U.S. patent application number 15/982141 was filed with the patent office on 2018-09-20 for compositions and methods relating to universal glycoforms for enhanced antibody efficacy.
The applicant listed for this patent is Academia Sinica. Invention is credited to Che MA, Chi-Huey WONG, Chung-Yi WU, Han-Chung WU.
Application Number | 20180265590 15/982141 |
Document ID | / |
Family ID | 56974948 |
Filed Date | 2018-09-20 |
United States Patent
Application |
20180265590 |
Kind Code |
A1 |
WONG; Chi-Huey ; et
al. |
September 20, 2018 |
COMPOSITIONS AND METHODS RELATING TO UNIVERSAL GLYCOFORMS FOR
ENHANCED ANTIBODY EFFICACY
Abstract
The present disclosure relates to compositions and methods of
use comprising antibodies or binding fragments thereof further
comprising universal Fc glycoforms.
Inventors: |
WONG; Chi-Huey; (Rancho
Santa Fe, CA) ; WU; Chung-Yi; (New Taipei City,
TW) ; MA; Che; (Taipei, TW) ; WU;
Han-Chung; (Taipei, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Academia Sinica |
Taipei |
|
TW |
|
|
Family ID: |
56974948 |
Appl. No.: |
15/982141 |
Filed: |
May 17, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15011544 |
Jan 30, 2016 |
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15982141 |
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14723297 |
May 27, 2015 |
10023892 |
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15011544 |
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14798312 |
Jul 13, 2015 |
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14723297 |
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62110338 |
Jan 30, 2015 |
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62003136 |
May 27, 2014 |
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62003104 |
May 27, 2014 |
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62003908 |
May 28, 2014 |
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62020199 |
Jul 2, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
C07K 16/241 20130101; C12Y 302/01 20130101; C07K 16/18 20130101;
C12N 9/24 20130101; C07K 2317/24 20130101; C12Y 302/01051 20130101;
C07K 2317/52 20130101; C07K 2317/41 20130101; C07K 2317/622
20130101; C12P 21/005 20130101; C07K 16/2887 20130101; C07K 16/30
20130101; C07K 2317/732 20130101; C07K 2319/00 20130101; C07K
2317/734 20130101; C07K 2317/21 20130101; C07K 16/1018 20130101;
C07K 16/32 20130101 |
International
Class: |
C07K 16/30 20060101
C07K016/30; A61K 45/06 20060101 A61K045/06; C07K 16/28 20060101
C07K016/28; C12P 21/00 20060101 C12P021/00; C07K 16/18 20060101
C07K016/18 |
Claims
1. An isolated monoclonal antibody or a binding fragment thereof
that binds to
Neu5Ac.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3GalNAc.beta.1.fwdarw.3-
Gal.alpha.1.fwdarw.4Gal.beta.1.fwdarw.4Glc.beta.1 wherein the
antibody or the fragment thereof comprises a Fc glycoform for
enhancing binding/effector activity in monoclonal antibody, wherein
said antibody comprising a glycoform having the formula:
##STR00004##
2. The isolated antibody of claim 1, wherein the antibody is an
IgG1 and the binding to
Neu5Ac.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3GalNAc.beta.1.fwdarw.3Gal.alpha-
.1.fwdarw.4Gal.beta.1 is specific binding.
3. The isolated antibody of claim 2 wherein the antibody comprised
V.sub.H having SEQ ID NO: 147 or SEQ ID No:137 and V.sub.L having
SEQ ID No: 148 or SEQ ID No:138.
4. The isolated antibody, or antigen-binding fragment thereof of
claim 3, comprising H-CDR1, H-CDR2, and H-CDR3 selected from
(i)-(iii): (i) H-CDR1 selected from SEQ ID NO:152 (GFSLTSYG); (ii)
H-CDR2 selected from SEQ ID NO: 153 (IWGEGST); (iii) H-CDR3
selected from SEQ ID NO:154 (AMTGTAY), respectively; and comprising
L-CDR1, L-CDR2 and L-CDR3 selected from (iv)-(vi): (iv) L-CDR1
selected from SEQ ID NO: 149 (SSVSY); (v) L-CDR2 selected from SEQ
ID NO:150 (DTS); and (vi) L-CDR3 selected from SEQ ID NO: 151
(HQWSSSPHT), respectively.
5. The isolated antibody or antigen-binding fragment of claim 4
wherein the antibody or the antigen binding fragment further
comprising H-FR1, H-FR2, H-FR3, and HFR4 selected from (i)-(iv):
(i) H-FR1 selected from SEQ ID NO:159 (QVQLKESGPGLVAPSQSLSITCTVS);
(ii) H-FR2 selected from SEQ ID NO:160 (VSWIRQPPGKGLEWIGV); (iii)
H-FR3 selected from SEQ ID NO:161
(NYHSVLISRLTISKDNSKSQVFLKLNSLQTDDTATYYC); (iv) H-FR4 selected from
SEQ ID NO:162 (WGQGTLVTVSS); respectively; and comprising L-FR1,
L-FR2, L-FR3 and L-FR4 selected from (v)-(viii): (v) L-FR1 selected
from SEQ ID NO: 155 (QIVLTQSPAIMSASPGEKVTMTCSAS); (vi) L-FR2
selected from SEQ ID NO:156 (MHWYQQKSGTSPKRWIY); (vii) L-FR3
selected from SEQ ID NO: 157
(KLSSGVPGRFSGSGSGTSYSLTISRLEAEDAATYYC); (viii) L-FR4 selected from
SEQ ID NO: 158 (FGGGTKVEIKR); respectively.
6. The antibody of claim 5 wherein the antibody is a human
antibody.
7. The antibody of claim 5 wherein the antibody is a humanized
antibody.
8. The isolated antibody of claim 2 wherein the antibody comprised
V.sub.H having SEQ ID NO: 202, SEQ ID No. 212 or SEQ ID No: 222 and
V.sub.L having SEQ ID No: 203 SEQ ID No. 213 or SEQ ID No: 223.
9. The isolated antibody, or antigen-binding fragment thereof of
claim 8, comprising H-CDR1, H-CDR2, and H-CDR3 selected from
(i)-(iii): (i) H-CDR1 selected from SEQ ID NO:207, SEQ ID NO: 217,
SEQ ID NO: 227; (ii) H-CDR2 selected from SEQ ID NO: 208; SEQ ID
NO: 218, SEQ ID NO: 228; (iii) H-CDR3 selected from SEQ ID NO: 209,
SEQ ID NO: 219, SEQ ID NO: 229; respectively; and comprising
L-CDR1, L-CDR2 and L-CDR3 selected from (iv)-(vi): (iv) L-CDR1
selected from SEQ ID NO: 204; SEQ ID NO: 214, and SEQ ID NO: 224;
(v) L-CDR2 selected from SEQ ID NO:205; SEQ ID NO: 215 and SEQ ID
NO: 225; (vi) L-CDR3 selected from SEQ ID NO: 206, SEQ ID NO: 216
and SEQ ID NO: 226; respectively.
10. The antibody of claim 9 wherein the antibody is a human
antibody.
11. The antibody of claim 9 wherein the antibody is a humanized
antibody.
12. The isolated antibody of claim 1, wherein the antigen binding
fragment is a Fab fragment, a F(ab')2 fragment, or a single-chain
Fv fragment.
13. A pharmaceutical composition comprising the monoclonal antibody
or binding fragment thereof of any one of claim 6, 7, 10, or 11 and
a pharmaceutically acceptable carrier.
14. The pharmaceutical composition of claim 13 wherein the
composition is useful in the treatment against a hyperproliferative
disease.
15. A method of treating cancer in a subject in need thereof,
wherein the method comprises administering to the subject a
therapeutically effective amount of the pharmaceutical composition
of claim 13 whereby the administered antibody enhances ADCC
activity in said subject.
16. The method of claim 15, 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.
17. The composition of claim 16 wherein the method comprising
optionally administering a combined pharmaceutical formulation with
at least one other chemotherapeutic agent.
18. A method for making a population of homogeneous antibodies of
claim 13 comprising: (a) contacting a monoclonal antibody with an
.alpha.-fucosidase and at least one endoglycosidase; (b) generating
a defucosylated antibody having a single N-acetylglucosamine
(GlcNAc); and (c) adding the universal glycan to GlcNAc of Fc
region of antibody to form the homogeneous antibody with said
glycoform.
19. The antibody or binding fragment thereof of claim 1, wherein
the antibodies includes antibodies or binding fragments thereof
specifically bind to one or more of the antigens selected from the
group consisting of Globo H, SSEA-3 and SSEA-4.
Description
RELATED APPLICATION
[0001] This application claims the benefit of priority of, and is a
Continuation of, U.S. application Ser. No. 15/011,544, filed on
Jan. 30, 2016, which is a Continuation-in-Part of, U.S. application
Ser. No. 14/723,297, filed on May 27, 2015; and a
Continuation-in-Part of, U.S. application Ser. No. 14/798,312,
filed on Jul. 13, 2015. U.S. application Ser. No. 15/011,544 also
claims priority to U.S. Ser. No. 62/110,338, filed on Jan. 30,
2015; U.S. Ser. No. 62/003,136, filed on May 27, 2014; U.S. Ser.
No. 62/003,104, filed on May 27, 2014; U.S. Ser. No. 62/003,908,
filed on May 28, 2014; and U.S. Ser. No. 62/020,199, filed on Jul.
2, 2014. The content of each of which is incorporated herein.
FIELD
[0002] The present disclosure relates to selected universal Fc
glycoforms tuned to the desired binding/effector activity for
enhancing the therapeutic efficacies of antibodies directed against
many diseases, including cancers, inflammatory disorders and
infectious diseases. Particularly, the selected and/or directed
optimized universal Fc glycoforms can be generated and/or
incorporated to the design and/or the generation of monoclonal
antibodies for enhanced therapeutic efficacy.
BACKGROUND
[0003] Antibody-based therapies have a proven record of efficacy
against many diseases including inflammatory disorders, cancers,
infectious diseases, and solid organ transplant rejection.
Currently, more than 40 therapeutic monoclonal antibodies (mAbs)
are approved for clinical use in USA, EU and several other
countries. Most of those are for therapy of cancer and immune
diseases. Examples of therapeutic antibodies with anti-tumor
activities include anti-CD20, anti-Her2, anti-EGFR, anti-CD40,
anti-CTLA-4, and anti-PD-1 antibodies.
[0004] The majority of approved biopharmaceuticals are produced in
mammalian cell culture systems to deliver proteins with desired
glycosylation patterns and thus ensure reduced immunogenicity and
higher in vivo efficacy and stability. Non-human mammalian
expression systems such as CHO or NS0 cells have the machinery
required to add complex, human-type glycans. However, glycans
produced in these systems can differ from glycans produced in
humans. Their glycosylation machinery often adds undesired
carbohydrate determinants which may alter protein folding, induce
immunogenicity, and reduce circulatory life span of the drug.
[0005] Furthermore, mammalian cell culture delivers a heterogeneous
mixture of glycosylation patterns which do not all have the same
properties. Properties like safety, efficacy and the serum
half-life of therapeutic proteins can be affected by these
glycosylation patterns. The mammalian cell culture system delivers
heterogeneous mixtures of glycosylation patterns which do not all
have the same properties.
SUMMARY
[0006] Fc glycosylation has been an important subject in the field
of therapeutic monoclonal antibodies. Fc glycosylation can
significantly modify Fc effector functions such as Fc receptor
binding and complement activation, and thus affect the in vivo
safety and efficacy profiles of therapeutic antibodies. Diversity
in Fc glycosylation within an antibody will correspond to diversity
in Fc effector functions. Thus, this heterogeneity in Fc glycans
has a functional consequence as it influences binding of IgG
molecules to Fc receptors and thereby impacts antibody effector
functions, and may trigger undesired effects in patients thus
deeming them a safety concern.
[0007] There is a need for improved monoclonal antibody therapy
against many diseases including inflammatory disorders, cancers and
infectious diseases. Some specific glycoforms in Fc can confer
desired biological functions with improved effector functions, such
as antibody-dependent cellular cytotoxicity (ADCC). Thus, it is
useful to generate therapeutic antibodies with optimized Fc
glycoforms.
[0008] Accordingly, the present disclosure provide selected
universal Fc glycoforms tuned to the desired binding/effector
activity for enhancing the efficacy of therapeutic antibodies
against many diseases, including cancers, inflammatory disorders
and infectious diseases. The selected and/or directed optimized
universal Fc glycoforms can be applied and/or incorporated to the
design and/or the generation of monoclonal antibodies (preferably,
therapeutic monoclonal antibodies) for enhanced therapeutic
efficacy.
[0009] In one aspect, the present disclosure provided a Fc
glycoform for enhancing binding/effector activity in monoclonal
antibody, wherein said antibody comprising a glycoform having the
formula:
##STR00001##
[0010] In some embodiments, the present disclosure provided a
pharmaceutical composition comprising the glycoform of FIG. 1 and a
pharmaceutically acceptable carrier. In one aspect, the present
disclosure provided a method of treating an infectious,
hyperproliferative disease and/or condition, wherein the method
comprises administering to a subject in need thereof a
pharmaceutical composition comprising the glycoform having the
Sia2(.alpha.2-6)Gal2GlcNAc2Man3GlcNAc2.
[0011] In some embodiments, the antibody is a mouse, chimeric,
humanized, and/or human MC41 antibody comprising the following
sequences:
TABLE-US-00001 TABLE 1-1 Amino acid and nucleotide sequences of
anti-SSEA-4 murine, MC41. SEQ ID NO DESCRIPTION SEQUENCE 200 MC41
VH CAGGTGCAGCTGAAGGAAAGCGGACCCGGACTG nucleotide
GTCGCCCCCTCTAAGTCTCTGTCTATTACTTGT sequence
ACTGTGAGCGGATTCTCTCTGAGCTCCCAGGGC GTGTACTGGGTGAGGCAGCCACCTGGCAAGGGC
CTGGAGTGGCTGGGAGCCATCTGGGCAGGAGGC AGCACCAACTATAATTCCGCCCTGATGTCTCGC
CTGTCTATCAGCAAGGACAACTCCAAGTCTCAG GTGTTCCTGAAGATGAACAGCCTGCAGACCGAC
GATACAGCCATGTACTATTGCGCCCGGGTGGAC GGCTACAGAGGCTATAACATGGATTACTGGGGC
CAGGGCACCAGCGTGACAGTGTCTAGC 201 MC41 VL
GAGAATGTGCTGACACAGTCCCCAGCAATCATG nucleotide
AGCGCCTCCCCAGGAGAGAAGGTGACCATGACA sequence
TGTTCCGCCTCCTCTAGCGTGTCTTACATGCAC TGGTATCAGCAGAAGTCCTCTACCAGCCCTAAG
CTGTGGATCTACGACACAAGCAAGCTGGCCTCC GGCGTGCCCGGCCGGTTTTCTGGCAGCGGCTCC
GGCAACTCTTATAGCCTGACCATCAGCAGCATG GAGGCCGAGGATGTGGCCACATACTATTGCTTT
CAGGGCTCTGGCTACCCACTGACATTCGGGGCT GGAACTAAACTGGAACTGAAGCGA 202 MC41
VEI QVQLKESGPGLVAPSKSLSITCTVSGFSLSSQG amino acid
VYWVRQPPGKGLEWLGAIWAGGSTNYNSALMSR sequence
LSISKDNSKSQVFLKMNSLQTDDTAMYYCARVD GYRGYNMDYWGQGTSVTVSS 203 MC41 VL
ENVLTQSPAIMSASPGEKVTMTCSASSSVSYMH amino acid
WYQQKSSTSPKLWIYDTSKLASGVPGRFSGSGS sequence
GNSYSLTISSMEAEDVATYYCFQGSGYPLTFGA GTKLELKR 204 MC41 VL SSVSY CDR1
205 MC41 VL DTS CDR2 206 MC41 VL FQGSGYPLT CDR3 207 MC41 VH
GFSLSSQG CDR1 208 MC41 VH IWAGGST CDR2 209 MC41 VH ARVDGYRGYNMDY
CDR3
TABLE-US-00002 TABLE 1-2 Amino acid and nucleotide sequences of
2.sup.nd humanized monoclonal antibody, hMC41. 2.sup.nd SEQ ID NO
DESCRIPTION SEQUENCE 210 MC41 VH CAGGTGCAGCTGAAGGAGTCCGGACCAGGACT
nucleotide GGTGGCACCATCTAAGACCCTGAGCCTGACCT sequence
GCACAGTGAGCGGCTTCTCCCTGAGCTCCCAG GGCGTGTACTGGATCAGGCAGCCACCTGGCAA
GGGATGGAGTGGATCGGCGCCATCTGGGCCGG CGGCTCTACAAACTATAATTCCGCCCTGATGT
CTCGCCTGTCTATCAGCAAGGACAACTCCAAG TCTCAGGTGTTTCTGAAGATGAATAGCCTGCA
GACCGACGATACAGCCATGTACTATTGCGCCC GGGTGGACGGCTACAGAGGCTATAACATGGAT
TATTGGGGCCAGGGCACCCTGGTGACAGTGTC TAGC 211 MC41 VL
GAGAATGTGCTGACCCAGTCTCCTGCCATCAT nucleotide
GAGCGCCACACCAGGCGAGAAGGTGACCATGA sequence
CATGTTCCGCCTCCTCTAGCGTGTCTTACCTG CACTGGTATCAGCAGAAGTCCTCTACCAGCCC
CAAGCTGTGGATCTACGACACAAGCAAGCTGG CATCCGGAGTGCCTGGCCGGTTCAGCGGATCC
GGATCTGGAAACAGCTATACCCTGACAATCAG CTCCATGGAGGCCGAGGATGTGGCCACCTACT
ATTGTTTCCAGGGATCCGGATACCCACTGACC TTTGGCGCCGGCACAAAGCTGGAGATCAAGCG T
212 MC41 VH QVQLKESGPGLVAPSKTLSLTCTVSGFSLSSQ amino acid
GVYWIRQPPGKGLEWIGAIWAGGSTNYNSALM sequence
SRLSISKDNSKSQVFLKMNSLQTDDTAMYYCA RVDGYRGYNMDYWGQGTLVTVSS 213 MC41
VL ENVLTQSPAIMSATPGEKVTMTCSASSSVSYL amino acid
HWYQQKSSTSPKLWIYDTSKLASGVPGRFSGS sequence
GSGNSYTLTISSMEAEDVATYYCFQGSGYPLT FGAGTKLEIKR 214 MC41 VL SSVSY CDR1
215 MC41 VL DTS CDR2 216 MC41 VL FQGSGYPLT CDR3 217 MC41 VH
GFSLSSQG CDR1 218 MC41 VH IWAGGST CDR2 219 MC41 VH ARVDGYRGYNMDY
CDR3
TABLE-US-00003 TABLE 1-3 Amino acid and nucleotide sequences of
3.sup.rd humanized monoclonal antibody, hMC41. 3.sup.rd SEQ ID NO
DESCRIPTION SEQUENCE 220 MC41 VH CAGGTGCAGCTGAAGGAGTCCGGACCAGGACT
nucleotide GGTGGCACCATCTAAGACCCTGAGCCTGACCT sequence
GCACAGTGAGCGGCTTCTCCCTGAGCTCCCAG GGCGTGTACTGGATCAGGCAGCCACCTGGCAA
GGGACTGGAGTGGATCGGCGCCATCTGGGCCG GCGGCTCTACAAACTATAATTCCGCCCTGATG
TCTCGCCTGTCTATCAGCAAGGACAACTCCAA GTCTCAGGTGTTTCTGAAGATGAATAGCCTGC
AGACCGACGATACAGCCATGTACTATTGCGCC CGGGTGGACGGCTACAGAGGCTATAACATGGA
TTATTGGGGCCAGGGCACCtcGGTGACAGTGT CTAGC 221 MC41 VL
GAGAATGTGCTGACCCAGTCTCCTGCCATCAT nucleotide
GAGCGCCACACCAGGCGAGAAGGTGACCATGA sequence
CATGTTCCGCCTCCTCTAGCGTGTCTTACATG CACTGGTATCAGCAGAAGTCCTCTACCAGCCC
CAAGCTGTGGATCTACGACACAAGCAAGCTGG CATCCGGAGTGCCTGGCCGGTTCAGCGGATCC
GGATCTGGAAACAGCTATACCCTGACAATCAG CTCCATGGAGGCCGAGGATGTGGCCACCTACT
ATTGTTTCCAGGGATCCGGATACCCACTGACC TTTGGCGCCGGCACAAAGCTGGAGATCAAGCG T
222 MC41 VA QVQLKESGPGLVAPSKTLSLTCTVSGFSLSSQ amino acid
GVYWIRQPPGKGLEWIGAIWAGGSTNYNSALM sequence
SRLSISKDNSKSQVFLKMNSLQTDDTAMYYCA RVDGYRGYNMDYWGQGTSVTVSS 223 MC41
VL ENVLTQSPAIMSATPGEKVTMTCSASSSVSYM amino acid
HWYQQKSSTSPKLWIYDTSKLASGVPGRFSGS sequence
GSGNSYTLTISSMEAEDVATYYCFQGSGYPLT FGAGTKLEIKR 224 MC41 VL SSVSY CDR1
225 MC41 VL DTS CDR2 226 MC41 VL FQGSGYPLT CDR3 227 MC41 VH
GFSLSSQG CDR1 228 MC41 VH IWAGGST CDR2 229 MC41 VH ARVDGYRGYNMDY
CDR3
[0012] In one aspect, the present disclosure provides an isolated
monoclonal antibody or a binding fragment thereof that binds to
Neu5Ac.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3GalNAc.beta.1.fwdarw.3Gal.alpha-
.1.fwdarw.4Gal.beta.1.fwdarw.4Glc.beta.1 wherein the antibody or
the fragment thereof comprises a Fc glycoform for enhancing
binding/effector activity in monoclonal antibody, wherein said
antibody comprising a glycoform having the formula:
##STR00002##
[0013] In one embodiment, the antibody is an IgG1 and the binding
to
Neu5Ac.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3GalNAc.beta.1.fwdarw.3Gal.alpha-
.1.fwdarw.4Gal.beta.1.fwdarw. is specific binding.
[0014] In one embodiment, the antibody comprised VH having SEQ ID
NO: 147 or SEQ ID No:137 and VL having SEQ ID No: 148 or SEQ ID
No:138.
[0015] In one embodiment, the isolated antibody, or antigen-binding
fragment thereof comprising H-CDR1, H-CDR2, and H-CDR3 selected
from (i)-(iii): [0016] (i) H-CDR1 selected from SEQ ID NO:152
(GFSLTSYG); [0017] (ii) H-CDR2 selected from SEQ ID NO: 153
(IWGEGST); [0018] (iii) H-CDR3 selected from SEQ ID NO:154
(AMTGTAY), respectively; [0019] and comprising L-CDR1, L-CDR2 and
L-CDR3 selected from (iv)-(vi): [0020] (iv) L-CDR1 selected from
SEQ ID NO: 149 (SSVSY); [0021] (v) L-CDR2 selected from SEQ ID
NO:150 (DTS); and [0022] (vi) L-CDR3 selected from SEQ ID NO: 151
(HQWSSSPHT), respectively.
[0023] In one embodiment, the isolated antibody or antigen-binding
fragment further comprising H-FR1, H-FR2, H-FR3, and HFR4 selected
from (i)-(iv): [0024] (i) H-FR1 selected from SEQ ID NO:159
(QVQLKESGPGLVAPSQSLSITCTVS); [0025] (ii) H-FR2 selected from SEQ ID
NO:160 (VSWIRQPPGKGLEWIGV); [0026] (iii) H-FR3 selected from SEQ ID
NO:161 (NYHSVLISRLTISKDNSKSQVFLKLNSLQTDDTATYYC); [0027] (iv) H-FR4
selected from SEQ ID NO:162 (WGQGTLVTVSS); respectively; [0028] and
comprising L-FR1, L-FR2, L-FR3 and L-FR4 selected from (v)-(viii):
[0029] (v) L-FR1 selected from SEQ ID NO: 155
(QIVLTQSPAIMSASPGEKVTMTCSAS);
[0030] (vi) L-FR2 selected from SEQ ID NO:156
(MHWYQQKSGTSPKRWIY);
[0031] (vii) L-FR3 selected from SEQ ID NO: 157
(KLSSGVPGRFSGSGSGTSYSLTISRLEAEDAATYYC);
[0032] (viii) L-FR4 selected from SEQ ID NO: 158 (FGGGTKVEIKR);
respectively.
[0033] In one embodiment, the antibody is a human antibody.
[0034] In one embodiment, the antibody is a humanized antibody.
[0035] In one embodiment, the antibody comprised VH having SEQ ID
NO: 200, SEQ ID No. 210 or SEQ ID No:137 and VL having SEQ ID No:
201 SEQ ID No. 211 or SEQ ID No: 221.
[0036] In one embodiment, the isolated antibody, or antigen-binding
fragment thereof comprises H-CDR1, H-CDR2, and H-CDR3 selected from
(i)-(iii): [0037] (i) H-CDR1 selected from SEQ ID NO:207, SEQ ID
NO: 217, SEQ ID NO: 227; [0038] (ii) H-CDR2 selected from SEQ ID
NO: 208; SEQ ID NO: 218, SEQ ID NO: 228; [0039] (iii) H-CDR3
selected from SEQ ID NO: 209, SEQ ID NO: 219, SEQ ID NO: 229;
respectively; [0040] and comprising L-CDR1, L-CDR2 and L-CDR3
selected from (iv)-(vi): [0041] (iv) L-CDR1 selected from SEQ ID
NO: SEQ ID NO: 204; SEQ ID NO: 214, and SEQ ID NO: 224; [0042] (v)
L-CDR2 selected from SEQ ID NO:205; SEQ ID NO: 215 and SEQ ID NO:
225; [0043] (vi) L-CDR3 selected from SEQ ID NO: 206, SEQ ID NO:
216 and SEQ ID NO: 226; respectively.
[0044] In one embodiment, the antibody of claim 9 wherein the
antibody is a human antibody.
[0045] In one embodiment, the antibody of claim 9 wherein the
antibody is a humanized antibody.
[0046] In one embodiment, the antigen binding fragment is a Fab
fragment, a F(ab')2 fragment, or a single-chain Fv fragment.
[0047] In one aspect, the present disclosure provides a
pharmaceutical composition comprising the monoclonal antibody or
binding fragment thereof of any one of claim 6, 7, 10, or 11 and a
pharmaceutically acceptable carrier.
[0048] In one embodiment, the pharmaceutical composition is useful
in the treatment against a hyperproliferative disease.
[0049] In one aspect, the present disclosure provides a method of
treating cancer in a subject in need thereof, wherein the method
comprises administering to the subject a therapeutically effective
amount of the pharmaceutical composition of claim 13 whereby the
administered antibody enhances ADCC activity in said subject.
[0050] In one embodiment, the method of treatment for 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.
[0051] In one embodiment, the method comprising optionally
administering a combined pharmaceutical formulation with at least
one other chemotherapeutic agent.
[0052] In another aspect, the present disclosure also provides a
method for making a population of homogeneous antibodies
comprising:
(a) contacting a monoclonal antibody with an .alpha.-fucosidase and
at least one endoglycosidase; (b) generating a defucosylated
antibody having a single N-acetylglucosamine (GlcNAc); and (c)
adding the universal glycan to GlcNAc of Fc region of antibody to
form the homogeneous antibody with said glycoform.
[0053] In one embodiment, the antibody or binding fragment thereof
includes antibodies or binding fragments thereof specifically bind
to one or more of the antigens selected from the group consisting
of Globo H, SSEA-3 and SSEA-4.
[0054] One other aspect of the present disclosure provides
humanized glycoantibodies based on the modification of the MC48.
Exemplars and their amino acid and nucleic acid
structures/sequences are provided below:
TABLE-US-00004 TABLE 17-0 Amino Acid and Nucleotide Sequences of
Mouse Monoclonal Antibody MC48. SEQ ID NO DESCRIPTION SEQUENCE 41
MC48 VH CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCT nucleotide
GGTGGCGCCCTCACAGAGCCTGTCCATCACAT sequence
GCACTGTCTCAGGGTTCTCATTAACCAGCTAT GGTGTAAGCTGGGTTCGCCAGCCTCCAGGAAA
GGGTCTGGAGTGGCTGGGAGTAATATGGGGTG AGGGGAGCACAAATTATCATTCAGTTCTCATA
TCCAGACTGACCATTAGTAAGGATAACTCCAA GAGCCAAGTTTTCTTAAAACTGAACAGTCTGC
AAACTGATGACACAGCCACGTACTACTGTGCC ATGACTGGGACAGCTTACTGGGGCCAAGGGAC
TCTGGTCACTGTCTCTGCA 42 MC48 VL CAAATTGTTCTCACCCAGTCTCCAGCAATCAT
nucleotide GTCTGCATCTCCAGGGGAGAAGGTCACCATGA sequence
CCTGCAGTGCCAGCTCAAGTGTAAGTTACATG CACTGGTACCAGCAGAAGTCAGGCACCTCCCC
CAAAAGATGGATTTATGACACATCCAAACTGT CTTCTGGAGTCCCTGGTCGCTTCAGTGGCAGT
GGGTCTGGGACCTCTTACTCTCTCACAATCAG CAGGTTGGAGGCTGAAGATGCTGCCACTTATT
ACTGCCATCAGTGGAGTAGTAGTCCACACACG TTCGGAGGGGGGACCAAGTTGGAGATAAAA 43
MC48 VH QVQLKESGPGLVAPSQSLSITCTVSGFSLTSY amino acid
GVSWVRQPPGKGLEWLGVIWGEGSTNYHSVLI sequence
SRLTISKDNSKSQVFLKLNSLQTDDTATYYCA MTGTAYWGQGTLVTVSA 44 MC48 VL
QIVLTQSPAIMSASPGEKVTMTCSASSSVSYM amino acid
HWYQQKSGTSPKRWIYDTSKLSSGVPGRFSGS sequence
GSGTSYSLTISRLEAEDAATYYCHQWSSSPHT FGGGTKLEIK 45 MC48 VL SSVSY CDR1
46 MC48 VL DTS CDR2 47 MC48 VL HQWSSSPHT CDR3 48 MC48 VH GFSLTSYG
CDR1 49 MC48 VH IWGEGST CDR2 50 MC48 VH AMTGTAY CDR3
TABLE-US-00005 TABLE 17-1 Amino Acid and Nucleotide Sequences of
Humanized Monoclonal Antibody MC48 (1.sup.st) SEQ ID NO DESCRIPTION
SEQUENCE 115 hMC48 VH CAGGTGCAGCTGCAAGAGTCAGGACCTGGCCTG nucleotide
GTGAAACCCTCAGAAACTCTGTCCCTTACATGC sequence
ACTGTCTCAGGGTTCTCATTAACCAGCTATGGT GTAAGCTGGATTCGCCAGCCTCCAGGAAAGGGT
CTGGAGTGGATTGGAGTAATATGGGGTGAGGGG AGCACAAATTATCATTCAGTTCTCATATCCAGA
CTGACCATTAGTGTGGATACCTCCAAGAATCAA TTTAGCTTAAAACTGAGCAGTGTTACCGCTGCT
GACACAGCCGTTTACTACTGTGCCATGACTGGG ACAGCTTACTGGGGCCAAGGGACTCTGGTCACT
GTCTCTAGC 116 hMC48 VL GAGATTGTGCTGACCCAGAGCCCTGCCACACTG nucleotide
TCACTGAGCCCAGGCGAGCGAGCCACACTGTCC sequence
TGTTCTGCTAGCTCCTCTGTCTCCTACATGCAT TGGTATCAGCAGAAGCCAGGACTGGCACCACGA
CTGCTGATCTATGACACTTCTAAACTGAGTTCA GGCATTCCCGCCAGATTCAGTGGCTCAGGGAGC
GGAACCGACTTTACTCTGACCATTAGCTCCCTG GAGCCTGAAGATTTCGCCGTGTACTATTGCCAT
CAGTGGTCATCAAGCCCTCATACCTTCGGGGGG GGGACTAAGGTGGAAATCAAACGC 117
hMC48 VH QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYG amino acid
VSWIRQPPGKGLEWIGVIWGEGSTNYHSVLISR sequence
LTISVDTSKNQFSLKLSSVTAADTAVYYCAMTG TAYWGQGTLVTVSS 118 hMC48 VL
EIVLTQSPATLSLSPGERATLSCSASSSVSYMH amino acid
WYQQKPGLAPRLLIYDTSKLSSGIPARFSGSGS sequence
GTDFTLTISSLEPEDFAVYYCHQWSSSPHTFGG GTKVEIKR 119 hMC48 VL SSVSY CDR1
120 hMC48 VL DTS CDR2 121 hMC48 VL HQWSSSPHT CDR3 122 hMC48 VH
GFSLTSYG CDR1 123 hMC48 VH IWGEGST CDR2 124 hMC48 VH AMTGTAY
CDR3
TABLE-US-00006 TABLE 17-2 Amino Acid and Nucleotide Sequences of
Humanized Monoclonal Antibody MC48 (2.sup.nd) SEQ ID NO DESCRIPTION
SEQUENCE 125 hMC48 VH CAGGTGCAGCTGAAGCAGAGCGGACCTGGCC nucleotide
TGGTGCAGCCCTCACAGAGCCTGAGCATCAC sequence
TTGTACCGTCAGTGGATTCTCCCTGACATCT TACGGCGTGTCTTGGGTCAGGCAGAGCCCTG
GCAAGGGGCTGGAGTGGCTGGGCGTGATCTG GGGAGAAGGCTCAACTAACTATCACAGCGTC
CTGATCAGTCGCCTGTCAATTAACAAGGACA ATTCTAAAAGTCAGGTGTTCTTTAAAATGAA
CAGCCTGCAGTCCAATGATACCGCCATCTAC TATTGCGCTATGACCGGCACAGCATACTGGG
GGCAGGGAACACTGGTGACTGTCTCCGCT 126 hMC48 VL
GAGATTGTGCTGACCCAGAGCCCTGCCACAC nucleotide
TGTCACTGAGCCCAGGCGAGCGAGCCACACT sequence
GTCCTGTTCTGCTAGCTCCTCTGTCTCCTAC ATGCATTGGTATCAGCAGAAGCCAGGACTGG
CACCACGACTGCTGATCTATGACACTTCTAA ACTGAGTTCAGGCATTCCCGCCAGATTCAGT
GGCTCAGGGAGCGGAACCGACTTTACTCTGA CCATTAGCTCCCTGGAGCCTGAAGATTTCGC
CGTGTACTATTGCCATCAGTGGTCATCAAGC CCTCATACCTTCGGGGGGGGGACTAAGCTGG
AAATCAAACGC 127 hMC48 VH QVQLKQSGPGLVQPSQSLSITCTVSGFSLTS amino acid
YGVSWVRQSPGKGLEWLGVIWGEGSTNYHSV sequence
LISRLSINKDNSKSQVFFKMNSLQSNDTAIY YCAMTGTAYWGQGTLVTVSA 128 hMC48 VL
EIVLTQSPATLSLSPGERATLSCSASSSVSY amino acid
MHWYQQKPGLAPRLLIYDTSKLSSGIPARFS sequence
GSGSGTDFTLTISSLEPEDFAVYYCHQWSSS PHTFGGGTKVLEIKR 129 hMC48 VL SSVSY
CDR1 130 hMC48 VL DTS CDR2 131 hMC48 VL HQWSSSPHT CDR3 132 hMC48 VH
GFSLTSYG CDR1 133 hMC48 VH IWGEGST CDR2 134 hMC48 VH AMTGTAY
CDR3
TABLE-US-00007 TABLE 17-3 Amino Acid and Nucleotide Sequences of
Humanized Monoclonal Antibody MC48 (3.sup.rd) SEQ ID NO DESCRIPTION
SEQUENCE 135 hMC48 VH CAGGTGCAGCTGCAGGAAAGCGGACCCGGAC nucleotide
TGGTGAAACCTAGCGAAACACTGAGCCTGAC sequence
TTGTACCGTGAGCGGATTTTCCCTGACCTCT TATGGAGTGAGCTGGATCAGACAGCCCCCTG
GCAAGGGACTGGAGTGGATCGGCGTGATTTG GGGAGAAGGCTCCACAAACTATCACAGTGTC
CTGATCTCACGACTGACTATTTCTAAGGACA ACTCTAAAAGTCAGGTCTTCCTGAAACTGAA
TAGTCTGCAGACTGACGATACCGCTACATAC TATTGCGCAATGACAGGGACAGCATACTGGG
GACAGGGAACCCTGGTGACAGTCAGCTCC 136 hMC48 VL
CAGATCGTGCTGACACAGTCCCCTGCAATTA nucleotide
TGTCAGCCAGCCCAGGGGAAAAGGTGACAAT sequence
GACTTGTAGTGCTTCTAGTTCAGTCTCATAC ATGCATTGGTATCAGCAGAAGCCAGGCCTGG
CCCCCAGACTGCTGATCTACGACACCTCCAA ACTGAGCTCCGGCGTGCCCGGGAGATTTTCC
GGCTCTGGGAGTGGAACTTCATATAGCCTGA CCATTTCTAGGCTGGAGGCCGAAGATGCCGC
TACATACTATTGCCACCAGTGGAGCAGTAGC CCCCATACATTCGGAGGCGGGACCAAAGTGG
AAATCAAACGC 137 hMC48 VH QVQLQESGPGLVKPSETLSLTCTVSGFSLTS amino acid
YGVSWIRQPPGKGLEWIGVIWGEGSTNYHSV sequence
LISRLTISKDNSKSQVFLKLNSLQTDDTATY YCAMTGTAYWGQGTLVTVSS 138 hMC48 VL
QIVLTQSPAIMSASPGEKVTMTCSASSSVSY amino acid
MHWYQQKPGLAPRLLIYDTSKLSSGVPGRFS sequence
GSGSGTSYSLTISRLEAEDAATYYCHQWSSS PHTFGGGTKVEIKR 139 hMC48 VL SSVSY
CDR1 140 hMC48 VL DTS CDR2 141 hMC48 VL HQWSSSPHT CDR3 142 hMC48 VH
GFSLTSYG CDR1 143 hMC48 VH IWGEGST CDR2 144 hMC48 VH AMTGTAY
CDR3
TABLE-US-00008 TABLE 17-4 Amino Acid and Nucleotide Sequences of
Humanized Monoclonal Antibody MC48 (4.sup.th) SEQ ID NO DESCRIPTION
SEQUENCE 145 hMC48 VH CAGGTCCAGCTGAAAGAGAGCGGCCCCGGACT nucleotide
GGTCGCCCCTTCACAGAGCCTGAGCATTACTT sequence
GCACCGTGAGCGGATTTTCACTGACCAGCTAC GGAGTGAGCTGGATTAGACAGCCTCCTGGCAA
GGGACTGGAGTGGATCGGCGTGATTTGGGGAG AAGGCAGCACCAACTATCACAGTGTCCTGATC
TCACGCCTGACAATTTCCAAGGACAACAGCAA ATCCCAGGTCTTCCTGAAACTGAATTCTCTGC
AGACTGACGATACCGCTACATACTATTGCGCA ATGACAGGGACAGCATACTGGGGACAGGGAAC
CCTGGTGACAGTCAGTAGT 146 hMC48 VL CAGATCGTGCTGACACAGTCCCCAGCAATTAT
nucleotide GTCTGCCAGTCCCGGGGAGAAGGTGACAATGA sequence
CTTGTAGTGCCAGCTCCTCTGTCTCATACATG CATTGGTATCAGCAGAAGTCCGGCACATCTCC
TAAACGGTGGATCTACGACACTTCTAAACTGA GTTCAGGCGTGCCCGGGAGATTTTCAGGCAGC
GGGTCCGGAACTTCTTATAGTCTGACCATTTC CCGACTGGAGGCCGAAGATGCCGCTACCTACT
ATTGCCATCAGTGGTCTTCAAGCCCTCATACT TTTGGGGGGGGAACTAAGGTGGAAATCAAGCG A
147 hMC48 VH QVQLKESGPGLVAPSQSLSITCTVSGFSLTSY amino acid
GVSWIRQPPGKGLEWIGVIWGEGSTNYHSVLI sequence
SRLTISKDNSKSQVFLKLNSLQTDDTATYYCA MTGTAYWGQGTLVTVSS 148 hMC48 VL
QIVLTQSPAIMSASPGEKVTMTCSASSSVSYM amino acid
HWYQQKSGTSPKRWIYDTSKLSSGVPGRFSGS sequence
GSGTSYSLTISRLEAEDAATYYCHQWSSSPHT FGGGTKVEIKR 149 hMC48 VL SSVSY
CDR1 150 hMC48 VL DTS CDR2 151 hMC48 VL HQWSSSPHT CDR3 152 hMC48 VH
GFSLTSYG CDR1 153 hMC48 VH IWGEGST CDR2 154 hMC48 VH AMTGTAY CDR3
155 hMC48 VL QIVLTQSPAIMSASPGEKVTMTCSAS FR1 156 hMC48 VL
MHWYQQKSGTSPKRWIY FR2 157 hMC48 VL KLSSGVPGRFSGSGSGTSYSLTISRLEAEDAA
FR3 TYYC 158 hMC48 VL FGGGTKVEIKR FR4 159 hMC48 VH
QVQLKESGPGLVAPSQSLSITCTVS FR1 160 hMC48 VH VSWIRQPPGKGLEWIGV FR2
161 hMC48 VH NYHSVLISRLTISKDNSKSQVFLKLNSLQTDD FR3 TATYYC 162 hMC48
VH WGQGTLVTVSS FR4
[0055] Antibodies Specific to SSEA4 and Fragment Thereof
[0056] One aspect of the present disclosure features the new
antibodies that bind to SSEA-4 and fragments thereof. The
anti-SSEA-4 antibody binds to
Neu5Ac.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3GalNAc.beta.1.fwdarw.3Gal.al-
pha.1.fwdarw.4Gal.beta.1.fwdarw.4Glc.beta.1 (SSEA-4 hexasaccharide)
and
Neu5Ac.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3GalNAc.beta.1.fwdarw.3Gal.alpha-
.1 (fragment of SSEA-4 hexasaccharide). In some examples, the
antibody is capable of
Neu5Ac.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3GalNAc.beta.1.fwdarw.3Gal.beta.-
1. In some examples, the antibody is capable of
Neu5Gc.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3GalNAc.beta.1.fwdarw.3Gal.alpha-
.1.fwdarw.4Gal.beta.1.fwdarw.4Glc.beta.1 (an analogue of SSEA-4
hexasaccharide).
[0057] In some embodiments, the method enhances ADCC.
[0058] 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.
[0059] 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 composition as described
herein.
[0060] 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.
[0061] In some embodiments, the composition described herein is
formulated an injectible. In some embodiments, the composition is
administered subcutaneously.
[0062] 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
[0063] FIG. 1. Structure of optimized universal Fc glycan of
therapeutic antibodies.
[0064] FIG. 2. General strategy for the preparation of homogeneous
antibody with optimized universal glycan at the Fc region for the
improvement of its therapeutic activity.
[0065] FIG. 3. Demonstrates the enhanced anti-viral
antibody-dependent cell-mediated cytotoxicity (ADCC) results of
anti-influenza virus antibodies.
[0066] FIG. 4. Table listing exemplary enhanced ADCC activities of
anti-CD20 GAbs as compared to Rituximab.
[0067] FIG. 5. Six anti-CD20 GAbs
[0068] FIGS. 6A and 6B. FIG. 6A is top of table, FIG. 6B is bottom
of table. Table lists exemplary Fc.gamma.RIIIA binding of anti-CD20
GAbs and Rituximab. Fc.gamma.RIIIA binding may be measured using
assays known in the art. Exemplary assays are described in the
examples. The Fc receptor binding may be determined as the relative
ratio of anti-CD20 GAb vs Rituximab. Fc receptor binding in
exemplary embodiments is increased by at least 1.2-fold, 2-fold,
3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,
15-fold or 20-fold, 30-fold, 40-fold, 50-fold, 100-fold or
higher.
[0069] FIG. 7. Binding activities of different homogeneous
antibodies with different cells with CD20. FIG. 7 shows CDC effects
of Rituxan-SCT (Gab101) and Rituxan mono-GlcNAc to Ramos cells.
[0070] FIG. 8. Binding activities of different homogeneous
antibodies with different cells with CD20. FIG. 8 shows CDC effects
of Rituxan-SCT (Gab101) and Rituxan mono-GlcNAc to Raji cells.
[0071] FIG. 9. Binding activities of different homogeneous
antibodies with different cells with CD20. FIG. 9 shows CDC effects
of Rituxan-SCT (Gab101) and Rituxan mono-GlcNAc to SU-DHL-4
cells.
[0072] FIG. 10. Depletion of human SU-DHL-4 B cells as analyzed on
FACS. Cells were cultured in the absence or presence of 15%
autologous plasma with anti-CD20 Gabs Rituxan-SCT, Rituxan-GlcNAc
and Rituximab at different concentrations. After wash cells were
stained with anti-CD2-PE and anti-CD19-FITC. B cell depletion was
analyzed on FACS, based on the CD19+CD2- B cells (FIG. 13).
[0073] FIG. 11. Depletion of human Ramos B cells as analyzed on
FACS. Cells were cultured in the absence or presence of 15%
autologous plasma with anti-CD20 Gabs Rituxan-SCT, Rituxan-GlcNAc
and Rituximab at different concentrations. After wash cells were
stained with anti-CD2-PE and anti-CD19-FITC. B cell depletion was
analyzed on FACS, based on the CD19+CD2- B cells (FIG. 13).
[0074] FIG. 12. Depletion of human Raji B cells as analyzed on
FACS. Cells were cultured in the absence or presence of 15%
autologous plasma with anti-CD20 Gabs Rituxan-SCT, Rituxan-GlcNAc
and Rituximab at different concentrations. After wash cells were
stained with anti-CD2-PE and anti-CD19-FITC. B cell depletion was
analyzed on FACS, based on the CD19+CD2- B cells (FIG. 13).
[0075] FIG. 13. Depletion of human B cells by different homogeneous
antibodies.
[0076] FIG. 14. Table listing exemplary enhanced ADCC activities of
anti-HER2 GAbs as compared to Trastuzumab.
[0077] FIG. 15. Table listing exemplary Fc.gamma.RIIIA binding of
anti-HER2 GAbs and Rituximab.
[0078] FIG. 16A. Solid-based ELISA coating SSEA-4 to determine the
binding activity of humanized MC41 phage clones
[0079] FIG. 16B. Solid-based ELISA coating BSA to determine the
binding activity of humanized MC41 phage clones
[0080] FIG. 17A. To evaluate the binding activity by intact
humanized MC41 IgG, intact IgGs of 1st, 2nd, 3rd humanized MC41,
and chimeric MC41 (chMC41) are constructed. The ELISA results show
that the humanized 2nd and 3rd MC41 could react to SSEA-4 (FIG.
17A) but not to BSA (FIG. 17B) in a dose-dependent pattern, same
results were observed for chMC41.
[0081] FIG. 17B. To evaluate the binding activity by intact
humanized MC41 IgG, intact IgGs of 1st, 2nd, 3rd humanized MC41,
and chimeric MC41 (chMC41) are constructed. The ELISA results show
that the humanized 2nd and 3rd MC41 could react to SSEA-4 (FIG.
17A) but not to BSA (FIG. 17B) in a dose-dependent pattern, same
results were observed for chMC41.
[0082] FIG. 18A and FIG. 18B. FIG. 18A shows the legend for bar
graph of FIG. 18B. In order to determine the binding specificity of
chMC41 and hMC41, glycan array is performed. Results are shown in
FIG. 18B. The chimeric and humanized MC41 show more specific
binding than commercial SSEA4 antibody (MC813). They only
recognized SSEA4 or glycolyl modified SSEA4.
[0083] FIGS. 19A and 19B. FIG. 19A shows the legend for the bar
graph of FIG. 19B. In order to determine the binding specificity of
chMC41 and hMC41, glycan array is performed. Results are shown in
FIG. 19B. The chimeric and humanized MC41 show more specific
binding than commercial SSEA4 antibody (MC813). They only
recognized SSEA4 or glycolyl modified SSEA4.
[0084] FIG. 20A. To investigate the effector function of chMC41 and
hMC41, ADCC and CDC assays were performed. HPAC pancreatic cancer
cell line was used to evaluate the ADCC and CDC activities of
chMC41, hMC41, positive control MC813 or negative controls NHIgG
and NHIgG.
[0085] FIG. 20B. To investigate the effector function of chMC41 and
hMC41, ADCC and CDC assays were performed. HPAC pancreatic cancer
cell line was used to evaluate the ADCC and CDC activities of
chMC41, hMC41, positive control MC813 or negative controls NHIgG
and NHIgG.
[0086] FIG. 21A and FIG. 21B. To investigate the effector function
of chMC41 and hMC41, ADCC and CDC assays were performed. HPAC
pancreatic cancer cell line was used to evaluate the ADCC and CDC
activities of chMC41, hMC41, positive control MC813 or negative
controls NHIgG and NHIgG. FIG. 21A shows cancer cell killing
activity through ADCC. FIG. 21B shows cancer cell killing activity
through CDC.
[0087] FIG. 22A. To identify the antibodies that bind to SSEA-4, we
used phage-displayed human naive scFv library containing
2.times.1010 members, which was established as described in our
previous report (Lu et al., 2011). This library was first removed
by Dynabeads-binding phages, and then SSEA-4-binding phages were
selected by SSEA-4-PEG-conjugated Dynabeads. We used two buffer
systems, PBS and PBS containing 0.01% Tween20 (PBST0.01), during
biopanning. After five rounds of affinity selection, the phage
recovery of the fifth round increased by about 55-fold and 80-fold,
compared to that of the first round in PBS and PBST0.01 system,
respectively.
[0088] FIG. 22B. To identify the antibodies that bind to SSEA-4, we
used phage-displayed human naive scFv library containing
2.times.1010 members, which was established as described in our
previous report (Lu et al., 2011). This library was first removed
by Dynabeads-binding phages, and then SSEA-4-binding phages were
selected by SSEA-4-PEG-conjugated Dynabeads. We used two buffer
systems, PBS and PBS containing 0.01% Tween20 (PBST0.01), during
biopanning. After five rounds of affinity selection, the phage
recovery of the fifth round increased by about 55-fold and 80-fold,
compared to that of the first round in PBS and PBST0.01 system,
respectively.
[0089] FIG. 23A. The phage clones were randomly selected and tested
for SSEA-4 binding by ELISA
[0090] FIG. 23B. The phage clones were randomly selected and tested
for SSEA-4 binding by ELISA
[0091] FIG. 23C. The phage clones were randomly selected and tested
for SSEA-4 binding by ELISA
[0092] FIG. 23D. The phage clones were randomly selected and tested
for SSEA-4 binding by ELISA
[0093] FIG. 24. To examine the specificity and binding affinity of
the two phage clones, we performed a comparative ELISA using the
same phage titer to Globo-series glycans including SSEA-4-BSA,
Globo H-BSA and SSEA-3-BSA.
[0094] FIG. 25A. To establish the fully human antibody (hAb)
against SSEA-4, we molecularly engineered the VH and VL coding
sequences of p2-78 scFv into human IgG1 backbone, respectively. The
anti-SSEA-4 p2-78 hAb was produced using FreeStyle 293 expression
system and then purified through the protein G sepharose column. We
examined the purity of antibody by SDS-PAGE analysis with coomassie
blue staining
[0095] FIG. 25B. ELISA to investigate the binding activity of p2-78
hAb for Globo-series glycans.
[0096] FIG. 26A. Positive control of commercially available IgM
antibody, MC631. Glycan array containing 203 different glycans to
further confirm the specificity of p2-78 hAb.
[0097] FIG. 26B. Glycans recognized by p2-78 hAb.
[0098] FIG. 26C. Glycan array containing 203 different glycans to
further confirm the specificity of p2-78 hAb.
[0099] FIG. 27A. After alignment of VH and VL variable region of
MC48 and MC41 with the NCBI IgBLAST or IMGT database, we generated
1st, 2nd, 3rd and 4th humanized MC48 sequences and 1st, 2nd and 3rd
humanized MC41 sequences. We next constructed and generated the
phage-displayed scFv formats according to these humanized MC48 and
MC41 sequences. To determine the binding activity of the humanized
MC48 and MC41 phage clones, we carried out solid-based ELISA
coating SSEA-4-BSA. We found that the 3rd and 4th humanized MC48,
and 2nd and 3rd humanized MC41 scFv phages could recognize SSEA-4
in a dose-dependent manner, whereas the 1st and 2nd humanized MC48
and 1st MC41 scFv lost the binding activity to SSEA-4. The data
showed that the binding affinities of the 4th humanized MC48, and
3rd humanized MC41 scFv phage clones were maintained, compared to
that of the murine mAbs MC48 or MC41.
[0100] FIG. 27B. After alignment of VH and VL variable region of
MC48 and MC41 with the NCBI IgBLAST or IMGT database, we generated
1st, 2nd, 3rd and 4th humanized MC48 sequences and 1st, 2nd and 3rd
humanized MC41 sequences. We next constructed and generated the
phage-displayed scFv formats according to these humanized MC48 and
MC41 sequences. To determine the binding activity of the humanized
MC48 and MC41 phage clones, we carried out solid-based ELISA
coating SSEA-4-BSA. We found that the 3rd and 4th humanized MC48,
and 2nd and 3rd humanized MC41 scFv phages could recognize SSEA-4
in a dose-dependent manner, whereas the 1st and 2nd humanized MC48
and 1st MC41 scFv lost the binding activity to SSEA-4. The data
showed that the binding affinities of the 4th humanized MC48, and
3rd humanized MC41 scFv phage clones were maintained, compared to
that of the murine mAbs MC48 or MC41.
[0101] FIG. 28A. After alignment of VH and VL variable region of
MC48 and MC41 with the NCBI IgBLAST or IMGT database, we generated
1st, 2nd, 3rd and 4th humanized MC48 sequences and 1st, 2nd and 3rd
humanized MC41 sequences. We next constructed and generated the
phage-displayed scFv formats according to these humanized MC48 and
MC41 sequences. To determine the binding activity of the humanized
MC48 and MC41 phage clones, we carried out solid-based ELISA
coating SSEA-4-BSA. We found that the 3rd and 4th humanized MC48,
and 2nd and 3rd humanized MC41 scFv phages could recognize SSEA-4
in a dose-dependent manner, whereas the 1st and 2nd humanized MC48
and 1st MC41 scFv lost the binding activity to SSEA-4. The data
showed that the binding affinities of the 4th humanized MC48, and
3rd humanized MC41 scFv phage clones were maintained, compared to
that of the murine mAbs MC48 or MC41.
[0102] FIG. 28B. After alignment of VH and VL variable region of
MC48 and MC41 with the NCBI IgBLAST or IMGT database, we generated
1st, 2nd, 3rd and 4th humanized MC48 sequences and 1st, 2nd and 3rd
humanized MC41 sequences. We next constructed and generated the
phage-displayed scFv formats according to these humanized MC48 and
MC41 sequences. To determine the binding activity of the humanized
MC48 and MC41 phage clones, we carried out solid-based ELISA
coating SSEA-4-BSA. We found that the 3rd and 4th humanized MC48,
and 2nd and 3rd humanized MC41 scFv phages could recognize SSEA-4
in a dose-dependent manner, whereas the 1st and 2nd humanized MC48
and 1st MC41 scFv lost the binding activity to SSEA-4. The data
showed that the binding affinities of the 4th humanized MC48, and
3rd humanized MC41 scFv phage clones were maintained, compared to
that of the murine mAbs MC48 or MC41.
[0103] FIG. 29A and FIG. 29B. After alignment of VH and VL variable
region of MC48 and MC41 with the NCBI IgBLAST or IMGT database, we
generated 1st, 2nd, 3rd and 4th humanized MC48 sequences and 1st,
2nd and 3rd humanized MC41 sequences. We next constructed and
generated the phage-displayed scFv formats according to these
humanized MC48 and MC41 sequences. To determine the binding
activity of the humanized MC48 and MC41 phage clones, we carried
out solid-based ELISA coating SSEA-4-BSA. We found that the 3rd and
4th humanized MC48, and 2nd and 3rd humanized MC41 scFv phages
could recognize SSEA-4 in a dose-dependent manner, whereas the 1st
and 2nd humanized MC48 and 1st MC41 scFv lost the binding activity
to SSEA-4. The data showed that the binding affinities of the 4th
humanized MC48, and 3rd humanized MC41 scFv phage clones were
maintained, compared to that of the murine mAbs MC48 or MC41.
[0104] FIG. 29B. After alignment of VH and VL variable region of
MC48 and MC41 with the NCBI IgBLAST or IMGT database, we generated
1st, 2nd, 3rd and 4th humanized MC48 sequences and 1st, 2nd and 3rd
humanized MC41 sequences. We next constructed and generated the
phage-displayed scFv formats according to these humanized MC48 and
MC41 sequences. To determine the binding activity of the humanized
MC48 and MC41 phage clones, we carried out solid-based ELISA
coating SSEA-4-BSA. We found that the 3rd and 4th humanized MC48,
and 2nd and 3rd humanized MC41 scFv phages could recognize SSEA-4
in a dose-dependent manner, whereas the 1st and 2nd humanized MC48
and 1st MC41 scFv lost the binding activity to SSEA-4. The data
showed that the binding affinities of the 4th humanized MC48, and
3rd humanized MC41 scFv phage clones were maintained, compared to
that of the murine mAbs MC48 or MC41.
[0105] FIGS. 30A and 30B. To evaluate the binding activity by
intact humanized MC41 IgG, we constructed intact IgGs of 1st, 2nd,
3rd humanized MC41 and chimeric MC41 (chMC41). The ELISA results
showed that the humanized 2nd and 3rd MC41 could react to SSEA-4
(FIG. 30A) but not to BSA (FIG. 30B) in a dose-dependent pattern,
same results were observed for chMC41.
[0106] FIG. 31A and FIG. 31B. In order to determine the binding
specificity of chMC41 and hMC41, glycan array was performed. The
chimeric and humanized MC41 showed more specific binding than
commercial SSEA4 antibody (MC813). They only recognized SSEA4 or
glycolyl modified SSEA4. FIG. 31A shows the glycans that were
recognized and FIG. 31B shows the array results.
[0107] FIG. 32A and FIG. 32B. In order to determine the binding
specificity of chMC41 and hMC41, glycan array was performed. The
chimeric and humanized MC41 showed more specific binding than
commercial SSEA4 antibody (MC813). They only recognized SSEA4 or
glycolyl modified SSEA4. FIG. 32A shows the glycans that were
recognized and FIG. 32B shows the array results.
[0108] FIG. 33A and FIG. 33B. To investigate the effector function
of hMC48, chMC41 and hMC41, ADCC and CDC assays were performed.
HPAC, BxPC3 and PL45 pancreatic cancer cell lines were used to
evaluate the ADCC and CDC activities at the concentration of 10
.mu.g/ml for hMC48 or NHIgG.
[0109] FIG. 34A. HPAC cells were treated with chMC41, hMC41,
positive control MC813 or negative control NHIgG.
[0110] FIG. 34B. HPAC cells were treated with chMC41, hMC41,
positive control MC813 or negative control NHIgG.
[0111] FIGS. 35A and 35B. The data showed that the effector
function of hMC41 and chMC41 was superior to that of hMC48.
Interestingly, the humanized MC41 not only maintain its original
activity, but it also showed stronger cancer cell killing activity
than MC813 through ADCC and CDC.
[0112] FIG. 36. The binding abilities of hMC41 and hMC48 to SSEA-4
were examined by ELISA. The result showed that the binding of hMC41
to SSEA-4 was much better than hMC48. The humanized MC41 has a
higher binding maximum and a smaller Kd (0.2 .mu.g/ml and 4.6
.mu.g/ml for hMC41 and hMC48, respectively) value as compared to
hMC48.
DETAILED DESCRIPTIONS
Chemical Definitions
[0113] 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, 75.sup.th 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, 5.sup.th 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.
[0114] 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, N Y, 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.
[0115] When a range of values is listed, it is intended to
encompass each value and sub-range within the range. For example
"C.sub.1-6" is intended to encompass C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, C.sub.6, C.sub.1-6, C.sub.1-5, C.sub.1-4,
C.sub.1-3, C.sub.1-2, C.sub.2-6, C.sub.2-5, C.sub.2-4, C.sub.2-3,
C.sub.3-6, C.sub.3-5, C.sub.3-4, C.sub.4-6, C.sub.4-5, and
C.sub.5-6.
[0116] 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).
[0117] 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 -1,4-linked D-glucose, and
chitin is a glycan composed of -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.
[0118] 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.
[0119] As used herein, the term "Flow cytometry" or "FACS" means a
technique for examining the physical and chemical properties of
particles or cells suspended in a stream of fluid, through optical
and electronic detection devices.
[0120] A non-naturally occurring or an "isolated" antibody is one
which has been identified and separated and/or recovered from a
component of its native environment. Contaminant components of its
native 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.
[0121] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group. F(ab')2
antibody fragments originally were produced as pairs of Fab'
fragments which have hinge cysteines between them. Other chemical
couplings of antibody fragments are also known.
[0122] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa (.kappa.) and lambda (.lamda.), based on the
amino acid sequences of their constant domains.
[0123] Depending on the amino acid sequences of the constant
domains of their heavy chains, antibodies (immunoglobulins) can be
assigned to different classes. There are five major classes of
immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these
may be further divided into subclasses (isotypes), e.g., IgG1,
IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains
that correspond to the different classes of immunoglobulins are
called .alpha., .delta., .epsilon., .gamma., and .mu.,
respectively. The subunit structures and three-dimensional
configurations of different classes of immunoglobulins are well
known and described generally in, for example, Abbas et al.
Cellular and Mol. Immunology, 4th ed. (2000). An antibody may be
part of a larger fusion molecule, formed by covalent or
non-covalent association of the antibody with one or more other
proteins or peptides.
[0124] "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.
[0125] Identity or homology with respect to a specified amino acid
sequence of this invention is defined herein as the percentage of
amino acid residues in a candidate sequence that are identical with
the specified residues, after aligning the sequences and
introducing gaps, if necessary, to achieve the maximum percent
homology, and not considering any conservative substitutions as
part of the sequence identity. None of N-terminal, C-terminal or
internal extensions, deletions, or insertions into the specified
sequence shall be construed as affecting homology. All sequence
alignments called for in this invention are such maximal homology
alignments. Generally, the nucleic acid sequence homology between
the polynucleotides, oligonucleotides, and fragments of the
invention and a nucleic acid sequence of interest will be at least
80%>, and more typically with preferably increasing homologies
of at least 85%, 90%, 91%, 92%, 92%, 94%, 95%, 96%, 97%, 98%, 99%,
and/or 100%. Two amino acid sequences are homologous if there is a
partial or complete identity between their sequences.
[0126] The term "globoseries-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.
[0127] 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.
[0128] 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).
[0129] 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 -1,4-linked D-glucose, and
chitin is a glycan composed of -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.
[0130] As used herein, the term "antigen" is defined as any
substance capable of eliciting an immune response.
[0131] As used herein, the term "immunogenicity" refers to the
ability of an immunogen, antigen, or vaccine to stimulate an immune
response.
[0132] 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.
[0133] 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.
[0134] 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.
[0135] 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.
[0136] 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.
[0137] 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.
[0138] The phrase "substantially similar," "substantially the
same", "equivalent", or "substantially equivalent", as used herein,
denotes a sufficiently high degree of similarity between two
numeric values (for example, one associated with a molecule and the
other associated with a reference/comparator molecule) such that
one of skill in the art would consider the difference between the
two values to be of little or no biological and/or statistical
significance within the context of the biological characteristic
measured by said values (e.g., Kd values, anti-viral effects,
etc.). The difference between said two values is, for example, less
than about 50%, less than about 40%, less than about 30%, less than
about 20%, and/or less than about 10% as a function of the value
for the reference/comparator molecule.
[0139] The phrase "substantially reduced," or "substantially
different", as used herein, denotes a sufficiently high degree of
difference between two numeric values (generally one associated
with a molecule and the other associated with a
reference/comparator molecule) such that one of skill in the art
would consider the difference between the two values to be of
statistical significance within the context of the biological
characteristic measured by said values (e.g., Kd values). The
difference between said two values is, for example, greater than
about 10%, greater than about 20%, greater than about 30%, greater
than about 40%, and/or greater than about 50% as a function of the
value for the reference/comparator molecule.
[0140] "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.
[0141] In one embodiment, the "Kd" or "Kd value" according to this
invention is measured by a radiolabeled antigen binding assay (RIA)
performed with the Fab version of an antibody of interest and its
antigen as described by the following assay. Solution binding
affinity of Fabs for antigen is measured by equilibrating Fab with
a minimal concentration of (125I)-labeled antigen in the presence
of a titration series of unlabeled antigen, then capturing bound
antigen with an anti-Fab antibody-coated plate (Chen, et al.,
(1999) J. Mol Biol 293:865-881). To establish conditions for the
assay, microtiter plates (Dynex) are coated overnight with 5
.mu.g/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM
sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v)
bovine serum albumin in PBS for two to five hours at room
temperature (approximately 23.degree. C.). In a non-adsorbent plate
(Nunc #269620), 100 pM or 26 pM [125I]-antigen are mixed with
serial dilutions of a Fab of interest (e.g., consistent with
assessment of an anti-VEGF antibody, Fab-12, in Presta et al.,
(1997) Cancer Res. 57:4593-4599). The Fab of interest is then
incubated overnight; however, the incubation may continue for a
longer period (e.g., 65 hours) to insure that equilibrium is
reached. Thereafter, the mixtures are transferred to the capture
plate for incubation at room temperature (e.g., for one hour). The
solution is then removed and the plate washed eight times with 0.1%
Tween-20 in PBS. When the plates have dried, 150 .mu.l/well of
scintillant (MicroScint-20; Packard) is added, and the plates are
counted on a Topcount gamma counter (Packard) for ten minutes.
Concentrations of each Fab that give less than or equal to 20% of
maximal binding are chosen for use in competitive binding assays.
According to another embodiment the Kd or Kd value is measured by
using surface plasmon resonance assays using a BIAcore.TM.-2000 or
a BIAcore.TM.-3000 (BIAcore, Inc., Piscataway, N.J.) at 25.degree.
C. with immobilized antigen CM5 chips at .sup..about.10 response
units (RU). Briefly, carboxymethylated dextran biosensor chips
(CM5, BIAcore Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
to 5 .mu.g/ml (.sup..about.0.2 .mu.M) before injection at a flow
rate of 5 .mu.l/minute to achieve approximately 10 response units
(RU) of coupled protein. Following the injection of antigen, 1 M
ethanolamine is injected to block unreacted groups. In each
experiment, a spot was activated and ethanolamine blocked without
immobilizing protein, to be used for reference subtraction. For
kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to
500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at
25.degree. C. at a flow rate of approximately 25 .mu.l/min.
Association rates (kon) and dissociation rates (koff) are
calculated using a simple one-to-one Langmuir binding model
(BIAcore Evaluation Software version 3.2) by simultaneously fitting
the association and dissociation sensorgrams. The equilibrium
dissociation constant (Kd) is calculated as the ratio koff/kon.
See, e.g., Chen, Y., et al., (1999) J. Mol Biol 293:865-881. If the
on-rate exceeds 106 M-1s-1 by the surface plasmon resonance assay
above, then the on-rate can be determined by using a fluorescent
quenching technique that measures the increase or decrease in
fluorescence emission intensity (excitation=295 nm; emission=340
nm, 16 nm band-pass) at 25.degree. C. of a 20 nM anti-antigen
antibody (Fab form) in PBS, pH 7.2, in the presence of increasing
concentrations of antigen as measured in a spectrometer, such as a
stop-flow equipped spectrophometer (Aviv Instruments) or a
8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with a
stirred cuvette.
[0142] An "on-rate" or "rate of association" or "association rate"
or "kon" according to this invention can also be determined with
the same surface plasmon resonance technique described above using
a BIAcore.TM.-2000 or a BIAcore.TM.-3000 (BIAcore, Inc.,
Piscataway, N.J.) at 25.degree. C. with immobilized antigen CM5
chips at .sup..about.10 response units (RU). Briefly,
carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) are
activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the
supplier's instructions. Antigen is diluted with 10 mM sodium
acetate, pH 4.8, to 5 .mu.g/ml (.sup..about.0.2 .mu.M) before
injection at a flow rate of 5 .mu.l/minute to achieve approximately
10 response units (RU) of coupled protein. Following the injection
of antigen, 1 M ethanolamine is injected to block unreacted groups.
For kinetics measurements, two-fold serial dilutions of Fab (0.78
nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at
25.degree. C. at a flow rate of approximately 25 .mu.l/min.
Association rates (kon) and dissociation rates (koff) are
calculated using a simple one-to-one Langmuir binding model
(BIAcore Evaluation Software version 3.2) by simultaneously fitting
the association and dissociation sensorgram. The equilibrium
dissociation constant (Kd) was calculated as the ratio koff/kon.
See, e.g., Chen, Y., et al., (1999) J. Mol Biol 293:865-881.
However, if the on-rate exceeds 106 M-1 s-1 by the surface plasmon
resonance assay above, then the on-rate can be determined by using
a fluorescent quenching technique that measures the increase or
decrease in fluorescence emission intensity (excitation=295 nm;
emission=340 nm, 16 nm band-pass) at 25.degree. C. of a 20 nM
anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of
increasing concentrations of antigen as measured in a spectrometer,
such as a stop-flow equipped spectrophometer (Aviv Instruments) or
a 8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with
a stirred cuvette.
[0143] The term "vector," as used herein, is intended to refer to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a "plasmid",
which refers to a circular double stranded DNA loop into which
additional DNA segments may be ligated. Another type of vector is a
phage vector. Another type of vector is a viral vector, wherein
additional DNA segments may be ligated into the viral genome.
Certain vectors are capable of autonomous replication in a host
cell into which they are introduced (e.g., bacterial vectors having
a bacterial origin of replication and episomal mammalian vectors).
Other vectors (e.g., non-episomal mammalian vectors) can be
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors are capable of directing the
expression of genes to which they are operatively linked. Such
vectors are referred to herein as "recombinant expression vectors"
(or simply, "recombinant vectors"). In general, expression vectors
of utility in recombinant DNA techniques are often in the form of
plasmids. In the present specification, "plasmid" and "vector" may
be used interchangeably as the plasmid is the most commonly used
form of vector.
[0144] "Polynucleotide," or "nucleic acid," as used interchangeably
herein, refer to polymers of nucleotides of any length, and include
DNA and RNA. The nucleotides can be deoxyribonucleotides,
ribonucleotides, modified nucleotides or bases, and/or their
analogs, or any substrate that can be incorporated into a polymer
by DNA or RNA polymerase, or by a synthetic reaction. A
polynucleotide may comprise modified nucleotides, such as
methylated nucleotides and their analogs. If present, modification
to the nucleotide structure may be imparted before or after
assembly of the polymer. The sequence of nucleotides may be
interrupted by non-nucleotide components. A polynucleotide may be
further modified after synthesis, such as by conjugation with a
label. Other types of modifications include, for example, "caps,"
substitution of one or more of the naturally occurring nucleotides
with an analog, internucleotide modifications such as, for example,
those with uncharged linkages (e.g., methyl phosphonates,
phosphotriesters, phosphoamidates, carbamates, etc.) and with
charged linkages (e.g., phosphorothioates, phosphorodithioates,
etc.), those containing pendant moieties, such as, for example,
proteins (e.g., nucleases, toxins, antibodies, signal peptides,
ply-L-lysine, etc.), those with intercalators (e.g., acridine,
psoralen, etc.), those containing chelators (e.g., metals,
radioactive metals, boron, oxidative metals, etc.), those
containing alkylators, those with modified linkages (e.g., alpha
anomeric nucleic acids, etc.), as well as unmodified forms of the
polynucleotides(s). Further, any of the hydroxyl groups ordinarily
present in the sugars may be replaced, for example, by phosphonate
groups, phosphate groups, protected by standard protecting groups,
or activated to prepare additional linkages to additional
nucleotides, or may be conjugated to solid or semi-solid supports.
The 5' and 3' terminal OH can be phosphorylated or substituted with
amines or organic capping group moieties of from 1 to 20 carbon
atoms. Other hydroxyls may also be derivatized to standard
protecting groups. Polynucleotides can also contain analogous forms
of ribose or deoxyribose sugars that are generally known in the
art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-
or 2'-azido-ribose, carbocyclic sugar analogs, .alpha.-anomeric
sugars, epimeric sugars such as arabinose, xyloses or lyxoses,
pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs
and basic nucleoside analogs such as methyl riboside. One or more
phosphodiester linkages may be replaced by alternative linking
groups. These alternative linking groups include, but are not
limited to, embodiments wherein phosphate is replaced by P(O)S
("thioate"), P(S)S ("dithioate"), "(O)NR2 ("amidate"), P(O)R,
P(O)OR', CO or CH2 ("formacetal"), in which each R or R' is
independently H or substituted or unsubstituted alkyl (1-20 C)
optionally containing an ether (--O--) linkage, aryl, alkenyl,
cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a
polynucleotide need be identical. The preceding description applies
to all polynucleotides referred to herein, including RNA and
DNA.
[0145] "Oligonucleotide," as used herein, generally refers to
short, generally single-stranded, generally synthetic
polynucleotides that are generally, but not necessarily, less than
about 200 nucleotides in length. The terms "oligonucleotide" and
"polynucleotide" are not mutually exclusive. The description above
for polynucleotides is equally and fully applicable to
oligonucleotides.
[0146] "Antibodies" (Abs) and "immunoglobulins" (Igs) are
glycoproteins having the same structural characteristics. While
antibodies exhibit binding specificity to a specific antigen,
immunoglobulins include both antibodies and other antibody-like
molecules which generally lack antigen specificity. Polypeptides of
the latter kind are, for example, produced at low levels by the
lymph system and at increased levels by myelomas.
[0147] The terms "antibody" and "immunoglobulin" are used
interchangeably in the broadest sense and include monoclonal
antibodies (e.g., full length or intact monoclonal antibodies),
polyclonal antibodies, monovalent, multivalent antibodies, multi
specific antibodies (e.g., bispecific antibodies so long as they
exhibit the desired biological activity) and may also include
certain antibody fragments (as described in greater detail herein).
An antibody can be chimeric, human, humanized and/or affinity
matured.
[0148] The "variable region" or "variable domain" of an antibody
refers to the amino-terminal domains of heavy or light chain of the
antibody. These domains are generally the most variable parts of an
antibody and contain the antigen-binding sites.
[0149] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
complementarity-determining regions (CDRs) or hypervariable regions
both in the light-chain and the heavy-chain variable domains. The
more highly conserved portions of variable domains are called the
framework (FR). The variable domains of native heavy and light
chains each comprise four FR regions, largely adopting a beta-sheet
configuration, connected by three CDRs, which form loops
connecting, and in some cases forming part of, the beta-sheet
structure. The CDRs in each chain are held together in close
proximity by the FR regions and, with the CDRs from the other
chain, contribute to the formation of the antigen-binding site of
antibodies (see Kabat et al., Sequences of Proteins of
Immunological Interest, Fifth Edition, National Institute of
Health, Bethesda, Md. (1991)). The constant domains are not
involved directly in binding an antibody to an antigen, but exhibit
various effector functions, such as participation of the antibody
in antibody-dependent cellular toxicity.
[0150] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab')2 fragment that has two antigen-combining sites and
is still capable of cross-linking antigen.
[0151] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and -binding site. In a two-chain Fv
species, this region consists of a dimer of one heavy- and one
light-chain variable domain in tight, non-covalent association. In
a single-chain Fv species, one heavy- and one light-chain variable
domain can be covalently linked by a flexible peptide linker such
that the light and heavy chains can associate in a "dimeric"
structure analogous to that in a two-chain Fv species. It is in
this configuration that the three CDRs of each variable domain
interact to define an antigen-binding site on the surface of the
VH-VL dimer. Collectively, the six CDRs confer antigen-binding
specificity to the antibody. However, even a single variable domain
(or half of an Fv comprising only three CDRs specific for an
antigen) has the ability to recognize and bind antigen, although at
a lower affinity than the entire binding site.
[0152] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group. F(ab')2
antibody fragments originally were produced as pairs of Fab'
fragments which have hinge cysteines between them. Other chemical
couplings of antibody fragments are also known.
[0153] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa (.kappa.) and lambda (.lamda.), based on the
amino acid sequences of their constant domains.
[0154] Depending on the amino acid sequences of the constant
domains of their heavy chains, antibodies (immunoglobulins) can be
assigned to different classes. There are five major classes of
immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these
may be further divided into subclasses (isotypes), e.g., IgG1,
IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains
that correspond to the different classes of immunoglobulins are
called .alpha., .delta., .epsilon., .gamma., and .mu.,
respectively. The subunit structures and three-dimensional
configurations of different classes of immunoglobulins are well
known and described generally in, for example, Abbas et al.
Cellular and Mol. Immunology, 4th ed. (2000). An antibody may be
part of a larger fusion molecule, formed by covalent or
non-covalent association of the antibody with one or more other
proteins or peptides.
[0155] The terms "full length antibody," "intact antibody" and
"whole antibody" are used herein interchangeably, to refer to an
antibody in its substantially intact form, not antibody fragments
as defined below. The terms particularly refer to an antibody with
heavy chains that contain the Fc region.
[0156] "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.
[0157] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, e.g., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts or comprising only
homogeneous glycoform profile (having only a single glycan or
single glycan profile on a glycoantibody in a population). Examples
of homogeneous antibody composition to enhance the effector
functions by using the 2,3- and 2,6-sialyl and defucosylated
complex bi-antennary glycans at the Fc-297 position are described
in U.S. Ser. No. 12/959,351. Thus, the modifier "monoclonal"
indicates the character of the antibody as not being a mixture of
discrete antibodies. Such monoclonal antibody typically includes an
antibody comprising a polypeptide sequence that binds a target,
wherein the target-binding polypeptide sequence was obtained by a
process that includes the selection of a single target binding
polypeptide sequence from a plurality of polypeptide sequences. For
example, the selection process can be the selection of a unique
clone from a plurality of clones, such as a pool of hybridoma
clones, phage clones or recombinant DNA clones. It should be
understood that the selected target binding sequence can be further
altered, for example, to improve affinity for the target, to
humanize the target binding sequence, to improve its production in
cell culture, to reduce its immunogenicity in vivo, to create a
multispecific antibody, etc., and that an antibody comprising the
altered target binding sequence is also a monoclonal antibody of
this invention. In contrast to polyclonal antibody preparations
which typically include different antibodies directed against
different determinants (epitopes), each monoclonal antibody of a
monoclonal antibody preparation is directed against a single
determinant on an antigen. In addition to their specificity, the
monoclonal antibody preparations are advantageous in that they are
typically uncontaminated by other immunoglobulins. The modifier
"monoclonal" indicates the character of the antibody as being
obtained from a substantially homogeneous population of antibodies,
and is not to be construed as requiring production of the antibody
by any particular method. For example, the monoclonal antibodies to
be used in accordance with the present invention may be made by a
variety of techniques, including, for example, the hybridoma method
(e.g., Kohler et al., Nature, 256: 495 (1975); Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies
and T-Cell hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant
DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display
technologies (See, e.g., Clackson et al., Nature, 352: 624-628
(1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et
al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol.
Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci.
USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol.
Methods 284(1-2): 119-132 (2004), and technologies for producing
human or human-like antibodies in animals that have parts or all of
the human immunoglobulin loci or genes encoding human
immunoglobulin sequences (see, e.g., WO98/24893; WO96/34096;
WO96/33735; WO91/10741; Jakobovits et al., Proc. Natl. Acad. Sci.
USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993);
Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos.
5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016;
Marks et al., Bio. Technology 10: 779-783 (1992); Lonberg et al.,
Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994);
Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger,
Nature Biotechnol. 14: 826 (1996) and Lonberg and Huszar, Intern.
Rev. Immunol. 13: 65-93 (1995).
[0158] 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)).
[0159] 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).
[0160] The term "hypervariable region", "HVR", or "HV", when used
herein refers to the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops. Generally, antibodies comprise six hypervariable regions;
three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). A
number of hypervariable region delineations are in use and are
encompassed herein. The Kabat Complementarity Determining Regions
(CDRs) are based on sequence variability and are the most commonly
used (Kabat et al., Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md. (1991)). Chothia refers instead to the
location of the structural loops (Chothia and Lesk J. Mol. Biol.
196:901-917 (1987)). The AbM hypervariable regions represent a
compromise between the Kabat CDRs and Chothia structural loops, and
are used by Oxford Molecular's AbM antibody modeling software. The
"contact" hypervariable regions are based on an analysis of the
available complex crystal structures. The residues from each of
these hypervariable regions are noted below.
[0161] Loop Kabat AbM Chothia Contact
[0162] L1 L24-L34 L26-L32 L30-L36
[0163] L2 L50-L56 L50-L52 L46-L55
[0164] L3 L89-L97 L91-L96 L89-L96
[0165] H1 H31-H35B H26-H35B H26-H32 H30-H35B
[0166] (Kabat Numbering)
[0167] H1 H31-H35 H26-H35 H26-H32 H30-H35
[0168] (Chothia Numbering)
[0169] H2 H50-H65 H50-H58 H53-H55 H47-H58
[0170] H3 H95-H102 H96-H101 H93-H101
[0171] Hypervariable regions may comprise "extended hypervariable
regions" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 or 49-56
(L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or
49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The
variable domain residues are numbered according to Kabat et al.,
supra, for each of these definitions.
[0172] "Framework" or "FR" residues are those variable domain
residues other than the hypervariable region residues as herein
defined.
[0173] The term "variable domain residue numbering as in Kabat" or
"amino acid position numbering as in Kabat," and variations
thereof, refers to the numbering system used for heavy chain
variable domains or light chain variable domains of the compilation
of antibodies in Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991). Using this numbering
system, the actual linear amino acid sequence may contain fewer or
additional amino acids corresponding to a shortening of, or
insertion into, a FR or HVR of the variable domain. For example, a
heavy chain variable domain may include a single amino acid insert
(residue 52a according to Kabat) after residue 52 of H2 and
inserted residues (e.g. residues 82a, 82b, and 82c, etc. according
to Kabat) after heavy chain FR residue 82. The Kabat numbering of
residues may be determined for a given antibody by alignment at
regions of homology of the sequence of the antibody with a
"standard" Kabat numbered sequence.
[0174] "Single-chain Fv" or "scFv" antibody fragments comprise the
VH and VL domains of antibody, wherein these domains are present in
a single polypeptide chain. Generally, the scFv polypeptide further
comprises a polypeptide linker between the VH and VL domains which
enables the scFv to form the desired structure for antigen binding.
For a review of scFv see Pluckthun, in The Pharmacology of
Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
Springer-Verlag, New York, pp. 269-315 (1994).
[0175] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VL) in the same polypeptide chain (VH-VL). By using a linker that
is too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites.
Diabodies are described more fully in, for example, EP 404,097;
WO93/1161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:
6444-6448 (1993).
[0176] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human and/or has been made using any of the techniques for making
human antibodies as disclosed herein. This definition of a human
antibody specifically excludes a humanized antibody comprising
non-human antigen-binding residues.
[0177] An "affinity matured" antibody is one with one or more
alterations in one or more HVRs thereof which result in an
improvement in the affinity of the antibody for antigen, compared
to a parent antibody which does not possess those alteration(s). In
one embodiment, an affinity matured antibody has nanomolar or even
picomolar affinities for the target antigen. Affinity matured
antibodies are produced by procedures known in the art. Marks et
al. Bio/Technology 10:779-783 (1992) describes affinity maturation
by VH and VL domain shuffling. Random mutagenesis of CDR and/or
framework residues is described by: Barbas et al. Proc Nat. Acad.
Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155
(1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et
al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol.
Biol. 226:889-896 (1992).
[0178] 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.
[0179] An "agonist antibody", as used herein, is an antibody which
mimics at least one of the functional activities of a polypeptide
of interest.
[0180] 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.
[0181] 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.
[0182] "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.
[0183] 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.
[0184] 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.
[0185] 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.
[0186] "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.
[0187] An "effective amount" refers to an amount effective, at
dosages and for periods of time necessary, to achieve the desired
therapeutic or prophylactic result.
[0188] 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.
[0189] 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, I131, I125, Y90, Re186, Re188,
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.
[0190] 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
gamma1I and calicheamicin omegaI1 (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 antibiotic 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.
[0191] Pharmaceutical Formulations
[0192] The pharmaceutical composition is administered in a manner
compatible with the dosage formulation, and in an amount that is
therapeutically effective, protective and therapeutic. 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.
[0193] 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).
[0194] 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.
[0195] 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.
[0196] Clinical Applications
[0197] The present invention provides selected and directed
optimized glycoantibodies 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.
[0198] The compositions described herein can also be used in both
cancer treatment and diagnosis. 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).
[0199] These compositions may further comprise suitable carriers,
such as pharmaceutically acceptable excipients including buffers,
which are well known in the art.
[0200] Non naturally occurring and or isolated antibodies and
polynucleotides are also provided. In certain embodiments, the
isolated antibodies and polynucleotides are substantially pure.
[0201] The antigen-binding domain of an antibody is formed from two
variable (V) regions of about 110 amino acids, one each from the
light (VL) and heavy (VH) chains, that both present three
hypervariable loops or complementarity-determining regions (CDRs).
Variable domains can be displayed functionally on phage, either as
single-chain Fv (scFv) fragments, in which VH and VL are covalently
linked through a short, flexible peptide, or as Fab fragments, in
which they are each fused to a constant domain and interact
non-covalently, as described in Winter et al., Ann. Rev. Immunol.,
12: 433-455 (1994). As used herein, scFv encoding phage clones and
Fab encoding phage clones are collectively referred to as "Fv phage
clones" or "Fv clones".
[0202] Repertoires of VH and VL genes can be separately cloned by
polymerase chain reaction (PCR) and recombined randomly in phage
libraries, which can then be searched for antigen-binding clones as
described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).
Libraries from immunized sources provide high-affinity antibodies
to the immunogen without the requirement of constructing
hybridomas. Alternatively, the naive repertoire can be cloned to
provide a single source of human antibodies to a wide range of
non-self and also self antigens without any immunization as
described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally,
naive libraries can also be made synthetically by cloning the
unrearranged V-gene segments from stem cells, and using PCR primers
containing random sequence to encode the highly variable CDR3
regions and to accomplish rearrangement in vitro as described by
Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
[0203] Filamentous phage is used to display antibody fragments by
fusion to the minor coat protein pIII. The antibody fragments can
be displayed as single chain Fv fragments, in which VH and VL
domains are connected on the same polypeptide chain by a flexible
polypeptide spacer, e.g. as described by Marks et al., J. Mol.
Biol., 222: 581-597 (1991), or as Fab fragments, in which one chain
is fused to pIII and the other is secreted into the bacterial host
cell periplasm where assembly of a Fab-coat protein structure which
becomes displayed on the phage surface by displacing some of the
wild type coat proteins, e.g. as described in Hoogenboom et al.,
Nucl. Acids Res., 19: 4133-4137 (1991).
[0204] Nucleic acid encoding antibody variable gene segments
(including VH and VL segments) are recovered from the cells of
interest and amplified. In the case of rearranged VH and VL gene
libraries, the desired DNA can be obtained by isolating genomic DNA
or mRNA from lymphocytes followed by polymerase chain reaction
(PCR) with primers matching the 5' and 3' ends of rearranged VH and
VL genes as described in Orlandi et al., Proc. Natl. Acad. Sci.
(USA), 86: 3833-3837 (1989), thereby making diverse V gene
repertoires for expression. The V genes can be amplified from cDNA
and genomic DNA, with back primers at the 5' end of the exon
encoding the mature V-domain and forward primers based within the
J-segment as described in Orlandi et al. (1989) and in Ward et al.,
Nature, 341: 544-546 (1989). However, for amplifying from cDNA,
back primers can also be based in the leader exon as described in
Jones et al., Biotechnol., 9: 88-89 (1991), and forward primers
within the constant region as described in Sastry et al., Proc.
Natl. Acad. Sci. (USA), 86: 5728-5732 (1989). To maximize
complementarity, degeneracy can be incorporated in the primers as
described in Orlandi et al. (1989) or Sastry et al. (1989). In
certain embodiments, the library diversity is maximized by using
PCR primers targeted to each V-gene family in order to amplify all
available VH and VL arrangements present in the immune cell nucleic
acid sample, e.g. as described in the method of Marks et al., J.
Mol. Biol., 222: 581-597 (1991) or as described in the method of
Orum et al., Nucleic Acids Res., 21: 4491-4498 (1993). For cloning
of the amplified DNA into expression vectors, rare restriction
sites can be introduced within the PCR primer as a tag at one end
as described in Orlandi et al. (1989), or by further PCR
amplification with a tagged primer as described in Clackson et al.,
Nature, 352: 624-628 (1991).
[0205] Repertoires of synthetically rearranged V genes can be
derived in vitro from V gene segments. Most of the human VH-gene
segments have been cloned and sequenced (reported in Tomlinson et
al., J. Mol. Biol., 227: 776-798 (1992)), and mapped (reported in
Matsuda et al., Nature Genet., 3: 88-94 (1993); these cloned
segments (including all the major conformations of the H1 and H2
loop) can be used to generate diverse VH gene repertoires with PCR
primers encoding H3 loops of diverse sequence and length as
described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388
(1992). VH repertoires can also be made with all the sequence
diversity focused in a long H3 loop of a single length as described
in Barbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992).
Human V.kappa. and V.lamda., segments have been cloned and
sequenced (reported in Williams and Winter, Eur. J. Immunol., 23:
1456-1461 (1993)) and can be used to make synthetic light chain
repertoires. Synthetic V gene repertoires, based on a range of VH
and VL folds, and L3 and H3 lengths, will encode antibodies of
considerable structural diversity. Following amplification of
V-gene encoding DNAs, germline V-gene segments can be rearranged in
vitro according to the methods of Hoogenboom and Winter, J. Mol.
Biol., 227: 381-388 (1992).
[0206] Repertoires of antibody fragments can be constructed by
combining VH and VL gene repertoires together in several ways. Each
repertoire can be created in different vectors, and the vectors
recombined in vitro, e.g., as described in Hogrefe et al., Gene,
128: 119-126 (1993), or in vivo by combinatorial infection, e.g.,
the loxP system described in Waterhouse et al., Nucl. Acids Res.,
21: 2265-2266 (1993). The in vivo recombination approach exploits
the two-chain nature of Fab fragments to overcome the limit on
library size imposed by E. coli transformation efficiency. Naive VH
and VL repertoires are cloned separately, one into a phagemid and
the other into a phage vector. The two libraries are then combined
by phage infection of phagemid-containing bacteria so that each
cell contains a different combination and the library size is
limited only by the number of cells present (about 1012 clones).
Both vectors contain in vivo recombination signals so that the VH
and VL genes are recombined onto a single replicon and are
co-packaged into phage virions. These huge libraries provide large
numbers of diverse antibodies of good affinity (Kd-1 of about 10-8
M).
[0207] Alternatively, the repertoires may be cloned sequentially
into the same vector, e.g. as described in Barbas et al., Proc.
Natl. Acad. Sci. USA, 88: 7978-7982 (1991), or assembled together
by PCR and then cloned, e.g. as described in Clackson et al.,
Nature, 352: 624-628 (1991). PCR assembly can also be used to join
VH and VL DNAs with DNA encoding a flexible peptide spacer to form
single chain Fv (scFv) repertoires. In yet another technique, "in
cell PCR assembly" is used to combine VH and VL genes within
lymphocytes by PCR and then clone repertoires of linked genes as
described in Embleton et al., Nucl. Acids Res., 20: 3831-3837
(1992).
[0208] Screening of the libraries can be accomplished by any
art-known technique. Targets can be used to coat the wells of
adsorption plates, expressed on host cells affixed to adsorption
plates or used in cell sorting, or conjugated to biotin for capture
with streptavidin-coated beads, or used in any other art-known
method for panning phage display libraries.
[0209] The phages bound to the solid phase are washed and then
eluted by acid, e.g. as described in Barbas et al., Proc. Natl.
Acad. Sci. USA, 88: 7978-7982 (1991), or by alkali, e.g. as
described in Marks et al., J. Mol. Biol., 222: 581-597 (1991), or
by SSEA-3/SSEA-4/GLOBO H antigen competition, e.g. in a procedure
similar to the antigen competition method of Clackson et al.,
Nature, 352: 624-628 (1991). Phages can be enriched 20-1,000-fold
in a single round of selection. Moreover, the enriched phages can
be grown in bacterial culture and subjected to further rounds of
selection.
[0210] The efficiency of selection depends on many factors,
including the kinetics of dissociation during washing, and whether
multiple antibody fragments on a single phage can simultaneously
engage with antigen. Antibodies with fast dissociation kinetics
(and weak binding affinities) can be retained by use of short
washes, multivalent phage display and high coating density of
antigen in solid phase. The high density not only stabilizes the
phage through multivalent interactions, but favors rebinding of
phage that has dissociated. The selection of antibodies with slow
dissociation kinetics (and good binding affinities) can be promoted
by use of long washes and monovalent phage display as described in
Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and a
low coating density of antigen as described in Marks et al.,
Biotechnol., 10: 779-783 (1992).
[0211] However, random mutation of a selected antibody (e.g. as
performed in some of the affinity maturation techniques described
above) is likely to give rise to many mutants, most binding to
antigen, and a few with higher affinity. With limiting
SSEA-3/SSEA-4/GLOBO H, rare high affinity phage could be competed
out. To retain all the higher affinity mutants, phages can be
incubated with excess biotinylated SSEA-3/SSEA-4/GLOBO H, but with
the biotinylated SSEA-3/SSEA-4/GLOBO H at a concentration of lower
molarity than the target molar affinity constant for
SSEA-3/SSEA-4/GLOBO H. The high affinity-binding phages can then be
captured by streptavidin-coated paramagnetic beads. Such
"equilibrium capture" allows the antibodies to be selected
according to their affinities of binding, with sensitivity that
permits isolation of mutant clones with as little as two-fold
higher affinity from a great excess of phages with lower affinity.
Conditions used in washing phages bound to a solid phase can also
be manipulated to discriminate on the basis of dissociation
kinetics.
[0212] DNA encoding the Fv clones of the invention is readily
isolated and sequenced using conventional procedures (e.g. by using
oligonucleotide primers designed to specifically amplify the heavy
and light chain coding regions of interest from hybridoma or phage
DNA template). Once isolated, the DNA can be placed into expression
vectors, which are then transfected into host cells such as E. coli
cells, simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of the desired monoclonal antibodies in the
recombinant host cells. Review articles on recombinant expression
in bacteria of antibody-encoding DNA include Skerra et al., Curr.
Opinion in Immunol., 5: 256 (1993) and Pluckthun, Immunol. Revs,
130: 151 (1992).
[0213] DNA encoding the Fv clones of the invention can be combined
with known DNA sequences encoding heavy chain and/or light chain
constant regions (e.g. the appropriate DNA sequences can be
obtained from Kabat et al., supra) to form clones encoding full or
partial length heavy and/or light chains. It will be appreciated
that constant regions of any isotype can be used for this purpose,
including IgG, IgM, IgA, IgD, and IgE constant regions, and that
such constant regions can be obtained from any human or animal
species. A Fv clone derived from the variable domain DNA of one
animal (such as human) species and then fused to constant region
DNA of another animal species to form coding sequence(s) for
"hybrid", full length heavy chain and/or light chain is included in
the definition of "chimeric" and "hybrid" antibody as used herein.
In one embodiment, a Fv clone derived from human variable DNA is
fused to human constant region DNA to form coding sequence(s) for
all human, full or partial length heavy and/or light chains.
[0214] The antibodies produced by naive libraries (either natural
or synthetic) can be of moderate affinity (Kd-1 of about 106 to 107
M-1), but affinity maturation can also be mimicked in vitro by
constructing and reselecting from secondary libraries as described
in Winter et al. (1994), supra. For example, mutation can be
introduced at random in vitro by using error-prone polymerase
(reported in Leung et al., Technique, 1: 11-15 (1989)) in the
method of Hawkins et al., J. Mol. Biol., 226: 889-896 (1992) or in
the method of Gram et al., Proc. Natl. Acad. Sci. USA, 89:
3576-3580 (1992). Additionally, affinity maturation can be
performed by randomly mutating one or more CDRs, e.g. using PCR
with primers carrying random sequence spanning the CDR of interest,
in selected individual Fv clones and screening for higher affinity
clones. WO 9607754 (published 14 Mar. 1996) described a method for
inducing mutagenesis in a complementarity determining region of an
immunoglobulin light chain to create a library of light chain
genes. Another effective approach is to recombine the VH or VL
domains selected by phage display with repertoires of naturally
occurring V domain variants obtained from unimmunized donors and
screen for higher affinity in several rounds of chain reshuffling
as described in Marks et al., Biotechnol., 10: 779-783 (1992). This
technique allows the production of antibodies and antibody
fragments with affinities in the 10-9 M range.
[0215] Other methods of generating and assessing the affinity of
antibodies are well known in the art and are described, e.g., in
Kohler et al., Nature 256: 495 (1975); U.S. Pat. No. 4,816,567;
Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press, 1986; Kozbor, J. Immunol., 133:3001 (1984);
Brodeur et al., Monoclonal Antibody Production Techniques and
Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987;
Munson et al., Anal. Biochem., 107:220 (1980); Engels et al.,
Agnew. Chem. Int. Ed. Engl., 28: 716-734 (1989); Abrahmsen et al.,
EMBO J., 4: 3901 (1985); Methods in Enzymology, vol. 44 (1976);
Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855
(1984).
[0216] General Methods
[0217] Generation of antibodies can be achieved using routine
skills in the art, including those described herein, such as the
hybridoma technique and screening of phage displayed libraries of
binder molecules. These methods are well-established in the
art.
[0218] Briefly, antibodies of the invention can be made by using
combinatorial libraries to screen for synthetic antibody clones
with the desired activity or activities. In principle, synthetic
antibody clones are selected by screening phage libraries
containing phage that display various fragments of antibody
variable region (Fv) fused to phage coat protein. Such phage
libraries are panned by affinity chromatography against the desired
antigen. Clones expressing Fv fragments capable of binding to the
desired antigen are adsorbed to the antigen and thus separated from
the non-binding clones in the library. The binding clones are then
eluted from the antigen, and can be further enriched by additional
cycles of antigen adsorption/elution. Any of the antibodies of the
invention can be obtained by designing a suitable antigen screening
procedure to select for the phage clone of interest followed by
construction of a full length antibody clone using the Fv sequences
from the phage clone of interest and suitable constant region (Fc)
sequences described in Kabat et al., Sequences of Proteins of
Immunological Interest, Fifth Edition, NIH Publication 91-3242,
Bethesda Md. (1991), vols. 1-3.
[0219] Monoclonal antibodies can be obtained from a population of
substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical except for
possible naturally occurring mutations that may be present in minor
amounts. Thus, the modifier "monoclonal" indicates the character of
the antibody as not being a mixture of discrete antibodies.
[0220] The monoclonal antibodies of the invention can be made using
a variety of methods known in the art, including the hybridoma
method first described by Kohler et al., Nature, 256:495 (1975), or
alternatively they may be made by recombinant DNA methods (e.g.,
U.S. Pat. No. 4,816,567).
[0221] Vectors, Host Cells and Recombinant Methods
[0222] For recombinant production of an antibody of the invention,
the nucleic acid encoding it is isolated and inserted into a
replicable vector for further cloning (amplification of the DNA) or
for expression. DNA encoding the antibody is readily isolated and
sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the antibody). Many
vectors are available. The choice of vector depends in part on the
host cell to be used. Host cells include, but are not limited to,
cells of either prokaryotic or eukaryotic (generally mammalian)
origin. It will be appreciated that constant regions of any isotype
can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE
constant regions, and that such constant regions can be obtained
from any human or animal species.
[0223] Generating Antibodies Using Prokaryotic Host Cells
[0224] Vector Construction
[0225] Polynucleotide sequences encoding polypeptide components of
the antibody of the invention can be obtained using standard
recombinant techniques. Desired polynucleotide sequences may be
isolated and sequenced from antibody producing cells such as
hybridoma cells. Alternatively, polynucleotides can be synthesized
using nucleotide synthesizer or PCR techniques. Once obtained,
sequences encoding the polypeptides are inserted into a recombinant
vector capable of replicating and expressing heterologous
polynucleotides in prokaryotic hosts. Many vectors that are
available and known in the art can be used for the purpose of the
present invention. Selection of an appropriate vector will depend
mainly on the size of the nucleic acids to be inserted into the
vector and the particular host cell to be transformed with the
vector. Each vector contains various components, depending on its
function (amplification or expression of heterologous
polynucleotide, or both) and its compatibility with the particular
host cell in which it resides. The vector components generally
include, but are not limited to: an origin of replication, a
selection marker gene, a promoter, a ribosome binding site (RBS), a
signal sequence, the heterologous nucleic acid insert and a
transcription termination sequence.
[0226] In general, plasmid vectors containing replicon and control
sequences which are derived from species compatible with the host
cell are used in connection with these hosts. The vector ordinarily
carries a replication site, as well as marking sequences which are
capable of providing phenotypic selection in transformed cells. For
example, E. coli is typically transformed using pBR322, a plasmid
derived from an E. coli species. pBR322 contains genes encoding
ampicillin (Amp) and tetracycline (Tet) resistance and thus
provides easy means for identifying transformed cells. pBR322, its
derivatives, or other microbial plasmids or bacteriophage may also
contain, or be modified to contain, promoters which can be used by
the microbial organism for expression of endogenous proteins.
Examples of pBR322 derivatives used for expression of particular
antibodies are described in detail in Carter et al., U.S. Pat. No.
5,648,237.
[0227] In addition, phage vectors containing replicon and control
sequences that are compatible with the host microorganism can be
used as transforming vectors in connection with these hosts. For
example, bacteriophage such as .lamda.GEM.TM.-11 may be utilized in
making a recombinant vector which can be used to transform
susceptible host cells such as E. coli LE392.
[0228] The expression vector of the invention may comprise two or
more promoter-cistron pairs, encoding each of the polypeptide
components. A promoter is an untranslated regulatory sequence
located upstream (5') to a cistron that modulates its expression.
Prokaryotic promoters typically fall into two classes, inducible
and constitutive. Inducible promoter is a promoter that initiates
increased levels of transcription of the cistron under its control
in response to changes in the culture condition, e.g. the presence
or absence of a nutrient or a change in temperature.
[0229] A large number of promoters recognized by a variety of
potential host cells are well known. The selected promoter can be
operably linked to cistron DNA encoding the light or heavy chain by
removing the promoter from the source DNA via restriction enzyme
digestion and inserting the isolated promoter sequence into the
vector of the invention. Both the native promoter sequence and many
heterologous promoters may be used to direct amplification and/or
expression of the target genes. In some embodiments, heterologous
promoters are utilized, as they generally permit greater
transcription and higher yields of expressed target gene as
compared to the native target polypeptide promoter.
[0230] Promoters suitable for use with prokaryotic hosts include
the PhoA promoter, the .beta.-galactamase and lactose promoter
systems, a tryptophan (trp) promoter system and hybrid promoters
such as the tac or the trc promoter. However, other promoters that
are functional in bacteria (such as other known bacterial or phage
promoters) are suitable as well. Their nucleotide sequences have
been published, thereby enabling a skilled worker operably to
ligate them to cistrons encoding the target light and heavy chains
(Siebenlist et al. (1980) Cell 20: 269) using linkers or adaptors
to supply any required restriction sites.
[0231] In one aspect of the invention, each cistron within the
recombinant vector comprises a secretion signal sequence component
that directs translocation of the expressed polypeptides across a
membrane. In general, the signal sequence may be a component of the
vector, or it may be a part of the target polypeptide DNA that is
inserted into the vector. The signal sequence selected for the
purpose of this invention should be one that is recognized and
processed (i.e. cleaved by a signal peptidase) by the host cell.
For prokaryotic host cells that do not recognize and process the
signal sequences native to the heterologous polypeptides, the
signal sequence is substituted by a prokaryotic signal sequence
selected, for example, from the group consisting of the alkaline
phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II
(STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment
of the invention, the signal sequences used in both cistrons of the
expression system are STII signal sequences or variants
thereof.
[0232] In another aspect, the production of the immunoglobulins
according to the invention can occur in the cytoplasm of the host
cell, and therefore does not require the presence of secretion
signal sequences within each cistron. In that regard,
immunoglobulin light and heavy chains are expressed, folded and
assembled to form functional immunoglobulins within the cytoplasm.
Certain host strains (e.g., the E. coli trxB- strains) provide
cytoplasm conditions that are favorable for disulfide bond
formation, thereby permitting proper folding and assembly of
expressed protein subunits. Proba and Pluckthun Gene, 159:203
(1995).
[0233] Antibodies of the invention can also be produced by using an
expression system in which the quantitative ratio of expressed
polypeptide components can be modulated in order to maximize the
yield of secreted and properly assembled antibodies of the
invention. Such modulation is accomplished at least in part by
simultaneously modulating translational strengths for the
polypeptide components.
[0234] One technique for modulating translational strength is
disclosed in Simmons et al., U.S. Pat. No. 5,840,523. It utilizes
variants of the translational initiation region (TIR) within a
cistron. For a given TIR, a series of amino acid or nucleic acid
sequence variants can be created with a range of translational
strengths, thereby providing a convenient means by which to adjust
this factor for the desired expression level of the specific chain.
TIR variants can be generated by conventional mutagenesis
techniques that result in codon changes which can alter the amino
acid sequence. In certain embodiments, changes in the nucleotide
sequence are silent. Alterations in the TIR can include, for
example, alterations in the number or spacing of Shine-Dalgarno
sequences, along with alterations in the signal sequence. One
method for generating mutant signal sequences is the generation of
a "codon bank" at the beginning of a coding sequence that does not
change the amino acid sequence of the signal sequence (i.e., the
changes are silent). This can be accomplished by changing the third
nucleotide position of each codon; additionally, some amino acids,
such as leucine, serine, and arginine, have multiple first and
second positions that can add complexity in making the bank. This
method of mutagenesis is described in detail in Yansura et al.
(1992) METHODS: A Companion to Methods in Enzymol. 4:151-158.
[0235] In one embodiment, a set of vectors is generated with a
range of TIR strengths for each cistron therein. This limited set
provides a comparison of expression levels of each chain as well as
the yield of the desired antibody products under various TIR
strength combinations. TIR strengths can be determined by
quantifying the expression level of a reporter gene as described in
detail in Simmons et al. U.S. Pat. No. 5,840,523. Based on the
translational strength comparison, the desired individual TIRs are
selected to be combined in the expression vector constructs of the
invention.
[0236] Prokaryotic host cells suitable for expressing antibodies of
the invention include Archaebacteria and Eubacteria, such as
Gram-negative or Gram-positive organisms. Examples of useful
bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B.
subtilis), Enterobacteria, Pseudomonas species (e.g., P.
aeruginosa), Salmonella typhimurium, Serratia marcescans,
Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or
Paracoccus. In one embodiment, gram-negative cells are used. In one
embodiment, E. coli cells are used as hosts for the invention.
Examples of E. coli strains include strain W3110 (Bachmann,
Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American
Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No.
27,325) and derivatives thereof, including strain 33D3 having
genotype W3110 .DELTA.fhu.DELTA. (.DELTA.tonA) ptr3 lac Iq lacL8
.DELTA.ompT.DELTA.(nmpc-fepE) degP41 kanR (U.S. Pat. No.
5,639,635). Other strains and derivatives thereof, such as E. coli
294 (ATCC 31,446), E. coli B, E. coli .lamda.1776 (ATCC 31,537) and
E. coli RV308 (ATCC 31,608) are also suitable. These examples are
illustrative rather than limiting. Methods for constructing
derivatives of any of the above-mentioned bacteria having defined
genotypes are known in the art and described in, for example, Bass
et al., Proteins, 8:309-314 (1990). It is generally necessary to
select the appropriate bacteria taking into consideration
replicability of the replicon in the cells of a bacterium. For
example, E. coli, Serratia, or Salmonella species can be suitably
used as the host when well known plasmids such as pBR322, pBR325,
pACYC177, or pKN410 are used to supply the replicon. Typically the
host cell should secrete minimal amounts of proteolytic enzymes,
and additional protease inhibitors may desirably be incorporated in
the cell culture.
[0237] Antibody Production
[0238] Host cells are transformed with the above-described
expression vectors and cultured in conventional nutrient media
modified as appropriate for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired
sequences.
[0239] Transformation means introducing DNA into the prokaryotic
host so that the DNA is replicable, either as an extrachromosomal
element or by chromosomal integrant. Depending on the host cell
used, transformation is done using standard techniques appropriate
to such cells. The calcium treatment employing calcium chloride is
generally used for bacterial cells that contain substantial
cell-wall barriers. Another method for transformation employs
polyethylene glycol/DMSO. Yet another technique used is
electroporation.
[0240] Prokaryotic cells used to produce the polypeptides of the
invention are grown in media known in the art and suitable for
culture of the selected host cells. Examples of suitable media
include luria broth (LB) plus necessary nutrient supplements. In
some embodiments, the media also contains a selection agent, chosen
based on the construction of the expression vector, to selectively
permit growth of prokaryotic cells containing the expression
vector. For example, ampicillin is added to media for growth of
cells expressing ampicillin resistant gene.
[0241] Any necessary supplements besides carbon, nitrogen, and
inorganic phosphate sources may also be included at appropriate
concentrations introduced alone or as a mixture with another
supplement or medium such as a complex nitrogen source. Optionally
the culture medium may contain one or more reducing agents selected
from the group consisting of glutathione, cysteine, cystamine,
thioglycollate, dithioerythritol and dithiothreitol.
[0242] The prokaryotic host cells are cultured at suitable
temperatures. For E. coli growth, for example, growth occurs at a
temperature range including, but not limited to, about 20.degree.
C. to about 39.degree. C., about 25.degree. C. to about 37.degree.
C., and at about 30.degree. C. The pH of the medium may be any pH
ranging from about 5 to about 9, depending mainly on the host
organism. For E. coli, the pH can be from about 6.8 to about 7.4,
or about 7.0.
[0243] If an inducible promoter is used in the expression vector of
the invention, protein expression is induced under conditions
suitable for the activation of the promoter. In one aspect of the
invention, PhoA promoters are used for controlling transcription of
the polypeptides. Accordingly, the transformed host cells are
cultured in a phosphate-limiting medium for induction. In one
embodiment, the phosphate-limiting medium is the C.R.A.P medium
(see, e.g., Simmons et al., J. Immunol. Methods (2002),
263:133-147). A variety of other inducers may be used, according to
the vector construct employed, as is known in the art.
[0244] In one embodiment, the expressed polypeptides of the present
invention are secreted into and recovered from the periplasm of the
host cells. Protein recovery typically involves disrupting the
microorganism, generally by such means as osmotic shock, sonication
or lysis. Once cells are disrupted, cell debris or whole cells may
be removed by centrifugation or filtration. The proteins may be
further purified, for example, by affinity resin chromatography.
Alternatively, proteins can be transported into the culture media
and isolated therein. Cells may be removed from the culture and the
culture supernatant being filtered and concentrated for further
purification of the proteins produced. The expressed polypeptides
can be further isolated and identified using commonly known methods
such as polyacrylamide gel electrophoresis (PAGE) and Western blot
assay.
[0245] In one aspect of the invention, antibody production is
conducted in large quantity by a fermentation process. Various
large-scale fed-batch fermentation procedures are available for
production of recombinant proteins. Large-scale fermentations have
at least 1000 liters of capacity, for example about 1,000 to
100,000 liters of capacity. These fermentors use agitator impellers
to distribute oxygen and nutrients, especially glucose (a common
carbon/energy source). Small scale fermentation refers generally to
fermentation in a fermentor that is no more than approximately 100
liters in volumetric capacity, and can range from about 1 liter to
about 100 liters.
[0246] In a fermentation process, induction of protein expression
is typically initiated after the cells have been grown under
suitable conditions to a desired density, e.g., an OD550 of about
180-220, at which stage the cells are in the early stationary
phase. A variety of inducers may be used, according to the vector
construct employed, as is known in the art and described above.
Cells may be grown for shorter periods prior to induction. Cells
are usually induced for about 12-50 hours, although longer or
shorter induction time may be used.
[0247] To improve the production yield and quality of the
polypeptides of the invention, various fermentation conditions can
be modified. For example, to improve the proper assembly and
folding of the secreted antibody polypeptides, additional vectors
overexpressing chaperone proteins, such as Dsb proteins (DsbA,
DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl
cis,trans-isomerase with chaperone activity) can be used to
co-transform the host prokaryotic cells. The chaperone proteins
have been demonstrated to facilitate the proper folding and
solubility of heterologous proteins produced in bacterial host
cells. Chen et al. (1999) J Bio Chem 274:19601-19605; Georgiou et
al., U.S. Pat. No. 6,083,715; Georgiou et al., U.S. Pat. No.
6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem.
275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem.
275:17106-17113; Arie et al. (2001) Mol. Microbiol. 39:199-210.
[0248] To minimize proteolysis of expressed heterologous proteins
(especially those that are proteolytically sensitive), certain host
strains deficient for proteolytic enzymes can be used for the
present invention. For example, host cell strains may be modified
to effect genetic mutation(s) in the genes encoding known bacterial
proteases such as Protease III, OmpT, DegP, Tsp, Protease I,
Protease Mi, Protease V, Protease VI and combinations thereof. Some
E. coli protease-deficient strains are available and described in,
for example, Joly et al. (1998), supra; Georgiou et al., U.S. Pat.
No. 5,264,365; Georgiou et al., U.S. Pat. No. 5,508,192; Hara et
al., Microbial Drug Resistance, 2:63-72 (1996).
[0249] In one embodiment, E. coli strains deficient for proteolytic
enzymes and transformed with plasmids overexpressing one or more
chaperone proteins are used as host cells in the expression system
of the invention.
[0250] Antibody Purification
[0251] In one embodiment, the antibody protein produced herein is
further purified to obtain preparations that are substantially
homogeneous for further assays and uses. Standard protein
purification methods known in the art can be employed. The
following procedures are exemplary of suitable purification
procedures: fractionation on immunoaffinity or ion-exchange
columns, ethanol precipitation, reverse phase HPLC, chromatography
on silica or on a cation-exchange resin such as DEAE,
chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel
filtration using, for example, Sephadex G-75.
[0252] In one aspect, Protein A immobilized on a solid phase is
used for immunoaffinity purification of the antibody products of
the invention. Protein A is a 41 kD cell wall protein from
Staphylococcus aureas which binds with a high affinity to the Fc
region of antibodies. Lindmark et al (1983) J. Immunol. Meth.
62:1-13. The solid phase to which Protein A is immobilized can be a
column comprising a glass or silica surface, or a controlled pore
glass column or a silicic acid column. In some applications, the
column is coated with a reagent, such as glycerol, to possibly
prevent nonspecific adherence of contaminants.
[0253] As the first step of purification, the preparation derived
from the cell culture as described above can be applied onto a
Protein A immobilized solid phase to allow specific binding of the
antibody of interest to Protein A. The solid phase would then be
washed to remove contaminants non-specifically bound to the solid
phase. Finally the antibody of interest is recovered from the solid
phase by elution.
[0254] Generating Antibodies Using Eukaryotic Host Cells
[0255] The vector components generally include, but are not limited
to, one or more of the following: a signal sequence, an origin of
replication, one or more marker genes, an enhancer element, a
promoter, and a transcription termination sequence.
[0256] (i) Signal Sequence Component
[0257] A vector for use in a eukaryotic host cell may also contain
a signal sequence or other polypeptide having a specific cleavage
site at the N-terminus of the mature protein or polypeptide of
interest. The heterologous signal sequence selected generally is
one that is recognized and processed (i.e., cleaved by a signal
peptidase) by the host cell. In mammalian cell expression,
mammalian signal sequences as well as viral secretory leaders, for
example, the herpes simplex gD signal, are available.
[0258] The DNA for such precursor region is ligated in reading
frame to DNA encoding the antibody.
[0259] (ii) Origin of Replication
[0260] Generally, an origin of replication component is not needed
for mammalian expression vectors. For example, the SV40 origin may
typically be used only because it contains the early promoter.
[0261] (iii) Selection Gene Component
[0262] Expression and cloning vectors may contain a selection gene,
also termed a selectable marker. Typical selection genes encode
proteins that (a) confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic deficiencies, where relevant, or (c) supply
critical nutrients not available from complex media.
[0263] One example of a selection scheme utilizes a drug to arrest
growth of a host cell. Those cells that are successfully
transformed with a heterologous gene produce a protein conferring
drug resistance and thus survive the selection regimen. Examples of
such dominant selection use the drugs neomycin, mycophenolic acid
and hygromycin.
[0264] Another example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the antibody nucleic acid, such as DHFR, thymidine
kinase, metallothionein-I and -II (e.g., primate metallothionein
genes), adenosine deaminase, ornithine decarboxylase, etc.
[0265] For example, cells transformed with the DHFR selection gene
may first be identified by culturing all of the transformants in a
culture medium that contains methotrexate (Mtx), a competitive
antagonist of DHFR. Appropriate host cells when wild-type DHFR is
employed include, for example, the Chinese hamster ovary (CHO) cell
line deficient in DHFR activity (e.g., ATCC CRL-9096).
[0266] Alternatively, host cells (particularly wild-type hosts that
contain endogenous DHFR) transformed or co-transformed with DNA
sequences encoding an antibody, wild-type DHFR protein, and another
selectable marker such as aminoglycoside 3'-phosphotransferase
(APH) can be selected by cell growth in medium containing a
selection agent for the selectable marker such as an
aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See
U.S. Pat. No. 4,965,199.
[0267] (iv) Promoter Component
[0268] Expression and cloning vectors usually contain a promoter
that is recognized by the host organism and is operably linked to
nucleic acid encoding a polypeptide of interest (e.g., an
antibody). Promoter sequences are known for eukaryotes. Virtually
all eukaryotic genes have an AT-rich region located approximately
25 to 30 bases upstream from the site where transcription is
initiated. Another sequence found 70 to 80 bases upstream from the
start of transcription of many genes is a CNCAAT region where N may
be any nucleotide. At the 3' end of most eukaryotic genes is an
AATAAA sequence that may be the signal for addition of the poly A
tail to the 3' end of the coding sequence. All of these sequences
are suitably inserted into eukaryotic expression vectors.
[0269] Antibody polypeptide transcription from vectors in mammalian
host cells can be controlled, for example, by promoters obtained
from the genomes of viruses such as polyoma virus, fowlpox virus,
adenovirus (such as Adenovirus 2), bovine papilloma virus, avian
sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and
Simian Virus 40 (SV40), from heterologous mammalian promoters,
e.g., the actin promoter or an immunoglobulin promoter, or from
heat-shock promoters, provided such promoters are compatible with
the host cell systems.
[0270] The early and late promoters of the SV40 virus are
conveniently obtained as an SV40 restriction fragment that also
contains the SV40 viral origin of replication. The immediate early
promoter of the human cytomegalovirus is conveniently obtained as a
HindIII E restriction fragment. A system for expressing DNA in
mammalian hosts using the bovine papilloma virus as a vector is
disclosed in U.S. Pat. No. 4,419,446. A modification of this system
is described in U.S. Pat. No. 4,601,978. See also Reyes et al.,
Nature 297:598-601 (1982) on expression of human .beta.-interferon
cDNA in mouse cells under the control of a thymidine kinase
promoter from herpes simplex virus. Alternatively, the Rous Sarcoma
Virus long terminal repeat can be used as the promoter.
[0271] (v) Enhancer Element Component
[0272] Transcription of DNA encoding an antibody polypeptide of the
invention by higher eukaryotes can often be increased by inserting
an enhancer sequence into the vector. Many enhancer sequences are
now known from mammalian genes (globin, elastase, albumin,
.alpha.-fetoprotein, and insulin). Typically, however, one will use
an enhancer from a eukaryotic cell virus. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270),
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing
elements for activation of eukaryotic promoters. The enhancer may
be spliced into the vector at a position 5' or 3' to the antibody
polypeptide-encoding sequence, but is generally located at a site
5' from the promoter.
[0273] (vi) Transcription Termination Component
[0274] Expression vectors used in eukaryotic host cells will
typically also contain sequences necessary for the termination of
transcription and for stabilizing the mRNA. Such sequences are
commonly available from the 5' and, occasionally 3', untranslated
regions of eukaryotic or viral DNAs or cDNAs. These regions contain
nucleotide segments transcribed as polyadenylated fragments in the
untranslated portion of the mRNA encoding an antibody. One useful
transcription termination component is the bovine growth hormone
polyadenylation region. See WO94/11026 and the expression vector
disclosed therein.
[0275] (vii) Selection and Transformation of Host Cells
[0276] Suitable host cells for cloning or expressing the DNA in the
vectors herein include higher eukaryote cells described herein,
including vertebrate host cells. Propagation of vertebrate cells in
culture (tissue culture) has become a routine procedure. Examples
of useful mammalian host cell lines are monkey kidney CV1 line
transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney
line (293 or 293 cells subcloned for growth in suspension culture,
Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney
cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO,
Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse
sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980));
monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney
cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells
(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);
buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells
(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse
mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,
Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells;
and a human hepatoma line (Hep G2).
[0277] Host cells are transformed with the above-described
expression or cloning vectors for antibody production and cultured
in conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences.
[0278] (viii) Culturing the Host Cells
[0279] The host cells used to produce an antibody of this invention
may be cultured in a variety of media. Commercially available media
such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM),
(Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium
((DMEM), Sigma) are suitable for culturing the host cells. In
addition, any of the media described in Ham et al., Meth. Enz.
58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.
Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;
WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as
culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as GENTAMYCIN.TM. drug), trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0280] (ix) Purification of Antibody
[0281] When using recombinant techniques, the antibody can be
produced intracellularly, or directly secreted into the medium. If
the antibody is produced intracellularly, as a first step, the
particulate debris, either host cells or lysed fragments, are
generally removed, for example, by centrifugation or
ultrafiltration. Where the antibody is secreted into the medium,
supernatants from such expression systems are generally first
concentrated using a commercially available protein concentration
filter, for example, an Amicon or Millipore Pellicon
ultrafiltration unit. A protease inhibitor such as PMSF may be
included in any of the foregoing steps to inhibit proteolysis and
antibiotics may be included to prevent the growth of adventitious
contaminants.
[0282] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being a generally acceptable purification
technique. The suitability of affinity reagents such as protein A
as an affinity ligand depends on the species and isotype of any
immunoglobulin Fc domain that is present in the antibody. Protein A
can be used to purify antibodies that are based on human .gamma.1,
.gamma.2, or .gamma.4 heavy chains (Lindmark et al., J. Immunol.
Meth. 62:1-13 (1983)). Protein G is recommended for all mouse
isotypes and for human .gamma.3 (Guss et al., EMBO J. 5:15671575
(1986)). The matrix to which the affinity ligand is attached is
most often agarose, but other matrices are available. Mechanically
stable matrices such as controlled pore glass or
poly(styrenedivinyl)benzene allow for faster flow rates and shorter
processing times than can be achieved with agarose. Where the
antibody comprises a CH3 domain, the Bakerbond ABX.TM. resin (J. T.
Baker, Phillipsburg, N.J.) is useful for purification. Other
techniques for protein purification such as fractionation on an
ion-exchange column, ethanol precipitation, Reverse Phase HPLC,
chromatography on silica, chromatography on heparin SEPHAROSE.TM.
chromatography on an anion or cation exchange resin (such as a
polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium
sulfate precipitation are also available depending on the antibody
to be recovered.
[0283] Following any preliminary purification step(s), the mixture
comprising the antibody of interest and contaminants may be
subjected to further purification steps, as necessary, for example
by low pH hydrophobic interaction chromatography using an elution
buffer at a pH between about 2.5-4.5, generally performed at low
salt concentrations (e.g., from about 0-0.25M salt).
[0284] It should be noted that, in general, techniques and
methodologies for preparing antibodies for use in research, testing
and clinical use are well-established in the art, consistent with
the above and/or as deemed appropriate by one skilled in the art
for the particular antibody of interest.
[0285] Activity Assays
[0286] Antibodies of the invention can be characterized for their
physical/chemical properties and biological functions by various
assays known in the art.
[0287] Purified antibodies can be further characterized by a series
of assays including, but not limited to, N-terminal sequencing,
amino acid analysis, non-denaturing size exclusion high pressure
liquid chromatography (HPLC), mass spectrometry, ion exchange
chromatography and papain digestion.
[0288] Where necessary, antibodies are analyzed for their
biological activity. In some embodiments, antibodies of the
invention are tested for their antigen binding activity. The
antigen binding assays that are known in the art and can be used
herein include without limitation any direct or competitive binding
assays using techniques such as western blots, radioimmunoassays,
ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays,
immunoprecipitation assays, fluorescent immunoassays, and protein A
immunoassays.
[0289] In one embodiment, the invention contemplates an altered
antibody that possesses some but not all effector functions, which
make it a desirable candidate for many applications in which the
half life of the antibody in vivo is important yet certain effector
functions (such as complement and ADCC) are unnecessary or
deleterious. In certain embodiments, the Fc activities of the
antibody are measured to ensure that only the desired properties
are maintained. In vitro and/or in vivo cytotoxicity assays can be
conducted to confirm the reduction/depletion of CDC and/or ADCC
activities. For example, Fc receptor (FcR) binding assays can be
conducted to ensure that the antibody lacks Fc.gamma.R binding
(hence likely lacking ADCC activity), but retains FcRn binding
ability. The primary cells for mediating ADCC, NK cells, express
Fc.gamma.RIII only, whereas monocytes express Fc.gamma.RI,
Fc.gamma.RII and Fc.gamma.RIII. FcR expression on hematopoietic
cells is summarized in Table 3 on page 464 of Ravetch and Kinet,
Annu. Rev. Immunol 9:457-92 (1991). An example of an in vitro assay
to assess ADCC activity of a molecule of interest is described in
U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337. Useful effector
cells for such assays include peripheral blood mononuclear cells
(PBMC) and Natural Killer (NK) cells. Alternatively, or
additionally, ADCC activity of the molecule of interest may be
assessed in vivo, e.g., in an animal model such as that disclosed
in Clynes et al. PNAS (USA) 95:652-656 (1998). C1q binding assays
may also be carried out to confirm that the antibody is unable to
bind C1q and hence lacks CDC activity. To assess complement
activation, a CDC assay, e.g. as described in Gazzano-Santoro et
al., J. Immunol. Methods 202:163 (1996), may be performed. FcRn
binding and in vivo clearance/half life determinations can also be
performed using methods known in the art.
[0290] Antibody Fragments
[0291] The present invention encompasses antibody fragments. In
certain circumstances there are advantages of using antibody
fragments, rather than whole antibodies. The smaller size of the
fragments allows for rapid clearance, and may lead to improved
access to solid tumors.
[0292] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992); and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
Fab, Fv and ScFv antibody fragments can all be expressed in and
secreted from E. coli, thus allowing the facile production of large
amounts of these fragments. Antibody fragments can be isolated from
the antibody phage libraries discussed above. Alternatively,
Fab'-SH fragments can be directly recovered from E. coli and
chemically coupled to form F(ab')2 fragments (Carter et al.,
Bio/Technology 10: 163-167 (1992)). According to another approach,
F(ab')2 fragments can be isolated directly from recombinant host
cell culture. Fab and F(ab')2 fragment with increased in vivo
half-life comprising salvage receptor binding epitope residues are
described in U.S. Pat. No. 5,869,046. Other techniques for the
production of antibody fragments will be apparent to the skilled
practitioner. In other embodiments, the antibody of choice is a
single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos.
5,571,894; and 5,587,458. Fv and sFv are the only species with
intact combining sites that are devoid of constant regions; thus,
they are suitable for reduced nonspecific binding during in vivo
use. sFv fusion proteins may be constructed to yield fusion of an
effector protein at either the amino or the carboxy terminus of an
sFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody
fragment may also be a "linear antibody", e.g., as described in
U.S. Pat. No. 5,641,870 for example. Such linear antibody fragments
may be monospecific or bispecific.
[0293] Humanized Antibodies
[0294] Any of the antibodies described herein can be a full length
antibody or an antigen-binding fragment thereof. In some examples,
the antigen binding fragment is a Fab fragment, a F(ab')2 fragment,
or a single-chain Fv fragment. In some examples, the antigen
binding fragment is a Fab fragment, a F(ab')2 fragment, or a
single-chain Fv fragment. In some examples, the isolated antibody
is a human antibody, a humanized antibody, a chimeric antibody, or
a single-chain antibody.
[0295] Any of the antibodies described herein has one or more
characteristics of:
[0296] a) is a recombinant antibody, a monoclonal antibody, a
chimeric antibody, a humanized antibody, a human antibody, an
antibody fragment, a bispecific antibody, a monospecific antibody,
a monovalent antibody, an IgG1 antibody, an IgG2 antibody, or
derivative of an antibody; b) is a human, murine, humanized, or
chimeric antibody, antigen-binding fragment, or derivative of an
antibody; c) is a single-chain antibody fragment, a multibody, a
Fab fragment, and/or an immunoglobulin of the IgG, IgM, IgA, IgE,
IgD isotypes and/or subclasses thereof; d) has one or more of the
following characteristics: (i) mediates ADCC and/or CDC of cancer
cells; (ii) induces and/or promotes apoptosis of cancer cells;
(iii) inhibits proliferation of target cells of cancer cells; (iv)
induces and/or promotes phagocytosis of cancer cells; and/or (v)
induces and/or promotes the release of cytotoxic agents; e)
specifically binds the tumor-associated carbohydrate antigen, which
is a tumor-specific carbohydrate antigen; f) does not bind an
antigen expressed on non-cancer cells, non-tumor cells, benign
cancer cells and/or benign tumor cells; and/or g) specifically
binds a tumor-associated carbohydrate antigen expressed on cancer
stem cells and on normal cancer cells.
[0297] Preferably the binding of the antibodies to their respective
antigens is specific. The term "specific" is generally used to
refer to the situation in which one member of a binding pair will
not show any significant binding to molecules other than its
specific binding partner (s) and e.g. has less than about 30%,
preferably 20%, 10%, or 1% cross-reactivity with any other molecule
other than those specified herein.
[0298] The antibodies are suitable bind to its target epitopes with
a high affinity (low KD value), and preferably KD is in the
nanomolar range or lower. Affinity can be measured by methods known
in the art, such as, for example; surface plasmon resonance.
[0299] Exemplary Antibody Preparation
[0300] Exemplary Antibodies capable of binding to the Globo H
epitopes and SSEA-4 epitopes described herein can be made by any
method known in the art. See, for example, Harlow and Lane, (1988)
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New
York.
[0301] Immunization of Host Animals and Hybridoma Technology
[0302] Exemplary Polyclonal antibodies against the anti-Globo Hand
anti-SSEA-4 antibodies may be prepared by collecting blood from the
immunized mammal examined for the increase of desired antibodies in
the serum, and by separating serum from the blood by any
conventional method. Polyclonal antibodies include serum containing
the polyclonal antibodies, as well as the fraction containing the
polyclonal antibodies may be isolated from the serum.
[0303] Polyclonal antibodies are generally raised in host animals
(e.g., rabbit, mouse, horse, or goat) by multiple subcutaneous (sc)
or intraperitoneal (ip) injections of the relevant antigen and an
adjuvant. It may be useful to conjugate the relevant antigen to a
protein that is immunogenic in the species to be immunized, e.g.,
keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or
soybean trypsin inhibitor using a bifunctional or derivatizing
agent, for example, maleimidobenzoyl sulfosuccinimide ester
(conjugation through cysteine residues), N-hydroxysuccinimide
(through lysine residues), glutaraldehyde, succinic anhydride,
SOCl.sub.2, etc.
[0304] Any mammalian animal may be immunized with the antigen for
producing the desired antibodies. In general, animals of Rodentia,
Lagomorpha, or Primates can be used. Animals of Rodentia include,
for example, mouse, rat, and hamster. Animals of Lagomorpha
include, for example, rabbit. Animals of Primates include, for
example, a monkey of Catarrhini (old world monkey) such as Macaca
fascicularis, rhesus monkey, baboon, and chimpanzees.
[0305] Methods for immunizing animals with antigens are known in
the art. Intraperitoneal injection or subcutaneous injection of
antigens is a standard method for immunization of mammals. More
specifically, antigens may be diluted and suspended in an
appropriate amount of phosphate buffered saline (PBS),
physiological saline, etc. If desired, the antigen suspension may
be mixed with an appropriate amount of a standard adjuvant, such as
Freund's complete adjuvant, made into emulsion, and then
administered to mammalian animals. Animals are immunized against
the antigen, immunogenic conjugates, or derivatives by combining 1
mg or 1 .mu.g of the peptide or conjugate (for rabbits or mice,
respectively) with 3 volumes of Freund's incomplete adjuvant.
[0306] Animals can be boosted until the titer plateaus by several
administrations of antigen mixed with an appropriately amount of
Freund's incomplete adjuvant every 4 to 21 days. Animals are
boosted with 1/5 to 1/10 the original amount of peptide or
conjugate in Freund's complete adjuvant by subcutaneous injection
at multiple sites. Seven to 14 days later the animals are bled and
the serum is assayed for antibody titer. An appropriate carrier may
also be used for immunization. After immunization as above, serum
is examined by a standard method for an increase in the amount of
desired antibodies. Preferably, the animal is boosted with the
conjugate of the same antigen, but conjugated to a different
protein and/or through a different cross-linking reagent.
Conjugates also can be made in recombinant cell culture as protein
fusions. Also, aggregating agents such as alum are suitably used to
enhance the immune response.
[0307] Over the past two to three decades, a number of
methodologies have been developed to prepare chimeric, humanized or
human antibodies for human in-vivo therapeutic applications. The
most used and proven methodology is to prepare mouse mAbs using
hybridoma methodology and then to humanize the mAbs by converting
the framework regions of the V.sub.H and V.sub.L domains and
constant domains of the mAbs into most homologous human framework
regions of human V.sub.H and V.sub.L domains and constant regions
of a desirable human .gamma. immunoglobulin isotype and subclass.
Many mAbs, such as Xolair, used clinically are humanized mAbs of
human .gamma.1, .kappa. isotype and subclass and prepared using
this methodology.
[0308] In some embodiments, antibodies can be made by the
conventional hybridoma technology. Kohler et al., Nature, 256:495
(1975). In the hybridoma method, a mouse or other appropriate host
animal, such as a hamster or rabbit, is immunized as hereinabove
described to elicit lymphocytes that produce or are capable of
producing antibodies that will specifically bind to the protein
used for immunization. Alternatively, lymphocytes may be immunized
in vitro.
[0309] To prepare monoclonal antibodies, immune cells are collected
from the mammal immunized with the antigen and checked for the
increased level of desired antibodies in the serum as described
above, and are subjected to cell fusion. The immune cells used for
cell fusion are preferably obtained from spleen. Other preferred
parental cells to be fused with the above immunocyte include, for
example, myeloma cells of mammalians, and more preferably myeloma
cells having an acquired property for the selection of fused cells
by drugs.
[0310] Preferred myeloma cells are those that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these, preferred myeloma cell lines are murine
myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse
tumors available from the Salk Institute Cell Distribution Center,
San Diego, Calif. USA, and SP-2 cells available from the American
Type Culture Collection, Rockville, Md. USA. Human myeloma and
mouse-human heteromyeloma cell lines also have been described for
the production of human monoclonal antibodies (Kozbor, J. Immunol.,
133:3001 (1984); Brodeur et al., Monoclonal Antibody Production
Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New
York, 1987)).
[0311] The above immunocyte and myeloma cells can be fused
according to known methods, for example, the method of Milstein et
al. (Galfre et al., Methods Enzymol. 73:3-46, 1981). Lymphocytes
are fused with myeloma cells using a suitable fusing agent, such as
polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal
Antibodies: Principles and Practice, pp. 59-103 (Academic Press,
1986)). Resulting hybridomas obtained by the cell fusion may be
selected by cultivating them in a standard selection medium, such
as HAT medium (hypoxanthine, aminopterin, and thymidine containing
medium). The cell culture is typically continued in the HAT medium
for several days to several weeks, the time being sufficient to
allow all the other cells, with the exception of the desired
hybridoma (non-fused cells), to die. Then, the standard limiting
dilution is performed to screen and clone a hybridoma cell
producing the desired antibody.
[0312] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
[0313] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay. Measurement of
absorbance in enzyme-linked immunosorbent assay (ELISA), enzyme
immunoassay (EIA), radioimmunoassay (MA), and/or immunofluorescence
may be used to measure the antigen binding activity of the antibody
of the invention. In ELISA, the antibody of the present invention
is immobilized on a plate, protein of the invention is applied to
the plate, and then a sample containing a desired antibody, such as
culture supernatant of antibody producing cells or purified
antibodies, is applied. Then, a secondary antibody that recognizes
the primary antibody and is labeled with an enzyme, such as
alkaline phosphatase, is applied, and the plate is incubated. Next,
after washing, an enzyme substrate, such as p-nitrophenyl
phosphate, is added to the plate, and the absorbance is measured to
evaluate the antigen binding activity of the sample. A fragment of
the protein, such as a C-terminal or N-terminal fragment may be
used in this method. BIAcore (Pharmacia) may be used to evaluate
the activity of the antibody according to the present invention.
The binding affinity of the monoclonal antibody can, for example,
be determined by the Scatchard analysis of Munson et al., Anal.
Biochem., 107:220 (1980).
[0314] Applying any of the conventional methods, including those
described above, hybridoma cells producing antibodies that bind to
epitopes described herein can be identified and selected for
further characterization.
[0315] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture
media for this purpose include, for example, D-MEM or RPMI-1640
medium. The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0316] In addition, the hybridoma cells may be grown in vivo as
ascites tumors in an animal. For example, the obtained hybridomas
can be subsequently transplanted into the abdominal cavity of a
mouse and the ascites are harvested.
[0317] The obtained monoclonal antibodies can be purified by, for
example, ammonium sulfate precipitation, a protein A or protein G
column, DEAE ion exchange chromatography, or an affinity column to
which the protein of the present invention is coupled. The antibody
of the present invention can be used not only for purification and
detection of the protein of the present invention, but also as a
candidate for agonists and antagonists of the protein of the
present invention. In addition, this antibody can be applied to the
antibody treatment for diseases related to the protein of the
present invention.
[0318] Recombinant Technology
[0319] The monoclonal antibodies thus obtained can be also
recombinantly prepared using genetic engineering techniques (see,
for example, Borrebaeck C. A. K. and Larrick J. W. Therapeutic
Monoclonal Antibodies, published in the United Kingdom by MacMillan
Publishers LTD, 1990). A DNA encoding an antibody may be cloned
from an immune cell, such as a hybridoma or an immunized lymphocyte
producing the antibody, inserted into an appropriate vector, and
introduced into host cells to prepare a recombinant antibody. The
present invention also provides recombinant antibodies prepared as
described above.
[0320] When the obtained antibody is to be administered to the
human body (antibody treatment), a human antibody or a humanized
antibody is preferable for reducing immunogenicity. For example,
transgenic animals having a repertory of human antibody genes may
be immunized with an antigen selected from a protein, protein
expressing cells, or their lysates. Antibody producing cells are
then collected from the animals and fused with myeloma cells to
obtain hybridoma, from which human antibodies against the protein
can be prepared. Alternatively, an immune cell, such as an
immunized lymphocyte, producing antibodies may be immortalized by
an oncogene and used for preparing monoclonal antibodies.
[0321] DNA encoding the monoclonal antibodies can be readily
isolated and sequenced using conventional procedures (e.g., by
using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells serve as a preferred source of
such DNA. Once isolated, the DNA may be placed into expression
vectors, which are then transfected into host cells such as E. coli
cells, simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. Review articles on recombinant expression in bacteria
of DNA encoding the antibody include Skerra et al., Curr. Opinion
in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Rev.,
130:151-188 (1992).
[0322] DNAs encoding the antibodies produced by the hybridoma cells
described above can be genetically modified, via routine
technology, to produce genetically engineered antibodies.
Genetically engineered antibodies, such as humanized antibodies,
chimeric antibodies, single-chain antibodies, and bi-specific
antibodies, can be produced via, e.g., conventional recombinant
technology. The DNA can then be modified, for example, by
substituting the coding sequence for human heavy and light chain
constant domains in place of the homologous murine sequences,
Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851, or by
covalently joining to the immunoglobulin coding sequence all or
part of the coding sequence for a non-immunoglobulin polypeptide.
In that manner, genetically engineered antibodies, such as
"chimeric" or "hybrid" antibodies; can be prepared that have the
binding specificity of a target antigen.
[0323] Techniques developed for the production of "chimeric
antibodies" are well known in the art. See, e.g., Morrison et al.
(1984) Proc. Natl. Acad. Sci. USA 81, 6851; Neuberger et al. (1984)
Nature 312, 604; and Takeda et al. (1984) Nature 314:452.
[0324] Typically such non-immunoglobulin polypeptides are
substituted for the constant domains of an antibody, or they are
substituted for the variable domains of one antigen-combining site
of an antibody to create a chimeric bivalent antibody comprising
one antigen-combining site having specificity for an antigen and
another antigen-combining site having specificity for a different
antigen.
[0325] Chimeric or hybrid antibodies also may be prepared in vitro
using known methods in synthetic protein chemistry, including those
involving crosslinking agents. For example, immunotoxins may be
constructed using a disulfide-exchange reaction or by forming a
thioether bond. Examples of suitable reagents for this purpose
include iminothiolate and methyl-4-mercaptobutyrimidate.
[0326] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567),
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0327] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework (FR) for the
humanized antibody (Sims et al., J. Immunol., 151:2296 (1993);
Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses
a particular framework derived from the consensus sequence of all
human antibodies of a particular subgroup of light or heavy chains.
The same framework may be used for several different humanized
antibodies (Carter et al., Proc. Natl. Acad Sci. USA, 89:4285
(1992); Presta et al., J. Immnol., 151:2623 (1993)).
[0328] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, according to a
preferred method, humanized antibodies are prepared by a process of
analysis of the parental sequences and various conceptual humanized
products using three-dimensional models of the parental and
humanized sequences. Three-dimensional immunoglobulin models are
commonly available and are familiar to those skilled in the art.
Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i. e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
CDR residues are directly and most substantially involved in
influencing antigen binding.
[0329] Alternatively, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (JO gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array in such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al.,
Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al.,
Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno.,
7:33 (1993). Human antibodies can also be derived from
phage-display libraries (Hoogenboom et al., J. Mol. Biol., 227:381
(1991); Marks et al., J. Mol. Biol., 222:581-597 (1991)).
[0330] Any of the nucleic acid encoding the anti-Globo Hand
anti-SSEA-4 antibodies described herein (including heavy chain,
light chain, or both), vectors such as expression vectors
comprising one or more of the nucleic acids, and host cells
comprising one or more of the vectors are also within the scope of
the present disclosure. In some examples, a vector comprising a
nucleic acid comprising a nucleotide sequence encoding either the
heavy chain variable region or the light chain variable region of
an anti-Globo H antibody as described herein. In some examples, a
vector comprising a nucleic acid comprising a nucleotide sequence
encoding either the heavy chain variable region or the light chain
variable region of an anti-SSEA-4 antibody as described herein. In
other examples, the vector comprises nucleotide sequences encoding
both the heavy chain variable region and the light chain variable
region, the expression of which can be controlled by a single
promoter or two separate promoters. Also provided here are methods
for producing any of the anti-Globo Hand anti-SSEA-4 antibodies as
described herein, e.g., via the recombinant technology described in
this section.
[0331] Other Technology for Preparing Antibodies
[0332] In other embodiments, fully human antibodies can be obtained
by using commercially available mice that have been engineered to
express specific human immunoglobulin proteins. Transgenic animals
that are designed to produce a more desirable (e.g., fully human
antibodies) or more robust immune response may also be used for
generation of humanized or human antibodies. Examples of such
technology are Xenomouse.RTM. from Amgen, Inc. (Fremont, Calif.)
and HuMAb-Mouse.RTM. and TC Mouse.TM. from Medarex, Inc.
(Princeton, N.J.). In another alternative, antibodies may be made
recombinantly by phage display technology. See, for example, U.S.
Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and
Winter et al., (1994) Annu. Rev. Immunol. 12:433-455.
Alternatively, the phage display technology (McCafferty et al.,
(1990) Nature 348:552-553) can be used to produce human antibodies
and antibody fragments in vitro, from immunoglobulin variable (V)
domain gene repertoires from unimmunized donors.
[0333] Antigen-binding fragments of an intact antibody (full-length
antibody) can be prepared via routine methods. For example, F(ab')2
fragments can be produced by pepsin digestion of an antibody
molecule, and Fab fragments that can be generated by reducing the
disulfide bridges of F(ab')2 fragments.
[0334] Alternatively, the anti-Globo Hand anti-SSEA-4 antibodies
described herein can be isolated from antibody phage libraries
(e.g., single-chain antibody phage libraries) generated using the
techniques described in McCafferty et al., Nature, 348:552-554
(1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et
al., J. Mol Biol., 222:581-597 (1991). Subsequent publications
describe the production of high affinity (nM range) human
antibodies by chain shuffling (Marks et al., Bio/Technology,
10:779-783 (1992)), as well as combinatorial infection and in vivo
recombination as a strategy for constructing very large phage
libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266
(1993)). Thus, these techniques are viable alternatives to
traditional monoclonal antibody hybridoma techniques for isolation
of monoclonal antibodies.
[0335] Antibodies obtained as described herein may be purified to
homogeneity. For example, the separation and purification of the
antibody can be performed according to separation and purification
methods used for general proteins. For example, the antibody may be
separated and isolated by the appropriately selected and combined
use of column chromatographies, such as affinity chromatography,
filter, ultrafiltration, salting-out, dialysis, SDS polyacrylamide
gel electrophoresis, isoelectric focusing, and others (Antibodies:
A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor
Laboratory, 1988), but are not limited thereto. The concentration
of the antibodies obtained as above may be determined by the
measurement of absorbance, Enzyme-linked immunosorbent assay
(ELISA), or so on. Exemplary chromatography, with the exception of
affinity includes, for example, ion-exchange chromatography,
hydrophobic chromatography, gel filtration, reverse-phase
chromatography, adsorption chromatography, and the like (Strategies
for Protein Purification and Characterization: A Laboratory Course
Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory
Press, 1996). The chromatographic procedures can be carried out by
liquid-phase chromatography, such as HPLC, FPLC.
[0336] The antibodies can be characterized using methods well known
in the art. For example, one method is to identify the epitope to
which the antigen binds, or "epitope mapping." There are many
methods known in the art for mapping and characterizing the
location of epitopes on proteins, including solving the crystal
structure of an antibody-antigen complex, competition assays, gene
fragment expression assays, and synthetic peptide-based assays, as
described, for example, in Chapter 11 of Harlow and Lane, Using
Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1999. In an additional example,
epitope mapping can be used to determine the sequence to which an
antibody binds. The epitope can be a linear epitope, i.e.,
contained in a single stretch of amino acids, or a conformational
epitope formed by a three-dimensional interaction of amino acids
that may not necessarily be contained in a single stretch (primary
structure linear sequence). Peptides of varying lengths (e.g., at
least 4-6 amino acids long) can be isolated or synthesized (e.g.,
recombinantly) and used for binding assays with an antibody. In
another example, the epitope to which the antibody binds can be
determined in a systematic screening by using overlapping peptides
derived from the target antigen sequence and determining binding by
the antibody. According to the gene fragment expression assays, the
open reading frame encoding the target antigen is fragmented either
randomly or by specific genetic constructions and the reactivity of
the expressed fragments of the antigen with the antibody to be
tested is determined. The gene fragments may, for example, be
produced by PCR and then transcribed and translated into protein in
vitro, in the presence of radioactive amino acids. The binding of
the antibody to the radioactively labeled antigen fragments is then
determined by immunoprecipitation and gel electrophoresis. Certain
epitopes can also be identified by using large libraries of random
peptide sequences displayed on the surface of phage particles
(phage libraries). Alternatively, a defined library of overlapping
peptide fragments can be tested for binding to the test antibody in
simple binding assays.
[0337] In an additional example, mutagenesis of an antigen binding
domain, domain swapping experiments and alanine scanning
mutagenesis can be performed to identify residues required,
sufficient, and/or necessary for epitope binding. For example,
domain swapping experiments can be performed using a mutant of a
target antigen in which various residues in the binding epitope for
the candidate antibody have been replaced (swapped) with sequences
from a closely related, but antigenically distinct protein (such as
another member of the neurotrophin protein family). By assessing
binding of the antibody to the mutant target protein, the
importance of the particular antigen fragment to antibody binding
can be assessed.
[0338] Alternatively, competition assays can be performed using
other antibodies known to bind to the same antigen to determine
whether an antibody binds to the same epitope (e.g., the MC45
antibody described herein) as the other antibodies. Competition
assays are well known to those of skill in the art.
[0339] Additional Aspects of Exemplary Suitable General Antibody
Production Methods
[0340] 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).
[0341] 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.
[0342] 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.
[0343] Compositions comprising at least one
anti-SSEA-3/SSEA-4/Globo H antibody or at least one polynucleotide
comprising sequences encoding an anti-SSEA-3/SSEA-4/Globo H
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 SSEA-3/SSEA-4/Globo
H and/or one or more polynucleotides comprising sequences encoding
one or more antibodies that bind to one or more SSEA-3/SSEA-4/Globo
H. These compositions may further comprise suitable carriers, such
as pharmaceutically acceptable excipients including buffers, which
are well known in the art.
[0344] Isolated antibodies and polynucleotides are also provided.
In certain embodiments, the isolated antibodies and polynucleotides
are substantially pure.
[0345] In one embodiment, anti-SSEA-3/SSEA-4/Globo H antibodies are
monoclonal. In another embodiment, fragments of the
anti-SSEA-3/SSEA-4/Globo H 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.
[0346] A variety of methods are known in the art for generating
phage display libraries from which an antibody of interest can be
obtained. One method of generating antibodies of interest is
through the use of a phage antibody library as described in Lee et
al., J. Mol. Biol. (2004), 340(5): 1073-93.
[0347] The anti-SSEA-3/SSEA-4/Globo H antibodies of the invention
can be made by using combinatorial libraries to screen for
synthetic antibody clones with the desired activity or activities.
In principle, synthetic antibody clones are selected by screening
phage libraries containing phage that display various fragments of
antibody variable region (Fv) fused to phage coat protein. Such
phage libraries are panned by affinity chromatography against the
desired antigen. Clones expressing Fv fragments capable of binding
to the desired antigen are adsorbed to the antigen and thus
separated from the non-binding clones in the library. The binding
clones are then eluted from the antigen, and can be further
enriched by additional cycles of antigen adsorption/elution. Any of
the anti-SSEA-3/SSEA-4/Globo H antibodies of the invention can be
obtained by designing a suitable antigen screening procedure to
select for the phage clone of interest followed by construction of
a full length anti-SSEA-3/SSEA-4/Globo H antibody clone using the
Fv sequences from the phage clone of interest and suitable constant
region (Fc) sequences described in Kabat et al., Sequences of
Proteins of Immunological Interest, Fifth Edition, NIH Publication
91-3242, Bethesda Md. (1991), vols. 1-3.
[0348] The antigen-binding domain of an antibody is formed from two
variable (V) regions of about 110 amino acids, one each from the
light (VL) and heavy (VH) chains, that both present three
hypervariable loops or complementarity-determining regions (CDRs).
Variable domains can be displayed functionally on phage, either as
single-chain Fv (scFv) fragments, in which VH and VL are covalently
linked through a short, flexible peptide, or as Fab fragments, in
which they are each fused to a constant domain and interact
non-covalently, as described in Winter et al., Ann. Rev. Immunol.,
12: 433-455 (1994). As used herein, scFv encoding phage clones and
Fab encoding phage clones are collectively referred to as "Fv phage
clones" or "Fv clones".
[0349] Repertoires of VH and VL genes can be separately cloned by
polymerase chain reaction (PCR) and recombined randomly in phage
libraries, which can then be searched for antigen-binding clones as
described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).
Libraries from immunized sources provide high-affinity antibodies
to the immunogen without the requirement of constructing
hybridomas. Alternatively, the naive repertoire can be cloned to
provide a single source of human antibodies to a wide range of
non-self and also self antigens without any immunization as
described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally,
naive libraries can also be made synthetically by cloning the
unrearranged V-gene segments from stem cells, and using PCR primers
containing random sequence to encode the highly variable CDR3
regions and to accomplish rearrangement in vitro as described by
Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
[0350] Filamentous phage is used to display antibody fragments by
fusion to the minor coat protein pIII. The antibody fragments can
be displayed as single chain Fv fragments, in which VH and VL
domains are connected on the same polypeptide chain by a flexible
polypeptide spacer, e.g. as described by Marks et al., J. Mol.
Biol., 222: 581-597 (1991), or as Fab fragments, in which one chain
is fused to pIII and the other is secreted into the bacterial host
cell periplasm where assembly of a Fab-coat protein structure which
becomes displayed on the phage surface by displacing some of the
wild type coat proteins, e.g. as described in Hoogenboom et al.,
Nucl. Acids Res., 19: 4133-4137 (1991).
[0351] In general, nucleic acids encoding antibody gene fragments
are obtained from immune cells harvested from humans or animals. If
a library biased in favor of anti-SSEA-3/SSEA-4/Globo H clones is
desired, the subject is immunized with SSEA-3/SSEA-4/Globo H to
generate an antibody response, and spleen cells and/or circulating
B cells or other peripheral blood lymphocytes (PBLs) are recovered
for library construction. In one embodiment, a human antibody gene
fragment library biased in favor of anti-human SSEA-3/SSEA-4/Globo
H clones is obtained by generating an anti-human
SSEA-3/SSEA-4/Globo H antibody response in transgenic mice carrying
a functional human immunoglobulin gene array (and lacking a
functional endogenous antibody production system) such that
SSEA-3/SSEA-4/Globo H immunization gives rise to B cells producing
human antibodies against SSEA-3/SSEA-4/Globo H. The generation of
human antibody-producing transgenic mice is described below.
[0352] Additional enrichment for anti-SSEA-3/SSEA-4/Globo H
reactive cell populations can be obtained by using a suitable
screening procedure to isolate B cells expressing
SSEA-3/SSEA-4/Globo H-specific antibody, e.g., by cell separation
with SSEA-3/SSEA-4/Globo H affinity chromatography or adsorption of
cells to fluorochrome-labeled SSEA-3/SSEA-4/Globo H followed by
flow-activated cell sorting (FACS).
[0353] Alternatively, the use of spleen cells and/or B cells or
other PBLs from an unimmunized donor provides a better
representation of the possible antibody repertoire, and also
permits the construction of an antibody library using any animal
(human or non-human) species in which SSEA-3/SSEA-4/Globo H is not
antigenic. For libraries incorporating in vitro antibody gene
construction, stem cells are harvested from the subject to provide
nucleic acids encoding unrearranged antibody gene segments. The
immune cells of interest can be obtained from a variety of animal
species, such as human, mouse, rat, lagomorpha, luprine, canine,
feline, porcine, bovine, equine, and avian species, etc.
[0354] Nucleic acid encoding antibody variable gene segments
(including VH and VL segments) are recovered from the cells of
interest and amplified. In the case of rearranged VH and VL gene
libraries, the desired DNA can be obtained by isolating genomic DNA
or mRNA from lymphocytes followed by polymerase chain reaction
(PCR) with primers matching the 5' and 3' ends of rearranged VH and
VL genes as described in Orlandi et al., Proc. Natl. Acad. Sci.
(USA), 86: 3833-3837 (1989), thereby making diverse V gene
repertoires for expression. The V genes can be amplified from cDNA
and genomic DNA, with back primers at the 5' end of the exon
encoding the mature V-domain and forward primers based within the
J-segment as described in Orlandi et al. (1989) and in Ward et al.,
Nature, 341: 544-546 (1989). However, for amplifying from cDNA,
back primers can also be based in the leader exon as described in
Jones et al., Biotechnol., 9: 88-89 (1991), and forward primers
within the constant region as described in Sastry et al., Proc.
Natl. Acad. Sci. (USA), 86: 5728-5732 (1989). To maximize
complementarity, degeneracy can be incorporated in the primers as
described in Orlandi et al. (1989) or Sastry et al. (1989). In
certain embodiments, the library diversity is maximized by using
PCR primers targeted to each V-gene family in order to amplify all
available VH and VL arrangements present in the immune cell nucleic
acid sample, e.g. as described in the method of Marks et al., J.
Mol. Biol., 222: 581-597 (1991) or as described in the method of
Orum et al., Nucleic Acids Res., 21: 4491-4498 (1993). For cloning
of the amplified DNA into expression vectors, rare restriction
sites can be introduced within the PCR primer as a tag at one end
as described in Orlandi et al. (1989), or by further PCR
amplification with a tagged primer as described in Clackson et al.,
Nature, 352: 624-628 (1991).
[0355] Repertoires of synthetically rearranged V genes can be
derived in vitro from V gene segments. Most of the human VH-gene
segments have been cloned and sequenced (reported in Tomlinson et
al., J. Mol. Biol., 227: 776-798 (1992)), and mapped (reported in
Matsuda et al., Nature Genet., 3: 88-94 (1993); these cloned
segments (including all the major conformations of the H1 and H2
loop) can be used to generate diverse VH gene repertoires with PCR
primers encoding H3 loops of diverse sequence and length as
described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388
(1992). VH repertoires can also be made with all the sequence
diversity focused in a long H3 loop of a single length as described
in Barbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992).
Human V.kappa. and V.lamda. segments have been cloned and sequenced
(reported in Williams and Winter, Eur. J. Immunol., 23: 1456-1461
(1993)) and can be used to make synthetic light chain repertoires.
Synthetic V gene repertoires, based on a range of VH and VL folds,
and L3 and H3 lengths, will encode antibodies of considerable
structural diversity. Following amplification of V-gene encoding
DNAs, germline V-gene segments can be rearranged in vitro according
to the methods of Hoogenboom and Winter, J. Mol. Biol., 227:
381-388 (1992).
[0356] Repertoires of antibody fragments can be constructed by
combining VH and VL gene repertoires together in several ways. Each
repertoire can be created in different vectors, and the vectors
recombined in vitro, e.g., as described in Hogrefe et al., Gene,
128: 119-126 (1993), or in vivo by combinatorial infection, e.g.,
the loxP system described in Waterhouse et al., Nucl. Acids Res.,
21: 2265-2266 (1993). The in vivo recombination approach exploits
the two-chain nature of Fab fragments to overcome the limit on
library size imposed by E. coli transformation efficiency. Naive VH
and VL repertoires are cloned separately, one into a phagemid and
the other into a phage vector. The two libraries are then combined
by phage infection of phagemid-containing bacteria so that each
cell contains a different combination and the library size is
limited only by the number of cells present (about 1012 clones).
Both vectors contain in vivo recombination signals so that the VH
and VL genes are recombined onto a single replicon and are
co-packaged into phage virions. These huge libraries provide large
numbers of diverse antibodies of good affinity (Kd-1 of about 10-8
M).
[0357] Alternatively, the repertoires may be cloned sequentially
into the same vector, e.g. as described in Barbas et al., Proc.
Natl. Acad. Sci. USA, 88: 7978-7982 (1991), or assembled together
by PCR and then cloned, e.g. as described in Clackson et al.,
Nature, 352: 624-628 (1991). PCR assembly can also be used to join
VH and VL DNAs with DNA encoding a flexible peptide spacer to form
single chain Fv (scFv) repertoires. In yet another technique, "in
cell PCR assembly" is used to combine VH and VL genes within
lymphocytes by PCR and then clone repertoires of linked genes as
described in Embleton et al., Nucl. Acids Res., 20: 3831-3837
(1992).
[0358] Screening of the libraries can be accomplished by any
art-known technique. For example, SSEA-3/SSEA-4/Globo H targets can
be used to coat the wells of adsorption plates, expressed on host
cells affixed to adsorption plates or used in cell sorting, or
conjugated to biotin for capture with streptavidin-coated beads, or
used in any other art-known method for panning phage display
libraries.
[0359] The phage library samples are contacted with immobilized
SSEA-3/SSEA-4/Globo H under conditions suitable for binding of at
least a portion of the phage particles with the adsorbent.
Normally, the conditions, including pH, ionic strength, temperature
and the like are selected to mimic physiological conditions. The
phages bound to the solid phase are washed and then eluted by acid,
e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88:
7978-7982 (1991), or by alkali, e.g. as described in Marks et al.,
J. Mol. Biol., 222: 581-597 (1991), or by SSEA-3/SSEA-4/Globo H
antigen competition, e.g. in a procedure similar to the antigen
competition method of Clackson et al., Nature, 352: 624-628 (1991).
Phages can be enriched from about 20.times. to about 1,000-fold in
a single round of selection. Moreover, the enriched phages can be
grown in bacterial culture and subjected to further rounds of
selection.
[0360] The efficiency of selection depends on many factors,
including the kinetics of dissociation during washing, and whether
multiple antibody fragments on a single phage can simultaneously
engage with antigen. Antibodies with fast dissociation kinetics
(and weak binding affinities) can be retained by use of short
washes, multivalent phage display and high coating density of
antigen in solid phase. The high density not only stabilizes the
phage through multivalent interactions, but favors rebinding of
phage that has dissociated. The selection of antibodies with slow
dissociation kinetics (and good binding affinities) can be promoted
by use of long washes and monovalent phage display as described in
Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and a
low coating density of antigen as described in Marks et al.,
Biotechnol., 10: 779-783 (1992).
[0361] It is possible to select between phage antibodies of
different affinities, even with affinities that differ slightly,
for SSEA-3/SSEA-4/Globo H. However, random mutation of a selected
antibody (e.g. as performed in some of the affinity maturation
techniques described above) is likely to give rise to many mutants,
most binding to antigen, and a few with higher affinity. With
limiting SSEA-3/SSEA-4/Globo H, rare high affinity phage could be
competed out. To retain all the higher affinity mutants, phages can
be incubated with excess biotinylated SSEA-3/SSEA-4/Globo H, but
with the biotinylated SSEA-3/SSEA-4/Globo H at a concentration of
lower molarity than the target molar affinity constant for
SSEA-3/SSEA-4/Globo H. The high affinity-binding phages can then be
captured by streptavidin-coated paramagnetic beads. Such
"equilibrium capture" allows the antibodies to be selected
according to their affinities of binding, with sensitivity that
permits isolation of mutant clones with as little as two-fold
higher affinity from a great excess of phages with lower affinity.
Conditions used in washing phages bound to a solid phase can also
be manipulated to discriminate on the basis of dissociation
kinetics.
[0362] Anti-SSEA-3/SSEA-4/Globo H clones may be activity selected.
In one embodiment, the invention provides anti-SSEA-3/SSEA-4/Globo
H antibodies that block the binding between a SSEA-3/SSEA-4/Globo H
ligand and SSEA-3/SSEA-4/Globo H, but do not block the binding
between a SSEA-3/SSEA-4/Globo H ligand and a second protein. Fv
clones corresponding to such anti-SSEA-3/SSEA-4/Globo H antibodies
can be selected by (1) isolating anti-SSEA-3/SSEA-4/Globo H clones
from a phage library as described in Section B(I)(2) above, and
optionally amplifying the isolated population of phage clones by
growing up the population in a suitable bacterial host; (2)
selecting SSEA-3/SSEA-4/Globo H and a second protein against which
blocking and non-blocking activity, respectively, is desired; (3)
adsorbing the anti-SSEA-3/SSEA-4/Globo H phage clones to
immobilized SSEA-3/SSEA-4/Globo H; (4) using an excess of the
second protein to elute any undesired clones that recognize
SSEA-3/SSEA-4/Globo H-binding determinants which overlap or are
shared with the binding determinants of the second protein; and (5)
eluting the clones which remain adsorbed following step (4).
Optionally, clones with the desired blocking/non-blocking
properties can be further enriched by repeating the selection
procedures described herein one or more times.
[0363] DNA encoding the Fv clones of the invention is readily
isolated and sequenced using conventional procedures (e.g. by using
oligonucleotide primers designed to specifically amplify the heavy
and light chain coding regions of interest from hybridoma or phage
DNA template). Once isolated, the DNA can be placed into expression
vectors, which are then transfected into host cells such as E. coli
cells, simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of the desired monoclonal antibodies in the
recombinant host cells. Review articles on recombinant expression
in bacteria of antibody-encoding DNA include Skerra et al., Curr.
Opinion in Immunol., 5: 256 (1993) and Pluckthun, Immunol. Revs,
130: 151 (1992).
[0364] DNA encoding the Fv clones of the invention can be combined
with known DNA sequences encoding heavy chain and/or light chain
constant regions (e.g. the appropriate DNA sequences can be
obtained from Kabat et al., supra) to form clones encoding full or
partial length heavy and/or light chains. It will be appreciated
that constant regions of any isotype can be used for this purpose,
including IgG, IgM, IgA, IgD, and IgE constant regions, and that
such constant regions can be obtained from any human or animal
species. A Fv clone derived from the variable domain DNA of one
animal (such as human) species and then fused to constant region
DNA of another animal species to form coding sequence(s) for
"hybrid", full length heavy chain and/or light chain is included in
the definition of "chimeric" and "hybrid" antibody as used herein.
In one embodiment, a Fv clone derived from human variable DNA is
fused to human constant region DNA to form coding sequence(s) for
all human, full or partial length heavy and/or light chains.
[0365] The antibodies produced by naive libraries (either natural
or synthetic) can be of moderate affinity (Kd-1 of about 106 to 107
M-1), but affinity maturation can also be mimicked in vitro by
constructing and reselecting from secondary libraries as described
in Winter et al. (1994), supra. For example, mutation can be
introduced at random in vitro by using error-prone polymerase
(reported in Leung et al., Technique, 1: 11-15 (1989)) in the
method of Hawkins et al., J. Mol. Biol., 226: 889-896 (1992) or in
the method of Gram et al., Proc. Natl. Acad. Sci. USA, 89:
3576-3580 (1992). Additionally, affinity maturation can be
performed by randomly mutating one or more CDRs, e.g. using PCR
with primers carrying random sequence spanning the CDR of interest,
in selected individual Fv clones and screening for higher affinity
clones. WO 9607754 (published 14 Mar. 1996) described a method for
inducing mutagenesis in a complementarity determining region of an
immunoglobulin light chain to create a library of light chain
genes. Another effective approach is to recombine the VH or VL
domains selected by phage display with repertoires of naturally
occurring V domain variants obtained from unimmunized donors and
screen for higher affinity in several rounds of chain reshuffling
as described in Marks et al., Biotechnol., 10: 779-783 (1992). This
technique allows the production of antibodies and antibody
fragments with affinities in the 10-9 M range.
[0366] Other Methods of Generating Anti-SSEA-3/SSEA-4/Globo H
Antibodies
[0367] Other methods of generating and assessing the affinity of
antibodies are well known in the art and are described, e.g., in
Kohler et al., Nature 256: 495 (1975); U.S. Pat. No. 4,816,567;
Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press, 1986; Kozbor, J. Immunol., 133:3001 (1984);
Brodeur et al., Monoclonal Antibody Production Techniques and
Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987;
Munson et al., Anal. Biochem., 107:220 (1980); Engels et al.,
Agnew. Chem. Int. Ed. Engl., 28: 716-734 (1989); Abrahmsen et al.,
EMBO J., 4: 3901 (1985); Methods in Enzymology, vol. 44 (1976);
Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855
(1984).
[0368] General Methods
[0369] Accordingly, one aspect of the present disclosure features
an isolated antibody triple-targeting Globo H, SSEA3 and SSEA-4.
The triple-targeting antibody specifically binds to
Fuc.alpha.1.fwdarw.2Gal.beta.1.fwdarw.3GalNAc.beta.1.fwdarw.3Gal.alpha.1.-
fwdarw.4Gal.beta.1.fwdarw.4Glc.beta.1 (Globo H hexasaccharide) and
Gal.beta.1.fwdarw.3GalNAc.beta.1.fwdarw.3Gal.alpha.1.fwdarw.4Gal.beta.1.f-
wdarw.4Glc.beta.1 (SSEA-3 pentasaccharide) and
Neu5Ac.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3GalNAc.beta.1.fwdarw.3Gal.alpha-
.1.fwdarw.4Gal.beta.1.fwdarw.4Glc.beta.1 (SSEA-4 hexasaccharide).
In one example, the triple-targeting antibody is mAb 651.
[0370] Another aspect of the present disclosure features an
isolated antibody dual-targeting Globo H and SSEA3. The
dual-targeting antibody specifically binds to
Fuc.alpha.1.fwdarw.2Gal.beta.1.fwdarw.3GalNAc.beta.1.fwdarw.3Gal.alpha.1.-
fwdarw.4Gal.beta.1.fwdarw.4Glc.beta.1 (Globo H hexasaccharide) and
Gal.beta.1.fwdarw.3GalNAc.beta.1.fwdarw.3Gal.alpha.1.fwdarw.4Gal.beta.1.f-
wdarw.4Glc.beta.1 (SSEA-3 pentasaccharide), In one example, the
dual-targeting antibody is mAb 273.
[0371] In yet another aspect, the present disclosure features an
isolated antibody specific to SSEA-4. The anti-SSEA-4 antibody
binds to
Neu5Ac.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3GalNAc.beta.1.fwdarw.3Gal.alpha-
.1.fwdarw.4Gal.beta.1.fwdarw.4Glc.beta.1 (SSEA-4 hexasaccharide).
In some examples, the antibody is capable of binding
Neu5Gc.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3GalNAc.beta.1.fwdarw.3Gal.alpha-
.1.fwdarw.4Gal.beta.1.fwdarw.4Glc.beta.1 (an analogue of SSEA-4
hexasaccharide). Preferably, the antibody is not a mouse IgG3
(e.g., mAb MC-831-70), and the antibody is not a mouse IgM (e.g.,
anti-RM1). Examples of the antibodies include, but are not limited
to, mAbs 45 and 48.
[0372] Another aspect of the present disclosure features an
isolated antibody specific to SSEA-4 and fragments thereof. The
anti-SSEA-4 antibody binds to
Neu5Ac.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3GalNAc.beta.1.fwdarw.3Gal.alpha-
.1.fwdarw.4Gal.beta.1.fwdarw.4Glc.beta.1 (SSEA-4 hexasaccharide)
and
Neu5Ac.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3GalNAc.beta.1.fwdarw.3Gal.alpha-
.1 (fragment of SSEA-4 hexasaccharide). In some examples, the
antibody is capable of
Neu5Ac.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3GalNAc.beta.1.fwdarw.3Gal.beta.-
1. In some examples, the antibody is capable of
Neu5Gc.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3GalNAc.beta.1.fwdarw.3Gal.alpha-
.1.fwdarw.4Gal.beta.1.fwdarw.4Glc.beta.1 (an analogue of SSEA-4
hexasaccharide), In one example, the antibody is mAb 46.
[0373] Antibodies triple-targeting Globo H, SSEA-3 and SSEA-4,
antibodies dual-targeting Globo H and SSEA-3, and anti-SSEA-4
antibodies were developed and disclosed herein. The antibodies
according to the disclosure can be used in therapeutics, diagnosis
or as a research tool.
[0374] Accordingly, one aspect of the present disclosure relates to
a composition of a homogeneous population of monoclonal antibodies
comprising a single, uniform N-glycan on Fc, wherein the structure
is an optimized N-glycan structure for enhancing the efficacy of
effector cell function.
[0375] In preferred embodiments, the N-glycan is attached to the
Asn-297 of the Fc region.
[0376] In preferred embodiments, wherein the N-glycan consists of
the structure of
Sia.sub.2(.alpha.2-6)Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2.
[0377] The glycoantibodies described herein may be produced in
vitro. The glycoantibodies may be generated by Fc glycoengineering.
In certain embodiments, the glycoantibodies are enzymatically or
chemoenzymatically engineered from the monoclonal antibodies
obtained by mammalian cell culturing.
[0378] In some embodiments, the Fc region of the glycoantibodies
described herein exhibits an increased binding affinity for
Fc.gamma.RIIA or Fc.gamma.RIIIA relative to a wild-type Fc region
in the corresponding monoclonal antibodies.
[0379] In some embodiments, the glycoantibodies described herein
exhibit an enhanced antibody-dependent cell mediated cytotoxicity
(ADCC) activity relative to wild-type immunoglobulins.
[0380] In some embodiments, the glycoantibodies are selected from a
group consisting of human IgG1, IgG2, IgG3, and IgG4. The
monoclonal antibodies may be humanized, human or chimeric.
[0381] The glycoantibodies described herein may bind to an antigen
associated with cancers, autoimmune disorders, inflammatory
disorders or infectious diseases. Exemplary cancer associated
antigens can include, for example, Globo-H, SSEA-3, SSEA-4.
[0382] In other aspects, the antibodies disclosed herein can detect
glycan variants and derivatives. For example, the reducing end of
the glycan is free or linked to a tail which is natural (e.g. SSEA4
glycolipid) or non-natural (e.g. a linker for making glycan array
or for conjugation for diagnostic purposes). All these derivatives
can be recognized by the antibody.
[0383] In certain diagnostic and array embodiments, the antibodies
of this invention can therefore detect not only the glycan
described herein, but also oxidized variants thereof. The
antibodies of this invention can also detect conjugation products
to said oxidized variants.
[0384] In certain aspects, the disclosure provides isolated
humanized monoclonal glycoantibody that specifically binds to
Neu5Ac.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3GalNAc.beta.1.fwdarw.3Gal.alpha-
.1.fwdarw.4Gal.beta.1.fwdarw.4Glc.beta.1, and oxidized variants
thereof, and conjugation products to said oxidized variants, and
oxidized variants thereof, and conjugation products to said
oxidized variants; wherein said oxidized variants are the
conversion products of the glycan primary alcohols to carbonyls,
and wherein the conjugation products are the conversion products of
carbonyls to imines with a primary or secondary amine moiety.
[0385] For example, the glycans comprising primary alcohols can be
converted to an oxidized variant by methods known to those skilled
in the art. As a non-limiting example, a primary alcohol on a
galactose can be converted to an aldehyde by contacting the glycan
with an oxidant, e.g. sodium periodate (sodium m-periodate), or
another salt of periodate (e.g., potassium, ammonium, manganese,
lithium). One or a plurality of sugar moieties in the glycan can be
oxidized. The concentration of oxidant can be 1 micromolar, 5
micromolar, 10 micromolar, 25 micromolar, 50 micromolar, 100
micromolar, 200 micromolar, 500 micromolar, 750 micromolar, 1
millimolar, 5 millimolar, 10 millimolar, 25 millimolar, 50
millimolar, 100 millimolar, or 500 millimolar in water or a
suitable buffer. The temperature can be from 5 to 45 degrees
Celsius, preferably 15 to 40 degrees Celsius, more preferably 35 to
40 degrees Celsius. The reaction time can be from 10 seconds to 20
minutes, preferably from 30 seconds to 10 minutes. Suitable buffers
can include or exclude saline, phosphate, CHES, MES, borate,
acetate, carbonate, formate, citrate, oxalate. Preferably, mildly
acidic buffers are used. Preferably, buffers without TRIS or
glycine or free sugars are used as these will compete in the
reaction. The conversion can be purified by dialysis or centrifugal
dialysis by methods known those skilled in the art.
[0386] The conjugation products can be formed from the reaction of
the oxidized products with an appropriate amine, hydrazine,
hydrazide, or oxo-amine by methods known to those skilled in the
art, and as described in G. Hermanson, Bioconjugate Techniques,
3.sup.rd Ed., ISBN: 978-0-12-382239-0, Academic Press, 2013, herein
incorporated by reference. As a non-limiting example, a primary
amine can be reacted to a glycan with a single aldehyde functional
group formed from the periodate-oxidized primary alcohol of a
galactose within the glycan. The net product would be an imine. The
imine can be optionally further reduced to an alcohol by methods
known the those skilled in the art, e.g. cyanoborohydride
reduction, to form a more stable conjugation product to hydrolysis.
In some aspects, the amine, hydrazine, hydrazide, or oxo-amine can
be further covalently linked to an array, a reporter molecule, or a
biotin for further modification of the conjugation product. In some
aspects, the reporter molecule can be a fluorescent molecule. In
some aspects, the reporter molecule can be a radiolabelled
molecule. In some aspects, the reporter molecule can be a molecule
with a unique spectral characteristic (e.g., IR spectra, Raman
spectra, or NMR spectra). In some aspects, the array can be a solid
surface, a chemically modified surface, a polymer-coated surface, a
bead, a gel, a particle, or a nanoparticle. In some aspects, the
nanoparticle can be fluorescent or exhibit photoluminescence. In
some aspects, the conjugation products can be the conversion
products of carbonyls to imines with a primary or secondary amine
moiety.
[0387] In general, the invention provides affinity-matured
SSEA-3/SSEA-4/Globo H antibodies. These antibodies have increased
affinity and specificity for SSEA-3/SSEA-4/Globo H. This increase
in affinity and sensitivity permits the molecules of the invention
to be used for applications and methods that are benefited by (a)
the increased sensitivity of the molecules of the invention and/or
(b) the tight binding of SSEA-3/SSEA-4/Globo H by the molecules of
the invention.
[0388] In one aspect, SSEA4/SSEA3/GloboH are three glycans that are
specifically expressed for cancer cells and cancer stem cells.
Knockdown of beta-3-GalT5, the key enzyme for the synthesis of
these three glycolipids, causes apoptosis of cancer cells, but not
normal cells. Antibodies, especially glycoantibodies against SSEA4
preferentially or specifically and/or against SSEA3/SSEA4/GloboH
simultaneously are effective cancer therapeutic agents. In another
aspect, the three glycans, SSEA4/SSEA3/GloboH, especially SSEA3,
are useful as cancer stem cell markers.
[0389] In one aspect, SSEA4 and/or SSEA4/SSEA3/GloboH in
combination are useful as therapeutic targets for the treatment of
different cancers, including for example, 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 (osteosarcoma), skin cancer,
cervical cancer, ovarian cancer, and prostate cancer.
[0390] In one embodiment, human or humanized therapeutic antibodies
against SSEA4 expressed on the cell surface of these exemplary
cancer types are provided.
[0391] In another embodiment, human or humanized therapeutic
antibodies against SSEA3/SSEA4/Globo-H simultaneously expressed on
the cell surface of these exemplary cancer types are provided.
[0392] Additionally, the present disclosure is also directed to
immunogenic conjugate compositions targeting the
SSEA-3/SSEA-4/Globo H associated epitopes (natural and modified)
which can elicit antibodies and/or binding fragment production
useful for modulating the globoseries 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.
[0393] In one embodiment, SSEA-3/SSEA-4/Globo H antibodies that are
useful for treatment of SSEA-3/SSEA-4/Globo H-mediated disorders in
which a partial or total blockade of one or more
SSEA-3/SSEA-4/Globo H activities is desired. In one embodiment, the
anti SSEA-3/SSEA-4/Globo H antibodies of the invention are used to
treat cancer.
[0394] The anti-SSEA-3/SSEA-4/Globo H antibodies of the invention
permit the sensitive and specific detection of the epitopes in
immunoassays such as sandwich assays, immunoprecipitations, ELISAs,
or immunomicroscopy without the need for mass spectrometry or
genetic manipulation. In turn, this provides a significant
advantage in both observing and elucidating the normal functioning
of these pathways and in detecting when the pathways are
functioning aberrantly.
[0395] The SSEA-3/SSEA-4/Globo H antibodies of the invention can
also be used to determine the role in the development and
pathogenesis of disease. For example, as described above, the
SSEA-3/SSEA-4/Globo H antibodies of the invention can be used to
determine whether the TACAs are normally temporally expressed which
can be correlated with one or more disease states.
[0396] The SSEA-3/SSEA-4/Globo H antibodies of the invention can
further be used to treat diseases in which one or more
SSEA-3/SSEA-4/Globo Hs are aberrantly regulated or aberrantly
functioning without interfering with the normal activity of
SSEA-3/SSEA-4/Globo Hs for which the anti-SSEA-3/SSEA-4/Globo H
antibodies of the invention are not specific.
[0397] In another aspect, the anti-SSEA-3/SSEA-4/Globo H antibodies
of the invention find utility as reagents for detection of cancer
states in various cell types and tissues.
[0398] In yet another aspect, the present anti-SSEA-3/SSEA-4/Globo
H antibodies are useful for the development of SSEA-3/SSEA-4/Globo
H antagonists with blocking activity patterns similar to those of
the subject antibodies of the invention. For example,
anti-SSEA-3/SSEA-4/Globo H antibodies of the invention can be used
to determine and identify other antibodies that have the same
SSEA-3/SSEA-4/Globo H binding characteristics and/or capabilities
of blocking SSEA-3/SSEA-4/Globo H- pathways.
[0399] As a further example, anti-SSEA-3/SSEA-4/Globo H antibodies
of the invention can be used to identify other
anti-SSEA-3/SSEA-4/Globo H antibodies that bind substantially the
same antigenic determinant(s) of SSEA-3/SSEA-4/Globo H as the
antibodies exemplified herein, including linear and conformational
epitopes.
[0400] The anti-SSEA-3/SSEA-4/Globo H antibodies of the invention
can be used in assays based on the physiological pathways in which
SSEA-3/SSEA-4/Globo H is involved to screen for small molecule
antagonists of SSEA-3/SSEA-4/Globo H which will exhibit similar
pharmacological effects in blocking the binding of one or more
binding partners to SSEA-3/SSEA-4/Globo H as the antibody does.
[0401] Generation of antibodies can be achieved using routine
skills in the art, including those described herein, such as the
hybridoma technique and screening of phage displayed libraries of
binder molecules. These methods are well-established in the
art.
[0402] Briefly, the anti-SSEA-3/SSEA-4/Globo H antibodies of the
invention can be made by using combinatorial libraries to screen
for synthetic antibody clones with the desired activity or
activities. In principle, synthetic antibody clones are selected by
screening phage libraries containing phage that display various
fragments of antibody variable region (Fv) fused to phage coat
protein. Such phage libraries are panned by affinity chromatography
against the desired antigen. Clones expressing Fv fragments capable
of binding to the desired antigen are adsorbed to the antigen and
thus separated from the non-binding clones in the library. The
binding clones are then eluted from the antigen, and can be further
enriched by additional cycles of antigen adsorption/elution. Any of
the anti-SSEA-3/SSEA-4/Globo H antibodies of the invention can be
obtained by designing a suitable antigen screening procedure to
select for the phage clone of interest followed by construction of
a full length anti-SSEA-3/SSEA-4/Globo H antibody clone using the
Fv sequences from the phage clone of interest and suitable constant
region (Fc) sequences described in Kabat et al., Sequences of
Proteins of Immunological Interest, Fifth Edition, NIH Publication
91-3242, Bethesda Md. (1991), vols. 1-3.
[0403] In one embodiment, anti-SSEA-3/SSEA-4/Globo H antibodies of
the invention are monoclonal. Also encompassed within the scope of
the invention are antibody fragments such as Fab, Fab', Fab'-SH and
F(ab')2 fragments, and variations thereof, of the
anti-SSEA-3/SSEA-4/Globo H antibodies provided herein. 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, human or humanized. These
fragments are useful for the experimental, diagnostic, and
therapeutic purposes set forth herein.
[0404] Monoclonal antibodies can be obtained from a population of
substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical except for
possible naturally occurring mutations that may be present in minor
amounts. Thus, the modifier "monoclonal" indicates the character of
the antibody as not being a mixture of discrete antibodies.
[0405] The anti-SSEA-3/SSEA-4/Globo H monoclonal antibodies of the
invention can be made using a variety of methods known in the art,
including the hybridoma method first described by Kohler et al.,
Nature, 256:495 (1975), or alternatively they may be made by
recombinant DNA methods (e.g., U.S. Pat. No. 4,816,567).
[0406] Vectors, Host Cells and Recombinant Methods
[0407] For recombinant production of an antibody of the invention,
the nucleic acid encoding it is isolated and inserted into a
replicable vector for further cloning (amplification of the DNA) or
for expression. DNA encoding the antibody is readily isolated and
sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the antibody). Many
vectors are available. The choice of vector depends in part on the
host cell to be used. Host cells include, but are not limited to,
cells of either prokaryotic or eukaryotic (generally mammalian)
origin. It will be appreciated that constant regions of any isotype
can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE
constant regions, and that such constant regions can be obtained
from any human or animal species.
[0408] Generating Antibodies Using Prokaryotic Host Cells
[0409] Vector Construction
[0410] Polynucleotide sequences encoding polypeptide components of
the antibody of the invention can be obtained using standard
recombinant techniques. Desired polynucleotide sequences may be
isolated and sequenced from antibody producing cells such as
hybridoma cells. Alternatively, polynucleotides can be synthesized
using nucleotide synthesizer or PCR techniques. Once obtained,
sequences encoding the polypeptides are inserted into a recombinant
vector capable of replicating and expressing heterologous
polynucleotides in prokaryotic hosts. Many vectors that are
available and known in the art can be used for the purpose of the
present invention. Selection of an appropriate vector will depend
mainly on the size of the nucleic acids to be inserted into the
vector and the particular host cell to be transformed with the
vector. Each vector contains various components, depending on its
function (amplification or expression of heterologous
polynucleotide, or both) and its compatibility with the particular
host cell in which it resides. The vector components generally
include, but are not limited to: an origin of replication, a
selection marker gene, a promoter, a ribosome binding site (RB S),
a signal sequence, the heterologous nucleic acid insert and a
transcription termination sequence.
[0411] In general, plasmid vectors containing replicon and control
sequences which are derived from species compatible with the host
cell are used in connection with these hosts. The vector ordinarily
carries a replication site, as well as marking sequences which are
capable of providing phenotypic selection in transformed cells. For
example, E. coli is typically transformed using pBR322, a plasmid
derived from an E. coli species. pBR322 contains genes encoding
ampicillin (Amp) and tetracycline (Tet) resistance and thus
provides easy means for identifying transformed cells. pBR322, its
derivatives, or other microbial plasmids or bacteriophage may also
contain, or be modified to contain, promoters which can be used by
the microbial organism for expression of endogenous proteins.
Examples of pBR322 derivatives used for expression of particular
antibodies are described in detail in Carter et al., U.S. Pat. No.
5,648,237.
[0412] In addition, phage vectors containing replicon and control
sequences that are compatible with the host microorganism can be
used as transforming vectors in connection with these hosts. For
example, bacteriophage such as .lamda.GEM.TM.-11 may be utilized in
making a recombinant vector which can be used to transform
susceptible host cells such as E. coli LE392.
[0413] The expression vector of the invention may comprise two or
more promoter-cistron pairs, encoding each of the polypeptide
components. A promoter is an untranslated regulatory sequence
located upstream (5') to a cistron that modulates its expression.
Prokaryotic promoters typically fall into two classes, inducible
and constitutive. Inducible promoter is a promoter that initiates
increased levels of transcription of the cistron under its control
in response to changes in the culture condition, e.g. the presence
or absence of a nutrient or a change in temperature.
[0414] A large number of promoters recognized by a variety of
potential host cells are well known. The selected promoter can be
operably linked to cistron DNA encoding the light or heavy chain by
removing the promoter from the source DNA via restriction enzyme
digestion and inserting the isolated promoter sequence into the
vector of the invention. Both the native promoter sequence and many
heterologous promoters may be used to direct amplification and/or
expression of the target genes. In some embodiments, heterologous
promoters are utilized, as they generally permit greater
transcription and higher yields of expressed target gene as
compared to the native target polypeptide promoter.
[0415] Promoters suitable for use with prokaryotic hosts include
the PhoA promoter, the .beta.-galactamase and lactose promoter
systems, a tryptophan (trp) promoter system and hybrid promoters
such as the tac or the trc promoter. However, other promoters that
are functional in bacteria (such as other known bacterial or phage
promoters) are suitable as well. Their nucleotide sequences have
been published, thereby enabling a skilled worker operably to
ligate them to cistrons encoding the target light and heavy chains
(Siebenlist et al. (1980) Cell 20: 269) using linkers or adaptors
to supply any required restriction sites.
[0416] In one aspect of the invention, each cistron within the
recombinant vector comprises a secretion signal sequence component
that directs translocation of the expressed polypeptides across a
membrane. In general, the signal sequence may be a component of the
vector, or it may be a part of the target polypeptide DNA that is
inserted into the vector. The signal sequence selected for the
purpose of this invention should be one that is recognized and
processed (i.e. cleaved by a signal peptidase) by the host cell.
For prokaryotic host cells that do not recognize and process the
signal sequences native to the heterologous polypeptides, the
signal sequence is substituted by a prokaryotic signal sequence
selected, for example, from the group consisting of the alkaline
phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II
(STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment
of the invention, the signal sequences used in both cistrons of the
expression system are STII signal sequences or variants
thereof.
[0417] In another aspect, the production of the immunoglobulins
according to the invention can occur in the cytoplasm of the host
cell, and therefore does not require the presence of secretion
signal sequences within each cistron. In that regard,
immunoglobulin light and heavy chains are expressed, folded and
assembled to form functional immunoglobulins within the cytoplasm.
Certain host strains (e.g., the E. coli trxB- strains) provide
cytoplasm conditions that are favorable for disulfide bond
formation, thereby permitting proper folding and assembly of
expressed protein subunits. Proba and Pluckthun Gene, 159:203
(1995).
[0418] Antibodies of the invention can also be produced by using an
expression system in which the quantitative ratio of expressed
polypeptide components can be modulated in order to maximize the
yield of secreted and properly assembled antibodies of the
invention. Such modulation is accomplished at least in part by
simultaneously modulating translational strengths for the
polypeptide components.
[0419] One technique for modulating translational strength is
disclosed in Simmons et al., U.S. Pat. No. 5,840,523. It utilizes
variants of the translational initiation region (TIR) within a
cistron. For a given TIR, a series of amino acid or nucleic acid
sequence variants can be created with a range of translational
strengths, thereby providing a convenient means by which to adjust
this factor for the desired expression level of the specific chain.
TIR variants can be generated by conventional mutagenesis
techniques that result in codon changes which can alter the amino
acid sequence. In certain embodiments, changes in the nucleotide
sequence are silent. Alterations in the TIR can include, for
example, alterations in the number or spacing of Shine-Dalgarno
sequences, along with alterations in the signal sequence. One
method for generating mutant signal sequences is the generation of
a "codon bank" at the beginning of a coding sequence that does not
change the amino acid sequence of the signal sequence (i.e., the
changes are silent). This can be accomplished by changing the third
nucleotide position of each codon; additionally, some amino acids,
such as leucine, serine, and arginine, have multiple first and
second positions that can add complexity in making the bank. This
method of mutagenesis is described in detail in Yansura et al.
(1992) METHODS: A Companion to Methods in Enzymol. 4:151-158.
[0420] In one embodiment, a set of vectors is generated with a
range of TIR strengths for each cistron therein. This limited set
provides a comparison of expression levels of each chain as well as
the yield of the desired antibody products under various TIR
strength combinations. TIR strengths can be determined by
quantifying the expression level of a reporter gene as described in
detail in Simmons et al. U.S. Pat. No. 5,840,523. Based on the
translational strength comparison, the desired individual TIRs are
selected to be combined in the expression vector constructs of the
invention.
[0421] Prokaryotic host cells suitable for expressing antibodies of
the invention include Archaebacteria and Eubacteria, such as
Gram-negative or Gram-positive organisms. Examples of useful
bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B.
subtilis), Enterobacteria, Pseudomonas species (e.g., P.
aeruginosa), Salmonella typhimurium, Serratia marcescans,
Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or
Paracoccus. In one embodiment, gram-negative cells are used. In one
embodiment, E. coli cells are used as hosts for the invention.
Examples of E. coli strains include strain W3110 (Bachmann,
Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American
Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No.
27,325) and derivatives thereof, including strain 33D3 having
genotype W3110 .DELTA.fhu.DELTA. (.DELTA.tonA) ptr3 lac Iq lacL8
.DELTA.ompT.DELTA.(nmpc-fepE) degP41 kanR (U.S. Pat. No.
5,639,635). Other strains and derivatives thereof, such as E. coli
294 (ATCC 31,446), E. coli B, E. coli .lamda.1776 (ATCC 31,537) and
E. coli RV308 (ATCC 31,608) are also suitable.
[0422] These examples are illustrative rather than limiting.
Methods for constructing derivatives of any of the above-mentioned
bacteria having defined genotypes are known in the art and
described in, for example, Bass et al., Proteins, 8:309-314 (1990).
It is generally necessary to select the appropriate bacteria taking
into consideration replicability of the replicon in the cells of a
bacterium. For example, E. coli, Serratia, or Salmonella species
can be suitably used as the host when well known plasmids such as
pBR322, pBR325, pACYC177, or pKN410 are used to supply the
replicon. Typically the host cell should secrete minimal amounts of
proteolytic enzymes, and additional protease inhibitors may
desirably be incorporated in the cell culture.
[0423] Antibody Production
[0424] Host cells are transformed with the above-described
expression vectors and cultured in conventional nutrient media
modified as appropriate for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired
sequences.
[0425] Transformation means introducing DNA into the prokaryotic
host so that the DNA is replicable, either as an extrachromosomal
element or by chromosomal integrant. Depending on the host cell
used, transformation is done using standard techniques appropriate
to such cells. The calcium treatment employing calcium chloride is
generally used for bacterial cells that contain substantial
cell-wall barriers. Another method for transformation employs
polyethylene glycol/DMSO. Yet another technique used is
electroporation.
[0426] Prokaryotic cells used to produce the polypeptides of the
invention are grown in media known in the art and suitable for
culture of the selected host cells. Examples of suitable media
include luria broth (LB) plus necessary nutrient supplements. In
some embodiments, the media also contains a selection agent, chosen
based on the construction of the expression vector, to selectively
permit growth of prokaryotic cells containing the expression
vector. For example, ampicillin is added to media for growth of
cells expressing ampicillin resistant gene.
[0427] Any necessary supplements besides carbon, nitrogen, and
inorganic phosphate sources may also be included at appropriate
concentrations introduced alone or as a mixture with another
supplement or medium such as a complex nitrogen source. Optionally
the culture medium may contain one or more reducing agents selected
from the group consisting of glutathione, cysteine, cystamine,
thioglycollate, dithioerythritol and dithiothreitol.
[0428] The prokaryotic host cells are cultured at suitable
temperatures. For E. coli growth, for example, growth occurs at a
temperature range including, but not limited to, about 20.degree.
C. to about 39.degree. C., about 25.degree. C. to about 37.degree.
C., and at about 30.degree. C. The pH of the medium may be any pH
ranging from about 5 to about 9, depending mainly on the host
organism. For E. coli, the pH can be from about 6.8 to about 7.4,
or about 7.0.
[0429] If an inducible promoter is used in the expression vector of
the invention, protein expression is induced under conditions
suitable for the activation of the promoter. In one aspect of the
invention, PhoA promoters are used for controlling transcription of
the polypeptides. Accordingly, the transformed host cells are
cultured in a phosphate-limiting medium for induction. In one
embodiment, the phosphate-limiting medium is the C.R.A.P medium
(see, e.g., Simmons et al., J. Immunol. Methods (2002),
263:133-147). A variety of other inducers may be used, according to
the vector construct employed, as is known in the art.
[0430] In one embodiment, the expressed polypeptides of the present
invention are secreted into and recovered from the periplasm of the
host cells. Protein recovery typically involves disrupting the
microorganism, generally by such means as osmotic shock, sonication
or lysis. Once cells are disrupted, cell debris or whole cells may
be removed by centrifugation or filtration. The proteins may be
further purified, for example, by affinity resin chromatography.
Alternatively, proteins can be transported into the culture media
and isolated therein. Cells may be removed from the culture and the
culture supernatant being filtered and concentrated for further
purification of the proteins produced. The expressed polypeptides
can be further isolated and identified using commonly known methods
such as polyacrylamide gel electrophoresis (PAGE) and Western blot
assay.
[0431] In one aspect of the invention, antibody production is
conducted in large quantity by a fermentation process. Various
large-scale fed-batch fermentation procedures are available for
production of recombinant proteins. Large-scale fermentations have
at least 1000 liters of capacity, for example about 1,000 to
100,000 liters of capacity. These fermentors use agitator impellers
to distribute oxygen and nutrients, especially glucose (a common
carbon/energy source). Small scale fermentation refers generally to
fermentation in a fermentor that is no more than approximately 100
liters in volumetric capacity, and can range from about 1 liter to
about 100 liters.
[0432] In a fermentation process, induction of protein expression
is typically initiated after the cells have been grown under
suitable conditions to a desired density, e.g., an OD550 of about
180-220, at which stage the cells are in the early stationary
phase. A variety of inducers may be used, according to the vector
construct employed, as is known in the art and described above.
Cells may be grown for shorter periods prior to induction. Cells
are usually induced for about 12-50 hours, although longer or
shorter induction time may be used.
[0433] To improve the production yield and quality of the
polypeptides of the invention, various fermentation conditions can
be modified. For example, to improve the proper assembly and
folding of the secreted antibody polypeptides, additional vectors
overexpressing chaperone proteins, such as Dsb proteins (DsbA,
DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl
cis,trans-isomerase with chaperone activity) can be used to
co-transform the host prokaryotic cells. The chaperone proteins
have been demonstrated to facilitate the proper folding and
solubility of heterologous proteins produced in bacterial host
cells. Chen et al. (1999) J Bio Chem 274:19601-19605; Georgiou et
al., U.S. Pat. No. 6,083,715; Georgiou et al., U.S. Pat. No.
6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem.
275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem.
275:17106-17113; Arie et al. (2001) Mol. Microbiol. 39:199-210.
[0434] To minimize proteolysis of expressed heterologous proteins
(especially those that are proteolytically sensitive), certain host
strains deficient for proteolytic enzymes can be used for the
present invention. For example, host cell strains may be modified
to effect genetic mutation(s) in the genes encoding known bacterial
proteases such as Protease III, OmpT, DegP, Tsp, Protease I,
Protease Mi, Protease V, Protease VI and combinations thereof. Some
E. coli protease-deficient strains are available and described in,
for example, Joly et al. (1998), supra; Georgiou et al., U.S. Pat.
No. 5,264,365; Georgiou et al., U.S. Pat. No. 5,508,192; Hara et
al., Microbial Drug Resistance, 2:63-72 (1996).
[0435] In one embodiment, E. coli strains deficient for proteolytic
enzymes and transformed with plasmids overexpressing one or more
chaperone proteins are used as host cells in the expression system
of the invention.
[0436] Antibody Purification
[0437] In one embodiment, the antibody protein produced herein is
further purified to obtain preparations that are substantially
homogeneous for further assays and uses. Standard protein
purification methods known in the art can be employed. The
following procedures are exemplary of suitable purification
procedures: fractionation on immunoaffinity or ion-exchange
columns, ethanol precipitation, reverse phase HPLC, chromatography
on silica or on a cation-exchange resin such as DEAE,
chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel
filtration using, for example, Sephadex G-75.
[0438] In one aspect, Protein A immobilized on a solid phase is
used for immunoaffinity purification of the antibody products of
the invention. Protein A is a 41 kD cell wall protein from
Staphylococcus aureas which binds with a high affinity to the Fc
region of antibodies. Lindmark et al (1983) J. Immunol. Meth.
62:1-13. The solid phase to which Protein A is immobilized can be a
column comprising a glass or silica surface, or a controlled pore
glass column or a silicic acid column. In some applications, the
column is coated with a reagent, such as glycerol, to possibly
prevent nonspecific adherence of contaminants.
[0439] As the first step of purification, the preparation derived
from the cell culture as described above can be applied onto a
Protein A immobilized solid phase to allow specific binding of the
antibody of interest to Protein A. The solid phase would then be
washed to remove contaminants non-specifically bound to the solid
phase. Finally the antibody of interest is recovered from the solid
phase by elution.
[0440] Generating Antibodies Using Eukaryotic Host Cells
[0441] The vector components generally include, but are not limited
to, one or more of the following: a signal sequence, an origin of
replication, one or more marker genes, an enhancer element, a
promoter, and a transcription termination sequence.
[0442] (i) Signal Sequence Component
[0443] A vector for use in a eukaryotic host cell may also contain
a signal sequence or other polypeptide having a specific cleavage
site at the N-terminus of the mature protein or polypeptide of
interest. The heterologous signal sequence selected generally is
one that is recognized and processed (i.e., cleaved by a signal
peptidase) by the host cell. In mammalian cell expression,
mammalian signal sequences as well as viral secretory leaders, for
example, the herpes simplex gD signal, are available.
[0444] The DNA for such precursor region is ligated in reading
frame to DNA encoding the antibody.
[0445] (ii) Origin of Replication
[0446] Generally, an origin of replication component is not needed
for mammalian expression vectors. For example, the SV40 origin may
typically be used only because it contains the early promoter.
[0447] (iii) Selection Gene Component
[0448] Expression and cloning vectors may contain a selection gene,
also termed a selectable marker. Typical selection genes encode
proteins that (a) confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic deficiencies, where relevant, or (c) supply
critical nutrients not available from complex media.
[0449] One example of a selection scheme utilizes a drug to arrest
growth of a host cell. Those cells that are successfully
transformed with a heterologous gene produce a protein conferring
drug resistance and thus survive the selection regimen. Examples of
such dominant selection use the drugs neomycin, mycophenolic acid
and hygromycin.
[0450] Another example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the antibody nucleic acid, such as DHFR, thymidine
kinase, metallothionein-I and -II (e.g., primate metallothionein
genes), adenosine deaminase, ornithine decarboxylase, etc.
[0451] For example, cells transformed with the DHFR selection gene
may first be identified by culturing all of the transformants in a
culture medium that contains methotrexate (Mtx), a competitive
antagonist of DHFR. Appropriate host cells when wild-type DHFR is
employed include, for example, the Chinese hamster ovary (CHO) cell
line deficient in DHFR activity (e.g., ATCC CRL-9096).
[0452] Alternatively, host cells (particularly wild-type hosts that
contain endogenous DHFR) transformed or co-transformed with DNA
sequences encoding an antibody, wild-type DHFR protein, and another
selectable marker such as aminoglycoside 3'-phosphotransferase
(APH) can be selected by cell growth in medium containing a
selection agent for the selectable marker such as an
aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See
U.S. Pat. No. 4,965,199.
[0453] (iv) Promoter Component
[0454] Expression and cloning vectors usually contain a promoter
that is recognized by the host organism and is operably linked to
nucleic acid encoding a polypeptide of interest (e.g., an
antibody). Promoter sequences are known for eukaryotes. Virtually
all eukaryotic genes have an AT-rich region located approximately
25 to 30 bases upstream from the site where transcription is
initiated. Another sequence found 70 to 80 bases upstream from the
start of transcription of many genes is a CNCAAT region where N may
be any nucleotide. At the 3' end of most eukaryotic genes is an
AATAAA sequence that may be the signal for addition of the poly A
tail to the 3' end of the coding sequence. All of these sequences
are suitably inserted into eukaryotic expression vectors.
[0455] Antibody polypeptide transcription from vectors in mammalian
host cells can be controlled, for example, by promoters obtained
from the genomes of viruses such as polyoma virus, fowlpox virus,
adenovirus (such as Adenovirus 2), bovine papilloma virus, avian
sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and
Simian Virus 40 (SV40), from heterologous mammalian promoters,
e.g., the actin promoter or an immunoglobulin promoter, or from
heat-shock promoters, provided such promoters are compatible with
the host cell systems.
[0456] The early and late promoters of the SV40 virus are
conveniently obtained as an SV40 restriction fragment that also
contains the SV40 viral origin of replication. The immediate early
promoter of the human cytomegalovirus is conveniently obtained as a
HindIII E restriction fragment. A system for expressing DNA in
mammalian hosts using the bovine papilloma virus as a vector is
disclosed in U.S. Pat. No. 4,419,446. A modification of this system
is described in U.S. Pat. No. 4,601,978. See also Reyes et al.,
Nature 297:598-601 (1982) on expression of human .beta.-interferon
cDNA in mouse cells under the control of a thymidine kinase
promoter from herpes simplex virus. Alternatively, the Rous Sarcoma
Virus long terminal repeat can be used as the promoter.
[0457] (v) Enhancer Element Component
[0458] Transcription of DNA encoding an antibody polypeptide of the
invention by higher eukaryotes can often be increased by inserting
an enhancer sequence into the vector. Many enhancer sequences are
now known from mammalian genes (globin, elastase, albumin,
.alpha.-fetoprotein, and insulin). Typically, however, one will use
an enhancer from a eukaryotic cell virus. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270),
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing
elements for activation of eukaryotic promoters. The enhancer may
be spliced into the vector at a position 5' or 3' to the antibody
polypeptide-encoding sequence, but is generally located at a site
5' from the promoter.
[0459] (vi) Transcription Termination Component
[0460] Expression vectors used in eukaryotic host cells will
typically also contain sequences necessary for the termination of
transcription and for stabilizing the mRNA. Such sequences are
commonly available from the 5' and, occasionally 3', untranslated
regions of eukaryotic or viral DNAs or cDNAs. These regions contain
nucleotide segments transcribed as polyadenylated fragments in the
untranslated portion of the mRNA encoding an antibody. One useful
transcription termination component is the bovine growth hormone
polyadenylation region. See WO94/11026 and the expression vector
disclosed therein.
[0461] (vii) Selection and Transformation of Host Cells
[0462] Suitable host cells for cloning or expressing the DNA in the
vectors herein include higher eukaryote cells described herein,
including vertebrate host cells. Propagation of vertebrate cells in
culture (tissue culture) has become a routine procedure. Examples
of useful mammalian host cell lines are monkey kidney CV1 line
transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney
line (293 or 293 cells subcloned for growth in suspension culture,
Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney
cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO,
Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse
sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980));
monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney
cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells
(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);
buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells
(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse
mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,
Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells;
and a human hepatoma line (Hep G2).
[0463] Host cells are transformed with the above-described
expression or cloning vectors for antibody production and cultured
in conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences.
[0464] (viii) Culturing the Host Cells
[0465] The host cells used to produce an antibody of this invention
may be cultured in a variety of media. Commercially available media
such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM),
(Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium
((DMEM), Sigma) are suitable for culturing the host cells. In
addition, any of the media described in Ham et al., Meth. Enz.
58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.
Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;
WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as
culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as GENTAMYCIN.TM. drug), trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0466] (ix) Purification of Antibody
[0467] When using recombinant techniques, the antibody can be
produced intracellularly, or directly secreted into the medium. If
the antibody is produced intracellularly, as a first step, the
particulate debris, either host cells or lysed fragments, are
generally removed, for example, by centrifugation or
ultrafiltration. Where the antibody is secreted into the medium,
supernatants from such expression systems are generally first
concentrated using a commercially available protein concentration
filter, for example, an Amicon or Millipore Pellicon
ultrafiltration unit. A protease inhibitor such as PMSF may be
included in any of the foregoing steps to inhibit proteolysis and
antibiotics may be included to prevent the growth of adventitious
contaminants.
[0468] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being a generally acceptable purification
technique. The suitability of affinity reagents such as protein A
as an affinity ligand depends on the species and isotype of any
immunoglobulin Fc domain that is present in the antibody. Protein A
can be used to purify antibodies that are based on human .gamma.1,
.gamma.2, or .gamma.4 heavy chains (Lindmark et al., J. Immunol.
Meth. 62:1-13 (1983)). Protein G is recommended for all mouse
isotypes and for human .gamma.3 (Guss et al., EMBO J. 5:15671575
(1986)). The matrix to which the affinity ligand is attached is
most often agarose, but other matrices are available. Mechanically
stable matrices such as controlled pore glass or
poly(styrenedivinyl)benzene allow for faster flow rates and shorter
processing times than can be achieved with agarose. Where the
antibody comprises a CH3 domain, the Bakerbond ABX.TM. resin (J. T.
Baker, Phillipsburg, N.J.) is useful for purification. Other
techniques for protein purification such as fractionation on an
ion-exchange column, ethanol precipitation, Reverse Phase HPLC,
chromatography on silica, chromatography on heparin SEPHAROSE.TM.
chromatography on an anion or cation exchange resin (such as a
polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium
sulfate precipitation are also available depending on the antibody
to be recovered.
[0469] Following any preliminary purification step(s), the mixture
comprising the antibody of interest and contaminants may be
subjected to further purification steps, as necessary, for example
by low pH hydrophobic interaction chromatography using an elution
buffer at a pH between about 2.5-4.5, generally performed at low
salt concentrations (e.g., from about 0-0.25M salt).
[0470] It should be noted that, in general, techniques and
methodologies for preparing antibodies for use in research, testing
and clinical use are well-established in the art, consistent with
the above and/or as deemed appropriate by one skilled in the art
for the particular antibody of interest.
[0471] Activity Assays
[0472] Antibodies of the invention can be characterized for their
physical/chemical properties and biological functions by various
assays known in the art.
[0473] Purified antibodies can be further characterized by a series
of assays including, but not limited to, N-terminal sequencing,
amino acid analysis, non-denaturing size exclusion high pressure
liquid chromatography (HPLC), mass spectrometry, ion exchange
chromatography and papain digestion.
[0474] Where necessary, antibodies are analyzed for their
biological activity. In some embodiments, antibodies of the
invention are tested for their antigen binding activity. The
antigen binding assays that are known in the art and can be used
herein include without limitation any direct or competitive binding
assays using techniques such as western blots, radioimmunoassays,
ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays,
immunoprecipitation assays, fluorescent immunoassays,
chemiluminescent immunoassays, nanoparticle immunoassays, aptamer
immunoassays, and protein A immunoassays.
[0475] Antibody Fragments
[0476] The present invention encompasses antibody fragments. In
certain circumstances there are advantages of using antibody
fragments, rather than whole antibodies. The smaller size of the
fragments allows for rapid clearance, and may lead to improved
access to solid tumors.
[0477] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992); and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
Fab, Fv and ScFv antibody fragments can all be expressed in and
secreted from E. coli, thus allowing the facile production of large
amounts of these fragments. Antibody fragments can be isolated from
the antibody phage libraries discussed above. Alternatively,
Fab'-SH fragments can be directly recovered from E. coli and
chemically coupled to form F(ab')2 fragments (Carter et al.,
Bio/Technology 10: 163-167 (1992)). According to another approach,
F(ab')2 fragments can be isolated directly from recombinant host
cell culture. Fab and F(ab')2 fragment with increased in vivo
half-life comprising salvage receptor binding epitope residues are
described in U.S. Pat. No. 5,869,046. Other techniques for the
production of antibody fragments will be apparent to the skilled
practitioner. In other embodiments, the antibody of choice is a
single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos.
5,571,894; and 5,587,458. Fv and sFv are the only species with
intact combining sites that are devoid of constant regions; thus,
they are suitable for reduced nonspecific binding during in vivo
use. sFv fusion proteins may be constructed to yield fusion of an
effector protein at either the amino or the carboxy terminus of an
sFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody
fragment may also be a "linear antibody", e.g., as described in
U.S. Pat. No. 5,641,870 for example. Such linear antibody fragments
may be monospecific or bispecific.
[0478] Humanized Antibodies
[0479] The invention encompasses humanized antibodies. Various
methods for humanizing non-human antibodies are known in the art.
For example, a humanized antibody can have one or more amino acid
residues introduced into it from a source which is non-human. These
non-human amino acid residues are often referred to as "import"
residues, which are typically taken from an "import" variable
domain. Humanization can be essentially performed following the
method of Winter and co-workers (Jones et al. (1986) Nature
321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen
et al. (1988) Science 239:1534-1536), by substituting hypervariable
region sequences for the corresponding sequences of a human
antibody. Accordingly, such "humanized" antibodies are chimeric
antibodies (U.S. Pat. No. 4,816,567) wherein substantially less
than an intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
hypervariable region residues and possibly some FR residues are
substituted by residues from analogous sites in rodent
antibodies.
[0480] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies can be important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework for the humanized
antibody (Sims et al. (1993) J. Immunol. 151:2296; Chothia et al.
(1987) J. Mol. Biol. 196:901. Another method uses a particular
framework derived from the consensus sequence of all human
antibodies of a particular subgroup of light or heavy chains. The
same framework may be used for several different humanized
antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA,
89:4285; Presta et al. (1993) J. Immunol., 151:2623.
[0481] It is further generally desirable that antibodies be
humanized with retention of high affinity for the antigen and other
favorable biological properties. To achieve this goal, according to
one method, humanized antibodies are prepared by a process of
analysis of the parental sequences and various conceptual humanized
products using three-dimensional models of the parental and
humanized sequences. Three-dimensional immunoglobulin models are
commonly available and are familiar to those skilled in the art.
Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
hypervariable region residues are directly and most substantially
involved in influencing antigen binding.
[0482] Human Antibodies
[0483] Human anti-SSEA-3/SSEA-4/Globo H antibodies of the invention
can be constructed by combining Fv clone variable domain
sequence(s) selected from human-derived phage display libraries
with known human constant domain sequences(s) as described above.
Alternatively, human monoclonal anti-SSEA-3/SSEA-4/Globo H
antibodies of the invention can be made by the hybridoma method.
Human myeloma and mouse-human heteromyeloma cell lines for the
production of human monoclonal antibodies have been described, for
example, by Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al.,
Monoclonal Antibody Production Techniques and Applications, pp.
51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.
Immunol., 147: 86 (1991).
[0484] It is now possible to produce transgenic animals (e.g. mice)
that are capable, upon immunization, of producing a full repertoire
of human antibodies in the absence of endogenous immunoglobulin
production. For example, it has been described that the homozygous
deletion of the antibody heavy-chain joining region (JH) gene in
chimeric and germ-line mutant mice results in complete inhibition
of endogenous antibody production. Transfer of the human germ-line
immunoglobulin gene array in such germ-line mutant mice will result
in the production of human antibodies upon antigen challenge. See,
e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551
(1993); Jakobovits et al., Nature, 362: 255 (1993); Bruggermann et
al., Year in Immunol., 7: 33 (1993).
[0485] Gene shuffling can also be used to derive human antibodies
from non-human, e.g. rodent, antibodies, where the human antibody
has similar affinities and specificities to the starting non-human
antibody. According to this method, which is also called "epitope
imprinting", either the heavy or light chain variable region of a
non-human antibody fragment obtained by phage display techniques as
described above is replaced with a repertoire of human V domain
genes, creating a population of non-human chain/human chain scFv or
Fab chimeras. Selection with antigen results in isolation of a
non-human chain/human chain chimeric scFv or Fab wherein the human
chain restores the antigen binding site destroyed upon removal of
the corresponding non-human chain in the primary phage display
clone, i.e. the epitope governs (imprints) the choice of the human
chain partner. When the process is repeated in order to replace the
remaining non-human chain, a human antibody is obtained (see PCT WO
93/06213 published Apr. 1, 1993). Unlike traditional humanization
of non-human antibodies by CDR grafting, this technique provides
completely human antibodies, which have no FR or CDR residues of
non-human origin.
[0486] Bispecific Antibodies
[0487] Bispecific antibodies are monoclonal antibodies that have
binding specificities for at least two different antigens. In
certain embodiments, bispecific antibodies are human or humanized
antibodies. In certain embodiments, one of the binding
specificities is for SSEA-3/SSEA-4/Globo H including a specific
lysine linkage and the other is for any other antigen. In certain
embodiments, bispecific antibodies may bind to two different
SSEA-3/SSEA-4/Globo Hs having two different lysine linkages.
Bispecific antibodies can be prepared as full length antibodies or
antibody fragments (e.g. F(ab')2 bispecific antibodies).
[0488] Methods for making bispecific antibodies are known in the
art. Traditionally, the recombinant production of bispecific
antibodies is based on the co-expression of two immunoglobulin
heavy chain-light chain pairs, where the two heavy chains have
different specificities (Milstein and Cuello, Nature, 305: 537
(1983)). Because of the random assortment of immunoglobulin heavy
and light chains, these hybridomas (quadromas) produce a potential
mixture of 10 different antibody molecules, of which only one has
the correct bispecific structure. The purification of the correct
molecule, which is usually done by affinity chromatography steps,
is rather cumbersome, and the product yields are low. Similar
procedures are disclosed in WO 93/08829 published May 13, 1993, and
in Traunecker et al., EMBO J., 10: 3655 (1991).
[0489] According to a different embodiment, antibody variable
domains with the desired binding specificities (antibody-antigen
combining sites) are fused to immunoglobulin constant domain
sequences. The fusion, for example, is with an immunoglobulin heavy
chain constant domain, comprising at least part of the hinge, CH2,
and CH3 regions. In certain embodiments, the first heavy-chain
constant region (CH1), containing the site necessary for light
chain binding, is present in at least one of the fusions. DNAs
encoding the immunoglobulin heavy chain fusions and, if desired,
the immunoglobulin light chain, are inserted into separate
expression vectors, and are co-transfected into a suitable host
organism. This provides for great flexibility in adjusting the
mutual proportions of the three polypeptide fragments in
embodiments when unequal ratios of the three polypeptide chains
used in the construction provide the optimum yields. It is,
however, possible to insert the coding sequences for two or all
three polypeptide chains in one expression vector when the
expression of at least two polypeptide chains in equal ratios
results in high yields or when the ratios are of no particular
significance.
[0490] In one embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. It was found that this asymmetric
structure facilitates the separation of the desired bispecific
compound from unwanted immunoglobulin chain combinations, as the
presence of an immunoglobulin light chain in only one half of the
bispecific molecule provides for a facile way of separation. This
approach is disclosed in WO 94/04690. For further details of
generating bispecific antibodies see, for example, Suresh et al.,
Methods in Enzymology, 121:210 (1986).
[0491] According to another approach, the interface between a pair
of antibody molecules can be engineered to maximize the percentage
of heterodimers which are recovered from recombinant cell culture.
The interface comprises at least a part of the CH3 domain of an
antibody constant domain. In this method, one or more small amino
acid side chains from the interface of the first antibody molecule
are replaced with larger side chains (e.g. tyrosine or tryptophan).
Compensatory "cavities" of identical or similar size to the large
side chain(s) are created on the interface of the second antibody
molecule by replacing large amino acid side chains with smaller
ones (e.g. alanine or threonine). This provides a mechanism for
increasing the yield of the heterodimer over other unwanted
end-products such as homodimers.
[0492] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360, WO 92/00373, and EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0493] Techniques for generating bispecific antibodies from
antibody fragments have also been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science, 229: 81 (1985) describe a
procedure wherein intact antibodies are proteolytically cleaved to
generate F(ab')2 fragments. These fragments are reduced in the
presence of the dithiol complexing agent sodium arsenite to
stabilize vicinal dithiols and prevent intermolecular disulfide
formation. The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0494] Recent progress has facilitated the direct recovery of
Fab'-SH fragments from E. coli, which can be chemically coupled to
form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:
217-225 (1992) describe the production of a fully humanized
bispecific antibody F(ab')2 molecule. Each Fab' fragment was
separately secreted from E. coli and subjected to directed chemical
coupling in vitro to form the bispecific antibody. The bispecific
antibody thus formed was able to bind to cells overexpressing the
HER2 receptor and normal human T cells, as well as trigger the
lytic activity of human cytotoxic lymphocytes against human breast
tumor targets.
[0495] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (VH) connected to a light-chain
variable domain (VL) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the
VH and VL domains of one fragment are forced to pair with the
complementary VL and VH domains of another fragment, thereby
forming two antigen-binding sites. Another strategy for making
bispecific antibody fragments by the use of single-chain Fv (sFv)
dimers has also been reported. See Gruber et al., J. Immunol.,
152:5368 (1994).
[0496] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al. J.
Immunol. 147: 60 (1991).
[0497] Multivalent Antibodies
[0498] A multivalent antibody may be internalized (and/or
catabolized) faster than a bivalent antibody by a cell expressing
an antigen to which the antibodies bind. The antibodies of the
present invention can be multivalent antibodies (which are other
than of the IgM class) with three or more antigen binding sites
(e.g. tetravalent antibodies), which can be readily produced by
recombinant expression of nucleic acid encoding the polypeptide
chains of the antibody. The multivalent antibody can comprise a
dimerization domain and three or more antigen binding sites. The
dimerization domain comprises (or consists of), for example, an Fc
region or a hinge region. In this scenario, the antibody will
comprise an Fc region and three or more antigen binding sites
amino-terminal to the Fc region. In one embodiment, a multivalent
antibody comprises (or consists of), for example, three to about
eight, or four antigen binding sites. The multivalent antibody
comprises at least one polypeptide chain (for example, two
polypeptide chains), wherein the polypeptide chain(s) comprise two
or more variable domains. For instance, the polypeptide chain(s)
may comprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first
variable domain, VD2 is a second variable domain, Fc is one
polypeptide chain of an Fc region, X1 and X2 represent an amino
acid or polypeptide, and n is 0 or 1. For instance, the polypeptide
chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fc region
chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibody
herein may further comprise at least two (for example, four) light
chain variable domain polypeptides. The multivalent antibody herein
may, for instance, comprise from about two to about eight light
chain variable domain polypeptides. The light chain variable domain
polypeptides contemplated here comprise a light chain variable
domain and, optionally, further comprise a CL domain. Antibody
Variants
[0499] In some embodiments, amino acid sequence modification(s) of
the antibodies described herein are contemplated. For example, it
may be desirable to improve the binding affinity and/or other
biological properties of the antibody. Amino acid sequence variants
of the antibody are prepared by introducing appropriate nucleotide
changes into the antibody nucleic acid, or by peptide synthesis.
Such modifications include, for example, deletions from, and/or
insertions into and/or substitutions of, residues within the amino
acid sequences of the antibody. Any combination of deletion,
insertion, and substitution can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics. The amino acid alterations may be introduced in
the subject antibody amino acid sequence at the time that sequence
is made.
[0500] A useful method for identification of certain residues or
regions of the antibody that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis" as described
by Cunningham and Wells (1989) Science, 244:1081-1085. Here, a
residue or group of target residues are identified (e.g., charged
residues such as Arg, Asp, His, Lys, and Glu) and replaced by a
neutral or negatively charged amino acid (e.g., alanine or
polyalanine) to affect the interaction of the amino acids with
antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to analyze the performance of a
mutation at a given site, ala scanning or random mutagenesis is
conducted at the target codon or region and the expressed
immunoglobulins are screened for the desired activity.
[0501] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue or the antibody fused to a cytotoxic
polypeptide. Other insertional variants of the antibody molecule
include the fusion to the N- or C-terminus of the antibody to an
enzyme (e.g. for ADEPT) or a polypeptide which increases the serum
half-life of the antibody.
[0502] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
antibody molecule replaced by a different residue. The sites of
greatest interest for substitutional mutagenesis include the
hypervariable regions, but FR alterations are also contemplated.
Conservative substitutions are shown in Table A under the heading
of "preferred substitutions". If such substitutions result in a
change in biological activity, then more substantial changes,
denominated "exemplary substitutions" in Table A, or as further
described below in reference to amino acid classes, may be
introduced and the products screened.
TABLE-US-00009 TABLE A Original Exemplary Preferred Residue
Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys
Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C)
Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala
His (H) Asn; Gln; Lys; Arg Ile (I) Leu; Val; Met; Ala; Leu Phe;
Norleucine Leu (L) Norleucine; Ile; Val; Ile Met; Ala; Phe Lys (K)
Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val;
Ile; Ala; Tyr Pro (P) Ala Ser (S) Thr Thr (T) Val; Ser Trp (W) Tyr;
Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe;
Leu Ala; Norleucine
[0503] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Amino acids may be grouped
according to similarities in the properties of their side chains
(in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth
Publishers, New York (1975)): [0504] (1) non-polar: Ala (A), Val
(V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M) [0505]
(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y),
Asn (N), Gln (O) [0506] (3) acidic: Asp (D), Glu (E) [0507] (4)
basic: Lys (K), Arg (R), His (H)
[0508] Alternatively, naturally occurring residues may be divided
into groups based on common side-chain properties:
[0509] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
[0510] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0511] (3) acidic: Asp, Glu;
[0512] (4) basic: His, Lys, Arg;
[0513] (5) residues that influence chain orientation: Gly, Pro;
[0514] (6) aromatic: Trp, Tyr, Phe.
[0515] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class. Such substituted
residues also may be introduced into the conservative substitution
sites or, into the remaining (non-conserved) sites.
[0516] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g. a
humanized or human antibody). Generally, the resulting variant(s)
selected for further development will have modified (e.g.,
improved) biological properties relative to the parent antibody
from which they are generated. A convenient way for generating such
substitutional variants involves affinity maturation using phage
display. Briefly, several hypervariable region sites (e.g. 6-7
sites) are mutated to generate all possible amino acid
substitutions at each site. The antibodies thus generated are
displayed from filamentous phage particles as fusions to at least
part of a phage coat protein (e.g., the gene III product of M13)
packaged within each particle. The phage-displayed variants are
then screened for their biological activity (e.g. binding affinity)
as herein disclosed. In order to identify candidate hypervariable
region sites for modification, scanning mutagenesis (e.g., alanine
scanning) can be performed to identify hypervariable region
residues contributing significantly to antigen binding.
Alternatively, or additionally, it may be beneficial to analyze a
crystal structure of the antigen-antibody complex to identify
contact points between the antibody and antigen. Such contact
residues and neighboring residues are candidates for substitution
according to techniques known in the art, including those
elaborated herein. Once such variants are generated, the panel of
variants is subjected to screening using techniques known in the
art, including those described herein, and antibodies with superior
properties in one or more relevant assays may be selected for
further development.
[0517] Nucleic acid molecules encoding amino acid sequence variants
of the antibody are prepared by a variety of methods known in the
art. These methods include, but are not limited to, isolation from
a natural source (in the case of naturally occurring amino acid
sequence variants) or preparation by oligonucleotide-mediated (or
site-directed) mutagenesis, PCR mutagenesis, and cassette
mutagenesis of an earlier prepared variant or a non-variant version
of the antibody.
[0518] It may be desirable to introduce one or more amino acid
modifications in an Fc region of antibodies of the invention,
thereby generating an Fc region variant. The Fc region variant may
comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3
or IgG4 Fc region) comprising an amino acid modification (e.g. a
substitution) at one or more amino acid positions including that of
a hinge cysteine.
[0519] Immunoconjugates
[0520] In another aspect, the invention provides immunoconjugates,
or antibody-drug conjugates (ADC), comprising an antibody
conjugated to a cytotoxic agent such as a chemotherapeutic agent, a
drug, a growth inhibitory agent, a toxin (e.g., an enzymatically
active toxin of bacterial, fungal, plant, or animal origin, or
fragments thereof), or a radioactive isotope (i.e., a
radioconjugate).
[0521] The use of antibody-drug conjugates for the local delivery
of cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit
tumor cells in the treatment of cancer (Syrigos and Epenetos (1999)
Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997)
Adv. Drg Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278) allows
targeted delivery of the drug moiety to tumors, and intracellular
accumulation therein, where systemic administration of these
unconjugated drug agents may result in unacceptable levels of
toxicity to normal cells as well as the tumor cells sought to be
eliminated (Baldwin et al., (1986) Lancet pp. (Mar. 15,
1986):603-05; Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents
In Cancer Therapy: A Review," in Monoclonal Antibodies '84:
Biological And Clinical Applications, A. Pinchera et al. (ed.s),
pp. 475-506). Maximal efficacy with minimal toxicity is sought
thereby. Both polyclonal antibodies and monoclonal antibodies have
been reported as useful in these strategies (Rowland et al., (1986)
Cancer Immunol. Immunother., 21:183-87). Drugs used in these
methods include daunomycin, doxorubicin, methotrexate, and
vindesine (Rowland et al., (1986) supra). Toxins used in
antibody-toxin conjugates include bacterial toxins such as
diphtheria toxin, plant toxins such as ricin, small molecule toxins
such as geldanamycin (Mandler et al (2000) Jour. of the Nat. Cancer
Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med.
Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.
13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc.
Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al
(1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res.
53:3336-3342). The toxins may effect their cytotoxic and cytostatic
effects by mechanisms including tubutin binding, DNA binding, or
topoisomerase inhibition. Some cytotoxic drugs tend to be inactive
or less active when conjugated to large antibodies or protein
receptor ligands.
[0522] Antibody Derivatives
[0523] Antibodies of the invention can be further modified to
contain additional nonproteinaceous moieties that are known in the
art and readily available. In one embodiment, the moieties suitable
for derivatization of the antibody are water soluble polymers.
Non-limiting examples of water soluble polymers include, but are
not limited to, polyethylene glycol (PEG), copolymers of ethylene
glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl
alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,
poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer,
polyaminoacids (either homopolymers or random copolymers), and
dextran or poly(n-vinyl pyrrolidone)polyethylene glycol,
propropylene glycol homopolymers, prolypropylene oxide/ethylene
oxide co-polymers, polyoxyethylated polyols (e.g., glycerol),
polyvinyl alcohol, and mixtures thereof. Polyethylene glycol
propionaldehyde may have advantages in manufacturing due to its
stability in water. The polymer may be of any molecular weight, and
may be branched or unbranched. The number of polymers attached to
the antibody may vary, and if more than one polymer is attached,
the polymers can be the same or different molecules. In general,
the number and/or type of polymers used for derivatization can be
determined based on considerations including, but not limited to,
the particular properties or functions of the antibody to be
improved, whether the antibody derivative will be used in a therapy
under defined conditions, etc.
[0524] In another embodiment, conjugates of an antibody and
nonproteinaceous moiety that may be selectively heated by exposure
to radiation are provided. In one embodiment, the nonproteinaceous
moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci.
102: 11600-11605 (2005)). The radiation may be of any wavelength,
and includes, but is not limited to, wavelengths that do not harm
ordinary cells, but which heat the nonproteinaceous moiety to a
temperature at which cells proximal to the
antibody-nonproteinaceous moiety are killed.
[0525] Pharmaceutical Formulations
[0526] Therapeutic formulations comprising an antibody 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).
[0527] 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.
[0528] 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).
[0529] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0530] 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
thio-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.
[0531] Uses
[0532] An antibody of the invention may be used in, for example, in
vitro, ex vivo and in vivo therapeutic methods. Antibodies of the
invention can be used as an antagonist to partially or fully block
the specific antigen activity in vitro, ex vivo and/or in vivo.
Moreover, at least some of the antibodies of the invention can
neutralize antigen activity from other species. Accordingly,
antibodies of the invention can be used to inhibit a specific
antigen activity, e.g., in a cell culture containing the antigen,
in human subjects or in other mammalian subjects having the antigen
with which an antibody of the invention cross-reacts (e.g.
chimpanzee, baboon, marmoset, cynomolgus and rhesus, pig or mouse).
In one embodiment, an antibody of the invention can be used for
inhibiting antigen activities by contacting the antibody with the
antigen such that antigen activity is inhibited. In one embodiment,
the antigen is a human protein molecule.
[0533] In one embodiment, an antibody of the invention can be used
in a method for inhibiting an antigen in a subject suffering from a
disorder in which the antigen activity is detrimental, comprising
administering to the subject an antibody of the invention such that
the antigen activity in the subject is inhibited. In one
embodiment, the antigen is a human protein molecule and the subject
is a human subject. Alternatively, the subject can be a mammal
expressing the antigen with which an antibody of the invention
binds. Still further the subject can be a mammal into which the
antigen has been introduced (e.g., by administration of the antigen
or by expression of an antigen transgene). An antibody of the
invention can be administered to a human subject for therapeutic
purposes. Moreover, an antibody of the invention can be
administered to a non-human mammal expressing an antigen with which
the antibody cross-reacts (e.g., a primate, pig or mouse) for
veterinary purposes or as an animal model of human disease.
Regarding the latter, such animal models may be useful for
evaluating the therapeutic efficacy of antibodies of the invention
(e.g., testing of dosages and time courses of administration).
Antibodies 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 SSEA-3/SSEA-4/Globo Hs and
SSEA-3/SSEA-4/Globo Hated 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.
[0534] In one aspect, a blocking antibody of the invention is
specific for a SSEA-3/SSEA-4/Globo H.
[0535] In certain embodiments, an immunoconjugate comprising an
antibody of the invention conjugated with a cytotoxic agent is
administered to the patient. In some embodiments, the
immunoconjugate and/or antigen to which it is bound is/are
internalized by cells expressing one or more proteins on their cell
surface which are associated with SSEA-3/SSEA-4/Globo H, resulting
in increased therapeutic efficacy of the immunoconjugate in killing
the target cell with which it is associated. In one embodiment, the
cytotoxic agent targets or interferes with nucleic acid in the
target cell. Examples of such cytotoxic agents include any of the
chemotherapeutic agents noted herein (such as a maytansinoid or a
calicheamicin), a radioactive isotope, or a ribonuclease or a DNA
endonuclease.
[0536] Antibodies 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.
[0537] An antibody 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 antibody is 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.
[0538] 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. One or more nucleic acids encoding
all or a portion of an anti-SSEA-3/SSEA-4/Globo H antibody of the
invention can be delivered to a target cell, such that one or more
intrabodies are expressed which are capable of intracellular
binding to a SSEA-3/SSEA-4/Globo H and modulation of one or more
SSEA-3/SSEA-4/Globo H-mediated cellular pathways.
[0539] In another embodiment, internalizing antibodies are
provided. Antibodies can possess certain characteristics that
enhance delivery of antibodies into cells, or can be modified to
possess such characteristics. Techniques for achieving this are
known in the art. For example, cationization of an antibody is
known to facilitate its uptake into cells (see, e.g., U.S. Pat. No.
6,703,019). Lipofections or liposomes can also be used to deliver
the antibody into cells. Where antibody fragments are used, the
smallest inhibitory fragment that specifically binds to the binding
domain of the target protein is generally advantageous. For
example, based upon the variable-region sequences of an antibody,
peptide molecules can be designed that retain the ability to bind
the target protein sequence. Such peptides can be synthesized
chemically and/or produced by recombinant DNA technology. See,
e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893
(1993).
[0540] Entry of modulator polypeptides into target cells can be
enhanced by methods known in the art. For example, certain
sequences, such as those derived from HIV Tat or the Antennapedia
homeodomain protein are able to direct efficient uptake of
heterologous proteins across cell membranes. See, e.g., Chen et
al., Proc. Natl. Acad. Sci. USA (1999), 96:4325-4329.
[0541] When the binding target is located in the brain, certain
embodiments of the invention provide for the antibody or
antigen-binding fragment thereof to traverse the blood-brain
barrier. Certain neurodegenerative diseases are associated with an
increase in permeability of the blood-brain barrier, such that the
antibody or antigen-binding fragment can be readily introduced to
the brain. When the blood-brain barrier remains intact, several
art-known approaches exist for transporting molecules across it,
including, but not limited to, physical methods, lipid-based
methods, and receptor and channel-based methods.
[0542] Physical methods of transporting the antibody or
antigen-binding fragment across the blood-brain barrier include,
but are not limited to, circumventing the blood-brain barrier
entirely, or by creating openings in the blood-brain barrier.
Circumvention methods include, but are not limited to, direct
injection into the brain (see, e.g., Papanastassiou et al., Gene
Therapy 9: 398-406 (2002)), interstitial
infusion/convection-enhanced delivery (see, e.g., Bobo et al.,
Proc. Natl. Acad. Sci. USA 91: 2076-2080 (1994)), and implanting a
delivery device in the brain (see, e.g., Gill et al., Nature Med.
9: 589-595 (2003); and Gliadel Wafers.TM., Guildford
Pharmaceutical). Methods of creating openings in the barrier
include, but are not limited to, ultrasound (see, e.g., U.S. Patent
Publication No. 2002/0038086), osmotic pressure (e.g., by
administration of hypertonic mannitol (Neuwelt, E. A., Implication
of the Blood-Brain Barrier and its Manipulation, Vols 1 & 2,
Plenum Press, N.Y. (1989))), permeabilization by, e.g., bradykinin
or permeabilizer A-7 (see, e.g., U.S. Pat. Nos. 5,112,596,
5,268,164, 5,506,206, and 5,686,416), and transfection of neurons
that straddle the blood-brain barrier with vectors containing genes
encoding the antibody or antigen-binding fragment (see, e.g., U.S.
Patent Publication No. 2003/0083299).
[0543] Lipid-based methods of transporting the antibody or
antigen-binding fragment across the blood-brain barrier include,
but are not limited to, encapsulating the antibody or
antigen-binding fragment in liposomes that are coupled to antibody
binding fragments that bind to receptors on the vascular
endothelium of the blood-brain barrier (see, e.g., U.S. Patent
Application Publication No. 20020025313), and coating the antibody
or antigen-binding fragment in low-density lipoprotein particles
(see, e.g., U.S. Patent Application Publication No. 20040204354) or
apolipoprotein E (see, e.g., U.S. Patent Application Publication
No. 20040131692).
[0544] Receptor and channel-based methods of transporting the
antibody or antigen-binding fragment across the blood-brain barrier
include, but are not limited to, using glucocorticoid blockers to
increase permeability of the blood-brain barrier (see, e.g., U.S.
Patent Application Publication Nos. 2002/0065259, 2003/0162695, and
2005/0124533); activating potassium channels (see, e.g., U.S.
Patent Application Publication No. 2005/0089473), inhibiting ABC
drug transporters (see, e.g., U.S. Patent Application Publication
No. 2003/0073713); coating antibodies with a transferrin and
modulating activity of the one or more transferrin receptors (see,
e.g., U.S. Patent Application Publication No. 2003/0129186), and
cationizing the antibodies (see, e.g., U.S. Pat. No.
5,004,697).
[0545] The antibody composition 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.
[0546] For the prevention or treatment of disease, the appropriate
dosage of an antibody 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.
[0547] Articles of Manufacture
[0548] 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.
[0549] Pharmaceutical Compositions and Formulations
[0550] After preparation of the antibodies as described herein,
"pre-lyophilized formulation" can be produced. The antibody for
preparing the formulation is preferably essentially pure and
desirably essentially homogeneous (i.e. free from contaminating
proteins etc.). "Essentially pure" protein means a composition
comprising at least about 90% by weight of the protein, based on
total weight of the composition, preferably at least about 95% by
weight. "Essentially homogeneous" protein means a composition
comprising at least about 99% by weight of protein, based on total
weight of the composition. In certain embodiments, the protein is
an antibody.
[0551] The amount of antibody in the pre-lyophilized formulation is
determined taking into account the desired dose volumes, mode(s) of
administration etc. Where the protein of choice is an intact
antibody (a full-length antibody), from about 2 mg/mL to about 50
mg/mL, preferably from about 5 mg/mL to about 40 mg/mL and most
preferably from about 20-30 mg/mL is an exemplary starting protein
concentration. The protein is generally present in solution. For
example, the protein may be present in a pH-buffered solution at a
pH from about 4-8, and preferably from about 5-7. Exemplary buffers
include histidine, phosphate, Tris, citrate, succinate and other
organic acids. The buffer concentration can be from about 1 mM to
about 20 mM, or from about 3 mM to about 15 mM, depending, for
example, on the buffer and the desired isotonicity of the
formulation (e.g. of the reconstituted formulation). The preferred
buffer is histidine in that, as demonstrated below, this can have
lyoprotective properties. Succinate was shown to be another useful
buffer.
[0552] The lyoprotectant is added to the pre-lyophilized
formulation. In preferred embodiments, the lyoprotectant is a
non-reducing sugar such as sucrose or trehalose. The amount of
lyoprotectant in the pre-lyophilized formulation is generally such
that, upon reconstitution, the resulting formulation will be
isotonic. However, hypertonic reconstituted formulations may also
be suitable. In addition, the amount of lyoprotectant must not be
too low such that an unacceptable amount of degradation/aggregation
of the protein occurs upon lyophilization. Where the lyoprotectant
is a sugar (such as sucrose or trehalose) and the protein is an
antibody, exemplary lyoprotectant concentrations in the
pre-lyophilized formulation are from about 10 mM to about 400 mM,
and preferably from about 30 mM to about 300 mM, and most
preferably from about 50 mM to about 100 mM.
[0553] The ratio of protein to lyoprotectant is selected for each
protein and lyoprotectant combination. In the case of an antibody
as the protein of choice and a sugar (e.g., sucrose or trehalose)
as the lyoprotectant for generating an isotonic reconstituted
formulation with a high protein concentration, the molar ratio of
lyoprotectant to antibody may be from about 100 to about 1500 moles
lyoprotectant to 1 mole antibody, and preferably from about 200 to
about 1000 moles of lyoprotectant to 1 mole antibody, for example
from about 200 to about 600 moles of lyoprotectant to 1 mole
antibody.
[0554] In preferred embodiments of the invention, it has been found
to be desirable to add a surfactant to the pre-lyophilized
formulation. Alternatively, or in addition, the surfactant may be
added to the lyophilized formulation and/or the reconstituted
formulation. Exemplary surfactants include nonionic surfactants
such as polysorbates (e.g. polysorbates 20 or 80); poloxamers (e.g.
poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurel
sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or
stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or
stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;
lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,
myristamidopropyl-, palnidopropyl-, or isostearamidopropyl-betaine
(e.g. lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or
isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or
disodium methyl oleyl-taurate; and the MONAQUAT.TM. series (Mona
Industries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl
glycol, and copolymers of ethylene and propylene glycol (e.g.
Pluronics, PF68 etc.). The amount of surfactant added is such that
it reduces aggregation of the reconstituted protein and minimizes
the formation of particulates after reconstitution. For example,
the surfactant may be present in the pre-lyophilized formulation in
an amount from about 0.001-0.5%, and preferably from about
0.005-0.05%.
[0555] In certain embodiments of the invention, a mixture of the
lyoprotectant (such as sucrose or trehalose) and a bulking agent
(e.g. mannitol or glycine) is used in the preparation of the
pre-lyophilization formulation. The bulking agent may allow for the
production of a uniform lyophilized cake without excessive pockets
therein etc.
[0556] Other pharmaceutically acceptable carriers, excipients or
stabilizers such as those described in Remington's Pharmaceutical
Sciences 16th edition, Osol, A. Ed. (1980) may be included in the
pre-lyophilized formulation (and/or the lyophilized formulation
and/or the reconstituted formulation) provided that they do not
adversely affect the desired characteristics of the formulation.
Acceptable carriers, excipients or stabilizers are nontoxic to
recipients at the dosages and concentrations employed and include;
additional buffering agents; preservatives; co-solvents;
antioxidants including ascorbic acid and methionine; chelating
agents such as EDTA; metal complexes (e.g. Zn-protein complexes);
biodegradable polymers such as polyesters; and/or salt-forming
counterions such as sodium.
[0557] The pharmaceutical compositions and formulations described
herein are preferably stable. A "stable" formulation/composition is
one in which the antibody therein essentially retains its physical
and chemical stability and integrity upon storage. Various
analytical techniques for measuring protein stability are available
in the art and are reviewed in Peptide and Protein Drug Delivery,
247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y.,
Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90
(1993). Stability can be measured at a selected temperature for a
selected time period.
[0558] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes, prior to, or following,
lyophilization and reconstitution. Alternatively, sterility of the
entire mixture may be accomplished by autoclaving the ingredients,
except for protein, at about 120.degree. C. for about 30 minutes,
for example.
[0559] After the protein, lyoprotectant and other optional
components are mixed together, the formulation is lyophilized. Many
different freeze-dryers are available for this purpose such as
Hull50.RTM. (Hull, USA) or GT20.RTM. (Leybold-Heraeus, Germany)
freeze-dryers. Freeze-drying is accomplished by freezing the
formulation and subsequently subliming ice from the frozen content
at a temperature suitable for primary drying. Under this condition,
the product temperature is below the eutectic point or the collapse
temperature of the formulation. Typically, the shelf temperature
for the primary drying will range from about -30 to 25.degree. C.
(provided the product remains frozen during primary drying) at a
suitable pressure, ranging typically from about 50 to 250 mTorr.
The formulation, size and type of the container holding the sample
(e.g., glass vial) and the volume of liquid will mainly dictate the
time required for drying, which can range from a few hours to
several days (e.g. 40-60 hrs). A secondary drying stage may be
carried out at about 0-40.degree. C., depending primarily on the
type and size of container and the type of protein employed.
However, it was found herein that a secondary drying step may not
be necessary. For example, the shelf temperature throughout the
entire water removal phase of lyophilization may be from about
15-30.degree. C. (e.g., about 20.degree. C.). The time and pressure
required for secondary drying will be that which produces a
suitable lyophilized cake, dependent, e.g., on the temperature and
other parameters. The secondary drying time is dictated by the
desired residual moisture level in the product and typically takes
at least about 5 hours (e.g. 10-15 hours). The pressure may be the
same as that employed during the primary drying step. Freeze-drying
conditions can be varied depending on the formulation and vial
size.
[0560] In some instances, it may be desirable to lyophilize the
protein formulation in the container in which reconstitution of the
protein is to be carried out in order to avoid a transfer step. The
container in this instance may, for example, be a 3, 5, 10, 20, 50
or 100 cc vial. As a general proposition, lyophilization will
result in a lyophilized formulation in which the moisture content
thereof is less than about 5%, and preferably less than about
3%.
[0561] At the desired stage, typically when it is time to
administer the protein to the patient, the lyophilized formulation
may be reconstituted with a diluent such that the protein
concentration in the reconstituted formulation is at least 50
mg/mL, for example from about 50 mg/mL to about 400 mg/mL, more
preferably from about 80 mg/mL to about 300 mg/mL, and most
preferably from about 90 mg/mL to about 150 mg/mL. Such high
protein concentrations in the reconstituted formulation are
considered to be particularly useful where subcutaneous delivery of
the reconstituted formulation is intended. However, for other
routes of administration, such as intravenous administration, lower
concentrations of the protein in the reconstituted formulation may
be desired (for example from about 5-50 mg/mL, or from about 10-40
mg/mL protein in the reconstituted formulation). In certain
embodiments, the protein concentration in the reconstituted
formulation is significantly higher than that in the
pre-lyophilized formulation. For example, the protein concentration
in the reconstituted formulation may be about 2-40 times,
preferably 3-10 times and most preferably 3-6 times (e.g. at least
three fold or at least four fold) that of the pre-lyophilized
formulation.
[0562] Reconstitution generally takes place at a temperature of
about 25.degree. C. to ensure complete hydration, although other
temperatures may be employed as desired. The time required for
reconstitution will depend, e.g., on the type of diluent, amount of
excipient(s) and protein. Exemplary diluents include sterile water,
bacteriostatic water for injection (BWFI), a pH buffered solution
(e.g. phosphate-buffered saline), sterile saline solution, Ringer's
solution or dextrose solution. The diluent optionally contains a
preservative. Exemplary preservatives have been described above,
with aromatic alcohols such as benzyl or phenol alcohol being the
preferred preservatives. The amount of preservative employed is
determined by assessing different preservative concentrations for
compatibility with the protein and preservative efficacy testing.
For example, if the preservative is an aromatic alcohol (such as
benzyl alcohol), it can be present in an amount from about 0.1-2.0%
and preferably from about 0.5-1.5%, but most preferably about
1.0-1.2%. Preferably, the reconstituted formulation has less than
6000 particles per vial which are >10 .mu.m size.
[0563] Therapeutic Applications
[0564] Described herein are therapeutic methods that include
administering to a subject in need of such treatment a
therapeutically effective amount of a composition that includes one
or more antibodies described herein.
[0565] 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.
[0566] In some embodiments, the subject (e.g., a human patient) in
need of the treatment is diagnosed with, suspected of having, or at
risk for cancer. 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. In some preferred
embodiments, the cancer is brain cancer or glioblastoma multiforme
(GBM) cancer.
[0567] In preferred embodiments, the antibody is capable of
targeting Globo H, SSEA-3 and SSEA-4-expressing cancer cells. In
some embodiments, the antibody is capable of targeting Globo H and
SSEA on cancer cells. In some embodiments, the antibody is capable
of targeting SSEA in cancers.
[0568] Accordingly, the antibody is a triple-targeting antibody
against Globo H, SSEA-3 and SSEA-4. In some embodiments, the
antibodies are a mixture of a dual-targeting antibody against Globo
H and SSEA-3, and an anti-SSEA-4 antibody. In some embodiments, the
antibodies are a mixture of a triple-targeting antibody against
Globo H, SSEA-3 and SSEA-4, and an anti-SSEA-4 antibody. In some
embodiments, the antibody is a mixture of an anti-Globo H, an
anti-SSEA-3 and an anti-SSEA-4 antibody. In some embodiments, the
antibody is a mixture of an anti-Globo H and an anti-SSEA-4
antibody. In some embodiments, the antibody is an anti-SSEA-4
antibody.
[0569] 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.
[0570] In some embodiments, the treatment further comprises
administering an additional therapy to said subject prior to,
during or subsequent to said administering of the antibodies. In
some embodiments, the additional therapy is treatment with a
chemotherapeutic agent. In some embodiments, the additional therapy
is radiation therapy.
[0571] The methods of the invention are particularly advantageous
in treating and preventing early stage tumors, thereby preventing
progression to the more advanced stages resulting in a reduction in
the morbidity and mortality associated with advanced cancer. The
methods of the invention are also advantageous in preventing the
recurrence of a tumor or the regrowth of a tumor, for example, a
dormant tumor that persists after removal of the primary tumor, or
in reducing or preventing the occurrence of a tumor.
[0572] In some embodiments, the methods as disclosed herein are
useful for the treatment or prevention of a cancer, for example
where a cancer is characterized by increased Globo H, SSEA-3 and/or
SSEA-4 expression. In some embodiments the cancer comprises a
cancer stem cell. In some embodiments, the cancer is a pre-cancer,
and/or a malignant cancer and/or a therapy resistant cancer. In
some embodiments, the cancer is a brain cancer.
[0573] For the methods of the invention, the cancer may be a solid
tumor, e.g., such as, breast cancer, colorectal cancer, rectal
cancer, lung cancer, renal cell cancer, a glioma (e.g., anaplastic
astrocytoma, anaplastic oligoastrocytoma, anaplastic
oligodendroglioma, glioblastoma multiforme (GBM)), kidney cancer,
prostate cancer, liver cancer, pancreatic cancer, soft-tissue
sarcoma, carcinoid carcinoma, head and neck cancer, melanoma, and
ovarian cancer. In one embodiment, the cancer is a brain cancer or
GBM. To practice the method disclosed herein, an effective amount
of the pharmaceutical composition/formulation described above,
containing at least one antibody described herein, can be
administered to a subject (e.g., a human) in need of the treatment
via a suitable route, such as intravenous administration, e.g., as
a bolus or by continuous infusion over a period of time, by
intramuscular, intraperitoneal, intracerebrospinal, subcutaneous,
intra-articular, intrasynovial, intrathecal, oral, inhalation or
topical routes. Commercially available nebulizers for liquid
formulations, including jet nebulizers and ultrasonic nebulizers
are useful for administration. Liquid formulations can be directly
nebulized and lyophilized powder can be nebulized after
reconstitution. Alternatively, the antibodies can be aerosolized
using a fluorocarbon formulation and a metered dose inhaler, or
inhaled as a lyophilized and milled powder.
[0574] The subject to be treated by the methods described herein
can be a mammal, more preferably a human. Mammals include, but are
not limited to, farm animals, sport animals, pets, primates,
horses, dogs, cats, mice and rats. A human subject who needs the
treatment may be a human patient having, at risk for, or suspected
of having cancer, which include, but 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. A subject having cancer can be identified by routine
medical examination.
[0575] "An effective amount" as used herein refers to the amount of
each active agent required to confer therapeutic effect on the
subject, either alone or in combination with one or more other
active agents. Effective amounts vary, as recognized by those
skilled in the art, depending on the particular condition being
treated, the severity of the condition, the individual patient
parameters including age, physical condition, size, gender and
weight, the duration of the treatment, the nature of concurrent
therapy (if any), the specific route of administration and like
factors within the knowledge and expertise of the health
practitioner. These factors are well known to those of ordinary
skill in the art and can be addressed with no more than routine
experimentation. It is generally preferred that a maximum dose of
the individual components or combinations thereof be used, that is,
the highest safe dose according to sound medical judgment. It will
be understood by those of ordinary skill in the art, however, that
a patient may insist upon a lower dose or tolerable dose for
medical reasons, psychological reasons or for virtually any other
reasons.
[0576] Empirical considerations, such as the half-life, generally
will contribute to the determination of the dosage. For example,
antibodies that are compatible with the human immune system, such
as humanized antibodies or fully human antibodies, may be used to
prolong half-life of the antibody and to prevent the antibody being
attacked by the host's immune system. Frequency of administration
may be determined and adjusted over the course of therapy, and is
generally, but not necessarily, based on treatment and/or
suppression and/or amelioration and/or delay of cancer.
Alternatively, sustained continuous release formulations of the
antibodies described herein may be appropriate. Various
formulations and devices for achieving sustained release are known
in the art.
[0577] In one example, dosages for an antibody as described herein
may be determined empirically in individuals who have been given
one or more administration(s) of the antibody. Individuals are
given incremental dosages of the antibody. To assess efficacy of
the antibody, an indicator of the disease (e.g., cancer) can be
followed according to routine practice.
[0578] Generally, for administration of any of the antibodies
described herein, an initial candidate dosage can be about 2 mg/kg.
For the purpose of the present disclosure, a typical daily dosage
might range from about any of 0.1 .mu.g/kg to 3 .mu.g/kg to 30
.mu.g/kg to 300 .mu.g/kg to 3 mg/kg, to 30 mg/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 is sustained until a desired suppression
of symptoms occurs or until sufficient therapeutic levels are
achieved to alleviate cancer, or a symptom thereof. An exemplary
dosing regimen comprises administering an initial dose of about 2
mg/kg, followed by a weekly maintenance dose of about 1 mg/kg of
the antibody, or followed by a maintenance dose of about 1 mg/kg
every other week. However, other dosage regimens may be useful,
depending on the pattern of pharmacokinetic decay that the
practitioner wishes to achieve. For example, dosing from one-four
times a week is contemplated. In some embodiments, dosing ranging
from about 3 .mu.g/mg to about 2 mg/kg (such as about 3 .mu.g/mg,
about 10 .mu.g/mg, about 30 .mu.g/mg, about 100 .mu.g/mg, about 300
.mu.g/mg, about 1 mg/kg, and about 2 mg/kg) may be used. In some
embodiments, dosing frequency is once every week, every 2 weeks,
every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8
weeks, every 9 weeks, or every 10 weeks; or once every month, every
2 months, or every 3 months, or longer. The progress of this
therapy is easily monitored by conventional techniques and assays.
The dosing regimen (including the antibody used) can vary over
time.
[0579] For the purpose of the present disclosure, the appropriate
dosage of an antibody described herein will depend on the specific
antibody (or compositions thereof) employed, the type and severity
of the cancer, 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 administration of the antibodies described
herein may be essentially continuous over a preselected period of
time or may be in a series of spaced dose, e.g., either before,
during, or after developing cancer.
[0580] As used herein, the term "treating" refers to the
application or administration of a composition including one or
more active agents to a subject, who has cancer, a symptom of
cancer, or a predisposition toward cancer, with the purpose to
cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve,
or affect cancer, the symptom of cancer, or the predisposition
toward cancer.
[0581] Alleviating cancer includes delaying the development or
progression of cancer, or reducing cancer severity. Alleviating
cancer does not necessarily require curative results. As used
therein, "delaying" the development of cancer means to defer,
hinder, slow, retard, stabilize, and/or postpone progression of
cancer. This delay can be of varying lengths of time, depending on
the history of cancer and/or individuals being treated. A method
that "delays" or alleviates the development of cancer, or delays
the onset of cancer, is a method that reduces probability (the
risk) of developing one or more symptoms of cancer in a given time
frame and/or reduces extent of the symptoms in a given time frame,
when compared to not using the method. Such comparisons are
typically based on clinical studies, using a number of subjects
sufficient to give a statistically significant result.
[0582] "Development" or "progression" of cancer means initial
manifestations and/or ensuing progression of cancer. Development of
cancer can be detectable and assessed using standard clinical
techniques as well known in the art. However, development also
refers to progression that may be undetectable. For purpose of this
disclosure, development or progression refers to the biological
course of the symptoms. "Development" includes occurrence,
recurrence, and onset. As used herein "onset" or "occurrence" of
cancer includes initial onset and/or recurrence.
[0583] Conventional methods, known to those of ordinary skill in
the art of medicine, can be used to administer the pharmaceutical
composition to the subject, depending upon the type of disease to
be treated or the site of the disease. This composition can also be
administered via other conventional routes, e.g., administered
orally, parenterally, by inhalation spray, topically, rectally,
nasally, buccally, vaginally or via an implanted reservoir. The
term "parenteral" as used herein includes subcutaneous,
intracutaneous, intravenous, intramuscular, intraarticular,
intraarterial, intrasynovial, intrasternal, intrathecal,
intralesional, and intracranial injection or infusion techniques.
In addition, it can be administered to the subject via injectable
depot routes of administration such as using 1-, 3-, or 6-month
depot injectable or biodegradable materials and methods.
[0584] Injectable compositions may contain various carriers such as
vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate,
ethyl carbonate, isopropyl myristate, ethanol, and polyols
(glycerol, propylene glycol, liquid polyethylene glycol, and the
like). For intravenous injection, water soluble antibodies can be
administered by the drip method, whereby a pharmaceutical
formulation containing the antibody and a physiologically
acceptable excipients is infused. Physiologically acceptable
excipients may include, for example, 5% dextrose, 0.9% saline,
Ringer's solution or other suitable excipients. Intramuscular
preparations, e.g., a sterile formulation of a suitable soluble
salt form of the antibody, can be dissolved and administered in a
pharmaceutical excipient such as Water-for-Injection, 0.9% saline,
or 5% glucose solution.
[0585] Diagnostic Applications
[0586] Described herein is a method for diagnosing cancer in a
subject, comprising (a) applying a composition that includes one or
more monoclonal antibodies that detect expression of a panel of
markers consisting of GM3, GM2, GM1, GD1, GD1a, GD3, GD2, GT1b,
A2B5, LeX, sLeX, LeY, SSEA-3, SSEA-4, Globo H, TF, Tn, sTn, CD44,
CD24, CD45, CD90, CD133 to a cell or tissue sample obtained from
the subject; (b) assaying the binding of the monoclonal antibody to
the cell or the tissue sample; and (c) comparing the binding with a
normal control to determine the presence of the cancer in the
subject.
[0587] Examples of the cancer for detection and diagnosis 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.
[0588] In some embodiments, the markers consist of GM2, GM1, GD1a,
GT1b, A2B5, Tf, Tn, Globo H, SSEA3, SSEA4, CD24, CD44 and CD90. In
some embodiments, the composition includes a plurality of
monoclonal antibodies capable of detecting GM2, GM1, GD1a, GT1b,
A2B5, Tf, Tn, Globo H, SSEA3, SSEA4, CD24, CD44 and CD90.
[0589] In some embodiments, the antibody is capable of detecting
Globo H, SSEA-3 and SSEA-4-expressing cancer cells. In some
embodiments, the antibody is capable of detecting Globo H and SSEA
on cancer cells. In some embodiments, the antibody is capable of
detecting SSEA in cancers. In some embodiments, the cancer is brain
cancer or glioblastoma multiforme (GBM) cancer, and the antibody is
an anti-SSEA-4 monoclonal antibody.
[0590] Globo H, SSEA-3 and/or SSEA-4-specific monoclonal antibodies
can be used alone or in combination for in vitro and in vivo
diagnostic assays to detect Globo H, SSEA-3 and SSEA-4-expressing
cancer cells (e.g., GBM, certain solid tumor cells, and
hematopoietic cancer cells as indicated herein). For example, a
sample (e.g., blood sample or tissue biopsy) can be obtained from a
patient and contacted with a triple-targeting antibody against
Globo H, SSEA-3 and SSEA-4, or a Globo H/SSEA-3 dual-targeting
antibody in combination with an anti-SSEA-4, and the presence of
Globo H, SSEA-3 and SSEA-4 expressing cancer cells in the patient
sample can be determined by detecting antibody binding. Antibody
binding can be detected directly (e.g., where the antibody itself
is labeled) or by using a second detection agent, such as a
secondary antibody. The detectable label can be associated with an
antibody of the invention, either directly, or indirectly, e.g.,
via a chelator or linker.
[0591] In some embodiments, Globo H, SSEA-3 and/or SSEA-4 specific
monoclonal antibodies are contacted with a biological sample from
an individual having or suspected of having cancer, and antibody
binding to a cell in the sample is determined when higher or lower
than normal antibody binding indicates that the individual has
cancer. In some embodiments, the biological sample is a blood
sample or blood fraction (e.g., serum, plasma, platelets, red blood
cells, white blood cells). In some embodiments, the biological
sample is a tissue sample (biopsy), e.g., from a suspected tumor
site, or from a tissue that is known to be affected, e.g., to
determine the boundaries of a known tumor. In some embodiments, the
biological sample is obtained from a site of inflammation.
[0592] Biopsies are typically performed to obtain samples from
tissues, i.e., non-fluid cell types. The biopsy technique applied
will depend on the tissue type to be evaluated (e.g., breast, skin,
colon, prostate, kidney, lung, bladder, lymph node, liver, bone
marrow, airway or lung). In the case of a cancer the technique will
also depend on the size and type of the tumor (e.g., solid,
suspended, or blood), among other factors. Biopsy techniques are
discussed, for example, in Harrison's Principles of Internal
Medicine, Kasper, et al., eds., 16th ed., 2005, Chapter 70, and
throughout Part V.
[0593] Any method of detecting antibody binding to a cell in a
sample can be used for the present diagnostic assays. Methods of
detecting antibody binding are well known in the art, e.g., flow
cytometry, fluorescent microscopy, ELISAs, etc. In some
embodiments, the method comprises preparing the biological sample
for detection prior to the determining step. For example, a
subpopulation of cells (e.g., white blood cells) can be separated
from the rest of the sample from the individual (e.g., other blood
components) or cells in a tissue can be suspended for easier
detection.
[0594] In some embodiments, the percentage of Globo H/SSEA-3/SSEA-4
expressing cells in the sample is determined and compared to a
control, e.g., a sample from an individual or group of individuals
that are known to have cancer (positive control) or from an
individual or group of individuals that are known not to have
cancer (normal, non-disease, or negative control). In some
embodiments, the control is a standard range of Globo
H/SSEA-3/SSEA-4 expression established for a given tissue. A higher
or lower than normal percentage of Globo H/SSEA-3/SSEA-4 expressing
cells, or higher or lower expression level, indicates that the
individual has cancer.
[0595] In one embodiment, a kit is provided for detecting Globo H,
SSEA-3 and SSEA-4 in a biological sample, such as a blood sample or
tissue sample. For example, to confirm a cancer diagnosis in a
subject, a biopsy can be performed to obtain a tissue sample for
histological examination. Alternatively, a blood sample can be
obtained to detect the presence of Globo H, SSEA-3 and SSEA-4. Kits
for detecting a polypeptide will typically comprise one or more
antibodies that specifically bind Globo H, SSEA-3 and SSEA-4, such
as any of the antibodies disclosed herein. In a further embodiment,
the antibodies are labeled (for example, with a fluorescent,
radioactive, or an enzymatic label).
[0596] In one embodiment, a kit includes instructional materials
disclosing means of use of one or more antibodies that specifically
bind Globo H, SSEA-3 and SSEA-4. The instructional materials may be
written, in an electronic form (such as a computer diskette or
compact disk) or may be visual (such as video files). The kits may
also include additional components to facilitate the particular
application for which the kit is designed. Thus, for example, the
kit may additionally contain means of detecting a label (such as
enzyme substrates for enzymatic labels, filter sets to detect
fluorescent labels, appropriate secondary labels such as a
secondary antibody, or the like). The kits may additionally include
buffers and other reagents routinely used for the practice of a
particular method. Such kits and appropriate contents are well
known to those of skill in the art.
[0597] Methods of determining the presence or absence of a cell
surface marker are well known in the art. For example, the
antibodies can be conjugated to other compounds including, but not
limited to, enzymes, magnetic beads, colloidal magnetic beads,
haptens, fluorochromes, metal compounds, radioactive compounds or
drugs. The antibodies can also be utilized in immunoassays such as
but not limited to radioimmunoassays (RIAs), enzyme linked
immunosorbent assays (ELISA), or immunohistochemical assays. The
antibodies can also be used for fluorescence activated cell sorting
(FACS). A FACS employs a plurality of color channels, low angle and
obtuse light-scattering detection channels, and impedance channels,
among other more sophisticated levels of detection, to separate or
sort cells (see U.S. Pat. No. 5,061,620). Any of the monoclonal
antibodies that bind to Globo H, SSEA-3 and SSEA-4, as disclosed
herein, can be used in these assays. Thus, the antibodies can be
used in a conventional immunoassay, including, without limitation,
an ELISA, an RIA, FACS, tissue immunohistochemistry, Western blot
or immunoprecipitation.
[0598] Methods for Staging and/or Determining Prognosis of
Tumors
[0599] Another aspect of the present disclosure features a method
for staging and/or determining prognosis of tumorsin a human
patient, the method comprising: (a) applying a composition that
includes one or more antibodies that detect the expression of
markers consisting of SSEA-3, SSEA-4 and Globo H to a cell or
tissue sample obtained from the patient; (b) assaying the binding
of the monoclonal antibodies to the cell or the tissue sample; (c)
comparing the expression level of the markers in the test sample
with the level in a reference sample, and (d) determining the stage
and/or prognosis of tumors in the patient based upon the outcome
identified in step (c).
[0600] In some embodiments, the cancer is brain cancer, lung
cancer, breast cancer, ovarian cancer, prostate cancer, colon
cancer, or pancreas cancer. In some preferred embodiments, the
cancer is brain cancer or GBM.
[0601] In some embodiments, the antibody is capable of detecting
Globo H, SSEA-3 and SSEA-4 expressing cancer cells. In some
embodiments, the antibody is capable of detecting Globo H and SSEA
on cancer cells. In some embodiments, the antibody is capable of
detecting SSEA in cancers. In some embodiments, the cancer is brain
cancer or glioblastoma multiforme (GBM) cancer, and the antibody is
an anti-SSEA-4 monoclonal antibody. In some embodiments, the
antibody is an anti-SSEA-4 when the cancer is brain cancer or
GBM.
[0602] In some embodiments, the provided glycan conjugates,
immunogenic compositions 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 (CIVIL) (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 (CIVIL), 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
(WE), 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.
[0603] To perform the treatment methods described herein, an
effective amount of any of the glycan compositions 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. In some embodiments, the amount
of the glycan conjugate or immunogenic composition is sufficient to
elicit responses leading to the inhibition of cancer growth and/or
reduction of tumor mass. In other embodiments, the amount of the
glycan 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 compositions 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).
[0604] In certain embodiments, an effective amount, of the provided
glycan compositions 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.
[0605] In certain embodiments, the provided glycan compositions 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.
[0606] 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.
[0607] It will be also appreciated that the provided glycan
compositions 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.
[0608] The provided glycan compositions 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.
[0609] In certain embodiments, the provided glycan composition 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.
[0610] Exemplary biotherapeutic anti-cancer agents include, but are
not limited to, interferons, cytokines (e.g., tumor necrosis
factor, interferon .alpha., interferon .gamma.), 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), RITUXAN (rituximab),
BEXXAR (tositumomab)).
[0611] 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), Ca.sup.2+ 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 (TOVOK.TM.),
SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869,
MP470, BIM 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.
[0612] 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.
EXAMPLES
[0613] 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 Structure of Optimized Universal Fc Glycan
[0614] The glycan structure of an optimized universal Fc glycan for
therapeutic antibodies is
Sia.sub.2(.alpha.2-6)Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2
(FIG. 1).
##STR00003##
Example 2: Exemplary General Procedure for the Preparation of
Homogeneous Antibodies with the Optimized Universal Glycan at the
Fc Region
[0615] The present disclosure provides exemplary improved method
for making a population of homogeneous antibodies with the
optimized universal glycan at the Fc region comprising the steps of
(a) contacting a monoclonal antibody with an .alpha.-fucosidase and
at least one endoglycosidase, thereby yielding a defucosylated
antibody having a single N-acetylglucosamine (GlcNAc), and (b)
adding the universal glycan to GlcNAc of Fc region of antibody to
form the homogeneous antibody with the FIG. 1 showed optimized
glycan form (FIG. 2).
[0616] See FIG. 2. General strategy for the preparation of
homogeneous antibody with optimized universal glycan at the Fc
region for the improvement of its therapeutic activity.
[0617] Endoglycosidase is used to trim off the variable portions of
an oligosaccharide in N-glycan. Examples of endoglycosidases used
herein include, but not limited to, EndoA, EndoF, EndoF1, EndoF2,
EndoH, EndoM, EndoS, and variants thereof.
Example 3: Preparation of Homogeneous Antibody with Universal
Glycan at the Fc Region Toward Enhancing Monoclonal Antibody
Mediated Antiviral Therapeutics
[0618] Exemplary method for the preparation of homogeneous
anti-influenza virus antibody with universal glycan at the Fc
region to increase the its ADCC effect.
[0619] Broadly neutralizing monoclonal antibodies targeting the
conserved stalk region of hemagglutinin (HA) can be facilitated by
the interactions between the antibody Fc and Fc receptors to
trigger its function. The anti-influenza virus antibody FI6 was
chosen based on its demonstrated ADCC effects, and the other
anti-influenza virus antibody F10 that target stalk region of (HA)
for the preparation of homogeneous antibody with optimized
universal glycan by using the general strategy and methods
disclosed herein. In brief, FI6 and F10 antibodies were prepared by
the literature reported methods (ref). The home-made heterogeneous
monoclonal antibodies FI6 or F10 was used as the starting material
and modified with endoglycosidase endo S to yield a mixture of
di-sugar mAb of GlcNAc-Fuc, and mono-sugar mAb of GlcNAc.
Subsequently a homogeneous mono-sugar mAb was obtained with
application of fucosidase; or the mono-sugar species was obtained
with combination of Endo S and fucosidase in one step.
[0620] FI6/F10 (0.25 mg) in a sodium phosphate buffer (50 mM, pH
7.0, 0.125 mL) was incubated with Endo S (12.5 .mu.g) and BfFucH
(0.25 mg) at 37.degree. C. for 22 h. LC-MS and SDS-PAGE analyses
indicated the complete cleavage of the N-glycans on the heavy
chain. The reaction mixture was subject to affinity chromatography
on a column of protein A-agarose resin (0.1 mL) that was
pre-equilibrated with a sodium phosphate buffer (20 mM, pH 7.0).
The column was washed with a sodium phosphate buffer (20 mM, pH
7.0, 1.0 mL). The bound IgG was released with glycine-HCl (50 mM,
pH 3.0, 1.0 mL), and the elution fractions were immediately
neutralized with Tris-Cl buffer (1.0 M, pH 8.3). The fractions
containing the Fc fragments were combined and concentrated by
centrifugal filtration (Amicon Ultra centrifugal filter, Millipore,
Billerica, Mass.) to give mono-GlcNAc homogeneous antibody (0.193
mg). The product was trypsinized, and the glycopeptides,
TKPREEQYNSTYR and EEQYNSTYR, were analyzed using nanospray LC/MS to
confirm the glycosylation pattern of glycan engineering
FI6/F10.
[0621] Isolation of the sialylglycan (SCT) from hen's egg yolk was
according to the published method with some modification. Briefly,
the ethanol extraction of hen's egg yolk was centrifuged,
filtrated, and the treated with endo M, after reaction complete,
the SCT was purified by gel filtration and ion exchange
chromatography, the purified SCT was lyophilized to give pure SCT
product as a white powder (82%).
[0622] A solution of SCT (Sia.sub.2Gal2GlcNAc2Man3GlcNAc) (3.0 mg),
2-chloro-1,3-dimethylimidazolinium chloride (DMC) (6.3 mg) and Et3N
(9.0 .mu.L) in water (60.0 .mu.L) was stirred at 4.degree. C. for 1
h. The reaction mixture was subjected to gel filtration
chromatography on a Sephadex G-25 column eluted by 0.05% aqueous
Et3N. The fractions containing the product (SCT oxazoline) were
combined and lyophilized to give a white powder (2.6 mg, yield
87.4%).
[0623] SCT oxazoline was added to a mixture of glycosynthase and
mono-GlcNAc Fi6 or F10 in 50 mM Tris buffer (pH 7.8) and incubated
for an hour at room temperature. The reaction mixture was purified
with protein A affinity column, followed by amanion exchange column
capto Q to collect the desired product, optimized anti-influenza
virus homogeneous antibody FI6-M or F10-M. The product was
trypsinized, and the glycopeptides, TKPREEQYNSTYR and EEQYNSTYR,
were analyzed using nanospray LC/MS to confirm the glycosylation
pattern of FI6-M or F10-M.
Example 4
[0624] ADCC assay of FI6/F10 and glycoengineering FI6-M/F10-M. See
FIG. 3 demonstrated the enhanced ADCC results of anti-influenza
virus antibodies.
[0625] Anti-viral antibody-dependent cell-mediated cytotoxicity
(ADCC) enhancement is demonstrated with anti-influenza monoclonal
antibodies FI6 and F10 with glycan modification. Human HEK293T
cells were transiently transfected with plasmid to express
full-length Cal/09 HA on the cell surface to mimic influenza virus
infected cells. These cells were mixed with freshly prepared human
peripheral blood mononuclear cells (PBMC) isolated from health
donors with ratio of infected cells to PBMC of 1:20 or 1:50.
Antibodies FI6 and F10 in different concentrations with and without
glycan modification are then added into the mixtures. After 5
hours, the result of FI6 and F10 induced ADCC was monitored by
HEK293T cell lysis (LDH release). The results show that the ADCC
induced by antibodies FI6 and F10 with glycan modification is
enhanced 1.5-3 folds.
Example 5: Exemplary Methods and Materials for ADCC Assay
Example: Anti-Stem Monoclonal Antibodies FI6 and F10
[0626] The F10 and FI6 antibody expression plasmids were
transfected to HEK293F cell by using polyethyleneimine and cultured
in Freestyle 293 expression medium (Invitrogen). After 7 days
incubation, the supernatants were collected by centrifugation and
the antibodies were purified by protein A beads (Roche
Diagnostics). The antibody was further purified by gel filtration
chromatography on Superdex 200 (GE Healthcare) in PBS buffer.
Example 6
[0627] In Vitro Antibody-Dependent Cellular Cytotoxicity (ADCC)
Assay
[0628] HEK293T cells were transfected with pVax-Cal/09
hemagglutinin (HA) expression plasmid for 48 hour. The
HA-expressing HEK293T cells were trypsinized and seeded in 96-well
U-bottom plates, 5,000 cells per well in 50 ul DMEM medium
(Gibco).
[0629] Peripheral blood mononuclear cells (PBMCs) were prepared by
Ficoll-Paque separation of whole blood obtained from healthy
volunteers and used as effector cells in the ADCC assay. Briefly,
whole blood was diluted with an equal volume of HBSS, layered over
Ficoll-Paque plus (GE Healthcare) and centrifuged at 400 g for 40
min. The PBMC cells were harvested, washed twice with HBSS and
mixed with HA-expressing HEK293T cells using an effector-to-target
ratio of 50/1.
[0630] Mixture of PBMCs and HA-expressing HEK293T cells were
treated with different concentrations of antibodies FI6 and F10 and
incubated at 37.degree. C. for 5 hours.
[0631] After 5 hour incubation, ADCC was monitored by measuring the
lactate dehydrogenase (LDH) released using cytoTox96
Non-Radioactive Cytotoxicity Assay kit (Promega).
Example 7: Preparation of Homogeneous Antibody with Universal
Glycan at the Fc Region Toward Enhancing Monoclonal Antibody
Mediated Anti-Cancer Therapeutics
REPRESENTATIVE EXAMPLES
[0632] Commercial available Rituxan and Herceptin were used as
starting material, after the same methods described previously for
the preparation of homogeneous anti-influenza virus antibody with
universal glycan at the Fc region. The homogeneous Rituxan and
Herceptin with the optimized universal glycan
Sia.sub.2(.alpha.2-6)Gal2GlcNAc2Man3GlcNAc2 at the Fc region can be
obtained. Using the same methods, we have also prepared different
homogeneous Rituxan.RTM. and Herceptin.RTM. antibodies with
different glycanform at their Fc region for the comparison of
antibodies activities with different glycans.
[0633] Biological Characteristic of Anti-CD20 Homogeneous
Antibody
[0634] Glycosylation on Fc can affect a variety of immunoglobulin
effector-mediated functions, including ADCC, CDC and circulating
half-life. ADCC enhancement is a key strategy for improving
therapeutic antibody drug efficacy. It can lowering effective drug
dosage for benefits of lower drug cost. The anti-CD20 homogeneous
antibodies described herein can be characterized by functional
properties. The anti-CD20 GAb has cell growth inhibitory activities
including apoptosis against human CD20 expressing cells. In some
embodiments, the anti-CD20 GAb exhibits more potent cell growth
inhibitory activities as compared to its patent antibody.
Example 8
[0635] ADCC Activities of Anti-CD20 Glycoantibodies
[0636] The ADCC activity of the homogeneous antibody according to
the invention is at least 8 fold increased, preferably at least 15
fold, more preferably at least 35 fold increased ADCC activity,
preferably at least 50 fold increased ADCC activity, preferably at
least 60 fold increased ADCC activity, most preferred at least 80
fold increased ADCC activity compared to the ADCC activity of the
parental antibody.
[0637] The ADCC lysis activity of the inventive homogeneous
antibody can be measured in comparison to the parental antibody
using target cancer cell lines such as, for example, SKBR5, SKBR3,
LoVo, MCF7, OVCAR3 and/or Kato III.
[0638] A number of anti-CD20 GAbs described herein, in particular
GAb101, and GAb104, exhibited enhanced ADCC activity compared to it
parental antibody, Rituximab. The homogeneous antibodies of the
invention can exhibit superior effect as therapeutic agents for B
cell-mediated malignant tumors and immunological diseases in which
B cells or antibodies produced by B cells are involved, and an
object of the present invention is to use the anti-CD20 GAb in
development of therapeutic agents.
Example 9: CDC Activities of Anti-CD20 Glycoantibodies
[0639] The homogeneous antibodies described herein are surprisingly
able to provide improved ADCC without affecting CDC. Exemplary CDC
assays are described in the examples. In exemplary embodiments,
ADCC of the glycoantibody is increased but other
immunoglobulin-type effector functions such as complement-dependent
cytoxicity (CDC) remain similar or are not significantly
affected.
[0640] Binding Between Fc.gamma.RIII and Anti-CD20
Glycoantibodies
[0641] Fc.gamma.RIIIA was transfected into HEK-293 cell line to
express recombinant protein. The secreted Fc.gamma.RIIIA
recombinant protein was purified and then diluted to serial
concentration in HBS-EP buffer (200 nM, 100 nM, 50 nM, 25 nM, and
12.5 nM). Each of anti-CD20 GAbs101, 102, 104, 105, 106, 107, 108,
109, 110 and 111 was diluted in HBS-EP buffer to the concentration
of 10 mg/ml, and then captured to the CM5 chip in which anti-human
Fab domain antibodies were pre-immobilized. A serial titration of
Fc.gamma.RIIIA was injected and bound at the flow rate of 30
ml/min. Single cycle kinetics data was fitted into 1:1 binding
model using Biacore T200 evaluation software to measure the
equilibrium constant (Ka/Kd). The results were shown in Table
2.
[0642] Table 2 lists exemplary Fc.gamma.RIIIA binding of anti-CD20
GAbs and Rituximab. Fc.gamma.RIIIA binding may be measured using
assays known in the art. Exemplary assays are described in the
examples. The Fc receptor binding may be determined as the relative
ratio of anti-CD20 GAb vs Rituximab. Fc receptor binding in
exemplary embodiments is increased by at least 1.2-fold, 2-fold,
3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,
15-fold or 20-fold, 30-fold, 40-fold, 50-fold, 100-fold or
higher.
[0643] As compared to Rituximab, the binding data showed that the
anti-CD20 GAbs, in particular GAb101 and GAb104, exhibit stronger
binding affinity for the target molecule CD20.
[0644] Taken together, anti-CD20 GAbs, in particular GAb101, and
GAb104, exhibited enhanced ADCC activity and stronger
Fc.gamma.RIIIA binding affinity as compared to Rituximab. The
homogeneous antibodies of the invention can provide a superior
clinical response either alone or, preferably, in a composition
comprising two or more such antibodies, and optionally in
combination with other treatments such as chemotherapy. The
ADCC-enhanced anti-CD20 glycoantibody can provide an alternative
therapeutic for B-cell lymphoma and other diseases. The
glycoantibodies of the present invention can be used to alter
current routes of administration and current therapeutic regimens,
as their increased effector function means they can be dosed at
lower concentrations and with less frequency, thereby reducing the
potential for antibody toxicity and/or development of antibody
tolerance. Furthermore, their improved effector function yields new
approaches to treating clinical indications that have previously
been resistant or refractory to treatment with the corresponding
anti-CD20 monoclonal antibody produced in recombinant host
systems.
Example 10: Binding to B-Lymphoma Cells
[0645] The binding activities of Rituxan-SCT (GAb101) and Rituxan
mono-GlcNAc to Ramos cells, Raji and SU-DHL-4 cells were examined,
and the results showed both have similar binding activities as
Rituximab (FIG. 4).
[0646] FIG. 4. Binding activities of different homogeneous
antibodies with different cells with CD20.
Example 11: CDC to B-Lymphoma Cells
[0647] The CDC effects of Rituxan-SCT (GAb101) and Rituxan
mono-GlcNAc to Ramos cells, Raji and SU-DHL-4 cells were tested.
The comparative CDC profiles seen with Ramos cells (enhanced by
GAb101 and reduced by Riruxan-GlcNAc) were confirmed in the other
B-lymphoma cell line SU-DHL-4 (FIG. 5 right panel). Reproducible
results were obtained when conducted on a second occasion using
different cell passages.
Example 11
[0648] See FIG. 5. Depletion of Human B Cells
[0649] The depletion of human B cells was conducted using human
PBMC cells freshly prepared from human blood. The cells at
2.times.10.sup.6 in RPMI 1640-5% FBS cultured on microplates were
incubated, in the absence or presence of 15% autologous plasma, at
37.degree. C. for 4 hr with the anti-CD20 GAbs Rituxan-SCT,
Rituxan-GlcNAc and Rituximab at different concentrations. The cells
after wash were stained with anti-CD2-PE and anti-CD19-FITC on ice
for 5 min. B cells depletion was analyzed on FACS, based on the
CD19.sup.+ CD2.sup.- B cells. (FIG. 6) See FIG. 6. Depletion of
human B cells by different homogeneous antibodies.
Example 12: Binding to B-Lymphoma Cells
[0650] The binding of the antibodies was investigated in CD20.sup.+
B lymphoma cell lines (Ramos, Raji, and) and analyzed on flow
cytometry. The cells in PBS containing 1% fetal bovine serum at
2.times.10.sup.5/well on microplate were incubated on ice for 1 hr
with antibodies of interest at different concentrations. The cells
are washed, re-suspended in the PBS buffer, and incubated with the
detecting goat anti-hIgG-Fc.gamma.-PE on ice for 30 min. The cells
are washed and subjected to analysis on FACS.
Example 13: Binding to FcRIIIa-Expressing CHO Cells
[0651] The binding of the antibodies to the FcRIIIa receptors
(CD16a), which is a precursor event known to be correlative with
the induction of antibody-dependent cellular cytotoxicity (ADCC),
was investigated in CHO cells transfected with the high-affinity
CD16a (158Val) and analyzed on flow cytometry. The cells in PBS
containing 1% fetal bovine serum at 1.times.10.sup.5/well on
microplate were incubated on ice for 1 hr with antibodies of
interest at different concentrations. The cells are washed,
re-suspended in the PBS buffer, and incubated with the detecting
goat anti-hIgG-Fc.gamma.-PE on ice for 30 min. The cells are washed
and subjected to analysis on FACS.
[0652] Complement-dependent cytotoxicity (CDC) to B-lymphoma cells.
The CDC effect induced by the antibodies were investigated in
CD20+B lymphoma cell lines (Ramos and SKW6.4) and analyzed on flow
cytometry. The cells in RPMI 1640 culture medium at
2.0.times.105/well on microplates were incubated on ice for 30 min
with antibodies of interest at different concentrations. The cells
were washed and incubated at 37.degree. C. for 30 min with 10%
human serum in RPMI 1640. The cells were washed and incubated in
the dark for 5 min with the PI reagent. The cell deaths by CDC were
analyzed on FACS.
[0653] Antibody-dependent cellular cytotoxicity (ADCC) to
B-lymphoma cells. The ADCC effect induced by the glyco-antibodies
were investigated in CD20-containing B lymphoma cell lines (Ramos
and SKW6.4), using freshly prepared human PBMC as effector cells,
and the results analyzed on flow cytometry. The target B cells in
PBS-0.1% BSA were first labeled with CFSE at 37.degree. C. for 5
min. After wash the CFSE-labeled cells in RPMI 1640 medium were
incubated at 37.degree. C. for 4 hr on microplates with the
glyco-antibodies of interest at different concentrations and PBMC
effector cells. The ratio of target cells to effector cells was set
at 25:1. The resultant mixtures were stained in the dark for 5 min
with the PI reagent. The cell deaths by ADDC were analyzed on
FACS.
[0654] Depletion of human B cells. The depletion of human B cells
was conducted using human PBMC cells freshly prepared from human
blood. The cells at 2.times.106 in RPMI 1640-5% FBS cultured on
microplates were incubated, in the absence or presence of 15%
autologous plasma, at 37.degree. C. for 4 hr with the antibodies of
interest at different concentrations. The cells after wash were
stained with anti-CD2-PE and anti-CD19-FITC on ice for 5 min. B
cells depletion was analyzed on FACS, based on the CD19+CD2- B
cells.
[0655] Preparation of Homogeneous Herceptinx by the Strategy of
Glycan Engineering.
[0656] Methods to Prepare Different Glycan Modified Homogeneous
Herceptin.RTM..
[0657] Biological Characteristic of Anti-HER2 Homogeneous
Antibodies
[0658] Glycosylation on Fc can affect a variety of immunoglobulin
effector-mediated functions, including ADCC, CDC and circulating
half-life. ADCC enhancement is a key strategy for improving
therapeutic antibody drug efficacy. It has the potential of
lowering effective drug dosage for benefits of lower drug cost. The
anti-HER2 glycoantibodies described herein can be characterized by
functional properties. The anti-HER2 GAb has cell growth inhibitory
activities including apoptosis against human HER2 expressing cells.
In some embodiments, the anti-HER2 GAb exhibits more potent cell
growth inhibitory activities as compared to its patent
antibody.
[0659] ADCC Activities of Anti-HER2 Glycoantibodies
[0660] The ADCC activity of the glycoantibody according to the
invention is at least 3 fold increased, preferably at least 9 fold,
more preferably at least 10 fold increased ADCC activity,
preferably at least 12 fold increased ADCC activity, preferably at
least 20 fold increased ADCC activity, most preferred at least 30
fold increased ADCC activity compared to the ADCC activity of the
parental antibody.
[0661] The ADCC lysis activity of the inventive glycoantibody can
be measured in comparison to the parental antibody using target
cancer cell lines such as SKBR5, SKBR3, LoVo, MCF7, OVCAR3 and/or
Kato III.
[0662] Table 3 lists exemplary enhanced ADCC activities of
anti-HER2 GAbs as compared to Trastuzumab. Exemplary assays are
described in the examples.
TABLE-US-00010 TABLE 3 Anti- HER2 Trastuzumab GAb101 GAb104 GAb105
GAb107 GAb108 GAb111 ADCC (fold) 1 30 14.3 9.5 10 6.5 3
[0663] A number of anti-HER2 GAbs described herein, in particular
GAb101, and GAb104, exhibit enhanced ADCC activity compared to it
parental antibody, Rituximab. It is contemplated that the
glycoantibodies of the invention may exhibit superior effect as
therapeutic agents for HER2-positive diseases, and an object of the
present invention is to use the anti-HER2 GAb in development of
therapeutic agents.
[0664] CDC Activities of Anti-HER2 Glycoantibodies
[0665] The glycoantibody described herein is surprisingly able to
provide improved ADCC without affecting CDC. Exemplary CDC assays
are described in the examples. In exemplary embodiments, ADCC of
the glycoantibody is increased but other immunoglobulin-type
effector functions such as complement-dependent cytoxicity (CDC)
remain similar or are not significantly affected.
[0666] Binding Between Fc.gamma.RIII and Anti-HER2
Glycoantibodies
[0667] Table 4 lists exemplary Fc.gamma.RIIIA binding of anti-HER2
GAbs and Rituximab. Table 4.
[0668] Fc.gamma.RIIIA binding may be measured using assays known in
the art. Exemplary assays are described in the examples. The Fc
receptor binding may be determined as the relative ratio of
anti-HER2 GAb vs Trastuzumab. Fc receptor binding in exemplary
embodiments is increased by at least 2.5-fold, 3-fold, 4-fold,
5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold or
20-fold, 30-fold, 40-fold, 50-fold or higher.
[0669] As compared to Trastuzumab, the binding data showed that the
anti-HER2 GAbs, in particular GAb101 and GAb104, exhibit stronger
binding affinity for the target molecule HER2.
[0670] Taken together, anti-HER2 GAbs, in particular GAb101, and
GAb104, exhibit enhanced ADCC activity and stronger Fc.gamma.RIIIA
binding affinity as compared to Trastuzumab. It is contemplated
that the glycoantibodies of the invention may provide a superior
clinical response either alone or, preferably, in a composition
comprising two or more such antibodies, and optionally in
combination with other treatments such as chemotherapy. It is
contemplated that the ADCC-enhanced anti-HER2 glycoantibody may
provide an alternative therapeutic for HER2-positive diseases. The
glycoantibodies of the present invention advantageously can be used
to alter current routes of administration and current therapeutic
regimens, as their increased effector function means they can be
dosed at lower concentrations and with less frequency, thereby
reducing the potential for antibody toxicity and/or development of
antibody tolerance. Furthermore, their improved effector function
yields new approaches to treating clinical indications that have
previously been resistant or refractory to treatment with the
corresponding anti-HER2 monoclonal antibody produced in recombinant
host systems.
[0671] Preparation of Homogeneous Antibody with Universal Glycan
(SCT) at the Fc Region Toward Enhancing Monoclonal Antibody
Mediated Anti-Inflammation Therapeutics
[0672] The Fc region with siaa2,6Gal structure can increase the
activities of anti-inflammation. Here we prepare the homogeneous
Humira with SCT glycan at the Fc region to improve its
anti-inflammation activities.
[0673] General Procedure for Analysis of N-Glycosylation of
Anti-TNF.alpha.
[0674] We developed a mass spectrometric method to monitor the
yield of oligosaccharide-derived fragment ions (oxonium ions) over
a collision induced dissociation (CID) energy applied to a
glycopeptides precursor. Multiple Reaction Monitoring (MRM) of
oxonium ions method could fulfill the regulatory requirement on the
routine quality control analysis of forthcoming biosimilar
therapeutics.
[0675] 5 ug of Adalimumab (Humira.RTM.) (purchased from Abbvie) was
dissolved in 25 ul of 2M Guanidine-HCl, and dithiothreitol (DTT)
were added to a final concentration of 5 mM. After 10 minutes
incubation in 110.degree. C., reduced cysteine residues were
alkylated in 10 mM Iodoacetamide (IAA) at 37.degree. C. for 1 hour.
Add 5 mM DTT to quench excess IAA at RT for 10 minutes. The product
was diluted 15 times in 50 mM ammonium bicarbonate before
microcentrifugation with spin column (10 kDa protein MW cut-off).
The trypsin digestion was performed for 4 hours at 37.degree. C.
using an enzyme: protein ratio of 1:25 (w/w). Sample was frozen at
-20.degree. C. for LC-MS/MS analysis.
[0676] Instrumentation
[0677] The glycopeptide quantification by m/z 204 oxonium ion
(HexNAc) monitoring was performed using a 4000 QTrap triple
quadrupole mass spectrometer (AB Sciex) with Aglient 1200 HPLC
system. For relative quantification of glycopeptide
microheterogeneity, precursor ion m/z was derived in-silico,
covering all possible glycan compositions, and a single
quantitative transition was monitored for each precursor ion (Q3
m/z=204).
[0678] MS Data Analysis
[0679] The acquired raw data was processed with Analyst 1.5 (AB
Sciex). The mass chromatogram of each transition was integrated and
quantified by peak area. The percentage composition of each
component was calculated with respect to the sum of all components
combined.
[0680] Preparation of Anti-TNF.alpha. Antibody Humira-SCT
[0681] Isolation of the sialylglycopeptide (SGP) from hen's egg
yolk was according to the published method. Briefly, the phenol
extraction of hen's egg yolk was centrifuged, filtrated, and
purified by the chromatographic columns, including Sephadex G-50,
Sephadex G-25, DEAE-Toyoperarl 650M, CM-Sephadex C-25 and Sephadex
G-25. A solution of sialylglycopeptide (SGP) (52 mg) in a sodium
phosphate buffer (50 mM, pH 6.0, 5 mM) was incubated with the Endo
M (53 .mu.g) at 37.degree. C. After 7 hour, the reaction mixture
was subjected to gel filtration chromatography on a Sephadex G-25
column eluted by water. The fractions containing the product were
combined and lyophilized to give the product (glycan-101) as a
white powder (30 mg, yield 82%).
[0682] A solution of glycan-101
(Sia.sub.2(.alpha.2-6)Gal2GlcNAc2Man3GlcNAc) (30 mg),
2-chloro-1,3-dimethylimidazolinium chloride (DMC) (62.7 mg) and
Et3N (89 .mu.L) in water was stirred at 4.degree. C. for 1 h. The
reaction mixture was subjected to gel filtration chromatography on
a Sephadex G-25 column and eluted by 0.05% aqueous Et3N. The
fractions containing the product (SCT oxazoline) were combined and
lyophilized to give a white powder.
[0683] SCT oxazoline was added to a mixture of endoglycosidase and
GAb Humira-GlcNAc in 50 mM Tris buffer (pH 7.8) and incubated for
an hour at room temperature. The reaction mixture was purified with
protein A affinity column, followed by amanion exchange column
capto Q to collect the desired product, anti-TNF.alpha. GAb101. The
product was trypsinized, and the glycopeptides, TKPREEQYNSTYR and
EEQYNSTYR, were analyzed using nanospray LC/MS to confirm the
glycosylation pattern of Humira-SCT.
[0684] Binding Affinity of Anti-TNF.alpha.
[0685] Human recombinant TNF-.alpha. containing 158 amino acids
(MW=17.5 kDa) was produced in E. coli (PROSPEC) and purified.
Recombinant human TNF-.alpha. protein was titrated and a serial
dilution of 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM was
prepared in HBS-EP buffer. Adalimumab and anti-TNF.alpha. GAb 200
and 401 were diluted in HBS-EP buffer to a concentration of 10
.mu.g/ml, and then captured to the CM5 chip where anti-human Fc
domain antibodies were pre-immobilized. Serial concentration of
recombinant human TNF-alpha as the analyte and then injected and
bound to the captured antibody on chip at the flow rate of 30
.mu.l/min. After binding, the antibody-analyte complex were washed
by regeneration buffer, 10 mM glycine-HCl pH1.5 at the flow rate of
50 .mu.l/min. CM5 chip was maintained in PBS pH7.4 at 4.degree. C.
for further use. Single cycle kinetics data was fitted into 1:1
binding model using Biacore T200 evaluation software to measure the
equilibrium constant (Ka/Kd).
Example 13: Generation of Anti-SSEA-4 Monoclonal Antibodies
[0686] Hybridoma methodology was employed for the development of
mAbs specific to SSEA-4. Female BALB/c mice, aged 6-8 weeks old,
were immunized three times subcutaneously with the SSEA-4 vaccine.
Three immunizations were given at 2-wk intervals. Each vaccination
contained 2 .mu.g of SSEA-4. All of the sera were obtained by
centrifugation at 4,000.times.g for 10 min. The serologic responses
were analyzed by glycan microarray. A final boost was given
intraperitoneally with 2 .mu.g of SSEA-4, and 3 days later, the
spleen cells from immunized mice were used for generating
hybridomas.
[0687] Hybridoma cells secreting antibodies with the desired
antigen-binding activities were screened as follows. Microtiter
plates were coated by incubating with 4 .mu.g/mL of neutravidin in
carbonate buffer, 0.1M, pH 9.6, overnight at 4.degree. C. The wells
were blocked with 1% BSA in PBS, pH=7.3 for 1 hour and incubated
with 4 .mu.g/mL SSEA-4-biotin for 1 hour. The antisera were at
various dilutions for 1 hour at 37.degree. C. After washing, the
ligand-bound antibodies were detected by HRP-conjugated goat
anti-mouse IgG or IgM antibody (Jackson ImmunoResearch) at 1:10,000
and incubated for 1 hour at 37.degree. C., followed by incubation
with TMB substrate. The OD was determined at 450 nm. Positive
clones were selected for further characterization. Three exemplary
clones 45, 46 and 48, were identified in this study as specifically
binding to SSEA-4. For mouse monoclonal isotyping, the IsoQuick
Strips and Kits was used (sigma, 19535). Add hybridoma medium to
the reaction vial. Insert the strip into the sample making sure the
strips are upright. The sample will travel up the strip. Allow the
strip to develop for 5 minutes before making final
interpretations.
[0688] The V.sub.H and V.sub.L gene segments of the mAbs 45, 46 and
48 were amplified by PCR from the hybridoma clone secreting the
antibody. The gene segments thus obtained were sequenced to
determine the V.sub.H and V.sub.L sequences of mAbs 45, 46 and 48,
which are shown in Tables 3-5.
Example 14: Generations of Chimeric Antibodies
[0689] The V.sub.H and V.sub.L gene segments of the mAb 273 and 651
were amplified by PCR from the hybridoma clone secreting the
antibody. The gene segments thus obtained were sequenced to
determine the V.sub.H and V.sub.L sequences of mAb 273 and 651,
which are shown in Tables 1 and 2. The heavy chain and light chain
variable region were cloned to human IgG1 antibody expression
vector show as FIG. 9. VH was using enzyme site BsiWI and ApaI, and
VL was using enzyme site BsPEI and NheI. Vectors were transiently
transfected into either 293F or CHO-S cells. Recombinant chimeric
Ab was purified and further study for binding assay and
complement-dependent tumor cell lysis assay.
[0690] The V.sub.H and V.sub.L gene segments of the mAb 46 and 48
were amplified by PCR from the hybridoma clone secreting the
antibody. The gene segments thus obtained were sequenced to
determine the V.sub.H and V.sub.L sequences of mAb 46 and 48, which
are shown in Tables 5 and 4. The heavy chain and light chain
variable region were cloned to human IgG1 antibody expression
vector show as FIG. 9. V.sub.H was using enzyme site BsiWI and
ApaI, and VL was using enzyme site BsPEI and NheI. Vectors were
transiently transfected into either 293F or CHO-S cells.
Recombinant chimeric Ab was purified and further study for binding
assay and complement-dependent tumor cell lysis assay.
Example 15: Binding Analysis of Antibodies to Cancer Cells by Flow
Cytometry
[0691] Binding of mAb 273 and anti-SSEA-4 (mAbs 45, 46 and 48) to
cancer cell lines were examined. Cells (1.times.10.sup.5) were
resuspended in 100 .mu.L FACS buffer (1% BSA/PBS solution)
containing various concentration antibody and incubated on ice for
30 min. After being washed twice with FACS buffer, cells were
incubated with 649-labeled goat anti-mouse antibody (1:100; Jackson
ImmunoResearch) for 30 min on ice before analysis on a FACSCalibur
system (BD Biosciences). The results are shown in FIGS. 7A-D.
Breast cancer cells MCF-7 were stained with mAb 273 (FIG. 7A).
Pancreatic cancer cells (HPAC and BxPC3) and breast cancer cells
MCF-7 were stained with mAb 45 (FIG. 7B). Pancreatic cancer cells
(HPAC and BxPC3) and breast cancer cells MCF-7 were stained with
mAb 46 (FIG. 7C). Pancreatic cancer cells (HPAC and BxPC3) and
breast cancer cells MCF-7 were stained with mAb 48 (FIG. 7D).
[0692] We also used the glycan array to determine the dissociation
constants of MC45, MC48 and MC813-70 with SSEA-4 hexasaccharide on
surface, and the Kd values for MC45, 48 and 813 are shown below.
These results showed that these mAbs are highly specific for
SSEA4.
TABLE-US-00011 Kd (nM) .+-. SD(nM) MC45 0.37 .+-. 0.08 MC48 0.46
.+-. 0.1 MC813-70 4.21 .+-. 0.26
Example 16
[0693] The ability of exemplary mAbs 46 and 48 to mediate CDC of
SSEA-4 expressing cells was examined. Homo sapiens pancreas
adenocarcinoma cell (BxPC3) in the presence of rabbit serum as a
source of complement. Cell death was assessed by the addition of
the viability probe 7-AAD. Based on the results of the 7-AAD
measurement, percentage-specific lysis was calculated using a
FACScan flow cytometer. The antibodies showed about 20% killing
activity at 40 .mu.g/mL. As shown in FIG. 5(C), mAbs 46 and 48
successfully mediated CDC of SSEA-4 expressing cells.
Example 15 Exemplary Phage Display Biopanning Procedures
[0694] The phage-displayed human naive scFv library contained
2.5.times.10.sup.10 clones (Lu et al., 2011) was subtracted with
non-specific binding in PEG-conjugated carboxyl Dynabeads
(Invitrogen) at room temperature (RT) for 1 hour, and subsequently
incubated with SSEA-4-PEG immobilized Dynabeads at 4.degree. C. for
1 hour. After washing with PBS or PBS containing 0.01% Tween 20
(PBST0.01), the phages that bound to SSEA-4-PEG-Dynabeads were
recovered by infection with E-coli TG1 cells at 37.degree. C. for
0.5 hour. Some of the infected cells were serially diluted to
determine titer, and the others were rescued by M13KO7 phage and
amplified. After determination of rescued phages titer, the next
round of biopanning was performed. In the fourth and fifth round of
biopanning, the phage clones were randomly selected to culture for
ELISA screening.
[0695] ELISA Screening of Selected Phage Clones
[0696] For detection of antigen recognition, microwell plates
(Nunc) were coated with 0.2 .mu.g/ml of SSEA-4-BSA, Globo H-BSA,
SSEA-3-BSA and BSA, respectively. The selected phage clones were
diluted 1:2 in PBS containing 3% BSA and added to each well. The
plates were incubated at RT for 1 hour, washed with PBST0.1, and
incubated with horseradish peroxidase (HRP)-conjugated mouse
anti-M13 phage antibody (GE Healthcare). The plates were washed
again, and OPD and H2O2 were added. After termination of reaction
by 3 N HCl, the absorbance was measured using a 490 nm using
microplate reader (Model 680, BioRad). We extracted phagemids from
ELISA-positive phage clones to identity scFv coding regions by
auto-sequencing.
[0697] Construction and Expression of Anti-SSEA-4 Human IgG
[0698] The VH region of selected scFv was cloned with AgeI and NheI
site into modified expression vector pcDNA5-FRT-Gamma1 containing a
signal peptide and the constant region of human immunoglobulin
gamma 1 heavy chain. The VL region of selected scFv was cloned with
AgeI and EcoRV site into modified expression vector p-Kappa-HuGs
containing a signal peptide and constant region of human
immunoglobulin kappa light chain. Both plasmids were transfected
into FreeStyle293 cells (Invitrogen) and continuously incubated in
serum-free medium at 37.degree. C. for 1 week to produce human
antibody.
[0699] Purification of Anti-SSEA-4 Human IgG
[0700] The culture medium was collected, centrifuged and filtrated
with 0.45 .mu.m pore-size membrane. The supernatant then was
subjected to protein G column chromatography (GE healthcare) for
purification of anti-SSEA-4 human IgG. After dialysis of eluents
with PBS, the antibody was examined by SDS-PAGE analysis with
coomassie blue staining as usual. The concentration of antibody was
assessed by Bradford reagent (Thermo Scientific) and
spectrophotometer.
[0701] Humanization of MC48
[0702] Two human genes, GenBank accession Q9UL73 and AY577298, were
the most similar to MC48 VH and VL, respectively. We humanized
three sequences of MC48, including the 1st humanized MC48 (hMC48)
VH consisted of modified framework (FR) 1 to FR4 of Q9UL73 gene and
the 1st hMC48 VL consisted of four FRs from the accession AY577298,
the 2nd hMC48 FRs of VH followed 1YY8 from PDB, while the 2nd hMC48
VL same as 1st sequence, and the 3rd hMC48 VH sequence modified
FR1, 2 and 4 of Q9UL73 gene and the 3rd hMC48 VL changed FR2 and
FR4 to human AY577298 gene. All of these humanized sequences were
conserved CDR1 to CDR3 of VH and VL of MC48.
[0703] Construction of Single Chain Fragments Variable (scFv) of
Humanized MC48 Variants
[0704] The scFv form of humanized MC48 sequences
(VH-GGGGSGGGGSGGGGS-VL (SEQ ID NO: 115)) were gene synthesized
(Genomics) and cut by Sfi I and Not I (Fermentas). After gel
extraction, the digested products were cloned to pCANTAB-5E
phagemid (GE Healthcare).
[0705] Generation of Humanized MC48 (hMC48) scFv Phage Clones.
[0706] hMC48 variant phagemids were transformed to TG1 E-coli and
recovered in 2.times.YT medium (BD Pharmingen) containing 100
.mu.g/ml ampicillin and 2% glucose and rescued by M13KO7 helper
phage (NEB) for 1 hour at 37.degree. C. After centrifugation by
1,500.times.g for 10 min, these pellets were resuspended in
2.times.YT medium containing 100 .mu.g/ml ampicillin and 50
.mu.g/ml kanamycin overnight to generate scFv-phages.
[0707] Binding Assay of hMC48 scFv Phage Clones by ELISA
[0708] SSEA-4-BSA was coated on an ELISA plate at the concentration
of 0.2 .mu.g/ml. After washing and blocking, the serial diluted
phages were incubated at RT for 1.5 hour. After washing, 1:1000
diluted HRP-conjugated anti-M13 antibody (GE Healthcare) was added
at RT for 1 hour. Then, liquid substrate
3,3',5,5'-tetramethylbenzidine (TMB) developed and was terminated
with 3N HCl. Optical density was measured at 450 nm.
[0709] Results
[0710] Identification of Phage-Displayed scFv that Binds to
SSEA-4
[0711] To identify the antibodies that bind to SSEA-4, we used
phage-displayed human naive scFv library containing
2.5.times.10.sup.10 members which was established as our previous
report described (Lu et al., 2011). This library was first removed
Dynabeads-binding phages and then selected for SSEA-4-binding
phages by SSEA-4-PEG-conjugated Dynabeads. We used two buffer
systems, PBS and PBS containing 0.01% Tween20 (PBST0.01), during
biopanning. After five rounds of affinity selection, the phage
recovery of the fifth round had increased about 55-fold and 80-fold
than that of the first round in PBS and PBST0.01 system,
respectively (FIG. 10). The phage clones were randomly selected and
tested for SSEA-4 binding by ELISA (FIG. 11). We found seven clones
that specifically bound to SSEA-4-BSA, but not to BSA control
protein. By sequencing all 8 individual clones, we identified two
unique anti-SSEA-4 phage clones (p1-52 and p2-'78) which contain
distinct human VH and VL coding regions (FIG. 16A).
[0712] To examine the specificity and binding affinity of the two
phage clones, we performed a comparative ELISA using the same phage
titer to Globo-series glycans including SSEA-4-BSA, Globo H-BSA and
SSEA-3-BSA (FIG. 12). The p2-78 phage clone showed the strong
binding to SSEA-4-BSA and SSEA-3-BSA, and slightly weaker binding
to Globo H-BSA. However, we found that the binding activity of
p1-52 phage clone to SSEA-4-BSA is very weak. Thus we focused on
p2-78 clone for further study.
[0713] To establish the fully human antibody (hAb) against SSEA-4,
we molecularly engineered the VH and VL coding sequences of p2-78
scFv into human IgG1 backbone, respectively. The anti-SSEA-4 p2-78
hAb was produced using FreeStyle 293 expression system and then
purified through the protein G sepharose column. We examined the
purity of antibody by SDS-PAGE analysis with coomassie blue
staining (FIG. 13A). The result shows the purity level of antibody
exceed 95%. Subsequently, we performed ELISA to investigate the
binding activity of p2-78 hAb for Globo-series glycans (FIG. 13B).
We found that p2-78 hAb bound to SSEA-4 and SSEA-3, but not to
Globo H, which demonstrates the human IgG version of p2-78 retains
the activity of its parental scFv version to recognize the binding
epitope of SSEA-4.
[0714] We used glycan array containing 203 different glycans to
further confirm the specificity of p2-78 hAb. The results showed
that p2-78 hAb recognized SSEA4, Sialyl-SSEA4, SSEA4Gc, and Gb5
(SSEA3) (FIG. 14B). Interestingly, p2-78 hAb also recognized
GloboH, similar to the results from ELISA assay (FIG. 12). The
commercially available IgM antibody, MC631, was used as a positive
control (FIG. 14A).
[0715] Development of Humanized MC48 mAbs
[0716] Non-humanized Murine mAbs may have certain limitations in
clinical settings, including their short serum half-life, inability
to trigger human effector functions and the production of human
anti-murine antibodies (HAMA) response (LoBuglio et al., 1989).
Therefore, mAbs can be humanized by grafting their CDRs onto the VH
and VL FRs of human Ig molecules (Roguska et al., 1994).
[0717] To develop humanized MC48, we sequenced V.sub.H and V.sub.L
variable region of MC48 from a hybridoma cell (Table 4). After
alignment of V.sub.H and V.sub.L variable region of MC48 with the
NCBI IgBLAST database, we modified FRs of MC48 and generated
1.sup.st, 2.sup.nd, 3.sup.rd and 4.sup.th humanized MC48 sequences
(Table 17, FIG. 17). We next constructed and generated the
phage-displayed scFv formats according to these humanized MC48
sequences. To determine the binding activity of the humanized MC48
phage clones, we carried out solid-based ELISA coating SSEA-4-BSA
(FIGS. 15 and 18). We found that the humanized MC48 scFv phage
could recognize SSEA-4 in a dose-dependent manner. The data
indicated that the 4.sup.th humanized MC48 scFv phage maintained
its binding affinity compared with the murine mAb MC48.
Example 16
[0718] Complement-Dependent Cytotoxicity (CDC) Assay.
[0719] The ability of exemplary humanized MC 48 to mediate CDC of
SSEA-4 expressing cells is examined. Homo sapiens breast or
pancreatic carcinoma cells were plated in each well of 96-well
plates for growth of overnight prior to the assay. The cells were
then incubated with serially diluted concentrations of humanized MC
48 or human IgG1 isotype control in RPMI in the presence of rabbit
serum as a source of complement (dilution of 1:5; Life
Technologies). Cell death is assessed by the addition of the
viability probe 7-AAD. Based on the results of the 7-AAD
measurement, percentage-specific lysis is calculated using a
FACScan flow cytometer. The antibodies show significant killing
activity at 10 .mu.g/mL compared to isotype control. As shown,
humanized MC48-4 successfully mediates CDC of SSEA-4 expressing
cells.
Example 17
[0720] Materials and Methods
[0721] Construction of Exemplary Single Chain Fragments Variable
(scFv) of MC41, 1.sup.st-hMC41, 2.sup.nd-hMC41 and 3.sup.rd-hMC41
Phage Clones
[0722] The scFv form of MC41, 1.sup.st-hMC41, 2.sup.nd-hMC41 and
3.sup.rd-hMC41 sequences (V.sub.H-GGGGSGGGGSGGGGS-V.sub.L) were
gene synthesized (Genomics) and cut by Sfi I and Not I (Fermentas).
After gel extraction, the digested products were cloned to
pCANTAB-5E phagemid (GE Healthcare). hMC41 variant phagemids were
transformed to TG1 E-coli and recovered in 2.times.YT medium (BD
Pharmingen) containing 100 .mu.g/ml ampicillin and 2% glucose and
rescued by M13KO7 helper phage (NEB) for 1 hour at 37.degree. C.
After centrifugation by 1,500.times.g for 10 min, these pellets
were resuspended in 2.times.YT medium containing 100 .mu.g/ml
ampicillin and 50 .mu.g/ml kanamycin overnight to generate
scFv-phages.
[0723] Demonstration of Efficacy: Binding Assay of MC41 and hMC41
scFv Phage Clones or IgGs by ELISA
[0724] SSEA-4-BSA was coated on an ELISA plate at the concentration
of 0.2 .mu.g/ml. After washing and blocking, the serial diluted
phages or IgGs were incubated at RT for 1.5 hour. After washing,
1:1000 diluted HRP-conjugated anti-M13 antibody (GE Healthcare),
1:2000 diluted HRP-conjugated anti-human or -mouse IgG antibodies
were added at RT for 1 hour. Then, liquid substrate
3,3',5,5'-tetramethylbenzidine (TMB) developed and was terminated
with 3N HCl. Optical density was measured at 450 nm.
[0725] Demonstration of Efficacy: Humanization of MC41
[0726] The two human genes, IGHJ4*08 and IGKV6-21*02, were the most
similar to MC41 V.sub.H and V.sub.L. As such, we chose FRs from
these two genes for humanization of MC41. CDR1 to CDR3 of V.sub.H
and V.sub.L in all of the humanized MC41 were conserved.
[0727] Demonstration of Efficacy: Construction and Expression of
Anti-SSEA-4 Humanized IgG
[0728] The V.sub.H region of humanized MC41 was cloned with AgeI
and NheI site into modified expression vector pcDNA5-FRT-Gamma1
containing a signal peptide and the constant region of human
immunoglobulin gamma 1 heavy chain. The V.sub.L region of humanized
MC41 was cloned with AgeI and EcoRV site into modified expression
vector p-Kappa-HuGs containing a signal peptide and constant region
of human immunoglobulin kappa light chain. Both plasmids were
transfected into FreeStyle293 cells (Invitrogen) and continuously
incubated in serum-free medium at 37.degree. C. for 1 week to
produce humanized antibody.
[0729] Demonstration of Efficacy: Purification of Anti-SSEA-4
Humanized IgG
[0730] The culture medium was collected, centrifuged and filtrated
with 0.45 .mu.m pore-size membrane. The supernatant then was
subjected to protein G column chromatography (GE healthcare) for
purification of anti-SSEA-4 humanized IgG. After dialysis of
eluents with PBS, the antibody was examined by SDS-PAGE analysis
with coomassie blue staining as usual. The concentration of
antibody was assessed by Bradford reagent (Thermo Scientific) and
spectrophotometer.
[0731] Demonstration of Efficacy: Binding Specificity of chMC41 and
hMC41 by Glycan Array
[0732] Glycan array slides were blocked by 1% BSA for 45 min and
then incubated with serially diluted chMC41 or hMC41 IgGs for
another 45 mins at RT. After washing, donkey anti-human IgG
Fc.gamma.-F674 was used as second antibody for 40 min at RT.
Finally, the slides were washed, dried and subsequently scanned
with wavelength 674 nm.
[0733] Demonstration of Efficacy: Antibody-Dependent Cell Mediated
Cytotoxicity (ADCC) Assay
[0734] HPAC (5.times.10.sup.3 cells) pancreatic cancer cell were
seeded in a 96-well plate and cultured until .about.80% confluent.
These cells were then incubated with antibodies chMC41, hMC41,
MC813, NHIgG or NHIgG, together with PBMCs (effectors, E) at
37.degree. C. for 16 hours. After treatment, the LDH expression
level was detected by CytoTox-ONE.TM. Homogeneous Membrane
Integrity Assay Kit (Promega). The reaction was read by
fluorescence with an excitation wavelength of 560 nm and emission
wavelength of 590 nm (Molecular Device, SpectraMax M5).
[0735] Demonstration of Efficacy: Complement-Dependent Cytotoxicity
(CDC) Assay
[0736] HPAC (5.times.10.sup.3 cells) pancreatic cancer cell lines
were cultured overnight to .about.80% confluent and reacted with
mixture containing antibodies chMC41, hMC41, MC813, NHIgG or NHIgG
and rabbit complement (20%) (Low-Tox-M rabbit complement,
Cedarlane) at 37.degree. C. for 16 hours. Then, cell viability was
measured by CytoTox-ONE.TM. Homogeneous Membrane Integrity Assay
Kit (Promega), following the same procedures as that of ADCC
assay.
[0737] Demonstration of Efficacy: Development of Humanized MC41
mAbs
[0738] Murine mAbs have limited clinical use, including their short
serum half-life, inability to trigger human effector functions and
the production of human anti-murine antibodies (HAMA) response
(LoBuglio et al., 1989). Therefore, mAbs have to humanize by
grafting their CDRs onto the V.sub.H and V.sub.L FRs of human Ig
molecules (Roguska et al., 1994).
[0739] After alignment of V.sub.H and V.sub.L variable region of
MC41 with the NCBI IgBLAST or IMGT database, we generated 1.sup.st,
2.sup.nd and 3.sup.rd humanized MC41 sequences. We next constructed
and generated the phage-displayed scFv formats according to these
humanized MC41 sequences. To determine the binding activity of the
humanized MC41 phage clones, we carried out solid-based ELISA
coating SSEA-4-BSA (FIG. 1). We found 2nd and 3rd humanized MC41
scFv phages could recognize SSEA-4 in a dose-dependent manner,
whereas the 1.sup.st MC41 scFv lost the binding activity to SSEA-4
(FIG. 1). To evaluate the binding activity by intact humanized MC41
IgG, we constructed intact IgGs of 1.sup.st, 2.sup.nd, 3.sup.rd
humanized MC41, and chimeric MC41 (chMC41). The ELISA results
showed that the humanized 2.sup.nd and 3.sup.rd MC41 could react to
SSEA-4 (FIG. 2A) but not to BSA (FIG. 2B) in a dose-dependent
pattern, same results were observed for chMC41. The binding
affinity of the 2.sup.nd and 3.sup.rd humanized MC41 was
maintained, compared to that of the murine MC41. We named humanized
2.sup.nd IgG as hMC41. In order to determine the binding
specificity of chMC41 and hMC41, glycan array was performed. The
chimeric and humanized MC41 showed more specific binding than
commercial SSEA4 antibody (MC813). They only recognized SSEA4 or
glycolyl modified SSEA4 (FIG. 3).
[0740] Demonstration of Efficacy: ADCC and CDC of chMC41 and
hMC41.
[0741] To demonstrate the effector function of chMC41 and hMC41,
ADCC and CDC assays were performed. HPAC pancreatic cancer cell
line was used to evaluate the ADCC and CDC activities of chMC41,
hMC41, positive control MC813 or negative controls NHIgG and NHIgG
(FIGS. 4 and 5). The data showed that the effector function of
hMC41 was similar to chMC41. Interestingly, the humanized MC41 not
only maintain its original activity, but it also showed stronger
cancer cell killing activity than MC813 through ADCC and CDC (FIG.
5).
REFERENCES
[0742] LoBuglio, A. F., Wheeler, R. H., Trang, J., Haynes, A.,
Rogers, K., Harvey, E. B., Sun, L., Ghrayeb, J., and Khazaeli, M.
B. (1989). Mouse/human chimeric monoclonal antibody in man:
kinetics and immune response. Proc Natl Acad Sci USA 86, 4220-4224.
[0743] Roguska, M. A., Pedersen, J. T., Keddy, C. A., Henry, A. H.,
Searle, S. J., Lambert, J. M., Goldmacher, V. S., Blattler, W. A.,
Rees, A. R., and Guild, B. C. (1994). Humanization of murine
monoclonal antibodies through variable domain resurfacing. Proc
Natl Acad Sci USA 91, 969-973.
Example 18
[0744] Demonstration of Efficacy: Materials and Methods
[0745] Phage Display Biopanning Procedures
[0746] The phage-displayed human naive scFv library containing
2.5.times.10.sup.10 clones (Lu et al., 2011) was subtracted with
non-specific binding in PEG-conjugated carboxyl Dynabeads
(Invitrogen) at room temperature (RT) for 1 hour, and subsequently
incubated with SSEA-4-PEG immobilized Dynabeads at 4.degree. C. for
1 hour. After washing with PBS or PBS containing 0.01% Tween 20
(PBST.sub.0.01), the phages that bound to SSEA-4-PEG-Dynabeads were
recovered by infection with E-coli TG1 cells at 37.degree. C. for
0.5 hour. Some of the infected cells were serially diluted to
determined titer, and the others were rescued by M13KO7 phage and
amplified. After determination of rescued phages titer, the next
round of biopanning was performed. In the fourth and fifth round of
biopanning, the phage clones were randomly selected to culture for
ELISA screening.
[0747] ELISA Screening of Selected Phage Clones
[0748] For detection of antigen recognition, microwell plates
(Nunc) were coated with 0.2 .mu.g/ml of SSEA-4-BSA, Globo H-BSA,
SSEA-3-BSA and BSA, respectively. The selected phage clones were
diluted 1:2 in PBS containing 3% BSA and added to each well. The
plates were incubated at RT for 1 hour, washed with PBST.sub.0.1,
and incubated with horseradish peroxidase (HRP)-conjugated mouse
anti-M13 phage antibody (GE Healthcare). The plates were washed
again, and OPD and H.sub.2O.sub.2 were added. After termination of
reaction by 3 N HCl, the absorbance was measured using a 490 nm
using microplate reader (Model 680, BioRad). We extracted phagemids
from ELISA-positive phage clones to identity scFv coding regions by
auto-sequencing.
[0749] Demonstration of Efficacy: Construction and Expression of
Anti-SSEA-4 Human IgG
[0750] The V.sub.H region of selected scFv was cloned with AgeI and
NheI site into modified expression vector pcDNA5-FRT-Gamma1
containing a signal peptide and the constant region of human
immunoglobulin gamma 1 heavy chain. The V.sub.L region of selected
scFv was cloned with AgeI and EcoRV site into modified expression
vector p-Kappa-HuGs containing a signal peptide and constant region
of human immunoglobulin kappa light chain. Both plasmids were
transfected into FreeStyle293 cells (Invitrogen) and continuously
incubated in serum-free medium at 37.degree. C. for 1 week to
produce human antibody.
[0751] Demonstration of Efficacy: Purification of Anti-SSEA-4 Human
IgG
[0752] The culture medium was collected, centrifuged and filtrated
with 0.45 .mu.m pore-size membrane. The supernatant then was
subjected to protein G column chromatography (GE healthcare) for
purification of anti-SSEA-4 human IgG. After dialysis of eluents
with PBS, the antibody was examined by SDS-PAGE analysis with
coomassie blue staining as usual. The concentration of antibody was
assessed by Bradford reagent (Thermo Scientific) and
spectrophotometer.
[0753] Demonstration of Efficacy: Humanization of MC48 and MC41
[0754] Two human genes, GenBank accession Q9UL73 and AY577298, were
the most similar to MC48 V.sub.H and V.sub.L, respectively. We
humanized three sequences of MC48, including the 1.sup.st humanized
MC48 (hMC48) V.sub.H consisted of modified framework (FR) 1 to FR4
of Q9UL73 gene, the 1.sup.st hMC48 V.sub.L consisted of four FRs
from the accession AY577298, the 2.sup.nd hMC48 FRs of V.sub.H
followed by 1YY8 from PDB, while the 2.sup.nd hMC48 V.sub.L same as
1.sup.st sequence, and the 3.sup.rd hMC48 V.sub.H sequence modified
FR1, 2 and 4 of Q9UL73 gene and the 3.sup.rd hMC48 V.sub.L only
changed FR2 and FR4 to human AY577298 gene. The other two human
genes, IGHJ4*08 and IGKV6-21*02, were the most similar to MC41
V.sub.H and V.sub.L. As such, we chose FRs from these two genes for
humanization of MC41. CDR1 to CDR3 of V.sub.H and V.sub.L in all of
the humanized MC48 and MC41 were conserved.
[0755] Demonstration of Efficacy: Construction of Single Chain
Fragments Variable (scFv) of Humanized MC48 and MC41 Phage
Clones
[0756] The scFv form of humanized MC48 (hMC48) and MC41 (hMC41)
sequences (V.sub.H-GGGGSGGGGSGGGGS-V.sub.L) were gene synthesized
(Genomics) and cut by Sfi I and Not I (Fermentas). After gel
extraction, the digested products were cloned to pCANTAB-5E
phagemid (GE Healthcare). hMC48 and hMC41 variant phagemids were
transformed to TG1 E-coli and recovered in 2.times.YT medium (BD
Pharmingen) containing 100 .mu.g/ml ampicillin and 2% glucose and
rescued by M13KO7 helper phage (NEB) for 1 hour at 37.degree. C.
After centrifugation by 1,500.times.g for 10 min, these pellets
were resuspended in 2.times.YT medium containing 100 .mu.g/ml
ampicillin and 50 .mu.g/ml kanamycin overnight to generate
scFv-phages.
[0757] Demonstration of Efficacy: Binding Assay of hMC48 and hMC41
scFv Phage Clones or IgGs by ELISA
[0758] SSEA-4-BSA was coated on an ELISA plate at the concentration
of 0.2 .mu.g/ml. After washing and blocking, the serial diluted
phages or IgGs were incubated at RT for 1.5 hour. After washing,
1:1000 diluted HRP-conjugated anti-M13 antibody (GE Healthcare),
1:2000 diluted HRP-conjugated anti-human or -mouse IgG antibodies
were added at RT for 1 hour. Then, liquid substrate
3,3',5,5'-tetramethylbenzidine (TMB) developed and was terminated
with 3N HCl. Optical density was measured at 450 nm.
[0759] Demonstration of Efficacy: Binding Specificity of p2-78 hAb,
chMC41 and hMC41 by Glycan Array
[0760] Glycan array slides were blocked by 1% BSA for 45 min and
then incubated with serially diluted p2-78 hAb, chMC41 or hMC41
IgGs for another 45 mins at RT. After washing, donkey anti-human
IgG Fc.gamma.-F674 was second antibody for 40 min at RT. Finally,
the slides were washed, dried and subsequently scanned with
wavelength 674 nm.
[0761] Demonstration of Efficacy: Antibody-Dependent Cell Mediated
Cytotoxicity (ADCC) Assay
[0762] HPAC, BxPC3 or PL45 (5.times.10.sup.3 cells) pancreatic
cancer cell were seeded in a 96-well plate and cultured until
.about.80% confluent. Then, these cells were incubated with
antibodies hMC48, hMC41 or NHIgG, together with PBMCs (effectors,
E) at 37.degree. C. for 16 hours. After treatment, the LDH
expression level was detected by CytoTox-ONE.TM. Homogeneous
Membrane Integrity Assay Kit (Promega). The reaction was read by
fluorescence with an excitation wavelength of 560 nm and emission
wavelength of 590 nm (Molecular Device, SpectraMax M5).
[0763] Demonstration of Efficacy: Complement-Dependent Cytotoxicity
(CDC) Assay
[0764] HPAC, BxPC3 or PL45 (5.times.10.sup.3 cells) pancreatic
cancer cell lines were cultured overnight to .about.80% confluent
and reacted with mixture containing antibodies hMC48, hMC41 or
NHIgG and rabbit complement (10% and 20%) (Low-Tox-M rabbit
complement, Cedarlane) at 37.degree. C. for 16 hours. Then, cell
viability was measured by CytoTox-ONE.TM. Homogeneous Membrane
Integrity Assay Kit (Promega), following the same procedures as
that of ADCC assay.
[0765] Demonstration of Efficacy:
[0766] Identification of Phage-Displayed scFv that Binds to
SSEA-4
[0767] To identify the antibodies that bind to SSEA-4, we used
phage-displayed human naive scFv library containing
2.times.10.sup.10 members, which was established as described in
our previous report (Lu et al., 2011). This library was first
removed by Dynabeads-binding phages, and then SSEA-4-binding phages
were selected by SSEA-4-PEG-conjugated Dynabeads. We used two
buffer systems, PBS and PBS containing 0.01% Tween20
(PBST.sub.0.01), during biopanning. After five rounds of affinity
selection, the phage recovery of the fifth round increased by about
55-fold and 80-fold, compared to that of the first round in PBS and
PBST.sub.0.01 system, respectively (FIG. 1). The phage clones were
randomly selected and tested for SSEA-4 binding by ELISA (FIG. 2).
We found seven clones that specifically bound to SSEA-4-BSA, but
not to BSA control protein. By sequencing all 8 individual clones,
we identified two unique anti-SSEA-4 phage clones (p1-52 and
p2-'78) which contained distinct human V.sub.H and V.sub.L coding
regions (Table 1).
[0768] To examine the specificity and binding affinity of the two
phage clones, we performed a comparative ELISA using the same phage
titer to Globo-series glycans including SSEA-4-BSA, Globo H-BSA and
SSEA-3-BSA (FIG. 3). The p2-78 phage clone showed the strong
binding to SSEA-4-BSA and SSEA-3-BSA, and more slight binding to
Globo H-BSA. However, we found that the binding activity of p1-52
phage clone to SSEA-4-BSA was very weak. Thus we focused on p2-78
clone for further study.
[0769] To establish the fully human antibody (hAb) against SSEA-4,
we molecularly engineered the V.sub.H and V.sub.L coding sequences
of p2-78 scFv into human IgG.sub.1 backbone, respectively. The
anti-SSEA-4 p2-78 hAb was produced using FreeStyle 293 expression
system and then purified through the protein G sepharose column. We
examined the purity of antibody by SDS-PAGE analysis with coomassie
blue staining (FIG. 4A). The result shows the purity level of
antibody exceed 95%. Subsequently, we performed ELISA to
investigate the binding activity of p2-78 hAb for Globo-series
glycans (FIG. 4B). We found that p2-78 hAb bound to SSEA-4 and
SSEA-3, but not to Globo H, demonstrating that the human IgG
version of p2-78 retains the activity of its parental scFv version
to recognize the binding epitope of SSEA-4.
[0770] We used glycan array containing 203 different glycans to
further confirm the specificity of p2-78 hAb. The results showed
that p2-78 hAb recognized SSEA4, Sialyl-SSEA4, SSEA4Gc, and Gb5
(SSEA3) (FIG. 5B). Interestingly, p2-78 hAb also slightly
recognized Globo H, similar to the results from ELISA assay (FIG.
3). The commercially available IgM antibody, MC631, was used as a
positive control (FIG. 5A).
[0771] Demonstration of Efficacy: Development of Humanized MC48 and
MC41 mAbs
[0772] Murine mAbs have limited clinical use, including their short
serum half-life, inability to trigger human effector functions and
the production of human anti-murine antibodies (HAMA) response
(LoBuglio et al., 1989). Therefore, mAbs have to humanize by
grafting their CDRs onto the V.sub.H and V.sub.L FRs of human Ig
molecules (Roguska et al., 1994).
[0773] After alignment of V.sub.H and V.sub.L variable region of
MC48 and MC41 with the NCBI IgBLAST or IMGT database, we generated
1.sup.st, 2.sup.nd, 3.sup.rd and 4.sup.th humanized MC48 sequences
and 1.sup.st 2.sup.nd and 3.sup.rd humanized MC41 sequences. We
next constructed and generated the phage-displayed scFv formats
according to these humanized MC48 and MC41 sequences. To determine
the binding activity of the humanized MC48 and MC41 phage clones,
we carried out solid-based ELISA coating SSEA-4-BSA (FIGS. 6, 7 and
8). We found that the 3.sup.rd and 4.sup.th humanized MC48, and
2.sup.nd and 3.sup.rd humanized MC41 scFv phages could recognize
SSEA-4 in a dose-dependent manner, whereas the 1.sup.st and
2.sup.nd humanized MC48 and 1.sup.st MC41 scFv lost the binding
activity to SSEA-4 (FIGS. 6, 7 and 8). The data showed that the
binding affinities of the 4.sup.th humanized MC48, and 3.sup.rd
humanized MC41 scFv phage clones were maintained, compared to that
of the murine mAbs MC48 or MC41. To evaluate the binding activity
by intact humanized MC41 IgG, we constructed intact IgGs of
1.sup.st, 2.sup.nd, 3.sup.rd humanized MC41 and chimeric MC41
(chMC41). The ELISA results showed that the humanized 2.sup.nd and
3.sup.rd MC41 could react to SSEA-4 (FIG. 9A) but not to BSA (FIG.
9B) in a dose-dependent pattern, same results were observed for
chMC41. We named humanized 2.sup.nd IgG as hMC41. In order to
determine the binding specificity of chMC41 and hMC41, glycan array
was performed. The chimeric and humanized MC41 showed more specific
binding than commercial SSEA4 antibody (MC813). They only
recognized SSEA4 or glycolyl modified SSEA4 (FIG. 10).
[0774] Demonstration of Efficacy: ADCC and CDC Test of hMC48,
chMC41 and hMC41
[0775] To investigate the effector function of hMC48, chMC41 and
hMC41, ADCC and CDC assays were performed. HPAC, BxPC3 and PL45
pancreatic cancer cell lines were used to evaluate the ADCC and CDC
activities at the concentration of 10 .mu.g/ml for hMC48 or NHIgG
(FIG. 11). Further, HPAC cells were treated with chMC41, hMC41,
positive control MC813 or negative control NHIgG (FIGS. 12 and 13).
The data showed that the effector function of hMC41 and chMC41 was
superior to that of hMC48. Interestingly, the humanized MC41 not
only maintain its original activity, but it also showed stronger
cancer cell killing activity than MC813 through ADCC and CDC (FIG.
13).
Example 19
[0776] Binding of MC41 vs MC 48
[0777] The binding abilities of hMC41 and hMC48 to SSEA-4 were
examined by ELISA. The result showed that the binding of hMC41 to
SSEA-4 was much better than hMC48. The humanized MC41 has a higher
binding maximum and a smaller Kd (0.2 .mu.g/ml and 4.6 .mu.g/ml for
hMC41 and hMC48, respectively) value as compared to hMC48.
REFERENCES
[0778] LoBuglio, A. F., Wheeler, R. H., Trang, J., Haynes, A.,
Rogers, K., Harvey, E. B., Sun, L., Ghrayeb, J., and Khazaeli, M.
B. (1989). Mouse/human chimeric monoclonal antibody in man:
kinetics and immune response. Proc Natl Acad Sci USA 86, 4220-4224.
[0779] Lu, R.-M., Chang, Y.-L., Chen, M.-S., and Wu, H.-C. (2011).
Single chain anti-c-Met antibody conjugated nanoparticles for in
vivo tumor-targeted imaging and drug delivery. Biomaterials 32,
3265-3274. [0780] Roguska, M. A., Pedersen, J. T., Keddy, C. A.,
Henry, A. H., Searle, S. J., Lambert, J. M., Goldmacher, V S.,
Blattler, W. A., Rees, A. R., and Guild, B. C. (1994). Humanization
of murine monoclonal antibodies through variable domain
resurfacing. Proc Natl Acad Sci USA 91, 969-973.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 229 <210> SEQ ID NO 1 <211> LENGTH: 351
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic polynucleotide <400> SEQUENCE:
1 caggtgcagc tgaagcagtc tggacctgag ctagtgaaga ctggggcttc agtgaagata
60 tcctgcaagg cttctggtta ctcattcact ggttactaca tgcactgggt
caagcagagc 120 catggaaaga gccttgagtg gattggatat attagttgtt
acaatggtgg tactaggtac 180 aacctgaagt tcaagggcaa ggccacattt
actgtagaca catcctccac cacagcctac 240 atgcagttca acaacctgac
atctgaagac tctgcggtct attactgtgc aagagggggg 300 tacgacgagg
gtgactactg gggccaaggc accactctca cagtctcctc a 351 <210> SEQ
ID NO 2 <211> LENGTH: 321 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polynucleotide <400> SEQUENCE: 2 gatattgtaa tgacacagtc
tcccaaatcc atattcatgt cagttggaga gagggtcacc 60 ttgagctgca
aggccagtga gaatgtgggt acttatgtat cctggtatca acagaaacca 120
gagcagtctc ctaaactgat gatatacggg gcatccaacc ggaacactgg ggtccccgat
180 cgcttcacag gcagtggatc tgcaacagat ttcactctga ccatcagcag
tgtgcaggct 240 gaagaccttg cagattatca ctgtggacag agttacacct
atccgtacac gttcggaggg 300 gggaccaagc tggaaatcaa a 321 <210>
SEQ ID NO 3 <211> LENGTH: 117 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polypeptide <400> SEQUENCE: 3 Gln Val Gln Leu Lys
Gln Ser Gly Pro Glu Leu Val Lys Thr Gly Ala 1 5 10 15 Ser Val Lys
Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Tyr
Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40
45 Gly Tyr Ile Ser Cys Tyr Asn Gly Gly Thr Arg Tyr Asn Leu Lys Phe
50 55 60 Lys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Thr Thr
Ala Tyr 65 70 75 80 Met Gln Phe Asn Asn Leu Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Asp Glu Gly Asp Tyr
Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115
<210> SEQ ID NO 4 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polypeptide <400> SEQUENCE: 4 Asp Ile Val Met Thr
Gln Ser Pro Lys Ser Ile Phe Met Ser Val Gly 1 5 10 15 Glu Arg Val
Thr Leu Ser Cys Lys Ala Ser Glu Asn Val Gly Thr Tyr 20 25 30 Val
Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Met Ile 35 40
45 Tyr Gly Ala Ser Asn Arg Asn Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val
Gln Ala 65 70 75 80 Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Ser Tyr
Thr Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys 100 105 <210> SEQ ID NO 5 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 5 Glu
Asn Val Gly Thr Tyr 1 5 <210> SEQ ID NO 6 <400>
SEQUENCE: 6 000 <210> SEQ ID NO 7 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 7 Gly
Gln Ser Tyr Thr Tyr Pro Tyr Thr 1 5 <210> SEQ ID NO 8
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 8 Gly Tyr Ser Phe Thr Gly Tyr Tyr 1 5
<210> SEQ ID NO 9 <211> LENGTH: 8 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 9 Ile Ser Cys Tyr Asn Gly
Gly Thr 1 5 <210> SEQ ID NO 10 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 10 Ala
Arg Gly Gly Tyr Asp Glu Gly Asp Tyr 1 5 10 <210> SEQ ID NO 11
<211> LENGTH: 348 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic
polynucleotide <400> SEQUENCE: 11 gaggtccagc tgcaacaatc
tgggtctgtg ctggtgaggc ctggagcttc agtgaagctg 60 tcctgcaagg
cttctggcta caccttcacc aactcctgga tgcactgggc gaagcagagg 120
cctggacaag gccttgtgtg gattggagag attgatccta atactggtaa tactaactac
180 aatgagaact tcaagggcaa ggccacactg actgtagaca catcctccac
cacagcctac 240 gtggatctca gcagcctgac atctgaagac tctgcggtct
attactgtgc aagaggactc 300 gggctacttg tttactgggg ccaagggact
ctggtcactg tctctgca 348 <210> SEQ ID NO 12 <211>
LENGTH: 318 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic polynucleotide
<400> SEQUENCE: 12 caaattgttc tcacccagtc tccagcaatc
ctgtctgcat ctccagggga gaaggtcaca 60 atgacttgca gggccagctc
aagtgtaagt tacatgcact ggtaccagca gaagccagga 120 tcctccccca
aaccctggat ttatgtcaca tccaacctga cttctggagt ccctgttcgc 180
ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagagt ggaggctgaa
240 gatgctgcca cttattactg ccagcagtgg agtaataacc cgtggacgtt
cggtggaggc 300 accaagctgg aaatcaaa 318 <210> SEQ ID NO 13
<211> LENGTH: 116 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 13 Glu Val Gln Leu Gln Gln Ser
Gly Ser Val Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Ser 20 25 30 Trp Met His
Trp Ala Lys Gln Arg Pro Gly Gln Gly Leu Val Trp Ile 35 40 45 Gly
Glu Ile Asp Pro Asn Thr Gly Asn Thr Asn Tyr Asn Glu Asn Phe 50 55
60 Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Thr Thr Ala Tyr
65 70 75 80 Val Asp Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Gly Leu Gly Leu Leu Val Tyr Trp Gly Gln
Gly Thr Leu Val 100 105 110 Thr Val Ser Ala 115 <210> SEQ ID
NO 14 <211> LENGTH: 106 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 14 Gln Ile Val Leu Thr Gln Ser
Pro Ala Ile Leu Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met
Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr
Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Val
Thr Ser Asn Leu Thr Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55
60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu
65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Asn Asn Pro
Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
<210> SEQ ID NO 15 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 15 Ser Ser Val Ser Tyr 1 5
<210> SEQ ID NO 16 <400> SEQUENCE: 16 000 <210>
SEQ ID NO 17 <211> LENGTH: 9 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 17 Gln Gln Trp Ser Asn Asn
Pro Trp Thr 1 5 <210> SEQ ID NO 18 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 18 Gly
Tyr Thr Phe Thr Asn Ser Trp 1 5 <210> SEQ ID NO 19
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 19 Ile Asp Pro Asn Thr Gly Asn Thr 1 5
<210> SEQ ID NO 20 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 20 Ala Arg Gly Leu Gly Leu
Leu Val Tyr 1 5 <210> SEQ ID NO 21 <211> LENGTH: 339
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic polynucleotide <400> SEQUENCE:
21 caggtgcagc tgaaggagtc aggacctggc ctggtggcgc cctcacagag
cctgtccatc 60 acatgcactg tctcagggtt ctcattaagc agatatggtg
taagctgggt tcgccagcct 120 ccaggaaagg gtctggagtg gctgggagta
atatggggtg acgggagcac aaattatcat 180 tcagctctca tatccagact
gagcatcagc aaggataact ccaagagcca agttttctta 240 aaactgaaca
gtctgcaaac tgatgacaca gccacgtact actgtgccat gactgggaca 300
gcttactggg gccaagggac tctggtcact gtctctgca 339 <210> SEQ ID
NO 22 <211> LENGTH: 318 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polynucleotide <400> SEQUENCE: 22 caaattgttc tcacccagtc
tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60 atgacctgca
gtgccagctc aagtgtaaat tacatgcact ggtaccagca gaagtcaggc 120
acctccccca aaagatggat ttatgacaca tccaaactgg cttctggagt ccctgctcgc
180 ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcggcat
ggaggctgaa 240 gatgctgcca cttattactg ccaccagtgg aatagtagcc
cacacacgtt cggagggggg 300 accaagctgg aaataaaa 318 <210> SEQ
ID NO 23 <211> LENGTH: 113 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 23 Gln Val Gln Leu Lys Glu Ser
Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr
Cys Thr Val Ser Gly Phe Ser Leu Ser Arg Tyr 20 25 30 Gly Val Ser
Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly
Val Ile Trp Gly Asp Gly Ser Thr Asn Tyr His Ser Ala Leu Ile 50 55
60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80 Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr Tyr Tyr
Cys Ala 85 90 95 Met Thr Gly Thr Ala Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser 100 105 110 Ala <210> SEQ ID NO 24
<211> LENGTH: 106 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 24 Gln Ile Val Leu Thr Gln Ser
Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met
Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Met 20 25 30 His Trp Tyr
Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp
Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55
60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu
65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys His Gln Trp Asn Ser Ser Pro
His Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
<210> SEQ ID NO 25 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 25 Ser Ser Val Asn Tyr 1 5
<210> SEQ ID NO 26 <400> SEQUENCE: 26 000 <210>
SEQ ID NO 27 <211> LENGTH: 9 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 27 His Gln Trp Asn Ser Ser
Pro His Thr 1 5 <210> SEQ ID NO 28 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 28 Gly
Phe Ser Leu Ser Arg Tyr Gly 1 5 <210> SEQ ID NO 29
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 29 Ile Trp Gly Asp Gly Ser Thr 1 5
<210> SEQ ID NO 30 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 30 Ala Met Thr Gly Thr Ala
Tyr 1 5 <210> SEQ ID NO 31 <211> LENGTH: 339
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic polynucleotide <400> SEQUENCE:
31 caggtgcagc tgaaggagtc aggacctggc ctggtggcgc cctcacagag
cctgtccatc 60 acatgcactg tctcaggatt ctcattaacc agctatggta
taagctgggt tcgccagcct 120 ccaggaaagg gtctggagtg gctgggagta
atatggggtg acgggagcac aaattatcat 180 tcagctctcg tatccagact
gagcatcagc aaggataact ccaagagcca agttttctta 240 aaactgaaca
gtctgcaaac tgatgacaca gccacgtact actgtgccaa aactgggaca 300
tcttactggg gccaagggac tctggtcact gtctctgca 339 <210> SEQ ID
NO 32 <211> LENGTH: 318 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polynucleotide <400> SEQUENCE: 32 caaattgttc tcacccagtc
tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60 atgacctgca
gtgccagctc aagtgtaagt tacatgcact ggtaccagca gaagtcaggc 120
acctccccca aaagatggat ttatgacaca tccaaactgg cttctggagt ccctgctcgc
180 ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat
ggaggctgaa 240 gatgctgcca cttattactg ccagcagtgg agtagtgccc
cacacacgtt cggagggggg 300 accaagctgg aaataaaa 318 <210> SEQ
ID NO 33 <211> LENGTH: 113 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 33 Gln Val Gln Leu Lys Glu Ser
Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr
Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Ile Ser
Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly
Val Ile Trp Gly Asp Gly Ser Thr Asn Tyr His Ser Ala Leu Val 50 55
60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80 Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr Tyr Tyr
Cys Ala 85 90 95 Lys Thr Gly Thr Ser Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser 100 105 110 Ala <210> SEQ ID NO 34
<211> LENGTH: 106 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 34 Gln Ile Val Leu Thr Gln Ser
Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met
Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr
Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp
Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55
60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Ala Pro
His Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
<210> SEQ ID NO 35 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 35 Ser Ser Val Ser Tyr 1 5
<210> SEQ ID NO 36 <400> SEQUENCE: 36 000 <210>
SEQ ID NO 37 <211> LENGTH: 9 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 37 Gln Gln Trp Ser Ser Ala
Pro His Thr 1 5 <210> SEQ ID NO 38 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 38 Gly
Phe Ser Leu Thr Ser Tyr Gly 1 5 <210> SEQ ID NO 39
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 39 Ile Trp Gly Asp Gly Ser Thr 1 5
<210> SEQ ID NO 40 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 40 Ala Lys Thr Gly Thr Ser
Tyr 1 5 <210> SEQ ID NO 41 <211> LENGTH: 339
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic polynucleotide <400> SEQUENCE:
41 caggtgcagc tgaaggagtc aggacctggc ctggtggcgc cctcacagag
cctgtccatc 60 acatgcactg tctcagggtt ctcattaacc agctatggtg
taagctgggt tcgccagcct 120 ccaggaaagg gtctggagtg gctgggagta
atatggggtg aggggagcac aaattatcat 180 tcagttctca tatccagact
gaccattagt aaggataact ccaagagcca agttttctta 240 aaactgaaca
gtctgcaaac tgatgacaca gccacgtact actgtgccat gactgggaca 300
gcttactggg gccaagggac tctggtcact gtctctgca 339 <210> SEQ ID
NO 42 <211> LENGTH: 318 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polynucleotide <400> SEQUENCE: 42 caaattgttc tcacccagtc
tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60 atgacctgca
gtgccagctc aagtgtaagt tacatgcact ggtaccagca gaagtcaggc 120
acctccccca aaagatggat ttatgacaca tccaaactgt cttctggagt ccctggtcgc
180 ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcaggtt
ggaggctgaa 240 gatgctgcca cttattactg ccatcagtgg agtagtagtc
cacacacgtt cggagggggg 300 accaagttgg agataaaa 318 <210> SEQ
ID NO 43 <211> LENGTH: 113 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 43 Gln Val Gln Leu Lys Glu Ser
Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr
Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val Ser
Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly
Val Ile Trp Gly Glu Gly Ser Thr Asn Tyr His Ser Val Leu Ile 50 55
60 Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80 Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr Tyr Tyr
Cys Ala 85 90 95 Met Thr Gly Thr Ala Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser 100 105 110 Ala <210> SEQ ID NO 44
<211> LENGTH: 106 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 44 Gln Ile Val Leu Thr Gln Ser
Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met
Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr
Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp
Thr Ser Lys Leu Ser Ser Gly Val Pro Gly Arg Phe Ser Gly Ser 50 55
60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Leu Glu Ala Glu
65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys His Gln Trp Ser Ser Ser Pro
His Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
<210> SEQ ID NO 45 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 45 Ser Ser Val Ser Tyr 1 5
<210> SEQ ID NO 46 <400> SEQUENCE: 46 000 <210>
SEQ ID NO 47 <211> LENGTH: 9 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 47 His Gln Trp Ser Ser Ser
Pro His Thr 1 5 <210> SEQ ID NO 48 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 48 Gly
Phe Ser Leu Thr Ser Tyr Gly 1 5 <210> SEQ ID NO 49
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 49 Ile Trp Gly Glu Gly Ser Thr 1 5
<210> SEQ ID NO 50 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 50 Ala Met Thr Gly Thr Ala
Tyr 1 5 <210> SEQ ID NO 51 <211> LENGTH: 25 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of artificial
sequence: synthetic peptide <400> SEQUENCE: 51 Gln Val Asn
Leu Arg Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr
Leu Ser Leu Thr Cys Ala Ile Ser 20 25 <210> SEQ ID NO 52
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 52 Gly Asp Ser Val Ser Ser Ser Ser Ala Ala 1
5 10 <210> SEQ ID NO 53 <211> LENGTH: 17 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of artificial
sequence: synthetic peptide <400> SEQUENCE: 53 Trp Asn Trp
Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu Trp Leu Gly 1 5 10 15 Arg
<210> SEQ ID NO 54 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 54 Thr Tyr Tyr Lys Ser Thr
Trp His Asn 1 5 <210> SEQ ID NO 55 <211> LENGTH: 25
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 55 Gln
Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Gly Ala Ser 20 25 <210> SEQ ID NO
56 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
peptide <400> SEQUENCE: 56 Gly Phe Thr Phe Arg Lys Ser Trp 1
5 <210> SEQ ID NO 57 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 57 Met Gly Trp Val Arg Gln
Ser Pro Gly Lys Gly Leu Glu Trp Val Ala 1 5 10 15 Asn <210>
SEQ ID NO 58 <211> LENGTH: 8 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 58 Thr Asn Asp Asp Ala Lys
Glu Lys 1 5 <210> SEQ ID NO 59 <211> LENGTH: 38
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic polypeptide <400> SEQUENCE: 59
Asp Tyr Ala Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr 1 5
10 15 Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu
Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> SEQ ID NO 60
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 60 Ala Arg Glu Lys Gly Arg Val Arg Ala Phe
Asp Ile 1 5 10 <210> SEQ ID NO 61 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 61 Trp
Gly Gln Gly Thr Met Val Thr Val Ser Ser 1 5 10 <210> SEQ ID
NO 62 <211> LENGTH: 38 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 62 Tyr Tyr Gly Asp Ser Val Lys
Gly Arg Phe Thr Ile Phe Arg Asp Asn 1 5 10 15 Asp Lys Asn Ser Leu
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr Ala Val
Tyr Phe Cys 35 <210> SEQ ID NO 63 <211> LENGTH: 23
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 63 Ala
Ser Leu Ser Ser Val Ser Ala Gly Asp Pro Pro Arg Gly Pro Glu 1 5 10
15 Asn Ile Glu Tyr Phe Glu His 20 <210> SEQ ID NO 64
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 64 Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 1 5 10 <210> SEQ ID NO 65 <211> LENGTH: 26
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <400> SEQUENCE: 65 Glu
Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ala Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser 20 25 <210> SEQ ID
NO 66 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
peptide <400> SEQUENCE: 66 Gln Ser Val Gly Asn Lys Tyr 1 5
<210> SEQ ID NO 67 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 67 Val Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 1 5 10 15 Tyr <210>
SEQ ID NO 68 <400> SEQUENCE: 68 000 <210> SEQ ID NO 69
<211> LENGTH: 26 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 69 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser 20 25 <210> SEQ ID NO 70 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 70 Gln
Ser Ile Ser Asn Tyr 1 5 <210> SEQ ID NO 71 <211>
LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 71 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 1 5 10 15 Tyr <210> SEQ ID NO 72 <400>
SEQUENCE: 72 000 <210> SEQ ID NO 73 <211> LENGTH: 36
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic polypeptide <400> SEQUENCE: 73
Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 1 5
10 15 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe
Ala 20 25 30 Val Tyr Tyr Cys 35 <210> SEQ ID NO 74
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 74 Gln Gln Tyr Gly Arg Ser Arg Arg Leu Thr 1
5 10 <210> SEQ ID NO 75 <211> LENGTH: 11 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of artificial
sequence: synthetic peptide <400> SEQUENCE: 75 Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys Arg 1 5 10 <210> SEQ ID NO 76
<211> LENGTH: 36 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 76 Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 20 25 30 Thr Tyr Tyr
Cys 35 <210> SEQ ID NO 77 <211> LENGTH: 9 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of artificial
sequence: synthetic peptide <400> SEQUENCE: 77 Gln Gln Ser
Tyr Ser Thr Pro Arg Thr 1 5 <210> SEQ ID NO 78 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 78 Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg 1 5 10
<210> SEQ ID NO 79 <211> LENGTH: 26 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 79 Gln Val Gln Leu Lys Glu
Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile
Thr Cys Thr Val Ser Gly 20 25 <210> SEQ ID NO 80 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 80 Phe Ser Leu Thr Ser Tyr Gly Val Ser 1 5 <210>
SEQ ID NO 81 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 81 Trp Val Arg Gln Pro Pro
Gly Lys Gly Leu Glu Trp Leu 1 5 10 <210> SEQ ID NO 82
<211> LENGTH: 19 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 82 Gly Val Ile Trp Gly Glu Gly Ser Thr Asn
Tyr His Ser Val Leu Ile 1 5 10 15 Ser Arg Leu <210> SEQ ID NO
83 <211> LENGTH: 26 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
peptide <400> SEQUENCE: 83 Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly 20 25 <210> SEQ ID NO 84 <211> LENGTH:
13 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 84 Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 1 5 10 <210>
SEQ ID NO 85 <211> LENGTH: 26 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 85 Gln Val Gln Leu Lys Gln
Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile
Thr Cys Thr Val Ser Gly 20 25 <210> SEQ ID NO 86 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 86 Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 1
5 10 <210> SEQ ID NO 87 <211> LENGTH: 26 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of artificial
sequence: synthetic peptide <400> SEQUENCE: 87 Gln Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr
Leu Ser Leu Thr Cys Thr Val Ser Gly 20 25 <210> SEQ ID NO 88
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 88 Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu
Glu Trp Ile 1 5 10 <210> SEQ ID NO 89 <211> LENGTH: 28
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 89 Thr
Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Leu Asn 1 5 10
15 Ser Leu Gln Thr Asp Asp Thr Ala Thr Tyr Tyr Cys 20 25
<210> SEQ ID NO 90 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 90 Ala Met Thr Gly Thr Ala
Tyr 1 5 <210> SEQ ID NO 91 <211> LENGTH: 11 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of artificial
sequence: synthetic peptide <400> SEQUENCE: 91 Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ala 1 5 10 <210> SEQ ID NO 92
<211> LENGTH: 28 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 92 Thr Ile Ser Val Asp Thr Ser Lys Asn Gln
Phe Ser Leu Lys Leu Ser 1 5 10 15 Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys 20 25 <210> SEQ ID NO 93 <211> LENGTH:
11 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 93 Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> SEQ ID
NO 94 <211> LENGTH: 28 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
peptide <400> SEQUENCE: 94 Ser Ile Asn Lys Asp Asn Ser Lys
Ser Gln Val Phe Phe Lys Met Asn 1 5 10 15 Ser Leu Gln Ser Asn Asp
Thr Ala Ile Tyr Tyr Cys 20 25 <210> SEQ ID NO 95 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 95 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 1 5 10
<210> SEQ ID NO 96 <211> LENGTH: 28 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 96 Thr Ile Ser Lys Asp Asn
Ser Lys Ser Gln Val Phe Leu Lys Leu Asn 1 5 10 15 Ser Leu Gln Thr
Asp Asp Thr Ala Thr Tyr Tyr Cys 20 25 <210> SEQ ID NO 97
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 97 Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 1 5 10 <210> SEQ ID NO 98 <211> LENGTH: 23
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 98 Gln
Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10
15 Glu Lys Val Thr Met Thr Cys 20 <210> SEQ ID NO 99
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 99 Ser Ala Ser Ser Ser Val Ser Tyr Met His 1
5 10 <210> SEQ ID NO 100 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of artificial
sequence: synthetic peptide <400> SEQUENCE: 100 Trp Tyr Gln
Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 1 5 10 15
<210> SEQ ID NO 101 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 101 Asp Thr Ser Lys Leu Ser
Ser 1 5 <210> SEQ ID NO 102 <211> LENGTH: 23
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 102
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5
10 15 Glu Arg Ala Thr Leu Ser Cys 20 <210> SEQ ID NO 103
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 103 Trp Tyr Gln Gln Lys Pro Gly Leu Ala Pro
Arg Leu Leu Ile Tyr 1 5 10 15 <210> SEQ ID NO 104 <211>
LENGTH: 23 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 104 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu
Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys 20 <210>
SEQ ID NO 105 <211> LENGTH: 15 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 105 Trp Tyr Gln Gln Lys Pro
Gly Leu Ala Pro Arg Leu Leu Ile Tyr 1 5 10 15 <210> SEQ ID NO
106 <211> LENGTH: 23 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
peptide <400> SEQUENCE: 106 Gln Ile Val Leu Thr Gln Ser Pro
Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr
Cys 20 <210> SEQ ID NO 107 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of artificial
sequence: synthetic peptide <400> SEQUENCE: 107 Trp Tyr Gln
Gln Lys Pro Gly Leu Ala Pro Arg Leu Leu Ile Tyr 1 5 10 15
<210> SEQ ID NO 108 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polypeptide <400> SEQUENCE: 108 Gly Val Pro Gly Arg
Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser 1 5 10 15 Leu Thr Ile
Ser Arg Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys 20 25 30
<210> SEQ ID NO 109 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 109 His Gln Trp Ser Ser Ser
Pro His Thr 1 5 <210> SEQ ID NO 110 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 110
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 1 5 10 <210> SEQ
ID NO 111 <211> LENGTH: 32 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 111 Gly Ile Pro Ala Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser
Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys 20 25 30 <210>
SEQ ID NO 112 <211> LENGTH: 11 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 112 Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys Arg 1 5 10 <210> SEQ ID NO 113 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic polypeptide
<400> SEQUENCE: 113 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Glu Pro
Glu Asp Phe Ala Val Tyr Tyr Cys 20 25 30 <210> SEQ ID NO 114
<211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 114 Gly Val Pro Gly Arg Phe Ser
Gly Ser Gly Ser Gly Thr Ser Tyr Ser 1 5 10 15 Leu Thr Ile Ser Arg
Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys 20 25 30 <210>
SEQ ID NO 115 <211> LENGTH: 339 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polynucleotide <400> SEQUENCE: 115 caggtgcagc
tgcaagagtc aggacctggc ctggtgaaac cctcagaaac tctgtccctt 60
acatgcactg tctcagggtt ctcattaacc agctatggtg taagctggat tcgccagcct
120 ccaggaaagg gtctggagtg gattggagta atatggggtg aggggagcac
aaattatcat 180 tcagttctca tatccagact gaccattagt gtggatacct
ccaagaatca atttagctta 240 aaactgagca gtgttaccgc tgctgacaca
gccgtttact actgtgccat gactgggaca 300 gcttactggg gccaagggac
tctggtcact gtctctagc 339 <210> SEQ ID NO 116 <211>
LENGTH: 321 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic polynucleotide
<400> SEQUENCE: 116 gagattgtgc tgacccagag ccctgccaca
ctgtcactga gcccaggcga gcgagccaca 60 ctgtcctgtt ctgctagctc
ctctgtctcc tacatgcatt ggtatcagca gaagccagga 120 ctggcaccac
gactgctgat ctatgacact tctaaactga gttcaggcat tcccgccaga 180
ttcagtggct cagggagcgg aaccgacttt actctgacca ttagctccct ggagcctgaa
240 gatttcgccg tgtactattg ccatcagtgg tcatcaagcc ctcatacctt
cggggggggg 300 actaaggtgg aaatcaaacg c 321 <210> SEQ ID NO
117 <211> LENGTH: 113 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 117 Gln Val Gln Leu Gln Glu Ser
Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr
Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val Ser
Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly
Val Ile Trp Gly Glu Gly Ser Thr Asn Tyr His Ser Val Leu Ile 50 55
60 Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95 Met Thr Gly Thr Ala Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser 100 105 110 Ser <210> SEQ ID NO 118
<211> LENGTH: 107 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 118 Glu Ile Val Leu Thr Gln Ser
Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr
Gln Gln Lys Pro Gly Leu Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Asp
Thr Ser Lys Leu Ser Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55
60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu
65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys His Gln Trp Ser Ser Ser Pro
His Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100
105 <210> SEQ ID NO 119 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of artificial
sequence: synthetic peptide <400> SEQUENCE: 119 Ser Ser Val
Ser Tyr 1 5 <210> SEQ ID NO 120 <400> SEQUENCE: 120 000
<210> SEQ ID NO 121 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 121 His Gln Trp Ser Ser Ser
Pro His Thr 1 5 <210> SEQ ID NO 122 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 122
Gly Phe Ser Leu Thr Ser Tyr Gly 1 5 <210> SEQ ID NO 123
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 123 Ile Trp Gly Glu Gly Ser Thr 1 5
<210> SEQ ID NO 124 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 124 Ala Met Thr Gly Thr Ala
Tyr 1 5 <210> SEQ ID NO 125 <211> LENGTH: 339
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic polynucleotide <400> SEQUENCE:
125 caggtgcagc tgaagcagag cggacctggc ctggtgcagc cctcacagag
cctgagcatc 60 acttgtaccg tcagtggatt ctccctgaca tcttacggcg
tgtcttgggt caggcagagc 120 cctggcaagg ggctggagtg gctgggcgtg
atctggggag aaggctcaac taactatcac 180 agcgtcctga tcagtcgcct
gtcaattaac aaggacaatt ctaaaagtca ggtgttcttt 240 aaaatgaaca
gcctgcagtc caatgatacc gccatctact attgcgctat gaccggcaca 300
gcatactggg ggcagggaac actggtgact gtctccgct 339 <210> SEQ ID
NO 126 <211> LENGTH: 321 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polynucleotide <400> SEQUENCE: 126 gagattgtgc tgacccagag
ccctgccaca ctgtcactga gcccaggcga gcgagccaca 60 ctgtcctgtt
ctgctagctc ctctgtctcc tacatgcatt ggtatcagca gaagccagga 120
ctggcaccac gactgctgat ctatgacact tctaaactga gttcaggcat tcccgccaga
180 ttcagtggct cagggagcgg aaccgacttt actctgacca ttagctccct
ggagcctgaa 240 gatttcgccg tgtactattg ccatcagtgg tcatcaagcc
ctcatacctt cggggggggg 300 actaagctgg aaatcaaacg c 321 <210>
SEQ ID NO 127 <211> LENGTH: 113 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polypeptide <400> SEQUENCE: 127 Gln Val Gln Leu Lys
Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser
Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly
Val Ser Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40
45 Gly Val Ile Trp Gly Glu Gly Ser Thr Asn Tyr His Ser Val Leu Ile
50 55 60 Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val
Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile
Tyr Tyr Cys Ala 85 90 95 Met Thr Gly Thr Ala Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser 100 105 110 Ala <210> SEQ ID NO 128
<211> LENGTH: 108 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 128 Glu Ile Val Leu Thr Gln Ser
Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr
Gln Gln Lys Pro Gly Leu Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Asp
Thr Ser Lys Leu Ser Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55
60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu
65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys His Gln Trp Ser Ser Ser Pro
His Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Leu Glu Ile Lys Arg
100 105 <210> SEQ ID NO 129 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of artificial
sequence: synthetic peptide <400> SEQUENCE: 129 Ser Ser Val
Ser Tyr 1 5 <210> SEQ ID NO 130 <400> SEQUENCE: 130 000
<210> SEQ ID NO 131 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 131 His Gln Trp Ser Ser Ser
Pro His Thr 1 5 <210> SEQ ID NO 132 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 132
Gly Phe Ser Leu Thr Ser Tyr Gly 1 5 <210> SEQ ID NO 133
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 133 Ile Trp Gly Glu Gly Ser Thr 1 5
<210> SEQ ID NO 134 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 134 Ala Met Thr Gly Thr Ala
Tyr 1 5 <210> SEQ ID NO 135 <211> LENGTH: 339
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic polynucleotide <400> SEQUENCE:
135 caggtgcagc tgcaggaaag cggacccgga ctggtgaaac ctagcgaaac
actgagcctg 60 acttgtaccg tgagcggatt ttccctgacc tcttatggag
tgagctggat cagacagccc 120 cctggcaagg gactggagtg gatcggcgtg
atttggggag aaggctccac aaactatcac 180 agtgtcctga tctcacgact
gactatttct aaggacaact ctaaaagtca ggtcttcctg 240 aaactgaata
gtctgcagac tgacgatacc gctacatact attgcgcaat gacagggaca 300
gcatactggg gacagggaac cctggtgaca gtcagctcc 339 <210> SEQ ID
NO 136 <211> LENGTH: 321 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polynucleotide <400> SEQUENCE: 136 cagatcgtgc tgacacagtc
ccctgcaatt atgtcagcca gcccagggga aaaggtgaca 60 atgacttgta
gtgcttctag ttcagtctca tacatgcatt ggtatcagca gaagccaggc 120
ctggccccca gactgctgat ctacgacacc tccaaactga gctccggcgt gcccgggaga
180 ttttccggct ctgggagtgg aacttcatat agcctgacca tttctaggct
ggaggccgaa 240 gatgccgcta catactattg ccaccagtgg agcagtagcc
cccatacatt cggaggcggg 300 accaaagtgg aaatcaaacg c 321 <210>
SEQ ID NO 137 <211> LENGTH: 113 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polypeptide <400> SEQUENCE: 137 Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly
Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45 Gly Val Ile Trp Gly Glu Gly Ser Thr Asn Tyr His Ser Val Leu Ile
50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val
Phe Leu 65 70 75 80 Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr
Tyr Tyr Cys Ala 85 90 95 Met Thr Gly Thr Ala Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser 100 105 110 Ser <210> SEQ ID NO 138
<211> LENGTH: 107 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 138 Gln Ile Val Leu Thr Gln Ser
Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met
Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr
Gln Gln Lys Pro Gly Leu Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Asp
Thr Ser Lys Leu Ser Ser Gly Val Pro Gly Arg Phe Ser Gly Ser 50 55
60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Leu Glu Ala Glu
65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys His Gln Trp Ser Ser Ser Pro
His Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100
105 <210> SEQ ID NO 139 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of artificial
sequence: synthetic peptide <400> SEQUENCE: 139 Ser Ser Val
Ser Tyr 1 5 <210> SEQ ID NO 140 <400> SEQUENCE: 140 000
<210> SEQ ID NO 141 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 141 His Gln Trp Ser Ser Ser
Pro His Thr 1 5 <210> SEQ ID NO 142 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 142
Gly Phe Ser Leu Thr Ser Tyr Gly 1 5 <210> SEQ ID NO 143
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 143 Ile Trp Gly Glu Gly Ser Thr 1 5
<210> SEQ ID NO 144 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 144 Ala Met Thr Gly Thr Ala
Tyr 1 5 <210> SEQ ID NO 145 <211> LENGTH: 339
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic polynucleotide <400> SEQUENCE:
145 caggtccagc tgaaagagag cggccccgga ctggtcgccc cttcacagag
cctgagcatt 60 acttgcaccg tgagcggatt ttcactgacc agctacggag
tgagctggat tagacagcct 120 cctggcaagg gactggagtg gatcggcgtg
atttggggag aaggcagcac caactatcac 180 agtgtcctga tctcacgcct
gacaatttcc aaggacaaca gcaaatccca ggtcttcctg 240 aaactgaatt
ctctgcagac tgacgatacc gctacatact attgcgcaat gacagggaca 300
gcatactggg gacagggaac cctggtgaca gtcagtagt 339 <210> SEQ ID
NO 146 <211> LENGTH: 321 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polynucleotide <400> SEQUENCE: 146 cagatcgtgc tgacacagtc
cccagcaatt atgtctgcca gtcccgggga gaaggtgaca 60 atgacttgta
gtgccagctc ctctgtctca tacatgcatt ggtatcagca gaagtccggc 120
acatctccta aacggtggat ctacgacact tctaaactga gttcaggcgt gcccgggaga
180 ttttcaggca gcgggtccgg aacttcttat agtctgacca tttcccgact
ggaggccgaa 240 gatgccgcta cctactattg ccatcagtgg tcttcaagcc
ctcatacttt tgggggggga 300 actaaggtgg aaatcaagcg a 321 <210>
SEQ ID NO 147 <211> LENGTH: 113 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polypeptide <400> SEQUENCE: 147 Gln Val Gln Leu Lys
Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser
Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly
Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45 Gly Val Ile Trp Gly Glu Gly Ser Thr Asn Tyr His Ser Val Leu Ile
50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val
Phe Leu 65 70 75 80 Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr
Tyr Tyr Cys Ala 85 90 95 Met Thr Gly Thr Ala Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser 100 105 110 Ser <210> SEQ ID NO 148
<211> LENGTH: 107 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 148 Gln Ile Val Leu Thr Gln Ser
Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met
Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr
Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp
Thr Ser Lys Leu Ser Ser Gly Val Pro Gly Arg Phe Ser Gly Ser 50 55
60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Leu Glu Ala Glu
65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys His Gln Trp Ser Ser Ser Pro
His Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100
105 <210> SEQ ID NO 149 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of artificial
sequence: synthetic peptide <400> SEQUENCE: 149 Ser Ser Val
Ser Tyr 1 5 <210> SEQ ID NO 150 <400> SEQUENCE: 150 000
<210> SEQ ID NO 151 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 151 His Gln Trp Ser Ser Ser
Pro His Thr 1 5 <210> SEQ ID NO 152 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 152
Gly Phe Ser Leu Thr Ser Tyr Gly 1 5 <210> SEQ ID NO 153
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 153 Ile Trp Gly Glu Gly Ser Thr 1 5
<210> SEQ ID NO 154 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 154 Ala Met Thr Gly Thr Ala
Tyr 1 5 <210> SEQ ID NO 155 <211> LENGTH: 26
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 155
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5
10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser 20 25 <210> SEQ
ID NO 156 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
peptide <400> SEQUENCE: 156 Met His Trp Tyr Gln Gln Lys Ser
Gly Thr Ser Pro Lys Arg Trp Ile 1 5 10 15 Tyr <210> SEQ ID NO
157 <211> LENGTH: 36 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 157 Lys Leu Ser Ser Gly Val Pro
Gly Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Ser Tyr Ser Leu
Thr Ile Ser Arg Leu Glu Ala Glu Asp Ala Ala 20 25 30 Thr Tyr Tyr
Cys 35 <210> SEQ ID NO 158 <211> LENGTH: 11 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of artificial
sequence: synthetic peptide <400> SEQUENCE: 158 Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys Arg 1 5 10 <210> SEQ ID NO 159
<211> LENGTH: 25 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 159 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly
Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val
Ser 20 25 <210> SEQ ID NO 160 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <400> SEQUENCE: 160
Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 1 5
10 15 Val <210> SEQ ID NO 161 <211> LENGTH: 38
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic polypeptide <400> SEQUENCE:
161 Asn Tyr His Ser Val Leu Ile Ser Arg Leu Thr Ile Ser Lys Asp Asn
1 5 10 15 Ser Lys Ser Gln Val Phe Leu Lys Leu Asn Ser Leu Gln Thr
Asp Asp 20 25 30 Thr Ala Thr Tyr Tyr Cys 35 <210> SEQ ID NO
162 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
peptide <400> SEQUENCE: 162 Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 1 5 10 <210> SEQ ID NO 163 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 163
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10
15 <210> SEQ ID NO 164 <400> SEQUENCE: 164 000
<210> SEQ ID NO 165 <400> SEQUENCE: 165 000 <210>
SEQ ID NO 166 <400> SEQUENCE: 166 000 <210> SEQ ID NO
167 <400> SEQUENCE: 167 000 <210> SEQ ID NO 168
<400> SEQUENCE: 168 000 <210> SEQ ID NO 169 <400>
SEQUENCE: 169 000 <210> SEQ ID NO 170 <400> SEQUENCE:
170 000 <210> SEQ ID NO 171 <400> SEQUENCE: 171 000
<210> SEQ ID NO 172 <400> SEQUENCE: 172 000 <210>
SEQ ID NO 173 <400> SEQUENCE: 173 000 <210> SEQ ID NO
174 <400> SEQUENCE: 174 000 <210> SEQ ID NO 175
<400> SEQUENCE: 175 000 <210> SEQ ID NO 176 <400>
SEQUENCE: 176 000 <210> SEQ ID NO 177 <400> SEQUENCE:
177 000 <210> SEQ ID NO 178 <400> SEQUENCE: 178 000
<210> SEQ ID NO 179 <400> SEQUENCE: 179 000 <210>
SEQ ID NO 180 <400> SEQUENCE: 180 000 <210> SEQ ID NO
181 <400> SEQUENCE: 181 000 <210> SEQ ID NO 182
<400> SEQUENCE: 182 000 <210> SEQ ID NO 183 <400>
SEQUENCE: 183 000 <210> SEQ ID NO 184 <400> SEQUENCE:
184 000 <210> SEQ ID NO 185 <400> SEQUENCE: 185 000
<210> SEQ ID NO 186 <400> SEQUENCE: 186 000 <210>
SEQ ID NO 187 <400> SEQUENCE: 187 000 <210> SEQ ID NO
188 <400> SEQUENCE: 188 000 <210> SEQ ID NO 189
<400> SEQUENCE: 189 000 <210> SEQ ID NO 190 <400>
SEQUENCE: 190 000 <210> SEQ ID NO 191 <400> SEQUENCE:
191 000 <210> SEQ ID NO 192 <400> SEQUENCE: 192 000
<210> SEQ ID NO 193 <400> SEQUENCE: 193 000 <210>
SEQ ID NO 194 <400> SEQUENCE: 194 000 <210> SEQ ID NO
195 <400> SEQUENCE: 195 000 <210> SEQ ID NO 196
<400> SEQUENCE: 196 000 <210> SEQ ID NO 197 <400>
SEQUENCE: 197 000 <210> SEQ ID NO 198 <400> SEQUENCE:
198 000 <210> SEQ ID NO 199 <400> SEQUENCE: 199 000
<210> SEQ ID NO 200 <211> LENGTH: 357 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polynucleotide <400> SEQUENCE: 200 caggtgcagc
tgaaggaaag cggacccgga ctggtcgccc cctctaagtc tctgtctatt 60
acttgtactg tgagcggatt ctctctgagc tcccagggcg tgtactgggt gaggcagcca
120 cctggcaagg gcctggagtg gctgggagcc atctgggcag gaggcagcac
caactataat 180 tccgccctga tgtctcgcct gtctatcagc aaggacaact
ccaagtctca ggtgttcctg 240 aagatgaaca gcctgcagac cgacgataca
gccatgtact attgcgcccg ggtggacggc 300 tacagaggct ataacatgga
ttactggggc cagggcacca gcgtgacagt gtctagc 357 <210> SEQ ID NO
201 <211> LENGTH: 321 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polynucleotide <400> SEQUENCE: 201 gagaatgtgc tgacacagtc
cccagcaatc atgagcgcct ccccaggaga gaaggtgacc 60 atgacatgtt
ccgcctcctc tagcgtgtct tacatgcact ggtatcagca gaagtcctct 120
accagcccta agctgtggat ctacgacaca agcaagctgg cctccggcgt gcccggccgg
180 ttttctggca gcggctccgg caactcttat agcctgacca tcagcagcat
ggaggccgag 240 gatgtggcca catactattg ctttcagggc tctggctacc
cactgacatt cggggctgga 300 actaaactgg aactgaagcg a 321 <210>
SEQ ID NO 202 <211> LENGTH: 119 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polypeptide <400> SEQUENCE: 202 Gln Val Gln Leu Lys
Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Lys 1 5 10 15 Ser Leu Ser
Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Gln 20 25 30 Gly
Val Tyr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40
45 Gly Ala Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met
50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val
Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met
Tyr Tyr Cys Ala 85 90 95 Arg Val Asp Gly Tyr Arg Gly Tyr Asn Met
Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115
<210> SEQ ID NO 203 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polypeptide <400> SEQUENCE: 203 Glu Asn Val Leu Thr
Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val
Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His
Trp Tyr Gln Gln Lys Ser Ser Thr Ser Pro Lys Leu Trp Ile Tyr 35 40
45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser
50 55 60 Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu
Ala Glu 65 70 75 80 Asp Val Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly
Tyr Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
Arg 100 105 <210> SEQ ID NO 204 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 204
Ser Ser Val Ser Tyr 1 5 <210> SEQ ID NO 205 <211>
LENGTH: 3 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 205 Asp Thr Ser 1 <210> SEQ ID NO 206 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 206 Phe Gln Gly Ser Gly Tyr Pro Leu Thr 1 5 <210>
SEQ ID NO 207 <211> LENGTH: 8 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 207 Gly Phe Ser Leu Ser Ser
Gln Gly 1 5 <210> SEQ ID NO 208 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 208
Ile Trp Ala Gly Gly Ser Thr 1 5 <210> SEQ ID NO 209
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 209 Ala Arg Val Asp Gly Tyr Arg Gly Tyr Asn
Met Asp Tyr 1 5 10 <210> SEQ ID NO 210 <211> LENGTH:
357 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic polynucleotide <400> SEQUENCE:
210 caggtgcagc tgaaggagtc cggaccagga ctggtggcac catctaagac
cctgagcctg 60 acctgcacag tgagcggctt ctccctgagc tcccagggcg
tgtactggat caggcagcca 120 cctggcaagg gactggagtg gatcggcgcc
atctgggccg gcggctctac aaactataat 180 tccgccctga tgtctcgcct
gtctatcagc aaggacaact ccaagtctca ggtgtttctg 240 aagatgaata
gcctgcagac cgacgataca gccatgtact attgcgcccg ggtggacggc 300
tacagaggct ataacatgga ttattggggc cagggcaccc tggtgacagt gtctagc 357
<210> SEQ ID NO 211 <211> LENGTH: 321 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polynucleotide <400> SEQUENCE: 211 gagaatgtgc
tgacccagtc tcctgccatc atgagcgcca caccaggcga gaaggtgacc 60
atgacatgtt ccgcctcctc tagcgtgtct tacctgcact ggtatcagca gaagtcctct
120 accagcccca agctgtggat ctacgacaca agcaagctgg catccggagt
gcctggccgg 180 ttcagcggat ccggatctgg aaacagctat accctgacaa
tcagctccat ggaggccgag 240 gatgtggcca cctactattg tttccaggga
tccggatacc cactgacctt tggcgccggc 300 acaaagctgg agatcaagcg t 321
<210> SEQ ID NO 212 <211> LENGTH: 119 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polypeptide <400> SEQUENCE: 212 Gln Val Gln Leu Lys
Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Lys 1 5 10 15 Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Gln 20 25 30 Gly
Val Tyr Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45 Gly Ala Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met
50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val
Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met
Tyr Tyr Cys Ala 85 90 95 Arg Val Asp Gly Tyr Arg Gly Tyr Asn Met
Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
<210> SEQ ID NO 213 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polypeptide <400> SEQUENCE: 213 Glu Asn Val Leu Thr
Gln Ser Pro Ala Ile Met Ser Ala Thr Pro Gly 1 5 10 15 Glu Lys Val
Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Leu 20 25 30 His
Trp Tyr Gln Gln Lys Ser Ser Thr Ser Pro Lys Leu Trp Ile Tyr 35 40
45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser
50 55 60 Gly Ser Gly Asn Ser Tyr Thr Leu Thr Ile Ser Ser Met Glu
Ala Glu 65 70 75 80 Asp Val Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly
Tyr Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys
Arg 100 105 <210> SEQ ID NO 214 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 214
Ser Ser Val Ser Tyr 1 5 <210> SEQ ID NO 215 <211>
LENGTH: 3 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 215 Asp Thr Ser 1 <210> SEQ ID NO 216 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 216 Phe Gln Gly Ser Gly Tyr Pro Leu Thr 1 5 <210>
SEQ ID NO 217 <211> LENGTH: 8 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 217 Gly Phe Ser Leu Ser Ser
Gln Gly 1 5 <210> SEQ ID NO 218 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic polypeptide <400> SEQUENCE:
218 Ile Trp Ala Gly Gly Ser Thr 1 5 <210> SEQ ID NO 219
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 219 Ala Arg Val Asp Gly Tyr Arg Gly Tyr Asn
Met Asp Tyr 1 5 10 <210> SEQ ID NO 220 <211> LENGTH:
357 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic polynucleotide <400> SEQUENCE:
220 caggtgcagc tgaaggagtc cggaccagga ctggtggcac catctaagac
cctgagcctg 60 acctgcacag tgagcggctt ctccctgagc tcccagggcg
tgtactggat caggcagcca 120 cctggcaagg gactggagtg gatcggcgcc
atctgggccg gcggctctac aaactataat 180 tccgccctga tgtctcgcct
gtctatcagc aaggacaact ccaagtctca ggtgtttctg 240 aagatgaata
gcctgcagac cgacgataca gccatgtact attgcgcccg ggtggacggc 300
tacagaggct ataacatgga ttattggggc cagggcacct cggtgacagt gtctagc 357
<210> SEQ ID NO 221 <211> LENGTH: 321 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polynucleotide <400> SEQUENCE: 221 gagaatgtgc
tgacccagtc tcctgccatc atgagcgcca caccaggcga gaaggtgacc 60
atgacatgtt ccgcctcctc tagcgtgtct tacatgcact ggtatcagca gaagtcctct
120 accagcccca agctgtggat ctacgacaca agcaagctgg catccggagt
gcctggccgg 180 ttcagcggat ccggatctgg aaacagctat accctgacaa
tcagctccat ggaggccgag 240 gatgtggcca cctactattg tttccaggga
tccggatacc cactgacctt tggcgccggc 300 acaaagctgg agatcaagcg t 321
<210> SEQ ID NO 222 <211> LENGTH: 119 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polypeptide <400> SEQUENCE: 222 Gln Val Gln Leu Lys
Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Lys 1 5 10 15 Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Gln 20 25 30 Gly
Val Tyr Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45 Gly Ala Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met
50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val
Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met
Tyr Tyr Cys Ala 85 90 95 Arg Val Asp Gly Tyr Arg Gly Tyr Asn Met
Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115
<210> SEQ ID NO 223 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polypeptide <400> SEQUENCE: 223 Glu Asn Val Leu Thr
Gln Ser Pro Ala Ile Met Ser Ala Thr Pro Gly 1 5 10 15 Glu Lys Val
Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His
Trp Tyr Gln Gln Lys Ser Ser Thr Ser Pro Lys Leu Trp Ile Tyr 35 40
45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser
50 55 60 Gly Ser Gly Asn Ser Tyr Thr Leu Thr Ile Ser Ser Met Glu
Ala Glu 65 70 75 80 Asp Val Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly
Tyr Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys
Arg 100 105 <210> SEQ ID NO 224 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 224
Ser Ser Val Ser Tyr 1 5 <210> SEQ ID NO 225 <211>
LENGTH: 3 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 225 Asp Thr Ser 1 <210> SEQ ID NO 226 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 226 Phe Gln Gly Ser Gly Tyr Pro Leu Thr 1 5 <210>
SEQ ID NO 227 <211> LENGTH: 8 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 227 Gly Phe Ser Leu Ser Ser
Gln Gly 1 5 <210> SEQ ID NO 228 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 228
Ile Trp Ala Gly Gly Ser Thr 1 5 <210> SEQ ID NO 229
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 229 Ala Arg Val Asp Gly Tyr Arg Gly Tyr Asn
Met Asp Tyr 1 5 10
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 229
<210> SEQ ID NO 1 <211> LENGTH: 351 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polynucleotide <400> SEQUENCE: 1 caggtgcagc
tgaagcagtc tggacctgag ctagtgaaga ctggggcttc agtgaagata 60
tcctgcaagg cttctggtta ctcattcact ggttactaca tgcactgggt caagcagagc
120 catggaaaga gccttgagtg gattggatat attagttgtt acaatggtgg
tactaggtac 180 aacctgaagt tcaagggcaa ggccacattt actgtagaca
catcctccac cacagcctac 240 atgcagttca acaacctgac atctgaagac
tctgcggtct attactgtgc aagagggggg 300 tacgacgagg gtgactactg
gggccaaggc accactctca cagtctcctc a 351 <210> SEQ ID NO 2
<211> LENGTH: 321 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic
polynucleotide <400> SEQUENCE: 2 gatattgtaa tgacacagtc
tcccaaatcc atattcatgt cagttggaga gagggtcacc 60 ttgagctgca
aggccagtga gaatgtgggt acttatgtat cctggtatca acagaaacca 120
gagcagtctc ctaaactgat gatatacggg gcatccaacc ggaacactgg ggtccccgat
180 cgcttcacag gcagtggatc tgcaacagat ttcactctga ccatcagcag
tgtgcaggct 240 gaagaccttg cagattatca ctgtggacag agttacacct
atccgtacac gttcggaggg 300 gggaccaagc tggaaatcaa a 321 <210>
SEQ ID NO 3 <211> LENGTH: 117 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polypeptide <400> SEQUENCE: 3 Gln Val Gln Leu Lys
Gln Ser Gly Pro Glu Leu Val Lys Thr Gly Ala 1 5 10 15 Ser Val Lys
Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Tyr
Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40
45 Gly Tyr Ile Ser Cys Tyr Asn Gly Gly Thr Arg Tyr Asn Leu Lys Phe
50 55 60 Lys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Thr Thr
Ala Tyr 65 70 75 80 Met Gln Phe Asn Asn Leu Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Asp Glu Gly Asp Tyr
Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115
<210> SEQ ID NO 4 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polypeptide <400> SEQUENCE: 4 Asp Ile Val Met Thr
Gln Ser Pro Lys Ser Ile Phe Met Ser Val Gly 1 5 10 15 Glu Arg Val
Thr Leu Ser Cys Lys Ala Ser Glu Asn Val Gly Thr Tyr 20 25 30 Val
Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Met Ile 35 40
45 Tyr Gly Ala Ser Asn Arg Asn Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val
Gln Ala 65 70 75 80 Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Ser Tyr
Thr Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys 100 105 <210> SEQ ID NO 5 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 5 Glu
Asn Val Gly Thr Tyr 1 5 <210> SEQ ID NO 6 <400>
SEQUENCE: 6 000 <210> SEQ ID NO 7 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 7 Gly
Gln Ser Tyr Thr Tyr Pro Tyr Thr 1 5 <210> SEQ ID NO 8
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 8 Gly Tyr Ser Phe Thr Gly Tyr Tyr 1 5
<210> SEQ ID NO 9 <211> LENGTH: 8 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 9 Ile Ser Cys Tyr Asn Gly
Gly Thr 1 5 <210> SEQ ID NO 10 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 10 Ala
Arg Gly Gly Tyr Asp Glu Gly Asp Tyr 1 5 10 <210> SEQ ID NO 11
<211> LENGTH: 348 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic
polynucleotide <400> SEQUENCE: 11 gaggtccagc tgcaacaatc
tgggtctgtg ctggtgaggc ctggagcttc agtgaagctg 60 tcctgcaagg
cttctggcta caccttcacc aactcctgga tgcactgggc gaagcagagg 120
cctggacaag gccttgtgtg gattggagag attgatccta atactggtaa tactaactac
180 aatgagaact tcaagggcaa ggccacactg actgtagaca catcctccac
cacagcctac 240 gtggatctca gcagcctgac atctgaagac tctgcggtct
attactgtgc aagaggactc 300 gggctacttg tttactgggg ccaagggact
ctggtcactg tctctgca 348 <210> SEQ ID NO 12 <211>
LENGTH: 318 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic polynucleotide
<400> SEQUENCE: 12 caaattgttc tcacccagtc tccagcaatc
ctgtctgcat ctccagggga gaaggtcaca 60 atgacttgca gggccagctc
aagtgtaagt tacatgcact ggtaccagca gaagccagga 120 tcctccccca
aaccctggat ttatgtcaca tccaacctga cttctggagt ccctgttcgc 180
ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagagt ggaggctgaa
240 gatgctgcca cttattactg ccagcagtgg agtaataacc cgtggacgtt
cggtggaggc 300
accaagctgg aaatcaaa 318 <210> SEQ ID NO 13 <211>
LENGTH: 116 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic polypeptide
<400> SEQUENCE: 13 Glu Val Gln Leu Gln Gln Ser Gly Ser Val
Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Asn Ser 20 25 30 Trp Met His Trp Ala Lys
Gln Arg Pro Gly Gln Gly Leu Val Trp Ile 35 40 45 Gly Glu Ile Asp
Pro Asn Thr Gly Asn Thr Asn Tyr Asn Glu Asn Phe 50 55 60 Lys Gly
Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Thr Thr Ala Tyr 65 70 75 80
Val Asp Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Gly Leu Gly Leu Leu Val Tyr Trp Gly Gln Gly Thr Leu
Val 100 105 110 Thr Val Ser Ala 115 <210> SEQ ID NO 14
<211> LENGTH: 106 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 14 Gln Ile Val Leu Thr Gln Ser
Pro Ala Ile Leu Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met
Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr
Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Val
Thr Ser Asn Leu Thr Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55
60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu
65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Asn Asn Pro
Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
<210> SEQ ID NO 15 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 15 Ser Ser Val Ser Tyr 1 5
<210> SEQ ID NO 16 <400> SEQUENCE: 16 000 <210>
SEQ ID NO 17 <211> LENGTH: 9 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 17 Gln Gln Trp Ser Asn Asn
Pro Trp Thr 1 5 <210> SEQ ID NO 18 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 18 Gly
Tyr Thr Phe Thr Asn Ser Trp 1 5 <210> SEQ ID NO 19
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 19 Ile Asp Pro Asn Thr Gly Asn Thr 1 5
<210> SEQ ID NO 20 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 20 Ala Arg Gly Leu Gly Leu
Leu Val Tyr 1 5 <210> SEQ ID NO 21 <211> LENGTH: 339
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic polynucleotide <400> SEQUENCE:
21 caggtgcagc tgaaggagtc aggacctggc ctggtggcgc cctcacagag
cctgtccatc 60 acatgcactg tctcagggtt ctcattaagc agatatggtg
taagctgggt tcgccagcct 120 ccaggaaagg gtctggagtg gctgggagta
atatggggtg acgggagcac aaattatcat 180 tcagctctca tatccagact
gagcatcagc aaggataact ccaagagcca agttttctta 240 aaactgaaca
gtctgcaaac tgatgacaca gccacgtact actgtgccat gactgggaca 300
gcttactggg gccaagggac tctggtcact gtctctgca 339 <210> SEQ ID
NO 22 <211> LENGTH: 318 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polynucleotide <400> SEQUENCE: 22 caaattgttc tcacccagtc
tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60 atgacctgca
gtgccagctc aagtgtaaat tacatgcact ggtaccagca gaagtcaggc 120
acctccccca aaagatggat ttatgacaca tccaaactgg cttctggagt ccctgctcgc
180 ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcggcat
ggaggctgaa 240 gatgctgcca cttattactg ccaccagtgg aatagtagcc
cacacacgtt cggagggggg 300 accaagctgg aaataaaa 318 <210> SEQ
ID NO 23 <211> LENGTH: 113 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 23 Gln Val Gln Leu Lys Glu Ser
Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr
Cys Thr Val Ser Gly Phe Ser Leu Ser Arg Tyr 20 25 30 Gly Val Ser
Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly
Val Ile Trp Gly Asp Gly Ser Thr Asn Tyr His Ser Ala Leu Ile 50 55
60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80 Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr Tyr Tyr
Cys Ala 85 90 95 Met Thr Gly Thr Ala Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser 100 105 110 Ala <210> SEQ ID NO 24
<211> LENGTH: 106 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 24 Gln Ile Val Leu Thr Gln Ser
Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met
Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Met
20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp
Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg
Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile
Ser Gly Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys His
Gln Trp Asn Ser Ser Pro His Thr 85 90 95 Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105 <210> SEQ ID NO 25 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 25 Ser Ser Val Asn Tyr 1 5 <210> SEQ ID NO 26
<400> SEQUENCE: 26 000 <210> SEQ ID NO 27 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 27 His Gln Trp Asn Ser Ser Pro His Thr 1 5 <210>
SEQ ID NO 28 <211> LENGTH: 8 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 28 Gly Phe Ser Leu Ser Arg
Tyr Gly 1 5 <210> SEQ ID NO 29 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 29 Ile
Trp Gly Asp Gly Ser Thr 1 5 <210> SEQ ID NO 30 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 30 Ala Met Thr Gly Thr Ala Tyr 1 5 <210> SEQ ID NO
31 <211> LENGTH: 339 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polynucleotide <400> SEQUENCE: 31 caggtgcagc tgaaggagtc
aggacctggc ctggtggcgc cctcacagag cctgtccatc 60 acatgcactg
tctcaggatt ctcattaacc agctatggta taagctgggt tcgccagcct 120
ccaggaaagg gtctggagtg gctgggagta atatggggtg acgggagcac aaattatcat
180 tcagctctcg tatccagact gagcatcagc aaggataact ccaagagcca
agttttctta 240 aaactgaaca gtctgcaaac tgatgacaca gccacgtact
actgtgccaa aactgggaca 300 tcttactggg gccaagggac tctggtcact
gtctctgca 339 <210> SEQ ID NO 32 <211> LENGTH: 318
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic polynucleotide <400> SEQUENCE:
32 caaattgttc tcacccagtc tccagcaatc atgtctgcat ctccagggga
gaaggtcacc 60 atgacctgca gtgccagctc aagtgtaagt tacatgcact
ggtaccagca gaagtcaggc 120 acctccccca aaagatggat ttatgacaca
tccaaactgg cttctggagt ccctgctcgc 180 ttcagtggca gtgggtctgg
gacctcttac tctctcacaa tcagcagcat ggaggctgaa 240 gatgctgcca
cttattactg ccagcagtgg agtagtgccc cacacacgtt cggagggggg 300
accaagctgg aaataaaa 318 <210> SEQ ID NO 33 <211>
LENGTH: 113 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic polypeptide
<400> SEQUENCE: 33 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly
Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val
Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp
Gly Asp Gly Ser Thr Asn Tyr His Ser Ala Leu Val 50 55 60 Ser Arg
Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80
Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala 85
90 95 Lys Thr Gly Thr Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser 100 105 110 Ala <210> SEQ ID NO 34 <211> LENGTH:
106 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic polypeptide <400> SEQUENCE: 34
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5
10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr
Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg
Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr
Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys
Gln Gln Trp Ser Ser Ala Pro His Thr 85 90 95 Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys 100 105 <210> SEQ ID NO 35 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 35 Ser Ser Val Ser Tyr 1 5 <210> SEQ ID NO 36
<400> SEQUENCE: 36 000 <210> SEQ ID NO 37 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 37 Gln Gln Trp Ser Ser Ala Pro His Thr 1 5
<210> SEQ ID NO 38 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 38 Gly Phe Ser Leu Thr Ser
Tyr Gly 1 5 <210> SEQ ID NO 39 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 39 Ile
Trp Gly Asp Gly Ser Thr 1 5 <210> SEQ ID NO 40 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 40 Ala Lys Thr Gly Thr Ser Tyr 1 5 <210> SEQ ID NO
41 <211> LENGTH: 339 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polynucleotide <400> SEQUENCE: 41 caggtgcagc tgaaggagtc
aggacctggc ctggtggcgc cctcacagag cctgtccatc 60 acatgcactg
tctcagggtt ctcattaacc agctatggtg taagctgggt tcgccagcct 120
ccaggaaagg gtctggagtg gctgggagta atatggggtg aggggagcac aaattatcat
180 tcagttctca tatccagact gaccattagt aaggataact ccaagagcca
agttttctta 240 aaactgaaca gtctgcaaac tgatgacaca gccacgtact
actgtgccat gactgggaca 300 gcttactggg gccaagggac tctggtcact
gtctctgca 339 <210> SEQ ID NO 42 <211> LENGTH: 318
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic polynucleotide <400> SEQUENCE:
42 caaattgttc tcacccagtc tccagcaatc atgtctgcat ctccagggga
gaaggtcacc 60 atgacctgca gtgccagctc aagtgtaagt tacatgcact
ggtaccagca gaagtcaggc 120 acctccccca aaagatggat ttatgacaca
tccaaactgt cttctggagt ccctggtcgc 180 ttcagtggca gtgggtctgg
gacctcttac tctctcacaa tcagcaggtt ggaggctgaa 240 gatgctgcca
cttattactg ccatcagtgg agtagtagtc cacacacgtt cggagggggg 300
accaagttgg agataaaa 318 <210> SEQ ID NO 43 <211>
LENGTH: 113 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic polypeptide
<400> SEQUENCE: 43 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly
Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val
Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val Ser Trp Val Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp
Gly Glu Gly Ser Thr Asn Tyr His Ser Val Leu Ile 50 55 60 Ser Arg
Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80
Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala 85
90 95 Met Thr Gly Thr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser 100 105 110 Ala <210> SEQ ID NO 44 <211> LENGTH:
106 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic polypeptide <400> SEQUENCE: 44
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5
10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr
Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg
Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ser Ser Gly Val Pro Gly
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr
Ile Ser Arg Leu Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys
His Gln Trp Ser Ser Ser Pro His Thr 85 90 95 Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys 100 105 <210> SEQ ID NO 45 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 45 Ser Ser Val Ser Tyr 1 5 <210> SEQ ID NO 46
<400> SEQUENCE: 46 000 <210> SEQ ID NO 47 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 47 His Gln Trp Ser Ser Ser Pro His Thr 1 5 <210>
SEQ ID NO 48 <211> LENGTH: 8 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 48 Gly Phe Ser Leu Thr Ser
Tyr Gly 1 5 <210> SEQ ID NO 49 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 49 Ile
Trp Gly Glu Gly Ser Thr 1 5 <210> SEQ ID NO 50 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 50 Ala Met Thr Gly Thr Ala Tyr 1 5 <210> SEQ ID NO
51 <211> LENGTH: 25 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
peptide
<400> SEQUENCE: 51 Gln Val Asn Leu Arg Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile
Ser 20 25 <210> SEQ ID NO 52 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 52 Gly
Asp Ser Val Ser Ser Ser Ser Ala Ala 1 5 10 <210> SEQ ID NO 53
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 53 Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg
Gly Leu Glu Trp Leu Gly 1 5 10 15 Arg <210> SEQ ID NO 54
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 54 Thr Tyr Tyr Lys Ser Thr Trp His Asn 1 5
<210> SEQ ID NO 55 <211> LENGTH: 25 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 55 Gln Val Gln Leu Gln Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Gly Ala Ser 20 25 <210> SEQ ID NO 56 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 56 Gly Phe Thr Phe Arg Lys Ser Trp 1 5 <210> SEQ ID
NO 57 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
peptide <400> SEQUENCE: 57 Met Gly Trp Val Arg Gln Ser Pro
Gly Lys Gly Leu Glu Trp Val Ala 1 5 10 15 Asn <210> SEQ ID NO
58 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
peptide <400> SEQUENCE: 58 Thr Asn Asp Asp Ala Lys Glu Lys 1
5 <210> SEQ ID NO 59 <211> LENGTH: 38 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polypeptide <400> SEQUENCE: 59 Asp Tyr Ala Val Ser
Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr 1 5 10 15 Ser Lys Asn
Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp 20 25 30 Thr
Ala Val Tyr Tyr Cys 35 <210> SEQ ID NO 60 <211> LENGTH:
12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 60 Ala
Arg Glu Lys Gly Arg Val Arg Ala Phe Asp Ile 1 5 10 <210> SEQ
ID NO 61 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
peptide <400> SEQUENCE: 61 Trp Gly Gln Gly Thr Met Val Thr
Val Ser Ser 1 5 10 <210> SEQ ID NO 62 <211> LENGTH: 38
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic polypeptide <400> SEQUENCE: 62
Tyr Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Phe Arg Asp Asn 1 5
10 15 Asp Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp 20 25 30 Thr Ala Val Tyr Phe Cys 35 <210> SEQ ID NO 63
<211> LENGTH: 23 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 63 Ala Ser Leu Ser Ser Val Ser Ala Gly Asp
Pro Pro Arg Gly Pro Glu 1 5 10 15 Asn Ile Glu Tyr Phe Glu His 20
<210> SEQ ID NO 64 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 64 Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 1 5 10 <210> SEQ ID NO 65 <211>
LENGTH: 26 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic peptide <400>
SEQUENCE: 65 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ala Leu
Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser 20 25
<210> SEQ ID NO 66 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial
sequence:
synthetic peptide <400> SEQUENCE: 66 Gln Ser Val Gly Asn Lys
Tyr 1 5 <210> SEQ ID NO 67 <211> LENGTH: 17 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of artificial
sequence: synthetic peptide <400> SEQUENCE: 67 Val Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 1 5 10 15 Tyr
<210> SEQ ID NO 68 <400> SEQUENCE: 68 000 <210>
SEQ ID NO 69 <211> LENGTH: 26 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 69 Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser 20 25 <210> SEQ ID NO 70 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 70 Gln Ser Ile Ser Asn Tyr 1 5 <210> SEQ ID NO 71
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 71 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> SEQ ID NO 72
<400> SEQUENCE: 72 000 <210> SEQ ID NO 73 <211>
LENGTH: 36 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic polypeptide
<400> SEQUENCE: 73 Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Glu Pro Glu Asp Phe Ala 20 25 30 Val Tyr Tyr Cys 35
<210> SEQ ID NO 74 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 74 Gln Gln Tyr Gly Arg Ser
Arg Arg Leu Thr 1 5 10 <210> SEQ ID NO 75 <211> LENGTH:
11 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 75 Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 1 5 10 <210> SEQ ID
NO 76 <211> LENGTH: 36 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 76 Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 20 25 30 Thr Tyr Tyr
Cys 35 <210> SEQ ID NO 77 <211> LENGTH: 9 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of artificial
sequence: synthetic peptide <400> SEQUENCE: 77 Gln Gln Ser
Tyr Ser Thr Pro Arg Thr 1 5 <210> SEQ ID NO 78 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 78 Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg 1 5 10
<210> SEQ ID NO 79 <211> LENGTH: 26 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 79 Gln Val Gln Leu Lys Glu
Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile
Thr Cys Thr Val Ser Gly 20 25 <210> SEQ ID NO 80 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 80 Phe Ser Leu Thr Ser Tyr Gly Val Ser 1 5 <210>
SEQ ID NO 81 <211> LENGTH: 13 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 81 Trp Val Arg Gln Pro Pro
Gly Lys Gly Leu Glu Trp Leu 1 5 10 <210> SEQ ID NO 82
<211> LENGTH: 19 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 82
Gly Val Ile Trp Gly Glu Gly Ser Thr Asn Tyr His Ser Val Leu Ile 1 5
10 15 Ser Arg Leu <210> SEQ ID NO 83 <211> LENGTH: 26
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 83 Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10
15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly 20 25 <210> SEQ ID
NO 84 <211> LENGTH: 13 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
peptide <400> SEQUENCE: 84 Trp Ile Arg Gln Pro Pro Gly Lys
Gly Leu Glu Trp Ile 1 5 10 <210> SEQ ID NO 85 <211>
LENGTH: 26 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 85 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln
Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly 20 25
<210> SEQ ID NO 86 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 86 Trp Val Arg Gln Ser Pro
Gly Lys Gly Leu Glu Trp Leu 1 5 10 <210> SEQ ID NO 87
<211> LENGTH: 26 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 87 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val
Ser Gly 20 25 <210> SEQ ID NO 88 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 88 Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 1 5 10 <210>
SEQ ID NO 89 <211> LENGTH: 28 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 89 Thr Ile Ser Lys Asp Asn
Ser Lys Ser Gln Val Phe Leu Lys Leu Asn 1 5 10 15 Ser Leu Gln Thr
Asp Asp Thr Ala Thr Tyr Tyr Cys 20 25 <210> SEQ ID NO 90
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 90 Ala Met Thr Gly Thr Ala Tyr 1 5
<210> SEQ ID NO 91 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 91 Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ala 1 5 10 <210> SEQ ID NO 92 <211>
LENGTH: 28 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 92 Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu
Lys Leu Ser 1 5 10 15 Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
Cys 20 25 <210> SEQ ID NO 93 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 93 Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> SEQ ID
NO 94 <211> LENGTH: 28 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
peptide <400> SEQUENCE: 94 Ser Ile Asn Lys Asp Asn Ser Lys
Ser Gln Val Phe Phe Lys Met Asn 1 5 10 15 Ser Leu Gln Ser Asn Asp
Thr Ala Ile Tyr Tyr Cys 20 25 <210> SEQ ID NO 95 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 95 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 1 5 10
<210> SEQ ID NO 96 <211> LENGTH: 28 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 96 Thr Ile Ser Lys Asp Asn
Ser Lys Ser Gln Val Phe Leu Lys Leu Asn 1 5 10 15 Ser Leu Gln Thr
Asp Asp Thr Ala Thr Tyr Tyr Cys 20 25 <210> SEQ ID NO 97
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 97 Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 1 5 10
<210> SEQ ID NO 98 <211> LENGTH: 23 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 98 Gln Ile Val Leu Thr Gln
Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr
Met Thr Cys 20 <210> SEQ ID NO 99 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 99 Ser
Ala Ser Ser Ser Val Ser Tyr Met His 1 5 10 <210> SEQ ID NO
100 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
peptide <400> SEQUENCE: 100 Trp Tyr Gln Gln Lys Ser Gly Thr
Ser Pro Lys Arg Trp Ile Tyr 1 5 10 15 <210> SEQ ID NO 101
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 101 Asp Thr Ser Lys Leu Ser Ser 1 5
<210> SEQ ID NO 102 <211> LENGTH: 23 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 102 Glu Ile Val Leu Thr Gln
Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr
Leu Ser Cys 20 <210> SEQ ID NO 103 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 103
Trp Tyr Gln Gln Lys Pro Gly Leu Ala Pro Arg Leu Leu Ile Tyr 1 5 10
15 <210> SEQ ID NO 104 <211> LENGTH: 23 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of artificial
sequence: synthetic peptide <400> SEQUENCE: 104 Glu Ile Val
Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu
Arg Ala Thr Leu Ser Cys 20 <210> SEQ ID NO 105 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 105 Trp Tyr Gln Gln Lys Pro Gly Leu Ala Pro Arg Leu Leu
Ile Tyr 1 5 10 15 <210> SEQ ID NO 106 <211> LENGTH: 23
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 106
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5
10 15 Glu Lys Val Thr Met Thr Cys 20 <210> SEQ ID NO 107
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 107 Trp Tyr Gln Gln Lys Pro Gly Leu Ala Pro
Arg Leu Leu Ile Tyr 1 5 10 15 <210> SEQ ID NO 108 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic polypeptide
<400> SEQUENCE: 108 Gly Val Pro Gly Arg Phe Ser Gly Ser Gly
Ser Gly Thr Ser Tyr Ser 1 5 10 15 Leu Thr Ile Ser Arg Leu Glu Ala
Glu Asp Ala Ala Thr Tyr Tyr Cys 20 25 30 <210> SEQ ID NO 109
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 109 His Gln Trp Ser Ser Ser Pro His Thr 1 5
<210> SEQ ID NO 110 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 110 Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys Arg 1 5 10 <210> SEQ ID NO 111 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic polypeptide
<400> SEQUENCE: 111 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Glu Pro
Glu Asp Phe Ala Val Tyr Tyr Cys 20 25 30 <210> SEQ ID NO 112
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 112 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
Arg 1 5 10 <210> SEQ ID NO 113 <211> LENGTH: 32
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic
polypeptide <400> SEQUENCE: 113 Gly Ile Pro Ala Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser
Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys 20 25 30 <210>
SEQ ID NO 114 <211> LENGTH: 32 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polypeptide <400> SEQUENCE: 114 Gly Val Pro Gly Arg
Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser 1 5 10 15 Leu Thr Ile
Ser Arg Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys 20 25 30
<210> SEQ ID NO 115 <211> LENGTH: 339 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polynucleotide <400> SEQUENCE: 115 caggtgcagc
tgcaagagtc aggacctggc ctggtgaaac cctcagaaac tctgtccctt 60
acatgcactg tctcagggtt ctcattaacc agctatggtg taagctggat tcgccagcct
120 ccaggaaagg gtctggagtg gattggagta atatggggtg aggggagcac
aaattatcat 180 tcagttctca tatccagact gaccattagt gtggatacct
ccaagaatca atttagctta 240 aaactgagca gtgttaccgc tgctgacaca
gccgtttact actgtgccat gactgggaca 300 gcttactggg gccaagggac
tctggtcact gtctctagc 339 <210> SEQ ID NO 116 <211>
LENGTH: 321 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic polynucleotide
<400> SEQUENCE: 116 gagattgtgc tgacccagag ccctgccaca
ctgtcactga gcccaggcga gcgagccaca 60 ctgtcctgtt ctgctagctc
ctctgtctcc tacatgcatt ggtatcagca gaagccagga 120 ctggcaccac
gactgctgat ctatgacact tctaaactga gttcaggcat tcccgccaga 180
ttcagtggct cagggagcgg aaccgacttt actctgacca ttagctccct ggagcctgaa
240 gatttcgccg tgtactattg ccatcagtgg tcatcaagcc ctcatacctt
cggggggggg 300 actaaggtgg aaatcaaacg c 321 <210> SEQ ID NO
117 <211> LENGTH: 113 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 117 Gln Val Gln Leu Gln Glu Ser
Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr
Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val Ser
Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly
Val Ile Trp Gly Glu Gly Ser Thr Asn Tyr His Ser Val Leu Ile 50 55
60 Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95 Met Thr Gly Thr Ala Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser 100 105 110 Ser <210> SEQ ID NO 118
<211> LENGTH: 107 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 118 Glu Ile Val Leu Thr Gln Ser
Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr
Gln Gln Lys Pro Gly Leu Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Asp
Thr Ser Lys Leu Ser Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55
60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu
65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys His Gln Trp Ser Ser Ser Pro
His Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100
105 <210> SEQ ID NO 119 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of artificial
sequence: synthetic peptide <400> SEQUENCE: 119 Ser Ser Val
Ser Tyr 1 5 <210> SEQ ID NO 120 <400> SEQUENCE: 120 000
<210> SEQ ID NO 121 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 121 His Gln Trp Ser Ser Ser
Pro His Thr 1 5 <210> SEQ ID NO 122 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 122
Gly Phe Ser Leu Thr Ser Tyr Gly 1 5 <210> SEQ ID NO 123
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 123 Ile Trp Gly Glu Gly Ser Thr 1 5
<210> SEQ ID NO 124 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 124 Ala Met Thr Gly Thr Ala
Tyr 1 5 <210> SEQ ID NO 125 <211> LENGTH: 339
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic polynucleotide <400> SEQUENCE:
125 caggtgcagc tgaagcagag cggacctggc ctggtgcagc cctcacagag
cctgagcatc 60 acttgtaccg tcagtggatt ctccctgaca tcttacggcg
tgtcttgggt caggcagagc 120 cctggcaagg ggctggagtg gctgggcgtg
atctggggag aaggctcaac taactatcac 180 agcgtcctga tcagtcgcct
gtcaattaac aaggacaatt ctaaaagtca ggtgttcttt 240 aaaatgaaca
gcctgcagtc caatgatacc gccatctact attgcgctat gaccggcaca 300
gcatactggg ggcagggaac actggtgact gtctccgct 339 <210> SEQ ID
NO 126
<211> LENGTH: 321 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic
polynucleotide <400> SEQUENCE: 126 gagattgtgc tgacccagag
ccctgccaca ctgtcactga gcccaggcga gcgagccaca 60 ctgtcctgtt
ctgctagctc ctctgtctcc tacatgcatt ggtatcagca gaagccagga 120
ctggcaccac gactgctgat ctatgacact tctaaactga gttcaggcat tcccgccaga
180 ttcagtggct cagggagcgg aaccgacttt actctgacca ttagctccct
ggagcctgaa 240 gatttcgccg tgtactattg ccatcagtgg tcatcaagcc
ctcatacctt cggggggggg 300 actaagctgg aaatcaaacg c 321 <210>
SEQ ID NO 127 <211> LENGTH: 113 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polypeptide <400> SEQUENCE: 127 Gln Val Gln Leu Lys
Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser
Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly
Val Ser Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40
45 Gly Val Ile Trp Gly Glu Gly Ser Thr Asn Tyr His Ser Val Leu Ile
50 55 60 Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val
Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile
Tyr Tyr Cys Ala 85 90 95 Met Thr Gly Thr Ala Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser 100 105 110 Ala <210> SEQ ID NO 128
<211> LENGTH: 108 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 128 Glu Ile Val Leu Thr Gln Ser
Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr
Gln Gln Lys Pro Gly Leu Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Asp
Thr Ser Lys Leu Ser Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55
60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu
65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys His Gln Trp Ser Ser Ser Pro
His Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Leu Glu Ile Lys Arg
100 105 <210> SEQ ID NO 129 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of artificial
sequence: synthetic peptide <400> SEQUENCE: 129 Ser Ser Val
Ser Tyr 1 5 <210> SEQ ID NO 130 <400> SEQUENCE: 130 000
<210> SEQ ID NO 131 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 131 His Gln Trp Ser Ser Ser
Pro His Thr 1 5 <210> SEQ ID NO 132 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 132
Gly Phe Ser Leu Thr Ser Tyr Gly 1 5 <210> SEQ ID NO 133
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 133 Ile Trp Gly Glu Gly Ser Thr 1 5
<210> SEQ ID NO 134 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 134 Ala Met Thr Gly Thr Ala
Tyr 1 5 <210> SEQ ID NO 135 <211> LENGTH: 339
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic polynucleotide <400> SEQUENCE:
135 caggtgcagc tgcaggaaag cggacccgga ctggtgaaac ctagcgaaac
actgagcctg 60 acttgtaccg tgagcggatt ttccctgacc tcttatggag
tgagctggat cagacagccc 120 cctggcaagg gactggagtg gatcggcgtg
atttggggag aaggctccac aaactatcac 180 agtgtcctga tctcacgact
gactatttct aaggacaact ctaaaagtca ggtcttcctg 240 aaactgaata
gtctgcagac tgacgatacc gctacatact attgcgcaat gacagggaca 300
gcatactggg gacagggaac cctggtgaca gtcagctcc 339 <210> SEQ ID
NO 136 <211> LENGTH: 321 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polynucleotide <400> SEQUENCE: 136 cagatcgtgc tgacacagtc
ccctgcaatt atgtcagcca gcccagggga aaaggtgaca 60 atgacttgta
gtgcttctag ttcagtctca tacatgcatt ggtatcagca gaagccaggc 120
ctggccccca gactgctgat ctacgacacc tccaaactga gctccggcgt gcccgggaga
180 ttttccggct ctgggagtgg aacttcatat agcctgacca tttctaggct
ggaggccgaa 240 gatgccgcta catactattg ccaccagtgg agcagtagcc
cccatacatt cggaggcggg 300 accaaagtgg aaatcaaacg c 321 <210>
SEQ ID NO 137 <211> LENGTH: 113 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polypeptide <400> SEQUENCE: 137 Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly
Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45 Gly Val Ile Trp Gly Glu Gly Ser Thr Asn Tyr His Ser Val Leu Ile
50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val
Phe Leu 65 70 75 80 Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr
Tyr Tyr Cys Ala 85 90 95 Met Thr Gly Thr Ala Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser 100 105 110
Ser <210> SEQ ID NO 138 <211> LENGTH: 107 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of artificial
sequence: synthetic polypeptide <400> SEQUENCE: 138 Gln Ile
Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20
25 30 His Trp Tyr Gln Gln Lys Pro Gly Leu Ala Pro Arg Leu Leu Ile
Tyr 35 40 45 Asp Thr Ser Lys Leu Ser Ser Gly Val Pro Gly Arg Phe
Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser
Arg Leu Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys His Gln
Trp Ser Ser Ser Pro His Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys Arg 100 105 <210> SEQ ID NO 139 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 139 Ser Ser Val Ser Tyr 1 5 <210> SEQ ID NO 140
<400> SEQUENCE: 140 000 <210> SEQ ID NO 141 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 141 His Gln Trp Ser Ser Ser Pro His Thr 1 5 <210>
SEQ ID NO 142 <211> LENGTH: 8 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 142 Gly Phe Ser Leu Thr Ser
Tyr Gly 1 5 <210> SEQ ID NO 143 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 143
Ile Trp Gly Glu Gly Ser Thr 1 5 <210> SEQ ID NO 144
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 144 Ala Met Thr Gly Thr Ala Tyr 1 5
<210> SEQ ID NO 145 <211> LENGTH: 339 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polynucleotide <400> SEQUENCE: 145 caggtccagc
tgaaagagag cggccccgga ctggtcgccc cttcacagag cctgagcatt 60
acttgcaccg tgagcggatt ttcactgacc agctacggag tgagctggat tagacagcct
120 cctggcaagg gactggagtg gatcggcgtg atttggggag aaggcagcac
caactatcac 180 agtgtcctga tctcacgcct gacaatttcc aaggacaaca
gcaaatccca ggtcttcctg 240 aaactgaatt ctctgcagac tgacgatacc
gctacatact attgcgcaat gacagggaca 300 gcatactggg gacagggaac
cctggtgaca gtcagtagt 339 <210> SEQ ID NO 146 <211>
LENGTH: 321 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic polynucleotide
<400> SEQUENCE: 146 cagatcgtgc tgacacagtc cccagcaatt
atgtctgcca gtcccgggga gaaggtgaca 60 atgacttgta gtgccagctc
ctctgtctca tacatgcatt ggtatcagca gaagtccggc 120 acatctccta
aacggtggat ctacgacact tctaaactga gttcaggcgt gcccgggaga 180
ttttcaggca gcgggtccgg aacttcttat agtctgacca tttcccgact ggaggccgaa
240 gatgccgcta cctactattg ccatcagtgg tcttcaagcc ctcatacttt
tgggggggga 300 actaaggtgg aaatcaagcg a 321 <210> SEQ ID NO
147 <211> LENGTH: 113 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 147 Gln Val Gln Leu Lys Glu Ser
Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr
Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val Ser
Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly
Val Ile Trp Gly Glu Gly Ser Thr Asn Tyr His Ser Val Leu Ile 50 55
60 Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80 Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr Tyr Tyr
Cys Ala 85 90 95 Met Thr Gly Thr Ala Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser 100 105 110 Ser <210> SEQ ID NO 148
<211> LENGTH: 107 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 148 Gln Ile Val Leu Thr Gln Ser
Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met
Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr
Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp
Thr Ser Lys Leu Ser Ser Gly Val Pro Gly Arg Phe Ser Gly Ser 50 55
60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Leu Glu Ala Glu
65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys His Gln Trp Ser Ser Ser Pro
His Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100
105 <210> SEQ ID NO 149 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of artificial
sequence: synthetic peptide <400> SEQUENCE: 149 Ser Ser Val
Ser Tyr 1 5 <210> SEQ ID NO 150 <400> SEQUENCE: 150
000 <210> SEQ ID NO 151 <211> LENGTH: 9 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of artificial
sequence: synthetic peptide <400> SEQUENCE: 151 His Gln Trp
Ser Ser Ser Pro His Thr 1 5 <210> SEQ ID NO 152 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 152 Gly Phe Ser Leu Thr Ser Tyr Gly 1 5 <210> SEQ
ID NO 153 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
peptide <400> SEQUENCE: 153 Ile Trp Gly Glu Gly Ser Thr 1 5
<210> SEQ ID NO 154 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 154 Ala Met Thr Gly Thr Ala
Tyr 1 5 <210> SEQ ID NO 155 <211> LENGTH: 26
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 155
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5
10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser 20 25 <210> SEQ
ID NO 156 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
peptide <400> SEQUENCE: 156 Met His Trp Tyr Gln Gln Lys Ser
Gly Thr Ser Pro Lys Arg Trp Ile 1 5 10 15 Tyr <210> SEQ ID NO
157 <211> LENGTH: 36 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 157 Lys Leu Ser Ser Gly Val Pro
Gly Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Ser Tyr Ser Leu
Thr Ile Ser Arg Leu Glu Ala Glu Asp Ala Ala 20 25 30 Thr Tyr Tyr
Cys 35 <210> SEQ ID NO 158 <211> LENGTH: 11 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of artificial
sequence: synthetic peptide <400> SEQUENCE: 158 Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys Arg 1 5 10 <210> SEQ ID NO 159
<211> LENGTH: 25 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 159 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly
Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val
Ser 20 25 <210> SEQ ID NO 160 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <400> SEQUENCE: 160
Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 1 5
10 15 Val <210> SEQ ID NO 161 <211> LENGTH: 38
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic polypeptide <400> SEQUENCE:
161 Asn Tyr His Ser Val Leu Ile Ser Arg Leu Thr Ile Ser Lys Asp Asn
1 5 10 15 Ser Lys Ser Gln Val Phe Leu Lys Leu Asn Ser Leu Gln Thr
Asp Asp 20 25 30 Thr Ala Thr Tyr Tyr Cys 35 <210> SEQ ID NO
162 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
peptide <400> SEQUENCE: 162 Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 1 5 10 <210> SEQ ID NO 163 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 163
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10
15 <210> SEQ ID NO 164 <400> SEQUENCE: 164 000
<210> SEQ ID NO 165 <400> SEQUENCE: 165 000 <210>
SEQ ID NO 166 <400> SEQUENCE: 166 000 <210> SEQ ID NO
167 <400> SEQUENCE: 167 000 <210> SEQ ID NO 168
<400> SEQUENCE: 168 000 <210> SEQ ID NO 169 <400>
SEQUENCE: 169 000 <210> SEQ ID NO 170 <400> SEQUENCE:
170 000 <210> SEQ ID NO 171 <400> SEQUENCE: 171 000
<210> SEQ ID NO 172 <400> SEQUENCE: 172 000 <210>
SEQ ID NO 173 <400> SEQUENCE: 173 000 <210> SEQ ID NO
174 <400> SEQUENCE: 174 000 <210> SEQ ID NO 175
<400> SEQUENCE: 175 000 <210> SEQ ID NO 176 <400>
SEQUENCE: 176 000 <210> SEQ ID NO 177 <400> SEQUENCE:
177 000 <210> SEQ ID NO 178 <400> SEQUENCE: 178 000
<210> SEQ ID NO 179 <400> SEQUENCE: 179 000 <210>
SEQ ID NO 180 <400> SEQUENCE: 180 000 <210> SEQ ID NO
181 <400> SEQUENCE: 181 000 <210> SEQ ID NO 182
<400> SEQUENCE: 182 000 <210> SEQ ID NO 183 <400>
SEQUENCE: 183 000 <210> SEQ ID NO 184 <400> SEQUENCE:
184 000 <210> SEQ ID NO 185 <400> SEQUENCE: 185 000
<210> SEQ ID NO 186 <400> SEQUENCE: 186 000 <210>
SEQ ID NO 187 <400> SEQUENCE: 187 000 <210> SEQ ID NO
188 <400> SEQUENCE: 188 000 <210> SEQ ID NO 189
<400> SEQUENCE: 189 000 <210> SEQ ID NO 190 <400>
SEQUENCE: 190 000 <210> SEQ ID NO 191 <400> SEQUENCE:
191 000 <210> SEQ ID NO 192 <400> SEQUENCE: 192 000
<210> SEQ ID NO 193 <400> SEQUENCE: 193 000 <210>
SEQ ID NO 194 <400> SEQUENCE: 194 000 <210> SEQ ID NO
195 <400> SEQUENCE: 195 000 <210> SEQ ID NO 196
<400> SEQUENCE: 196 000 <210> SEQ ID NO 197 <400>
SEQUENCE: 197 000 <210> SEQ ID NO 198 <400> SEQUENCE:
198 000 <210> SEQ ID NO 199 <400> SEQUENCE: 199 000
<210> SEQ ID NO 200 <211> LENGTH: 357 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polynucleotide <400> SEQUENCE: 200 caggtgcagc
tgaaggaaag cggacccgga ctggtcgccc cctctaagtc tctgtctatt 60
acttgtactg tgagcggatt ctctctgagc tcccagggcg tgtactgggt gaggcagcca
120 cctggcaagg gcctggagtg gctgggagcc atctgggcag gaggcagcac
caactataat 180 tccgccctga tgtctcgcct gtctatcagc aaggacaact
ccaagtctca ggtgttcctg 240 aagatgaaca gcctgcagac cgacgataca
gccatgtact attgcgcccg ggtggacggc 300 tacagaggct ataacatgga
ttactggggc cagggcacca gcgtgacagt gtctagc 357 <210> SEQ ID NO
201 <211> LENGTH: 321 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polynucleotide <400> SEQUENCE: 201 gagaatgtgc
tgacacagtc cccagcaatc atgagcgcct ccccaggaga gaaggtgacc 60
atgacatgtt ccgcctcctc tagcgtgtct tacatgcact ggtatcagca gaagtcctct
120 accagcccta agctgtggat ctacgacaca agcaagctgg cctccggcgt
gcccggccgg 180 ttttctggca gcggctccgg caactcttat agcctgacca
tcagcagcat ggaggccgag 240 gatgtggcca catactattg ctttcagggc
tctggctacc cactgacatt cggggctgga 300 actaaactgg aactgaagcg a 321
<210> SEQ ID NO 202 <211> LENGTH: 119 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polypeptide <400> SEQUENCE: 202 Gln Val Gln Leu Lys
Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Lys 1 5 10 15 Ser Leu Ser
Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Gln 20 25 30 Gly
Val Tyr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40
45 Gly Ala Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met
50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val
Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met
Tyr Tyr Cys Ala 85 90 95 Arg Val Asp Gly Tyr Arg Gly Tyr Asn Met
Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115
<210> SEQ ID NO 203 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polypeptide <400> SEQUENCE: 203 Glu Asn Val Leu Thr
Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val
Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His
Trp Tyr Gln Gln Lys Ser Ser Thr Ser Pro Lys Leu Trp Ile Tyr 35 40
45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser
50 55 60 Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu
Ala Glu 65 70 75 80 Asp Val Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly
Tyr Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
Arg 100 105 <210> SEQ ID NO 204 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 204
Ser Ser Val Ser Tyr 1 5 <210> SEQ ID NO 205 <211>
LENGTH: 3 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 205 Asp Thr Ser 1 <210> SEQ ID NO 206 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of artificial sequence: synthetic peptide <400>
SEQUENCE: 206 Phe Gln Gly Ser Gly Tyr Pro Leu Thr 1 5 <210>
SEQ ID NO 207 <211> LENGTH: 8 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 207 Gly Phe Ser Leu Ser Ser
Gln Gly 1 5 <210> SEQ ID NO 208 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 208
Ile Trp Ala Gly Gly Ser Thr 1 5 <210> SEQ ID NO 209
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 209 Ala Arg Val Asp Gly Tyr Arg Gly Tyr Asn
Met Asp Tyr 1 5 10 <210> SEQ ID NO 210 <211> LENGTH:
357 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic polynucleotide <400> SEQUENCE:
210 caggtgcagc tgaaggagtc cggaccagga ctggtggcac catctaagac
cctgagcctg 60 acctgcacag tgagcggctt ctccctgagc tcccagggcg
tgtactggat caggcagcca 120 cctggcaagg gactggagtg gatcggcgcc
atctgggccg gcggctctac aaactataat 180 tccgccctga tgtctcgcct
gtctatcagc aaggacaact ccaagtctca ggtgtttctg 240 aagatgaata
gcctgcagac cgacgataca gccatgtact attgcgcccg ggtggacggc 300
tacagaggct ataacatgga ttattggggc cagggcaccc tggtgacagt gtctagc 357
<210> SEQ ID NO 211 <211> LENGTH: 321 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polynucleotide <400> SEQUENCE: 211 gagaatgtgc
tgacccagtc tcctgccatc atgagcgcca caccaggcga gaaggtgacc 60
atgacatgtt ccgcctcctc tagcgtgtct tacctgcact ggtatcagca gaagtcctct
120 accagcccca agctgtggat ctacgacaca agcaagctgg catccggagt
gcctggccgg 180 ttcagcggat ccggatctgg aaacagctat accctgacaa
tcagctccat ggaggccgag 240 gatgtggcca cctactattg tttccaggga
tccggatacc cactgacctt tggcgccggc 300 acaaagctgg agatcaagcg t 321
<210> SEQ ID NO 212 <211> LENGTH: 119 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polypeptide <400> SEQUENCE: 212 Gln Val Gln Leu Lys
Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Lys 1 5 10 15 Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Gln 20 25 30 Gly
Val Tyr Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45 Gly Ala Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met
50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val
Phe Leu 65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala 85
90 95 Arg Val Asp Gly Tyr Arg Gly Tyr Asn Met Asp Tyr Trp Gly Gln
Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> SEQ ID
NO 213 <211> LENGTH: 107 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polypeptide <400> SEQUENCE: 213 Glu Asn Val Leu Thr Gln Ser
Pro Ala Ile Met Ser Ala Thr Pro Gly 1 5 10 15 Glu Lys Val Thr Met
Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Leu 20 25 30 His Trp Tyr
Gln Gln Lys Ser Ser Thr Ser Pro Lys Leu Trp Ile Tyr 35 40 45 Asp
Thr Ser Lys Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser 50 55
60 Gly Ser Gly Asn Ser Tyr Thr Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80 Asp Val Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro
Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg 100
105 <210> SEQ ID NO 214 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of artificial
sequence: synthetic peptide <400> SEQUENCE: 214 Ser Ser Val
Ser Tyr 1 5 <210> SEQ ID NO 215 <211> LENGTH: 3
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 215
Asp Thr Ser 1 <210> SEQ ID NO 216 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 216
Phe Gln Gly Ser Gly Tyr Pro Leu Thr 1 5 <210> SEQ ID NO 217
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 217 Gly Phe Ser Leu Ser Ser Gln Gly 1 5
<210> SEQ ID NO 218 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polypeptide <400> SEQUENCE: 218 Ile Trp Ala Gly Gly
Ser Thr 1 5 <210> SEQ ID NO 219 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 219
Ala Arg Val Asp Gly Tyr Arg Gly Tyr Asn Met Asp Tyr 1 5 10
<210> SEQ ID NO 220 <211> LENGTH: 357 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polynucleotide <400> SEQUENCE: 220 caggtgcagc
tgaaggagtc cggaccagga ctggtggcac catctaagac cctgagcctg 60
acctgcacag tgagcggctt ctccctgagc tcccagggcg tgtactggat caggcagcca
120 cctggcaagg gactggagtg gatcggcgcc atctgggccg gcggctctac
aaactataat 180 tccgccctga tgtctcgcct gtctatcagc aaggacaact
ccaagtctca ggtgtttctg 240 aagatgaata gcctgcagac cgacgataca
gccatgtact attgcgcccg ggtggacggc 300 tacagaggct ataacatgga
ttattggggc cagggcacct cggtgacagt gtctagc 357 <210> SEQ ID NO
221 <211> LENGTH: 321 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of artificial sequence: synthetic
polynucleotide <400> SEQUENCE: 221 gagaatgtgc tgacccagtc
tcctgccatc atgagcgcca caccaggcga gaaggtgacc 60 atgacatgtt
ccgcctcctc tagcgtgtct tacatgcact ggtatcagca gaagtcctct 120
accagcccca agctgtggat ctacgacaca agcaagctgg catccggagt gcctggccgg
180 ttcagcggat ccggatctgg aaacagctat accctgacaa tcagctccat
ggaggccgag 240 gatgtggcca cctactattg tttccaggga tccggatacc
cactgacctt tggcgccggc 300 acaaagctgg agatcaagcg t 321 <210>
SEQ ID NO 222 <211> LENGTH: 119 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polypeptide <400> SEQUENCE: 222 Gln Val Gln Leu Lys
Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Lys 1 5 10 15 Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Gln 20 25 30 Gly
Val Tyr Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45 Gly Ala Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met
50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val
Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met
Tyr Tyr Cys Ala 85 90 95 Arg Val Asp Gly Tyr Arg Gly Tyr Asn Met
Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115
<210> SEQ ID NO 223 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic polypeptide <400> SEQUENCE: 223 Glu Asn Val Leu Thr
Gln Ser Pro Ala Ile Met Ser Ala Thr Pro Gly 1 5 10 15 Glu Lys Val
Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His
Trp Tyr Gln Gln Lys Ser Ser Thr Ser Pro Lys Leu Trp Ile Tyr 35 40
45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser
50 55 60 Gly Ser Gly Asn Ser Tyr Thr Leu Thr Ile Ser Ser Met Glu
Ala Glu 65 70 75 80 Asp Val Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly
Tyr Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys
Arg 100 105
<210> SEQ ID NO 224 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 224 Ser Ser Val Ser Tyr 1 5
<210> SEQ ID NO 225 <211> LENGTH: 3 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 225 Asp Thr Ser 1
<210> SEQ ID NO 226 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 226 Phe Gln Gly Ser Gly Tyr
Pro Leu Thr 1 5 <210> SEQ ID NO 227 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
artificial sequence: synthetic peptide <400> SEQUENCE: 227
Gly Phe Ser Leu Ser Ser Gln Gly 1 5 <210> SEQ ID NO 228
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of artificial sequence: synthetic peptide
<400> SEQUENCE: 228 Ile Trp Ala Gly Gly Ser Thr 1 5
<210> SEQ ID NO 229 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of artificial sequence:
synthetic peptide <400> SEQUENCE: 229 Ala Arg Val Asp Gly Tyr
Arg Gly Tyr Asn Met Asp Tyr 1 5 10
* * * * *