc_SSEA4 70750 5.83 M3 Ac-Alkynyl_SSEA4 68965 5.94 M4
Ac-Fluoride_SSEA4 69190 4.59 M5 Ac-Phenyl_SSEA4 75454 8.10 M6
Ac-Azido_SSEA4 70274 5.30 M7 9'-Azido_SSEA4 76596 9.87 M8
Glc-azido_SSEA4 73047 8.00
Example 4
Syntheses of SSEA4-Gc-CRM197 Conjugates Via Sulfo-EMCS
Crosslink
##STR00023##
[0322] Step A: To modify SSEA4-Gc-NH2 into SSEA4-Gc-SH
[0323] DTSSP (5.0 mg, 8.22.times.10.sup.-6 mol) was added to a
flask of SSEA4-Gc-NH2 (5.0 mg, 4.01.times.10.sup.-6 mol) in PBS
buffer (pH 7.4, 1.0 mL) at room temperature. To monitor the pH
value of solution by pH paper, NaOH (1 M/water) was added to the
solution when the solution became neuter or acid. After the
reaction was stirred at room temperature for 12 hours, DTT (5.0 mg,
32.41.times.10.sup.-6 mol) was added to the solution at room
temperature. The solution was kept stirring at 40.degree. C. for 1
hour, and then the solvent was removed under reduced pressure. The
residue was purified by LH-20 column chromatography to give
SSEA4-Gc-SH (5.0 mg, 93%)
[0324] Step B: To modify CRM197 into CRM197-maleimide.
[0325] After the salt of commercial CRM197 (1.0 mg) was removed via
alternate dissolving in water and dialyzing (Amicon Ultra-0.5, 10
kDa,), the residue was dissolved in PBS buffer (pH 6.5, 1.0 mL) and
transferred into a sample vial. Sulfo-EMCS (1.0 mg,
8.22.times.10.sup.-6 mol) was added to the solution, and then the
reaction was kept stirring at room temperature for 2 hours. The
mixture was purified by Amicon Ultra-0.5 (10 kDa). After using
MALDI-TOF to check the molecular weight and BCA assay to calculate
the amount of protein, the CRM197-maleimid was stored in PBS buffer
(pH 7.2, 1.0 mg/mL) for next step. According to the data of
MALDI-TOF, the amount of maleimid function groups could be
calculated. For example, when the molecular weight of
CRM197-maleimid was 61841, the numbers of maleimide function groups
on CRM197-maleimid were (61841-58326)/193=18.2.
[0326] The CRM197-maleimids were dissolved in PBS buffer (pH 7.2,
the concentration was 1.0 mg/mL) and then different amount of
SSE4Gc-SH (5.0 mg/mL in PBS buffer, pH 7.2) were added into the
solution. The mixtures were stirred at room temperature for 2
hours. The SSEA4-Gc-CRM197 conjugates were purified by using Amicon
Ultra-0.5 (10 kDa) to remove the nonreactive SSEA4-Gc-SH and sodium
phosphate salt via dialysis. The obtained SSEA4-Gc-CRM197
conjugates could be characterized by MALDI-TOF analysis to
determine the carbohydrate incorporation rate as showing in Table
2. The nonreactive SSEA4-Gc-SH could be recovered after reacting
with DTT and purifying by LH-20 column chromatography.
[0327] Step C: To trap the nonreactive maleimides of
CRM197-maleimide
[0328] The SSEA4-Gc-CRM197 conjugates were dissolved in PBS buffer
(pH 7.2, the concentration was 1.0 mg/mL) and 10.0 equivalent of
2-mercaptoethanol (5 mg/mL, PBS buffer, pH 7.2) were added to the
solution. The mixtures were stirred at room temperature for 2
hours. The SSEA4-Gc-CRM197 conjugates were purified by using Amicon
Ultra-0.5 (10 kDa) to remove the nonreactive 2-mercaptoethanol and
sodium phosphate salt via dialysis and then lyphophilized to a
white powder.
TABLE-US-00002 TABLE 2 Conjugation of CRM197 with SSEA4-Gc Number
Amount of Number HSC.sub.2H.sub.4OH CRM197 of Linkers PBS Buffer
SSEA4-Gc Reaction of (5 mg/mL) CRM197 (.mu.g) Linkers (mol) (pH
7.4, .mu.L) (5 mg/mL) Time Sugars 10.0 eq. (.mu.g) 1 1388 21.9 5.21
.times. 10.sup.-7 1000 28.1 .mu.L 2 hr 2.0 81.4 .mu.L 1550.4 (0.2
eq.) 2 694 21.9 2.61 .times. 10.sup.-7 500 28.2 .mu.L 2 hr 4.2 40.8
.mu.L 657.4 (0.4 eq.) 3 694 21.9 2.61 .times. 10.sup.-7 500 56.4
.mu.L 2 hr 6.5 40.8 .mu.L 665.0 (0.8 eq.) 4 694 21.9 2.61 .times.
10.sup.-7 500 84.5 .mu.L 2 hr 6.9 40.8 .mu.L 627.0 (1.2 eq.) 5 694
21.9 2.61 .times. 10.sup.-7 500 140.9 .mu.L 2 hr 7.1 40.8 .mu.L
615.6 (2.0 eq.) 6 694 21.9 2.61 .times. 10.sup.-7 500 281.8 .mu.L 2
hr 7.0 40.8 .mu.L 665.0 (4.0 eq.) 7 694 21.9 2.61 .times. 10.sup.-7
500 704.4 .mu.L 2 hr 6.8 40.8 .mu.L 695.4 (10.0 eq.) a) M.W. of
CRM197 = 58326 .fwdarw. 1000 .mu.g = 0.1715 .times. 10.sup.-7 mol
b) M.W. of SSEA4-Gc-SH = 1349.479 .fwdarw. 5 mg/mL = 37.051 .times.
10.sup.-7 mol/mL c) M.W. of 2-Mercaptoethanol = 78.13 .fwdarw. 5
mg/mL = 639.91 .times. 10.sup.-7 mol/mL
Example 5
SSEA4 Analog-CRM197 Conjugate Via SBAP Crosslink
##STR00024##
[0330] CRM-197 was dissolved in 0.1 M phosphate buffer pH 7.4
(.about.1 mg/ml), and SBAP (1.0 mg) was added to the solution. The
solution was stirred gently for 2 hrs at room temperature. The
mixture was then diluted with PBS buffer and centrifuge against 5
changes of 0.1 M phosphate buffer pH 7.4 by Amicon Ultra-0.5 (10
kDa, 2.times.). The obtained modified CRM-197 can be characterized
by MALDI-TOF (positive mode, matrix was sinapinic acid, H.sub.2O)
analysis to determine the SBAP incorporation rate.
[0331] Modified CRM-197 was dissolved in 0.1 M phosphate buffer pH
8.0 (.about.1 mg/ml), and SSEA4-SH analog was added to the
solution. The mixture was stirred for 1 day at room temperature.
The mixture was then diluted with PBS buffer and centrifuge against
5 changes of 0.1 M PBS buffer pH 7.4 by Amicon Ultra-0.5 (10 kDa,
2.times.). The obtained sugar-protein conjugate could be
characterized by MALDI-TOF (positive mode, matrix was sinapinic
acid, H.sub.2O) analysis to determine the carbohydrate
incorporation rate. (Scheme 9)
TABLE-US-00003 TABLE 3 Carbohydrate incorporation rate of SSEA4
analog with CRM-197 via SBAP Molecular weight (x) average Code
Sugar after glycosylation incorporation rate S1 SSEA4 68212 4.79 S2
Neu5Gc_SSEA4 67651 4.84 S3 Ac-Alkynyl_SSEA4 70308 5.70 S4
Ac-Fluoride_SSEA4 69309 5.01 S5 Ac-Phenyl_SSEA4 68891 5.05 S6
Ac-Azido_SSEA4 68359 4.50 S7 5'-Azido_SSEA4 71638 7.06 S8
9'-Azido_SSEA4 72545 7.90 S9 Glc-azido_SSEA4 67131 3.9 S10
NHBz_SSEA4 69636 5.50
Example 6
SSEA4 Analog-CRM197 Conjugate Via Reductive Amination Crosslink
##STR00025##
[0333] In certain embodiments, CRM197 was dissolved in 0.1 M
phosphate buffer (pH 6-9) (.about.1 mg/ml), and enough quantity
SSEA4-aldehyde analogs and NaCNBH3 were added to the solution. The
solution was stirred gently for 3 days at room temperature. The
mixture was then diluted with deionized water and centrifuge
against 5 changes of 0.1 M phosphate buffer pH 7.4 by Amicon
Ultra-0.5 (10 kDa, 2.times.). The obtained sugar-protein conjugate
was characterized by MALDI-TOF (positive mode, matrix was sinapinic
acid, H.sub.2O) analysis to determine the carbohydrate
incorporation rate. (Scheme 10)
TABLE-US-00004 TABLE 4 Carbohydrate incorporation rate of SSEA4
analog with CRM-197 via reductive amination Molecular weight (m)
average Code Sugar after glycosylation incorporation rate R1 SSEA4
69025 8.89 R2 Neu5Gc_SSEA4 65154 5.6 R3 Ac-Fluoride_SSEA4 69315 9
R4 Ac-Phenyl_SSEA4 71329 10.1 R5 Ac-Azido_SSEA4 67765 7.6 R6
9'-Azido_SSEA4 67635 7.58 R7 NHBz_SSEA4 67124 6.95
Example 7
Immunization Determination of the SSEA4 Analog-CRM197
Conjugates
[0334] Exemplary Method
[0335] To demonstrate the efficacy/immunogenicity of the SSEA4
analog CRM197 conjugates (S1.about.S10), female C57BL/6 mice (n=5
for each group) were vaccinated intramuscularly with 0.5 .mu.g of
SSEA4 analog CRM197-conjugates combining the use of 2.0 .mu.g of
glycolipid adjuvant. Control mice were given only phosphate buffer
saline with 2.0 .mu.g of glycolipid adjuvant. The vaccination was
conducted at biweekly intervals for 2 months, and the antisera from
the immunized mice were collected one week after each vaccination.
The antibody titers against SSEA4 were examined by ELISA using
SSEA4 immobilized 96-well titer plates. ELISA was conducted using
SSEA4 immobilized 96-well titer plate. Briefly, the diluted
antisera were incubated with the immobilized SSEA4 at room
temperature for 2 hr. After the washing cycle, the captured
anti-SSEA4 antibodies were then detected using HPR-conjugated
anti-IgG or IgM specific antibody.
[0336] To determine if the glycan-protein conjugation method would
interfere the immune response, native SSEA4 was conjugated with
CRM197 through EMCS linker (M1), SBAP linker (S1) or reductive
amination (R1) and used for immunogenicity study as described
above.
[0337] Representative Result
[0338] After four times of immunization, native SSEA4, as well as
all eight SSEA4 analogs, could positively elicit both IgG (FIG. 3A)
and IgM (FIG. 3B) antibodies against SSEA4 when combining the use
of Gal-C34 adjuvant. There is no significant difference in the
titers of anti-SSEA4 IgG and IgM antibodies among different analog
groups. In addition, Glc-C34 can also be used as vaccine adjuvant
for inducing both IgG (FIG. 4A) and IgM (FIG. 4B) antibodies
against SSEA4 when co-administering with native SSEA4 and the other
analogs.
[0339] Furthermore, the results shown in FIG. 5 indicated that the
glycan-protein conjugation method can affect the immune response.
Combining the use of Gal-C34, the SSEA4-EMCS-CRM197 (M1) elicited a
higher anti-SSEA4 IgG antibody titer when comparing to
SSEA4-SBAP-CRM197 (S1) and SSEA4-CRM197 (conjugated through
reductive amination, R1).
Example 8
Immunogenicity Study of the SSEA4 Analogs CRM197-Conjugates
[0340] To demonstrate the immunogenicity of the SSEA4 analog
CRM197-conjugates, five female BALB/c mice were immunized
intramuscularly with 2 .mu.g of SSEA4 analog CRM197-conjugates and
2 .mu.g of the glycolipid adjuvant C34 three times at biweekly
intervals. In the previous study, the anti-GH antibodies titer was
low with SSEA4 analog-protein conjugates alone without any
adjuvants. The antisera from each immunogen were obtained ten days
after the third immunization and were tested on the glycan
microarray containing 94 chemically synthesized glycans, including
globo series glycans and other tumor-associated carbohydrate
antigens. Because some chemical modifications were carried out on
the glycan, some functional linkers were also included in the
glycan array to check the cross reactivity.
[0341] Antibodies induced by the SSEA4-Gc CRM197-conjugates were
specifically recognized by SSEA4-Gc, native SSEA4 or SSEA4
tetrasaccharide fragments but not by other TACAs and functional
linkers. The sera obtained from the glycoconjugates induced high
IgG antibody titers, indicating a T-cell-dependent immune response.
Interestingly, no significant IgM production was observed for
SSEA4-Gc or native SSEA4. Regarding the IgG level against GloboH,
the titers of antibodies induced by SSEA4-Gc CRM197 was much higher
than the nature form native SSEA4-CRM197 conjugate. Among them the
6.9 molecule of SSEA4-Gc conjugated with one molecule of CRM197 can
induce the highest antibody titers.
[0342] Mice Dosage and Immunization Schedule
[0343] For comparing the immunogenicity of SSEA4 analog CRM197, ten
groups of five mice (8-week-old female Balb/c mice, BioLASCO,
Taiwan) were immunized intramuscularly with glycolipid C34. Three
immunizations were given at 2-week intervals. Each vaccination
contained 2 .mu.g SSEA4 analog and 2 .mu.g C34. Control mice were
injected with phosphate buffer saline (PBS). Mice were bled before
the first immunization (preimmune) and 10 days after the third
immunization. All of the sera were obtained by centrifugation at
4,000.times.g for 10 min. The serologic responses were analyzed by
glycan microarray.
[0344] Serologic Assay with Glycan Array
[0345] Mouse sera were diluted with 1% BSA/PBST buffer (PBST
buffer: PBS and 0.05% Tween-20, pH 7.4). The glycan microarray was
blocked with Superblock blocking buffer (Pierce) for 1 h at
4.degree. C. and washed three times with PBST buffer before use.
The serum dilutions were then introduced to the glycan microarray
and incubated at 4.degree. C. for 1 h. Excess serum antibodies were
washed out and the microarrays were incubated individually with
Alexa Fluor 647-conjugated goat anti-mouse IgG antibody or DyLight
649-conjugated goat anti-mouse IgM antibody as the 2nd antibody at
4.degree. C. in dark for 1 h. The slides were then washed three
times with PBST and scanned at 635 nm wavelength with a microarray
fluorescence chip reader (GenePix 4300A; Molecular Devices
Corporation) and scanned images were analyzed with GenePix Pro-6.0
analysis software (Axon Instruments, Union City, Calif., USA).
OTHER EMBODIMENTS
[0346] All of the features disclosed in this specification may be
combined in any combination. Each feature disclosed in this
specification may be replaced by an alternative feature serving the
same, equivalent, or similar purpose. Thus, unless expressly stated
otherwise, each feature disclosed is only an example of a generic
series of equivalent or similar features. From the above
description, one skilled in the art can easily ascertain the
essential characteristics of the described embodiments, and without
departing from the spirit and scope thereof, can make various
changes and modifications of the embodiments to adapt it to various
usages and conditions. Thus, other embodiments are also within the
claims.
* * * * *