U.S. patent application number 17/658731 was filed with the patent office on 2022-08-04 for glycan-based antibody-drug conjugates.
This patent application is currently assigned to Merck Sharp & Dohme Corp.. The applicant listed for this patent is Merck Sharp & Dohme Corp.. Invention is credited to Robert Davidson, Bing Gong.
Application Number | 20220242970 17/658731 |
Document ID | / |
Family ID | 1000006272156 |
Filed Date | 2022-08-04 |
United States Patent
Application |
20220242970 |
Kind Code |
A1 |
Davidson; Robert ; et
al. |
August 4, 2022 |
GLYCAN-BASED ANTIBODY-DRUG CONJUGATES
Abstract
Genetically engineered antibodies containing non-native
N-glycosylated sites, preparation of the antibodies in yeast and
fungi, site-specific conjugation of drugs to these antibodies, and
methods of treatment utilizing these antibodies are described
herein.
Inventors: |
Davidson; Robert; (Enfield,
NH) ; Gong; Bing; (North Reading, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Merck Sharp & Dohme Corp. |
Rahway |
NJ |
US |
|
|
Assignee: |
Merck Sharp & Dohme
Corp.
Rahway
NJ
|
Family ID: |
1000006272156 |
Appl. No.: |
17/658731 |
Filed: |
April 11, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15493720 |
Apr 21, 2017 |
11332544 |
|
|
17658731 |
|
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|
62325497 |
Apr 21, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/52 20130101;
C07K 2317/14 20130101; A61K 47/6811 20170801; A61K 47/6851
20170801; A61K 47/6803 20170801; C07K 2317/24 20130101; A61K
2039/505 20130101; A61K 47/6889 20170801; C07K 2317/41 20130101;
C07K 2317/21 20130101; C07K 16/32 20130101; C07K 16/00 20130101;
C12P 21/005 20130101 |
International
Class: |
C07K 16/32 20060101
C07K016/32; C12P 21/00 20060101 C12P021/00; A61K 47/68 20060101
A61K047/68; C07K 16/00 20060101 C07K016/00 |
Claims
1. An engineered IgG antibody or heavy chain constant domain
fragment comprising an S134N mutation in the heavy chain constant
domain, which forms a first non-native N-glycosylation site having
the amino acid sequence NTS over positions 134-136 of the heavy
chain constant domain and a G161T or G161S mutation, which forms a
second non-native N-glycosylation site in the heavy chain constant
domain having the amino acid sequence NST over positions 159-161 of
the heavy chain constant domain, said non-native N-glycosylation
sites having an N-glycan attached to the N at position 134 and 159,
wherein the N-glycans have a
Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 or
GalGlcNAcMan.sub.3GlcNAc.sub.2 glycoform in which the terminal
galactose residues have been oxidized to a C-6 aldehyde group,
which is conjugated to a reactive amine group of a derivatized drug
by oxime bonds, and wherein the amino acid positions of the heavy
chain contant domain are according to Eu numbering.
2. An engineered IgG antibody or heavy chain constant domain
fragment of claim 1, wherein N-glycan comprises a
Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 glycoform.
3. An engineered IgG antibody or heavy chain constant domain
fragment of claim 1, wherein N-glycan comprises a
Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 glycoform.
4. An engineered IgG antibody or heavy chain constant domain
fragment of claim 1, wherein the engineered IgG antibody or heavy
chain constant domain fragment further comprises one to ten amino
acid mutations or pairs of mutations are selected from the group
consisting of N203 T, N203S, V363T, V363S, Q438N, S176N, A118N,
S132N, K133N, A162N, T195N, K210T, Y391T, F423T, F423S, Y436T,
Y436S, L193N, K392T, K392S, F423T, S176N/G178T, S176N/G178S,
Q419N/N421T, Q419N/N421S, S191N/L193T, S191N/L193S, G194N/Q196T,
and G194N/Q196S, wherein the amino acid positions of the heavy
chain contant domain are according to Eu numbering.
5. An engineered IgG antibody or heavy chain constant domain
fragment of claim 1, wherein the derivatized drug is selected from
the group consisting of a polymer, cytotoxic agent, a radionuclide,
fluorescent or chemiluminescent labels, steroid, steroid receptor
agonist, signal transduction inhibitor, a peptide and scFv.
6. An engineered IgG antibody or heavy chain constant domain
fragment of claim 1, wherein the derivatized drug comprises a
cytotoxic agent.
7. The engineered IgG antibody or antigen binding fragment of claim
1, wherein the IgG antibody further comprises one or two
N-glycosylation sites in the variable domain that have been
generated by amino acid mutations selected from the group
consisting of Q105N and S113N, wherein the numbering is according
to the Kabat numbering system.
8. The engineered IgG antibody or constant domain fragment of claim
1, wherein the IgG antibody is selected from the group consisting
of anti-Her2, anti-Her2/neu, anti-glycoprotein IIb/IIIa,
anti-TNF-.alpha., anti-CD52, anti-CD25, anti-BAFF,
anti-Vascularendothelial growth factor, anti-CD30, anti-IL-1.beta.,
anti-epidermal growth factor receptor, anti-RANK Ligand,
anti-Complement C5, anti-CD11a, anti-CD33, anti-CD20, anti-CTLA-4,
anti-T cell CD3 Receptor, anti-.alpha.-4 (.alpha.4) integrin, anti,
anti-Immunoglobulin E, anti-RSV F protein, anti-epidermal growth
factor receptor, anti-VEGF-A, anti-ErbB2, anti-IL-12/IL-23,
anti-integrin .alpha.4.beta.7, anti-CD274, anti-3-amyloid,
anti-4-1BB, anti-SAC, anti-5T4, anti-ACVR2B,
anti-adenocarcinomaantigen, anti-AGS-22M6,
anti-.alpha.-fetoprotein, anti-angiopoietin 2, anti-angiopoietin 3,
anti-anthrax toxin, anti-AOC3, anti-, anti-B7-H3, anti-Bacillus
anthracia, anti-.beta. amyloid, anti-B-lymphoma cell, anti-C242
antigen, anti-05, anti-CA-125, anti-carbonic anhydrase 9,
anti-cardiac myosin, anti-CCL11, anti-CCR4, anti-CCR5,
anti-CD11/CD18, anti-CD125, anti-CD140a, anti-CD147, anti-CD15,
anti-CD152, anti-CD154, anti-CD19, anti-CD2, anti-CD200, anti-CD22,
anti-CD221, anti-CD23, anti-CD27, anti-CD28, anti-CD3, anti-CD3
epsilon, anti-CD30, anti-CD37, anti-CD38, anti-CD4, anti-CD40,
anti-CD41, anti-CD44, anti-CD5, anti-CD51, anti-CD52, anti-CD56,
anti-CD6, anti-CD70, anti-CD74, anti-CD79B, anti-CD80, anti-CEA,
anti-CFD, anti-ch4D5, anti-CLDN18.2, anti-C. difficile,
anti-clumping factor A, anti-CSF2, anti-cytomegalovirus, anti-CMV
gp B, anti-DLL4, anti-DRS, anti-E. coli shiga toxin type-1 or 2,
anti-EGFL7, anti-endotoxin, anti-EpCAM, anti-EpCAM/CD3,
anti-episialin, anti-ERBB3, anti-Escherichia coli, anti-F protein,
anti-FAP, anti-fibrin II, anti-.beta.chain, anti-fibronectin extra
domain-B, anti-folate receptor 1, anti-Frizzled receptor, anti-GD2
ganglioside, anti-GD3 ganglioside, anti-GMCSF receptor
.alpha.-chain, anti-GPNMB, anti-Influenza, anti-Influenza
hemagglutinin, anti-hepatitis B, anti-surface antigen, anti-HER1,
anti-HER3, anti-HGF, anti-HHGFR, anti-HIV-1, anti-HLA-DR,
anti-HNGF, anti-Hsp90, anti-human scatter factor receptor kinase,
anti-human TNF, anti-human .beta.-amyloid, anti-CD54,
anti-IFN-.alpha., anti-IFN-.gamma., anti-IgE Fc region, anti-IGF-1
receptor, anti-IGF-I, anti-IgG4, anti-IGHE, anti-IL-13, anti-IL-17,
anti-IL-17A, anti-IL-10, anti-IL-22, anti-IL-23, anti-IL-4,
anti-IL-5, anti-IL-6, anti-IL-6 receptor, anti-IL9, anti-ILGF2,
anti-insulin-like growth factor I receptor anti-integrin .alpha.4,
anti-integrin .alpha.5.beta.1, anti-integrin .alpha.7.beta.7,
anti-integrin .alpha.IIb.beta.3, anti-integrin .alpha.v.beta.3,
anti-interferon receptor, anti-interferon .alpha./.beta. receptor,
anti-interferon .gamma.-induced protein, anti-ITGA2, anti-KIR2D,
anti-Lewis-Y antigen, anti-lipoteichoic acid, anti-LOXL2,
anti-L-selectin (CD62L), anti-LTA, anti-MCP-1, anti-mesothelin,
anti-MS4A1, anti-MUC1, anti-mucin CanAg, anti-myostatin,
anti-NARP-1, anti-NCA-90, anti-NGF, anti-N-glycolylneuraminic acid,
anti-NOGO-A, anti-Notch receptor, anti-NRP1, anti-Oryctolagus
cuniculus, anti-OX-40, anti-oxLDL, anti-PCSK9, anti-PD-1,
anti-PDCD1, anti-PDGF-R .alpha., anti-phosphate-sodium
co-transporter, anti-phosphatidylserine, anti-prostatic carcinoma
cells, anti-Pseudomonas aeruginosa, anti-rabies virus, anti-rabies
virus glycoprotein, anti-respiratory syncytial virus, anti-RHD,
anti-Rhesus factor, anti-RON, anti-RTN4, anti-sclerostin,
anti-SDC1, anti-selectin P, anti-SLAMF7, anti-SOST,
anti-sphingosine-1-phosphate, anti-TAG-72, anti-T-cell receptor,
anti-TEM1, anti-tenascin C, anti-TFPI, anti-TGF.beta.1,
anti-TGF.beta.2, anti-TGF-.beta., anti-TRAIL-R1, anti-TRAIL-R2,
anti-tumor antigen CTAA16.88, anti-TWEAK receptor, anti-TYRP1,
anti-VEGF-A, anti-VEGFR-1, anti-VEGFR2, anti-vimentin, anti-VWF,
anti-IL-1, anti-IL-2, anti-IL-5, anti-IL-8, anti-IL-12, anti-IL-15,
anti-IL-18, anti-IL-20, anti-IL-21, anti-IL-23R, anti-IL-25,
anti-IL-27, anti-IL-33, anti-CD14, anti-CD18, anti-CD64,
anti-CD200, anti-CD200R, anti-TSLP, anti-TSLPR, anti-PDL1,
anti-VLA-4, anti-E-selectin, anti-Fact II, anti-ICAM-3,
anti-.beta.2-integrin, anti-CBL, anti-LCAT, anti-CR3, anti-MDL-1,
anti-GITR, anti-CGRP, anti-TRKA, anti-IGF1R, and anti-GTC.
9. The engineered IgG antibody or constant domain fragment of claim
1, wherein the IgG antibody is selected from the group consisting
of abciximab, adalimumab, certolizumab pegol, golimumab,
infliximab, alemtuzumab, basiliximab), belimumab, bevacizumab,
brentuximab, canakinumab, cetuximab, daclizumab, denosumab,
eculizumab, efalizumab, gemtuzumab, ibritumomab tiuxetan,
ipilimumab, muromonab-cd3, natalizumab, ofatumumab, omalizumab,
palivizumab, panitumumab, ranibizumab, rituximab, tocilizumab,
atlizumab, tositumomab, trastuzumab, ustekinumab, and
vedolizumab.
10. A method of preparing a conjugated N-glycosylated IgG antibody
or fragment thereof comprising an N-glycan attached to the N at
position 134, wherein the N-glycan has a
Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 or
GalGlcNAcMan.sub.3GlcNAc.sub.2 glycoform in which the terminal
galactose residues are conjugated to a reactive amine group of a
derivatized drug by an oxime bond, the method comprising: (a)
transforming a yeast or filamentous fungus host cell genetically
engineered to produce N-glycans comprising terminal galactose
residues of the structure
Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2 or the structure
Gal.sub.(1-2)GlcNAc.sub.(1-2)Man.sub.5GlcNAc.sub.2 with a nucleic
acid encoding an IgG heavy chain constant domain having the amino
acid sequence NTS over positions 134-136 of the heavy chain
constant domain; b) culturing the transformed host cell under
conditions that allow the expression of the IgG heavy chain
constant domain comprising terminal galactose residues; (c)
contacting the expressed IgG heavy chain constant domain with a
reagent that oxidizes the terminal galactose residues to a C-6
aldehyde group; and (d) conjugating the reactive amine group of a
derivatized drug to the C-6 aldehyde group to produce the
conjugated N-glycosylated IgG antibody or fragment thereof, wherein
the amino acid positions of the heavy chain contant domain are
according to Eu numbering.
11. The method of claim 10, wherein the IgG heavy chain further
comprising a G161 T mutation, which forms a second non-native
N-glycosylation site in the heavy chain constant domain having the
amino acid sequence NST over positions 159-161 of the heavy chain
constant domain, wherein the amino acid positions of the heavy
chain contant domain are according to Eu numbering.
12. The method of claim 10, wherein the engineered IgG antibody or
heavy chain constant domain fragment further comprises one to ten
amino acid mutations or pairs of mutations are selected from the
group consisting of N203 T, N203 S, V363T, V363S, Q438N, S176N,
A118N, S132N, K133N, A162N, T195N, K2l10T, Y391T, F423T, F423S,
Y436T, Y436S, L193N, K392T, K392S, F423 T, S176N/G178T,
S176N/G178S, Q419N/N421T, Q419N/N421S, S191N/L193T, S191N/L193S,
G194N/Q196T, and G194N/Q196S, whereinthe amino acid positions of
the heavy chain contant domain are according to Eu numbering.
13. The method of claim 10, wherein the nucleic acid encodes an IgG
antibody and further comprises one or two N-glycosylation sites in
the variable domain that have been generated by amino acid
mutations selected from the group consisting of Q105N and S113N,
wherein the numbering is according to the Kabat numbering
system.
14. The method of claim 11, wherein the engineered IgG antibody or
heavy chain constant domain fragment further comprises one to ten
amino acid mutations or pairs of mutations are selected from the
group consisting of N203 T, N203 S, V363T, V363S, Q438N, S176N,
A118N, S132N, K133N, A162N, T195N, K210T, Y391T, F423T, F423S,
Y436T, Y436S, L193N, K392T, K392S, F423T, S176N/G178T, S176N/G178S,
Q419N/N421T, Q419N/N421S, S191N/L193T, S191N/L193S, G194N/Q196T,
andG194N/Q196S, wherein the amino acid positions of the heavy chain
contant domain are according to Eu numbering.
15. The method of claim 11, wherein the nucleic acid encodes an IgG
antibody and further comprises one or two N-glycosylation sites in
the variable domain that have been generated by amino acid
mutations selected from the group consisting of Q105N and S113N,
wherein the numbering is according to the Kabat numbering
system.
16. The method of claim 10, wherein the yeast host cell is selected
from the group consisting of Pichia pastoris (Komagataella
pastoris), Pichia finlandica, Pichia trehalophila, Pichia koclamae,
Pichia membranaefaciens, Pichia opuntiae, Pichiathermotolerans,
Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis,
Pichia methanolica, Pichia minuta (Ogataea minuta, Pichia
lindneri), Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp.,
Hansenula polymorphs, Kluyveromyces sp., Kluyveromyces lactis,
Candida albicans, Aspergillus nidulans, Aspergillus niger,
Aspergillus oryzae, Trichodermareesei, Chrysosporium lucknowense,
Fusarium sp., Fusarium gramineum, Fusarium venenatum, and
Neurospora crassa.
17. An engineered IgG antibody or heavy chain constant domain
fragment comprising a G161 T mutation, which forms a second
non-native N-glycosylation site in the heavy chain constant domain
having the amino acid sequence NST over positions 159-161 of the
heavy chain constant domain, said non-native N-glycosylation site
having an N-glycan attached to the N at position 134, wherein the
N-glycan has a Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 glycoform
or GalGlcNAcMan.sub.3GlcNAc.sub.2 glycoform in which the terminal
galactose residues have been oxidized to a C-6 aldehyde group,
which is conjugated to a reactive amine group of a derivatized drug
by an oxime bond, and wherein the amino acid positions of the heavy
chain contant domain are according to Eu numbering.
18. The engineered IgG antibody or constant domain fragment of
claim 17, wherein the engineered IgG antibody or heavy chain
constant domain fragment further comprises one to ten amino acid
mutations or pairs of mutations are selected from the group
consisting of N203 T, N203S, V363T, V363S, Q438N, S176N, A118N,
S132N, K133N, A162N, T195N, K210T, Y391T, F423T, F423S, Y436T,
Y436S, L193N, K392T, K392S, F423T, S176N/G178T, S176N/G178S,
Q419N/N421T, Q419N/N421S, S191N/L193T, S191N/L193S, G194N/Q196T,
and G194N/Q196S, wherein the amino acid positions of the heavy
chain contant domain are according to Eu numbering.
19. The engineered IgG antibody or constant domain fragment of
claim 17, wherein the IgG antibody and further comprises one or two
N-glycosylation sites in the variable domain that have been
generated by amino acid mutations selected from the group
consisting of Q105N and S113N, wherein the numbering is according
to the Kabat numbering system.
20. The engineered IgG antibody or constant domain fragment of
claim 17, wherein the IgG antibody is (a) selected from the group
consisting of anti-Her2, anti-Her2/neu, anti-glycoprotein IIb/IIIa,
anti-TNF-.alpha., anti-CD52, anti-CD25, anti-BAFF,
anti-Vascularendothelial growth factor, anti-CD30, anti-IL-1.beta.,
anti-epidermal growth factor receptor, anti-RANK Ligand,
anti-Complement C5, anti-CD11a, anti-CD33, anti-CD20, anti-CTLA-4,
anti-T cell CD3 Receptor, anti-.alpha.-4 (a4) integrin, anti,
anti-Immunoglobulin E, anti-RSV F protein, anti-epidermal growth
factor receptor, anti-VEGF-A, anti-ErbB2, anti-IL-12/IL-23,
anti-integrin .alpha.407, anti-CD274, anti-3-amyloid, anti-4-1BB,
anti-SAC, anti-5T4, anti-ACVR2B, anti-adenocarcinomaantigen,
anti-AGS-22M6, anti-.alpha.-fetoprotein, anti-angiopoietin 2,
anti-angiopoietin 3, anti-anthrax toxin, anti-AOC3, anti-,
anti-B7-H3, anti-Bacillus anthracia, anti-.beta. amyloid,
anti-B-lymphoma cell, anti-C242 antigen, anti-05, anti-CA-125,
anti-carbonic anhydrase 9, anti-cardiac myosin, anti-CCL11,
anti-CCR4, anti-CCR5, anti-CD11/CD18, anti-CD125, anti-CD140a,
anti-CD147, anti-CD15, anti-CD152, anti-CD154, anti-CD19, anti-CD2,
anti-CD200, anti-CD22, anti-CD221, anti-CD23, anti-CD27, anti-CD28,
anti-CD3, anti-CD3 epsilon, anti-CD30, anti-CD37, anti-CD38,
anti-CD4, anti-CD40, anti-CD41, anti-CD44, anti-CD5, anti-CD51,
anti-CD52, anti-CD56, anti-CD6, anti-CD70, anti-CD74, anti-CD79B,
anti-CD80, anti-CEA, anti-CFD, anti-ch4D5, anti-CLDN18.2, anti-C.
difficile, anti-clumping factor A, anti-CSF2, anti-cytomegalovirus,
anti-CMV gp B, anti-DLL4, anti-DRS, anti-E. coli shiga toxin type-1
or 2, anti-EGFL7, anti-endotoxin, anti-EpCAM, anti-EpCAM/CD3,
anti-episialin, anti-ERBB3, anti-Escherichia coli, anti-F protein,
anti-FAP, anti-fibrin II, anti-.beta. chain, anti-fibronectin extra
domain-B, anti-folate receptor 1, anti-Frizzled receptor, anti-GD2
ganglioside, anti-GD3 ganglioside, anti-GMCSF receptor
.alpha.-chain, anti-GPNMB, anti-Influenza, anti-Influenza
hemagglutinin, anti-hepatitis B, anti-surface antigen, anti-HER1,
anti-HER3, anti-HGF, anti-HHGFR, anti-HIV-1, anti-HLA-DR,
anti-HNGF, anti-Hsp90, anti-human scatter factor receptor kinase,
anti-human TNF, anti-human .beta.-amyloid, anti-CD54,
anti-IFN-.alpha., anti-IFN-.gamma., anti-IgE Fc region, anti-IGF-1
receptor, anti-IGF-I, anti-IgG4, anti-IGHE, anti-IL-13, anti-IL-17,
anti-IL-17A, anti-IL-10, anti-IL-22, anti-IL-23, anti-IL-4,
anti-IL-5, anti-IL-6, anti-IL-6 receptor, anti-IL9, anti-ILGF2,
anti-insulin-like growth factor I receptor, anti-integrin .alpha.4,
anti-integrin .alpha.5.beta.1, anti-integrin .alpha.7.beta.7,
anti-integrin .alpha.IIb.beta.3, anti-integrin .alpha.v.beta.3,
anti-interferon receptor, anti-interferon .alpha./.beta. receptor,
anti-interferon 7-induced protein, anti-ITGA2, anti-KIR2D,
anti-Lewis-Y antigen, anti-lipoteichoic acid, anti-LOXL2,
anti-L-selectin (CD62L), anti-LTA, anti-MCP-1, anti-mesothelin,
anti-MS4A1, anti-MUC1, anti-mucin CanAg, anti-myostatin,
anti-NARP-1, anti-NCA-90, anti-NGF, anti-N-glycolylneuraminic acid,
anti-NOGO-A, anti-Notch receptor, anti-NRP1, anti-Oryctolagus
cuniculus, anti-OX-40, anti-oxLDL, anti-PCSK9, anti-PD-1,
anti-PDCD1, anti-PDGF-R .alpha., anti-phosphate-sodium
co-transporter, anti-phosphatidylserine, anti-prostatic carcinoma
cells, anti-Pseudomonas aeruginosa, anti-rabies virus, anti-rabies
virus glycoprotein, anti-respiratory syncytial virus, anti-RHD,
anti-Rhesus factor, anti-RON, anti-RTN4, anti-sclerostin,
anti-SDC1, anti-selectin P, anti-SLAMF7, anti-SOST,
anti-sphingosine-1-phosphate, anti-TAG-72, anti-T-cell receptor,
anti-TEM1, anti-tenascin C, anti-TFPI, anti-TGF.beta.1,
anti-TGF.beta.2, anti-TGF-.beta., anti-TRAIL-R1, anti-TRAIL-R2,
anti-tumor antigen CTAA16.88, anti-TWEAK receptor, anti-TYRP1,
anti-VEGF-A, anti-VEGFR-1, anti-VEGFR2, anti-vimentin, anti-VWF,
anti-IL-1, anti-IL-2, anti-IL-5, anti-IL-8, anti-IL-12, anti-IL-15,
anti-IL-18, anti-IL-20, anti-IL-21, anti-IL-23R, anti-IL-25,
anti-IL-27, anti-IL-33, anti-CD14, anti-CD18, anti-CD64,
anti-CD200, anti-CD200R, anti-TSLP, anti-TSLPR, anti-PDL1,
anti-VLA-4, anti-E-selectin, anti-Fact II, anti-ICAM-3,
anti-02-integrin, anti-CBL, anti-LCAT, anti-CR3, anti-MDL-1,
anti-GITR, anti-CGRP, anti-TRKA, anti-IGF1R, and anti-GTC; or (b)
selected from the group consisting of abciximab, adalimumab,
certolizumab pegol, golimumab, infliximab, alemtuzumab,
basiliximab), belimumab, bevacizumab, brentuximab, canakinumab,
cetuximab, daclizumab, denosumab, eculizumab, efalizumab,
gemtuzumab, ibritumomab tiuxetan, ipilimumab, muromonab-cd3,
natalizumab, ofatumumab, omalizumab, palivizumab, panitumumab,
ranibizumab, rituximab, tocilizumab, atlizumab, tositumomab,
trastuzumab, ustekinumab, and vedolizumab.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 15/493,720, filed Apr. 21, 2017, which claims
the benefit of U.S. Provisional Patent Application No. 62/325,497,
filed Apr. 21, 2016, which is herein incorporated by referenced in
its entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] The sequence listing of the present application is submitted
electronically via EFS-Web as an ASCII formatted sequence listing
with a file name "24059-US-DIV-SEQTXT-07APRIL2022.txt", creation
date of Apr. 7, 2022, and a size of 229 Kb. This sequence listing
submitted via EFS-Web is part of the specification and is herein
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to engineered immunoglobulin
comprising mutations in the constant domains useful in
antibody-drug conjugates (ADCs), methods of treatment utilizing the
ADCs and methods of preparing the ADCs.
BACKGROUND
[0004] Monoclonal antibodies (mAbs) represent one of the fastest
growing and most important sectors of the pharmaceutical market
(Walsh, 2014). Antibodies (Abs) are unique molecules that provide
the ability to target cell-associated and soluble antigens in a
highly specific manner. This targeting can be used to block
activities such as receptor-ligand interactions, influence or
induce target-specific biological processes such as complement
activities and immune cell-mediated cytotoxic activities, or
modulate inflammation. However, efficient target cell killing,
especially in the context of a solid tumor remains a limitation for
many conventional mAbs (Schrama et al, 2006; Ricart, 2007).
Increasing the versatility and effectiveness of these molecules
will be a crucial focus as the next generation of antibody-based
therapeutics is developed. One of the primary advancements in
recent years is the resurgence of ADCs, particularly in the area of
oncology (Flygare, 2013; Mullard, 2013). ADCs are comprised of a
targeting vehicle and a linker that provides a stable support for
the drug to prevent off-target release but allow effective release
at the target location (Alley et al, 2010). Using the same
strategy, Abs can also be conjugated to radioisotopes, peptides, or
other macromolecules such as RNA (Ricart, 2011). However, to date,
ADCs have been most often used for the delivery of drug payloads to
improve cytotoxicity of a mAb to a known target (e.g. trastuzumab
in the case of Her2; also known as Herceptin), which concomitantly
increases the therapeutic index of an otherwise intolerable
cytotoxic therapy (Burris, 2013).
[0005] While important technical challenges such as an optimal
drug:antibody ratio (DAR) and methods of linker attachment
(cleavable vs. non-cleavable) have been addressed to improve the
characteristics of the current generation of ADCs, important
limitations still exist (Panowski, 2013; Boylan, 2013). These
hurdles include more efficient manufacture of ADCs, increased
product homogeneity, and improving the sophistication of the
chemistry available to allow broadening the scope of ADCs to
include combining Abs with peptides and hormones.
[0006] It has been shown that site-specific targeting of a
cytotoxic agent to an Ab not only improves the homogeneity of the
drug product but also the therapeutic index and efficacy of the ADC
(Junutula, 2008; Junutula, 2012). However, all current
site-specific targeting technologies have significant limitations.
Using engineered Cysteine (Cys) residues allows for a large degree
of site specificity but does not prevent targeting to native Cys
residues within the Ab protein, thus resulting in residual
heterogeneity while likely decreasing the stability of the molecule
through the disruption of native disulfide bonds, as well as
complicating manufacture of an Ab that now contains free thiol
residues (native Abs typically only contain paired Cys residues
that are engaged in disulfide bonds). Recent technologies that rely
on the incorporation of non-native amino acids are a step forward
from engineered Cys residues because they allow for unique
chemistry that is notpresent in the 20 amino acid code (Axup, 2012;
Zimmerman, 2014). Incorporation of non-native amino acids also
allows for more discrete control of the number of sites of
conjugation. However scale-up and the subsequent manufacture of
mAbs in mammalian cell lines, e.g., CHO cells, expressing
non-native amino acids and efficient incorporation of these
non-native amino acids introduces new challenges. Several other new
technologies have emerged, but each with its own limitations,
including introducing site specific tags in the mAb, which could
promote immunogenicity, or modifying the N-297 glycan, which limits
or abolishes immune effector function (Panowski, 2013). Also, in
each case, scalability is either challenging or an unknown. Thus,
an opportunity remains for a practical and scalable site-specific
modification technology that would permit linking Abs to a range of
different payloads.
SUMMARY OF THE INVENTION
[0007] The present invention relates to engineered Abs or fragments
thereof, and in particular IgG Abs possessing one to ten mutations
within the heavy chain constant domains, e.g, C.sub.H1, hinge,
C.sub.H2, C.sub.H3, and Fc. These mutations create non-native
N-glycosylation sites in the heavy chain constant domain. In one
embodiment, the engineered Abs of the invention are expressed in
yeast or filamentous fungal host cells, for example Pichia pastoris
host cells. When the engineered Abs are expressed in yeast and
filamentous fungi host cells genetically engineered to produce
human N-glycans, N-glycans are efficiently incorporated into the
non-native N-glycosylation site(s) of the heavy chain constant
domain. The engineered Abs expressed in genetically engineered
yeast and filamentous fungal host cells possess a high degree
ofN-glycan occupancy without disrupting the normal folding or
function of the Ab, and allow conjugation of a suitable amount of
payload/drug to the Ab to form an ADC. In particular, the
engineered Abs of the invention comprise galactose-terminated
N-glycans, which can be oxidized by an oxidizing reagent to produce
aldehyde groups. The reactive aldehyde groups, in the presence of a
derivatized drug containing a reactive amine form a bond (e.g., the
reactive amine, alkoxyamine, reacts with aldehyde groups to form an
oxime bond), thereby conjugating the drug to the Ab. In one
embodiment, the engineered IgG Ab or fragment thereof comprises a
human IgG1 constant domain.
[0008] In one embodiment, the present invention provides an
engineered IgG Ab or fragment thereof comprising one to ten
mutations (or pairs of mutations) in the heavy chain constant
domain which generate one to ten non-native N-glycosylation sites,
the mutations being selected from the group consisting of S134N,
G161T, G161S, N203 T, N203 S, V363T, V363S, Q438N, S176N, A118N,
S132N, K133N, A162N, T195N, K210T, Y391T, F423T, F423S, Y436T,
Y436S, L193N, K392T, K392S, F423T, S176N/G178T, S176N/G178S,
Q419N/N421T, Q419N/N421S, S191N/L193T, S191N/L193S, G194N/Q196T,
and G194N/Q196S, according to EU numbering. In one embodiment, the
engineered IgG Ab or fragment thereof comprises a human IgG1
constant domain.
[0009] In one embodiment, the engineered IgG Ab or fragment thereof
comprises at least one or two amino acid mutations (or a pair of
mutations) in the heavy chain constant domain selected from S134N,
G161T and S134N/G161T. In one embodiment, the engineered IgG Ab or
fragment thereof comprises a human IgG1 constant domain.
[0010] In another embodiment, the engineered IgG Ab or fragment
thereof comprises at least two amino acid mutations in the heavy
chain constant domain selected from G161T/S134T and G161S/S134T. In
one embodiment, the engineered Ab comprises a human IgG1 constant
domain.
[0011] In another embodiment, the N-glycosylated non-native site of
the engineered IgG Ab or fragment thereof is conjugated to a drug
selected from the group consisting of a polymer, cytotoxic agent, a
radionuclide, fluorescent or chemiluminescent labels, steroid,
steroid receptor agonist, signal transduction inhibitor, a peptide
and scFv.
[0012] The present invention also relates to engineered IgG Abs or
fragments thereof, and in particular Abs possessing one to two
mutations in the heavy chain variable framework domain which
generate one to two non-native N-glycosylation sites, the mutations
being selected from the group consisting of Q105N and S113N,
according to Kabat numbering.
[0013] In an embodiment, the engineered IgG Ab or fragment
comprising one to two mutations in the heavy chain variable
framework domain is conjugated to a drug and is selected from the
group consisting of a polymer, cytotoxic agent, a radionuclide,
fluorescent or chemiluminescent labels, steroid, signal
transduction inhibitor, a peptide and scFv.
[0014] In another embodiment, a method of treating a disease or
cancer in a patient suffering from the disease or cancer is
provided, the method comprising administering to the patient a
therapeutically effective amount of any of the aforementioned
engineered IgG Abs or fragments thereof which are conjugated to a
drug.
[0015] In another embodiment, a method of preparing a conjugated
N-glycosylated IgG Ab or fragment thereof containing one to ten
non-native N-glycosylation sites (or pairing of mutations) in the
heavy chain constant domain is provided, the method comprising:
[0016] (a) transforming a yeast or filamentous fungus host cell
genetically engineered to produce N-glycans comprising terminal
galactose residues of the structure
Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2 or the structure
Gal.sub.(1-2)GlcNAc.sub.(1-2)Man.sub.5GlcNAc.sub.2 with a nucleic
acid encoding an IgG heavy chain constant domain or fragment
thereof, wherein the IgG heavy chain constant domain comprises one
to ten amino acid mutations, and wherein the one to ten amino acid
mutations generate at least one N-glycosylation site in the IgG
heavy chain constant domain; [0017] (b) culturing the transformed
host cell under conditions that allow the expression of the IgG
heavy chain constant domain comprising terminal galactose residues,
[0018] (c) contacting the expressed IgG heavy chain constant domain
with a reagent that oxidizes the terminal galactose residues; and
[0019] (d) conjugating a drug to the oxidized moiety of the
terminal galactose residues.
[0020] In one embodiment, the yeast host cell used in the method of
preparing a conjugated N-glycosylated IgG Ab or fragment thereof
containing one to ten non-native N-glycosylation sites in the heavy
chain constant domain is selected from the group consisting of
Pichia pastoris (Komagataella pastoris), Pichia finlandica, Pichia
trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia
opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia
guercuum, Pichia piperi, Pichia stiptis, Pichia methanolica, Pichia
minuta (Ogataea minuta, Pichia lindneri), Pichia sp., Saccharomyces
cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces
sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans,
Aspergillus niger, Aspergillus oryzae, Trichoderma reesei,
Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum,
Fusarium venenatum, Neurospora crassa and Yarrowia lipolytica. In
another embodiment, the yeast host cell is Pichia pastoris.
[0021] In another embodiment, the IgG Ab or fragment thereof
prepared by the aforementioned method comprises one to ten
mutations (or pairs of mutations) in the heavy chain constant
domain polypeptide selected from the group consisting of S134N,
G161T, G161S, N203T, N203S, V363T, V363S, Q438N, S176N, A118N,
S132N, K133N, A162N, T195N, K210T, Y391T, F423T, F423S, Y436T,
Y436S, L193N, K392T, K392S, F423T, S176N/G178T, S176N/G178S,
Q419N/N421T, Q419N/N421S, S191N/L193T, S191N/L193S, G194N/Q196T,
and G194N/Q196S, according to EU numbering. In one embodiment, the
IgG heavy chain is a human IgG1 constant domain.
[0022] In another embodiment, methods of preparing a conjugated
N-glycosylated IgG Ab or fragment thereof containing one to two
non-native N-glycosylation sites in the heavy chain variable
framework domain are also provided.
[0023] In another embodiment, a method of preparing a conjugated
N-glycosylated IgG Ab or fragment thereof containing one to ten
non-native N-glycosylation sites in the heavy chain constant domain
is provided, the method comprising: [0024] (a) transforming a yeast
or filamentous fungus host cell genetically engineered to produce
N-glycans comprising terminal sialic acid residues of the structure
NANA.sub.(1-4)Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2
with a nucleic acid encoding an IgG heavy chain contain domain,
wherein the IgG heavy chain comprises one to ten amino acid
mutations (or pairs of mutations), and wherein the one to ten amino
acid mutation generates at least one non-native N-glycosylation
site in the IgG heavy chain constant domain; [0025] (b) culturing
the transformed host cell under conditions that allow the
expression of the IgG heavy chain constant domain comprising
terminal sialic acid residues, [0026] (c) contacting the expressed
IgG heavy chain constant domain with neuraminidase to remove the
terminal sialic acid residues to form N-glycosylated heavy chain
constant domain comprising terminal galactose residues; [0027] (d)
contacting the expressed glycosylated heavy chain constant domain
comprising terminal galactose residues of step (c), with a reagent
that oxidizes the terminal galactose residues; and [0028] (e)
conjugating a drug to the oxidized moiety of the terminal galactose
residues.
[0029] In one embodiment, the IgG heavy chain is a human IgG1
constant domain.
BRIEF DESCRIPTION OF THE FIGURES
[0030] FIG. 1. A comparison of the N-glycosylation machinery
between yeast and mammals. Yeast and mammals initiate glycosylation
similarly via the secretory pathway, both resulting in a
Man.sub.8GlcNAc.sub.2 N-glycan following protein folding and ER
maturation. N-glycosylation pathways differ in the Golgi with
mammals trimming mannose residues and adding GlcNAc to produce
hybrid and complex N-gly cans in bi-, tri-, or tetra-antennary
form, which are then terminated with varying amounts of galactose
and sialic acid. In contrast, fungi, such as P. pastoris, add
additional mannose with various linkages, including an outer chain
initiated by the Och1p .alpha.-1,6-mannosyltransferase, resulting
in glycans that in total can be comprised of dozens of mannose
residues. Man, mannose; GlcNAc, N-acetyl glucosamine; Gal,
galactose; MnT, mannosyltransferase; MNS, mannosidase; GnT, GlcNAc
transferase; GalT, galactosyl transferase.
[0031] FIG. 2. A flow diagram of an example of the basic molecular
genetic steps of yeast N-glycan engineering. OCH1 is knocked out
and, depending on the yeast species, other yeast N-glycan machinery
encoding genes are also knocked out. Mannosidases and glycosyl
transferases responsible for the successive steps in human N-glycan
biosynthesis are introduced, wherein each intermediate step can be
isolated via a strain producing that particular glycan structure
(GS). Man, mannose; GlcNAc, N-acetyl glucosamine; Gal, galactose;
MnT, mannosyltransferase; MNS, mannosidase; GnT, GlcNAc
transferase; GalT, galactosyl transferase; UDP, Uridine
diphosphate; CMP, Cytidine monophosphate.
[0032] FIG. 3: Restriction map of plasmid pGLY5883. The E. coli/P.
pastoris shuttle vector is depicted circularly as it is maintained
in E. coli. The plasmid contains the pUC19 Ori and AmpR region for
E. coli maintenance as well as the Sh ble gene encoding Zeocin
resistance (ZeoR) and the P. pastoris TRP2 gene, used as an
integration site. The genes encoding the trastuzumab anti-Her2 H
(heavy) chain and L (light) chain are contained as separate
cassettes, each with the P. pastoris AOX1 promoter and S.
cerevisiae CYC1 transcriptional terminator.
[0033] FIG. 4(A-B): Capillary electrophoretic analysis of N-glycan
modified antibodies. Gel animation image depicting protein bands
following separation by capillary electrophoresis of
glycan-engineered versions of the trastuzumab anti-Her2 antibody
under denatured non-reduced conditions. Antibodies are described in
Table 1 and were expressed in GS5.0 (FIG. 2) glycoengineered
Pichia, then resulting clones cultivated in 96 deep well plates,
and culture supernatant protein A purified. Arrows indicate the
presence of an antibody tetramer band. Sizes are indicated by the
markers at the far left and right.
[0034] FIG. 5(A-D): Capillary electrophoresis analysis of N-glycan
modified antibodies cultivated in micro24. Clones expressing
indicated plasmids were cultivated in micro24 5 ml fermenter
vessels. Following protein A purification, purified protein was
analyzed by Caliper GXII under denatured non-reducing conditions
and reducing conditions. Arrows indicate the presence of an
antibody tetramer band in the non-reduced samples and the antibody
H chain monomer in the reduced samples. C, control samples, are
samples from cultivation of strain YGLY13979, expressing the wild
type trastuzumab antibody sequence. Sizes are indicated by the
markers at the far left and right.
[0035] FIG. 6(A-G): Q-ToF Mass spectrometry analysis of N-glycan
modified antibodies. Deconvoluted mass spectra of reduced
antibodies isolated from the strains indicated after cultivation in
micro24 5 ml fermenters. The expected mass range for H chain with
0, 1, and 2 N-glycans is indicated. Where actual masses agree
closely with a predicted size for a modified glycosylated antibody,
the structure is indicated for the peak representing expected
modified protein. GS, glycan structure (see FIG. 2).
[0036] FIG. 7: Capillary electrophoretic analysis of N-glycan
modified antibodies cultivated in Dasgip 1 L fermenters. Clones
expressing the plasmids indicated in Table 3 were cultivated in
Dasgip (Shrewsbury, Mass.) 1 L fermenter vessels. Following protein
A purification, purified protein was analyzed by Caliper GXII under
denatured non-reducing and reducing conditions. Arrows indicate the
presence of an antibody tetramer band in the non-reduced samples
and the antibody H and L chain monomers in the reduced samples.
Sizes are indicated by the markers at the far left.
[0037] FIG. 8(A-C): Q-ToF Mass spectrometry analysis of N-glycan
engineered antibodies cultivated in Dasgip 1 L fermenters.
Deconvoluted mass spectra of reduced antibodies isolated and
purified by small scale high throughput protein A from strains
cultivated in Dasgip 1 L fermenters. The strain cultivated in each
fermenter and associated plasmid/antibody information is indicated
in Table 3. The expected mass range for H chain with 0, 1, and 2
N-glycans is indicated. Where actual masses agree closely with a
predicted size for a modified glycosylated antibody, the structure
is indicated for the peak representing expected modified protein.
A, samples D133201-08; B, samples D133401-04; C, samples
D133405-08. GS, glycan structure (see FIG. 2).
[0038] FIG. 9(A-B): Mass spectrometry analysis of highly purified
N-glycan engineered antibodies cultivated in Dasgip 1 L fermenters.
Deconvoluted mass spectra are shown for reduced antibodies isolated
and purified by larger scale protein A purification (Zha, 2013)
from 1 L fermentation samples D133202, D133203, D133208, D133404,
and D133406 (see Table 3). A) Mass spectra are gated to include
expectedL and H chain masses. The expected mass ranges for L chain
(LC) and glycosylated H chain (HC+glycans) are indicated. B) Mass
spectra gated to zoom in on H chain expected mass. Where actual
masses agree closely with a predicted size for a modified
glycosylated antibody, the structure is indicated for the peak
representing expected modified protein. GS, glycan structure (see
FIG. 2).
[0039] FIG. 10: Glycosidase digestion and analysis of N-glycan
modified antibodies. Purified antibody from batch D133404
(trastuzumab, S134N) was analyzed directly by Q-ToF MS, as
previously shown, then subjected to EndoS glycosidase digestion to
remove the N-297 glycan. The released N-glycans were analyzed by
MALDI-ToF MS and the remaining intact protein analyzed by Q-ToF MS
and deconvoluted. Finally, the Endo S digested protein was further
subjected to PNGase F glycosidase digestion and N-glycans and
protein were again separately analyzed by MALDI-TOF MS and Q-ToF,
respectively.
[0040] FIG. 11(A-F): Analysis of binding of glycan modified
anti-Her2 antibodies to Her2 antigen. Protein A purified N-glycan
modified antibodies analyzed for binding to Her2 protein using
surface plasmon resonance. Anti-human Fc antibody was immobilized
and used to capture purified glycoengineered Pichia-produced mutant
trastuzumab variants (A) S134N, (B) G161T, (C) N203T, and (D) V363T
as well as (E) commercial Herceptin (trastuzumab) and (F) S134N
conjugated with DM1 cytotoxin.
[0041] FIG. 12: Conjugation of a fluorescent dye to native
N-glycans on commercial trastuzumab by galactose oxidase treatment.
Commercial trastuzumab was subjected to galactose oxidase enzyme
treatment for 48 h in the presence of aminooxy CF633 fluorophore
and 50 mM aniline and the resulting protein was reduced and
analyzed by Q-ToF MS. The deconvoluted mass spectrum is shown with
the mass range expected for unconjugated (H chain) and conjugated
(H chain+CF633) indicated. Peaks corresponding to the expected mass
of trastuzumab glycan-containing heavy chain variants are labeled
with the canonical glycan structures.
[0042] FIG. 13: Glycan-mediated conjugation of a fluorescent dye
glycan-engineered antibodies using galactose oxidase.
Pichia-produced glycan-engineered versions of trastuzumab were
subjected to galactose oxidase enzyme treatment in the presence of
aminooxy CF633 fluorophore and the resulting protein was reduced
and analyzed by Q-ToF MS. The deconvoluted mass spectra are shown
with the mass range expected for unconjugated (HC), singly
conjugated (+1.times.CF633), and doubly conjugated (+2.times.CF633)
antibody indicated. The predominant peak corresponding to the
expected mass of the glycosylated mutated trastuzumab heavy chain
is labeled (HC+GS4.0+GS5.0) with glycan structures referenced in
FIG. 2.
[0043] FIG. 14: Q-ToF Mass spectrometry analysis of sialylated
N-glycan engineered antibodies. Deconvoluted mass spectra are shown
for reduced glycan engineered trastuzumab antibodies isolated and
purified by small scale high throughput protein A expressed in
GS6.0 glycoengineered Pichia strains cultivated in Dasgip 1 L
fermenters with batch numbers shown. The predominant peaks
corresponding to the expected masses of the glycosylated mutated
trastuzumab heavy chain is labeled (HC+2 GS6.0) with glycan
structures referenced in FIG. 2.
[0044] FIG. 15: Glycan-mediated conjugation of a fluorophore to
glycan-engineered antibodies with galactose oxidase in the presence
of reaction catalysts. Pichia-produced glycan-engineered versions
of trastuzumab were conjugated with an aminooxy activated CF633
fluorophore using galactose oxidase in the presence of reaction
catalysts 2-Amino-5-methoxybenzoic acid (AMB), 3,5-diaminobenzoic
acid (DAB), and aniline. The resulting conjugated protein was
reduced and analyzed by Q-ToF MS and deconvoluted mass spectra are
shown with the mass range expected for unconjugated (HC), singly
conjugated (+1.times.CF633), and doubly conjugated (+2.times.CF633)
antibody indicated. The predominant peak corresponding to the
expected mass of the glycosylated mutated trastuzumab heavy chain
is labeled (HC+GS4.0+GS5.0) with glycan structures referenced in
FIG. 2.
[0045] FIG. 16: Glycan-mediated conjugation of a fluorescent dye at
multiple different sites on glycan-engineered antibodies. Multiple
Pichia-produced glycan-engineered versions of trastuzumab (Sample
IDs and mutations as described in Table 2) were conjugated with an
aminooxy activated CF633 fluorophore in the presence of aniline as
a reaction catalyst. The resulting conjugated protein was reduced
and analyzed by Q-ToF MS and deconvoluted mass spectra are shown
with the mass range expected for unconjugated (HC), singly
conjugated (+1 Alexa488), and doubly conjugated (+2 Alexa488)
antibody indicated as well as triply conjugated (+3 Alexa488).
[0046] FIG. 17: Scale-up and quantification of conjugation to
glycan-engineered antibodies. Pichia-produced glycan-engineered
versions of trastuzumab were conjugated with an alkoxyamine
activated Biotin and an alkoxyamine activated fluorophore
(Alexa488) in the presence of aniline then reduced and the
resulting reaction products analyzed by Q-ToF MS. The peak areas
were then calculated and ratioed to determine the average number of
conjugates per whole mAb (Drug-antibody-ratio, DAR).
[0047] FIG. 18(A-B): IdeS digestion and mass spectrometry analysis
of a conjugated, glycan-engineered antibody. Glycan-engineered
(G161T) trastuzumab that was produced in glycoengineered Pichia and
conjugated with CF633 fluorescent dye was digested with IdeS enzyme
to separate the F(ab')2 and Fc domains and the resulting protein
analyzed by Q-ToF MS. Expected mass ranges for glycosylated,
conjugated Fab fragments (F(ab')2+3 CF633 and F(ab')2+4 CF633) and
glycosylated unconjuguated (Fc+GS4.0) as well as conjugated Fc
fragments (Fc+GS3.5+CF633) are identified.
[0048] FIG. 19: Size Exclusion Chromatography of a
glycan-engineered and conjugated antibody. Glycan-engineered
(G161T) trastuzumab that was produced in glycoengineered Pichia and
then conjugated with CF633 fluorescent dye was analyzed by native
size exclusion chromatography prior to and after conjugation
compared to the commercially available trastuzumab as a
control.
[0049] FIG. 20: Temperature stability of glycan-conjugated
antibodies. Glycan-engineered trastuzumab variants produced in
glycoengineered Pichia were analyzed by native size exclusion
chromatography compared commercially available trastuzumab as a
control prior to and after a two week incubation at 45.degree. C.
in 100 mM sodium phosphate pH 7.0.
[0050] FIG. 21(A-C): Conjugation of exendin-4 peptide to a
glycan-engineered antibody. A, Illustration of the site-specific
conjugation of alkoxyamine activated exendin-4 peptide to the Fab
glycan of the GS5.0 Pichia produced glycan-engineered antibody. B,
A deconvoluted Q-ToF mass spectrum of reduced antibody (H chain)
after conjugation with exendin-4 peptide, which was used as the
basis calculating the peptide:mAb ratio (DAR). C, GLP1-receptor
agonist activity assay demonstrating the activity of the
mAb/exendin-4 conjugate compared to native GLP-1 peptide,
calculated as the EC50 of intracellular cAMP change in GLP-1R
recombinant CHO cells.
[0051] FIG. 22(A-B): Conjugation of a modified DM1 cytotoxin to a
glycan-engineered antibody. A, Illustration of the conjugation
reaction of alkoxy-labeled, C5-linked, Mertansine (DM1) to the
galactose residues of the Fab glycans on a glycan-engineered
anti-Her2 antibody in one-pot with FgGalOx. B, Deconvoluted Q-ToF
mass spectrum of reduced antibody (H chain) after conjugation with
DM1 for two different glycan-engineered anti-Her2 antibodies.
[0052] FIG. 23(A-B): Q-ToF Mass spectrometry analysis of
DM1-conjugated antibodies. A, Deconvoluted Q-ToF mass spectrum of
reduced DM1-conjugated anti-Her2 G161T glycan-engineered antibody,
gated to include predicted heavy (Hc) and light (Lc) chain masses,
with conjugation efficiency calculated (DAR) based on integrated
peak areas. B, Deconvoluted Q-ToF mass spectrum of reduced
DM1-conjugated anti-Her2 (ado-trastuzumab emtansine) antibody,
gated to include predicted H and L chain masses.
[0053] FIG. 24(A-B): Q-ToF Mass spectrometry analysis of
glycan-engineered antibodies isolated from microfermenter
cultivation. Deconvoluted Q-ToF mass spectra of reduced antibodies
for A) one set of 7 glycan-engineered anti-Her2 antibodies with
F.sub.ab and C.sub.H1-localized muteins, and B) an additional set
of 7 diverse glycan-engineered anti-Her2 antibodies including
CH3-located and double mutant sequences. All spectra are gated to
include predicted heavy (H) chain masses. Unglycosylated, singly
glycosylated (+1 N-glycan) and doubly glycosylated (+2 N-glycans)
predicted masses are identified, while specific masses differ
slightly depending on the mutational change(s) made to incorporate
the respective N-glycan sequon. Sequence-related information for
each mutation is found in Table 4.
[0054] FIG. 25(A-B): Q-ToF Mass spectrometry analysis of
glycan-engineered antibodies with two additional N-glycan sites.
Deconvoluted Q-ToF mass spectra of reduced antibodies that have
been glycan-engineered to incorporate two non-native
N-glycosylation sites, in each case located on the C.sub.H1 domain
of the heavy chain. The spectra are gated to include: A) predicted
light and heavy chain masses, and B) zoomed to observe the
predicted H chain masses.
[0055] FIG. 26(A-F): Glycan-engineered antibodies with three or
more additional N-glycan sites. A, Illustration depicting the
single position glycan-engineered antibody (shown here in the
C.sub.H1 region), which may or may not also contain the C.sub.H2
Fc-297 N-glycan and B, single position glyco-conjugation, leading
to a distinct DAR of 2 or 4 depending the N-glycan chosen (or 6 or
8 if multiantennary N-glycans are employed). C, Illustration
showing a multi-position glycan-engineered antibody. D, Capillary
electrophoresis profile showing several non-reduced multi-position
glycan engineered antibodies compared to the one position-modified
and control (anti-Her2 Trasutuzmab sequence) antibodies. E,
MALDI-TOF MS of released N-glycans from multi-position
glycan-engineered antibody modified with ten additional N-glycan
sites and expressed in a GS5.0 glycoform strain (see FIG. 2). F,
Illustration showing conjugation to the exposed galactose residues
of a multi-position glycan-engineered mAb to achieve higher DAR
(Drug to Ab Ratio).
[0056] FIG. 27: Restriction map of plasmid pGLY11576. The E.
coli/P. pastoris shuttle vector is depicted circularly as it is
maintained in E. coli. The plasmid contains the pUC19 Ori and AmpR
region for E. coli maintenance as well as the Sh ble gene encoding
Zeocin resistance (ZeoR) and the P. pastoris TRP2 gene, used as an
integration site. The genes encoding the modified trastuzumab-based
"null-Her2" H chain and L chain that have been modified to no
longer bind the Her2 receptor are contained as separate cassettes.
Each antibody gene cassette is flanked with the P. pastoris AOX1
promoter and a transcriptional terminator, that from S. cerevisiae
CYC1 for the H chain and that from P. pastoris AOX1 for the L
chain.
[0057] FIG. 28(A-B): Q-ToF Mass spectrometry analysis of
glycan-engineered antibodies with more than two additional N-glycan
sites. Deconvoluted Q-ToF mass spectra of reduced antibodies that
have been glycan-engineered to incorporate from three to ten
non-native N-glycosylation sites resulting from transformation of
plasmids: A, pGLY14172-14175 or B, pGLY14176, 14177, and 14179 (see
Table 5) into a GS5.0 glycoengineered Pichia strain (see FIG. 2).
The resulting spectra are gated to include the predicted H chain
masses.
[0058] FIG. 29(A-B): Capillary electrophoresis analysis of
sialylated N-glycan modified antibodies containing three to ten
additional N-glycans. The plasmids indicated in Table 5 were
transformed into GS6.0 (see FIG. 2) glycoengineered strain
YGLY36472 and Zeocin resistant clones were cultivated in 96 deep
well plates. Protein A purified protein was analyzed by capillary
electrophoresis under denatured, non-reducing conditions. Arrows
indicate the presence of an antibody tetramer band in the
non-reduced samples and the antibody H and L chain monomers in the
reduced samples. Sizes are indicated by the markers at the far
left.
[0059] FIG. 30: MALDI-TOF MS of released N-glycans from a
multi-position glycan-engineered antibody. A MALDI-TOF mass
spectrum in negative ion mode of released N-glycans from an
antibody protein A purified from a clone of plasmid pGLY14179
expressed in GS6.0 strain YGLY36472 (see FIG. 2 and Table 5),
following cultivation of the clone in a 1 L fermenter. The spectrum
is gated to include masses consistent with N-glycans and the masses
representative of predicted GS6.0 and a singly sialylated version
are identified.
[0060] FIG. 31(A-D): Q-ToF Mass spectrometry analysis of
glycan-engineered antibodies with more than two additional N-glycan
sites expressed with GS6.0 N-glycans. Deconvoluted Q-ToF mass
spectra of reduced antibodies that have been glycan-engineered to
incorporate from three to ten non-native N-glycosylation sites
resulting from transformation of plasmids: A, pGLY14172 and 14173;
B, pGLY14174 and 14175; C, pGLY14176 and 14177; or D, pGLY14178 and
14179 (see Table 5) into a GS6.0 glycoengineered Pichia strain (see
FIG. 2), and following 1 L fermentation and protein A purification.
The resulting spectra are paired and gated to include first both
the predicted L and H chain masses and then zoomed to include only
the predicted H chain masses. Where individual peaks closely match
predicted masses, these glycosylated species are identified.
[0061] FIG. 32: Restriction map of plasmid pGLY13649. The E.
coli/P. pastoris shuttle vector is depicted circularly as it is
maintained in E. coli. The plasmid contains the pUC19 Ori and AmpR
region for E. coli maintenance as well as the Sh ble gene encoding
Zeocin resistance (not marked) and the P. pastoris TRP2 gene, used
as an integration site. The genes encoding an anti-murine PD1
antibody H chain and L chain are contained as separate cassettes,
each initiated with the P. pastoris AOX1 promoter.
[0062] FIG. 33: Q-ToF Mass spectrometry analysis of an N-glycan
engineered anti-PD1 antibody. Deconvoluted mass spectra of reduced
glycan-engineered anti-murine PD1 antibodies modified to
incorporate one or two non-native N-glycosylation sites, then
isolated and purified by protein A from clones cultivated in 1 L
fermenters. The incorporated mutations (EU numbering) are noted for
each modified antibody and the expected mass range for H chain with
two, three, and four N-glycans is indicated (the anti-murine mAb
clone contains a H chain CDR N-glycan in addition to the Fc-297
N-glycan in the native sequence).
[0063] FIG. 34: Restriction map of plasmid pGLY8040. The E. coli/P.
pastoris shuttle vector is depicted circularly as it is maintained
in E. coli. The plasmid contains the pUC19 Ori and AmpR region for
E. coli maintenance as well as the Sh ble gene encoding Zeocin
resistance (not marked) and the P. pastoris TRP2 gene, used as an
integration site. The genes encoding an anti-CS1 antibody H chain
and L chain are contained as separate cassettes, each flanked with
the P. pastoris AOX1 promoter and a transcriptional terminator (not
marked).
[0064] FIG. 35: Q-ToF Mass spectrometry analysis of an N-glycan
engineered anti-CS1 antibody. Deconvoluted mass spectra of reduced
glycan-engineered anti-murine PD1 antibodies modified to
incorporate two separate non-native N-glycosylation sites, then
isolated and purified by protein A from clones cultivated in 1 L
fermenters. The incorporated mutations (EU numbering) are noted for
each modified antibody and the expected mass range for H chain with
one or two N-glycans is indicated.
[0065] FIG. 36: Restriction map of plasmid pGLY5730. The E. coli/P.
pastoris shuttle vector is depicted circularly as itis maintained
in E. coli. The plasmid contains the pUC19 Ori and AmpR region for
E. coli maintenance as well as the Sh ble gene encoding Zeocin
resistance (not marked) and the P. pastoris TRP2 gene, used as an
integration site. The genes encoding an antibody H chain and L
chain (with the D2E7 anti-TNF.alpha. variable regions from
commercial antibody adalimumab) are contained as separate
cassettes, each flanked with the P. pastoris AOX1 promoter and a
transcriptional terminator (not marked).
[0066] FIG. 37: Restriction map of plasmid pGLY14148. The E.
coli/P. pastoris shuttle vector is depicted circularly as itis
maintained in E. coli. The plasmid contains the pUC19 Ori and AmpR
region for E. coli maintenance as well as the Sh ble gene encoding
Zeocin resistance (ZeocinR) and the P. pastoris TRP2 gene, used as
an integration site. The genes encoding an anti-CD70 antibody H
chain and L chain are contained as separate cassettes, each flanked
with the P. pastoris AOX1 promoter and a transcriptional terminator
(TT).
[0067] FIG. 38: Q-ToF Mass spectrometry analysis of an N-glycan
engineered anti-CD70 antibody. Deconvoluted mass spectra of reduced
glycan-engineered anti-CD70 antibodies modified to incorporate two
separate non-native N-glycosylation sites, then isolated and
purified by protein A from clones cultivated in 1 L fermenters. The
incorporated mutations (EU numbering) are noted for each modified
antibody and the expected mass range for H chain with one or two
N-glycans is indicated.
DESCRIPTION OF THE INVENTION
[0068] Patents, patent applications, publications, product
descriptions, and protocols are cited throughout this application,
the disclosures of which are incorporated herein by reference in
their entireties for all purposes. All references cited herein are
incorporated by reference to the same extent as if each individual
publication, database entry (e.g. GenBank sequences or GeneID
entries), patent application, or patent, was specifically and
individually indicated to be incorporated by reference. Citation of
the references herein is not intended as an admission that the
reference is pertinent prior art, nor does it constitute any
admission as to the contents or date of these publications or
documents.
Definitions
[0069] So that the invention may be more readily understood,
certain technical and scientific terms are specifically defined
below. Unless specifically defined elsewhere in this document, all
other technical and scientific terms used herein have the meaning
commonly understood by one of ordinary skill in the art to which
this invention belongs.
[0070] As used herein, including the appended claims, the singular
forms of words such as "a," "an," and "the," include their
corresponding plural references unless the context clearly dictates
otherwise.
[0071] By "position" as used herein is meant a location in the
sequence of a protein. Positions may be numbered sequentially, or
according to an established format, for example the EU index for
antibody numbering or the Kabat numbering.
[0072] Amino acid positions in a heavy chain constant domain
include amino acid positions in the C.sub.H1, hinge, C.sub.H2,
C.sub.H3, Fc, and are numbered according to the EU index numbering
system (also referred as the "EU index of Kabat" or the "EU index
for antibody numbering" or "EU numbering). See Kabat et al.,
"Sequence of proteins of Immunological interest", 5.sup.th edition,
U.S. Dept Health and Human Services, U.S. Gov. Printing Office,
1991.
[0073] Amino acid positions in a variable domain (for example, in
framework4 of the heavy chain) are numbered according to the Kabat
numbering system (Kabat 1991).
[0074] Kabat numbering is based on the seminal work of Kabat et al.
(1991) Sequences of Proteins of Immunological Interest, Publication
No. 91-3242, published as a three volume set by the National
Institutes of Health, National Technical Information Service
(hereinafter "Kabat"). Kabat provides multiple sequence alignments
of immunoglobulin chains from numerous species antibody isotypes.
The aligned sequences are numbered according to a single numbering
system, the Kabat numbering system. The Kabat sequences have been
updated since the 1991 publication and are available as an
electronic sequence database (latest downloadable version 1997).
Any immunoglobulin sequence can be numbered according to Kabat by
performing an alignment with the Kabat reference sequence. Herein
the heavy chain variable sequences are numbered according to the
Kabat reference sequence.
[0075] The term "C.sub.H1" domain as used herein refers to the
first constant domain of an IgG heavy chain that extends from about
amino acid position 118-215 of the EU numbering system.
[0076] The term "hinge region" as used herein refers to the portion
of a heavy chain that attaches the C.sub.H1 domain to the C.sub.H2
domain and comprises about 25 amino acid residues.
[0077] The term "C.sub.H2 domain" as used herein refers to the
portion of a heavy chain IgG constant domain that extends from
about amino acid positions 231-340 of the EU numbering system.
[0078] The term "C.sub.H3 domain" as used herein refers to the
heavy chain IgG constant domain that extends from the N-terminus of
the C.sub.H2 domain from about amino acid positions 341-445 of the
EU numbering system.
[0079] The term "cytotoxic agent" as used herein refers to the
effect of an agent that has a cytotoxic effect on a cell (i.e., an
agent that can cause cell death).
[0080] The term "drug" as used herein refers to a compound
including a compound, a pharmaceutically active compound, element,
agent, pharmaceutically active peptide or protein, or molecular
entity.
[0081] In general, the basic Ab structural unit comprises a
tetramer. Each tetramer includes two identical pairs of polypeptide
chains, each pair having one "light" (about 25 kDa) and one "heavy"
chain (about 50-70 kDa). The amino-terminal portion of each chain
includes a variable region of about 100 to 110 or more amino acids
primarily responsible for antigen recognition. The carboxy-terminal
portion of the heavy chain may define a constant region primarily
responsible for effector function.
[0082] As used herein "fragment" with respect to "antibody" or
"IgG" or "monoclonal antibody" refers to those fragments produced
by digestion with various proteases, those produced by chemical
cleavage and/or chemical dissociation and those produced by
recombination or recombinant DNA technology so long as the fragment
remains capable of specific binding to a target molecule. Examples
of fragments include, but are not limited to, Fc, Fab, Fab',
F(ab').sub.2, Fv, and scFv fragments.
[0083] In an embodiment, the Ab or fragment thereof comprises a
heavy chain constant region, e.g. a human constant region, such as
.gamma.1, .gamma.2, .gamma.3, or .gamma.4 human heavy chain
constant region or a variant thereof. In another embodiment, the Ab
or antigen binding fragment comprises a light chain constant
region, e.g. a human light chain constant region, such as lambda or
kappa human light chain region or variant thereof. By way of
example, and not limitation the human heavy chain constant region
can be .gamma.1 and the human light chain constant region can be
kappa.
[0084] A "Fab fragment" is comprised of one light chain and the
C.sub.H1 and variable regions of one heavy chain. The heavy chain
of a Fab molecule cannot form a disulfide bond with another heavy
chain molecule. A "Fab fragment" can be the product of papain
cleavage of an Ab.
[0085] A "Fab' fragment" contains one light chain and a portion or
fragment of one heavy chain that contains the V.sub.H domain and
the C.sub.H1 domain and also the region between the C.sub.H1 and
C.sub.H2 domains, such that an interchain disulfide bond can be
formed between the two heavy chains of two Fab' fragments to form a
F(ab').sub.2 molecule.
[0086] A "F(ab').sub.2 fragment" contains two light chains and two
heavy chains containing a portion of the constant region between
the C.sub.H1 and C.sub.H2 domains, such that an interchain
disulfide bond is formed between the two heavy chains. A F(ab')2
fragment thus is composed of two Fab' fragments that are held
together by a disulfide bond between the two heavy chains. An
"F(ab').sub.2 fragment" can be the product of pepsin cleavage of an
Ab.
[0087] The term "Fe domain" as used herein refers to the portion of
a heavy chain constant domain contains the hinge region (i.e.,
residue 216 in IgG, taking the first amino acid residue of the
heavy chain constant domain to be 114), and C.sub.H2, and C.sub.H3
domains and ending at the C-terminus of the Ab.
[0088] The "Fv region" comprises the variable regions from both the
heavy and light chains, but lacks the constant regions.
[0089] As used herein, a "chimeric Ab" is an Ab having the variable
domain from a first Ab and the constant domain from a second Ab,
where the first and second Abs are from different species. (U.S.
Pat. No. 4,816,567; and Morrison et al., (1984) Proc. Natl. Acad.
Sci. USA 81: 6851-6855). Typically the variable domains are
obtained from an antibody from an experimental animal (the
"parental Ab"), such as a rodent, and the constant domain sequences
are obtained from human Ab, so that the resulting chimeric Ab will
be less likely to elicit an adverse immune response in a human
subject than the parental (e.g. rodent) antibody.
[0090] Typically, human light chains are classified as kappa and
lambda light chains. Furthermore, human heavy chains are typically
classified as mu, delta, gamma, alpha, or epsilon, and define the
antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
Within light and heavy chains, the variable and constant regions
are joined by a "J" region of about 12 or more amino acids, with
the heavy chain also including a "D" region of about 10 more amino
acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed.,
2nd ed. Raven Press, N.Y. (1989).
[0091] The variable regions of each light/heavy chain pair form the
Ab binding site. Thus, in general, an intact Ab has two binding
sites. Except in bifunctional or bispecific Abs, the two binding
sites are, in general, the same.
[0092] Typically, the variable domains of both the heavy and light
chains comprise three hypervariable regions, also called
complementarity determining regions (CDRs), located within
relatively conserved framework regions (FR). The CDRs are usually
aligned by the framework regions, enabling binding to a specific
epitope. In general, from N-terminal to C-terminal, both light and
heavy chains variable domains comprise FR1, CDR1, FR2, CDR2, FR3,
CDR3 andFR4. The assignment of amino acids to each domain is,
generally, in accordance with the definitions of Sequences of
Proteins of Immunological Interest, Kabat, et al.; National
Institutes of Health, Bethesda, Md.; 5.sup.th ed.; NIH Publ. No.
91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et
al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J
Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature
342:878-883.
[0093] As used herein, the term "IgG" refers to IgG1, IgG2, IgG3
and IgG4. In an embodiment, IgG is IgG1. In one embodiment, the
IgG1 is human IgG1.
[0094] As used herein, the term "hypervariable region" refers to
the amino acid residues of an Ab that are responsible for
antigen-binding. The hypervariable region comprises amino acid
residues from a "complementarity determining region" or "CDR" (i.e.
CDRL1, CDRL2 and CDRL3 in the light chain variable domain and
CDRH1, CDRH2 and CDRH3 in the heavy chain variable domain). See
Kabat et al. (1991); see also Chothia and Lesk (1987) J Mol. Biol.
196: 901-917 (defining the CDR regions of an Ab by structure).
[0095] As used herein, the term "framework" or "FR" residues refers
to those variable domain residues other than the hypervariable
region residues defined herein as CDR residues.
[0096] The phrase "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to use promoters, polyadenylation
signals, and enhancers.
[0097] A nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0098] As used herein, the expressions "cell," "cell line," and
"cell culture" are used interchangeably and all such designations
include progeny. Thus, the words "transformants" and "transformed
cells" include the primary subject cell and cultures derived
therefrom without regard for the number of transfers. It is also
understood that not all progeny will have precisely identical DNA
content, due to deliberate or inadvertent mutations. Mutant progeny
that have the same function or biological activity as screened for
in the originally transformed cell are included. Where distinct
designations are intended, it will be clear from the context.
[0099] "Treat" or "treating" means to administer a therapeutic
agent, such as a composition containing any of the genetically
engineered Abs of the present invention, internally or externally
to a subject or patient having one or more disease symptoms, or
being suspected of having a disease, for which the agent has
therapeutic activity. Typically, the agent is administered in an
amount effective to alleviate one or more disease symptoms in the
treated subject or population, whether by inducing the regression
of or inhibiting the progression of such symptom(s) by any
clinically measurable degree. The term further includes a
postponement of development of the symptoms associated with a
disorder and/or a reduction in the severity of the symptoms of such
disorder. The terms further include ameliorating existing
uncontrolled or unwanted symptoms, preventing additional symptoms,
and ameliorating or preventing the underlying causes of such
symptoms. Thus, the terms denote that a beneficial result has been
conferred on a vertebrate subject with a disorder, disease or
symptom, or with the potential to develop such a disorder, disease
or symptom.
[0100] As used herein, the term "therapeutically effective amount"
or "effective amount" refers to an amount of an engineered Ab drug
conjugate or engineered Ab fragment thereof, that when administered
alone or in combination with an additional therapeutic agent to a
cell, tissue, or subject is effective to prevent or ameliorate the
disease or condition to be treated. A therapeutically effective
dose further refers to that amount of the compound sufficient to
result in amelioration of symptoms, e.g., treatment, healing,
prevention or amelioration of the relevant medical condition, or an
increase in rate of treatment, healing, prevention or amelioration
of such conditions. When applied to an individual active ingredient
administered alone, a therapeutically effective dose refers to that
ingredient alone. When applied to a combination, a therapeutically
effective dose refers to combined amounts of the active ingredients
that result in the therapeutic effect, whether administered in
combination, serially or simultaneously.
[0101] As used herein, the terms "N-glycan" refers to an N-linked
oligosaccharide, for example, one that is attached by an
asparagine-N-acetylglucosamine linkage to an asparagine residue of
a polypeptide. N-linked glycoproteins contain an
N-acetylglucosamine residue linked to the amide nitrogen of an
asparagine residue in the protein. The predominant sugars found on
glycoproteins are glucose, galactose, mannose, fucose,
N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and
sialic acid (e.g., N-acetyl-neuraminic acid (NANA)). The processing
of the sugar groups occurs co-translationally in the lumen of the
ER and continues post-translationally in the Golgi apparatus for
N-linked glycoproteins.
[0102] Typically, N-glycans have a common pentasaccharide core of
Man.sub.3GlcNAc.sub.2 ("Man" refers to mannose; "Glc" refers to
glucose; and "NAc" refers to N-acetyl; GlcNAc refers to
N-acetylglucosamine). Usually, N-glycan structures are presented
with the non-reducing end to the left and the reducing end to the
right. The reducing end of the N-glycan is the end that is attached
to the Asn residue comprising the glycosylation site on the
protein. N-glycans differ with respect to the number of branches
(antennae) comprising peripheral sugars (e.g., GlcNAc, galactose,
fucose and sialic acid) that are added to the
Man..sub.3GlcNAc.sub.2 ("Man3") core structure which is also
referred to as the "triammnose core", the "pentasaccharide core" or
the "paucimannose core". N-glycans are classified according to
their branched constituents (e.g., high mannose, complex or
hybrid). A "high mannose" type N-glycan has five or more mannose
residues. A "complex" type N-glycan typically has at least one
GlcNAc attached to the 1,3 mannose arm and at least one GlcNAc
attached to the 1,6 mannose arm of a "trimannose" core.
ComplexN-glycans may also have galactose ("Gal") or
N-acetylgalactosamine ("GalNAc") residues that are optionally
modified with sialic acid or derivatives (e.g., "NANA" or "NeuAc",
where "Neu" refers to neuraminic acid and "Ac" refers to acetyl).
ComplexN-glycans may also have intrachain substitutions comprising
bisecting GlcNAc and core fucose ("Fuc"). Complex N-glycans may
also have multiple antennae on the "trimannose core," often
referred to as "multiple antennary glycans." A "hybrid" N-glycan
has at least one GlcNAc on the terminal of the 1.3 mannose arm of
the trimannose core and zero or more mannoses on the 1,6 mannose
arm of the trimannose core. The various N-glycans are also referred
to as "glycoforms."
[0103] With respect to complex N-glycans, the terms "G-2", "G-1",
"G0", "G1", "G2", "A1", and "A2" mean the following. "G-2" refers
to an N-glycan structure that can be characterized as
Man.sub.3GlcNAc.sub.2; the term "G-1" refers to an N-glycan
structure that can be characterized as GlcNAcMan.sub.3GlcNAc.sub.2;
the term "G0" refers to an N-glycan structure that can be
characterized as GlcNAc.sub.2Man.sub.3GlcNAc.sub.2; the term "G1"
refers to an N-glycan structure that can be characterized as
GalGlcNAc.sub.2Man.sub.3GlcNAc.sub.2; the term "G2" refers to an
N-glycan structure that can be characterized as
Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2; the term "A1" refers to
an N-glycan structure that can be characterized as
NANAGal.sub.2Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2; and, the
term "A2" refers to an N-glycan structure that can be characterized
as NANA.sub.2Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2. Unless
otherwise indicated, the terms G-2", "G-1", "G0", "G1", "G2", "A1",
and "A2" refer to N-glycan species that lack fucose attached to the
GlcNAc residue at the reducing end of the N-glycan.
[0104] With respect to multiantennary N-glycans, the term
"multiantennary N-glycan" refers to N-glycans that further comprise
a GlcNAc residue on the mannose residue comprising the non-reducing
end of the 1,6 arm or the 1,3 arm of the N-glycan or a GlcNAc
residue on each of the mannose residues comprising the non-reducing
end of the 1,6 arm and the 1,3 arm of the N-glycan. Thus,
multiantennary N-glycans can be characterized by the formulas
GlcNAc.sub.(2-4)Man.sub.3GlcNAc.sub.2,
Gal.sub.(1-4)GlcNAc.sub.(2-4)Man.sub.3GlcNAc.sub.2, or
NANA.sub.(1-4)Gal.sub.(1-4)GlcNAc.sub.(2-4)Man.sub.3GlcNAc.sub.2.
The term "1-4" refers to 1, 2, 3, or 4 residues.
[0105] The term "GS3.5", when used herein refers to the
N-glycosylation structure GalGlcNAcMan.sub.5GlcNAc.sub.2.
[0106] The term "GS4.0", when used herein refers to the
N-glycosylation structure GlcNAc.sub.2Man.sub.3GlcNAc.sub.2.
[0107] The term "GS5.0", when used herein refers to the
N-glycosylation structure
Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2.
[0108] The term "GS6.0", when used herein refers to the
N-glycosylation structure
NANA.sub.2Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2.
[0109] The term "non-native N-glycosylation site" as used herein
refers to any consensus (N-X-S/T, wherein X is any amino acid
except proline) N-glycosylation site incorporated by mutation into
an IgG that is not observed on naturally occurring IgG molecules
(e.g. N-297). Moreover, while Asn-297 is the N-glycosylation site
typically found in murine and human IgG molecules (Kabat et al,
Sequences of Proteins of Immunological Interest, 1991), this site
doesn't necessarily have to be maintained for function. Using known
methods for mutagenesis, the skilled artisan can alter a DNA
molecule encoding an Ig of the present invention so that the
N-glycosylation site at Asn-297 is deleted.
[0110] As used herein, the term "predominantly" or variations such
as "the predominant" or "which is predominant" will be understood
to mean the glycan species or collective species that has the
highest mole percent (%) of total N-glycans after the glycoprotein
has been analyzed by mass spectrometry (e.g. Q-ToF) or
enzymatically released N-glycans analyzed by mass spectroscopy
(e.g. MALDI-TOF MS) or HPLC. For example, if a composition consists
of species A in 40 mole percent, species B in 35 mole percent and
species C in 25 mole percent, the composition comprises
predominantly species A, and species B would be the next most
predominant species. Furthermore, if a composition contains a
mixture of species where 60% contain terminal galactose and 40%
contain terminal sialic acid the composition will be defined has
having predominantly terminal galactose.
[0111] As used herein, a glycoprotein composition "lacks" or "is
lacking" a particular sugar residue, such as fucose or galactose,
when no detectable amount of such sugar residue is present on the
N-glycan structures at any time. For example, in preferred
embodiments of the present invention, the glycoprotein compositions
are produced by lower eukaryotic organisms, as defined above,
including yeast and fungi [e.g., Pichia sp.; Saccharomyces sp.;
Kluyveromyces sp.; Aspergillus sp.], and will "lack fucose,"
because the cells of these organisms do not have the enzymes needed
to produce fucosylated N-glycan structures. Thus, the term
"essentially free of fucose" encompasses the term "lacking fucose."
However, a composition may be "essentially free of fucose" even if
the composition at one time contained fucosylated N-glycan
structures or contains limited, but detectable amounts of
fucosylated N-glycan structures.
[0112] The term "mole percent" of a glycan present in a preparation
of a N-glycosylated Ab refers to the molar percent of a particular
glycan present in the pool of N-linked oligosaccharides released
when the N-glycosylated Ab preparation is treated with PNGase and
then quantified by a method that is not affected by glycoform
composition, for instance, labeling a PNGase released glycan pool
with a fluorescent tag such as 2-aminobenzamide and then separating
by high performance liquid chromatography or capillary
electrophoresis and then quantifying glycans by fluorescence
intensity, or MALDI-TOF mass spectrometry. For example, 50 mole
percent NANA.sub.2Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 means
that 50 percent of the released glycans are
NANA.sub.2Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 and the
remaining 50 percent are comprised of other N-linked
oligosaccharides. In embodiments, the mole percent of a particular
glycan in a preparation of glycoprotein will be between 20% and
100%, preferably above 25%, 30%, 35%, 40% or 45%, more preferably
above 50%, 55%, 60%, 65% or 70% and most preferably above 75%, 80%
85%, 90% or 95%.
Overview
[0113] The present invention provides engineered Abs, in particular
IgG Abs or fragments thereof comprising one to ten mutations (or
pairs of mutations) in the heavy chain constant domain which
generate one to ten non-native N-glycosylation sites. As shown in
the Examples (see Example 1 and Example 14), the non-native
N-glycosylation sites provide specific targeting sites for drug
conjugates. In one embodiment, the engineered Abs, or fragments
thereof of the invention are expressed in yeast and filamentous
fungi host cells that have been engineered to produce human like
N-glycans of the structure:
Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2,
Gal.sub.(1-2)GlcNAc.sub.(1-2)Man.sub.5GlcNAc.sub.2 or
NANA.sub.(1-4)Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2.
In some embodiments, engineered Abs or fragments thereof expressed
in these engineered yeast and filamentous fungi host cells comprise
N-glycans where the predominant glycoform comprise the following
structures: Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2 or
Gal.sub.(1-2)GlcNAc.sub.(1-2)Man.sub.5GlcNAc.sub.2. In some
embodiments, engineered Abs or fragments thereof expressed in
glycoengineered yeast and filamentous fungi host cells comprise
N-glycans in the non-native N-glycosylation sites comprising
predominantly terminal sialylated residues
(NANA.sub.(1-4)Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.-
2), which in turn, can be converted into terminal galactose
residues by an enzymatic reaction.
[0114] In some embodiments, the engineered Abs comprise N-glycans
at the non-native N-glycosylation sites which do not disrupt the
normal folding or function of the mAb, and allow the efficient,
uniform conjugation of drugs such as toxins, peptides or bioactive
sugar moieties. The DAR of the conjugated Ab can be modulated
through the number of non-native N-glycosylation sites engineered
into the Ab at specific sites as specified by the invention
examples and the number of terminal galactose residues, which is
driven by the N-glycan machinery of the recombinant host
strain.
[0115] As background, the N-glycans are important molecules in
biology that participate in a wide-range of different activities,
from protein folding and trafficking to immune cell and
host-pathogen interactions, owing in large part to the
heterogeneity available from the modular, non-template nature of
this protein modification, leading to an impressive breadth of
potential forms that can be produced from a single core structure.
N-glycans are also a key factor in biotherapeutic discovery and
manufacture and limit the choice of expression system in many cases
(Hossler, 2009; Schirrmann, 2008; Sethuraman, 2006). Examples of
the differences in N-glycan pathways between humans and yeast can
be found in FIG. 1. A unique biotherapeutic platform has been
developed in yeast, in particular, in the species Pichia pastoris,
in which the N-glycosylation pathway has been modified to generate
human-like N-glycans (See, e.g., Bobrowicz et al., 2004;
Sethuraman, 2006; Hamilton, 2006; Li et al., 2006; Nett et al.,
2010; Choi et al., 2009; U.S. Pat. Nos. 7,029,872 and 7,449,308).
Glycoengineered Pichia allows for unprecedented uniformity,
specificity and control of N-glycans on recombinantly produced
proteins, including Abs. A summary of glycoengineered Pichia can be
found in FIG. 2. The unique nature of this genetically engineered
system and process versatility allows for a tremendous level of
freedom over what sugars can be attached and in what arrangements,
ultimately allowing for modifications both prior to and after
addition to the growing N-glycan chain.
[0116] Directed and specific control of N-glycosylation can provide
unique control over biology (e.g. modulation of inflammation,
tissue and cell type targeting) but can also provide an opportunity
for creating unique macromolecule chemistry, particularly in the
case of the terminal sugars galactose and sialic acid (Ramya et al,
2013). Galactose oxidase (GO) is an enzyme that can specifically
modify terminal galactose sugars present on an N-glycan, thus
allowing for highly versatile and specific aldehyde-based chemistry
that is not otherwise available in the 20 amino acid repetoire.
However, the gly can heterogeneity that is confronted with
conventional expression systems such as mammalian cells could
complicate such an approach. For example, as demonstrated for human
Erythropoietin (Nett et al, 2010; Restelli et al, 2006), proteins
with exposed N-glycans can have huge variability in numbers of
antennae (bi-, tri-, and tetra-) and amount of terminal sialic
acid, whereas antibodies can contain differing amounts of fucose
and galactose in addition to trace amounts of sialic acid (Li et
al, 2006), all of which contributes to the considerable
heterogeneity of mammalian N-glycans.
[0117] In one embodiment, the present invention provides an
engineered IgG Ab or fragment thereof comprising one to ten
mutations (or pairs of mutations) in the heavy chain constant
domain, which generate one to ten non-native N-glycosylation sites,
the mutations being selected from the group consisting of S134N,
G161T, G161S, N203T, N203S, V363T, V363S, Q438N, S176N, A118N,
S132N, K133N, A162N, T195N, K210T, Y391T, F423T, F423S, Y436T,
Y436S, L193N, K392T, K392S, F423T, S176N/G178T, S176N/G178S,
Q419N/N421T, Q419N/N421S, S191N/L193T, S191N/L193S, G194N/Q196T,
and G194N/Q196S, according to EU numbering.
[0118] In one embodiment, the engineered IgG Ab or fragment thereof
comprises at least one or two amino acid mutations in the heavy
chain constant polypeptide selected from S134N, G161T and
S134N/G161T.
[0119] In another embodiment, the engineered IgG Ab or fragment
thereof comprises at least two amino acid mutations in the heavy
chain constant domain selected from G161T/S134T and G161
S/S134T.
[0120] In one embodiment, the engineered heavy chain constant
domains comprise, at the non-native N-glycosylated sites,
predominantly complex N-glycans having the structure
Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2. In one
embodiment, engineered heavy chain constant domains comprise, at
the non-native N-glycosylation sites, predominantly complex N-gly
cans having the structure
Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 (also referred to as
GS5.0 N-glycans in FIG. 2 and Example 3).
[0121] In another embodiment, the engineered heavy chain constant
domains comprise, atthe non-native N-glycosylated sites,
predominantly hybrid N-glycans having the structure
Gal.sub.(1-2)GlcNAc.sub.(1-2)Man.sub.5GlcNAc.sub.2.
[0122] In another embodiment, the engineered heavy chain constant
domains comprise, at the non-native N-glycosylated sites,
predominantly N-glycans having the structure
NANA.sub.(1-4)Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2.
[0123] In another embodiment, the engineered IgG Abs or fragments
thereof having non-native N-glycosylation sites comprise N-glycans
wherein about 50 to about 100 mole % of the N-glycans comprise the
structure:
Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2Gal.sub.(1-2)GlcNAc.sub.(1-2)Ma-
n.sub.5GlcNAc.sub.2 or
NANA.sub.(1-4)Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2.
In another embodiment, the engineered IgG Abs or fragments thereof
having non-native N-glycosylation sites comprise N-glycans where
about 80 to about 100 mole % of the N-glycans comprise the
structure: Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2,
Gal.sub.(1-2)GlcNAc.sub.(1-2)Man.sub.5GlcNAc.sub.2 or
NANA.sub.(1-4)Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2.
[0124] In one embodiment, the engineered Abs or fragments thereof
are expressed in host cells capable of producing a composition of
Abs or fragments thereof comprising N-glycans where the predominant
glycoform comprise a Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2
N-glycan structure lacking fucose, wherein said structure is
present at a level that is at least about 5 mole percent more than
the next predominant N-glycan structure in the composition. In one
embodiment, the engineered IgG Ab or fragments thereof comprise a
predominant Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2N-glycan
structure lacking fucose, wherein said structure is present at a
level of at least about 10 mole percent to about 25 mole percent
more than the next predominantN-glycan structure in the
composition. In one embodiment, the engineered IgG Abs or fragments
thereof comprise a predominant
Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 N-glycan structure
lacking fucose, wherein said structure is present at a level that
is at least about 25 mole percent to about 50 mole percent more
than the next predominant N-glycan structure in the composition. In
one embodiment, the engineered IgG Abs or fragments thereof
comprise a predominant Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2
glycan structure lacking fucose, wherein said structure is present
at a level that is greater than about 50 mole percent more than the
next predominant glycan structure in the composition. In one
embodiment, the engineered IgG Abs or fragments thereof comprise a
predominant Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 N-glycan
structure lacking fucose, wherein said structure is present at a
level that is greater than about 75 mole percent more than the next
predominant glycan structure in the composition. In still another
embodiment, the engineered IgG Abs or fragments thereof comprise a
predominant Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 N-glycan
structure lacking fucose wherein said structure is present at a
level that is greater than about 90 mole percent more than the next
predominant glycan structure in the composition. MALDI-TOF analysis
of N-glycans of an IgG having a predominant (greater than 95 mole
%) Gal2GlcNAc2Man3GlcNAc2 lacking fucose is shown in FIG. 26.
[0125] In another embodiment, the aforementioned IgG Ab or fragment
thereof used for genetic engineering in its heavy chain constant
domain is selected from the group consisting of anti-Her2,
anti-Her2/neu (Herceptin), anti-glycoprotein IIb/IIIa (Abciximab),
anti-TNF-.alpha. (Adalimumab, Certolizumab pegol, Golimumab,
Infliximab), anti-CD52 (Alemtuzumab), anti-IL-2R.alpha. (CD25)
(Basiliximab), anti-BAFF (Belimumab), anti-Vascular endothelial
growth factor (VEGF) (Bevacizumab), anti-CD30 (Brentuximab
vedotin), anti-IL-1.beta. (Canakinumab), anti-epidermal growth
factor receptor (EGFR) (Cetuximab), anti-IL-2R.alpha. receptor
(CD25) (Daclizumab), anti-RANK Ligand (Denosumab), anti-Complement
C5 (Eculizumab), anti-CD11a (Efalizumab), anti-CD33 (Gemtuzumab),
anti-CD20 (Ibritumomab tiuxetan), anti-CTLA-4 (Ipilimumab
(MDX-101)), anti-T cell CD3 Receptor (Muromonab-CD3), anti-alpha-4
(.alpha.4) integrin, anti-(Natalizumab), anti-CD20 (Ofatumumab),
anti-Immunoglobulin E (IgE) (Omalizumab), anti-RSV F protein
(Palivizumab), anti-epidermal growth factor receptor (Panitumumab),
anti-VEGF-A (Ranibizumab), anti-CD20 (Rituximab), anti-Anti-IL-6R
(Tocilizumab, Atlizumab), anti-CD20 (Tositumomab), anti-ErbB2
(Trastuzumab), anti-IL-12/IL-23 (Ustekinumab), anti-integrin
.alpha.4.beta.7 (Vedolizumab), anti-CD274, anti-.beta.-amyloid,
anti-4-1BB, anti-5AC, anti-5T4, anti-ACVR2B,
anti-adenocarcinomaantigen, anti-AGS-22M6, anti-alpha-fetoprotein,
anti-angiopoietin 2, anti-angiopoietin 3, anti-anthrax toxin,
anti-AOC3 (VAP-1), anti-, anti-B7-H3, anti-Bacillus anthracis,
anti-BAFF, anti-beta amyloid, anti-B-lymphoma cell, anti-C242
antigen, anti-C5, anti-CA-125, anti-carbonic anhydrase 9 (CA-IX),
anti-cardiac myosin, anti-CCL11 (eotaxin-1), anti-CCR4, anti-CCR5,
anti-CD11/CD18, anti-CD125, anti-CD140a, anti-CD147 (basigin),
anti-CD15, anti-CD152, anti-CD154 (CD40L), anti-CD19, anti-CD2,
anti-CD20, anti-CD200, anti-CD22, anti-CD221, anti-CD23, anti-CD25,
anti-CD27, anti-CD28, anti-CD28, anti-CD3, anti-CD3 epsilon,
anti-CD30 (TNFRSF8), anti-CD33, anti-CD37, anti-CD38, anti-CD4,
anti-CD40, anti-CD41, anti-CD44, anti-CD5, anti-CD51, anti-CD52,
anti-CD56, anti-CD6, anti-CD70, anti-CD74, anti-CD79B, anti-CD80,
anti-CEA, anti-CFD, anti-ch4D5, anti-CLDN18.2, anti-C. difficile,
anti-clumping factor A, anti-CSF2, anti-CTLA-4,
anti-cytomegalovirus, anti-CMV gp B, anti-DLL4, anti-DR5, anti-E.
coli shiga toxin type-1 or 2, anti-EGFL7, anti-EGFR,
anti-endotoxin, anti-EpCAM, anti-EpCAM/CD3, anti-episialin,
anti-ERBB3, anti-Escherichia coli, anti-F protein RSV, anti-FAP,
anti-fibrin II, beta chain, anti-fibronectin extra domain-B,
anti-folate receptor 1, anti-Frizzled receptor, anti-ganglioside
GD2, anti-GD2, anti-GD3 ganglioside, anti-GD3 ganglioside,
anti-GMCSF receptor .alpha.-chain, anti-GPNMB, anti-Influenza,
anti-Influenza hemagglutinin, anti-hepatitis B, anti-hepatitis B,
anti-surface antigen, HER1, anti-HER2/neu, anti-HER2, CD3,
anti-HER3, anti-HGF, anti-HGF, anti-HHGFR, anti-HIV-1, anti-HLA-DR,
anti-HNGF, anti-Hsp90, anti-human scatter factor receptor kinase,
anti-human TNF, anti-human beta-amyloid, anti-ICAM-1 (CD54),
anti-IFN-.alpha., anti-IFN-.gamma., anti-IgE, anti-IgE Fc region,
anti-IGF-1 receptor, anti-IGF-I, anti-IgG4, anti-IGHE, anti-IL20,
anti-IL-1beta, anti-IL-12/IL-23, anti-IL-13, anti-IL-17,
anti-IL-17A, anti-IL-1.beta., anti-IL-22, anti-IL-23, anti-IL-4,
anti-IL-5, anti-IL-6, anti-IL-6 receptor, anti-IL9, anti-ILGF2,
anti-insulin-like growth factor I receptor (IGF-1R), anti-integrin
.alpha.4.beta.7, anti-integrin .alpha.4, anti-integrin
.alpha.4.beta.7, anti-integrin .alpha.5.beta.1, anti-integrin
.alpha.7.beta.7, anti-integrin .alpha.IIb.beta.3, anti-integrin
.alpha.v.beta., anti-interferon receptor, anti-interferon
.alpha./.beta. receptor, anti-interferon gamma-induced protein,
anti-ITGA2, anti-ITGB2 (CD18), anti-KIR2D, anti-Lewis-Y antigen,
anti-LFA-1 (CD11a), anti-lipoteichoic acid, anti-LOXL2,
anti-L-selectin (CD62L), anti-LTA, anti-MCP-1, anti-mesothelin,
anti-MS4A1, anti-MUC1, anti-mucin CanAg, anti-myostatin,
anti-NARP-1, anti-NCA-90, anti-NGF, anti-N-glycolylneuraminic acid
(NGNA), anti-NOGO-A, anti-Notch receptor, anti-NRP1,
anti-Oryctolagus cuniculus, anti-OX-40, anti-oxLDL, anti-PCSK9,
anti-PD-1, anti-PDCD1, anti-PDCD1, anti-PDGF-R .alpha.,
anti-phosphate-sodium co-transporter, anti-phosphatidylserine,
anti-prostatic carcinoma cells, anti-Pseudomonas aeruginosa,
anti-rabies virus, anti-rabies virus glycoprotein, anti-RANKL,
anti-respiratory syncytial virus, anti-RHD, anti-Rhesus factor,
anti-RON, anti-RTN4, anti-sclerostin, anti-SDC1, anti-selectin P,
anti-SLAMF7, anti-SOST, anti-sphingosine-1-phosphate, anti-TAG-72,
anti-T-cell receptor, anti-TEM1, anti-tenascin C, anti-TFPI,
anti-TGF beta 1, anti-TGF beta 2, anti-TGF-0, anti-TNF-.alpha.,
anti-TRAIL-R1, anti-TRAIL-R2, anti-tumor antigen CTAA16.88,
anti-MUC1 (tumor-specific glycan), anti-TWEAK receptor, anti-TYRP1
(glycoprotein 75), anti-VEGF-A, anti-VEGFR-1, anti-VEGFR2,
anti-vimentin, anti-VWF, anti-IL-1, anti-IL-2, anti-IL-4,
anti-IL-5, anti-IL-6, anti-IL-8, anti-IL-9, anti-IL-10, anti-IL-12,
anti-IL-15, anti-IL-17, anti-IL-18, anti-IL-20, anti-IL-21,
anti-IL-22, anti-IL-23, anti-IL-23R, anti-IL-25, anti-IL-27,
anti-IL-33, anti-CD2, anti-CD4, anti-CD 11A, anti-CD14, anti-CD18,
anti-CD19, anti-CD23, anti-CD25, anti-CD40, anti-CD40L, anti-CD20,
anti-CD52, anti-CD64, anti-CD80, anti-CD147, anti-CD200,
anti-CD200R, anti-TSLP, anti-TSLPR, anti-PD-1, anti-PDL1,
anti-CTLA4, anti-VLA-4, anti-VEGF, anti-PCSK9,
anti-.alpha.4.beta.7-integrin, anti-E-selectin, anti-Fact II,
anti-ICAM-3, anti-beta2-integrin, anti-IFN.gamma., anti-C5,
anti-CBL, anti-LCAT, anti-CR3, anti-MDL-1, anti-GITR, anti-CGRP,
anti-TRKA, anti-IGF1R, anti-GTC.
[0126] In one embodiment, the engineered IgG Ab or fragment thereof
is human anti-Her2 Ab or a fragment thereof. In another embodiment,
the engineered IgG Ab is human anti-PD-1 Ab or a fragment
thereof.
[0127] In another embodiment, the engineered IgG Ab or fragment
thereof is an anti-Her2 Ab comprising a heavy chain mutant
polypeptide comprising an amino acid sequence selected from SEQ ID
NOs: 4-29, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40.
[0128] In another embodiment, the engineered IgG Ab or fragment
thereof is an anti-mouse-PD-1 Ab comprising a heavy chain mutant
polypeptide comprising an amino acid sequence selected from SEQ ID
NOs: 41-45.
[0129] In another embodiment, the engineered IgG Ab or fragment
thereof is an anti-CS1 Ab comprising a heavy chain mutant
polypeptide comprising an amino acid sequence selected from SEQ ID
NOs: 46-50.
[0130] In another embodiment, the engineered IgG Ab or fragment
thereof is an anti-CD70 Ab comprising a heavy chain mutant
polypeptide comprising an amino acid sequence selected from SEQ ID
NOs: 51-55.
[0131] In another embodiment, the present invention provides an
engineered IgG Ab or fragment thereof comprising one to two
mutations in the heavy chain framework domain which generate one to
two non-native N-glycosylation sites, the mutations being selected
from the group consisting of Q105N and S113N, according to Kabat
numbering.
[0132] In one embodiment, the engineered heavy chain constant
domain comprises, at the non-native N-glycosylated sites,
predominantly complex N-glycans having the structure
Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2. In one
embodiment, engineered heavy chain constant domain comprise, at the
non-native N-glycosylation sites, predominantly complex N-gly cans
having the structure Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2
(also referred to as GS5.0 N-glycans in FIG. 2 and Example 3).
[0133] In another embodiment, the engineered heavy chain constant
domain comprises, at the non-native N-glycosylated sites,
predominantly hybrid N-glycans having the structure
Gal.sub.(1-2)GlcNAc.sub.(1-2)Man.sub.5GlcNAc.sub.2.
[0134] In another embodiment, the engineered heavy chain constant
domain comprises, at the non-native N-glycosylated sites,
predominantly N-glycans having the structure
NANA.sub.(1-4)Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2
[0135] In another embodiment, the engineered IgG Ab having
non-native N-glycosylation sites comprises N-glycans wherein about
50 to about 100 mole % of the N-gly cans comprise the structure:
Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2,
Gal.sub.(1-2)GlcNAc.sub.1-2Man.sub.5GlcNAc.sub.2 or
NANA.sub.(1-4)Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2.
In another embodiment, the engineered Ab having non-native
N-glycosylation sites comprises N-glycans where about 80 to about
100 mole % of the N-glycans comprise the structure:
Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2,
Gal.sub.(1-2)GlcNAc.sub.1-2Man.sub.5GlcNAc.sub.2 or
NANA.sub.(1-4)Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2.
[0136] In one embodiment, the aforementioned IgG Ab or fragment
thereof used for genetic engineering in its heavy chain framework
domain is selected from the Abs as described above.
Ab-Drug Conjugates
[0137] In another embodiment, the engineered IgG Ab or fragment
thereof is conjugated to a drug via the Ab's non-native
N-glycosylated site. The drug is selected from the group consisting
of a polymer, cytotoxic agent, a radionuclide, fluorescent or
chemiluminescent labels, steroid, steroid receptor agonist, signal
transduction inhibitor, a peptide and scFv.
[0138] In one embodiment, the drug is a polymer which increases the
half-life of the engineered Ab or fragment thereof in the body of a
subject. Suitable polymers include, but are not limited to,
hydrophilic polymers which include but are not limited to
polyethylene glycol (PEG) (e.g., PEG with a molecular weight of 2
kDa, 5 kDa, 10 kDa, 12 kDa, 20 kDa, 30 kDa or 40 kDa), dextran and
monomethoxypolyethylene glycol (mPEG). Methods for pegylating
proteins are known in the art and can be applied to the Abs of the
invention. See, e.g., EP 0 154 316 and EP 0 401 384. Lee, et al.,
(1999) (Bioconj. Chem. 10:973-981) discloses PEG conjugated
single-chain Abs. Wen, et al., (2001) (Bioconj. Chem. 12:545-553)
disclose conjugating antibodies with PEG which is attached to a
radiometal chelator (diethylenetriaminpentaacetic acid (DTPA)). For
example, to pegylate an Ab, the Ab, or fragment thereof, typically
is reacted with a reactive form of polyethylene glycol (PEG), such
as a reactive ester or aldehyde derivative of PEG, under conditions
in which one or more PEG groups become attached to the antibody or
antibody fragment. In particular embodiments, the pegylation is
carried out via an acylation reaction or an alkylation reaction
with a reactive PEG molecule (or an analogous reactive
water-soluble polymer). As used herein, the term "polyethylene
glycol" is intended to encompass any of the forms of PEG that have
been used to derivatize other proteins, such as mono (C1-C10)
alkoxy- or aryloxy-polyethylene glycol or polyethylene
glycol-maleimide.
[0139] The engineered IgG Ab or fragments thereof may also be
conjugated to a cytotoxic agent such as diptheria toxin,
Pseudomonas aeruginosa exotoxin A chain, ricin A chain, abrin A
chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins
and compounds (e.g., fatty acids), dianthin proteins, Phytoiacca
americana proteins PAPI, PAPII, and PAP-S, Momordica charantia
inhibitor, curcin, crotin, Saponaria officinalis inhibitor,
mitogellin, restrictocin, phenomycin, and enomycin.
[0140] The engineered IgG Ab and fragments thereof may also be
conjugated with labels such as .sup.99Tc, .sup.90Y, .sup.111In,
.sup.32P, .sup.14C, .sup.125I, .sup.3H, .sup.131I, .sup.11C,
.sup.15O, .sup.13N, .sup.18F, .sup.35S, .sup.51Cr, .sup.57To,
.sup.226Ra, .sup.60Co, .sup.59Fe, .sup.57Se, .sup.152Eu, .sup.67CU,
.sup.217Ci, .sup.211At, .sup.212Pb, .sup.47Sc, .sup.109Pd,
.sup.234Th, and .sup.40K, .sup.157Gd, .sup.55Mn, .sup.52Tr, and
.sup.56Fe.
[0141] The engineered IgG Ab or fragment thereof may also be
conjugated with fluorescent or chemilluminescent labels, including
fluorophores such as rare earth chelates, fluorescein and its
derivatives, rhodamine and its derivatives, isothiocyanate,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaladehyde,
fluorescamine, .sup.152Eu, dansyl, umbelliferone, luciferin,
luminal label, isoluminal label, an aromatic acridinium ester
label, an imidazole label, an acridimium salt label, an oxalate
ester label, an aequorin label, 2,3-dihydrophthalazinediones,
biotin/avidin, spin labels and stable free radicals.
[0142] The engineered IgG Ab or fragment thereof may also be
conjugated to a steroid such as glucocorticoid.
[0143] The engineered IgG Ab or fragment thereof may also be
conjugated to a steroid receptor agonist such as a glucocorticoid
receptor agonist (e.g., mapracorat).
[0144] The engineered IgGAb or fragment thereof may also be
conjugated to a signal transduction inhibitor such as dasatinib
(see Wang et al., 2015).
[0145] The engineered IgG Ab or fragment thereof may also be
conjugated to a peptide, e.g., activated GLP-1 receptor agonistic
peptide (see Example 9)
[0146] The engineered IgG Ab or fragment thereof may also be
conjugated to a scFv.
[0147] Any method known in the art for conjugating the Ab molecules
to the various moieties may be employed (see e.g., Axup, 2012;
Tian, 2014; Jackson, 2014, WO2005047334, WO2005047336, WO200547337
and WO2006107124 (the disclosures of which are incorporated herein
by reference) disclose chemically conjugating peptides or drug
molecules to Fc fragment. Methods for conjugating Abs are
conventional and very well known in the art.
[0148] ADC delivery of a drug moiety to its intracellular target
occurs via a multistep sequence of events: binding to the cell
surface, endocytosis, trafficking (within an endosome) to a
lysosome, proteolytic degradation of the conjugate, and diffusion
of the released drug moiety across the lysosomal or endosomal
membrane toward its intracellular target and its interaction with
the target. Therefore, the linker should be sufficiently stable
while in circulation to allow delivery of the intact ADC to the
target cell but, on the other hand, sufficiently labile to allow
release of the drug moiety from the ADC once inside the targeted
cell.
[0149] In an embodiment as described below in the method of
preparing an engineered Ab-drug conjugate, the linker is comprised
of an oxime linkage. In this regard, the terminal galactose
residues of the human complex N-glycan or human hybrid N-glycan are
specifically oxidized to produce chemically-reactive aldehyde
groups utilizing an enzyme known as galactose oxidase (GO) as
described e.g., in Cooper et al., 1959. The chemically reactive
aldehyde group is receptive to direct conjugation with an
alkoxyamine substrate forming a stable oxime bond as described e.g,
in Ramya et al. 2013, and as described below and in Example 5.
Uses of Ab-Drug Conjugates
Immunoimaging
[0150] In another embodiment, the engineered IgG Ab-drug conjugates
of the present invention can be used for in vivo immunoimaging. For
this purpose, the Ab or fragment thereof is labeled by means which
permit external visualization of its position or location within a
subject or part thereof, such as an organ. Typically, an
immunoimaging agent will be an Ab labeled directly (as with
Technetium) or indirectly (as with chelated Indium) with a suitable
radioisotope. After injection into the patient, the location of the
conjugate may be tracked by a detector sensitive to particles
emitted by the radiolabel, e.g., a gamma-scintillation camera in
the case of a gamma emitter.
Immunotherapy
[0151] In another embodiment, the engineered IgG Ab-drug conjugate
of the present invention can be used to treat cancer or a disease,
such as an autoimmune disease or an infectious disease, in a
patient, such as a human or an animal (e.g., a dog or a cat)
suffering from the cancer or the disease. Accordingly, methods of
treating a disease or cancer in a patient suffering from the cancer
or disease are provided, the methods comprising administering to
the patient a therapeutically effective amount of the engineered
IgG Ab or fragment thereof, which is conjugated to a drug.
[0152] With respect to cancer, the engineered Ab-drug conjugates
can be used for inhibiting the multiplication of a tumor cell or
cancer cell, causing apoptosis in a tumor or cancer cell, or for
treating cancer in a patient by delivering a drug to a tumor cell
or cancer cell.
[0153] The specificity of the Ab for a particular tumor cell or
cancer cell can be important for determining those tumors or
cancers that are most effectively treated. For example, the
anti-HER2 mAb trastuzumab is known to be useful in treating HER+
tumors such as breast cancer and brain cancer.
[0154] Examples of cancers that can be treated with the engineered
Ab-drug conjugates include, but are not limited to, solid tumors,
including but not limited to: fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,
angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal
cancer, kidney cancer, pancreatic cancer, bone cancer, breast
cancer, ovarian cancer, prostate cancer, esophogeal cancer, stomach
cancer, oral cancer, nasal cancer, throat cancer, squamous cell
carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland
carcinoma, sebaceous gland carcinoma, papillary carcinoma,
papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms'
tumor, cervical cancer, uterine cancer, testicular cancer, small
cell lung carcinoma, bladder carcinoma, lung cancer, epithelial
carcinoma, glioma, glioblastoma multiforme, astrocytoma,
medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,
skin cancer, melanoma, neuroblastoma, retinoblastoma, blood-borne
cancers (including but not limited to: acute lymphoblastic leukemia
"ALL", acute lymphoblastic B-cell leukemia, acute lymphoblastic
T-cell leukemia, acute myeloblastic leukemia "AML", acute
promyelocytic leukemia "APL", acute monoblastic leukemia, acute
erythroleukemic leukemia, acute megakaryoblastic leukemia, acute
myelomonocytic leukemia, acute nonlymphocyctic leukemia, acute
undifferentiated leukemia, chronic myelocytic leukemia "CML",
chronic lymphocytic leukemia "CLL", hairy cell leukemia, multiple
myeloma), acute and chronic leukemias (e.g., lymphoblastic,
myelogenous, lymphocytic, and myelocytic leukemias), and Lymphomas
(e.g., Hodgkin's disease, non-Hodgkin's Lymphoma, Multiple myeloma,
Waldenstrom's macroglobulinemia, Heavy chain disease, and
Polycythemia vera).
[0155] In another embodiment, the engineered Ab-drug conjugate can
be administered concurrently with another anti-cancer agent such as
a chemotherapeutic agent or with radiation therapy. In another
embodiment, the chemotherapeutic agent or radiation therapy is
administered prior or subsequent to administration of the
engineered Ab-drug conjugate. Any one or a combination of the
chemotherapeutic agents listed below can be administered. With
respect to radiation, any radiation therapy protocol can be used
depending upon the type of cancer to be treated. For example, but
not by way of limitation, x-ray radiation can be administered; in
particular, high-energy megavoltage (radiation of greater that 1
MeV energy) can be used for deep tumors, and electron beam and
orthovoltage x-ray radiation can be used for skin cancers.
Gamma-ray emitting radioisotopes, such as radioactive isotopes of
radium, cobalt and other elements, can also be administered.
[0156] Examples of chemotherapeutic agents include, but are not
limited to, methotrexate, taxol, L-asparaginase, mercaptopurine,
thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide,
nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine,
procarbizine, topotecan, nitrogen mustards, cytoxan, etoposide,
5-fluorouracil, BCNU, irinotecan, camptothecins, bleomycin,
doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin,
mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine,
paclitaxel, and docetaxel. drugs such as an alkylating agents such
as a nitrogen mustard (e.g., cyclophosphamide, ifosfamide,
trofosfamide, chlorambucil, melphalan), nitrosoureas (e.g.,
carmustine (BCNU), lomustine (CCNU)), alkylsulphonates (e.g.,
busulfan, treosulfan), triazenes (e.g., decarbazine), Platinum
containing compounds (e.g., cisplatin, carboplatin); plant
alkaloids, such as vinca alkaloids (e.g, vincristine, vinblastine,
vindesine, vinorelbine), taxoids (e.g., paclitaxel, docetaxol); DNA
topoisomerase inhibitors such as epipodophyllins (e.g., etoposide,
teniposide, topotecan, 9-aminocamptothecin, camptothecin,
crisnatol, mitomycins (e.g., mitomycin C); anti-metabolites such as
anti-folates such as DHFR inhibitors (e.g., methotrexate,
trimetrexate), IMP dehydrogenase inhibitors (mycophenolic acid,
tiazofurin, ribavirin, EICAR) and ribonucleotide reductase
inhibitors (e.g., hydroxyurea, deferoxamine), pyrimidine analogs
such as uracil analogs (5-fluorouracil, floxuridine, doxifluridine,
ratitrexed), cytosine analogs (e.g., cytarabine (ara C), cytosine
arabinoside, fludarabine), and purine analogs (e.g.,
mercaptopurine, thioguanine); hormonal therapies, such as receptor
antagonists, such as anti-estrogens (e.g., tamoxifen, raloxifene,
megestrol), LHRH agonists (e.g., goscrclin, leuprolide acetate),
and anti-androgens (e.g., flutamide, bicalutamide;
retinoids/deltoids such as vitamin D3 analogs (e.g., EB 1089, CB
1093, KH 1060), photodynamic therapies (e.g., vertoporfin (BPD-MA),
phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A
(2BA-2-DMHA)), cytokines (e.g., interferon-.alpha.,
interferon-.gamma., tumor necrosis factor), as well as other drugs,
such as gemcitabine, velcade, revamid, thalamid, isoprenylation
inhibitors (e.g., lovastatin), dopaminergic neurotoxins (e.g.,
1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g.,
staurosporine), actinomycins (e.g., actinomycin D, dactinomycin),
bleomycins, bleomycin A2, bleomycin B2, peplomycin), anthracyclines
(daunorubicin, Doxorubicin (adriamycin), idarubicin, epirubicin,
pirarubicin, zorubicin, mtoxantrone), MDRinhibitors (e.g.,
verapamil), and Ca.sup.2+ATPase inhibitors (e.g, thapsigargin)
[0157] The engineered Ab-drug conjugates can also be used for
killing or inhibiting the replication of a cell that produces an
autoimmune disease or for treating an autoimmune disease.
Accordingly, the engineered Ab-drug conjugates can be used
accordingly in a variety of settings for treating an autoimmune
disease in a patient suffering from the autoimmune disease. For
example, the conjugates can be used to deliver a drug to a target
cell. Without being bound by theory, in one embodiment, the
engineered Ab-drug conjugates associate with an antigen on the
surface of a target cell, and the conjugate is then taken up inside
a target-cell through receptor-mediated endocytosis. Once inside
the cell, one or more specific peptide sequences (e.g., within a
linker) are enzymatically or hydrolytically cleaved, resulting in
release of a drug. The released drug is then free to migrate in the
cytosol and induce cytotoxic or cytostatic activities. In another
embodiment, the drug is cleaved from the engineered Ab-conjugate
outside the target cell, and the drug subsequently penetrates the
cell.
[0158] In another embodiment, the engineered Ab-drug conjugates
bind to an autoimmune antigen which is on the surface of a cell.
For example, the engineered Ab can bind to activated lymphocytes
that are associated with the autoimmune disease state. In a further
embodiment, the engineered Ab-drug conjugates kill or inhibit the
multiplication of cells that produce an autoimmune antibody
associated with a particular autoimmune disease.
[0159] Examples of autoimmune diseases that can be treated with the
engineered Ab-drug conjugates include, but are not limited to, Th2
lymphocyte related disorders (e.g., atopic dermatitis, atopic
asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome,
systemic sclerosis, and graft versus host disease); Th1 lymphocyte
related disorders (e.g., rheumatoid arthritis, multiple sclerosis,
psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis, Grave's
disease, primary biliary cirrhosis, Wegener's granulomatosis and
tuberculosis); and activated B lymphocyte related disorders (e.g.,
systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid
arthritis and type I diabetes). Other autoimmune diseasesinclude,
butare notlimited to, active chronic hepatitis, Addison's disease,
allergic alveolitis, allergic reaction, allergic rhinitis, Alport's
Syndrome, anaphlaxis, ankylosing spondylitis, anti-phosholipid
syndrome, arthritis, ascariasis, aspergillosis, atopic allergy,
atropic dermatitis, atropic rhinitis, Behcet's disease,
Bird-Fancier's Lung, bronchial asthma, Caplan's syndrome,
cardiomyopathy, Celiac disease, Chagas' disease, chronic
glomerulonephritis, Cogan's Syndrome, cold agglutinin disease,
congenital rubella infection, CREST syndrome, Crohn's disease,
cryoglobulinemia, Cushing's syndrome, dermatomyositis, discoid
lupus, Dressler's syndrome, Eaton-Lambert syndrome, echovirus
infection, encephalomyelitis, endocrine opthalmopathy, Epstein-Barr
virus infection, equine heaves, erythematosis, Evan's syndrome,
Felty's syndrome, fibromyalgia, Fuch's cyclitis, gastric atrophy,
gastrointestinal allergy, giant cell arteritis, glomerulonephritis,
goodpasture's syndrome, graft v. host disease, Graves' disease,
Guillain-Barre disease, Hashimoto's thyroiditis, hemolytic anemia,
Henoch-Schonlein Purpura, idiopathic adrenal atrophy, idiopathic
pulmonary fibritis, IgA nephropathy, inflammatory bowel disease,
insulin-dependent diabetes mellitus, juvenile arthritis, juvenile
diabetes mellitus (Type I), Lambert-Eaton syndrome, laminitis,
lichen planus, lupoid hepatitis, lupus, lymphopenia, Meniere's
disease, mixed connective tissue disease, multiple sclerosis,
myasthenia gravis, pernicious anemia, polyglandular syndromes,
presenile dementia, primary agammaglobulinemia, primary biliary
cirrhosis, psoriasis, psoriatic arthritis, Raynauds phenomenon,
recurrent abortion, Reiter's syndrome, rheumatic fever, rheumatoid
arthritis, Sampter's syndrome, schistosomiasis, Schmidt's syndrome,
scleroderma, Shulman's syndrome, Sjorgen's syndrome, stiff-man
syndrome, sympathetic ophthalmia, systemic lupus erythematosis,
Takayasu's arteritis, temporal arteritis, thyroiditis,
thrombocytopenia, thyrotoxicosis, toxic epidermalnecrolysis,
TypeBinsulinresistance, Type Idiabetesmellitus, ulcerative colitis,
uveitis, vitiligo, Waldenstrom's macroglobulemia, and Wegener's
granulomatosis.
[0160] In another embodiment, methods for treating an autoimmune
disease arealso provided that comprise administering to a patient
in need thereof an effective amount of an engineered IgG Ab-drug
conjugate alone or in combination another therapeutic agent known
for the treatment of an autoimmune disease. Examples of
anti-autoimmune disease agent include, but are not limited to, the
following: cyclosporine, cyclosporine A, mycophenylate mofetil,
sirolimus, tacrolimus, etanercept, prednisone, azathioprine,
methotrexate, cyclophosphamide, aminocaproic acid, chloroquine,
hydroxychloroquine, hydrocortisone, dexamethasone, chlorambucil,
DHEA, danazol, bromocriptine, meloxicam and infliximab.
[0161] In another embodiment, methods for treating an infectious
disease are provided which comprise administering to the patient
suffering from the infectious disease a therapeutically effective
amount of an engineered IgG Ab or fragment thereof conjugated to a
drug. The engineered Ab-drug conjugates can be used accordingly in
a variety of settings for the treatment of an infectious disease in
a patient. The ADCs can be used to deliver a drug to a target cell.
In one embodiment, the Ab binds to the infectious disease cell. In
another embodiment, the engineered Ab-drug conjugate kills or
inhibit the multiplication of cells that produce a particular
infectious disease. Examples of infectious diseases that can be
treated with the engineered Ab-drug conjugates include, but are not
limited to, the following: bacterial diseases, such as diphtheria,
pertussis, occult bacteremia, urinary tract infection,
gastroenteritis, cellulites, epiglottitis, tracheitis, adenoid
hypertrophy, retropharyngeal abcess, impetigo, ecthyma, pneumonia,
endocarditis, septic arthritis, pneumococcal, peritonitis,
bactermia, meningitis, acute purulent meningitis, urethritis,
cervicitis, proctitis, pharyngitis, salpingitis, epididymitis,
gonorrhea, syphilis, listeriosis, anthrax, nocardiosis, salmonella,
typhoid fever, dysentery, conjunctivitis, sinusitis, brucellosis,
tularemia, cholera, bubonic plague, tetanus, necrotizing enteritis,
and actinomycosis; mixed anaerobic infections, such as syphilis,
relapsing fever, leptospirosis, Lyme disease, rat bite fever,
tuberculosis, lymphadenitis, leprosy, chlamydia, chlamydial
pneumonia, trachoma, and inclusion conjunctivitis; systemic fungal
diseases such as histoplamosis, coccidiodomycosis, blastomycosis,
sporotrichosis, cryptococcsis, systemic candidiasis, aspergillosis,
mucormycosis, mycetoma, and chromomycosis; rickettsial diseases
such as typhus, Rocky Mountain Spotted Fever, ehrlichiosis, Eastern
Tick-Borne Rickettsioses, rickettsialpox, Q fever and
bartonellosis; parasitic diseases such as malaria, babesiosis,
African sleeping sickness, Chagas' disease, leishmaniasis, Dum-Dum
fever, toxoplasmosis, meningoencephalitis, keratitis, entamebiasis,
giardiasis, cryptosporidiosis, isosporiasis, cyclosporiasis,
microsporidiosis, ascariasis, whipworm infection, hookworm
infection, threadworm infection, ocular larva migrans, trichinosis,
Guinea worm disease, lymphatic Filariasis, loiasis, River
Blindness, canine heartworm infection, schistosomiasis, swimmer's
itch, Oriental lung fluke, Oriental liver fluke, fascioliasis,
fasciolopsiasis, opisthorchiasis, tapeworm infections, hydatid
disease, and alveolar hydatid disease; viral diseases such as
measles, subacute sclerosing panencephalitis, common cold, mumps,
rubella, roseola, Fifth Disease, chickenpox, respiratory syncytial
virus infection, croup, bronchiolitis, infectious mononucleosis,
poliomyelitis, herpangina, hand-foot-and-mouth disease, Bornholm
disease, genital herpes, genital warts, aseptic meningitis,
myocarditis, pericarditis, gastroenteritis, acquired
immunodeficiency syndrome (AIDS), human immunodeficiency vims
(HIV), Reye's syndrome, Kawasaki syndrome, influenza, bronchitis,
viral "Walking" pneumonia, acute febrile respiratory disease, acute
pharyngoconjunctival fever, epidemic keratoconjunctivitis, Herpes
Simplex Virus 1 (HSV-1), Herpes Simplex Virus 2 (HSV-2), shingles,
cytomegalic inclusion disease, rabies, progressive multifocal
leukoencephalopathy, kuru, fatal familial insomnia,
Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker disease,
tropical spastic paraparesis, western equine encephalitis,
California encephalitis, St. Louis encephalitis, Yellow Fever,
Dengue, lymphocytic choriomeningitis, Lassa fever, hemorrhagic
fever, Hantvims pulmonary syndrome, Marburg virus infections, Ebola
virus infections and smallpox.
[0162] In yet another embodiment, methods for treating an
infectious disease are provided which comprise administering to a
patient suffering from the infectious disease an engineered IgG
Ab-drug conjugate alone or in combination with another therapeutic
agent that is an anti-infectious disease agent. Examples of
anti-infectious disease agents include, but not limited to,
beta.-lactam antibiotics, such as penicillin G, penicillin V,
cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin,
ampicillin, amoxicillin, bacampicillin, azlocillin, carbenicillin,
mezlocillin, piperacillin and ticarcillin; aminoglycosides such as
amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin
and tobramycin; macrolides such as azithromycin, clarithromycin,
erythromycin, lincomycin and clindamycin; tetracyclines such as
demeclocycline, doxycycline, minocycline, oxytetracycline and
tetracycline; quinolones such as cinoxacin, and nalidixic acid;
fluoroquinolones such as ciprofloxacin, enoxacin, grepafloxacin,
levofloxacin, lomefloxacin, norfloxacin, ofloxacin, sparfloxacin
and trovafloxicin; polypeptides such as bacitracin, colistin and
polymyxin B; sulfonamides such as sulfisoxazole, sulfamethoxazole,
sulfadiazine, sulfamethizole and sulfacetamide; and other
antibacterial agents, such as trimethoprim, sulfamethazole,
chloramphenicol, vancomycin, metronidazole, quinupristin,
dalfopristin, rifampin, spectinomycin and nitrofurantoin; and
antiviral agents, such as general antiviral agents such as
idoxuradine, vidarabine, trifluridine, acyclovir, famcicyclovir,
pencicyclovir, valacyclovir, gancicyclovir, foscarnet, ribavirin,
amantadine, rimantadine, cidofovir; antisense oligonucleotides,
immunoglobulins and interferons; drugs for HIV infection such as
tenofovir, emtricitabine, zidovudine, didanosine, zalcitabine,
stavudine, lamivudine, nevirapine, delavirdine, saquinavir,
ritonavir, indinavir, and drugs for treatment of metabolic disease
such as nelfinavir.
Pharmaceutical Compositions Containing the Ab-Drug Conjugates
[0163] In another embodiment, pharmaceutical compositions are
provided comprising an effective amount of the engineered IgG
Ab-drug conjugate and a pharmaceutical acceptable carrier. The
pharmaceutical compositions can be in various forms for
administration to a patient. For example, the composition can be in
the form of a solid, liquid or gas (aerosol). Typical routes of
administration include, without limitation, oral, topical,
parenteral, sublingual, rectal, vaginal, ocular, intra-tumor, and
intranasal. Parenteral administration includes subcutaneous
injections, intravenous, intramuscular, intrasternal injection or
infusion techniques. In one embodiment, the compositions are
administered parenterally. In another embodiment, the conjugate or
compositions are administered intravenously. Such compositions are
suitable for veterinary or human administration.
[0164] Pharmaceutical compositions can be formulated so as to allow
a conjugate to be bioavailable upon administration of the
composition to a patient. Compositions can take the form of one or
more dosage units, where for example, a tablet can be a single
dosage unit, and a container of a conjugate in injectable form can
hold a plurality of dosage units.
[0165] Materials used in preparing the pharmaceutical compositions
can be non-toxic in the amounts used. As evident to those of
ordinary skill in the art that the optimal dosage of the active
ingredient(s) in the pharmaceutical composition will depend on a
variety of factors. Relevant factors include, without limitation,
the type of patient, e.g., human or animal, the particular form of
the engineered-Ab conjugate, the manner of administration, and the
composition employed.
[0166] The pharmaceutically acceptable carrier or vehicle can be
particulate, so that the compositions are, for example, in tablet
or powder form. The carrier(s) can be liquid, with the compositions
being, for example, an oral syrup or injectable liquid. In
addition, the carrier(s) can be gaseous or particulate, so as to
provide an aerosol composition useful in, e.g., inhalatory
administration.
[0167] When intended for oral administration, the composition is
preferably in solid or liquid form, where semi-solid, semi-liquid,
suspension and gel forms are included within the forms considered
herein as either solid or liquid.
[0168] As a solid composition for oral administration, the
composition can be formulated into a powder, granule, compressed
tablet, pill, capsule, chewing gum, wafer or the like. Such a solid
composition typically contains one or more inert diluents. In
addition, one or more of the following can be present: binders such
as carboxymethylcellulose, ethyl cellulose, microcrystalline
cellulose, or gelatin; excipients such as starch, lactose or
dextrins; disintegrating agents such as alginic acid, sodium
alginate, Primogel, corn starch and the like; lubricants such as
magnesium stearate or Sterotex; glidants such as colloidal silicon
dioxide; sweetening agents such as sucrose or saccharin, a
flavoring agent such as peppermint, methyl salicylate or orange
flavoring, and a coloring agent. When the composition is in the
form of a capsule, e.g., a gelatin capsule, it can contain, in
addition to materials of the above type, a liquid carrier such as
polyethylene glycol, cyclodextrin or a fatty oil.
[0169] The composition can be in the form of a liquid, e.g., an
elixir, syrup, solution, emulsion or suspension. The liquid can be
useful for oral administration or for delivery by injection. When
intended for oral administration, a composition can comprise one or
more of a sweetening agent, preservatives, dye/colorant and flavor
enhancer. In a composition for administration by injection, one or
more of a surfactant, preservative, wetting agent, dispersing
agent, suspending agent, buffer, stabilizer and isotonic agent can
also be included.
[0170] The liquid compositions, whether they are solutions,
suspensions or other like form, can also include one or more of the
following: sterile diluents such as water for injection, saline
solution, preferably physiological saline, Ringer's solution,
isotonic sodium chloride, fixed oils such as synthetic mono or
digylcerides which can serve as the solvent or suspending medium,
polyethylene glycols, glycerin, cyclodextrin, propylene glycol or
other solvents; antibacterial agents such as benzyl alcohol or
methyl paraben; 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.
A parenteral composition can be enclosed in ampoule, a disposable
syringe or a multiple-dose vial made of glass, plastic or other
material. Physiological saline is an exemplary adjuvant. An
injectable composition is preferably sterile.
[0171] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's, or fixed oils. Intravenous vehicles include
fluid and nutrient replenishers, electrolyte replenishers (such as
those based on Ringer's dextrose), and the like. Preservatives and
other additives may also be present such as, for example,
antimicrobials, anti-oxidants, chelating agents, and inert gases
and the like.
[0172] The effective amount of the engineered Ab-drug conjugate for
treatment of a particular cancer or disease will depend on the
nature of the cancer or disease and can be determined by standard
clinical techniques. The dosage ranges for the administration of
the disclosed engineered Ab-drug conjugates are those large enough
to produce the desired effect in which the symptoms of the
condition or disorder are ameliorated. The dosage should not be so
large as to cause adverse side effects, such as unwanted
cross-reactions, anaphylactic reactions, and the like. In addition,
in vitro or in vivo assays can optionally be employed to help
identify optimal dosage ranges.
[0173] The precise dose to be employed in the pharmaceutical
compositions will also depend on the age, condition, sex and extent
of the disease in the patient, route of administration, and the
seriousness of the disease or disorder, and should be decided
according to the judgment of the practitioner and each patient's
circumstances.
[0174] Typically, the effective amount of the engineered Ab-drug
conjugate is at least about 0.01% of a conjugate by weight of the
composition. When intended for oral administration, this amount can
be varied to range from about 0.1% to about 80% by weight of the
pharmaceutical composition. In one embodiment, oral pharmaceutical
compositions can comprise from about 4% to about 50% of the
conjugate by weight of the composition. In yet another embodiment,
present compositions are prepared so that a parenteral dosage unit
contains from about 0.011% to about 2% by weight of the
conjugate.
[0175] For intravenous administration, the composition can comprise
from about 0.01 to about 100 mg of a conjugate per kg of the
animal's body weight. In one embodiment, the composition can
include from about 1 to about 100 mg of a conjugate per kg of the
animal's body weight. In another embodiment, the amount
administered will be in the range from about 0.1 to about 25 mg/kg
of body weight of the conjugate.
[0176] Generally, the dosage of an Ab-conjugate administered to a
patient is typically about 0.01 mg/kg to about 2000 mg/kg of the
animal's body weight. In one embodiment, the dosage administered to
a patient is between about 0.01 mg/kg to about 10 mg/kg of the
animal's body weight. In another embodiment, the dosage
administered to a patient is between about 0.1 mg/kg and about 250
mg/kg of the animal's body weight. In yet another embodiment, the
dosage administered to a patient is between about 0.1 mg/kg and
about 20 mg/kg of the animal's body weight. In yet another
embodiment the dosage administered is between about 0.1 mg/kg to
about 10 mg/kg of the animal's body weight. In yet another
embodiment, the dosage administered is between about 1 mg/kg to
about 10 mg/kg of the animal's body weight.
[0177] The Ab-conjugates can be administered by any convenient
route, for example by infusion or bolus injection, by absorption
through epithelial or mucocutaneous linings (e.g., oral mucosa,
rectal and intestinal mucosa, etc.). Administration can be systemic
or local. Various delivery systems are known, e.g., encapsulation
in liposomes, microparticles, microcapsules, capsules, etc., and
can be used to administer a conjugate or composition. In certain
embodiments, more than one conjugate or composition is administered
to a patient.
[0178] The term "carrier" as used herein refers to a diluent,
adjuvant or excipient, with which a conjugate is administered. Such
pharmaceutical carriers can be liquids, such as water and oils,
including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like. The carriers can be saline, gum acacia, gelatin,
starch paste, talc, keratin, colloidal silica, urea, and the like.
In addition, auxiliary, stabilizing, thickening lubricating and
coloring agents can be used. In one embodiment, when administered
to a patient, the Ab-conjugate or compositions and pharmaceutically
acceptable carriers are sterile. Water is an exemplary carrier when
the conjugate are administered intravenously. Saline solutions and
aqueous dextrose and glycerol solutions can also be employed as
liquid carriers, particularly for injectable solutions. Suitable
pharmaceutical carriers also include excipients such as starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skim milk, glycerol, propylene, glycol, water,
ethanol and the like. The compositions, if desired, can also
contain minor amounts of wetting or emulsifying agents, or pH
buffering agents.
[0179] The compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, pellets, capsules, capsules
containing liquids, powders, sustained-release formulations,
suppositories, emulsions, aerosols, sprays, suspensions, or any
other form suitable for use. Other examples of suitable
pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin.
[0180] Typically, the carriers or vehicles for intravenous
administration are sterile isotonic aqueous buffer solutions.
Pharmaceutical compositions may also include a solubilizing agent
or a local anesthetic such as lignocaine to ease pain at the site
of the injection.
[0181] Pharmaceutical compositions for oral delivery can be in the
form of tablets, lozenges, aqueous or oily suspensions, granules,
powders, emulsions, capsules, syrups, or elixirs, for example.
Orally administered compositions can contain one or more optionally
agents, for example, sweetening agents such as fructose, aspartame
or saccharin; flavoring agents; coloring agents; and preserving
agents, to provide a pharmaceutically palatable preparation.
Moreover, where in tablet or pill form, the pharmaceutical
compositions can be coated to delay disintegration and absorption
in the gastrointestinal tract thereby providing a sustained action
over an extended period of time.
[0182] Pharmaceutical compositions can also be administered
topically, in which case the carrier may be in the form of a
solution, emulsion, ointment or gel base. For transdermal
administration, the pharmaceutical composition can be in the form
of a transdermal patch or an iontophoresis device. Topical
formulations can comprise a concentration of an engineered Ab-drug
conjugate of from about 0.05% to about 50% w/v (weight per unit
volume of composition), in another embodiment, from 0.1% to 10%
ow/v.
Methods of Preparing Engineered mAb-Drug Conjugates
[0183] In another embodiment, a method of preparing a conjugated
N-glycosylated IgG Ab or fragment thereof containing one to ten
non-native N-glycosylation sites in the heavy chain constant domain
is provided, the method comprising: [0184] (a) transforming a yeast
or filamentous fungus host cell genetically engineered to produce
N-glycans comprising terminal galactose residues of the structure
Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2) or the
structure Gal.sub.(1-2)GlcNAc.sub.(1-2)Man.sub.5GlcNAc.sub.2, with
a nucleic acid encoding an IgG heavy chain contain domain, wherein
the IgG heavy chain constant domain comprises one to ten amino acid
mutations, and wherein the amino acid mutations generate at least
one non-native N-glycosylation site in the IgG heavy chain constant
domain; [0185] (b) culturing the transformed host cell under
conditions that allow the expression of IgG heavy chain constant
domain comprising terminal galactose residues; [0186] (c)
contacting the expressed IgG heavy chain constant domain with a
reagent that oxidizes the terminal galactose residues; and [0187]
(d) conjugating a drug to the oxidized moiety of the terminal
galactose residues.
[0188] The yeast host cell used in step (a) of the method of
preparing a conjugated N-glycosylated IgG Ab or fragment thereof
containing one to ten non-native N-glycosylation sites in the heavy
chain constant domain is selected from the group consisting of
Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia
koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia
thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi,
Pichia stiptis, Pichia methanolica, Pichia minuta (Ogataea minuta,
Pichia lindneri), Pichia sp., Saccharomyces cerevisiae,
Saccharomyces sp., Hansenula polymorphs, Kluyveromyces sp.,
Kluyveromyces lactis, Candida albicans, Aspergillus nidulans,
Aspergillus niger, Aspergillus oryzae, Trichoderma reesei,
Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum,
Fusarium venenatum, Neurospora crassa and Yarrowia lipolitica. In
an embodiment, the yeast host cell is Pichia pastoris.
[0189] Methods for producing yeast host cells and filamentous
fungal host cells genetically engineered to produce human-like
complex N-glycans containing terminal galactose residues
(Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2), or human-like
hybrid N-gly cans containing galactose residues
(Gal.sub.(1-2)GlcNAc.sub.(1-2)Man.sub.5GlcNAc.sub.2) are described
in the art, e.g., in U.S. Pat. No. 8,815,544; Bobrowicz P, et al.,
Glycobiology 14: 757-766; Li et al., (2006) Nat. Biotechnol. 24:
210-215; Zha, 2013. In one embodiment, the yeast host cell is a
Pichia pastoris host cell that has been engineered to produce
N-glycans comprising predominantly the
Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2 glycoforms. In
another embodiment, the yeast host cell is a Pichia pastoris host
cell that has been engineered to produce N-glycans comprising
predominantly the
Gal.sub.(1-2)GlcNAc.sub.(1-2)Man.sub.5GlcNAc.sub.2 glycoforms. In
another embodiment, the yeast host cell is a Pichia pastoris host
cell that has been engineered to produce N-glycans comprising
predominantly the Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2
glycoforms.
[0190] The nucleic acid encoding a heavy chain constant domain, in
useful embodiments may comprise the complete heavy chain of the IgG
Ab e.g., anti-Her2 IgG1 (SEQ ID NO: 1), wherein the heavy chain
constant domain can be modified to effect the substitution of the
relevant amino acid residues by site directed mutagenesis as in
Example 1. Alternatively, the nucleic acid encoding the heavy chain
domain may be prepared by chemical synthesis, wherein
oligonucleotides are designed based on the specific amino acid
sequence of the antibody mutant.
[0191] In an embodiment, the aforementioned nucleic acid comprises
a nucleotide sequence encoding an anti-Her2 heavy chain mutant
polypeptide comprising an amino acid sequence selected from SEQ ID
NOs: 4-29, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40.
[0192] In another embodiment, the aforementioned nucleic acid
comprises a nucleotide sequence encoding an anti-mouse PD-1 heavy
chain mutant polypeptide comprising an amino acid sequence selected
from SEQ ID NOs: 41-45.
[0193] In another embodiment, the aforementioned nucleic acid
comprises a nucleotide sequence encoding an anti-CS1 heavy chain
mutant polypeptide comprising an amino acid sequence selected from
SEQ ID NOs: 46-50.
[0194] In another embodiment, the aforementioned nucleic acid
comprises a nucleotide sequence encoding an anti-CD70 heavy chain
mutant polypeptide comprising an amino acid sequence selected from
SEQ ID NOs: 51-55.
[0195] In another embodiment, the yeast or filamentous host cell is
transformed with the complete nucleotide sequences encoding one or
both of the heavy and light chain sequences of an IgG Ab, e.g.,
anti-Her2 IgG1 heavy and light chain sequences (SEQ ID NOs: 1 and
2, respectively) or a fragment thereof. Transformation is effected
by inserting the nucleotide sequences encoding the heavy and/or
light chains of the antibody into a recombinant vector and operably
linked to control sequences required for expression of the heavy
and light chain in the transformed host cell. One of skill in the
art may make a selection among vectors and expression control
sequences well known in the art. In an embodiment, the vector is an
expression vector in which the nucleotide sequence encoding the
heavy and light chain of the antibodies is operably linked to
additional segments required for transcription of the nucleotide
sequence. The vector is typically derived from plasmid (see Example
1) or viral DNA. A number of suitable expression vectors for
expression in the host cells mentioned herein are commercially
available or described in the literature.
[0196] In another embodiment of the foregoing method, the IgG Ab or
fragment thereof prepared by the aforementioned method comprises
one to ten mutations (or pairs of mutations) in the heavy chain
constant polypeptide selected from the group consisting of S134N,
G161T, G161S, N203T, N203S, V363T, V363S, Q438N, S176N, A118N,
S132N, K133N, A162N, T195N, K210T, Y391T, F423T, F423S, Y436T,
Y436S, L193N, K392T, K392S, F423T, S176N/G178T, S176N/G178S,
Q419N/N421T, Q419N/N421S, S191N/L193T, S191N/L193S, G194N/Q196T,
and G194N/Q196S, according to EU numbering.
[0197] Following transformation of the yeast or filamentous host
cells (step a), the transformed host cells are cultured (step b) in
a nutrient medium suitable for production of the engineered Ab or
fragment thereof using methods known in the art. For example, the
host cells may be cultured by shake flask, small-scale or
large-scale fermentation (including continuous, batch, fed-batch,
or solid state fermentations) in laboratory or industrial
fermenters performed in a suitable medium and under conditions
allowing the heavy and light chains to be expressed and isolated.
Suitable media are available from commercial suppliers or may be
prepared according to published composition. The produced Abs or
fragments thereof contain an engineered N-glycosylated heavy chain
constant domain comprising an N-glycan comprising terminal
galactose residues of the structure
Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2) or the
structure Gal.sub.(1-2)GlcNAc.sub.(1-2)Man.sub.5GlcNAc.sub.2.
[0198] In an embodiment, the engineered Ab prepared by the
foregoing method comprises about 50 to about 100 mole % of N-glycan
with terminal galactose residues of the structure
Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2) or the
structure Gal.sub.(1-2)GlcNAc.sub.(1-2)Man.sub.5GlcNAc.sub.2. In
another embodiment, the engineered antibody prepared by the
foregoing method comprises about 80 to about 100 mole % of N-gly
can with terminal galactose residues of the structure
Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2) or the
structure Gal.sub.(1-2)GlcNAc.sub.(1-2)Man.sub.5GlcNAc.sub.2.
[0199] The aforementioned expressed Ab comprising the
N-glycosylated heavy chain constant domain may be recovered from
the nutrient medium by methods known in the art, e.g.,
centrifugation, filtration, extraction, evaporation, or
precipitation.
[0200] The engineered Ab may then be purified by a variety of
procedures known in the art including, but not limited to,
chromatography (e.g. ion exchange, affinity, hydrophobic,
chromatofocusing, and size exclusion), electrophoretic procedures
(e.g. preparative isoelectric focusing), differential solubility
(e.g. ammonium sulfate precipitation), SDS-PAGE, or extraction. In
an embodiment, the engineered Ab is purified by protein A
chromatography (See Example 3.
[0201] The purified engineered Ab containing the expressed
N-glycosylated heavy chain constant domain is then contacted with a
reagent that oxidizes the terminal galactose residues of the
N-glycan to generate an aldehyde (step c).
[0202] In an embodiment, the reagent is the enzyme galactose
oxidase (GAL, D-galactose: oxygen 6-oxidoreductase; EC 1.1.3.9,
commercially available from Sigma purified from Dactylium
dendroites; also referred herein as GO) which specifically oxidizes
terminal galactose residues at the C-6 position to generate an
aldehyde group (see e.g., Cooper et al., 1959). The aldehyde group
is a chemically reactive group that can be directly conjugated with
a reactive amine group such as an alkoxyamine (also known as
aminooxy) present in a derivatized-drug to form a stable oxime bond
(Ramya et al., 2013) between the derivatized drug and the
engineered N-glycan.
[0203] The term "derivatived drug" refers to a drug that contains
or is modified to contain a reactive amine.
[0204] The term "reactive amine" refers to any nitrogen-containing
functional group that can be covalently attached or bonded through
a nitrogen atom to an aldehyde functional group by a simple
chemical condensation reaction. Examples of other reactive amines
include but are not limited to hydrazine, hydrazide,
phenylhydrazine, phenylhydrazide, phenoxyamine, semicarbazide and
thiosemicarbazide.
[0205] With respect to carrying out oxidation of the terminal
galactose residues of the engineered N-glycosylated Abs, the Ab is
present in aqueous solution at a concentration of about 0.1 to 100
mg/ml, 0.5 to 50 mg/ml, 1.0 to 20 mg/ml, or 0.5 to 20 mg/ml (see
e.g., Copper et al., and Rayma et al., 2013). The enzyme GO
generally is used at a pH about 5.5 to about 8.0. The influence of
pH, substrate concentration, buffers and buffer concentrations on
enzyme reaction are reported in Cooper et al., supra.
[0206] In another embodiment utilizing GO as the oxidizing reagent,
the purified engineered Abs containing the expressed N-glycosylated
heavy chain constant domain can be contacted with GO by producing
the GO in vivo in the yeast and filamentous fungi host cells that
have been genetically engineered to produce N-glycosylated mAbs
containing N-glycans comprising terminal galactose residues. For
example, a yeast or host cell can also be transformed with a
plasmid containing the nucleotide sequence encoding GO (e.g., GO
from Fusarium graminearum, SEQ ID NO: 57; see Paukner, 2014;
Anasontzis, 2014; Deacon, 2004). In another embodiment, the
terminal galactose residue of the engineered N-gly can of the Ab
can alsobe oxidized to form aldehyde groups utilizing chemical
oxidizing reagents. Examples of chemical oxidizing reagents
include, but are not limited to periodic acid, paraperiodic acid,
sodium metaperiodate and potassium metaperiodate. Among these,
oxygen acids and salts thereof are preferred since secondary or
undesirable side reactions are less frequent. For a general
discussion, see Jackson, 1944, in Organic Reactions 2, p. 341;
Bunton, 1965, Oxidation in Organic Chemistry, Vol. 1 Wiberg, ed.,
Academic Press, New York, p. 367.
[0207] Oxidation of the engineered Abs with these chemical
oxidizing reagents can be carried out by known methods. In the
oxidation, the engineered Ab is generally present in the form of an
aqueous solution at a concentration generally of less than 100
mg/ml, or 1 to 20 mg/ml. When an oxygen acid or salt thereof is
used as the oxidizing agent, it is used generally in the form of an
aqueous solution, and the concentration is generally 0.001 to 10 mM
and preferably 1.0 to 10 mM. The amount of the oxygen acid or salt
thereof depends on the kind of antibody, but generally it is used
in excess, for example, ten to 100 times as much as the amount of
the oxidizable N-glycan. The optimal amount, however, can be
determined by routine experimentation.
[0208] Following oxidation of the engineered N-glycosylated Ab, the
Ab can be conjugated to a drug by reacting the Ab with a drug
having a reactive amine group selected from the group consisting of
hydrazine, hydrazide, phenylhydrazine, phenylhydazide, alkoxyamine,
phenoxyamino, semicarbazide and thiosemicarbazide groups.
[0209] In a useful embodiment, the reactive amine group is an
alkoxyamine (aminooxy) group. Drugs modified to contain a reactive
amino group such as alkoxyamine can be synthesized by methods known
in the art (Jayasekara, 2014; Trimaille 2014; Su 2005; Singh 2005)
and are also commercially available (e.g., the amino oxy activated
C5-linker containing DMI (chemically synthesized by Concortis
Biosystems in San Diego, Calif., see Example 10; aminooxy activated
Exendin-4-peptide chemically synthesized by Biopeptekin Malvern,
Pa., see Example 9; and aminooxy activated CF633 dye chemically
synthesized by Biotium in Hayward, Calif., Example 5).
[0210] In an embodiment, a solution of the oxidized engineered Ab
at a concentration from about 0.5 to 20 mg/ml is mixed with an
amine derivative of a drug (molar ratios of reactive amine group to
antibody aldehyde ranging from about 1 to about 10,000) and the
solution incubated for from about 1 to 72 hours, preferably in the
dark. Suitable temperatures are from 0.degree. to 37.degree. C. and
pH may be from about 6 to 8.
[0211] The aforementioned method of preparing Ab-drug conjugate can
also be used to prepare Ab-drug conjugate, wherein the engineered
Ab contains one to two non-native N-glycosylation sites in the
heavy chain framework domain as is described above.
[0212] In another embodiment, a method of preparing a conjugated
N-glycosylated Ab or fragment thereof containing one to ten
non-native N-glycosylation sites in the heavy chain constant domain
is provided, the method comprising: [0213] (a) transforming a yeast
or filamentous fungus host cell genetically engineered to produce
N-glycans comprising terminal sialic acid residues
(NANA.sub.(1-4)Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2)
with a nucleic acid encoding a heavy chain contain domain, wherein
the heavy chain comprises one to ten amino acid mutations, and
wherein the one to ten amino acid mutation generates at least one
non-native N-glycosylation site in the heavy chain constant domain;
[0214] (b) culturing the transformed yeast host cell under
conditions that allow the expression of the heavy chain constant
domain comprising terminal sialic acid residues, [0215] (c)
contacting the expressed heavy chain constant domain with
neuraminidase to remove the terminal sialic acid residues to form
N-glycosylated heavy chain constant domain comprising terminal
galactose residues; [0216] (d) contacting the expressed
glycosylated heavy chain constant domain comprising terminal
galactose residues of step (c), with a reagent that oxidizes the
terminal galactose residues; [0217] (e) conjugating a drug to the
oxidized moiety of the terminal galactose residues.
[0218] Methods for producing yeast host cells and filamentous
fungal host cells genetically engineered to produce human-like
complex N-glycans containing terminal sialic acid residues are
described e.g. in U.S. Pat. Nos. 8,715,963; 7,863,020; Nett et al.,
(2011), Yeast 28(3):237-52 (2011); Hamilton et al., Curr Opin
Biotechnol. 18(5):387-92 (2007); Hamilton et al., (2006) Science
313: 1441-1443.
[0219] Methods of transforming the yeast strains to produce
N-glycans with terminal sialic acid residues, preparing mutated
nucleic acids containing the non-native N-glycosylation sites, and
culturing steps (steps a-b) are as described above (strains
containing N-gly cans with terminal galactose residues). With
respect to step (c), N-glycans comprising sialic acid residues can
be desialyated using the enzyme Acetyl-neuroaminyl hydrolase
(neuraminidase, New England Biolabs, Ipswich, Mass.) according to
the manufacturer's recommended reaction conditions, to efficiently
remove the sialic acid residues leaving predominantly terminal
galactose residues. Step (d) of contacting the expressed
glycosylated heavy chain constant domain with a reagent, e.g., GO
or a chemical reagent, that oxidizes the terminal galactose
residues, and step (e) of conjugating a drug to the oxidized moiety
of the terminal galactose residues is as described above.
TABLE-US-00001 Sequences (anti-Her2/trastuzumab IgG1 H chain) SEQ
ID NO: 1 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYT
RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT
LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (anti-HER2/trastuzumab Kappa
L chain) SEQ ID NO: 2
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVP
SRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPS
DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL
TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (pGLY5883 nucleotide sequence)
SEQ ID NO: 3
TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACG
GTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTC
AGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTG
TACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAA
TACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATC
GGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGC
GATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCA
GTGAATTGAGATCTAACATCCAAAGACGAAAGGTTGAATGAAACCTTTTTGCCATCC
GACATCCACAGGTCCATTCTCACACATAAGTGCCAAACGCAACAGGAGGGGATACA
CTAGCAGCAGACCGTTGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAACACCCAC
TTTTGCCATCGAAAAACCAGCCCAGTTATTGGGCTTGATTGGAGCTCGCTCATTCCA
ATTCCTTCTATTAGGCTACTAACACCATGACTTTATTAGCCTGTCTATCCTGGCCCCC
CTGGCGAGGTTCATGTTTGTTTATTTCCGAATGCAACAAGCTCCGCATTACACCCGA
ACATCACTCCAGATGAGGGCTTTCTGAGTGTGGGGTCAAATAGTTTCATGTTCCCCA
AATGGCCCAAAACTGACAGTTTAAACGCTGTCTTGGAACCTAATATGACAAAAGCG
TGATCTCATCCAAGATGAACTAAGTTTGGTTCGTTGAAATGCTAACGGCCAGTTGGT
CAAAAAGAAACTTCCAAAAGTCGGCATACCGTTTGTCTTGTTTGGTATTGATTGACG
AATGCTCAAAAATAATCTCATTAATGCTTAGCGCAGTCTCTCTATCGCTTCTGAACCC
CGGTGCACCTGTGCCGAAACGCAAATGGGGAAACACCCGCTTTTTGGATGATTATGC
ATTGTCTCCACATTGTATGCTTCCAAGATTCTGGTGGGAATACTGCTGATAGCCTAA
CGTTCATGATCAAAATTTAACTGTTCTAACCCCTACTTGACAGCAATATATAAACAG
AAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTTATCATCATTATTAGCTTACTTTCAT
AATTGCGACTGGTTCCAATTGACAAGCTTTTGATTTTAACGACTTTTAACGACAACTT
GAGAAGATCAAAAAACAACTAATTATTCGAAACGGAATTCGAAACGATGAGATTCC
CATCCATCTTCACTGCTGTTTTGTTCGCTGCTTCTTCTGCTTTGGCTGAGGTTCAGTTG
GTTGAATCTGGAGGAGGATTGGTTCAACCTGGTGGTTCTTTGAGATTGTCCTGTGCT
GCTTCCGGTTTCAACATCAAGGACACTTACATCCACTGGGTTAGACAAGCTCCAGGA
AAGGGATTGGAGTGGGTTGCTAGAATCTACCCAACTAACGGTTACACAAGATACGC
TGACTCCGTTAAGGGAAGATTCACTATCTCTGCTGACACTTCCAAGAACACTGCTTA
CTTGCAGATGAACTCCTTGAGAGCTGAGGATACTGCTGTTTACTACTGTTCCAGATG
GGGTGGTGATGGTTTCTACGCTATGGACTACTGGGGTCAAGGAACTTTGGTTACTGT
TTCCTCCGCTTCTACTAAGGGACCATCTGTTTTCCCATTGGCTCCATCTTCTAAGTCT
ACTTCCGGTGGTACTGCTGCTTTGGGATGTTTGGTTAAAGACTACTTCCCAGAGCCA
GTTACTGTTTCTTGGAACTCCGGTGCTTTGACTTCTGGTGTTCACACTTTCCCAGCTG
TTTTGCAATCTTCCGGTTTGTACTCTTTGTCCTCCGTTGTTACTGTTCCATCCTCTTCC
TTGGGTACTCAGACTTACATCTGTAACGTTAACCACAAGCCATCCAACACTAAGGTT
GACAAGAAGGTTGAGCCAAAGTCCTGTGACAAGACACATACTTGTCCACCATGTCC
AGCTCCAGAATTGTTGGGTGGTCCATCCGTTTTCTTGTTCCCACCAAAGCCAAAGGA
CACTTTGATGATCTCCAGAACTCCAGAGGTTACATGTGTTGTTGTTGACGTTTCTCAC
GAGGACCCAGAGGTTAAGTTCAACTGGTACGTTGACGGTGTTGAAGTTCACAACGCT
AAGACTAAGCCAAGAGAAGAGCAGTACAACTCCACTTACAGAGTTGTTTCCGTTTTG
ACTGTTTTGCACCAGGACTGGTTGAACGGTAAAGAATACAAGTGTAAGGTTTCCAAC
AAGGCTTTGCCAGCTCCAATCGAAAAGACTATCTCCAAGGCTAAGGGTCAACCAAG
AGAGCCACAGGTTTACACTTTGCCACCATCCAGAGAAGAGATGACTAAGAACCAGG
TTTCCTTGACTTGTTTGGTTAAAGGATTCTACCCATCCGACATTGCTGTTGAGTGGGA
ATCTAACGGTCAACCAGAGAACAACTACAAGACTACTCCACCAGTTTTGGATTCTGA
TGGTTCCTTCTTCTTGTACTCCAAGTTGACTGTTGACAAGTCCAGATGGCAACAGGG
TAACGTTTTCTCCTGTTCCGTTATGCATGAGGCTTTGCACAACCACTACACTCAAAAG
TCCTTGTCTTTGTCCCCTGGTTAATGAGGCCGGCCATTTAAATACAGGCCCCTTTTCC
TTTGTCGATATCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCTCCCACAT
CCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATT
TTTTTTAATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTT
TTCTGTACAAACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGG
TTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGGATCTAACATCCAAAGACGAAA
GGTTGAATGAAACCTTTTTGCCATCCGACATCCACAGGTCCATTCTCACACATAAGT
GCCAAACGCAACAGGAGGGGATACACTAGCAGCAGACCGTTGCAAACGCAGGACC
TCCACTCCTCTTCTCCTCAACACCCACTTTTGCCATCGAAAAACCAGCCCAGTTATTG
GGCTTGATTGGAGCTCGCTCATTCCAATTCCTTCTATTAGGCTACTAACACCATGACT
TTATTAGCCTGTCTATCCTGGCCCCCCTGGCGAGGTTCATGTTTGTTTATTTCCGAAT
GCAACAAGCTCCGCATTACACCCGAACATCACTCCAGATGAGGGCTTTCTGAGTGTG
GGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAAAACTGACAGTTTAAACGCTGTC
TTGGAACCTAATATGACAAAAGCGTGATCTCATCCAAGATGAACTAAGTTTGGTTCG
TTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAAAAGTCGGCATACCGT
TTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATAATCTCATTAATGCTTAGCG
CAGTCTCTCTATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGCAAATGGGGAA
ACACCCGCTTTTTGGATGATTATGCATTGTCTCCACATTGTATGCTTCCAAGATTCTG
GTGGGAATACTGCTGATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTAACCCC
TACTTGACAGCAATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTT
ATCATCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAATTGACAAGCTTTTGA
TTTTAACGACTTTTAACGACAACTTGAGAAGATCAAAAAACAACTAATTATTCGAAA
CGGAATTCGAAACGATGAGATTCCCATCCATCTTCACTGCTGTTTTGTTCGCTGCTTC
TTCTGCTTTGGCTGACATCCAAATGACTCAATCCCCATCTTCTTTGTCTGCTTCCGTT
GGTGACAGAGTTACTATCACTTGTAGAGCTTCCCAGGACGTTAATACTGCTGTTGCT
TGGTATCAACAGAAGCCAGGAAAGGCTCCAAAGTTGTTGATCTACTCCGCTTCCTTC
TTGTACTCTGGTGTTCCATCCAGATTCTCTGGTTCCAGATCCGGTACTGACTTCACTT
TGACTATCTCCTCCTTGCAACCAGAAGATTTCGCTACTTACTACTGTCAGCAGCACTA
CACTACTCCACCAACTTTCGGACAGGGTACTAAGGTTGAGATCAAGAGAACTGTTGC
TGCTCCATCCGTTTTCATTTTCCCACCATCCGACGAACAGTTGAAGTCTGGTACAGCT
TCCGTTGTTTGTTTGTTGAACAACTTCTACCCAAGAGAGGCTAAGGTTCAGTGGAAG
GTTGACAACGCTTTGCAATCCGGTAACTCCCAAGAATCCGTTACTGAGCAAGACTCT
AAGGACTCCACTTACTCCTTGTCCTCCACTTTGACTTTGTCCAAGGCTGATTACGAGA
AGCACAAGGTTTACGCTTGTGAGGTTACACATCAGGGTTTGTCCTCCCCAGTTACTA
AGTCCTTCAACAGAGGAGAGTGTTAATAGGGCCGGCCATTTAAATACAGGCCCCTTT
TCCTTTGTCGATATCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCTCCCA
CATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTT
ATTTTTTTTAATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTT
TTTTTCTGTACAAACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGA
AGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGGATCCGCGGCCGCTTACGC
GCCGATCCCCCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAG
ATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATAC
TAAATTTCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGG
AAAAGAAAAAAGAGACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAA
TTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTCTTTCGATGA
CCTCCCATTGATATTTAAGTTAATAAACGGTCTTCAATTTCTCAAGTTTCAGTTTCAT
TTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCA
ATCTAATCTAAGTTTTAATTACAAATTAATTAATGGCCAAGTTGACCAGTGCCGTTC
CGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTC
GGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTG
ACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTG
GGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCA
CGAACTTCCGGGACGCCTCCGGGCCTGCCATGACCGAGATCGGCGAGCAGCCGTGG
GGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGA
GGAGCAGGACTGATTAATTAACAGGCCCCTTTTCCTTTGTCGATATCATGTAATTAG
TTATGTCACGCTTACATTCACGCCCTCCTCCCACATCCGCTCTAACCGAAAAGGAAG
GAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTTAATAGTTATGTTAGTA
TTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAAACGCGTGTACGC
ATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTT
AATTTGCAAGCTGCGGCCTAAGGCGCGCCAGGCCATAATGGCCAAACGGTTTCTCA
ATTACTATATACTACTAACCATTTACCTGTAGCGTATTTCTTTTCCCTCTTCGCGAAA
GCTCAAGGGCATCTTCTTGACTCATGAAAAATATCTGGATTTCTTCTGACAGATCAT
CACCCTTGAGCCCAACTCTCTAGCCTATGAGTGTAAGTGATAGTCATCTTGCAACAG
ATTATTTTGGAACGCAACTAACAAAGCAGATACACCCTTCAGCAGAATCCTTTCTGG
ATATTGTGAAGAATGATCGCCAAAGTCACAGTCCTGAGACAGTTCCTAATCTTTACC
CCATTTACAAGTTCATCCAATCAGACTTCTTAACGCCTCATCTGGCTTATATCAAGCT
TACCAACAGTTCAGAAACTCCCAGTCCAAGTTTCTTGCTTGAAAGTGCGAAGAATGG
TGACACCGTTGACAGGTACACCTTTATGGGACATTCCCCCAGAAAAATAATCAAGAC
TGGGCCTTTAGAGGGTGCTGAAGTTGACCCCTTGGTGCTTCTGGAAAAAGAACTGAA
GGGCACCAGACAAGCGCAACTTCCTGGTATTCCTCGTCTAAGTGGTGGTGCCATAGG
ATACATCTCGTACGATTGTATTAAGTACTTTGAACCAAAAACTGAAAGAAAACTGAA
AGATGTTTTGCAACTTCCGGAAGCAGCTTTGATGTTGTTCGACACGATCGTGGCTTTT
GACAATGTTTATCAAAGATTCCAGGTAATTGGAAACGTTTCTCTATCCGTTGATGAC
TCGGACGAAGCTATTCTTGAGAAATATTATAAGACAAGAGAAGAAGTGGAAAAGAT
CAGTAAAGTGGTATTTGACAATAAAACTGTTCCCTACTATGAACAGAAAGATATTAT
TCAAGGCCAAACGTTCACCTCTAATATTGGTCAGGAAGGGTATGAAAACCATGTTCG
CAAGCTGAAAGAACATATTCTGAAAGGAGACATCTTCCAAGCTGTTCCCTCTCAAAG
GGTAGCCAGGCCGACCTCATTGCACCCTTTCAACATCTATCGTCATTTGAGAACTGT
CAATCCTTCTCCATACATGTTCTATATTGACTATCTAGACTTCCAAGTTGTTGGTGCT
TCACCTGAATTACTAGTTAAATCCGACAACAACAACAAAATCATCACACATCCTATT
GCTGGAACTCTTCCCAGAGGTAAAACTATCGAAGAGGACGACAATTATGCTAAGCA
ATTGAAGTCGTCTTTGAAAGACAGGGCCGAGCACGTCATGCTGGTAGATTTGGCCAG
AAATGATATTAACCGTGTGTGTGAGCCCACCAGTACCACGGTTGATCGTTTATTGAC
TGTGGAGAGATTTTCTCATGTGATGCATCTTGTGTCAGAAGTCAGTGGAACATTGAG
ACCAAACAAGACTCGCTTCGATGCTTTCAGATCCATTTTCCCAGCAGGAACCGTCTC
CGGTGCTCCGAAGGTAAGAGCAATGCAACTCATAGGAGAATTGGAAGGAGAAAAG
AGAGGTGTTTATGCGGGGGCCGTAGGACACTGGTCGTACGATGGAAAATCGATGGA
CACATGTATTGCCTTAAGAACAATGGTCGTCAAGGACGGTGTCGCTTACCTTCAAGC
CGGAGGTGGAATTGTCTACGATTCTGACCCCTATGACGAGTACATCGAAACCATGAA
CAAAATGAGATCCAACAATAACACCATCTTGGAGGCTGAGAAAATCTGGACCGATA
GGTTGGCCAGAGACGAGAATCAAAGTGAATCCGAAGAAAACGATCAATGAACGGA
GGACGTAAGTAGGAATTTATGGTTTGGCCATAATGGCCTAGCTTGGCGTAATCATGG
TCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGA
GCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATT
AATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCA
TTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGC
TTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGC
TCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGA
ACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCT
GGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAG
TCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAA
GCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTT
TCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCG
GTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGAC
CGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTA
TCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGG
TGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATT
TGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTG
ATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGAT
TACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGA
CGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAA
GGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTA
TATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCT
CAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAAC
TACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACC
CACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAG
CGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGG
GAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCT
ACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCC
AACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCT
TCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTA
TGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGAC
TGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTC
TTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCT
CATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAG
ATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTC
ACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAA
TAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAA
GCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAA
ATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAA
GAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTT CGTC
(anti-Her2/trastuzumab IgG1 H chain, Q105N (Kabat), pGLY10044) SEQ
ID NO: 4 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARTYPTNGYT
RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGNGT
LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (anti-Her2/trastuzumab IgG1
H chain, S134N (EU), pGLY10045) SEQ ID NO: 5
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARTYPTNGYT
RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT
LVTVSSASTKGPSVFPLAPSSKNTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (anti-Her2/trastuzumab IgG1
H chain, G161T (EU), pGLY10046) SEQ ID NO: 6
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYT
RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT
LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSTALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (anti-Her2/trastuzumab IgG1
H chain, Q175N (EU), pGLY10047) SEQ ID NO: 7
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYT
RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT
LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLNSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (anti-Her2/trastuzumab IgG1
H chain, N203T (EU), pGLY10048) SEQ ID NO: 8
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYT
RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT
LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVTHKPSNTKVDKKVEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (anti-Her2/trastuzumab IgG1
H chain, V363T (EU), pGLY10049) SEQ ID NO: 9
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYT
RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT
LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA
PELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQTSLTCLVKGEYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (anti-Her2/trastuzumab IgG1
H chain, Q386T (EU), pGLY10050)
SEQ ID NO: 10
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYT
RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT
LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA
PELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGTPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (anti-Her2/trastuzumab IgG1
H chain, Q438N (EU), pGLY10051) SEQ ID NO: 11
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYT
RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT
LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA
PELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTNKSLSLSPG (anti-Her2/trastuzumab IgG1
H chain, S113N (Kabat), pGLY14120) SEQ ID NO: 12
MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFYAMDYWGQGTLVTVSNASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(anti-Her2/trastuzumab IgG1 H chain, A118N (EU), pGLY14121) SEQ ID
NO: 13 MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVARTYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFYAMDYWGQGTLVTVSSNSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(anti-Her2/trastuzumab IgG1 H chain, S132N (EU), pGLY14122) SEQ ID
NO: 14 MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVARTYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSNKSTSGGTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(anti-Her2/trastuzumab IgG1 H chain, K133N (EU), pGLY14123) SEQ ID
NO: 15 MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSNSTSGGTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(anti-Her2/trastuzumab IgG1 H chain, A162N (EU), pGLY14124) SEQ ID
NO: 16 MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVARTYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV
TVSWNSGNLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(anti-Her2/trastuzumab IgG1 H chain, T195N (EU), pGLY14125) SEQ ID
NO: 17 MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGNQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(anti-Her2/trastuzumab IgG1 H chain, K210T (EU), pGLY14126) SEQ ID
NO: 18 MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTTVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(anti-Her2/trastuzumab IgG1 H chain, Y391T (EU), pGLY14127) SEQ ID
NO: 19 MREPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NTKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(anti-Her2/trastuzumab IgG1 H chain, F423T (EU), pGLY14128) SEQ ID
NO: 20 MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVTSCSVMHEALHNHYTQKSLSLSPG
(anti-Her2/trastuzumab IgG1 H chain, Y436T (EU), pGLY14129) SEQ ID
NO: 21 MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHTTQKSLSLSPG
(anti-Her2/trastuzumab IgG1 H chain, L193N (EU), pGLY14130) SEQ ID
NO: 22 MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSNGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(anti-Her2/trastuzumab IgG1 H chain, Q419N, N421T (EU), pGLY14131)
SEQ ID NO: 23
MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQNGTVFSCSVMHEALHNHYTQKSLSLSPG
(anti-Her2/trastuzumab IgG1 H chain, S176N, G178T (EU), pGLY14132)
SEQ ID NO: 24
MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQNSTLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(anti-Her2/trastuzumab IgG1 H chain, S191N, L193T (EU), pGLY14133)
SEQ ID NO: 25
MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSNSTGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(anti-Her2/trastuzumab IgG1 H chain, G194N, Q196T (EU), pGLY14134)
SEQ ID NO: 26
MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLNTTTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(anti-Her2/trastuzumab IgG1 H chain, G161T, S134N (EU), pGLY14135)
SEQ ID NO: 27
MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKNTSGGTAALGCLVKDYFPEPV
TVSWNSTALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(anti-Her2/trastuzumab IgG1 H chain, G161S, S134N (EU), pGLY14136)
SEQ ID NO: 28
MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKNTSGGTAALGCLVKDYFPEPV
TVSWNSSALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK
VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA
PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (null-Her2
IgG1 H chain, F243A, F264A (EU), pGLY11576) SEQ ID NO: 29
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAEIYPTNGYTR
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFGAMDYWGQGTL
VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP
ELLGGPSVFLAPPKPKDTLMISRTPEVTCVVADVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (null-Her2 L chain,
pGLY11576) SEQ ID NO: 30
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVP
SRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPS
DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL
TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (null-Her2 IgG1 H chain, S134N
(EU), pGLY14137) SEQ ID NO: 31
MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVAEIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKNTSGGTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLAPPKPKDTLMISRTPEVTCVVADVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(null-Her2 IgG1 H chain, G161T (EU), pGLY14138) SEQ ID NO: 32
MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVAEIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV
TVSWNSTALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLAPPKPKDTLMISRTPEVTCVVADVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(null-Her2 IgG1 H chain, S134N, G161T (EU), pGLY14139) SEQ ID NO:
33 MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVAEIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKNTSGGTAALGCLVKDYFPEPV
TVSWNSTALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLAPPKPKDTLMISRTPEVTCVVADVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(null-Her2 IgG1 H chain, S134N, G161T, N203T (EU), pGLY14172) SEQ
ID NO: 34
MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVAEIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKNTSGGTAALGCLVKDYFPEPV
TVSWNSTALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVTHKPSNTKVDKK
VEPKSCDKTHTCPPCPAPELLGGPSVFLAPPKPKDTLMISRTPEVTCVVADVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA
PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (null-Her2
IgG1 H chain, K30T, Y56T (Kabat), S134N (EU), G161T (EU),
pGLY14173) SEQ ID NO: 35
MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNITDTYIHWVRQAP
GKGLEWVAEIYPTNGTTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKNTSGGTAALGCLVKDYFPEPV
TVSWNSTALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLAPPKPKDTLMISRTPEVTCVVADVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(null-Her2 IgG1 H chain, K30T (Kabat), K64N/R66T (Kabat), S134N
(EU), G161T (EU), pGLY14174) SEQ ID NO: 36
MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNITDTYIHWVRQAP
GKGLEWVAEIYPTNGYTRYADSVNGTFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKNTSGGTAALGCLVKDYFPEPV
TVSWNSTALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLAPPKPKDTLMISRTPEVTCVVADVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(null-Her2 IgG1 H chain, Y56T, K64N/R66T (Kabat), S134N(EU), G161T
(EU), N203T (EU), pGLY14175) SEQ ID NO: 37
MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVAEIYPTNGTTRYADSVNGTFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKNTSGGTAALGCLVKDYFPEPV
TVSWNSTALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVTHKPSNTKVDKK
VEPKSCDKTHTCPPCPAPELLGGPSVFLAPPKPKDTLMISRTPEVTCVVADVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA
PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (null-Her2
IgG1 H chain, Y56T (Kabat), S134N (EU), G161T (EU), S176N/G178T
(EU), N203T (EU), pGLY14176) SEQ ID NO: 38
MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP
GKGLEWVAEIYPTNGTTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKNTSGGTAALGCLVKDYFPEPV
TVSWNSTALTSGVHTFPAVLQNSTLYSLSSVVTVPSSSLGTQTYICNVTHKPSNTKVDKK
VEPKSCDKTHTCPPCPAPELLGGPSVFLAPPKPKDTLMISRTPEVTCVVADVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA
PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (null-Her2
IgG1 H chain, K30T (Kabat), Y56T (Kabat), K64N/R66T (Kabat), S134N
(EU), G161T (EU), N203T (EU), pGLY14177) SEQ ID NO: 39
MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNITDTYIHWVRQAP
GKGLEWVAEIYPTNGTTRYADSVNGTFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKNTSGGTAALGCLVKDYFPEPV
TVSWNSTALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVTHKPSNTKVDKK
VEPKSCDKTHTCPPCPAPELLGGPSVFLAPPKPKDTLMISRTPEVTCVVADVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA
PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (null-Her2
IgG1 H chain, K30T, Y56T, K64N/R66T (Kabat), S134N, G161T,
S176N/G178T, N203T, V363T, K392T, F423T (EU), pGLY14178) SEQ ID NO:
40 MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNITDTYIHWVRQAP
GKGLEWVAEIYPTNGTTRYADSVNGTFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKNTSGGTAALGCLVKDYFPEPV
TVSWNSTALTSGVHTFPAVLQNSTLYSLSSVVTVPSSSLGTQTYICNVTHKPSNTKVDKK
VEPKSCDKTHTCPPCPAPELLGGPSVFLAPPKPKDTLMISRTPEVTCVVADVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA
PIEKTISKAKGQPREPQVYTLPPSREEMTKNQTSLTCLVKGFYPSDIAVEWESNGQPENN
YTTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVTSCSVMHEALHNHYTQKSLSLSPG (null-Her2
IgG1 H chain, K30T (Kabat), Y56T (Kabat), K64N/R66T (Kabat), S134N
(EU), G161T (EU), L193N (EU), N203T (EU), V363T (EU), K392T (EU),
F423T (EU), pGLY14179) SEQ ID NO: 41
MRFPSIFTAVLFAASSALAEVQLVESGGGLVQPGGSLRLSCAASGFNITDTYIHWVRQAP
GKGLEWVAEIYPTNGTTRYADSVNGTFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW
GGDGFGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKNTSGGTAALGCLVKDYFPEPV
TVSWNSTALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSNGTQTYICNVTHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLAPPKPKDTLMISRTPEVTCVVADVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQTSLTCLVKGFYPSDIAVEWESNGQPEN
NYTTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVTSCSVMHEALHNHYTQKSLSLSPG
(anti-mouse PD1 chimeric IgG1 H chain, pGLY13649) SEQ ID NO: 42
EVQLVESGGGLVQPGGSLKLSCAASGFTFSNSGLAWVRQAPEKGLEWVATITYNGTST
YYRDSVKGRFTISRDNAKNTLYLQMSSLRSEDTATYYCARWVPGSGNFDYWGQGTLV
TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (anti-mouse PD1 mouse L chain,
pGLY13649) SEQ ID NO: 43
DIVLTQSPASLAVSLGQRATISCRASQSVTISRYTLMHWYQQKPGQPPKLLIYRASNLAS
GIPARFSGSGSGTDFTLNIHPVEEDDAATYYCQQSRESPWTFGGGTKLEIKRADAAPTVS
IFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSM
SSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (anti-mouse PD1 chimeric
IgG1 H chain, S134N (EU), pGLY14163) SEQ ID NO: 44
EVQLVESGGGLVQPGGSLKLSCAASGFTFSNSGLAWVRQAPEKGLEWVATITYNGTST
YYRDSVKGRFTISRDNAKNTLYLQMSSLRSEDTATYYCARWVPGSGNFDYWGQGTLV
TVSSASTKGPSVFPLAPSSKNTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY
TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (anti-mouse PD1 chimeric IgG1
H chain, G161T (EU), pGLY14164) SEQ ID NO: 45
EVQLVESGGGLVQPGGSLKLSCAASGFTFSNSGLAWVRQAPEKGLEWVATITYNGTST
YYRDSVKGRFTISRDNAKNTLYLQMSSLRSEDTATYYCARWVPGSGNFDYWGQGTLV
TVSSASTKGPSVFPLAPSSKNTSGGTAALGCLVKDYFPEPVTVSWNSTALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (anti-mouse PD1 chimeric IgG1 H
chain, S134N, G161T (EU), pGLY14165) SEQ ID NO: 46
EVQLVESGGGLVQPGGSLKLSCAASGFTFSNSGLAWVRQAPEKGLEWVATITYNGTST
YYRDSVKGRFTISRDNAKNTLYLQMSSLRSEDTATYYCARWVPGSGNFDYWGQGTLV
TVSSASTKGPSVFPLAPSSKNTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY
TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (anti-CS1 IgG1 H chain,
pGLY8040) SEQ ID NO: 47
EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTIN
YAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWGQGTLVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (anti-CS1 L chain, pGLY8040) SEQ
ID NO: 48
DIQMTQSPSSLSASVGDRVTITCKASQDVGIAVAWYQQKPGKVPKLLIYWASTRHTGVP
DRFSGSGSGTDFTLTISSLQPEDVATYYCQQYSSYPYTFGQGTKVEIKRTVAAPSVFIFPP
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST
LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (anti-CS1 IgG1 H chain, S134N
(EU), pGLY14157) SEQ ID NO: 49
EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTIN
YAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWGQGTLVT
VSSASTKGPSVFPLAPSSKNTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (anti-CS1 IgG1 H chain, G161T
(EU), pGLY14158) SEQ ID NO: 50
EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTIN
YAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWGQGTLVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSTALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (anti-CS1 IgG1 H chain, S134N,
G161T (EU), pGLY14159) SEQ ID NO: 51
EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTIN
YAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWGQGTLVT
VSSASTKGPSVFPLAPSSKNTSGGTAALGCLVKDYFPEPVTVSWNSTALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (anti-CD70 IgG1 H chain,
pGLY14148) SEQ ID NO: 52
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYIMHWVRQAPGKGLEWVAVISYDGRNK
YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDTDGYDFDYWGQGTLVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG anti-CD70 L chain, pGLY14148)
SEQ ID NO: 53
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA
RFSGSGSGTDFTLTISSLEPEDFAVYYCQQRTNWPLTFGGGTKVEIKRTVAAPSVFIFPPS
DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL
TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (anti-CD70 IgG1 H chain, S134N
(EU), pGLY14149) SEQ ID NO: 54
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYIMHWVRQAPGKGLEWVAVISYDGRNK
YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDTDGYDFDYWGQGTLVT
VSSASTKGPSVFPLAPSSKNTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (anti-CD70 IgG1 H chain, G161T
(EU), pGLY14150) SEQ ID NO: 55
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYIMHWVRQAPGKGLEWVAVISYDGRNK
YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDTDGYDFDYWGQGTLVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSTALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (anti-CD70 IgG1 H chain, S134N,
G161T (EU), pGLY14151) SEQ ID NO: 56
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYIMHWVRQAPGKGLEWVAVISYDGRNK
YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDTDGYDFDYWGQGTLVT
VSSASTKGPSVFPLAPSSKNTSGGTAALGCLVKDYFPEPVTVSWNSTALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Protein sequence of Galactose
Oxidase from Fusarium graminearum Genbank: P0CS93) SEQ ID NO: 57
MKHLLTLALCFSSINAVAVTVPHKAVGTGIPEGSLQFLSLRASAPIGSAISRNNWAVTCD
SAQSGNECNKAIDGNKDTFWHTFYGANGDPKPPHTYTIDMKTTQNVNGLSMLPRQDG
NQNGWIGRHEVYLSSDGTNWGSPVASGSWFADSTTKYSNFETRPARYVRLVAITEANG
QPWTSIAEINVFQASSYTAPQPGLGRWGPTIDLPIVPAAAAIEPTSGRVLMWSSYRNDAF
GGSPGGITLTSSWDPSTGIVSDRTVTVTKHDMFCPGISMDGNGQIVVTGGNDAKKTSLY
DSSSDSWIPGPDMQVARGYQSSATMSDGRVFTIGGSWSGGVFEKNGEVYSPSSKTWTSL
PNAKVNPMLTADKQGLYRSDNHAWLFGWKKGSVFQAGPSTAMNWYYTSGSGDVKSA
GKRQSNRGVAPDAMCGNAVMYDAVKGKILTFGGSPDYQDSDATTNAHIITLGEPGTSP
NTVFASNGLYFARTFHTSVVLPDGSTFITGGQRRGIPFEDSTPVFTPEIYVPEQDTFYKQN
PNSIVRVYHSISLLLPDGRVFNGGGGLCGDCTTNHFDAQIFTPNYLYNSNGNLATRPKIT
RTSTQSVKVGGRITISTDSSISKASLIRYGTATHTVNTDQRRIPLTLTNNGGNSYSFQVPSD
SGVALPGYWMLFVMNSAGVPSVASTIRVTQ (Gene sequence of Galactose Oxidase
from Fusarium graminearum) SEQ ID NO: 58
ATGGCCGATCAGCAAACGGTCCTTAGTGTATCCGTACCTGGATATATAAGACTGGAA
GATATCAGTTGTTCTTCATCTGCCAGTATCACCTTCATTATCTATTCAAGTCACTCTC
TCAACTTATTCTTGCCTCTCTCTATGTCAATATGAAACACTTTTTATCACTCGCTCTTT
GCTTCAGCAGCATCAATGCTGTTGCTGTCACCGTCCCTCACAAGTCCGGAGGAACTG
GAAGTCCTGAAGGGAGTCTTCAGTTCCTGAGTCTTCGGGCCTCAGCACCTATCGGAA
GCGCTATTTCTCGCAACAACTGGGCCGTCACTTGCGACAGTGCACAGTCGGGAAATG
AATGCAACAAGGCCATCGATGGCAACAAGGATACCTTTTGGCACACATTCTATGGG
GCCAATGGAGATCCAAAGCCCCCTCACACATACACGATTGACATGAAGACAACTCA
GAATGTCAACGGCTTGTCTATGTTGCCTCGACAGGATGGTAACCAAAACGGCTGGAT
CGGTCGCCATGAGGTTTATCTAAGCTCAGATGGCACAAACTGGGGCAGCCCTGTTGC
GTCAGGTAGTTGGTTTGCCGACTCTACTACAAAATACTCCAACTTTGAAACTCGCCC
TGCTCGCTATGTTCGTCTTGTCGCTGTCACTGAAGCGAATGGCCAGCCTTGGACTAG
CATTGCAGAGATCAACGTCTTCCAAGCTAGTTCTTACACAGCCCCTCAGCCTGGCCT
TGGCCGCTGGGGTCCGACTATTGACTTGCCGATTGTTCCTGCGGCTGCAGCAATTGA
GCCGACATCGGGACGAGTCCTTATGTGGTCTTCGTATCGCAATGATGCATTTGGAGG
ATCCCCTGGTGGTATCACTTTGACGTCTTCGTGGGATCCATCCACTGGCATTGTTTCC
GACCGCACTGTGACAGTCACCAAGCATGATATGTTCTGCCCTGGTATCTCCATGGAT
GGTAACGGTCAGATCGTAGTCACAGGTGGCAACGACGCCAAGAAGACCAGTTTGTA
TGATTCATCTAGCGATAGCTGGATCCCGGGACCTGACATGCAAGTGGCTCGTGGGTA
TCAGTCATCAGCTACCATGTCAGACGGTCGTGTTTTTACCATTGGAGGCTCCTGGAG
CGGTGGCGTATTTGAGAAGAATGGCGAAGTCTATAGCCCATCTTCAAAGACATGGA
CGTCCCTACCCAATGCCAAGGTCAACCCAATGTTGACGGCTGACAAGCAAGGATTG
TACCGTTCAGACAACCACGCGTGGCTCTTTGGATGGAAGAAGGGTTCGGTGTTCCAA
GCGGGACCTAGTACAGCCATGAACTGGTACTATACCAGTGGAAGTGGCGATGTGAA
GTCAGCCGGAAAACGCCAGTCTAACCGTGGTGTAGCCCCTGATGCCATGTGCGGAA
ACGCTGTCATGTACGACGCCGTTAAAGGAAAGATCCTGACCTTTGGCGGCTCCCCAG
ACTATCAAGACTCTGACGCCACAACCAACGCCCACATCATCACCCTCGGTGAACCCG
GAACATCTCCCAACACTGTCTTTGCTAGCAATGGCTTGTACTTTGCTCGAACGTTCCA
CACCTCTGTTGTTCTTCCAGACGGAAGCACGTTCATTACAGGAGGCCAACGACGTGG
AATTCCGTTCGAGGATTCAACCCCGGTATTTACACCTGAGATCTACGTCCCTGAACA
AGACACTTTCTACAAGCAGAACCCCAACTCCATTGTTCGCGTCTACCACAGCATTTC
CCTTTTGTTACCTGATGGCAGGGTATTTAACGGTGGTGGTGGTCTTTGTGGCGATTGT
ACCACGAATCATTTCGACGCGCAAATCTTTACGCCAAACTATCTTTACAATAGCAAC
GGCAATCTCGCGACACGTCCCAAGATTACCAGAACCTCTACACAGAGCGTCAAGGT
CGGTGGCAGGATCACAATCTCGACGGACTCTTCGATTACAAAGGCGTCGTTGATTCG
CTATGGTACAGCGACACACACGGTTAATACTGACCAGCGTCGCATTCCCCTGACTCT
GACAAACAATGGAGGAAATAGCTATTCTTTCCAAGTTCCTAGCGACTCTGGTGTTGC
TTTGCCTGGCTACTGGATGTTGTTCGTGATGAACTCGGCCGGTGTTCCTAGTGTGGCT
TCGACGATTCGCGTTACTCAGTGA
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EXAMPLES
Example 1: Expression of Anti-Her2 with Non-Native
N-Glycan-Incorporating Site-Directed Alterations
[0271] The anti-HER2 (trastuzumab) IgG1 H chain and L chain
sequences (Seq ID NO: 1 and 2, respectively) were incorporated into
a single Zeo.sup.R-marked, TRP2-integrating Pichia pastoris roll-in
expression plasmid, each as part of separate AOX1-driven expression
cassettes containing the Saccharomyces cerevisiae alpha factor pre
signal sequence (Seq ID NO: 3) to generate plasmid pGLY5883 (FIG.
3; SEQ ID NO: 4). Site-directed mutations were then designed based
on desirable locations within the H chain. These locations were
chosen as sites that would: 1) be within or near loop or where a
side chain was solvent exposed to not disrupt an Ig-fold, 2) not be
near critical sites related to mAb function (e.g. Ag-binding, FcRN
binding, Fc.gamma.R-binding), and 3) be converted to an N-glycan
sequon with a minimum of primary sequence modification. Initially 8
sites were chosen to test whether non-native N-glycosylation sites
can be efficiently incorporated into an immunoglobulin with a high
degree of N-glycan occupancy without disrupting the normal folding
or function of the parent antibody. These 8 sites are indicated in
Table 1.
[0272] Plasmids pGLY10044-10051, constructed by Genewiz (South
Plainfield, N.J.), are derived from plasmid pGLY5883 and differ
only by the indicated non-native N-glycosylation site introducing
mutations in the anti-HER2 H chain cassette (Table 1) resulting in
SEQ ID NO: 4-11.
TABLE-US-00002 TABLE 1 Heavy chain Sequence Sequence Sequence
Secreted N-glycan Conjugated protein change change change and
folded site is with Plasmid Seq ID (EU) (anti-HER2) (Kabat) mAb?
occupied? Fluorophore? pGLY10044 Seq. ID NO: 4 N/A Q112N Q105N Yes
Yes No pGLY10045 Seq. ID NO: 5 S134N S137N S130N Yes Yes Yes
pGLY10046 Seq. ID NO: 6 G161T G164T G158T Yes Yes Yes pGLY10047
Seq. ID NO: 7 Q175N Q178N Q179N No N/A N/A pGLY10048 Seq. ID NO: 8
N203T N206T N211T Yes Yes Yes pGLY10049 Seq. ID NO: 9 V363T V366T
V386T Yes Yes Yes pGLY10050 Seq. ID NO: 10 Q386T Q389T Q414T Yes No
N/A pGLY10051 Seq. ID NO: 11 Q438N Q441N Q469N Yes Yes Yes
[0273] Each of these plasmids was transformed into strain
YGLY30329, a glycoengineered strain of P. pastoris that has been
genetically engineered to produce N-glycans of the human complex
type with terminal galactose acid residues (FIG. 1, GS5.0; see
also, e.g., Bobrowicz, 2004). Transformations were performed as
previously described (Cregg et al, 2000) and clones were selected
on YSD (100 yeast extract, 2% soytone, 2% dextrose) agar plates
containing 100 .mu.g/ml Zeocin (Life Technologies, Carlsbad,
Calif.). Clones were then cultivated in 96 deep well plates (DWP),
in BMGY liquid medium, and induced in BMMY containing methanol as a
sole carbon source, as previously described (Barnard et al, 2010).
Cultures were centrifuged at 2500.times.g for 10 minutes in a
Beckman swinging bucket centrifuge and supernatants were subjected
to protein A purification (Jiang et al, 2011). The protein
A-purified samples were subjected to capillary electrophoresis (CE)
using a Caliper GXII (Perkin Elmer, Waltham, Mass.) using the
standard HT Protein Express 200 method as detailed previously
(Gomathinayagam, 2012). Upon CE analysis, seven of the eight
constructs yielded bands consistent with heavy and light chain
under reducing conditions and a band consistent with a uniform,
well assembled antibody under non-reducing conditions when
visualized using the Caliper LabChip GX software version 4.1 (FIG.
4). The clones transformed with plasmid pGLY10047 (Q175N) did not
produce any antibody presumably because the additional
N-glycosylation site disrupted antibody folding and prevented
proper assembly. Thus, even though the engineered sites were
carefully chosen to be exposed and not affect antibody folding,
there is an empirical aspect to the N-glycan site engineering that
was not predicted.
Example 2: Microreactor Cultivation of N-Glycan Site Engineered
Anti-Her2 Antibody-Producing Clones
[0274] Representative clones from the seven constructs that yielded
fully folded antibody were cultivated in an Applikon (Foster City,
Calif.) micro24 5 ml mini-fermenter apparatus. Seed cultures were
prepared by inoculating strains from YSD plates to a Whatman
24-well Uniplate (10 ml, natural polypropylene) containing 3.5 ml
of 4% BMGY medium (Invitrogen, Carlsbad, Calif.) buffered to pH 6.0
with potassium phosphate buffer. The seed cultures were grown 65-72
hours in a temperature controlled shaker at 24.degree. C. and 650
rpm agitation. 1.0 ml of the 24 well plate grown seed culture and
4.0 ml of 4% BMGY medium was then used to inoculate each well of a
Micro24 plate (Type: REG2). 30 ml of Antifoam 204 (1:25 dilution,
Sigma Aldrich) was added to each well. The Micro24 was operated in
Microaerobic1 mode and the fermentations were controlled at 200%
dissolved oxygen, pH at 6.5, temperature at 24.degree. C. and
agitation at 800 rpm. The induction phase was initiated upon
observance of a dissolved oxygen (DO) spike after the growth phase
by adding bolus shots of methanol feed solution (100% [w/w]
methanol, 5 mg/l biotin and 12.5 ml/l PTM2 salts), 50p in the
morning and 125 .mu.l in the afternoon. After approximately 72
hours of methanol induction, the cell-free culture supernatant was
harvested by centrifugation at 2500.times. g in a Beckman swinging
bucket centrifuge and subjected to protein A purification by
standard methods (Jiang, 2011).
[0275] Antibody was quantified by reverse phase HPLC (Barnard et
al, 2010) and also analyzed by capillary electrophoresis as
described above, in this case both under non-reduced and reduced
conditions. In all cases upon analysis using the Caliper LabChip GX
software version 4.1, the selected clones produced protein
consistent with well-assembled antibody, and consisting of a single
heavy and light chain band in the reducing condition (FIG. 5).
[0276] The protein was further subjected to Quadrapole
Time-of-Flight Liquid chromatography/Mass spectrometry (Q-ToF mass
spectrometry or Q-ToF LCMS or Q-ToF MS) analysis under reduced
conditions as described previously (Lynaugh et al, 2013). Briefly,
5 .mu.l (1 mg/ml) was injected in an Agilent Q-TOF 6520 mass
spectrometer. The dual ESI ion source was set as follows: gas temp
at 350.degree. C.; drying gas at 13 L/min; nebulizer at 45 psig;
fragmentor at 150 V; skimmer at 65 V; Oct1 RF VPP at 750 V; Vcap at
3500 V. Data were analyzed using MassHunter software. Of the seven
constructs that were tested, six resulted in proteins where the
second engineered N-glycan site was occupied with a glycan in
addition to the canonical N-297 site (FIG. 6). The only exception
is in panel 5F where only a single N-glycan is added, the Fc N-297
glycan. Therefore it was concluded that the engineered N-glycan
site in pGLY10050 (resulting from the Q386T mutation) is not
efficiently occupied. The remaining samples resulted in profiles
consistent with a majority of the protein being occupied with two
N-glycans per H chain (4 N-glycans per mAb), one at N-297 and
another at each of the respective engineered sites.
[0277] Therefore, based on this evidence, it is not possible to
simply select a site and introduce an Asn-turn sequence through
mutagenesis that will lead to a well-folded protein and be
efficiently N-glycan occupied.
Example 3: Bioreactor Expression and Purification of Non-Native
N-Glycosylation Site Engineered Anti-HER2 Antibody
[0278] Representative clones from the six constructs that yielded
fully folded antibody with a well-occupied non-native
N-glycosylation site (See Table 1) were cultured in 1 L Fedbatch
Pro fermenters (DASGIP Biotools, Shrewsbury, Mass.) using a
glycerol fedbatch and methanol induction similarly to what has been
described previously (Hopkins, 2011), with the notable difference
of using a dissolved oxygen (DO) limited fed-batch induction
paradigm. Briefly, inocula derived from yeast patches (isolated
from a single colony) on agar plates were cultivated in 0.5 L
baffled seed flasks in 0.1 L of 4% BSGY (without maltitol, table
2). Seed flasks were grown at 180 rpm and 24.degree. C. (Innova 44,
New Brunswick Scientific) for 48 hours. Bioreactor vessels were
charged with 0.6 L of 0.2 .mu.m filtered 4% BSGY media (plus 4
drops/L Sigma 204 antifoam, Table 2) and autoclaved at 121.degree.
C. for 45 minutes.
TABLE-US-00003 TABLE 2 Media/Reagent Composition 4% BSGY 40 g/L
glycerol, 20 g/L soytone, 10 g/L yeast extract, medium: 11.9 g/L
KH2PO4, 2.3 g/L K2HPO4, 50 g/L maltitol, 13.4 g/L YNB with ammonium
sulfate without amino acids, 8 mg/L Biotin. PTM2 salts: 0.6 g/L
CuSO4--5H2O, 80 mg/L NaI, 1.8 g/L MnSO--4H2O, 20 mg/L H3BO4, 6.5
g/L FeSO4--7H2O, 2.0 g/L ZnCl2, 0.5 g/L CoCl2--6H2O, 0.2 g/L
Na2MoO4--2H2O, 0.2 g/L biotin, 5 mL/L H2SO4 (85%)
[0279] After sterilization and cooling, the aeration, agitation,
and temperatures were set to 0.7 vvm, 600 rpm, and 24.degree. C.
respectively. The pH was adjusted to and controlled at 6.5 using
15% ammonium hydroxide. Inoculation of a prepared bioreactor
occurred aseptically with 60 mL from a seed flask. Agitation was
ramped to maintain 20% DO saturation. After the initial glycerol
charge was consumed, denoted by a sharp increase in the DO, a 50%
w/w glycerol solution containing 5 mg/L biotin and 32.3 mg/L PMTi4
was triggered to feed at 7.7 g/L-h for 8 hours. During the glycerol
fed-batch phase 0.42 mL of PTM2 salts (Table 2) was injected
manually. After completion of the glycerol fed-batch phase, the
agitation rate was locked at 600 rpm and a bolus addition of 6.0 g
of methanol containing 5 mg/L biotin and 12.5 mL/L PTM2 salts was
added. During methanol induction phase the DO remains near 0% until
the methanol bolus is entirely consumed. Each time the DO increases
to >30% another 6.0 g bolus of the methanol feed solution is
added to prolong the induction time. After methanol adaptation, it
takes on average 9-10 hours to consume the 1% methanol boluses.
Injections of 0.25 mL of 2.1 mg/mL PMTi4 (in methanol) were added
each 24 hours of induction time. After 80-90 hours of methanol
induction, the cell-free culture supernatant was harvested by
centrifugation (Sorvall Evolution RC, Thermo Scientific) at 8500
rpm for 40 minutes and then subjected to small scale protein A
purification by standard methods (Jiang, 2011).
[0280] Antibody was quantified by reverse phase TIPLC and
calculated on a per liter basis (Barnard et al, 2010). Fermentation
titers indicated that the N-glycan sites that were occupied and
tolerated by the mAb structure based on small scale expression
resulted in no significant alteration in mAb titer at 1 L
fermentation scale (Table 3).
TABLE-US-00004 TABLE 3 Sequence mAb liter mAb titer Ferm. 1 L Ferm.
change (mg/L) (mg/L) # Sample ID Strain name (EU) HPLC Bradford 1
D133201 YGLY35490 N203T 332 ND 2 D133202* YGLY35491 N203T 499 440 3
D133203* YGLY35492 V363T 569 446 4 D133204 YGLY35493 V363T 279 ND 5
D133205 YGLY35494 V363T 275 ND 6 D133206 YGLY35495 Q438N 488 ND 7
D133207 YGLY35496 Q438N 269 ND 8 D133208* YGLY35497 Q438N 705 569 9
D133401 YGLY35516 Q109N.sup.# 443 ND 10 D133402 YGLY35517
Q109N.sup.# 512 ND 11 D133403 YGLY35518 S134N 276 ND 12 D133404*
YGLY35519 S134N 299 253 13 D133405 YGLY35520 G161T 646 ND 14
D133406* YGLY35521 G161T 384 305 15 D133407 YGLY35522 N203T 445 ND
16 D133408 YGLY35523 N203T 393 ND C Anti-HER2 multiple None 509 +/-
54 ND
[0281] The purified antibody was further subjected to capillary
electrophoresis and Q-ToF mass spectrometry analysis as outlined
above. As with smaller scale cultivation, the selected clones
produced protein consistent with well-assembled antibody, and
consisting of a single heavy and light chain band in the reducing
condition (FIG. 7). Based on reduced Q-T of, the clones selected
also yielded a majority of antibody with N-glycans fully occupied
at two sites, the N-297 canonical site and the respective
engineered non-native N-glycosylation site (FIG. 8A-8C). Some of
the additional sites resulted in better N-glycan uniformity than
others. For instance, the Q109N site resulted in poor conversion to
complex forms with the resulting N-glycans primarily of the hybrid
type whereas S134N resulted in predominantly complex terminally
galactosylated N-glycans, with the assumption of a mixture of
GS4.0, GS4.5 and GS5.0 at the Fc N-297 site based on previous mAb
N-glycan analysis (FIG. 8B).
[0282] Larger aliquots (500 ml) of fermentation supernatant were
purified by protein A chromatography for one each of the five best
mutations (Samples D133202, D133203, D133208, D133404, and D133406,
Table 3). The quantification of purified protein as measured by
Bradford assay agreed well with the HPLC measurements from
supernatant (Table 3). Purified protein was analyzed by Q-ToF and
revealed masses that correspond to the expected L chain mass and
several clustered masses that corresponded to the expected H chain
mass (FIG. 9A). Upon zooming to the H chain mass region on the
trace, the predominant H chain mass in each case corresponded to
the predicted anti-Her2 H chain with 2 N-glycans one comprised of a
GS4.0 glycoform and the other of a GS5.0 glycform (FIG. 9B).
Previous analysis of antibodies in the same host strain yielded
N-297 canonical Fc N-glycans consisting of primarily GS4.0 with a
minority of GS4.5 and GS5.0 glycoforms (Zhang et al, 2011). It was
therefore concluded that the additional N-linked site was occupied
with predominantly GS5.0 N-glycans. In support of this, sample
D133404 was digested with Endoglycosidase S (EndoS, Genovis,
Cambridge, Mass.) and subsequently digested with
Peptide-N-glycosidase F (PNGase, New England Biolabs, Ipswich,
Mass.). The EndoS enzyme will remove the N-297 canonical glycan,
cleaving between the core GlcNAc residues, and leave any other mAb
glycans intact. N-glycan analysis by MALDI-TOF MS of the EndoS
released N-glycans revealed the expected predominant GS4.0 mass
(less one GlcNAc which is the core GlcNAc not removed); while Q-ToF
analysis of the reduced H chain showed a mass consistent with the H
chain containing G2 and GlcNAc (FIG. 10). Moreover, PNGase
digestion of the EndoS digested sample and MALDI-TOF MS of the
released N-glycans revealed G2 and G2-GlcNAc (the EndoS enzyme,
still active in the mixture, can remove a GlcNAc from the released
G2 glycan; FIG. 10). Finally, Q-ToF analysis of the EndoS and
PNGase digested mAb yielded an expected mass consistent with the
deglycosylated H chain (FIG. 10). Taken together these data
demonstrate that the additional N-glycosylation site (atN134 in
sample D133404) is occupied with a predominant GS5.0 N-glycan while
the canonical N-297 glycan is occupied with the expected mixture of
predominantly GS4.0 plus a minority of GS4.5 and GS5.0.
Example 4: Antigen Binding of Anti-Her2 Modified Abs with
Non-Native N-Glycosylation Sites
[0283] To determine whether incorporation of non-canonical
N-glycans into the anti-Her2 mAb sequence impacts binding of the
antibody to the Her2 protein, the affinity of purifiedN-glycan
modified mAbs was measured by surface plasmon resonance using a
Biacore T-100 instrument (GE Healthcare, Little Chalfont, UK).
First, a Series S CM5 Chip (GE Healthcare) was immobilized via
amine coupling kit (GE Healthcare) to >10000 RU with an
anti-human Fc capture antibody kit (GE Healthcare). Purified
anti-Her2 antibody protein samples from batches D133202, D133203,
D133404, and D133406 along with the commercial (CHO-produced
trastuzumab) anti-Her2 were were captured to 30 RU on the active
flowcells and no antibody was captured on the reference flowcell.
Serially diluted human Her2 ectodomain (Biotang, Lexington, Mass.),
ranging in concentration from 50 nMto 0.39 nM, was injected for 5
minutes over all flowcells and dissociation was monitored for 10
minutes. Binding data was double referenced by subtracting the
reference flowcell signal and a 0 nM Her2 injection. All of the
reagents were prepared in 1.times. HBS-EP+(GE Healthcare, pH7.4)
running buffer and the binding measurements were performed on a
Biacore T100 at 25.degree. C. All data was fit with 1:1 Binding
Model in Biacore T100 Evaluation Software (v2.0.4). Analysis of the
binding curves, maximum binding capacity, and affinity, based on a
1:1 binding fit revealed no significant differences between
commercial anti-Her2, trastuzumab (FIG. 11, Panel E), and any of
the N-glycan modified, glycoengineered Pichia-produced Abs (FIG.
11, Panels A-D)
Example 5: Conjugation of an Activated Fluorophore to Enzymatically
Oxidized Terminal Galactose of N-Glycans
[0284] Next, we asked whether N-glycans at non-native
N-glycosylation sites on mAbs would be appropriate substrates and
locales for chemical conjugation. Galactose oxidase
(d-galactose:oxygen 6-oxidoreductase GO; EC 1.1.3.9)
fromFusariumgraminarium, aka Dactylium dendroides (Fg GO) is a
glycan-modifying enzyme previously shown to oxidize terminal
.beta.-1,4-galactose residues in the context of a protein (Cooper
et al, 1959). The result of this enzymatic galactose oxidation is a
chemically reactive aldehyde group that is receptive to direct
conjugation with an alkoxyamine substrate to form a stable oxime
bond (Ramya et al, 2013). However, attempts to oxidize and
efficiently conjugate to the asialylated complex Fc N-297 glycan of
a standard IgG, which typically contains a small but significant
amount of terminal .beta.-1,4-linked galactose (20-40% on one arm,
1-10% on both arms), have beenunsuccessful to date; a finding that
was recapitulated here with commercial trastuzumab (FIG. 12). Given
that the location of this canonical IgG glycan is known to be
buried between and closely tethered to the Ig folds of the Fc
C.sub.H2 domain, it is likely that steric hindrance prevents either
enzyme modification or addition of a conjugate to this N-glycan
site. As shown, when mAbs with non-native N-glycosylation sites are
produced in a GFI5.0 glycoengineered yeast strain (FIG. 2) the
additional non-native N-glycosylation sites are composed
predominantly of biantennary terminal .beta.-1,4-linked galactose
(FIG. 9). Therefore, we interrogated whether it is possible to
enzymatically oxidase and chemically conjugate an activated
fluorophore to an antibody via these engineered terminal galactose
sugars. In a one-pot reaction method, 200 .mu.g of the purified
anti-HER2 antibodies containing extra N-glycans (Table 1) was
incubated with 0.45 units of Fg GO (Sigma, St. Louis, Mo.), 800
units of catalase and an aminooxy activated CF633 dye (Biotium,
Hayward, Calif.) to a final concentration of 100 .mu.M in 50 mM
sodium phosphate buffer at pH 7 and 25.degree. C. in dark
conditions for 24 and 48 hours. For the mAb variants that were most
efficiently conjugated, the S134N and G161 T mutant anti-HER2
proteins, significant transfer of up to two fluorophore moieties
were observed per reduced H chain as determined by Q-ToF, with two
per H chain being the expected maximum number of available sites
given a theoretical 100% biantennary galactose structure and 100%
N-glycan occupancy and no conjugation at the Fe N-297 glycan (FIG.
13).
Example 6: Desialylation and Conjugation of mAbs Containing
Sialylated N-Glycans
[0285] For non-native N-glycosylation site containing mAbs produced
in GFI6.0 glycoengineered strains (FIG. 2) the N-glycans will be
predominantly sialylated and thus resistant to galactose oxidase
(which requires a terminal galactose sugar). Therefore, to generate
a substrate for enzymatic/chemical conjugation in such a strain,
plasmids expressing modified anti-Her2 mAbs containing non-native
N-glycosylation sites (pGLY14137 and pGLY14138) were transformed
into GFI6.0 strain YGLY36472 and clones were selected on 100 mg/ml
zeocin plates. The resulting clones were cultivated in micro24 5 mL
bioreactors as described earlier (see Example 2). Selected clones
were then cultivated in 1 L Dasgip bioreactors as described
(Example 3). The harvested supernatants were purified by protein A
chromatography also as referenced (Jiang, 2011). The resulting
protein was analyzed by Q-ToF under reducing conditions, which
resulted in masses consistent with the intactH chain of the
modified anti-Her2 mAb with the expected sialylated N-glycan
profile (FIG. 14). The purified protein was subsequently
desialylated using Acetyl-neuraminyl hydrolase (neuraminidase, New
England Biolab s, Ipswich, Mass.) according to the manufacturer's
recommended reaction conditions. The resulting protein was analyzed
by Q-ToF under reducing conditions and N-glycans were removed
enzymatically by PNGase digestion (New England Biolabs, Ipswich,
Mass.) and analyzed quantitatively by HPLC (Burnina, 2012). The
results indicated that as expected the sialic acid residues were
efficiently removed by the Neuraminidase enzyme leaving
predominantly terminal galactose residues (Prime, 1996), which have
been shown here and previously to be efficient substrates for the
GO enzyme to support oxime ligation.
Example 7: Optimization of Combined Enzymatic/Chemical Conjugation
Step
[0286] Conjugation reactions were next carried out with chemical
catalysts for the two most receptive acceptor position anti-Her2
muteins (S134N and G161T). Initially, three different catalysts
were used: 2-Amino-5-methoxybenzoic acid (AMB), 3,5-diaminobenzoic
acid (DAB), and aniline (Crisalli 2013). Both aniline and AMB
improved the conjugation efficiency, whereas addition of DAB did
not result in increased conjugation. The conjugation reaction was
improved most by the presence of 50 mM Aniline which, after 72 h
resulted in >90% of the available terminal galactose residues
having a fluorophore (FIG. 15). Also, temperature and pH
optimization can be applied to the initial reaction to reduce the
amount of aniline or time required for complete conjugation (not
shown).
[0287] Using these optimized conditions, conjugation reactions were
carried out for each of the five purified modified mAbs containing
non-native N-glycosylation sites (N206T, V363T, Q438N, S134N, and
G161T). The glycan modified antibodies were conjugated with
alkoxyamine-modified Alexafluor488 fluorophore (Alexa488,
Invitrogen, Claremont, Calif.). Conjugation proceeded highly
efficiently for 4 of the 5 with a significant proportion of singly
conjugated H chain remaining for the N203T variant. However, even
for this protein the plurality of resulting mAb contained two
conjugated fluorophores (FIG. 16). For the other four
glycan-modified mAbs, the predominant species (>80%) following
optimized conjugation was H chain containing two fluorophores,
where in each case the maximum number of available sites is two,
considering one extra N-glycan per H chain, each containing a
predominantly biantennary N-glycan with terminal galactose (FIG.
16). The heterogeneity of peaks observed in the Q-ToF arise from
the expected N-glycan profile atthe Fc N-297 site, which typically
contains a mixture of GS4.0, GS4.5 and GS5.0 structures with a
small amount of hybrid and mannose forms (Zha, 2013).
[0288] The minor conjugation of a 3.sup.rd site observed for three
of the glycan modified mAbs (N206T, V363T, and Q438N), can be
interpreted based on mass to be conjugation of the N-297 glycan on
hybrid galactosylated (GS3.5, FIG. 1) structures. These three
protein samples contained a larger degree of such hybrid structures
than the S134N and G161T sample preparations. While previous data
showed that conjugation was not possible to complex N-glycans at
the N-297 site, this result indicates that conjugation can be
directed to the N-297 site if the N-glycan is a hybrid structure.
This important finding demonstrates that for a mAb produced with
exclusively hybrid N-glycoforms at the N-297 site (an outcome that
is possible using glycoengineered yeast strain GFI3.5, FIG. 2),
conjugation could be performed at this site with a single available
site per N-glycan. However, confirming previous results, no
conjugation to complex N-glycan structures is observed at the N-297
site, which allows for discrete control over conjugation position
if glycosylation microheterogeneity is controlled.
[0289] Taken together, these data demonstrate that nearly
quantitative oxime conjugation can be achieved at certain
non-native N-glycosylation sites of glycan modified mAbs following
enzymatic oxidation of galactose residues to a reactive aldehyde
form. Moreover, even under conditions that promote highly efficient
conjugation to the desired site, no non-specific oxidation or
conjugation is observed. Finally under these conditions, complex
N-glycans at the N-297 of the Fc (a modest fraction of which are
galactosylated GS4.5 and GS5.0) are not oxidized by the GalOx
enzyme, thereby maintaining site-specificity of the conjugation
reaction irrespective of the presence of a complex glycan at the
canonical N-297 site. Thus, importantly, this glycan-based
conjugation is completely compatible with full effector
function-enabled antibodies.
Example 8: Scale-Up and Bioanalytical Characterization of
Glyco-Conjugated Abs
[0290] The conjugation reaction described in Example 5 (modified by
addition of aniline as described in Example 7) was scaled to larger
volume using the S134N and G161T modified anti-Her2 mAb sequences.
Alkoxyamine-modified Alexa488 and alkoxyamine modified biotin were
used as conjugation substrates (100 mM for each) and conjugation
reactions were carried out at 25.degree. C. for 72 h. The reaction
products were subjected to Q-ToF MS with the results shown in FIG.
17. In each case the DAR was calculated based on the relative
abundance of the peaks assigned to the bi-conjugated,
mono-conjugated, and unconjugated antibody (FIG. 17).
[0291] The Alexa488 conjugated G161T glycan modified anti-Her2
antibody was also subjected to IdeS (Fabricator, NEB, Ipswich,
Mass.) digestion and Q-ToF MS to confirm the location of the
conjugated dye. The IdeS digestion was carried out according to the
manufacturer's instructions. Upon digestion and MS analysis, it was
observed that the two non-native N-glycosylation sites residing on
the F(ab').sub.2 were modified with 3-4 Alexa 488 moieties while
the Fc-fragment was nearly completely unmodified.
[0292] The same Alexa488 conjugated G161T glycan modified anti-Her2
antibody (FIG. 19) was subjected to size exclusion chromatography
(SEC) along with the parental unconjugated G161 T anti-Her2 mAb
(FIG. 9B, D133406) and commercial anti-Her2 (Trastuzumab). SEC was
performed as previously described (Potgieter et al, 2009).
Retention times and peak analysis indicates that the glycan
modified anti-Her2 produced in glycoengineered Pichia and the
Alexa488 conjugated version retain a minimal propensity for
aggregation and in general have a comparable initial stability
compared to the commercial control. Thus, the addition of a
non-native N-glycosylation sites or conjugation to the glycan does
not increase propensity for aggregation or mis-folding of the
antibody.
[0293] In order to further probe stability, the glycan-modified
antibodies were incubated at 45.degree. C. for 2 weeks in 100 mM
sodium phosphate pH 7.0 at a concentration of 5 mg/ml and then
subjected to SEC. All samples retained intactness and resisted
aggregation except for the G161T modified antibody, which degraded
slightly more rapidly at 45.degree. C. than the commercial control
or other glycan-modified mAbs (FIG. 20).
Example 9: Covalent Attachment of an Aminooxy Activated GLP-1
Receptor Agonistic Peptide to an Ab Via Glyco-Conjugation
[0294] An aminooxy chemically activated Exendin-4 peptide modified
at the gamma amine of the C-terminal Lysine (FIG. 21) was
constructed by Biopeptek (Malvern, Pa.). This peptide was
conjugated to the purified S134N and G161T modified anti-Her2 Abs
using the one-pot combined enzymatic/chemical conjugation procedure
illustrated in Examples 5 (with a 100 .mu.M final concentration of
peptide and 50 mM aniline). The conjugated peptide was analyzed by
Q-ToF MS and shown to be intact and conjugated to a DAR of 3.3 out
of a theoretical maximum of 4 potential sites (FIG. 21). The
glyco-ADC exendin-4 conjugate was evaluated for its ability to bind
and activate the GLP-1 receptor (GLP-1R) using a recombinant
Chinese Hamster Ovary (CHO) cell-based assay. A recombinant CHO
stable cell line was generated by expression of the serpentine G
protein coupled receptor GLP-1R, which induces intracellular cAMP
production by native CHO Adenylyl Cyclase via G protein activation.
To evaluate glyco-ADC exendin-4 activity, CHO GLP-1R cells were
exposed to either glyco-ADC exendin-4 conjugate or native GLP-1 as
a control at a range of concentrations. Resulting changes in cAMP
levels were determined using the HitHunter cAMP XS+ system
(DiscoveRx Corporation, Fremont, Calif.) on a Tecan infinite 200Pro
reader. Results were compiled and plotted and EC50 valueswere
calculated using Graphpad Prism 5 software. The glyco-ADC exendin-4
conjugate was active with EC50 values ranging from 2-5 fold lower
than native GLP-1 peptide (FIG. 21). These data indicate that an
active peptide can be conjugated to antibodies at non-native
N-glycosylation sites via galactose oxidase and oxime ligation.
Example 10: Conjugation of a Cytotoxic Agent to an Antibody at
Non-Native N-Glycosylation Sites
[0295] An aminooxy activated C5-linker containing DM1
(alkoxy-C5-DM1, FIG. 22A) chemically synthesized by Concortis
Biosystems (San Diego, Calif.), was conjugated to the S134N and
G61T anti-Her2 mAbs using the protocol described in example 5 (with
100 pMDM1 and 50 mM aniline) to generate oxime ligated C5-DM1
conjugated mAbs. The C5-DM1 conjugated mAbs were subjected to Q-ToF
MS under reducing conditions and the G161T MS trace is shown as an
example (FIG. 22B). For both S134N and G161T C5-DM1 conjugated Abs,
masses were observed that are consistent with addition of a single
and two C5-DM1 molecules as well as a very small fraction of
unconjugated H chain. This is in contrast to the heterogeneity
observed in commercial ado-trastuzumab emtansine, which contains
from 0-8 DM1 molecules dispersed throughout the H and L chains as
evidenced by Q-ToF MS (FIG. 23). This diversity has been
demonstrated in other studies to contribute to lower serum
stability (PK) and activity (PD) compared to similar but more
homogeneous ADCs based on the same mAb scaffold, resulting in
better efficacy for the more homogeneous molecule, even with a
lower DAR (Jackson, 2014; Tian, 2014; Axup, 2012).
Example 11: Generation of Additional Non-Native N-Glycosylation
Site-Modified Antibodies for Site-Specific Glyco-Conjugation
[0296] With the knowledge that combined enzymatic/chemical
conjugation can occur efficiently at selected engineered N-glycan
sequons a further set of native N-glycosylation sites were
constructed by introducing site-directed mutants into an IgG to
determine whether these new structurally selected sites would be
suitable substrates for 1) efficient addition of N-glycans and 2)
conjugation of cargo. A list of mutations that were constructed and
the associated sequence references is found in Table 4.
TABLE-US-00005 TABLE 4 Plasmid Mutation Mutation Mutation H chain
name (EU numbering) (Herceptin numbering) (Kabat numbering)
Sequence pGLY14120 N/A S120N S113N SEQ ID NO: 12 pGLY14121 A118N
A121N A114N SEQ ID NO: 13 pGLY14122 S132N S135N S128M SEQ ID NO: 14
pGLY14123 K133N K136N K129N SEQ ID NO: 15 pGLY14124 A162N A165N
A165N SEQ ID NO: 16 pGLY14125 T195N T198N T200N SEQ ID NO: 17
pGLY14126 K210T K213T K218T SEQ ID NO: 18 pGLY14127 Y391T Y394T
Y419T SEQ ID NO: 19 pGLY14128 F423T F426T F454T SEQ ID NO: 20
pGLY14129 Y436T Y439T Y467T SEQ ID NO: 21 pGLY14130 L193N L196N
L198N SEQ ID NO: 22 pGLY14131 Q419N/N421T Q422N/N424T Q450N/N452T
SEQ ID NO: 23 pGLY14132 S176N/G178T S179N/G181T S180N/G183T SEQ ID
NO: 24 pGLY14133 S191N/L193T S194N/L196T S196N/L198T SEQ ID NO: 25
pGLY14134 G194N/Q196T G197N/Q199T G199N/Q203T SEQ ID NO: 26
[0297] Notably, at some sites more than a single mutation was
required to generate an efficient predicted NXS/T (where X is not
Pro) sequon. Mutations were introduced into the anti-Her2 IgG1 mAb
sequence in plasmid pGLY5883 (FIG. 3), generating plasmids
pGLY14120-pGLY14134, containing the AOX1 promoter and Zeocin
resistance cassette. The new anti-HER2 N-glycan mutein expressing
plasmids were transformed into glycoengineered Pichia strain
YGLY30329 by electroporation and clones were selected on medium
containing 100 .mu.g/mL and 300 .mu.g/mL Zeocin. Isolated clones
were screened for expression by 96 well plate cultivation as
described (Example 1 and Barnard, 2010). Clones demonstrated to
express mAb were then cultivated in microreactors as described
(Example 2). The supernatant was harvested and the produced
antibody from these clones was purified by protein A (Jiang et al,
2011). The purified protein was analyzed by Q-ToF MS and the
results are shown in FIGS. 24A and 24B. In each case the L chain is
the same and was observed as expected (data not shown). The MS
traces are zoomed to show the region where the expected H chain
masses would reside. The expected mass range for a naked H chain, a
H chain with a single N-glycan, and a H chain with two N-glycans is
indicated (masses vary slightly due to the mutations introduced).
For all Abs shown the predominant peaks observed correspond to H
chains with 2 N-linked glycans (the N-297 glycan plus one other
glycan at a different location in each case) with the masses
varying slightly due to amino acid changes for the introduced
mutations and glycan microheterogeneity (FIGS. 24A and 24B). Thus,
based on knowledge gained in the first round on mutagenesis,
mutations can be selected to add glycans at locations where the
glycan will be transferred efficiently and the protein folded and
secreted.
Example 12: Generation of Abs with Multiple Non-Native
N-Glycosylation Sites
[0298] The S134N and G161T mutations provide two efficient N-glycan
sites for conjugation (N134 and N159). We next sought to determine
if these sites could be combined on the same antibody scaffold to
generate an ADC with a DAR:8 at specific sites of conjugation. The
S134N mutated anti-Her2 antibody was mutated to incorporate G161T
or G161S mutations. The resulting plasmids (pGLY14135 and
pGLY14136) were transformed into strain YGLY30329 as described in
Example 2. Two resulting clones of each were cultivated in Dasgip 1
L fermenters as described in Example 3. The fermentation
supernatant was purified by protein A chromatography and the
resulting protein subjected to Q-ToF analysis. Both the S134N/G161T
and S134N/G161S double mutein containing mAbs were efficiently
glycosylated at 3 sites on each reduced H chain (N134, N159, and
N297) with minimal residual singly or doubly glycosylated protein
(FIG. 25).
[0299] Given that Abs can be produced accommodating two N-glycan
sites and the glycoengineered yeast system can modify these sites
efficiently to terminal galactose (Illustrated in FIGS. 26A and
26B), we sought to determine how many N-glycans could be added to a
structure, for example, to maximize DAR. To accomplish this, a
series of sites were chosen to combine that would each be in
disparate loops of the same antibody sequence. Between four and ten
extra N-glycan sites were introduced by site directed mutagenesis
(Illustrated in FIG. 26C) into an engineered version of the
anti-Her2 (trastuzumab) sequence, deemed null-HER2, that has been
mutated to eliminate Her2 antigen binding by mutation of two
residues in the variable region (one each in the VH and VL, See SEQ
ID 29 and 30, respectively).
[0300] Plasmids pGLYl4172-14179, constructed by Genewiz (South
Plainfield, N.J.), contain the null-Her2 H and L chain sequences as
derived from pGLY11576 (FIG. 27), and are each modified only by
introduction of the mutations as illustrated in Table 5.
TABLE-US-00006 TABLE 5 Plasmid Mutation Mutation Mutation H chain
name (EU numbering) (Herceptin numbering) (Kabat numbering)
Sequence pGLY14172 S134N, G161T, N203T S137, G164T, N206T S130N,
G158T, N211T SEQ ID NO: 34 pGLY14173 N/A, N/A, S134N, K30T, Y57T,
S137N, K30T, Y56T, S137N, SEQ ID NO: 35 G161T G164T G164T pGLY14174
N/A, N/A, S134N, K30T, K65N/R67T, K30T, K64N/R66T, SEQ ID NO: 36
G161T S137N, G164T S130N, G158T pGLY14175 N/A, N/A, S134N, Y57T,
K65N/R67T, Y56T, K64N/R66T, SEQ ID NO: 37 G161T, N203T S137N,
G164T, N206T S130N, G158T, N211T pGLY14176 N/A, S134N, G161T, Y57T,
S137N, G164T, Y56T, S130N, G158T, SEQ ID NO: 38 S176N/G178T, N203T
S179N/G181T, N206T S180N/G183T, N211T pGLY14177 N/A, N/A, N/A,
S134N, K30T, Y57T, K65N/R67T, K30T, Y56T, K64N/R66T, SEQ ID NO: 39
G161T, N203T S137N, G164T, N206T S130N, G158T, N211T PGLY14178 N/A,
N/A, N/A S134N, K30T, Y57T, K65N/R67T, K30T, Y56T, K64N/R66T, SEQ
ID NO: 40 G161T, S176N/G178T, S137N, G164T, S130N, G158T, N203T,
V363T, K392T, S179N/G181T, N206T, S180N/G183T, N211T, F423T V366T,
K395T, F246T V386T, K420T, F454T pGLY14179 N/A, N/A, N/A, S134N,
K30T, Y57T, K65N/R67T, K30T, Y56T, K64N/R66T, SEQ ID NO: 41 G161T,
L193N, N203T, S137N, G164T, L196T, S130N, G158T, L198N, V363T,
K392T, F423T N206T, V366T, K395T, N211T, V386T, K420T, F426T
F454T
[0301] Each of these plasmids was transformed into strain YGLY30329
and clones were selected and screened for secretion of antibody in
96 DWP format as described in Example 1. Following this, several
positive clones were cultivated in 5 ml micro24 reactors and the
supernatants were harvested by centrifugation and purified by
protein A chromatography as described in example 2 above. The
purified samples were subjected to capillary electrophoresis (CE)
on a Labchip GXII instrument (Caliper Life Sciences, Hopkinton,
Mass.) using the standard HT Protein Express 200 method as
described (Gomathinayagam, 2012). From this analysis it was
possible to conclude that most of the additional N-glycosylation
sites were indeed occupied due to the shifts in migration. To
illustrate this, a single representative purified non-reduced CE
sample from each of the plasmids was displayed using the Labchip
GXII visualization software version 4.1 (FIG. 26D). Moreover, these
samples were subjected to enzymatic N-glycan removal by PNGase
digestion and MALDI-TOF MS of the free N-glycans as previously
described (Choi, 2003). Most of the clones revealed a predominant
mass at 1660 or 1676 (Na or K adducts), identified to be the
biantennary terminally galactosylated human complex N-glycan
Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 (FIG. 2, GS5.0). A
representative sample from a clone resulting from introduction of
pGLY14179 into YGLY30329 is shown in FIG. 26E. The mAb expressed in
this strain contains a total of 22 N-glycosylation sites (11 per H
chain). It can also been observed in these non-reducing samples
that mAb assembly is of high integrity with mostly a single species
observed in each case. Poorly assembled mAb would often result in
poor resolution in CE under non-reducing conditions.
[0302] Several clones from strain YGLY30329 expressing plasmids
pGLY14172-14179 were cultivated in Dasgip IL fermenters as
described in Example 3 above. Following around 80-90 h of methanol
induction, supernatants were harvested by centrifugation and
purified by standard protein A chromatography (Example 3 above and
Jiang, 2011). The purified Abs from these strains were then
subjected to Q-ToF MS under reducing conditions (see example 2
above). The results, illustrated in FIGS. 28A and 28B, reveal again
that the non-native N-glycosylation sites are occupied in each of
the newly constructed antibody sequences. Here, with precise mass
identification, it can be determined that in each case at least the
majority of the antibody contains the number of N-glycans as
engineered. Inmost cases there is very little evidence of reduced
occupancy at any of the sites. Moreover, it can also be observed
that in most cases a single peak is predominantly visible,
corresponding to a mass of the H chain plus the corresponding
number of N-glycans with the GS5.0 structure (FIG. 2). Importantly,
this indicates that the Abs in each case are able to be occupied
with a specified number of N-glycans at preselected sites, properly
assembled in the ER, and fully glycan matured in the Golgi with
high integrity, all of which is required for the desired substrate
for efficient conjugation.
Example 13: Highly Sialylated mAbs
[0303] In addition to generating highly glycan-modified Abs with
terminal galactose for conjugation purposes, it could be desirable
to produce antibodies with a high degree of sialylation in the same
manner. Such Abs could be used for conjugation by chemical
modification of the sialic acid residues (Ramya, 2013).
[0304] To generate mAbs with a high degree of sialylation, the
plasmids illustrated in Example 12 (see table 5) were transformed
into Glycoengineered Pichia strain YGLY36472 capable of modifying
secreted proteins with the biantennary sialylated human N-glycan
(see, e.g., Hamilton, 2006; FIG. 2, GS6.0). Clones were selected
and screened for secretion of antibody in 96 DWP format as
described in Example 1. Supernatants from these 96 DWP cultures
were harvested by centrifugation at 2500.times.g in a Beckman
swinging bucket centrifuge and purified by protein A chromatography
as described previously (Barnard, 2010). The purified samples were
subjected to non-reducing CE analysis on a Labchip GXII instrument
(Caliper Life Sciences, Hopkinton, Mass.) using the standard HT
Protein Express 200 method as described (Gomathinayagam, 2012). As
illustrated in FIGS. 29A and 29B using the Labchip GX gel image
software version 4.1, it can be concluded that most of the
additional N-glycosylation sites were indeed occupied due to the
shifts in migration. Moreover, it can also be observed in these
non-reduced samples that the assembly, while somewhat clone- and
plasmid-dependent is very robust overall with generally a single
predominant band in each lane as illustrated by (FIGS. 29A and B).
Moreover, when the N-glycans from these Abs were released and
analyzed by MALDI-TOF as described (Choi, 2003). The predominant
N-glycan observed in most clones was consistent with
NANA.sub.2Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 (FIG. 2,
GS6.0) based on mass with the 2.sup.nd most predominant species
being NANAGal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2. A
representative MALDI-TOF MS trace from a clone resulting from
introducing plasmid pGLYl4179 into strain YGLY36472 is depicted in
FIG. 30. Moreover, the purified intact Abs from these strains were
subjected to Q-ToF MS under reducing conditions (see example 2
above). The results, illustrated in FIGS. 31A, 31B, 31C, and 31D,
reveal that the non-native N-glycosylation sites are occupied in
each of the constructed antibody sequences in GS6.0 strains similar
to in GS5.0 strains. Here, with precise mass identification, it can
be determined that in each case at vast the majority of the
antibody contains the number of N-glycans as engineered. In most
cases there is very little evidence of reduced occupancy at any of
the sites. Moreover, it can also be observed that in most cases a
single peak is predominantly visible, corresponding to a mass of
the H chain plus the corresponding number of N-glycans with the
GS6.0 structure (FIG. 2), indicating that antibodies modified with
non-native N-glycosylation sites can be expressed efficiently in
GS6.0 strains and are suitable for conjugation by the methods
described herein.
Example 14: Generation of Additional Abs with Non-Native
N-Glycosylation Sites
[0305] To assure that the efficient modification of the anti-HER2
antibody with non-canonical N-glycans is not restricted to the
anti-HER2 sequence we modified additional mAb sequences with
different antigen-binding Fab regions. A pair (H and L) of variable
domain sequences directed against the murine Programmed Cell Death
1 (PD-1) ligand was constructed as a human IgG1 chimera (Seq ID 42
and 43). This chimeric mAb sequence was further modified to
incorporate the S134N (EU, Seq ID 44) or G161T (EU, Seq ID 45)
mutations, which each add one additional N-glycan to the CHi domain
or the combined S137N/G161T mutations (EU, Seq ID 46), which adds
two additional N-glycans per H chain. The original anti-mPD-1 H
chain sequence (pGLY13649, FIG. 32) was modified using
site-directed mutagenesis by Genewiz (South Plainfield, N.J.) to
generate plasmids pGLY14163 (S134N), pGLY14164 (G161 T), and
pGLY14165 (S134N/G161T). These plasmids were transformed into
strain YGLY30329 and clones were selected and screened for mAb
secretion as described in Examples 1 and 2 above. Clones deemed to
be expressing antibody were then cultivated in 1 L Dasgip
fermenters as described in Example 3 above. The fermentation
supernatant was purified by protein A chromatography and the
resulting protein subjected to Q-ToF analysis. Each of the S134N,
and G161T single mutein mAbs was efficiently glycosylated at 3
sites on each reduced H chain, the canonical N-297 site, a variable
chain site that was part of the anti-PD-1 CDR sequence, and the
non-native N-glycosylation (either N134 orN159) site with a
majority of GS5.0 biantennary terminally galactosylated N-glycans
atboth the CDR N-glycan and at the non-native site and a mixture of
GS4.0, GS4.5 and GS5.0 N-glycans at the N-297 site (FIG. 33).
Similarly, the S134N/G161T double mutein containing mAbs were
efficiently glycosylated at 4 sites on each reduced H chain (FIG.
33).
[0306] In addition to the anti-mPD-1 Ab sequence, an anti-CS1 Ab
sequence H chain and L chain (Zha 2013, Seq ID 47 and Seq ID 48,
respectively) was modified to incorporate the same sets of
mutations, S134N (EU, Seq ID 49), G161T (EU, Seq ID 50), and the
double mutant S134N/G161T (EU, Seq ID 51). The original anti-CS-1 H
chain sequence (pGLY8040, FIG. 34) was modified using site-directed
mutagenesis by Genewiz (South Plainfield, N.J.) to generate
plasmids pGLY14157 (S134N), pGLY14158 (G161T), andpGLYl4159
(S134N/G161T). These plasmids were transformed into strain
YGLY30329 and clones were selected and screened for mAb secretion
as described in Examples 1 and 2 above. Clones deemed to be
expressing antibody were then cultivated in 1 L Dasgip fermenters
as described in Example 3 above. The fermentation supernatant was
purified by protein A chromatography and the resulting protein
subjected to Q-ToF analysis. Each of the S134N, and G161T single
mutein mAbs was efficiently glycosylated at 2 sites on each reduced
H chain, the canonical N-297 site, and the non-canonical (either
N134 orN159) site with a majority of GS5.0 biantennary terminally
galactosylated N-glycans at the non-canonical site and a mixture of
GS4.0, GS4.5 and GS5.0N-glycans at the N-297 site (FIG. 35).
Similarly, the S134N/G161T double mutein containing mAbs were
efficiently glycosylated at 3 sites on each reduced H chain (FIG.
35).
[0307] Finally, a previously published anti-CD70 antibody sequence
(Coccia, USapp 2010/0150950 A1) was modified to incorporate the
same sets of mutations, S134N (EU), G161T (EU), and the double
mutant (S134N/G161T). The anti-CD70 VH and VL sequences (Seq ID 52
and 53, respectively) were synthesized and constructed by Genewiz
(South Plainfield, N.J.) in a human IgG1 frameworkby cloning into
plasmid pGLY5730 (FIG. 36) to generate a P. pastoris IgG1 anti-CD70
expression plasmid named pGLY14148. This plasmid was then further
modified to incorporate three sets of N-glycan site-generating
muteins to generate plasmids pGLYl4149 (S134N, Seq ID 54),
pGLYl4150 (G161T, Seq ID 55), and pGLYl4151 (S134N/G161T, Seq ID
56). These plasmids were transformed into strain YGLY30329 and
clones were selected and screened for mAb secretion as described in
Examples 1 and 2 above. Clones deemed to be expressing antibody
were then cultivated in 1 L Dasgip fermenters as described in
Example 3 above. The fermentation supernatant was purified by
protein A chromatography and the resulting protein subjected to
Q-ToF analysis. Each of the S134N, and G161T single mutein mAbs was
efficiently glycosylated at 2 sites on each reduced H chain, the
canonical N-297 site, and the non-canonical (either N134 or N159)
site with a majority of GS5.0 biantennary terminally galactosylated
N-glycans at the non-canonical site and a mixture of GS4.0, GS4.5
and GS5.0 N-glycans at the N-297 site (FIG. 37). Similarly, the
S134N/G161T double mutein containing mAbs were efficiently
glycosylated at 3 sites on each reduced H chain (FIG. 37).
Sequence CWU 1
1
581449PRTArtificial Sequenceimmunoglobulin chain 1Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Ile
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala
Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200
205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315
320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445Gly2214PRTArtificial Sequenceimmunoglobulin chain 2Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr
Thr Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185
190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205Phe Asn Arg Gly Glu Cys 210310216DNAArtificial
Sequenceimmunoglobulin chain 3tcgcgcgttt cggtgatgac ggtgaaaacc
tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca
gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg
cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg
gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
240attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
tcttcgctat 300tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
aagttgggta acgccagggt 360tttcccagtc acgacgttgt aaaacgacgg
ccagtgaatt gagatctaac atccaaagac 420gaaaggttga atgaaacctt
tttgccatcc gacatccaca ggtccattct cacacataag 480tgccaaacgc
aacaggaggg gatacactag cagcagaccg ttgcaaacgc aggacctcca
540ctcctcttct cctcaacacc cacttttgcc atcgaaaaac cagcccagtt
attgggcttg 600attggagctc gctcattcca attccttcta ttaggctact
aacaccatga ctttattagc 660ctgtctatcc tggcccccct ggcgaggttc
atgtttgttt atttccgaat gcaacaagct 720ccgcattaca cccgaacatc
actccagatg agggctttct gagtgtgggg tcaaatagtt 780tcatgttccc
caaatggccc aaaactgaca gtttaaacgc tgtcttggaa cctaatatga
840caaaagcgtg atctcatcca agatgaacta agtttggttc gttgaaatgc
taacggccag 900ttggtcaaaa agaaacttcc aaaagtcggc ataccgtttg
tcttgtttgg tattgattga 960cgaatgctca aaaataatct cattaatgct
tagcgcagtc tctctatcgc ttctgaaccc 1020cggtgcacct gtgccgaaac
gcaaatgggg aaacacccgc tttttggatg attatgcatt 1080gtctccacat
tgtatgcttc caagattctg gtgggaatac tgctgatagc ctaacgttca
1140tgatcaaaat ttaactgttc taacccctac ttgacagcaa tatataaaca
gaaggaagct 1200gccctgtctt aaaccttttt ttttatcatc attattagct
tactttcata attgcgactg 1260gttccaattg acaagctttt gattttaacg
acttttaacg acaacttgag aagatcaaaa 1320aacaactaat tattcgaaac
ggaattcgaa acgatgagat tcccatccat cttcactgct 1380gttttgttcg
ctgcttcttc tgctttggct gaggttcagt tggttgaatc tggaggagga
1440ttggttcaac ctggtggttc tttgagattg tcctgtgctg cttccggttt
caacatcaag 1500gacacttaca tccactgggt tagacaagct ccaggaaagg
gattggagtg ggttgctaga 1560atctacccaa ctaacggtta cacaagatac
gctgactccg ttaagggaag attcactatc 1620tctgctgaca cttccaagaa
cactgcttac ttgcagatga actccttgag agctgaggat 1680actgctgttt
actactgttc cagatggggt ggtgatggtt tctacgctat ggactactgg
1740ggtcaaggaa ctttggttac tgtttcctcc gcttctacta agggaccatc
tgttttccca 1800ttggctccat cttctaagtc tacttccggt ggtactgctg
ctttgggatg tttggttaaa 1860gactacttcc cagagccagt tactgtttct
tggaactccg gtgctttgac ttctggtgtt 1920cacactttcc cagctgtttt
gcaatcttcc ggtttgtact ctttgtcctc cgttgttact 1980gttccatcct
cttccttggg tactcagact tacatctgta acgttaacca caagccatcc
2040aacactaagg ttgacaagaa ggttgagcca aagtcctgtg acaagacaca
tacttgtcca 2100ccatgtccag ctccagaatt gttgggtggt ccatccgttt
tcttgttccc accaaagcca 2160aaggacactt tgatgatctc cagaactcca
gaggttacat gtgttgttgt tgacgtttct 2220cacgaggacc cagaggttaa
gttcaactgg tacgttgacg gtgttgaagt tcacaacgct 2280aagactaagc
caagagaaga gcagtacaac tccacttaca gagttgtttc cgttttgact
2340gttttgcacc aggactggtt gaacggtaaa gaatacaagt gtaaggtttc
caacaaggct 2400ttgccagctc caatcgaaaa gactatctcc aaggctaagg
gtcaaccaag agagccacag 2460gtttacactt tgccaccatc cagagaagag
atgactaaga accaggtttc cttgacttgt 2520ttggttaaag gattctaccc
atccgacatt gctgttgagt gggaatctaa cggtcaacca 2580gagaacaact
acaagactac tccaccagtt ttggattctg atggttcctt cttcttgtac
2640tccaagttga ctgttgacaa gtccagatgg caacagggta acgttttctc
ctgttccgtt 2700atgcatgagg ctttgcacaa ccactacact caaaagtcct
tgtctttgtc ccctggttaa 2760tgaggccggc catttaaata caggcccctt
ttcctttgtc gatatcatgt aattagttat 2820gtcacgctta cattcacgcc
ctcctcccac atccgctcta accgaaaagg aaggagttag 2880acaacctgaa
gtctaggtcc ctatttattt tttttaatag ttatgttagt attaagaacg
2940ttatttatat ttcaaatttt tctttttttt ctgtacaaac gcgtgtacgc
atgtaacatt 3000atactgaaaa ccttgcttga gaaggttttg ggacgctcga
aggctttaat ttgcaagctg 3060gatctaacat ccaaagacga aaggttgaat
gaaacctttt tgccatccga catccacagg 3120tccattctca cacataagtg
ccaaacgcaa caggagggga tacactagca gcagaccgtt 3180gcaaacgcag
gacctccact cctcttctcc tcaacaccca cttttgccat cgaaaaacca
3240gcccagttat tgggcttgat tggagctcgc tcattccaat tccttctatt
aggctactaa 3300caccatgact ttattagcct gtctatcctg gcccccctgg
cgaggttcat gtttgtttat 3360ttccgaatgc aacaagctcc gcattacacc
cgaacatcac tccagatgag ggctttctga 3420gtgtggggtc aaatagtttc
atgttcccca aatggcccaa aactgacagt ttaaacgctg 3480tcttggaacc
taatatgaca aaagcgtgat ctcatccaag atgaactaag tttggttcgt
3540tgaaatgcta acggccagtt ggtcaaaaag aaacttccaa aagtcggcat
accgtttgtc 3600ttgtttggta ttgattgacg aatgctcaaa aataatctca
ttaatgctta gcgcagtctc 3660tctatcgctt ctgaaccccg gtgcacctgt
gccgaaacgc aaatggggaa acacccgctt 3720tttggatgat tatgcattgt
ctccacattg tatgcttcca agattctggt gggaatactg 3780ctgatagcct
aacgttcatg atcaaaattt aactgttcta acccctactt gacagcaata
3840tataaacaga aggaagctgc cctgtcttaa accttttttt ttatcatcat
tattagctta 3900ctttcataat tgcgactggt tccaattgac aagcttttga
ttttaacgac ttttaacgac 3960aacttgagaa gatcaaaaaa caactaatta
ttcgaaacgg aattcgaaac gatgagattc 4020ccatccatct tcactgctgt
tttgttcgct gcttcttctg ctttggctga catccaaatg 4080actcaatccc
catcttcttt gtctgcttcc gttggtgaca gagttactat cacttgtaga
4140gcttcccagg acgttaatac tgctgttgct tggtatcaac agaagccagg
aaaggctcca 4200aagttgttga tctactccgc ttccttcttg tactctggtg
ttccatccag attctctggt 4260tccagatccg gtactgactt cactttgact
atctcctcct tgcaaccaga agatttcgct 4320acttactact gtcagcagca
ctacactact ccaccaactt tcggacaggg tactaaggtt 4380gagatcaaga
gaactgttgc tgctccatcc gttttcattt tcccaccatc cgacgaacag
4440ttgaagtctg gtacagcttc cgttgtttgt ttgttgaaca acttctaccc
aagagaggct 4500aaggttcagt ggaaggttga caacgctttg caatccggta
actcccaaga atccgttact 4560gagcaagact ctaaggactc cacttactcc
ttgtcctcca ctttgacttt gtccaaggct 4620gattacgaga agcacaaggt
ttacgcttgt gaggttacac atcagggttt gtcctcccca 4680gttactaagt
ccttcaacag aggagagtgt taatagggcc ggccatttaa atacaggccc
4740cttttccttt gtcgatatca tgtaattagt tatgtcacgc ttacattcac
gccctcctcc 4800cacatccgct ctaaccgaaa aggaaggagt tagacaacct
gaagtctagg tccctattta 4860ttttttttaa tagttatgtt agtattaaga
acgttattta tatttcaaat ttttcttttt 4920tttctgtaca aacgcgtgta
cgcatgtaac attatactga aaaccttgct tgagaaggtt 4980ttgggacgct
cgaaggcttt aatttgcaag ctggatccgc ggccgcttac gcgccgatcc
5040cccacacacc atagcttcaa aatgtttcta ctcctttttt actcttccag
attttctcgg 5100actccgcgca tcgccgtacc acttcaaaac acccaagcac
agcatactaa atttcccctc 5160tttcttcctc tagggtgtcg ttaattaccc
gtactaaagg tttggaaaag aaaaaagaga 5220ccgcctcgtt tctttttctt
cgtcgaaaaa ggcaataaaa atttttatca cgtttctttt 5280tcttgaaaat
tttttttttt gatttttttc tctttcgatg acctcccatt gatatttaag
5340ttaataaacg gtcttcaatt tctcaagttt cagtttcatt tttcttgttc
tattacaact 5400ttttttactt cttgctcatt agaaagaaag catagcaatc
taatctaagt tttaattaca 5460aattaattaa tggccaagtt gaccagtgcc
gttccggtgc tcaccgcgcg cgacgtcgcc 5520ggagcggtcg agttctggac
cgaccggctc gggttctccc gggacttcgt ggaggacgac 5580ttcgccggtg
tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg
5640gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct
gtacgccgag 5700tggtcggagg tcgtgtccac gaacttccgg gacgcctccg
ggcctgccat gaccgagatc 5760ggcgagcagc cgtgggggcg ggagttcgcc
ctgcgcgacc cggccggcaa ctgcgtgcac 5820ttcgtggccg aggagcagga
ctgattaatt aacaggcccc ttttcctttg tcgatatcat 5880gtaattagtt
atgtcacgct tacattcacg ccctcctccc acatccgctc taaccgaaaa
5940ggaaggagtt agacaacctg aagtctaggt ccctatttat tttttttaat
agttatgtta 6000gtattaagaa cgttatttat atttcaaatt tttctttttt
ttctgtacaa acgcgtgtac 6060gcatgtaaca ttatactgaa aaccttgctt
gagaaggttt tgggacgctc gaaggcttta 6120atttgcaagc tgcggcctaa
ggcgcgccag gccataatgg ccaaacggtt tctcaattac 6180tatatactac
taaccattta cctgtagcgt atttcttttc cctcttcgcg aaagctcaag
6240ggcatcttct tgactcatga aaaatatctg gatttcttct gacagatcat
cacccttgag 6300cccaactctc tagcctatga gtgtaagtga tagtcatctt
gcaacagatt attttggaac 6360gcaactaaca aagcagatac acccttcagc
agaatccttt ctggatattg tgaagaatga 6420tcgccaaagt cacagtcctg
agacagttcc taatctttac cccatttaca agttcatcca 6480atcagacttc
ttaacgcctc atctggctta tatcaagctt accaacagtt cagaaactcc
6540cagtccaagt ttcttgcttg aaagtgcgaa gaatggtgac accgttgaca
ggtacacctt 6600tatgggacat tcccccagaa aaataatcaa gactgggcct
ttagagggtg ctgaagttga 6660ccccttggtg cttctggaaa aagaactgaa
gggcaccaga caagcgcaac ttcctggtat 6720tcctcgtcta agtggtggtg
ccataggata catctcgtac gattgtatta agtactttga 6780accaaaaact
gaaagaaaac tgaaagatgt tttgcaactt ccggaagcag ctttgatgtt
6840gttcgacacg atcgtggctt ttgacaatgt ttatcaaaga ttccaggtaa
ttggaaacgt 6900ttctctatcc gttgatgact cggacgaagc tattcttgag
aaatattata agacaagaga 6960agaagtggaa aagatcagta aagtggtatt
tgacaataaa actgttccct actatgaaca 7020gaaagatatt attcaaggcc
aaacgttcac ctctaatatt ggtcaggaag ggtatgaaaa 7080ccatgttcgc
aagctgaaag aacatattct gaaaggagac atcttccaag ctgttccctc
7140tcaaagggta gccaggccga cctcattgca ccctttcaac atctatcgtc
atttgagaac 7200tgtcaatcct tctccataca tgttctatat tgactatcta
gacttccaag ttgttggtgc 7260ttcacctgaa ttactagtta aatccgacaa
caacaacaaa atcatcacac atcctattgc 7320tggaactctt cccagaggta
aaactatcga agaggacgac aattatgcta agcaattgaa 7380gtcgtctttg
aaagacaggg ccgagcacgt catgctggta gatttggcca gaaatgatat
7440taaccgtgtg tgtgagccca ccagtaccac ggttgatcgt ttattgactg
tggagagatt 7500ttctcatgtg atgcatcttg tgtcagaagt cagtggaaca
ttgagaccaa acaagactcg 7560cttcgatgct ttcagatcca ttttcccagc
aggaaccgtc tccggtgctc cgaaggtaag 7620agcaatgcaa ctcataggag
aattggaagg agaaaagaga ggtgtttatg cgggggccgt 7680aggacactgg
tcgtacgatg gaaaatcgat ggacacatgt attgccttaa gaacaatggt
7740cgtcaaggac ggtgtcgctt accttcaagc cggaggtgga attgtctacg
attctgaccc 7800ctatgacgag tacatcgaaa ccatgaacaa aatgagatcc
aacaataaca ccatcttgga 7860ggctgagaaa atctggaccg ataggttggc
cagagacgag aatcaaagtg aatccgaaga 7920aaacgatcaa tgaacggagg
acgtaagtag gaatttatgg tttggccata atggcctagc 7980ttggcgtaat
catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
8040cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
agtgagctaa 8100ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
cgggaaacct gtcgtgccag 8160ctgcattaat gaatcggcca acgcgcgggg
agaggcggtt tgcgtattgg gcgctcttcc 8220gcttcctcgc tcactgactc
gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 8280cactcaaagg
cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
8340tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
ggcgtttttc 8400cataggctcc gcccccctga cgagcatcac aaaaatcgac
gctcaagtca gaggtggcga 8460aacccgacag gactataaag ataccaggcg
tttccccctg gaagctccct cgtgcgctct 8520cctgttccga ccctgccgct
taccggatac ctgtccgcct ttctcccttc gggaagcgtg 8580gcgctttctc
atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
8640ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
cggtaactat 8700cgtcttgagt ccaacccggt aagacacgac ttatcgccac
tggcagcagc cactggtaac 8760aggattagca gagcgaggta tgtaggcggt
gctacagagt tcttgaagtg gtggcctaac 8820tacggctaca ctagaaggac
agtatttggt atctgcgctc tgctgaagcc agttaccttc 8880ggaaaaagag
ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
8940tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
tcctttgatc 9000ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
gttaagggat tttggtcatg 9060agattatcaa aaaggatctt cacctagatc
cttttaaatt aaaaatgaag ttttaaatca 9120atctaaagta tatatgagta
aacttggtct gacagttacc aatgcttaat cagtgaggca 9180cctatctcag
cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
9240ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
accgcgagac 9300ccacgctcac cggctccaga tttatcagca ataaaccagc
cagccggaag ggccgagcgc 9360agaagtggtc ctgcaacttt atccgcctcc
atccagtcta ttaattgttg ccgggaagct 9420agagtaagta gttcgccagt
taatagtttg cgcaacgttg ttgccattgc tacaggcatc 9480gtggtgtcac
gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
9540cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
tcctccgatc 9600gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
ttatggcagc actgcataat 9660tctcttactg tcatgccatc cgtaagatgc
ttttctgtga ctggtgagta ctcaaccaag 9720tcattctgag aatagtgtat
gcggcgaccg agttgctctt gcccggcgtc aatacgggat 9780aataccgcgc
cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
9840cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
cactcgtgca 9900cccaactgat cttcagcatc ttttactttc accagcgttt
ctgggtgagc aaaaacagga 9960aggcaaaatg ccgcaaaaaa gggaataagg
gcgacacgga aatgttgaat actcatactc 10020ttcctttttc aatattattg
aagcatttat cagggttatt gtctcatgag cggatacata 10080tttgaatgta
tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
10140ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
taggcgtatc 10200acgaggccct ttcgtc 102164449PRTArtificial
Sequenceimmunoglobulin chain 4Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Tyr Pro Thr
Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ser Arg
Trp Gly Gly Asp Gly Phe Tyr Ala Met
Asp Tyr Trp Gly Asn 100 105 110Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170
175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp 210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295
300Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410
415Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro 435 440 445Gly5449PRTArtificial Sequenceimmunoglobulin
chain 5Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys
Asp Thr 20 25 30Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr
Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser
Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ser Arg Trp Gly Gly Asp Gly Phe
Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala
Pro Ser Ser Lys Asn Thr Ser Gly Gly Thr Ala Ala 130 135 140Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155
160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp 210 215 220Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly225 230 235 240Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280
285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390 395
400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His 420 425 430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro 435 440 445Gly6449PRTArtificial
Sequenceimmunoglobulin chain 6Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Tyr Pro Thr
Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ser Arg
Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser145 150 155 160Trp Asn Ser Thr Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly225 230
235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345
350Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445Gly7449PRTArtificial Sequenceimmunoglobulin chain 7Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr
Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp
Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu
Asn Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185
190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp 210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310
315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425
430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445Gly8449PRTArtificial Sequenceimmunoglobulin chain 8Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn
Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala
Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170
175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Thr
His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp 210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295
300Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410
415Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro 435 440 445Gly9449PRTArtificial Sequenceimmunoglobulin
chain 9Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys
Asp Thr 20 25 30Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr
Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser
Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ser Arg Trp Gly Gly Asp Gly Phe
Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155
160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp 210 215 220Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly225 230 235 240Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280
285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys Asn Gln Thr Ser Leu 355 360 365Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375
380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His 420 425 430Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445Gly10449PRTArtificial
Sequenceimmunoglobulin chain 10Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Tyr Pro Thr
Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ser Arg
Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly225 230
235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345
350Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp 370 375 380Glu Ser Asn Gly Thr Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445Gly11449PRTArtificial Sequenceimmunoglobulin chain 11Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr
Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp
Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185
190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp 210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310
315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425
430Glu Ala Leu His Asn His Tyr Thr Asn Lys Ser Leu Ser Leu Ser Pro
435 440 445Gly12468PRTArtificial Sequenceimmunoglobulin chain 12Met
Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser1 5 10
15Ala Leu Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn
Ile 35 40 45Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu 50 55 60Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr
Arg Tyr Ala65 70 75 80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala
Asp Thr Ser Lys Asn 85 90 95Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ser Arg Trp Gly Gly
Asp Gly Phe Tyr Ala Met Asp Tyr 115 120 125Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Asn Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly145 150 155 160Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170
175Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
180 185 190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val 195 200 205Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val 210 215 220Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys225 230 235 240Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295
300Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser305 310 315 320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu 325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala 340 345 350Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 370 375 380Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395 400Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410
415Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
420 425 430Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser 435 440 445Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser 450 455 460Leu Ser Pro Gly46513468PRTArtificial
Sequenceimmunoglobulin chain 13Met Arg Phe Pro Ser Ile Phe Thr Ala
Val Leu Phe Ala Ala Ser Ser1 5 10 15Ala Leu Ala Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Asn Ile 35 40 45Lys Asp Thr Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Arg
Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala65 70 75 80Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 85 90 95Thr Ala
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105
110Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr
115 120 125Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Asn Ser Thr
Lys Gly 130 135 140Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly145 150 155 160Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys225 230
235 240Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu 245 250 255Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr 260 265 270Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val 275 280 285Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val 290 295 300Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315 320Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345
350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460Leu
Ser Pro Gly46514468PRTArtificial Sequenceimmunoglobulin chain 14Met
Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser1 5 10
15Ala Leu Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn
Ile 35 40 45Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu 50 55 60Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr
Arg Tyr Ala65 70 75 80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala
Asp Thr Ser Lys Asn 85 90 95Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ser Arg Trp Gly Gly
Asp Gly Phe Tyr Ala Met Asp Tyr 115 120 125Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe
Pro Leu Ala Pro Ser Asn Lys Ser Thr Ser Gly Gly145 150 155 160Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170
175Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
180 185 190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val 195 200 205Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val 210 215 220Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys225 230 235 240Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295
300Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser305 310 315 320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu 325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala 340 345 350Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 370 375 380Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395 400Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410
415Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
420 425 430Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser 435 440 445Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser 450 455 460Leu Ser Pro Gly46515468PRTArtificial
Sequenceimmunoglobulin chain 15Met Arg Phe Pro
Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser1 5 10 15Ala Leu Ala
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30Pro Gly
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile 35 40 45Lys
Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55
60Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala65
70 75 80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
Asn 85 90 95Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val 100 105 110Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr
Ala Met Asp Tyr 115 120 125Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Asn Ser Thr Ser Gly Gly145 150 155 160Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200
205Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
210 215 220Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys225 230 235 240Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val 275 280 285Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315
320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala 340 345 350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro 355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440
445Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
450 455 460Leu Ser Pro Gly46516468PRTArtificial
Sequenceimmunoglobulin chain 16Met Arg Phe Pro Ser Ile Phe Thr Ala
Val Leu Phe Ala Ala Ser Ser1 5 10 15Ala Leu Ala Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Asn Ile 35 40 45Lys Asp Thr Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Arg
Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala65 70 75 80Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 85 90 95Thr Ala
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105
110Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr
115 120 125Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly 130 135 140Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly145 150 155 160Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr Val Ser Trp Asn Ser Gly
Asn Leu Thr Ser Gly Val His Thr Phe 180 185 190Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys225 230
235 240Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu 245 250 255Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr 260 265 270Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val 275 280 285Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val 290 295 300Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315 320Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345
350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460Leu
Ser Pro Gly46517468PRTArtificial Sequenceimmunoglobulin chain 17Met
Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser1 5 10
15Ala Leu Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn
Ile 35 40 45Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu 50 55 60Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr
Arg Tyr Ala65 70 75 80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala
Asp Thr Ser Lys Asn 85 90 95Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ser Arg Trp Gly Gly
Asp Gly Phe Tyr Ala Met Asp Tyr 115 120 125Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly145 150 155 160Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170
175Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
180 185 190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val 195 200 205Thr Val Pro Ser Ser Ser Leu Gly Asn Gln Thr Tyr
Ile Cys Asn Val 210 215 220Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys225 230 235 240Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295
300Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser305 310 315 320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu 325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala 340 345 350Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 370 375 380Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395 400Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410
415Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
420 425 430Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser 435 440 445Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser 450 455 460Leu Ser Pro Gly46518468PRTArtificial
Sequenceimmunoglobulin chain 18Met Arg Phe Pro Ser Ile Phe Thr Ala
Val Leu Phe Ala Ala Ser Ser1 5 10 15Ala Leu Ala Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Asn Ile 35 40 45Lys Asp Thr Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Arg
Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala65 70 75 80Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 85 90 95Thr Ala
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105
110Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr
115 120 125Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly 130 135 140Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly145 150 155 160Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220Asn
His Lys Pro Ser Asn Thr Thr Val Asp Lys Lys Val Glu Pro Lys225 230
235 240Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu 245 250 255Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr 260 265 270Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val 275 280 285Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val 290 295 300Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315 320Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345
350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460Leu
Ser Pro Gly46519468PRTArtificial Sequenceimmunoglobulin chain 19Met
Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser1 5 10
15Ala Leu Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn
Ile 35 40 45Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu 50 55 60Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr
Arg Tyr Ala65 70 75 80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala
Asp Thr Ser Lys Asn 85 90 95Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ser Arg Trp Gly Gly
Asp Gly Phe Tyr Ala Met Asp Tyr 115 120 125Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly145 150 155 160Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170
175Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
180 185 190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val 195 200 205Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val 210 215 220Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys225 230 235 240Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295
300Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser305 310 315 320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu 325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala 340 345 350Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 370 375 380Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395 400Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Thr Lys Thr Thr 405 410
415Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
420 425 430Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser 435 440 445Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser 450 455 460Leu Ser Pro Gly46520468PRTArtificial
Sequenceimmunoglobulin chain 20Met Arg Phe Pro Ser Ile Phe Thr Ala
Val Leu Phe Ala Ala Ser Ser1 5 10 15Ala Leu Ala Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Asn Ile 35 40 45Lys Asp Thr Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Arg
Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala65 70 75 80Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 85 90 95Thr Ala
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105
110Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr
115 120 125Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly 130 135 140Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly145 150 155 160Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys225 230
235 240Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu 245 250 255Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr 260 265 270Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val 275 280 285Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val 290 295 300Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315 320Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345
350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Thr Ser Cys Ser 435 440 445Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460Leu
Ser Pro Gly46521468PRTArtificial Sequenceimmunoglobulin chain 21Met
Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser1 5 10
15Ala Leu Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn
Ile 35 40 45Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu 50 55 60Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr
Arg Tyr Ala65 70 75 80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala
Asp Thr Ser Lys Asn 85 90 95Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ser Arg Trp Gly Gly
Asp Gly Phe Tyr Ala Met Asp Tyr 115 120 125Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly145 150 155 160Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170
175Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
180 185 190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val 195 200 205Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val 210 215 220Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys225 230 235 240Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295
300Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser305 310 315 320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu 325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala 340 345 350Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 370 375 380Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395 400Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410
415Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
420 425 430Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser 435 440 445Val Met His Glu Ala Leu His Asn His Thr Thr Gln
Lys Ser Leu Ser 450 455 460Leu Ser Pro Gly46522468PRTArtificial
Sequenceimmunoglobulin chain 22Met Arg Phe Pro Ser Ile Phe Thr Ala
Val Leu Phe Ala Ala Ser Ser1 5 10 15Ala Leu Ala Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Asn Ile 35 40 45Lys Asp Thr Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Arg
Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala65 70 75 80Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 85 90 95Thr Ala
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105
110Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr
115 120 125Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly 130 135 140Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly145 150 155 160Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205Thr Val Pro
Ser Ser Ser Asn Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys225 230
235 240Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu 245 250 255Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr 260 265 270Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val 275 280 285Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val 290 295 300Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315 320Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345
350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460Leu
Ser Pro Gly46523468PRTArtificial Sequenceimmunoglobulin chain 23Met
Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser1 5 10
15Ala Leu Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn
Ile 35 40 45Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu 50 55 60Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr
Arg Tyr Ala65 70 75 80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala
Asp Thr Ser Lys Asn 85 90 95Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ser Arg Trp Gly Gly
Asp Gly Phe Tyr Ala Met Asp Tyr 115 120 125Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly145 150 155 160Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170
175Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
180 185 190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val 195 200 205Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val 210 215 220Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys225 230 235 240Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295
300Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser305 310 315 320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu 325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala 340 345 350Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 370 375 380Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395 400Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410
415Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
420 425 430Thr Val Asp Lys Ser Arg Trp Gln Asn Gly Thr Val Phe Ser
Cys Ser 435 440 445Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser 450 455 460Leu Ser Pro Gly46524468PRTArtificial
Sequenceimmunoglobulin chain 24Met Arg Phe Pro Ser Ile Phe Thr Ala
Val Leu Phe Ala Ala Ser Ser1 5 10 15Ala Leu Ala Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Asn Ile 35 40 45Lys Asp Thr Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Arg
Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala65 70 75 80Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 85 90 95Thr Ala
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105
110Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr
115 120 125Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly 130 135 140Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly145 150 155 160Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190Pro Ala Val Leu Gln
Asn Ser Thr Leu Tyr Ser Leu Ser Ser Val Val 195 200 205Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys225 230
235 240Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu 245 250 255Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr 260 265 270Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val 275 280 285Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val 290 295 300Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315 320Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345
350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460Leu
Ser Pro Gly46525468PRTArtificial Sequenceimmunoglobulin chain 25Met
Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser1 5 10
15Ala Leu Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn
Ile 35 40 45Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu 50 55 60Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr
Arg Tyr Ala65 70 75 80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala
Asp Thr Ser Lys Asn 85 90 95Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ser Arg Trp Gly Gly
Asp Gly Phe Tyr Ala Met Asp Tyr 115 120 125Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly145 150 155 160Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170
175Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
180 185 190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val 195 200 205Thr Val Pro Ser Asn Ser Thr Gly Thr Gln Thr Tyr
Ile Cys Asn Val 210
215 220Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys225 230 235 240Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu 245 250 255Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val 275 280 285Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315 320Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330
335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
340 345 350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro 355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455
460Leu Ser Pro Gly46526468PRTArtificial Sequenceimmunoglobulin
chain 26Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser
Ser1 5 10 15Ala Leu Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln 20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Asn Ile 35 40 45Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly
Tyr Thr Arg Tyr Ala65 70 75 80Asp Ser Val Lys Gly Arg Phe Thr Ile
Ser Ala Asp Thr Ser Lys Asn 85 90 95Thr Ala Tyr Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ser Arg Trp
Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr 115 120 125Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly145 150 155
160Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
165 170 175Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe 180 185 190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val 195 200 205Thr Val Pro Ser Ser Ser Leu Asn Thr Thr
Thr Tyr Ile Cys Asn Val 210 215 220Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys225 230 235 240Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280
285Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
290 295 300Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser305 310 315 320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu 325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala 340 345 350Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 370 375 380Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395
400Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
405 410 415Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu 420 425 430Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser 435 440 445Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser 450 455 460Leu Ser Pro
Gly46527468PRTArtificial Sequenceimmunoglobulin chain 27Met Arg Phe
Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser1 5 10 15Ala Leu
Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30Pro
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile 35 40
45Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr
Ala65 70 75 80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr
Ser Lys Asn 85 90 95Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly
Phe Tyr Ala Met Asp Tyr 115 120 125Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys Asn Thr Ser Gly Gly145 150 155 160Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr
Val Ser Trp Asn Ser Thr Ala Leu Thr Ser Gly Val His Thr Phe 180 185
190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
195 200 205Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val 210 215 220Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys225 230 235 240Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310
315 320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu 325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala 340 345 350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro 355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425
430Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
435 440 445Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser 450 455 460Leu Ser Pro Gly46528468PRTArtificial
Sequenceimmunoglobulin chain 28Met Arg Phe Pro Ser Ile Phe Thr Ala
Val Leu Phe Ala Ala Ser Ser1 5 10 15Ala Leu Ala Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Asn Ile 35 40 45Lys Asp Thr Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Arg
Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala65 70 75 80Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 85 90 95Thr Ala
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105
110Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr
115 120 125Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly 130 135 140Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Asn
Thr Ser Gly Gly145 150 155 160Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr Val Ser Trp Asn Ser Ser
Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys225 230
235 240Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu 245 250 255Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr 260 265 270Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val 275 280 285Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val 290 295 300Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315 320Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345
350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460Leu
Ser Pro Gly46529449PRTArtificial Sequenceimmunoglobulin chain 29Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Glu Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn
Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ser Arg Trp Gly Gly Asp Gly Phe Gly Ala
Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170
175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp 210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly225 230 235 240Pro Ser Val Phe Leu Ala Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu
Val Thr Cys Val Val Ala Asp Val Ser His Glu 260 265 270Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295
300Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410
415Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro 435 440 445Gly30214PRTArtificial Sequenceimmunoglobulin
chain 30Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn
Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln His Tyr Thr Thr Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155
160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
21031468PRTArtificial Sequenceimmunoglobulin chain 31Met Arg Phe
Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser1 5 10 15Ala Leu
Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30Pro
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile 35 40
45Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60Glu Trp Val Ala Glu Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr
Ala65 70 75 80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr
Ser Lys Asn 85 90 95Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly
Phe Gly Ala Met Asp Tyr 115 120 125Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Asn Thr Ser Gly Gly145 150 155
160Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
165 170 175Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe 180 185 190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val 195 200 205Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val 210 215 220Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys225 230 235 240Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly
Pro Ser Val Phe Leu Ala Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Ala Asp Val 275 280
285Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
290 295 300Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser305 310 315 320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu 325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala 340 345 350Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 370 375 380Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395
400Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
405 410 415Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu 420 425 430Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser 435 440 445Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser 450 455 460Leu Ser Pro
Gly46532468PRTArtificial Sequenceimmunoglobulin chain 32Met Arg Phe
Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser1 5 10 15Ala Leu
Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30Pro
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile 35 40
45Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60Glu Trp Val Ala Glu Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr
Ala65 70 75 80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr
Ser Lys Asn 85 90 95Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly
Phe Gly Ala Met Asp Tyr 115 120 125Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly145 150 155 160Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr
Val Ser Trp Asn Ser Thr Ala Leu Thr Ser Gly Val His Thr Phe 180 185
190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
195 200 205Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val 210 215 220Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys225 230 235 240Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly Pro Ser Val Phe
Leu Ala Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Ala Asp Val 275 280 285Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310
315 320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu 325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala 340 345 350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro 355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425
430Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
435 440 445Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser 450 455 460Leu Ser Pro Gly46533468PRTArtificial
Sequenceimmunoglobulin chain 33Met Arg Phe Pro Ser Ile Phe Thr Ala
Val Leu Phe Ala Ala Ser Ser1 5 10 15Ala Leu Ala Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Asn Ile 35 40 45Lys Asp Thr Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Glu
Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala65 70 75 80Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 85 90 95Thr Ala
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105
110Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Gly Ala Met Asp Tyr
115 120 125Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly 130 135 140Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Asn
Thr Ser Gly Gly145 150 155 160Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr Val Ser Trp Asn Ser Thr
Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys225 230
235 240Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu 245 250 255Leu Gly Gly Pro Ser Val Phe Leu Ala Pro Pro Lys Pro
Lys Asp Thr 260 265 270Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Ala Asp Val 275 280 285Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val 290 295 300Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315 320Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345
350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460Leu
Ser Pro Gly46534468PRTArtificial Sequenceimmunoglobulin chain 34Met
Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser1 5 10
15Ala Leu Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn
Ile 35 40 45Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu 50 55 60Glu Trp Val Ala Glu Ile Tyr Pro Thr Asn Gly Tyr Thr
Arg Tyr Ala65 70 75 80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala
Asp Thr Ser Lys Asn 85 90 95Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ser Arg Trp Gly Gly
Asp Gly Phe Gly Ala Met Asp Tyr 115 120 125Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Asn Thr Ser Gly Gly145 150 155 160Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170
175Thr Val Ser Trp Asn Ser Thr Ala Leu Thr Ser Gly Val His Thr Phe
180 185 190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val 195 200 205Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val 210 215 220Thr His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys225 230 235 240Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly Pro Ser
Val Phe Leu Ala Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Ala Asp Val 275 280 285Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295
300Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser305 310 315 320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu 325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala 340 345 350Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 370 375 380Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395 400Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410
415Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
420 425 430Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser 435 440 445Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser 450 455 460Leu Ser Pro Gly46535468PRTArtificial
Sequenceimmunoglobulin chain 35Met Arg Phe Pro Ser Ile Phe Thr Ala
Val Leu Phe Ala Ala Ser Ser1 5 10 15Ala Leu Ala Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Asn Ile 35 40 45Thr Asp Thr Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Glu
Ile Tyr Pro Thr Asn Gly Thr Thr Arg Tyr Ala65 70 75 80Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 85 90 95Thr Ala
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105
110Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Gly Ala Met Asp Tyr
115 120 125Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly 130 135 140Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Asn
Thr Ser Gly Gly145 150 155 160Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr Val Ser Trp Asn Ser Thr
Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys225 230
235 240Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu 245 250 255Leu Gly Gly Pro Ser Val Phe Leu Ala Pro Pro Lys Pro
Lys Asp Thr 260 265 270Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Ala Asp Val 275 280 285Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val 290 295 300Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315 320Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345
350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460Leu
Ser Pro Gly46536468PRTArtificial Sequenceimmunoglobulin chain 36Met
Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser1 5 10
15Ala Leu Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn
Ile 35 40 45Thr Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu 50 55 60Glu Trp Val Ala Glu Ile Tyr Pro Thr Asn Gly Tyr Thr
Arg Tyr Ala65 70 75 80Asp Ser Val Asn Gly Thr Phe Thr Ile Ser Ala
Asp Thr Ser Lys Asn 85 90 95Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ser Arg Trp Gly Gly
Asp Gly Phe Gly Ala Met Asp Tyr 115 120 125Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Asn Thr Ser Gly Gly145 150 155 160Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170
175Thr Val Ser Trp Asn Ser Thr Ala Leu Thr Ser Gly Val His Thr Phe
180 185 190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val 195 200 205Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val 210 215 220Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys225 230 235
240Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
245 250 255Leu Gly Gly Pro Ser Val Phe Leu Ala Pro Pro Lys Pro Lys
Asp Thr 260 265 270Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Ala Asp Val 275 280 285Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val 290 295 300Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315 320Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345 350Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360
365Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
370 375 380Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala385 390 395 400Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr 405 410 415Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu 420 425 430Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460Leu Ser Pro
Gly46537468PRTArtificial Sequenceimmunoglobulin chain 37Met Arg Phe
Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser1 5 10 15Ala Leu
Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30Pro
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile 35 40
45Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60Glu Trp Val Ala Glu Ile Tyr Pro Thr Asn Gly Thr Thr Arg Tyr
Ala65 70 75 80Asp Ser Val Asn Gly Thr Phe Thr Ile Ser Ala Asp Thr
Ser Lys Asn 85 90 95Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly
Phe Gly Ala Met Asp Tyr 115 120 125Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys Asn Thr Ser Gly Gly145 150 155 160Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr
Val Ser Trp Asn Ser Thr Ala Leu Thr Ser Gly Val His Thr Phe 180 185
190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
195 200 205Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val 210 215 220Thr His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys225 230 235 240Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly Pro Ser Val Phe
Leu Ala Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Ala Asp Val 275 280 285Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310
315 320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu 325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala 340 345 350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro 355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425
430Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
435 440 445Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser 450 455 460Leu Ser Pro Gly46538468PRTArtificial
Sequenceimmunoglobulin chain 38Met Arg Phe Pro Ser Ile Phe Thr Ala
Val Leu Phe Ala Ala Ser Ser1 5 10 15Ala Leu Ala Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Asn Ile 35 40 45Lys Asp Thr Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Glu
Ile Tyr Pro Thr Asn Gly Thr Thr Arg Tyr Ala65 70 75 80Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 85 90 95Thr Ala
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105
110Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Gly Ala Met Asp Tyr
115 120 125Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly 130 135 140Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Asn
Thr Ser Gly Gly145 150 155 160Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr Val Ser Trp Asn Ser Thr
Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190Pro Ala Val Leu Gln
Asn Ser Thr Leu Tyr Ser Leu Ser Ser Val Val 195 200 205Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220Thr
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys225 230
235 240Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu 245 250 255Leu Gly Gly Pro Ser Val Phe Leu Ala Pro Pro Lys Pro
Lys Asp Thr 260 265 270Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Ala Asp Val 275 280 285Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val 290 295 300Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315 320Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345
350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460Leu
Ser Pro Gly46539468PRTArtificial Sequenceimmunoglobulin chain 39Met
Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser1 5 10
15Ala Leu Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn
Ile 35 40 45Thr Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu 50 55 60Glu Trp Val Ala Glu Ile Tyr Pro Thr Asn Gly Thr Thr
Arg Tyr Ala65 70 75 80Asp Ser Val Asn Gly Thr Phe Thr Ile Ser Ala
Asp Thr Ser Lys Asn 85 90 95Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ser Arg Trp Gly Gly
Asp Gly Phe Gly Ala Met Asp Tyr 115 120 125Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Asn Thr Ser Gly Gly145 150 155 160Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170
175Thr Val Ser Trp Asn Ser Thr Ala Leu Thr Ser Gly Val His Thr Phe
180 185 190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val 195 200 205Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val 210 215 220Thr His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys225 230 235 240Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly Pro Ser
Val Phe Leu Ala Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Ala Asp Val 275 280 285Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295
300Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser305 310 315 320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu 325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala 340 345 350Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 370 375 380Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395 400Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410
415Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
420 425 430Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser 435 440 445Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser 450 455 460Leu Ser Pro Gly46540468PRTArtificial
Sequenceimmunoglobulin chain 40Met Arg Phe Pro Ser Ile Phe Thr Ala
Val Leu Phe Ala Ala Ser Ser1 5 10 15Ala Leu Ala Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Asn Ile 35 40 45Thr Asp Thr Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Glu
Ile Tyr Pro Thr Asn Gly Thr Thr Arg Tyr Ala65 70 75 80Asp Ser Val
Asn Gly Thr Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn 85 90 95Thr Ala
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105
110Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Gly Ala Met Asp Tyr
115 120 125Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly 130 135 140Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Asn
Thr Ser Gly Gly145 150 155 160Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr Val Ser Trp Asn Ser Thr
Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190Pro Ala Val Leu Gln
Asn Ser Thr Leu Tyr Ser Leu Ser Ser Val Val 195 200 205Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220Thr
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys225 230
235 240Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu 245 250 255Leu Gly Gly Pro Ser Val Phe Leu Ala Pro Pro Lys Pro
Lys Asp Thr 260 265 270Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Ala Asp Val 275 280 285Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val 290 295 300Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315 320Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345
350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln 370 375 380Thr Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Thr Thr Thr 405 410 415Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Thr Ser Cys Ser 435 440 445Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460Leu
Ser Pro Gly46541468PRTArtificial Sequenceimmunoglobulin chain 41Met
Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser1 5 10
15Ala Leu Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn
Ile 35 40 45Thr Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu 50 55 60Glu Trp Val Ala Glu Ile Tyr Pro Thr Asn Gly Thr Thr
Arg Tyr Ala65 70 75 80Asp Ser Val Asn Gly Thr Phe Thr Ile Ser Ala
Asp Thr Ser Lys Asn 85 90 95Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ser Arg Trp Gly Gly
Asp Gly Phe Gly Ala Met Asp Tyr 115 120 125Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Asn Thr Ser Gly Gly145 150 155 160Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170
175Thr Val Ser Trp Asn Ser Thr Ala Leu Thr Ser Gly Val His Thr Phe
180 185 190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val 195 200 205Thr Val Pro Ser Ser Ser Asn Gly Thr Gln Thr Tyr
Ile Cys Asn Val 210 215 220Thr His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys225 230 235 240Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly Pro Ser
Val Phe Leu Ala Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Ala Asp Val 275 280 285Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295
300Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser305 310 315 320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu 325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala 340
345 350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro 355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln 370 375 380Thr Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Thr Thr Thr 405 410 415Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Thr Ser Cys Ser 435 440 445Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455
460Leu Ser Pro Gly46542448PRTArtificial Sequenceimmunoglobulin
chain 42Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Asn Ser 20 25 30Gly Leu Ala Trp Val Arg Gln Ala Pro Glu Lys Gly Leu
Glu Trp Val 35 40 45Ala Thr Ile Thr Tyr Asn Gly Thr Ser Thr Tyr Tyr
Arg Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Arg Ser Glu
Asp Thr Ala Thr Tyr Tyr Cys 85 90 95Ala Arg Trp Val Pro Gly Ser Gly
Asn Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155
160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280
285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395
400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly 435 440 44543218PRTArtificial
Sequenceimmunoglobulin chain 43Asp Ile Val Leu Thr Gln Ser Pro Ala
Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg
Ala Ser Gln Ser Val Thr Ile Ser 20 25 30Arg Tyr Thr Leu Met His Trp
Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Arg
Ala Ser Asn Leu Ala Ser Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His65 70 75 80Pro Val Glu
Glu Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Arg 85 90 95Glu Ser
Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105
110Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln
115 120 125Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn
Phe Tyr 130 135 140Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly
Ser Glu Arg Gln145 150 155 160Asn Gly Val Leu Asn Ser Trp Thr Asp
Gln Asp Ser Lys Asp Ser Thr 165 170 175Tyr Ser Met Ser Ser Thr Leu
Thr Leu Thr Lys Asp Glu Tyr Glu Arg 180 185 190His Asn Ser Tyr Thr
Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro 195 200 205Ile Val Lys
Ser Phe Asn Arg Asn Glu Cys 210 21544448PRTArtificial
Sequenceimmunoglobulin chain 44Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asn Ser 20 25 30Gly Leu Ala Trp Val Arg Gln
Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Thr Tyr Asn
Gly Thr Ser Thr Tyr Tyr Arg Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met
Ser Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95Ala Arg
Trp Val Pro Gly Ser Gly Asn Phe Asp Tyr Trp Gly Gln Gly 100 105
110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125Pro Leu Ala Pro Ser Ser Lys Asn Thr Ser Gly Gly Thr Ala
Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro225 230
235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345
350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
44545448PRTArtificial Sequenceimmunoglobulin chain 45Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ser 20 25 30Gly
Leu Ala Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40
45Ala Thr Ile Thr Tyr Asn Gly Thr Ser Thr Tyr Tyr Arg Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Thr
Tyr Tyr Cys 85 90 95Ala Arg Trp Val Pro Gly Ser Gly Asn Phe Asp Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Asn
Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Thr
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185
190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310
315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425
430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 44546448PRTArtificial Sequenceimmunoglobulin chain 46Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ser
20 25 30Gly Leu Ala Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp
Val 35 40 45Ala Thr Ile Thr Tyr Asn Gly Thr Ser Thr Tyr Tyr Arg Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr
Ala Thr Tyr Tyr Cys 85 90 95Ala Arg Trp Val Pro Gly Ser Gly Asn Phe
Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser
Lys Asn Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295
300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410
415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly 435 440 44547448PRTArtificial Sequenceimmunoglobulin chain
47Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg
Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Ile 35 40 45Gly Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Ala
Pro Ser Leu 50 55 60Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Pro Asp Gly Asn Tyr Trp Tyr
Phe Asp Val Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155
160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280
285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375
380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly 435 440 44548214PRTArtificial
Sequenceimmunoglobulin chain 48Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys
Ala Ser Gln Asp Val Gly Ile Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg
His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Val
Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Tyr 85 90 95Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105
110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg
Gly Glu Cys 21049448PRTArtificial Sequenceimmunoglobulin chain
49Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg
Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Ile 35 40 45Gly Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Ala
Pro Ser Leu 50 55 60Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Pro Asp Gly Asn Tyr Trp Tyr
Phe Asp Val Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser
Ser Lys Asn Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155
160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280
285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395
400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly 435 440 44550448PRTArtificial
Sequenceimmunoglobulin chain 50Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Asp Phe Ser Arg Tyr 20 25 30Trp Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Asn Pro Asp
Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu 50 55 60Lys Asp Lys Phe Ile
Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Pro Asp Gly Asn Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly 100 105
110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp145 150 155 160Asn Ser Thr Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro225 230
235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345
350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
44551448PRTArtificial Sequenceimmunoglobulin chain 51Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr 20 25 30Trp
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45Gly Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu
50 55 60Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Pro Asp Gly Asn Tyr Trp Tyr Phe Asp Val
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Asn
Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Thr
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185
190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310
315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425
430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 44552447PRTArtificial Sequenceimmunoglobulin chain 52Gln
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Ile Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Arg Asn Lys Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Thr Asp Gly Tyr Asp Phe Asp
Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170
175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr 210 215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295
300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr305 310 315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr 325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu 340 345 350Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410
415Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly 435 440 44553214PRTArtificial Sequenceimmunoglobulin chain
53Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile 35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Arg Thr Asn Trp Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155
160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
21054447PRTArtificial Sequenceimmunoglobulin chain 54Gln Val Gln
Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ile
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Val Ile Ser Tyr Asp Gly Arg Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Asp Thr Asp Gly Tyr Asp Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro 115 120 125Leu Ala Pro Ser Ser Lys Asn Thr Ser Gly Gly Thr
Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215
220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro 260 265 270Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315 320Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330
335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
44555447PRTArtificial Sequenceimmunoglobulin chain 55Gln Val Gln
Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ile
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Val Ile Ser Tyr Asp Gly Arg Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Asp Thr Asp Gly Tyr Asp Phe Asp Tyr Trp
Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Thr Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185
190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr 210 215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro 260 265 270Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310
315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr 325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu 340 345 350Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425
430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435
440 44556447PRTArtificial Sequenceimmunoglobulin chain 56Gln Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Ile Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Val Ile Ser Tyr Asp Gly Arg Asn Lys Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Asp Thr Asp Gly Tyr Asp Phe Asp Tyr
Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Ser Ser Lys Asn
Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Thr
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170
175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr 210 215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295
300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr305 310 315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr 325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu 340 345 350Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410
415Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly 435 440 44557680PRTFusarium graminearum 57Met Lys His Leu Leu
Thr Leu Ala Leu Cys Phe Ser Ser Ile Asn Ala1 5 10 15Val Ala Val Thr
Val Pro His Lys Ala Val Gly Thr Gly Ile Pro Glu 20 25 30Gly Ser Leu
Gln Phe Leu Ser Leu Arg Ala Ser Ala Pro Ile Gly Ser 35 40 45Ala Ile
Ser Arg Asn Asn Trp Ala Val Thr Cys Asp Ser Ala Gln Ser 50 55 60Gly
Asn Glu Cys Asn Lys Ala Ile Asp Gly Asn Lys Asp Thr Phe Trp65 70 75
80His Thr Phe Tyr Gly Ala Asn Gly Asp Pro Lys Pro Pro His Thr Tyr
85 90 95Thr Ile Asp Met Lys Thr Thr Gln Asn Val Asn Gly Leu Ser Met
Leu 100 105 110Pro Arg Gln Asp Gly Asn Gln Asn Gly Trp Ile Gly Arg
His Glu Val 115 120 125Tyr Leu Ser Ser Asp Gly Thr Asn Trp Gly Ser
Pro Val Ala Ser Gly 130 135 140Ser Trp Phe Ala Asp Ser Thr Thr Lys
Tyr Ser Asn Phe Glu Thr Arg145 150 155 160Pro Ala Arg Tyr Val Arg
Leu Val Ala Ile Thr Glu Ala Asn Gly Gln 165 170 175Pro Trp Thr Ser
Ile Ala Glu Ile Asn Val Phe Gln Ala Ser Ser Tyr 180 185 190Thr Ala
Pro Gln Pro Gly Leu Gly Arg Trp Gly Pro Thr Ile Asp Leu 195 200
205Pro Ile Val Pro Ala Ala Ala Ala Ile Glu Pro Thr Ser Gly Arg Val
210 215 220Leu Met Trp Ser Ser Tyr Arg Asn Asp Ala Phe Gly Gly Ser
Pro Gly225 230 235 240Gly Ile Thr Leu Thr Ser Ser Trp Asp Pro Ser
Thr Gly Ile Val Ser 245 250 255Asp Arg Thr Val Thr Val Thr Lys His
Asp Met Phe Cys Pro Gly Ile 260 265 270Ser Met Asp Gly Asn Gly Gln
Ile Val Val Thr Gly Gly Asn Asp Ala 275 280 285Lys Lys Thr Ser Leu
Tyr Asp Ser Ser Ser Asp Ser Trp Ile Pro Gly 290 295 300Pro Asp Met
Gln Val Ala Arg Gly Tyr Gln Ser Ser Ala Thr Met Ser305 310 315
320Asp Gly Arg Val Phe Thr Ile Gly Gly Ser Trp Ser Gly Gly Val Phe
325 330 335Glu Lys Asn Gly Glu Val Tyr Ser Pro Ser Ser Lys Thr Trp
Thr Ser 340 345 350Leu Pro Asn Ala Lys Val Asn Pro Met Leu Thr Ala
Asp Lys Gln Gly 355 360 365Leu Tyr Arg Ser Asp Asn His Ala Trp Leu
Phe Gly Trp Lys Lys Gly 370 375 380Ser Val Phe Gln Ala Gly Pro Ser
Thr Ala Met Asn Trp Tyr Tyr Thr385 390 395 400Ser Gly Ser Gly Asp
Val Lys Ser Ala Gly Lys Arg Gln Ser Asn Arg 405 410 415Gly Val Ala
Pro Asp Ala Met Cys Gly Asn Ala Val Met Tyr Asp Ala 420 425 430Val
Lys Gly Lys Ile Leu Thr Phe Gly Gly Ser Pro Asp Tyr Gln Asp 435 440
445Ser Asp Ala Thr Thr Asn Ala His Ile Ile Thr Leu Gly Glu Pro Gly
450 455 460Thr Ser Pro Asn Thr Val Phe Ala Ser Asn Gly Leu Tyr Phe
Ala Arg465 470 475 480Thr Phe His Thr Ser Val Val Leu Pro Asp Gly
Ser Thr Phe Ile Thr 485 490 495Gly Gly Gln Arg Arg Gly Ile Pro Phe
Glu Asp Ser Thr Pro Val Phe 500 505 510Thr Pro Glu Ile Tyr Val Pro
Glu Gln Asp Thr Phe Tyr Lys Gln Asn 515 520 525Pro Asn Ser Ile Val
Arg Val Tyr His Ser Ile Ser Leu Leu Leu Pro 530 535 540Asp Gly Arg
Val Phe Asn Gly Gly Gly Gly Leu Cys Gly Asp Cys Thr545 550 555
560Thr Asn His Phe Asp Ala Gln Ile Phe Thr Pro Asn Tyr Leu Tyr Asn
565 570 575Ser Asn Gly Asn Leu Ala Thr Arg Pro Lys Ile Thr Arg Thr
Ser Thr 580 585 590Gln Ser Val Lys Val Gly Gly Arg Ile Thr Ile Ser
Thr Asp Ser Ser 595 600 605Ile Ser Lys Ala Ser Leu Ile Arg Tyr Gly
Thr Ala Thr His Thr Val 610 615 620Asn Thr Asp Gln Arg Arg Ile Pro
Leu Thr Leu Thr Asn Asn Gly Gly625 630 635 640Asn Ser Tyr Ser Phe
Gln Val Pro Ser Asp Ser Gly Val Ala Leu Pro 645 650 655Gly Tyr Trp
Met Leu Phe Val Met Asn Ser Ala Gly Val Pro Ser Val 660 665 670Ala
Ser Thr Ile Arg Val Thr Gln 675 680582189DNAFusarium graminearum
58atggccgatc agcaaacggt ccttagtgta tccgtacctg gatatataag actggaagat
60atcagttgtt cttcatctgc cagtatcacc ttcattatct attcaagtca ctctctcaac
120ttattcttgc ctctctctat gtcaatatga aacacttttt atcactcgct
ctttgcttca 180gcagcatcaa tgctgttgct gtcaccgtcc ctcacaagtc
cggaggaact ggaagtcctg 240aagggagtct tcagttcctg agtcttcggg
cctcagcacc tatcggaagc gctatttctc 300gcaacaactg ggccgtcact
tgcgacagtg cacagtcggg aaatgaatgc aacaaggcca 360tcgatggcaa
caaggatacc ttttggcaca cattctatgg ggccaatgga gatccaaagc
420cccctcacac atacacgatt gacatgaaga caactcagaa tgtcaacggc
ttgtctatgt 480tgcctcgaca ggatggtaac caaaacggct ggatcggtcg
ccatgaggtt tatctaagct 540cagatggcac aaactggggc agccctgttg
cgtcaggtag ttggtttgcc gactctacta 600caaaatactc caactttgaa
actcgccctg ctcgctatgt tcgtcttgtc gctgtcactg 660aagcgaatgg
ccagccttgg actagcattg cagagatcaa cgtcttccaa gctagttctt
720acacagcccc tcagcctggc cttggccgct ggggtccgac tattgacttg
ccgattgttc 780ctgcggctgc agcaattgag ccgacatcgg gacgagtcct
tatgtggtct tcgtatcgca 840atgatgcatt tggaggatcc cctggtggta
tcactttgac gtcttcgtgg gatccatcca 900ctggcattgt ttccgaccgc
actgtgacag tcaccaagca tgatatgttc tgccctggta 960tctccatgga
tggtaacggt cagatcgtag tcacaggtgg caacgacgcc aagaagacca
1020gtttgtatga ttcatctagc gatagctgga tcccgggacc tgacatgcaa
gtggctcgtg 1080ggtatcagtc atcagctacc atgtcagacg gtcgtgtttt
taccattgga ggctcctgga 1140gcggtggcgt atttgagaag aatggcgaag
tctatagccc atcttcaaag acatggacgt 1200ccctacccaa tgccaaggtc
aacccaatgt tgacggctga caagcaagga ttgtaccgtt 1260cagacaacca
cgcgtggctc tttggatgga agaagggttc ggtgttccaa gcgggaccta
1320gtacagccat gaactggtac tataccagtg gaagtggcga tgtgaagtca
gccggaaaac 1380gccagtctaa ccgtggtgta gcccctgatg ccatgtgcgg
aaacgctgtc atgtacgacg 1440ccgttaaagg aaagatcctg acctttggcg
gctccccaga ctatcaagac tctgacgcca 1500caaccaacgc ccacatcatc
accctcggtg aacccggaac atctcccaac actgtctttg 1560ctagcaatgg
cttgtacttt gctcgaacgt tccacacctc tgttgttctt ccagacggaa
1620gcacgttcat tacaggaggc caacgacgtg gaattccgtt cgaggattca
accccggtat 1680ttacacctga gatctacgtc cctgaacaag acactttcta
caagcagaac cccaactcca 1740ttgttcgcgt ctaccacagc atttcccttt
tgttacctga tggcagggta tttaacggtg 1800gtggtggtct ttgtggcgat
tgtaccacga atcatttcga cgcgcaaatc tttacgccaa 1860actatcttta
caatagcaac ggcaatctcg cgacacgtcc caagattacc agaacctcta
1920cacagagcgt caaggtcggt ggcaggatca caatctcgac ggactcttcg
attacaaagg 1980cgtcgttgat tcgctatggt acagcgacac acacggttaa
tactgaccag cgtcgcattc 2040ccctgactct gacaaacaat ggaggaaata
gctattcttt ccaagttcct agcgactctg 2100gtgttgcttt gcctggctac
tggatgttgt tcgtgatgaa ctcggccggt gttcctagtg 2160tggcttcgac
gattcgcgtt actcagtga 2189
* * * * *