U.S. patent application number 17/403592 was filed with the patent office on 2022-03-24 for glycoengineered antibody drug conjugates.
The applicant listed for this patent is Genzyme Corporation. Invention is credited to Luis Z. Avila, Qun Zhou.
Application Number | 20220088214 17/403592 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-24 |
United States Patent
Application |
20220088214 |
Kind Code |
A1 |
Avila; Luis Z. ; et
al. |
March 24, 2022 |
GLYCOENGINEERED ANTIBODY DRUG CONJUGATES
Abstract
The current disclosure provides binding polypeptides (e.g.,
antibodies), and targeting moiety conjugates thereof, comprising a
site-specifically engineered glycan linkage within native or
engineered glycans of the binding polypeptide. The current
disclosure also provides nucleic acids encoding the antigen-binding
polypeptides, recombinant expression vectors and host cells for
making such antigen-binding polypeptides. Methods of using the
antigen-binding polypeptides disclosed herein to treat disease are
also provided.
Inventors: |
Avila; Luis Z.; (Arlington,
MA) ; Zhou; Qun; (Ashland, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genzyme Corporation |
Cambridge |
MA |
US |
|
|
Appl. No.: |
17/403592 |
Filed: |
August 16, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16055661 |
Aug 6, 2018 |
11160874 |
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17403592 |
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14878444 |
Oct 8, 2015 |
10064952 |
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16055661 |
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62061989 |
Oct 9, 2014 |
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International
Class: |
A61K 47/68 20060101
A61K047/68; C07K 16/28 20060101 C07K016/28; C07K 16/32 20060101
C07K016/32; C07K 16/40 20060101 C07K016/40; A61K 47/54 20060101
A61K047/54 |
Claims
1-16. (canceled)
17. A method of making an effector moiety conjugated binding
polypeptide comprising the steps of: (a) reacting a CMP-sialic acid
derivative with a glycan of a binding polypeptide to form a sialic
acid derivative-conjugated binding polypeptide; (b) reacting the
sialic acid derivative-conjugated binding polypeptide with an
effector moiety to form the effector moiety conjugated binding
polypeptide, wherein a thioether bond is formed.
18. The method of claim 17, wherein neither the binding polypeptide
nor the sialic acid derivative-conjugated binding polypeptide are
treated with an oxidizing agent.
19. The method of claim 17, wherein the sialic acid derivative
comprises a terminal thiol moiety.
20. The method of claim 17, wherein the effector moiety comprises a
maleimide moiety.
21. The method of claim 17, wherein the effector moiety is
bis-mannose-6-phosphate hexamannose maleimide, lactose maleimide,
or any other component comprising at least one maleimide moiety of
the following structural formula: ##STR00076##
22. The method of claim 17, wherein the effector moiety comprises
one or more proteins, nucleic acids, lipids, carbohydrates, or
combinations thereof.
23. The method of claim 17, wherein the effector moiety comprises a
glycan.
24. The method of claim 17, wherein the effector moiety comprises
one or more glycoproteins, glycopeptides, or glycolipids.
25. The method of claim 17, wherein the binding protein has one or
more native or engineered glycosylation sites.
26. The method of claim 17, further comprising achieving or
modifying the glycosylation of the binding protein using one or
more glycosyltransferases, one or more glycosidases, or a
combination thereof.
27. The method of claim 26, wherein step (a) occurs in a reaction
with sialyltransferase.
28. The method of claim 27, wherein the sialyltransferase is a
mammalian sialyltransferase.
29. The method of claim 28, wherein the sialyltransferase is
beta-galactoside alpha-2,6-sialyltransferase 1.
30. The method of claim 17, wherein the CMP-sialic acid derivative
has the following structure: ##STR00077##
31. The method of claim 17, wherein the binding polypeptide
comprises a S298N mutant.
32. The method of claim 17, wherein the binding polypeptide
comprises an A114N mutant in the CH1 domain.
33. The method of claim 17, wherein the binding polypeptide
comprises a Y300S mutant.
34. The method of claim 17, wherein the binding polypeptide
comprises a heavy chain of SEQ ID NO:14 and a light chain of SEQ ID
NO:12.
35. The method of claim 17, wherein the effector moiety comprises
polyethylene glycol (PEG).
36. The method of claim 17, wherein the effector moiety comprises
one of the following structures: ##STR00078## ##STR00079##
##STR00080## ##STR00081## ##STR00082##
Description
RELATED APPLICATIONS
[0001] This application is a division of U.S. patent application
Ser. No. 16/055,661, filed Aug. 6, 2018, which is a division of
U.S. patent application Ser. No. 14/878,444, filed Oct. 8, 2015,
now U.S. Pat. No. 10,064,952, which claims the benefit of priority
to U.S. Provisional Patent Application Ser. No. 62/061,989, filed
Oct. 9, 2014. The entire contents of the aforementioned
applications are incorporated by reference in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Aug. 16, 2021, is named 721364_SA9-166DIV2_ST25.txt and is
114,820 bytes in size.
BACKGROUND
[0003] Use of specific antibodies to treat people and other animals
is a powerful tool that has been very effective in treating many
conditions and disorders. However, there is great demand for more
effective targeted therapeutics, especially target specific
therapies with higher efficacy and greater therapeutic window. One
of these target specific treatments employs antibody-effector
moiety conjugates in which a targeting moiety directs a specific
antibody to a desired treatment site. These molecules have shown
improved therapeutic index--higher efficacy and/or lower toxicity
profiles than the un-targeted antibody in a clinical setting.
However, development of such therapeutics can be challenging as
many factors, including the antibody itself and linkage stability,
can have significant impact on the disease target (e.g. tumor)
specificity, thereby reducing efficacy. With high non-specific
binding and low stability in circulation, the antibody-effector
moiety conjugate would be cleared through normal tissues before
reaching the target site. Moreover, antibody-effector moiety
conjugates with significant subpopulations of high drug loading
could generate aggregates which would be eliminated by macrophages,
leading to shorter half-life. Thus, there are increasing needs for
critical process control and improvement as well as preventing
complications such as the product aggregation and nonspecific
toxicity from antibodies.
[0004] Although antibody-effector moiety conjugates generated
according to current methods are effective, development of such
therapeutics can be challenging as heterogeneous mixtures are often
a consequence of the conjugation chemistries used. For example,
effector moiety conjugation to antibody lysine residues is
complicated by the fact that there are many lysine residues
(.about.30) in an antibody available for conjugation. Since the
optimal number of conjugated effector moiety to antibody ratio
(DAR) is much lower to minimize loss of function of the antibody
(e.g., around 4:1), lysine conjugation often generates a very
heterogeneous profile. Furthermore, many lysines are located in
critical antigen binding sites of CDR region and drug conjugation
may lead to a reduction in antibody affinity. On the other hand,
while thiol mediated conjugation mainly targets the eight cysteines
involved in hinge disulfide bonds, it is still difficult to predict
and identify which four of eight cysteines are consistently
conjugated among the different preparations. More recently, genetic
engineering of free cysteine residues has enabled site-specific
conjugation with thiol-based chemistries, but such linkages often
exhibit highly variable stability, with the linker undergoing
exchange reactions with albumin and other thiol-containing serum
molecules. Finally, oxidizing agents (such as periodate oxidase and
galactose oxidase) used to treat antibodies in previously developed
conjugation protocols can cause over-oxidation and extraneous
oxidation of the binding polypeptide, reducing efficiency and
efficacy of the conjugation itself.
[0005] Therefore, a site-specific conjugation strategy which
generates an antibody conjugate with a defined conjugation site and
stable linkage without the use of oxidizing agents would be highly
useful in guaranteeing effector moiety conjugation while minimizing
adverse effects on antibody structure or function.
SUMMARY
[0006] The current disclosure provides methods of making effector
moiety conjugates (e.g., targeting moiety conjugates). These
methods involve the incorporation of sialic acid derivatives in the
glycan of a binding polypeptide to form a sialic acid
derivative-conjugated binding polypeptide, and a subsequent
reaction in which an effector moiety is reacted with the sialic
acid derivative-conjugated binding protein to create an effector
moiety-conjugated binding polypeptide.
[0007] In one aspect, the instant disclosure provides methods of
making an effector moiety conjugated binding polypeptide comprising
the steps of: (a) reacting a cytidine monophosphate-sialic acid
(CMP-sialic acid) derivative with a glycan of a binding polypeptide
to form a sialic acid derivative-conjugated binding polypeptide;
and (b) reacting the sialic acid derivative-conjugated binding
polypeptide with an effector moiety to form the effector moiety
conjugated binding polypeptide, wherein an imine bond is formed,
and wherein neither the binding polypeptide nor the sialic acid
derivative-conjugated binding polypeptide are treated with an
oxidizing agent.
[0008] In one embodiment, the sialic acid derivative-conjugated
binding polypeptide comprises a terminal keto or aldehyde moiety.
In another embodiment, the effector moiety comprises a terminal
aminooxy moiety or is bound to a moiety comprising an aminooxy
derivative. In a further embodiment, the effector moiety is
selected from those in FIGS. 45 and 46.
[0009] In one embodiment, step (b) results in the formation of an
oxime bond. In another embodiment, the effector moiety comprises a
terminal hydrazine. In a specific embodiment, step (b) results in
the formation of a hydrazone linkage. In a further embodiment, the
effector moiety has one or more of the following structural
formulas:
##STR00001## ##STR00002##
[0010] In one aspect, the instant application provides methods of
making an effector moiety conjugated binding polypeptide comprising
the steps of: (a) reacting a CMP-sialic acid derivative comprising
a terminal reactive moiety at the C5 position with a glycan of a
binding polypeptide to form a sialic acid derivative-conjugated
binding polypeptide; and (b) reacting the sialic acid
derivative-conjugated binding polypeptide with an effector moiety
to form the effector moiety conjugated binding polypeptide using
click chemistry.
[0011] In one embodiment, the terminal reactive moiety is an azide,
wherein the effector moiety comprises an alkyne or is bound to a
moiety comprising an alkyne, and wherein step (b) forms a triazole
ring at or linked to the C5 position of the sialic acid derivative.
In another embodiment, neither the binding polypeptide nor the
sialic acid derivative-conjugated binding polypeptide are treated
with an oxidizing agent. In another embodiment, the CMP-sialic acid
derivative has a structural formula selected from the
following:
##STR00003##
[0012] In another embodiment, the effector moiety comprises or is
bound to a cyclooctyne. In a specific embodiment, the cyclooctyne
is an azadibenzocyclooctyne. In another embodiment, step (b) occurs
at ambient temperatures. In another embodiment, step (b) is
performed in the absence of copper.
[0013] In one aspect, the instant application provides methods of
making an effector moiety conjugated binding polypeptide comprising
the steps of: (a) reacting a CMP-sialic acid derivative with a
glycan of a binding polypeptide to form a sialic acid
derivative-conjugated binding polypeptide; and (b) reacting the
sialic acid derivative-conjugated binding polypeptide with an
effector moiety to form the effector moiety conjugated binding
polypeptide, wherein a thioether bond is formed.
[0014] In one embodiment, neither the binding polypeptide nor the
sialic acid derivative-conjugated binding polypeptide are treated
with an oxidizing agent. In another embodiment, the sialic acid
derivative comprises a terminal thiol moiety. In another
embodiment, the effector moiety comprises a maleimide moiety. In
another embodiment, the effector moiety is bis-mannose-6-phosphate
hexamannose maleimide, lactose maleimide, or any other component
comprising at least one maleimide moiety of the following
structural formula:
##STR00004##
[0015] In one embodiment, the effector moiety comprises one or more
proteins, nucleic acids, lipids, carbohydrates, or combinations
thereof. In another embodiment, the effector moiety comprises a
glycan. In a specific embodiment, the effector moiety comprises one
or more glycoproteins, glycopeptides, or glycolipids.
[0016] In another embodiment, the binding protein has one or more
native or engineered glycosylation sites. In a further embodiment,
the method comprising achieving or modifying the glycosylation of
the binding protein using one or more glycosyltransferases, one or
more glycosidases, or a combination thereof. In another embodiment,
step (a) occurs in a reaction with sialyltransferase. In a further
embodiment, the sialyltransferase is a mammalian sialyltransferase.
In a specific embodiment, the sialyltransferase is beta-galactoside
alpha-2,6-sialyltransferase 1. In one embodiment, the effector
moiety binds to a cell. In a further embodiment, the cell is
selected from an immune cell, a liver cell, a tumor cell, a
vascular cell, an epithelial cell, or a mesenchymal cell. In
another embodiment, the cell is selected from a B cell, a T cell, a
dendritic cell, a natural killer (NK) cell, a macrophage, a
hepatocyte, a liver sinusoidal endothelial cell, or a hepatoma
cell.
[0017] In one embodiment, the effector moiety binds to a mannose 6
phosphate receptor on the cell. In a further embodiment, the
effector moiety comprises a mannose 6 phosphate moiety. In another
embodiment, the effector moiety binds to a Siglec on the cell. In a
further embodiment, the Siglec is sialoadhesin (Siglec-1), CD22
(Siglec-2), CD33 (Siglec-3), MAG (Siglec-4), Siglec-5, Siglec-6,
Siglec-7, Siglec-8, Siglec-9, Siglec-10, Siglec-11, Siglec-12,
Siglec-14, or Siglec-15. In another embodiment, the effector moiety
binds to a C-type lectin receptor, a galectin, or an L-type lectin
receptor on the cell. In a further embodiment, the effector moiety
binds to TDEC-205, macrophage mannose receptor (MMR), Dectin-1,
Dectin-2, macrophage-inducible C-type lectin (Mincle), dendritic
cell-specific ICAM3-grabbing nonintegrin (DC-SIGN, CD209), DC NK
lectin group receptor-1 (DNGR-1), Langerin (CD207), CD169, a
lectican, an asialoglycoprotein receptor (ASGPR), DCIR, MGL, a DC
receptor, a collectin, a selectin, an NK-cell receptor, a
multi-CTLD endocytic receptor, a Reg group (type VII) lectin,
chondrolectin, tetranectin, polycystin, attractin (ATRN),
eosinophil major basic protein (EMBP), DGCR2, Thrombomodulin,
Bimlec, SEEC, or CBCP/Frem1/QBRICK.
[0018] In one embodiment, the effector moiety is a glycopeptide
capable of binding ASGPR on a cell. In a further embodiment, the
effector moiety is a trivalent GalNAc glycan containing
glycopeptide or a trivalent galactose containing glycopeptide. In a
specific embodiment, the effector moiety is represented by Formula
V:
##STR00005##
In another specific embodiment, the effector moiety is represented
by Formula VI:
##STR00006##
[0019] In one embodiment, the binding polypeptide comprises an Fc
domain. In another embodiment, a modified glycan is N-linked to the
binding polypeptide via an asparagine residue at amino acid
position 297 of the Fc domain, according to EU numbering. In
another embodiment a modified glycan is N-linked to the binding
polypeptide via an asparagine residue at amino acid position 298 of
the Fc domain, according to EU numbering. In a further embodiment,
the Fc domain is human.
[0020] In another embodiment, the binding polypeptide comprises a
CH1 domain. In a further embodiment, a modified glycan is N-linked
to the binding polypeptide via an asparagine residue at amino acid
position 114 of the CH1 domain, according to Kabat numbering.
[0021] In a specific embodiment, the binding polypeptide is an
antibody or immunoadhesin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 is a schematic illustration of the formation of
exemplary CMP-sialic acid derivatives from sugar or sugar
derivatives.
[0023] FIG. 2 is a schematic illustration of exemplary CMP-sialic
acid derivatives.
[0024] FIGS. 3A-3E are a series of depictions of different chemical
reactions of the instant invention, the circles in combination with
the reactive moieties to which they are bonded represent sialic
acid derivative-conjugated binding polypeptides. The stars
represent targeting or effector moieties.
[0025] FIG. 4 depicts an example of an effector moiety-conjugated
binding polypeptide according to the methods illustrated in FIG. 3
(parts A-C) with a sialic acid derivative shown in FIG. 2.
[0026] FIG. 5 depicts an example of an effector moiety-conjugated
binding polypeptide according to the methods illustrated in FIG. 3D
with a sialic acid derivative shown in FIG. 2.
[0027] FIG. 6 is a schematic illustration of the synthesis of an
antibody drug conjugate where a toxin moiety is linked to an
oxidized sialic acid residue of the antibody glycan using an oxime
linkage.
[0028] FIG. 7 is a Coomassie-blue stained gel showing the
expression and purification of glycosylation mutants.
[0029] FIG. 8 depicts the results of surface plasmon resonance
experiments used to assess the binding of .alpha..beta.TCR HEBE1
IgG antibody mutants to recombinant human Fc.gamma.RIIIa (V158
& F158).
[0030] FIG. 9 depicts the results of surface plasmon resonance
experiments used to assess the binding of .alpha..beta.TCR HEBE1
IgG antibody mutants to recombinant human Fc.gamma.RT.
[0031] FIG. 10 depicts the cytokine release profile from PBMCs for
TNFa, GM-CSF, IFNy and IL10 in the presence of mutant
anti-.alpha..beta.TCR antibodies (day 2).
[0032] FIG. 11 depicts the cytokine release profile from PBMCs for
IL6, IL4 and IL2 in the presence of mutant anti-.alpha..beta.TCR
antibodies (day 2).
[0033] FIG. 12 depicts the cytokine release profile from PBMCs for
TNFa, GM-CSF, IFNy and IL10 in the presence of mutant
anti-.alpha..beta.TCR antibodies (day 4).
[0034] FIG. 13 depicts the cytokine release profile from PBMCs for
IL6, IL4 and IL2 in the presence of mutant anti-.alpha..beta.TCR
antibodies (day 4).
[0035] FIGS. 14A-14B depict the results of experiments
investigating the expression level of 2C3 mutants by Western
blotting (FIG. 14A) and surface plasmon resonance (FIG. 14B).
[0036] FIG. 15 depicts the results of experiments investigating
glycosylation of 2C3 mutants pre- and post-PNGase F treatment.
[0037] FIG. 16 depicts the results of SDS-PAGE experiments
investigating glycosylation sites on 2C3 mutants isolated from cell
culture.
[0038] FIGS. 17A-17C depict the results of surface plasmon
resonance experiments used to assess the binding of modified
anti-CD52 to recombinant human Fc.gamma.RIIIa (V158). Anti-CD52
comprising S298N/Y300S mutations in the Fc domain were used to
assess the effector function of the modified molecule. binding to
CD52 peptide (FIG. 17A), binding to Fc.gamma.RIIIa (V158, FIG.
17B), and control binding to mouse FcRn (FIG. 17C).
[0039] FIG. 18 depicts the results of surface plasmon resonance
experiments investigating the Fc binding properties of 2C3
mutants.
[0040] FIGS. 19A-19B depict the results of surface plasmon
resonance experiments investigating the binding of modified
anti-CD52 to both Fc.gamma.RIIIa (Val158) (as above) and
Fc.gamma.RIIIa (Phe158). Anti-CD52 antibodies comprising
S298N/Y300S mutations in the Fc domain were used to assess the
effector function of the modified molecule binding to
Fc.gamma.RIIIa (Val158, FIG. 19A) and Fc.gamma.RIIIa (Phe58, FIG.
19B).
[0041] FIGS. 20A-20B depict the analysis of C1q binding in the
S298N/Y300S mutant and the WT 2C3 control (FIG. 20A) and the
results of an Eliza analysis confirming equivalent coating of the
wells (FIG. 20B).
[0042] FIG. 21 depicts the results of plasmon resonance experiments
measuring the binding kinetics of 2C3 mutants to CD-52 peptide
741.
[0043] FIG. 22 depicts the results of plasmon resonance experiments
comparing the antigen binding affinity of WT anti-CD-52 2C3 and the
A114N hyperglycosylation mutant.
[0044] FIG. 23A-23D depict the results of isoelectric focusing and
mass spectrometry charge characterization experiments to determine
the glycan content of 2C3 mutants.
[0045] FIGS. 24A-24B depict the results of concentration (FIG. 24A;
Octet) and plasmon resonance experiments (FIG. 24B) comparing the
antigen binding affinity of WT anti-CD52 2C3 and mutants.
[0046] FIG. 25 depicts the results of SDS-PAGE experiments to
demonstrate the additional glycosylation of the anti-TEM1 A114N
mutant.
[0047] FIG. 26 depicts the results of SDS-PAGE and hydrophobic
interaction chromatography analysis of the A114N anti-Her2
mutant.
[0048] FIG. 27 depicts the results of SDS-PAGE experiments to
demonstrate the conjugation of PEG to the 2C3 A114N mutant through
an aminooxy linkage.
[0049] FIG. 28 depicts the results of LC-MS experiments to
determine the glycan contents of anti-TEM1 A114N hyperglycosylation
mutant.
[0050] FIGS. 29A-29B depict the results of LC-MS experiments to
determine the glycan contents of a wild-type HER2 antibody (FIG.
29A) and an A114N anti-Her2 hyperglycosylation mutant (FIG.
29B).
[0051] FIGS. 30A-30C depict an alternative method for performing
site-specific conjugation of an antibody comprising the use of
oxidizing agents.
[0052] FIG. 31 depicts a synthesis of exemplary effector moieties:
aminooxy-Cys-MC-VC-PABC-MMAE and
aminooxy-Cys-MC-VC-PABC-PEG8-Dol10.
[0053] FIGS. 32A-32C depict characterization information for a
sialylated HER2 antibody.
[0054] FIGS. 33A-33D depict characterization information for
oxidized sialylated anti-HER 2 antibody.
[0055] FIG. 34 depicts hydrophobic interaction chromatographs of
glycoconjugates prepared with three different sialylated antibodies
with two different aminooxy groups.
[0056] FIG. 35 shows a HIC chromatograph of anti-Her2 A114
glycosylation mutant conjugate with AO-MMAE prepared using GAM(+)
chemistry.
[0057] FIGS. 36A-36D depicts a comparison of the in vitro potency
of an anti-HER2 glycoconjugate and thiol conjugate.
[0058] FIG. 37 depicts a comparison of the in vitro potency of an
anti FAP B11 glycoconjugate and thiol conjugate.
[0059] FIGS. 38A-38D depicts a comparison of in vivo efficacy of
anti-HER2 glycoconjugates and thiol conjugates in a Her2+ tumor
cell xenograft model.
[0060] FIG. 39 depicts the results of LC-MS experiments to
determine the glycan content of a mutant anti-.alpha..beta.TCR
antibody containing the S298N/Y300S mutation. FIG. 39 discloses
"NNAS" as SEQ ID NO: 40.
[0061] FIG. 40 depicts the results of circular dichroism
experiments to determine the relative thermal stability of a
wild-type anti-.alpha..beta.TCR antibody and mutant
anti-.alpha..beta.TCR antibody containing the S298N/Y300S
mutation.
[0062] FIG. 41 depicts the results of a cell proliferation assay
for ADC prepared with the anti-HER antibody bearing the A114N
hyperglycosylation mutation and AO-MMAE.
[0063] FIG. 42 is a schematic illustration of an alternative
synthesis of an antibody drug conjugate where a targeting moiety is
linked to an oxidized sialic acid residue of the antibody glycan
using an oxime linkage. This alternative synthesis makes use of
oxidizing agents.
[0064] FIG. 43 is a schematic illustration depicting an alternative
method for performing site-specific conjugation of an antibody to a
glycopeptide through an aminooxy linkage according to the disclosed
methods. This alternative synthesis makes use of oxidizing
agents.
[0065] FIG. 44 is a schematic illustration depicting an alternative
method of site-specific conjugation of neoglycans to antibody
through sialic acid in native Fc glycans. This alternative
synthesis makes use of oxidizing agents.
[0066] FIG. 45 is a series of exemplary glycans that may be used
for conjugation including lactose aminooxy and bis M6P hexamannose
aminooxy (for aminooxy conjugation).
[0067] FIG. 46 is a schematic depiction the preparation of M-6-P
hexamannose maleimide.
[0068] FIG. 47 depicts SDS-PAGE and MALDI-TOF characterization of
Man-6-P hexamannose aminooxy conjugates made with rabbit polyclonal
antibody.
[0069] FIG. 48 depicts the results of surface plasmon resonance
experiments used to assess the binding of control and Man-6-P
hexamannose conjugated rabbit IgG antibodies to M6P receptor.
[0070] FIG. 49 depicts the uptake of Man-6-P conjugated rabbit IgG
antibody in HepG2 and RAW cells.
[0071] FIG. 50 depicts the characterization of control, Man-6-P
conjugated, and lactose conjugated antibodies through SDS-PAGE and
lectin blotting.
[0072] FIG. 51 depicts the results of MALDI-TOF intact protein
analyses for control, Man-6-P conjugated, and lactose conjugated
antibodies.
[0073] FIG. 52 depicts the characterization of polyclonal antibody
conjugated to Man-6-P hexamannose maleimide (thiol conjugation at
hinge cysteines) through SDS-PAGE (non-reducing and reducing),
lectin blot (reducing), and M6P quantitation.
[0074] FIG. 53 depicts the characterization of polyclonal antibody
conjugated to lactose maleimide (thio conjugation at hinge
cysteines) through SDS-PAGE and galactose quantitation.
[0075] FIG. 54 depicts the characterization of monoclonal antibody
conjugated to Man-6-P hexamannose maleimide (thiol conjugation at
hinge cysteines) through SDS-PAGE (non-reducing and reducing), and
glycan (M6P) quantitation.
[0076] FIG. 55 depicts the results of size exclusion chromatography
(SEC) analysis of a hinge cysteine polyclonal antibody
conjugate.
[0077] FIG. 56 depicts the results of size exclusion chromatography
(SEC) analysis of a hinge cysteine monoclonal antibody
conjugate.
[0078] FIG. 57 depicts the results of sialidase titration to
determine the amount of sialic acid release from NNAS ("NNAS"
disclosed as SEQ ID NO: 40), sialylated NNAS ("NNAS" disclosed as
SEQ ID NO: 40), and desialylated and galatosylated NNAS antibodies
("NNAS" disclosed as SEQ ID NO: 40). FIG. 57 discloses "NNAS" as
SEQ ID NO: 40.
[0079] FIG. 58 depicts the results of LC-MS experiments to
determine the glycan contents of an NNAS modified antibody ("NNAS"
disclosed as SEQ ID NO: 40) and a desialylated and galactosylated
NNAS modified antibody ("NNAS" disclosed as SEQ ID NO: 40). FIG. 58
discloses "NNAS" as SEQ ID NO: 40.
[0080] FIG. 59 depicts the results of LC-MS experiments to
determine the glycan contents of an NNAS modified antibody ("NNAS"
disclosed as SEQ ID NO: 40) and a sialylated NNAS modified antibody
("NNAS" disclosed as SEQ ID NO: 40). FIG. 59 discloses "NNAS" as
SEQ ID NO: 40.
[0081] FIG. 60 depicts the characterization of M6P Receptor bound
to bisM6P glycan-conjugated polyclonal and monoclonal antibodies
through native Fc glycan or hinge disulfides in solution.
[0082] FIG. 61 depicts the characterization of enzyme modified and
conjugated NNAS ("NNAS" disclosed as SEQ ID NO: 40) antibodies by
SDS-PAGE (4-12% NuPAGE; reducing and non-reducing) and ECL lectin
blotting (reducing).
[0083] FIG. 62 depicts the results of terminal galactose
quantitation in an NNAS antibody ("NNAS" disclosed as SEQ ID NO:
40), a disialylated/galactosylated NNAS antibody ("NNAS" disclosed
as SEQ ID NO: 40), and a conjugated NNAS antibody ("NNAS" disclosed
as SEQ ID NO: 40) in mol galactose or mol glycopeptide per mol
antibody. FIG. 62 discloses "NNAS" as SEQ ID NO: 40.
[0084] FIG. 63 depicts the examination of lactose maleimide that
had been modified with alpha-2,3-sialyltransferase and eluted from
QAE purification columns with 20 mM NaCl. The resultant eluate was
characterized using MALDI-TOF and Dionex HPLC.
[0085] FIGS. 64A-64B depict the characterization of rabbit antibody
conjugated with sialyllactose maleimide (thiol reaction) using
SDS-PAGE (FIG. 64A) and Dionex HPLC (FIG. 64B; sialic acid
quantitation).
[0086] FIGS. 65A-D depict the characterization of lactose maleimide
sialylated with alpha-2,6-sialyltransferase and purified using a
QAE-sepharose column. Analysis using Dionex HPLC is shown for (FIG.
65A) a lactose standard; (FIG. 65B) an alpha-2,6-sialyllactose
standard; (FIG. 65C) a lactose maleimide standard; and (FIG. 65D) a
fraction of alpha-2,6-sialyllactose maleimide eluted from a
QAE-sepharose column.
[0087] FIG. 66 depicts the characterization of a fraction of
alpha-2,6-sialyllactose maleimide eluted from a QAE-sepharose
column using MALDI-TOF.
[0088] FIGS. 67A-67B depicts the characterization of a control
antibody, an alpha-2,3-sialyllactose glycan conjugated polyclonal
antibody, and an alpha-2,6-sialyllactose glycan conjugated
polyclonal antibody through SDS-PAGE (FIG. 67A) and Dionex HPLC
(FIG. 67B; graph of sialic acid analysis shown).
[0089] FIG. 68 depicts the characterization of control and enzyme
modified (disalylated/galactosylated) NNAS mutant antibodies
("NNAS" disclosed as SEQ ID NO: 40) using SDS-PAGE and lectin
blotting.
[0090] FIG. 69 depicts the characterization through reducing
SDS-PAGE of the PEGylation of a control antibody and Gal NNAS
("NNAS" disclosed as SEQ ID NO: 40) with various amounts of
galactose oxidase. FIG. 69 discloses "NNAS" as SEQ ID NO: 40.
[0091] FIG. 70 depicts the results from a Protein Simple scan
characterizing the PEGylation of an antibody heavy chain.
[0092] FIG. 71 depicts the characterization through reducing
SDS-PAGE of the PEGylation of a control antibody and Gal NNAS
("NNAS" disclosed as SEQ ID NO: 40) with various molar excess of
PEG over antibody. FIG. 71 discloses "NNAS" as SEQ ID NO: 40.
[0093] FIG. 72 depicts the results from a Protein Simple scan
characterizing the PEGylation of an antibody heavy chain.
[0094] FIG. 73 is a structural drawing of
lactose.sub.3-Cys.sub.3Gly.sub.4.
[0095] FIGS. 74A-74B depict the characterization through reducing
SDS-PAGE of the PEGylation of a control antibody and Gal NNAS
("NNAS" disclosed as SEQ ID NO: 40) with galactose oxidase in the
absence of copper acetate (FIG. 74A) and in the presence of varying
amounts of copper acetate (FIGS. 74A and 74B). FIG. 74 discloses
"NNAS" as SEQ ID NO: 40.
[0096] FIG. 75 the characterization of enzyme modified and
conjugated wild type, A114N, NNAS ("NNAS" disclosed as SEQ ID NO:
40), and A114N/NNAS antibodies ("NNAS" disclosed as SEQ ID NO: 40)
by SDS-PAGE (4-12% NuPAGE; reducing and non-reducing) and ECL
lectin blotting (reducing) along with the results of terminal
galactose quantitation in mol galactose per mol antibody. FIG. 75
discloses "NNAS" as SEQ ID NO: 40.
[0097] FIG. 76 is a graph depicting the sialic acid content (in
mol/mol) of wild-type and mutant antibodies as measured using
Dionex HPLC. FIG. 76 discloses "NNAS" as SEQ ID NO: 40.
[0098] FIG. 77 depicts the characterization of the PEGylation of
wild-type and mutant antibodies through reducing and non-reducing
SDS-PAGE. FIG. 77 discloses "NNAS" as SEQ ID NO: 40.
[0099] FIG. 78 is a graph depicting the PEGylation (in mol/mol) of
wild-type and mutant antibodies. FIG. 78 discloses "NNAS" as SEQ ID
NO: 40.
[0100] FIG. 79 is a series of photos depicting immunofluorescence
staining results from the incubation of control, modified (with
galactosyltransferase), or conjugated (with lactose aminooxy or
lactose maleimide) antibodies with HepG2 cells.
[0101] FIG. 80 is a depiction of a trivalent GalNAc glycan
[0102] FIG. 81 depicts the results of surface plasmon resonance
experiments used to assess the binding of trivalent GalNAc
glycan-conjugated antibodies to ASGPR receptor subunit H1. FIG. 82
is a depiction of a trivalent GalNAc-containing glycopeptide and a
trivalent galactose-containing glycopeptide.
[0103] FIG. 83 depicts the results of surface plasmon resonance
experiments used to assess the binding of trivalent
GalNAc-conjugated and trivalent galactose-conjugated recombinant
lysosomal enzymes to ASGPR receptor subunit H1.
[0104] FIG. 84 is a graph depicting the titration of sialic acid
(0.2 .mu.mol) with various amounts of CMP-sialic acid synthetase
(N. mentingitidis) at 37.degree. C. as CMP-sialic acid synthesized
versus the amounts of enzyme used.
[0105] FIG. 85 is a graph depicting the synthesized sialic acid
(from ManNAc) versus the amounts of the sialic acid aldolase (E.
coli K-12) enzyme used at 37.degree. C.
[0106] FIG. 86 is a graph depicting the synthesized sialic acid
derivative (from ManLev) versus the amounts of the sialic acid
aldolase (E. coli K-12) enzyme used at 37.degree. C.
[0107] FIG. 87 is a graph depicting the released sialic acid
derivative after digestion of CMP-sialic acid derivative
(synthesized from ManLev) with sialidase at 37.degree. C. as
compared to the retention time of sialic acid standard monitored
using HPAEC-PAD.
[0108] FIG. 88 is a graph depicting the HPAEC-PAD profile of
CMP-sialic acid synthesized from ManNAc and CMP-sialic acid
derivative synthesized from ManLev as compared to the CMP-sialic
acid standard.
[0109] FIG. 89 is a graph depicting the HPAEC-PAD profile of
CMP-sialic acid derivatives (synthesized from ManLev, ManNAz and
ManAz) as compared to the CMP-sialic acid standard.
[0110] FIG. 90 is a schematic representation demonstrating the
sialylation of antibody using a CMP-sialic acid derivative prepared
from ManLev.
[0111] FIG. 91 is a graph showing the LC-MS analysis of
CH.sub.2CH.sub.3 fragments released by IdeS protease from antibody
Herceptin sialylated in vitro using .alpha.2,6 sialyltransferase
and CMP-sialic acid derivative prepared from ManLev.
[0112] FIG. 92 is a schematic representation demonstrating the
PEGylation of antibody sialylated with a sialic acid derivative
prepared from ManLev.
[0113] FIG. 93 depicts SDS-PAGE characterization of PEGylated
Herceptin pre-sialylated with a sialic acid derivative prepared
from ManLev. The PEGylation is performed using oxime chemistry.
[0114] FIG. 94 is a schematic representation demonstrating the
sialylation of antibody using a CMP-sialic acid derivative prepared
from ManNAz.
[0115] FIG. 95 depicts SDS-PAGE characterization of PEGylated
Herceptin pre-sialylated with a sialic acid derivative prepared
from ManNAz. The PEGylation was performed using click
chemistry.
DETAILED DESCRIPTION
[0116] The current disclosure provides methods of making effector
moiety conjugates (e.g., targeting moiety conjugates). These
methods involve the incorporation of sialic acid derivatives in the
glycan of a binding polypeptide to form a sialic acid
derivative-conjugated binding polypeptide, and a subsequent
reaction in which an effector moiety is reacted with the sialic
acid derivative-conjugated binding protein to create an effector
moiety-conjugated binding polypeptide.
I. Definitions
[0117] As used herein, the term "binding protein" or "binding
polypeptide" shall refer to a polypeptide (e.g., an antibody) that
contains at least one binding site which is responsible for
selectively binding to a target antigen of interest (e.g. a human
antigen). Exemplary binding sites include an antibody variable
domain, a ligand binding site of a receptor, or a receptor binding
site of a ligand. In certain aspects, the binding polypeptides
comprise multiple (e.g., two, three, four, or more) binding sites.
In certain aspects, the binding protein is not a therapeutic
enzyme.
[0118] As used herein, the term "native residue" shall refer to an
amino acid residue that occurs naturally at a particular amino acid
position of a binding polypeptide (e.g., an antibody or fragment
thereof) and which has not been modified, introduced, or altered by
the hand of man. As used herein, the term "altered binding protein"
or "altered binding polypeptide" includes binding polypeptides
(e.g., an antibody or fragment thereof) comprising at least one
non-native mutated amino acid residue.
[0119] The term "specifically binds" as used herein, refers to the
ability of an antibody or an antigen-binding fragment thereof to
bind to an antigen with a dissociation constant (Kd) of at most
about 1.times.10.sup.-6 M, 1.times.10.sup.-7 M, 1.times.10.sup.-8M,
1.times.10.sup.-9M, 1.times.10.sup.-1.degree. M,
1.times.10.sup.-11M, 1.times.10.sup.-12 M, or less, and/or to bind
to an antigen with an affinity that is at least two-fold greater
than its affinity for a nonspecific antigen.
[0120] As used herein, the term "antibody" refers to such
assemblies (e.g., intact antibody molecules, antibody fragments, or
variants thereof) which have significant known specific
immunoreactive activity to an antigen of interest (e.g. a tumor
associated antigen). Antibodies and immunoglobulins comprise light
and heavy chains, with or without an interchain covalent linkage
between them. Basic immunoglobulin structures in vertebrate systems
are relatively well understood.
[0121] As will be discussed in more detail below, the generic term
"antibody" comprises five distinct classes of antibody that can be
distinguished biochemically. While all five classes of antibodies
are clearly within the scope of the current disclosure, the
following discussion will generally be directed to the IgG class of
immunoglobulin molecules. With regard to IgG, immunoglobulins
comprise two identical light chains of molecular weight
approximately 23,000 Daltons, and two identical heavy chains of
molecular weight 53,000-70,000. The four chains are joined by
disulfide bonds in a "Y" configuration wherein the light chains
bracket the heavy chains starting at the mouth of the "Y" and
continuing through the variable region.
[0122] Light chains of immunoglobulin are classified as either
kappa or lambda (.kappa., .lamda.). Each heavy chain class may be
bound with either a kappa or lambda light chain. In general, the
light and heavy chains are covalently bonded to each other, and the
"tail" portions of the two heavy chains are bonded to each other by
covalent disulfide linkages or non-covalent linkages when the
immunoglobulins are generated either by hybridomas, B cells, or
genetically engineered host cells. In the heavy chain, the amino
acid sequences run from an N-terminus at the forked ends of the Y
configuration to the C-terminus at the bottom of each chain. Those
skilled in the art will appreciate that heavy chains are classified
as gamma, mu, alpha, delta, or epsilon, (.gamma., .mu., .alpha.,
.delta., .epsilon.) with some subclasses among them (e.g.,
.gamma.1-.gamma.4). It is the nature of this chain that determines
the "class" of the antibody as IgG, IgM, IgA IgG, or IgE,
respectively. The immunoglobulin isotype subclasses (e.g., IgG1,
IgG2, IgG3, IgG4, IgA1, etc.) are well characterized and are known
to confer functional specialization. Modified versions of each of
these classes and isotypes are readily discernable to the skilled
artisan in view of the instant disclosure and, accordingly, are
within the scope of the current disclosure.
[0123] Both the light and heavy chains are divided into regions of
structural and functional homology. The term "region" refers to a
part or portion of an immunoglobulin or antibody chain and includes
constant region or variable regions, as well as more discrete parts
or portions of said regions. For example, light chain variable
regions include "complementarity determining regions" or "CDRs"
interspersed among "framework regions" or "FRs", as defined
herein.
[0124] The regions of an immunoglobulin heavy or light chain may be
defined as "constant" (C) region or "variable" (V) regions, based
on the relative lack of sequence variation within the regions of
various class members in the case of a "constant region", or the
significant variation within the regions of various class members
in the case of a "variable regions". The terms "constant region"
and "variable region" may also be used functionally. In this
regard, it will be appreciated that the variable regions of an
immunoglobulin or antibody determine antigen recognition and
specificity. Conversely, the constant regions of an immunoglobulin
or antibody confer important effector functions such as secretion,
transplacental mobility, Fc receptor binding, complement binding,
and the like. The subunit structures and three dimensional
configurations of the constant regions of the various
immunoglobulin classes are well known.
[0125] The constant and variable regions of immunoglobulin heavy
and light chains are folded into domains. The term "domain" refers
to a globular region of a heavy or light chain comprising peptide
loops (e.g., comprising 3 to 4 peptide loops) stabilized, for
example, by .beta.-pleated sheet and/or intrachain disulfide bond.
Constant region domains on the light chain of an immunoglobulin are
referred to interchangeably as "light chain constant region
domains", "CL regions" or "CL domains". Constant domains on the
heavy chain (e.g. hinge, CH1, CH2 or CH3 domains) are referred to
interchangeably as "heavy chain constant region domains", "CH"
region domains or "CH domains". Variable domains on the light chain
are referred to interchangeably as "light chain variable region
domains", "VL region domains or "VL domains". Variable domains on
the heavy chain are referred to interchangeably as "heavy chain
variable region domains", "VH region domains" or "VH domains".
[0126] By convention the numbering of the variable constant region
domains increases as they become more distal from the antigen
binding site or amino-terminus of the immunoglobulin or antibody.
The N-terminus of each heavy and light immunoglobulin chain is a
variable region and at the C-terminus is a constant region; the CH3
and CL domains actually comprise the carboxy-terminus of the heavy
and light chain, respectively. Accordingly, the domains of a light
chain immunoglobulin are arranged in a VL-CL orientation, while the
domains of the heavy chain are arranged in the VH-CH1-hinge-CH2-CH3
orientation.
[0127] Amino acid positions in a heavy chain constant region,
including amino acid positions in the CH1, hinge, CH2, CH3, and CL
domains, may be numbered according to the Kabat index numbering
system (see Kabat et al, in "Sequences of Proteins of Immunological
Interest", U.S. Dept. Health and Human Services, 5th edition,
1991). Alternatively, antibody amino acid positions may be numbered
according to the EU index numbering system (see Kabat et al,
ibid).
[0128] As used herein, the term "VH domain" includes the amino
terminal variable domain of an immunoglobulin heavy chain, and the
term "VL domain" includes the amino terminal variable domain of an
immunoglobulin light chain.
[0129] As used herein, the term "CH1 domain" includes the first
(most amino terminal) constant region domain of an immunoglobulin
heavy chain that extends, e.g., from about positions 114-223 in the
Kabat numbering system (EU positions 118-215). The CH1 domain is
adjacent to the VH domain and amino terminal to the hinge region of
an immunoglobulin heavy chain molecule, and does not form a part of
the Fc region of an immunoglobulin heavy chain.
[0130] As used herein, the term "hinge region" includes the portion
of a heavy chain molecule that joins the CH1 domain to the CH2
domain. This hinge region comprises approximately 25 residues and
is flexible, thus allowing the two N-terminal antigen binding
regions to move independently. Hinge regions can be subdivided into
three distinct domains: upper, middle, and lower hinge domains
(Roux et al. J. Immunol. 1998, 161:4083).
[0131] As used herein, the term "CH2 domain" includes the portion
of a heavy chain immunoglobulin molecule that extends, e.g., from
about positions 244-360 in the Kabat numbering system (EU positions
231-340). The CH2 domain is unique in that it is not closely paired
with another domain. Rather, two N-linked branched carbohydrate
chains are interposed between the two CH2 domains of an intact
native IgG molecule. In one embodiment, a binding polypeptide of
the current disclosure comprises a CH2 domain derived from an IgG1
molecule (e.g. a human IgG1 molecule).
[0132] As used herein, the term "CH3 domain" includes the portion
of a heavy chain immunoglobulin molecule that extends approximately
110 residues from N-terminus of the CH2 domain, e.g., from about
positions 361-476 of the Kabat numbering system (EU positions
341-445). The CH3 domain typically forms the C-terminal portion of
the antibody. In some immunoglobulins, however, additional domains
may extend from CH3 domain to form the C-terminal portion of the
molecule (e.g. the CH4 domain in the .mu. chain of IgM and the e
chain of IgE). In one embodiment, a binding polypeptide of the
current disclosure comprises a CH3 domain derived from an IgG1
molecule (e.g. a human IgG1 molecule).
[0133] As used herein, the term "CL domain" includes the constant
region domain of an immunoglobulin light chain that extends, e.g.
from about Kabat position 107A-216. The CL domain is adjacent to
the VL domain. In one embodiment, a binding polypeptide of the
current disclosure comprises a CL domain derived from a kappa light
chain (e.g., a human kappa light chain).
[0134] As used herein, the term "Fc region" is defined as the
portion of a heavy chain constant region beginning in the hinge
region just upstream of the papain cleavage site (i.e. residue 216
in IgG, taking the first residue of heavy chain constant region to
be 114) and ending at the C-terminus of the antibody. Accordingly,
a complete Fc region comprises at least a hinge domain, a CH2
domain, and a CH3 domain.
[0135] The term "native Fc" as used herein refers to a molecule
comprising the sequence of a non-antigen-binding fragment resulting
from digestion of an antibody or produced by other means, whether
in monomeric or multimeric form, and can contain the hinge region.
The original immunoglobulin source of the native Fc can be of human
origin and can be any of the immunoglobulins, such as IgG1 or IgG2.
Native Fc molecules are made up of monomeric polypeptides that can
be linked into dimeric or multimeric forms by covalent (i.e.,
disulfide bonds) and non-covalent association. The number of
intermolecular disulfide bonds between monomeric subunits of native
Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA,
and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). One
example of a native Fc is a disulfide-bonded dimer resulting from
papain digestion of an IgG. The term "native Fc" as used herein is
generic to the monomeric, dimeric, and multimeric forms.
[0136] The term "Fc variant" as used herein refers to a molecule or
sequence that is modified from a native Fc but still comprises a
binding site for the salvage receptor, FcRn (neonatal Fc receptor).
Exemplary Fc variants, and their interaction with the salvage
receptor, are known in the art. Thus, the term "Fc variant" can
comprise a molecule or sequence that is humanized from a non-human
native Fc. Furthermore, a native Fc comprises regions that can be
removed because they provide structural features or biological
activity that are not required for the antibody-like binding
polypeptides. Thus, the term "Fc variant" comprises a molecule or
sequence that lacks one or more native Fc sites or residues, or in
which one or more Fc sites or residues has be modified, that affect
or are involved in: (1) disulfide bond formation, (2)
incompatibility with a selected host cell, (3) N-terminal
heterogeneity upon expression in a selected host cell, (4)
glycosylation, (5) interaction with complement, (6) binding to an
Fc receptor other than a salvage receptor, or (7)
antibody-dependent cellular cytotoxicity (ADCC).
[0137] The term "Fc domain" as used herein encompasses native Fc
and Fc variants and sequences as defined above. As with Fc variants
and native Fc molecules, the term "Fc domain" includes molecules in
monomeric or multimeric form, whether digested from whole antibody
or produced by other means.
[0138] As indicated above, the variable regions of an antibody
allow it to selectively recognize and specifically bind epitopes on
antigens. That is, the VL domain and VH domain of an antibody
combine to form the variable region (Fv) that defines a three
dimensional antigen binding site. This quaternary antibody
structure forms the antigen binding site present at the end of each
arm of the Y. More specifically, the antigen binding site is
defined by three complementary determining regions (CDRs) on each
of the heavy and light chain variable regions. As used herein, the
term "antigen binding site" includes a site that specifically binds
(immunoreacts with) an antigen (e.g., a cell surface or soluble
antigen). The antigen binding site includes an immunoglobulin heavy
chain and light chain variable region and the binding site formed
by these variable regions determines the specificity of the
antibody. An antigen binding site is formed by variable regions
that vary from one antibody to another. The altered antibodies of
the current disclosure comprise at least one antigen binding
site.
[0139] In certain embodiments, binding polypeptides of the current
disclosure comprise at least two antigen binding domains that
provide for the association of the binding polypeptide with the
selected antigen. The antigen binding domains need not be derived
from the same immunoglobulin molecule. In this regard, the variable
region may or be derived from any type of animal that can be
induced to mount a humoral response and generate immunoglobulins
against the desired antigen. As such, the variable region of the a
binding polypeptide may be, for example, of mammalian origin e.g.,
may be human, murine, rat, goat, sheep, non-human primate (such as
cynomolgus monkeys, macaques, etc.), lupine, or camelid (e.g., from
camels, llamas and related species).
[0140] In naturally occurring antibodies, the six CDRs present on
each monomeric antibody are short, non-contiguous sequences of
amino acids that are specifically positioned to form the antigen
binding site as the antibody assumes its three dimensional
configuration in an aqueous environment. The remainder of the heavy
and light variable domains show less intermolecular variability in
amino acid sequence and are termed the framework regions. The
framework regions largely adopt a .beta.-sheet conformation and the
CDRs form loops which connect, and in some cases form part of, the
.beta.-sheet structure. Thus, these framework regions act to form a
scaffold that provides for positioning the six CDRs in correct
orientation by inter-chain, non-covalent interactions. The antigen
binding domain formed by the positioned CDRs defines a surface
complementary to the epitope on the immunoreactive antigen. This
complementary surface promotes the non-covalent binding of the
antibody to the immunoreactive antigen epitope.
[0141] Exemplary binding polypeptides featured in the invention
include antibody variants. As used herein, the term "antibody
variant" includes synthetic and engineered forms of antibodies
which are altered such that they are not naturally occurring, e.g.,
antibodies that comprise at least two heavy chain portions but not
two complete heavy chains (such as, domain deleted antibodies or
minibodies); multispecific forms of antibodies (e.g., bispecific,
trispecific, etc.) altered to bind to two or more different
antigens or to different epitopes on a single antigen); heavy chain
molecules joined to scFv molecules and the like. In addition, the
term "antibody variant" includes multivalent forms of antibodies
(e.g., trivalent, tetravalent, etc., antibodies that bind to three,
four or more copies of the same antigen.
[0142] As used herein the term "valency" refers to the number of
potential target binding sites in a polypeptide. Each target
binding site specifically binds one target molecule or specific
site on a target molecule. When a polypeptide comprises more than
one target binding site, each target binding site may specifically
bind the same or different molecules (e.g., may bind to different
ligands or different antigens, or different epitopes on the same
antigen). The subject binding polypeptides typically have at least
one binding site specific for a human antigen molecule.
[0143] The term "specificity" refers to the ability to specifically
bind (e.g., immunoreact with) a given target antigen (e.g., a human
target antigen). A binding polypeptide may be monospecific and
contain one or more binding sites which specifically bind a target
or a polypeptide may be multispecific and contain two or more
binding sites which specifically bind the same or different
targets. In certain embodiments, a binding polypeptide is specific
for two different (e.g., non-overlapping) portions of the same
target. In certain embodiments, the binding polypeptide is specific
for more than one target. Exemplary binding polypeptides (e.g.,
antibodies) which comprise antigen binding sites that bind to
antigens expressed on tumor cells are known in the art and one or
more CDRs from such antibodies can be included in an antibody as
described herein.
[0144] The term "linking moiety" includes moieties which are
capable of linking the effector moiety to the binding polypeptides
disclosed herein. The linking moiety may be selected such that it
is cleavable (e.g., enzymatically cleavable or pH-sensitive) or
non-cleavable.
[0145] As used herein, the term "effector moiety" comprises agents
(e.g. proteins, nucleic acids, lipids, carbohydrates,
glycopeptides, and fragments thereof) with biological or other
functional activity. For example, a modified binding polypeptide
comprising an effector moiety conjugated to a binding polypeptide
has at least one additional function or property as compared to the
unconjugated antibody. For example, the conjugation of a cytotoxic
drug (e.g., an effector moiety) to binding polypeptide results in
the formation of a binding polypeptide with drug cytotoxicity as
second function (i.e. in addition to antigen binding). In another
example, the conjugation of a second binding polypeptide to the
binding polypeptide may confer additional binding properties. In
certain embodiments, where the effector moiety is a genetically
encoded therapeutic or diagnostic protein or nucleic acid, the
effector moiety may be synthesized or expressed by either peptide
synthesis or recombinant DNA methods that are well known in the
art. In another aspect, where the effector moiety is a
non-genetically encoded peptide, or a drug moiety, the effector
moiety may be synthesized artificially or purified from a natural
source. As used herein, the term "drug moiety" includes
anti-inflammatory, anticancer, anti-infective (e.g., anti-fungal,
antibacterial, anti-parasitic, anti-viral, etc.), and anesthetic
therapeutic agents. In a further embodiment, the drug moiety is an
anticancer or cytotoxic agent. Compatible drug moieties may also
comprise prodrugs. Exemplary effector moieties are set forth in
Table 1 herein.
[0146] In certain embodiments, an "effector moiety" comprises a
"targeting moiety." As used herein, the term "targeting moiety"
refers to an effector moiety that binds to a target molecule.
Targeting moieties can comprise, without limitation, proteins,
nucleic acids, lipids, carbohydrates (e.g., glycans), and
combinations thereof (e.g., glycoproteins, glycopeptides, and
glycolipids).
[0147] As used herein, the term "prodrug" refers to a precursor or
derivative form of a pharmaceutically active agent that is less
active, reactive or prone to side effects as compared to the parent
drug and is capable of being enzymatically activated or otherwise
converted into a more active form in vivo. Prodrugs compatible with
the compositions of the current disclosure include, but are not
limited to, phosphate-containing prodrugs, amino acid-containing
prodrugs, thiophosphate-containing prodrugs, sulfate-containing
prodrugs, peptide-containing prodrugs, .beta.-lactam-containing
prodrugs, optionally substituted phenoxyacetamide-containing
prodrugs or optionally substituted phenylacetamide-containing
prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs that
can be converted to the more active cytotoxic free drug. One
skilled in the art may make chemical modifications to the desired
drug moiety or its prodrug in order to make reactions of that
compound more convenient for purposes of preparing modified binding
polypeptides of the current disclosure. The drug moieties also
include derivatives, pharmaceutically acceptable salts, esters,
amides, and ethers of the drug moieties described herein.
Derivatives include modifications to drugs identified herein which
may improve or not significantly reduce a particular drug's desired
therapeutic activity.
[0148] As used herein, the term "anticancer agent" includes agents
which are detrimental to the growth and/or proliferation of
neoplastic or tumor cells and may act to reduce, inhibit or destroy
malignancy. Examples of such agents include, but are not limited
to, cytostatic agents, alkylating agents, antibiotics, cytotoxic
nucleosides, tubulin binding agents, hormones, hormone antagonists,
cytotoxic agents, and the like. Cytotoxic agents include tomaymycin
derivatives, maytansine derivatives, cryptophycine derivatives,
anthracycline derivatives, bisphosphonate derivatives, leptomycin
derivatives, streptonigrin derivatives, auristatine derivatives,
and duocarmycin derivatives. Any agent that acts to retard or slow
the growth of immunoreactive cells or malignant cells is within the
scope of the current disclosure.
[0149] The term "antigen" or "target antigen" as used herein refers
to a molecule or a portion of a molecule that is capable of being
bound by the binding site of a binding polypeptide. A target
antigen may have one or more epitopes.
[0150] The term "sialic acid derivative-conjugated binding
polypeptide" as used herein refers to the polypeptide formed by
reacting a CMP-sialic acid derivative with a glycan of a binding
peptide. For example, a sialic acid derivative-conjugated binding
polypeptide includes, but is not limited to, polypeptides of FIGS.
3A-E represented by circles in combination with the reactive
moieties to which they are bonded.
[0151] The term "trivalent glycopeptide" as used herein refers to a
targeting or effector moiety comprising three glycopeptides.
[0152] The term "trivalent aminooxy," as used herein, refers to an
aminooxy moiety further comprising three carbohydrates or
glycopeptides. The trivalent aminooxy may contain additional
functional groups, e.g., a linker.
[0153] As used herein, "click chemistry" refers to pairs of
terminal reactive moieties that rapidly and selectively react
("click") with each other to form a targeting or effector moiety
conjugated binding polypeptide. Click chemistry is discussed
further herein.
[0154] As used herein, the term "metal catalyst" refers to
catalysts that comprise a transition metal including, but not
limited to, ruthenium, nickel, palladium, platinum, and iron and
one or more ligands including, but not limited to, bipyridine
derivatives or terpyridine derivaties. A metal catalyst may also be
formed in situ. For example, a copper(II) compound may be added to
the reaction mixture in the presence of a reducing agent including,
but not limited to, copper sulfate (CuSO.sub.4) as the copper(II)
compound and sodium ascorbate as the reducing agent.
[0155] As used herein, the term "reactive moiety" refers to a
moiety comprising a portion or an entire functional group are
specific groups of one or more atoms and one or more bonds that are
responsible for characteristic chemical reactions. In example
embodiments, a reactive moiety includes, but is not limited to, an
aldehyde moiety, an alkyne, an aminooxy moiety, an azide, a
hydrazine, a keto moiety, and a thiol. In some embodiments, the
reactive moiety is a terminal reactive moiety. In the reacting
step, a first reactive moiety reacts with a second reactive moiety
to form an effector moiety conjugated binding polypeptide.
[0156] An "aldehyde" moiety, as used herein, refers to a formyl
functional group and is represented by the following structural
formula:
##STR00007##
For example, a CMP-sialic acid-derivative comprising a terminal
aldehyde moiety includes, but is not limited to, the following
structural formulas:
##STR00008##
[0157] An "alkyne" moiety, as used herein, refers to a
carbon-carbon triple bond.
[0158] An "aminooxy" moiety, as used herein, refers to a
nitrogen-oxygen single bond and is represented by the following
structural formula:
##STR00009##
[0159] An "azide" moiety, as used herein, refers to an RN.sub.3
moiety and may be represented by the following structural
formula:
##STR00010##
[0160] A "hydrazine" moiety, as used herein, refers to at least one
nitrogen-nitrogen single bond and is represented by the following
structural formula:
##STR00011##
For example, a hydrazine may have a structural formula of:
##STR00012##
[0161] As used herein, an "imine" moiety refers to a
carbon-nitrogen double bond and is represented by the following
structural formula:
##STR00013##
In some embodiments, a targeting or effector moiety conjugated
binding polypeptide comprises an imine. For example, a type of
imine includes, but is not limited to, an aldimine, a
hydroxylamine, a hydrazone, a ketamine, or an oxime.
[0162] A "hydrazone" moiety, as used herein, refers to a type of
imine and is represented by the following structural formula:
##STR00014##
In some embodiments, the hydrazone may be a terminal hydrazone. In
some embodiments, a hydrazone linkage comprises a hydrazone moiety
along with additional functional groups, e.g., a linker or a
portion of a linking moiety.
[0163] A "keto" or "ketone" moiety, as used herein, comprises a
carbonyl functional group and is represented by the following
structural formula:
##STR00015##
[0164] A "maleimide" moiety, as used herein, comprises an
unsaturated imide and is represented by the following structural
formula:
##STR00016##
[0165] An "oxime" moiety is a type of imine and is represented by
the following structural formula:
##STR00017##
[0166] The term "thioether" is represented by the following
structural formula:
##STR00018##
[0167] A "thiol" refers to a moiety comprising a --SH functional
group, which is also referred to as a sulfhydryl group. In some
embodiments, a thiol contains a carbon-bonded sulfhydryl group.
[0168] The term "terminal" when referring to a reactive moiety, as
used herein, describes a group bonded to a terminus of a straight
or branched-chain moiety. In some embodiments, the terminal
reactive moiety is a substituent of a functional group.
[0169] The term "oxidizing agent" refers to a compound or a reagent
that accepts or gains electrons from another compound or reagent
thereby undergoing a reduction while oxidizing the other compound
or reagent. For example, oxidizing agents include, but are not
limited to, sodium periodate, periodate oxidase, galactose oxidase,
hydrogen peroxide, and copper compounds (e.g., copper(II)
sulfate).
[0170] The term "ambient temperature," as used herein, is
equivalent to the term "room temperature" and denotes the range of
temperatures between 20.degree. C. and 26.degree. C. (equivalent to
68.degree. F. and 79.degree. F.), with an average temperature of
approximately of 23.degree. C. (73.degree. F.).
[0171] The term "effector moiety conjugated binding polypeptide,"
as used herein, refers to a structure comprising one or more
binding proteins linked or bonded to an effector moiety. There may
be a number of chemical moieties and functional groups that
comprise the linkage between the binding protein(s) and the
effector moieties(s) including, but not limited to, any glycan or
modified glycan (e.g. one or more sialic acid derivatives or
CMP-sialic acid derivatives).
II. Sialic acid derivatives
[0172] In one aspect, the current disclosure provides for a method
of making sialic acids or sialic acid derivatives from sugars or
sugar derivatives. The sugar or sugar derivative used may be but is
not limited to N-acetylmannosamine or its derivatives such as
N-acetyl mannosamine (ManNAc), N-levulinoyl mannosamine (ManLev),
N-azidoacetylmannosamine (ManHAz), azidomannosamine, and N-thio
acetylmannosamine (ManHS).
[0173] In example embodiments, the sugar or sugar derivative has
the following structural formula:
##STR00019##
[0174] wherein R.sub.1 is a reactive moiety including, but not
limited to, NH(C.dbd.O)CH.sub.3,
NH(C.dbd.O)CH.sub.2CH.sub.2(C.dbd.O)CH.sub.3,
NH(C.dbd.O)CH.sub.2OH, NH(C.dbd.O)CH.sub.2N.sub.3, NH(C.dbd.O)SH,
OH or N.sub.3. In some embodiments, the CMP-sialic acid derivative
has the following structural formula:
##STR00020##
[0175] wherein R.sub.1 is a reactive moiety including, but not
limited to, the groups listed above.
III. Binding Polypeptides
[0176] In one aspect, the current disclosure provides binding
polypeptides (e.g., antibodies, antibody fragments, antibody
variants, and fusion proteins) comprising a glycosylated domain,
e.g, a glycosylated constant domain. The binding polypeptides
disclosed herein encompass any binding polypeptide that comprises a
domain having an N-linked glycosylation site. In certain
embodiments, the binding polypeptide is an antibody, or fragment or
derivative thereof .DELTA.ny antibody from any source or species
can be employed in the binding polypeptides disclosed herein.
Suitable antibodies include without limitation, human antibodies,
humanized antibodies or chimeric antibodies.
[0177] In certain embodiments, the glycosylated domain is an Fc
domain. In certain embodiments, the glycosylation domain is a
native glycosylation domain at N297.
[0178] In other embodiments, the glycosylation domain is an
engineered glycosylation domain. Exemplary engineered glycosylation
domains in Fc domain comprise an asparagine residue at amino acid
position 298, according to EU numbering; and a serine or threonine
residue at amino acid position 300, according to EU numbering.
[0179] Fc domains from any immunoglobulin class (e.g., IgM, IgG,
IgD, IgA and IgE) and species can be used in the binding
polypeptides disclosed herein. Chimeric Fc domains comprising
portions of Fc domains from different species or Ig classes can
also be employed. In certain embodiments, the Fc domain is a human
IgG1 Fc domain. In the case of a human IgG1 Fc domain, mutation of
the wild type amino acid at Kabat position 298 to an asparagine and
Kabat position 300 to a serine or threonine results in the
formation of an N-linked glycosylation consensus site (i.e, the
N-X-T/S sequon, where X is any amino acid except proline). However,
in the case of Fc domains of other species and/or Ig classes or
isotypes, the skill artisan will appreciate that it may be
necessary to mutate Kabat position 299 of the Fc domain if a
proline residue is present to recreate an N-X-T/S sequon.
[0180] In other embodiments, the current disclosure provides
binding polypeptides (e.g., antibodies, antibody fragments,
antibody variants, and fusion proteins) comprising at least one CH1
domain having an N-linked glycosylation site. Such exemplary
binding polypeptides include may comprise, for example, and
engineered glycosylation site at position 114, according to Kabat
numbering.
[0181] CH1 domains from any immunoglobulin class (e.g., IgM, IgG,
IgD, IgA and IgE) and species can be used in the binding
polypeptides disclosed herein. Chimeric CHI domains comprising
portions of CHI domains from different species or Ig classes can
also be employed. In certain embodiments, the CH1 domain is a human
IgG1 CH1 domain. In the case of a human IgG1 domain, mutation of
the wild type amino acid at position 114 to an asparagine results
in the formation of an N-linked glycosylation consensus site (i.e,
the N-X-T/S sequon, where X is any amino acid except proline).
However, in the case of other CH1 domains of other species and/or
Ig classes or isotypes, the skilled artisan will appreciate that it
may be necessary to mutate positions 115 and/or 116 of the CH1
domain to create an N-X-T/S sequon.
[0182] In certain embodiments, the binding polypeptide of the
current disclosure may comprise an antigen binding fragment of an
antibody. The term "antigen-binding fragment" refers to a
polypeptide fragment of an immunoglobulin or antibody which binds
antigen or competes with intact antibody (i.e., with the intact
antibody from which they were derived) for antigen binding (i.e.,
specific binding). Antigen binding fragments can be produced by
recombinant or biochemical methods that are well known in the art.
Exemplary antigen-binding fragments include Fv, Fab, Fab', and
(Fab')2. In some embodiments, the antigen-binding fragment of the
current disclosure is an altered antigen-binding fragment
comprising at least one engineered glycosylation site. In one
exemplary embodiment, an altered antigen binding fragment of the
current disclosure comprises an altered VH domain described supra.
In another exemplary embodiment, an altered antigen binding
fragment of the current disclosure comprises an altered CH1 domain
described supra.
[0183] In exemplary embodiments, the binding polypeptide comprises
a single chain variable region sequence (ScFv). Single chain
variable region sequences comprise a single polypeptide having one
or more antigen binding sites, e.g., a VL domain linked by a
flexible linker to a VH domain. ScFv molecules can be constructed
in a VH-linker-VL orientation or VL-linker-VH orientation. The
flexible hinge that links the VL and VH domains that make up the
antigen binding site typically has from about 10 to about 50 amino
acid residues. Connecting peptides are known in the art. Binding
polypeptides may comprise at least one scFv and/or at least one
constant region. In one embodiment, a binding polypeptide of the
current disclosure may comprise at least one scFv linked or fused
to an antibody or fragment comprising a CH1 domain (e.g. a CH1
domain comprising an asparagine residue at Kabat position 114)
and/or a CH2 domain (e.g. a CH2 domain comprising an asparagine
residue at EU position 298, and a serine or threonine residue at EU
position 300).
[0184] In certain exemplary embodiments, a binding polypeptide of
the current disclosure is a multivalent (e.g., tetravalent)
antibody which is produced by fusing a DNA sequence encoding an
antibody with a ScFv molecule (e.g., an altered ScFv molecule). For
example, in one embodiment, these sequences are combined such that
the ScFv molecule (e.g., an altered ScFv molecule) is linked at its
N-terminus or C-terminus to an Fc fragment of an antibody via a
flexible linker (e.g., a gly/ser linker). In another embodiment a
tetravalent antibody of the current disclosure can be made by
fusing an ScFv molecule to a connecting peptide, which is fused to
a CH1 domain (e.g. a CH1 domain comprising an asparagine residue at
Kabat position 114) to construct an ScFv-Fab tetravalent
molecule.
[0185] In another embodiment, a binding polypeptide of the current
disclosure is an altered minibody. Altered minibodies of the
current disclosure are dimeric molecules made up of two polypeptide
chains each comprising an ScFv molecule (e.g., an altered ScFv
molecule comprising an altered VH domain described supra) which is
fused to a CH3 domain or portion thereof via a connecting peptide.
Minibodies can be made by constructing an ScFv component and
connecting peptide-CH3 components using methods described in the
art (see, e.g., U.S. Pat. No. 5,837,821 or WO 94/09817A1). In
another embodiment, a tetravalent minibody can be constructed.
Tetravalent minibodies can be constructed in the same manner as
minibodies, except that two ScFv molecules are linked using a
flexible linker. The linked scFv-scFv construct is then joined to a
CH3 domain.
[0186] In another embodiment, a binding polypeptide of the current
disclosure comprises a diabody. Diabodies are dimeric, tetravalent
molecules each having a polypeptide similar to scFv molecules, but
usually having a short (less than 10, e.g., 1-5) amino acid residue
linker connecting both variable domains, such that the VL and VH
domains on the same polypeptide chain cannot interact. Instead, the
VL and VH domain of one polypeptide chain interact with the VH and
VL domain (respectively) on a second polypeptide chain (see, for
example, WO 02/02781). Diabodies of the current disclosure comprise
an scFv molecule fused to a CH3 domain.
[0187] In other embodiments, the binding polypeptides include
multispecific or multivalent antibodies comprising one or more
variable domain in series on the same polypeptide chain, e.g.,
tandem variable domain (TVD) polypeptides. Exemplary TVD
polypeptides include the "double head" or "Dual-Fv" configuration
described in U.S. Pat. No. 5,989,830. In the Dual-Fv configuration,
the variable domains of two different antibodies are expressed in a
tandem orientation on two separate chains (one heavy chain and one
light chain), wherein one polypeptide chain has two VH domains in
series separated by a peptide linker (VH1-linker-VH2) and the other
polypeptide chain consists of complementary VL domains connected in
series by a peptide linker (VL1-linker-VL2). In the cross-over
double head configuration, the variable domains of two different
antibodies are expressed in a tandem orientation on two separate
polypeptide chains (one heavy chain and one light chain), wherein
one polypeptide chain has two VH domains in series separated by a
peptide linker (VH1-linker-VH2) and the other polypeptide chain
consists of complementary VL domains connected in series by a
peptide linker in the opposite orientation (VL2-linker-VL1).
Additional antibody variants based on the "Dual-Fv" format include
the Dual-Variable-Domain IgG (DVD-IgG) bispecific antibody (see
U.S. Pat. No. 7,612,181 and the TBTI format (see US 2010/0226923
A1). The addition of constant domains to respective chains of the
Dual-Fv (CH1-Fc to the heavy chain and kappa or lambda constant
domain to the light chain) leads to functional bispecific
antibodies without any need for additional modifications (i.e.,
obvious addition of constant domains to enhance stability).
[0188] In another exemplary embodiment, the binding polypeptide
comprises a cross-over dual variable domain IgG (CODV-IgG)
bispecific antibody based on a "double head" configuration (see
US20120251541 A1, which is incorporated by reference herein in its
entirety). CODV-IgG antibody variants have one polypeptide chain
with VL domains connected in series to a CL domain
(VL1-L1-VL2-L2-CL) and a second polypeptide chain with
complementary VH domains connected in series in the opposite
orientation to a CH1 domain (VH2-L3-VH1-L4-CH1), where the
polypeptide chains form a cross-over light chain-heavy chain pair.
In certain embodiment, the second polypeptide may be further
connected to an Fc domain (VH2-L3-VH1-L4-CH1-Fc). In certain
embodiments, linker L3 is at least twice the length of linker L1
and/or linker L4 is at least twice the length of linker L2. For
example, L1 and L2 may be 1-3 amino acid residues in length, L3 may
be 2 to 6 amino acid residues in length, and L4 may be 4 to 7 amino
acid residues in length. Examples of suitable linkers include a
single glycine (Gly) residue; a diglycine peptide (Gly-Gly); a
tripeptide (Gly-Gly-Gly); a peptide with four glycine residues
(Gly-Gly-Gly-Gly (SEQ ID NO: 17)); a peptide with five glycine
residues (Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 18)); a peptide with six
glycine residues (Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 19)); a
peptide with seven glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly
(SEQ ID NO: 20)); a peptide with eight glycine residues
(Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 21)). Other
combinations of amino acid residues may be used such as the peptide
Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 22) and the peptide
Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 39).
[0189] In certain embodiments, the binding polypeptide comprises an
immunoadhesin molecule comprising a non-antibody binding region
(e.g., a receptor, ligand, or cell-adhesion molecule) fused to an
antibody constant region (see e.g., Ashkenazi et al., Methods, 1995
8(2), 104-115, which is incorporated by reference herein in its
entirety)
[0190] In certain embodiments, the binding polypeptide comprises
immunoglobulin-like domains. Suitable immunoglobulin-like domains
include, without limitation, fibronectin domains (see, for example,
Koide et al. (2007), Methods Mol. Biol. 352: 95-109, which is
incorporated by reference herein in its entirety), DARPin (see, for
example, Stumpp et al. (2008) Drug Discov. Today 13 (15-16):
695-701, which is incorporated by reference herein in its
entirety), Z domains of protein A (see, Nygren et al. (2008) FEBS
J. 275 (11): 2668-76, which is incorporated by reference herein in
its entirety), Lipocalins (see, for example, Skerra et al. (2008)
FEBS J. 275 (11): 2677-83, which is incorporated by reference
herein in its entirety), Affilins (see, for example, Ebersbach et
al. (2007) J. Mol. Biol. 372 (1): 172-85, which is incorporated by
reference herein in its entirety), Affitins (see, for example,
Krehenbrink et al. (2008). J. Mol. Biol. 383 (5): 1058-68, which is
incorporated by reference herein in its entirety), Avimers (see,
for example, Silverman et al. (2005) Nat. Biotechnol. 23 (12):
1556-61, which is incorporated by reference herein in its
entirety), Fynomers, (see, for example, Grabulovski et al. (2007) J
Biol Chem 282 (5): 3196-3204, which is incorporated by reference
herein in its entirety), and Kunitz domain peptides (see, for
example, Nixon et al. (2006) Curr Opin Drug Discov Devel 9 (2):
261-8, which is incorporated by reference herein in its
entirety).
IV. N-linked Glycans
[0191] In certain embodiments, the binding polypeptides employs
N-linked glycans which are "N-linked" via an asparagine residue to
a glycosylation site in the polypeptide backbone of the binding
polypeptide. The glycosylation site may be a native or engineered
glycosylation site. Additionally or alternatively, the glycan may
be a native glycan or an engineered glycan containing non-native
linkages.
[0192] In certain exemplary embodiments, the binding polypeptide
includes the native glycosylation site of an antibody Fc domain.
This native glycosylation site comprises a wild-type asparagine
residue at position 297 of the Fc domain (N297), according to EU
numbering. The native N-linked glycan that resides at this position
is generally linked though a .beta.-glycosylamide linkage to the
nitrogen group of the N297 side chain. However, other suitable art
recognized linkages can also be employed. In other exemplary
embodiments, the binding polypeptides comprise one or more
engineered glycosylation sites. Such engineered glycosylation sites
comprise the substitution of one or more wild-type amino acids in
the polypeptide backbone of the binding polypeptide with an
asparagine residue that is capable of being N-glycosylated by the
glycosylation enzymes of a cell. Exemplary engineered glycosylation
sites include the introduction of asparagine mutation at amino acid
position 298 of the Fc domain (298N) or amino acid position 114 of
a CH1 domain (114N).
[0193] Any type of naturally occurring or synthetic (i.e.,
non-natural) N-linked glycan can be linked to a glycosylation site
of a binding polypeptide featured in the invention. In certain
embodiments, the glycan comprises a saccharide (e.g., a saccharide
residue located at terminus of an oligosaccharide) that can be
oxidized (e.g., by periodate treatment or galactose oxidase) to
produce a group suitable for conjugation to an effector moiety
(e.g., a reactive aldehyde group). Suitable oxidizable saccharides
included, without limitation, galactose and sialic acid (e.g.,
N-Acetylneuraminic acid). In other embodiments, the glycan
comprises a sialic acid or sialic acid derivative that does not
require further oxidation to produce a group suitable for
conjugation to an effector moiety (e.g., a reactive moiety
including, but not limited to, an aldehyde moiety, an alkyne, an
aminooxy moiety, an azide, a hydrazine, a keto moiety, and a
thiol). In specific embodiments, the glycan comprises a sialic acid
derivative. In one embodiment, the glycan comprising a sialic acid
derivative is formed by a reaction between a binding polypeptide
comprising a glycan and a CMP-sialic acid derivative. In one
embodiment, the sialic acid derivative or CMP-sialic acid
derivative may comprise a terminal azide moiety. In a further
embodiment, the CMP-sialic acid derivative may be a CMP-sialic acid
C5 azide. In another embodiment, the sialic acid derivative may
comprise a C5 azide. In certain embodiments, the CMP-sialic acid
derivative has the following structural formula:
##STR00021##
[0194] wherein R.sub.1 is a reactive moiety including, but not
limited to, NH(C.dbd.O)CH3,
NH(C.dbd.O)CH.sub.2CH.sub.2(C.dbd.O)CH.sub.3,
NH(C.dbd.O)CH.sub.2OH, NH(C.dbd.O)CH.sub.2N.sub.3, NH(C.dbd.O)SH,
OH or N.sub.3.
[0195] In certain embodiments, the glycan is a biantennary glycan.
In certain embodiments, the glycan is a naturally occurring
mammalian glycoform.
[0196] Glycosylation can be achieved through any means known in the
art. In certain embodiments, the glycosylation is achieved by
expression of the binding polypeptides in cells capable of N-linked
glycosylation. Any natural or engineered cell (e.g., prokaryotic or
eukaryotic) can be employed. In general, mammalian cells are
employed to effect glycosylation. The N-glycans that are produced
in mammalian cells are commonly referred to as complex, high
manose, hybrid-type N-glycans (see e.g., Drickamer K, Taylor M E
(2006). Introduction to Glycobiology, 2nd ed., which is
incorporated herein by reference in its entirety). These complex
N-glycans have a structure that typically has two to six outer
branches with a sialyllactosamine sequence linked to an inner core
structure Man.sub.3GlcNAc.sub.2. A complex N-glycan has at least
one branch, e.g., at least two, of alternating GlcNAc and galactose
(Gal) residues that terminate in oligosaccharides such as, for
example: NeuNAc--; NeuAc .alpha.2,6 GalNAc .alpha.1-; NeuAc
.alpha.2,3 Gal .beta.1,3 GalNAc .alpha.1-; and NeuAc .alpha.2,3/6
Gal .beta.1,4 GlcNAc .beta.1.; In addition, sulfate esters can
occur on galactose, GalNAc, and GlcNAc residues. NeuAc can be
O-acetylated or replaced by NeuGl (N-glycolylneuraminic acid).
Complex N-glycans may also have intrachain substitutions of
bisecting GlcNAc and core fucose (Fuc).
[0197] Additionally or alternatively, glycosylation can be achieved
or modified through enzymatic means, in vitro. For example, one or
more glycosyltransferases may be employed to add specific
saccharide residues to the native or engineered N-glycan of a
binding polypeptide, and one or more glycosidases may be employed
to remove unwanted saccharides from the N-linked glycan. Such
enzymatic means are well known in the art (see. e.g.,
WO2007/005786, which is incorporated herein by reference in its
entirety).
V. Immunological Effector Functions and Fc Modifications
[0198] In certain embodiments, binding polypeptides may include an
antibody constant region (e.g. an IgG constant region e.g., a human
IgG constant region, e.g., a human IgG1 or IgG4 constant region)
which mediates one or more effector functions. For example, binding
of the C1-complex to an antibody constant region may activate the
complement system. Activation of the complement system is important
in the opsonisation and lysis of cell pathogens. The activation of
the complement system also stimulates the inflammatory response and
may also be involved in autoimmune hypersensitivity. Further,
antibodies bind to receptors on various cells via the Fc region (Fc
receptor binding sites on the antibody Fc region bind to Fc
receptors (FcRs) on a cell). There are a number of Fc receptors
which are specific for different classes of antibody, including IgG
(gamma receptors), IgE (epsilon receptors), IgA (alpha receptors)
and IgM (mu receptors). Binding of antibody to Fc receptors on cell
surfaces triggers a number of important and diverse biological
responses including engulfment and destruction of antibody-coated
particles, clearance of immune complexes, lysis of antibody-coated
target cells by killer cells (called antibody-dependent
cell-mediated cytotoxicity, or ADCC), release of inflammatory
mediators, placental transfer and control of immunoglobulin
production. In some embodiments, the binding polypeptides (e.g.,
antibodies or antigen binding fragments thereof) featured in the
invention bind to an Fc-gamma receptor. In alternative embodiments,
binding polypeptides may include a constant region which is devoid
of one or more effector functions (e.g., ADCC activity) and/or is
unable to bind Fc.gamma. receptor.
[0199] Certain embodiments include antibodies in which at least one
amino acid in one or more of the constant region domains has been
deleted or otherwise altered so as to provide desired biochemical
characteristics such as reduced or enhanced effector functions, the
ability to non-covalently dimerize, increased ability to localize
at the site of a tumor, reduced serum half-life, or increased serum
half-life when compared with a whole, unaltered antibody of
approximately the same immunogenicity. For example, certain
antibodies for use in the diagnostic and treatment methods
described herein are domain deleted antibodies which comprise a
polypeptide chain similar to an immunoglobulin heavy chain, but
which lack at least a portion of one or more heavy chain domains.
For instance, in certain antibodies, one entire domain of the
constant region of the modified antibody will be deleted, for
example, all or part of the CH.sub.2 domain will be deleted.
[0200] In certain other embodiments, binding polypeptides comprise
constant regions derived from different antibody isotypes (e.g.,
constant regions from two or more of a human IgG1, IgG2, IgG3, or
IgG4). In other embodiments, binding polypeptides comprises a
chimeric hinge (i.e., a hinge comprising hinge portions derived
from hinge domains of different antibody isotypes, e.g., an upper
hinge domain from an IgG4 molecule and an IgG1 middle hinge
domain). In one embodiment, binding polypeptides comprise an Fc
region or portion thereof from a human IgG4 molecule and a
Ser228Pro mutation (EU numbering) in the core hinge region of the
molecule.
[0201] In certain embodiments, the Fc portion may be mutated to
increase or decrease effector function using techniques known in
the art. For example, the deletion or inactivation (through point
mutations or other means) of a constant region domain may reduce Fc
receptor binding of the circulating modified antibody thereby
increasing tumor localization. In other cases it may be that
constant region modifications consistent with the instant invention
moderate complement binding and thus reduce the serum half life and
nonspecific association of a conjugated cytotoxin. Yet other
modifications of the constant region may be used to modify
disulfide linkages or oligosaccharide moieties that allow for
enhanced localization due to increased antigen specificity or
flexibility. The resulting physiological profile, bioavailability
and other biochemical effects of the modifications, such as tumor
localization, biodistribution and serum half-life, may easily be
measured and quantified using well know immunological techniques
without undue experimentation.
[0202] In certain embodiments, an Fc domain employed in an antibody
featured in the invention is an Fc variant. As used herein, the
term "Fc variant" refers to an Fc domain having at least one amino
acid substitution relative to the wild-type Fc domain from which
said Fc domain is derived. For example, wherein the Fc domain is
derived from a human IgG1 antibody, the Fc variant of said human
IgG1 Fc domain comprises at least one amino acid substitution
relative to said Fc domain.
[0203] The amino acid substitution(s) of an Fc variant may be
located at any position (i.e., any EU convention amino acid
position) within the Fc domain. In one embodiment, the Fc variant
comprises a substitution at an amino acid position located in a
hinge domain or portion thereof. In another embodiment, the Fc
variant comprises a substitution at an amino acid position located
in a CH2 domain or portion thereof. In another embodiment, the Fc
variant comprises a substitution at an amino acid position located
in a CH3 domain or portion thereof. In another embodiment, the Fc
variant comprises a substitution at an amino acid position located
in a CH4 domain or portion thereof.
[0204] The binding polypeptides may employ any art-recognized Fc
variant which is known to impart an improvement (e.g., reduction or
enhancement) in effector function and/or FcR binding. Said Fc
variants may include, for example, any one of the amino acid
substitutions disclosed in International PCT Publications
WO88/07089A1, WO96/14339A1, WO98/05787A1, WO98/23289A1,
WO99/51642A1, WO99/58572A1, WO00/09560A2, WO00/32767A1,
WO00/42072A2, WO02/44215A2, WO02/060919A2, WO03/074569A2,
WO04/016750A2, WO04/029207A2, WO04/035752A2, WO04/063351A2,
WO04/074455A2, WO04/099249A2, WO05/040217A2, WO05/070963A1,
WO05/077981A2, WO05/092925A2, WO05/123780A2, WO06/019447A1,
WO06/047350A2, and WO06/085967A2 or U.S. Pat. Nos. 5,648,260;
5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551;
6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505;
6,998,253; and 7,083,784, each of which is incorporated in its
entirety by reference herein. In one exemplary embodiment, a
binding polypeptide may comprise an Fc variant comprising an amino
acid substitution at EU position 268 (e.g., H268D or H268E). In
another exemplary embodiment, a binding polypeptide may include an
amino acid substitution at EU position 239 (e.g., S239D or S239E)
and/or EU position 332 (e.g., I332D or I332Q).
[0205] In certain embodiments, a binding polypeptide may include an
Fc variant comprising an amino acid substitution which alters the
antigen-independent effector functions of the antibody, in
particular the circulating half-life of the binding polypeptide.
Such binding polypeptides exhibit either increased or decreased
binding to FcRn when compared to binding polypeptides lacking these
substitutions, therefore, have an increased or decreased half-life
in serum, respectively. Fc variants with improved affinity for FcRn
are anticipated to have longer serum half-lives, and such molecules
have useful applications in methods of treating mammals where long
half-life of the administered antibody is desired, e.g., to treat a
chronic disease or disorder. In contrast, Fc variants with
decreased FcRn binding affinity are expected to have shorter
half-lives, and such molecules are also useful, for example, for
administration to a mammal where a shortened circulation time may
be advantageous, e.g. for in vivo diagnostic imaging or in
situations where the starting antibody has toxic side effects when
present in the circulation for prolonged periods. Fc variants with
decreased FcRn binding affinity are also less likely to cross the
placenta and, thus, are also useful in the treatment of diseases or
disorders in pregnant women. In addition, other applications in
which reduced FcRn binding affinity may be desired include
applications localized to the brain, kidney, and/or liver. In one
exemplary embodiment, the altered binding polypeptides (e.g.,
antibodies or antigen binding fragments thereof) exhibit reduced
transport across the epithelium of kidney glomeruli from the
vasculature. In another embodiment, the altered binding
polypeptides (e.g., antibodies or antigen binding fragments
thereof) exhibit reduced transport across the blood brain barrier
(BBB) from the brain into the vascular space. In one embodiment, an
antibody with altered FcRn binding comprises an Fc domain having
one or more amino acid substitutions within the "FcRn binding loop"
of an Fc domain. The FcRn binding loop is comprised of amino acid
residues 280-299 (according to EU numbering). Exemplary amino acid
substitutions which alter FcRn binding activity are disclosed in
International PCT Publication No. WO05/047327 which is incorporated
in its entirety by reference herein. In certain exemplary
embodiments, the binding polypeptides (e.g., antibodies or antigen
binding fragments thereof) include an Fc domain having one or more
of the following substitutions: V284E, H285E, N286D, K290E and
S304D (EU numbering). In yet other exemplary embodiments, the
binding molecules include a human Fc domain with the double
mutation H433K/N434F (see, e.g., U.S. Pat. No. 8,163,881).
[0206] In other embodiments, binding polypeptides, for use in the
diagnostic and treatment methods described herein have a constant
region, e.g., an IgG1 or IgG4 heavy chain constant region, which is
altered to reduce or eliminate glycosylation. For example, binding
polypeptides (e.g., antibodies or antigen binding fragments
thereof) may also include an Fc variant comprising an amino acid
substitution which alters the glycosylation of the antibody Fc. For
example, said Fc variant may have reduced glycosylation (e.g., N-
or O-linked glycosylation). In exemplary embodiments, the Fc
variant comprises reduced glycosylation of the N-linked glycan
normally found at amino acid position 297 (EU numbering). In
another embodiment, the antibody has an amino acid substitution
near or within a glycosylation motif, for example, an N-linked
glycosylation motif that contains the amino acid sequence NXT or
NXS. In a particular embodiment, the antibody comprises an Fc
variant with an amino acid substitution at amino acid position 228
or 299 (EU numbering). In more particular embodiments, the antibody
comprises an IgG1 or IgG4 constant region comprising an S228P and a
T299A mutation (EU numbering).
[0207] Exemplary amino acid substitutions which confer reduced or
altered glycosylation are disclosed in International PCT
Publication No. WO05/018572, which is incorporated in its entirety
by reference herein. In some embodiments, the binding polypeptides
are modified to eliminate glycosylation. Such binding polypeptides
may be referred to as "agly" binding polypeptides (e.g. "agly"
antibodies). While not being bound by theory, it is believed that
"agly" binding polypeptides may have an improved safety and
stability profile in vivo. Agly binding polypeptides can be of any
isotype or subclass thereof, e.g., IgG1, IgG2, IgG3, or IgG4. In
certain embodiments, agly binding polypeptides comprise an
aglycosylated Fc region of an IgG4 antibody which is devoid of
Fc-effector function, thereby eliminating the potential for Fc
mediated toxicity to the normal vital organs that express IL-6. In
yet other embodiments, binding polypeptides include an altered
glycan. For example, the antibody may have a reduced number of
fucose residues on an N-glycan at Asn297 of the Fc region, i.e., is
afucosylated. Afucosylation increases Fc.gamma.RII binding on the
NK cells and potently increases ADCC. It has been shown that a
diabody comprising an anti-IL-6 scFv and an anti-CD3 scFv induces
killing of IL-6 expressing cells by ADCC. Accordingly, in one
embodiment, an afucosylated anti-IL-6 antibody is used to target
and kill IL-6-expressing cells. In another embodiment, the binding
polypeptide may have an altered number of sialic acid residues on
the N-glycan at Asn297 of the Fc region. Numerous art-recognized
methods are available for making "agly" antibodies or antibodies
with altered glycans. For example, genetically engineered host
cells (e.g., modified yeast, e.g., Picchia, or CHO cells) with
modified glycosylation pathways (e.g., glycosyl-transferase
deletions) can be used to produce such antibodies.
VI. Effector Moieties
[0208] In certain embodiments, the binding polypeptides of the
current disclosure comprise effector moieties (e.g., targeting
moieties). In general these effector moieties are conjugated
(either directly or through a linker moiety) to an N-linked glycan
on the binding polypeptide, (e.g., an N-linked glycan linked to
N298 (EU numbering) of the CH2 domain and/or N114 (Kabat numbering)
of a CH1 domain). In certain embodiments, the binding polypeptide
is full length antibody comprising two CH1 domains with a glycan at
Kabat position 114, wherein both of the glycans are conjugated to
one or more effector moieties.
[0209] Any effector moiety can be added to the binding polypeptides
disclosed herein. The effector moieties typically add a non-natural
function to an altered antibody or fragments thereof without
significantly altering the intrinsic activity of the binding
polypeptide. The effector moiety may be, for example but not
limited to, targeting moiety (e.g., a glycopeptide or neoglycan). A
modified binding polypeptide (e.g., an antibody) of the current
disclosure may comprise one or more effector moieties, which may be
the same of different.
[0210] In one embodiment, the effector moiety can be of Formula
(I):
H.sub.2N-Q-CON-X Formula (I),
wherein:
[0211] A) Q is NH or O; and
[0212] B) CON is a connector moiety; and
[0213] C) X is an effector moiety (e.g., a targeting moiety as
defined herein).
[0214] The connector moiety connects the therapeutic agent to
H.sub.2N-Q-. The connector moiety can include at least one of any
suitable components known to those skilled in the art, including,
for example, an alkylenyl component, a polyethylene glycol
component, a poly(glycine) component, a poly(oxazoline) component,
a carbonyl component, a component derived from cysteinamide, a
component derived from valine coupled with citruline, and a
component derived from 4-aminobenzyl carbamate, or any combination
thereof.
[0215] In some embodiments, the connector moiety (CON) may comprise
portions of the molecules formed in the reacting step whereby an
effector moiety conjugated binding polypeptide is formed. For
example, the connector moiety may comprise one or more of the
following structural formulas:
##STR00022##
[0216] In another embodiment, the effector moiety of Formula (I)
can be of Formula (Ia):
H.sub.2N-Q-CH.sub.2--C(O)--Z--X Formula (Ia),
wherein:
[0217] A) Q is NH or O; and
[0218] B) Z is
-Cys-(MC).sub.a-(VC).sub.b-(PABC).sub.c-(C.sub.16H.sub.32O.sub.8C.sub.2H.-
sub.4).sub.f, [0219] wherein [0220] i. Cys is a component derived
cysteinamide; [0221] ii. MC is a component derived from maleimide;
[0222] iii. VC is a component derived from valine coupled with
citruline; [0223] iv. PABC is a component derived from
4-aminobenzyl carbamate; [0224] v. X is an effector moiety (e.g., a
targeting moiety as defined herein); [0225] vi. a is 0 or 1; [0226]
vii. b is 0 or 1; [0227] viii. c is 0 or 1; and [0228] ix. f is 0
or 1
[0229] The "component derived from cysteinamide" is the point of
attachment to H.sub.2N-Q-CH.sub.2--C(O)--. In one embodiment, the
"component derived from cysteinamide" can refer to one or more
portions of the effector moiety having the structure:
##STR00023##
[0230] In one embodiment, the "Cys" component of an effector moiety
may include one such portion. For example, the following structure
shows an effector moiety with one such portion (wherein the "Cys"
component is indicated with the dotted line box):
##STR00024##
[0231] In another embodiment, the "Cys" component of an effector
moiety may include two or more such portions. For example, the
following moiety contains two such portions:
##STR00025##
[0232] As can be seen from the structure, each "Cys" component
bears an
-(MC).sub.a-(VC).sub.b-(PABC).sub.c-(C.sub.16H.sub.32O.sub.8C.sub.2H.sub.-
4).sub.f--X group.
[0233] In one embodiment, the phrase "component derived from
maleimide" can refer to any portion of the effector moiety having
the structure:
##STR00026##
[0234] wherein d is an integer from 2 to 5. The number of MC
components included in any
Cys-(MC).sub.a-(VC).sub.b-(PABC).sub.c-(C.sub.16H.sub.32O.sub.8C.sub.2H.s-
ub.4).sub.f--X group in the effector moiety is indicated by
subscript "a," and can be 0 or 1 In one embodiment, a is 1. In
another embodiment, b is 0.
[0235] In one embodiment, the "Cys" component can be connected to
the "MC" component via the sulfur atom in the "Cys" component, as
indicated with the dotted line box in the structure below:
##STR00027##
[0236] In one embodiment, the phrase "component derived from valine
coupled with citruline" can refer to any portion of the effector
moiety with the following structure:
##STR00028##
[0237] The number of VC components included in any
Cys-(MC).sub.a-(VC).sub.b-(PABC).sub.c-(C.sub.16H.sub.32O.sub.8C.sub.2H.s-
ub.4).sub.f--X group in the effector moiety is indicated by
subscript "b," and can be 0 or 1. In one embodiment, b is 1. In
another embodiment, b is 0.
[0238] In one embodiment, the phrase "component derived from
4-aminobenzyl carbamate" can refer to any portion of the effector
moiety with the following structure:
##STR00029##
[0239] The number of PABC components included in any
Cys-(MC).sub.a-(VC).sub.b-(PABC).sub.c-(C.sub.16H.sub.32O.sub.8C.sub.2H.s-
ub.4).sub.f--X group in the effector moiety is indicated by
subscript "c," and can be 0 or 1. In one embodiment, c is 1. In
another embodiment, c is 0.
[0240] In one embodiment, "C.sub.16H.sub.32O.sub.8C.sub.2H.sub.4"
refers to the following structure:
##STR00030##
[0241] The number of C.sub.16H.sub.32O.sub.8 units included in any
Cys-(MC).sub.a-(VC).sub.b-(PABC).sub.c-(C.sub.16H.sub.32O.sub.8C.sub.2H.s-
ub.4).sub.f--X group in the effector moiety is indicated by
subscript "f," In one embodiment, f is 1. In another embodiment, f
is 0.
[0242] In one embodiment, a is 1, b is 1, c is 1, and f is 0.
[0243] a) Therapeutic Effector Moieties
[0244] In certain embodiments, the binding polypeptides of the
current disclosure are conjugated to an effector moiety comprising
a therapeutic agent, e.g. a drug moiety (or prodrug thereof) or
radiolabeled compound. In one embodiment the therapeutic agent is a
cytotoxin. Exemplary cytotoxic therapeutic agents are set forth in
Table 1 herein.
TABLE-US-00001 TABLE 1 Exemplary cytotoxic therapeutic agents
##STR00031## ##STR00032## ##STR00033## ##STR00034## ##STR00035##
##STR00036## ##STR00037## ##STR00038## ##STR00039## ##STR00040##
##STR00041## ##STR00042## ##STR00043## ##STR00044## ##STR00045##
##STR00046## ##STR00047## ##STR00048## ##STR00049##
##STR00050##
[0245] Further exemplary drug moieties include anti-inflammatory,
anti-cancer, anti-infective (e.g., anti-fungal, antibacterial,
anti-parasitic, anti-viral, etc.), and anesthetic therapeutic
agents. In a further embodiment, the drug moiety is an anti-cancer
agent. Exemplary anti-cancer agents include, but are not limited
to, cytostatics, enzyme inhibitors, gene regulators, cytotoxic
nucleosides, tubulin binding agents or tubulin inhibitors,
proteasome inhibitors, hormones and hormone antagonists,
anti-angiogenesis agents, and the like. Exemplary cytostatic
anti-cancer agents include alkylating agents such as the
anthracycline family of drugs (e.g. adriamycin, carminomycin,
cyclosporin-A, chloroquine, methopterin, mithramycin, porfiromycin,
streptonigrin, porfiromycin, anthracenediones, and aziridines).
Other cytostatic anti-cancer agents include DNA synthesis
inhibitors (e.g., methotrexate and dichloromethotrexate,
3-amino-1,2,4-benzotriazine 1,4-dioxide, aminopterin, cytosine
.beta.-D-arabinofuranoside, 5-fluoro-5'-deoxyuridine,
5-fluorouracil, ganciclovir, hydroxyurea, actinomycin-D, and
mitomycin C), DNA-intercalators or cross-linkers (e.g., bleomycin,
carboplatin, carmustine, chlorambucil, cyclophosphamide,
cis-diammineplatinum(II) dichloride (cisplatin), melphalan,
mitoxantrone, and oxaliplatin), and DNA-RNA transcription
regulators (e.g., actinomycin D, daunorubicin, doxorubicin,
homoharringtonine, and idarubicin). Other exemplary cytostatic
agents that are compatible with the present disclosure include
ansamycin benzoquinones, quinonoid derivatives (e.g. quinolones,
genistein, bactacyclin), busulfan, ifosfamide, mechlorethamine,
triaziquone, diaziquone, carbazilquinone, indoloquinone EO9,
diaziridinyl-benzoquinone methyl DZQ, triethylenephosphoramide, and
nitrosourea compounds (e.g. carmustine, lomustine, semustine).
[0246] Exemplary cytotoxic nucleoside anti-cancer agents include,
but are not limited to: adenosine arabinoside, cytarabine, cytosine
arabinoside, 5-fluorouracil, fludarabine, floxuridine, ftorafur,
and 6-mercaptopurine. Exemplary anti-cancer tubulin binding agents
include, but are not limited to: taxoids (e.g. paclitaxel,
docetaxel, taxane), nocodazole, rhizoxin, dolastatins (e.g.
Dolastatin-10, -11, or -15), colchicine and colchicinoids (e.g.
ZD6126), combretastatins (e.g. Combretastatin A-4, AVE-6032), and
vinca alkaloids (e.g. vinblastine, vincristine, vindesine, and
vinorelbine (navelbine)). Exemplary anti-cancer hormones and
hormone antagonists include, but are not limited to:
corticosteroids (e.g. prednisone), progestins (e.g.
hydroxyprogesterone or medroprogesterone), estrogens, (e.g.
diethylstilbestrol), antiestrogens (e.g. tamoxifen), androgens
(e.g. testosterone), aromatase inhibitors (e.g. aminogluthetimide),
17-(allylamino)-17-demethoxygeldanamycin,
4-amino-1,8-naphthalimide, apigenin, brefeldin A, cimetidine,
dichloromethylene-diphosphonic acid, leuprolide (leuprorelin),
luteinizing hormone-releasing hormone, pifithrin-a, rapamycin, sex
hormone-binding globulin, and thapsigargin. Exemplary anti-cancer,
anti-angiogenesis compounds include, but are not limited to:
Angiostatin Kl-3, DL-a-difluoromethyl-ornithine, endostatin,
fumagillin, genistein, minocycline, staurosporine, and
(.+-.)-thalidomide.
[0247] Exemplary anti-cancer enzyme inhibitors include, but are not
limited to: S(+)-camptothecin, curcumin, (-)-deguelin,
5,6-diCH1orobenz-imidazole 1-.beta.-D-ribofuranoside, etoposide,
formestane, fostriecin, hispidin, 2-imino-1-imidazolidineacetic
acid (cyclocreatine), mevinolin, trichostatin A, tyrphostin AG 34,
and tyrphostin AG 879.
[0248] Exemplary anti-cancer gene regulators include, but are not
limited to: 5-aza-2'-deoxycytidine, 5-azacytidine, cholecalciferol
(vitamin D3), 4-hydroxytamoxifen, melatonin, mifepristone,
raloxifene, trans-retinal (vitamin A aldehydes), retinoic acid,
vitamin A acid, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol
(vitamin A), tamoxifen, and troglitazone.
[0249] Other classes of anti-cancer agents include, but are not
limited to: the pteridine family of drugs, diynenes, and the
podophyllotoxins. Particularly useful members of those classes
include, for example, methopterin, podophyllotoxin, or
podophyllotoxin derivatives such as etoposide or etoposide
phosphate, leurosidine, vindesine, leurosine and the like.
[0250] Still other anti-cancer agents that are compatible with the
teachings herein include auristatins (e.g. auristatin E and
monomethylauristan E), geldanamycin, calicheamicin, gramicidin D,
maytansanoids (e.g. maytansine), neocarzinostatin, topotecan,
taxanes, cytochalasin B, ethidium bromide, emetine, tenoposide,
colchicin, dihydroxy anthracindione, mitoxantrone, procaine,
tetracaine, lidocaine, propranolol, puromycin, and analogs or
homologs thereof.
[0251] Still other anti-cancer agents that are compatible with the
teachings herein include tomaymycin derivatives, maytansine
derivatives, cryptophycine derivatives, anthracycline derivatives,
bisphosphonate derivatives, leptomycin derivatives, streptonigrin
derivatives, auristatine derivatives, and duocarmycin
derivatives.
[0252] Another class of compatible anti-cancer agents that may be
used as drug moieties are radiosensitizing drugs that may be
effectively directed to tumor or immunoreactive cells. Such drug
moeities enhance the sensitivity to ionizing radiation, thereby
increasing the efficacy of radiotherapy. Not to be limited by
theory, but an antibody modified with a radiosensitizing drug
moiety and internalized by the tumor cell would deliver the
radiosensitizer nearer the nucleus where radiosensitization would
be maximal. Antibodies which lose the radiosensitizer moiety would
be cleared quickly from the blood, localizing the remaining
radiosensitization agent in the target tumor and providing minimal
uptake in normal tissues. After clearance from the blood, adjunct
radiotherapy could be administered by external beam radiation
directed specifically to the tumor, radioactivity directly
implanted in the tumor, or systemic radioimmunotherapy with the
same modified antibody.
[0253] In one embodiment, the therapeutic agent comprises
radionuclides or radiolabels with high-energy ionizing radiation
that are capable of causing multiple strand breaks in nuclear DNA,
leading to cell death. Exemplary high-energy radionuclides include:
.sup.90Y, .sup.125I, .sup.131I, .sup.123I, .sup.111In, .sup.105Rh,
.sup.153Sm, .sup.67Cu, .sup.67Ga, .sup.166Ho, .sup.177Lu,
.sup.186Re and .sup.188Re. These isotopes typically produce high
energy .alpha.- or .beta.-particles which have a short path length.
Such radionuclides kill cells to which they are in close proximity,
for example neoplastic cells to which the conjugate has attached or
has entered. They have little or no effect on non-localized cells
and are essentially non-immunogenic. Alternatively, high-energy
isotopes may be generated by thermal irradiation of an otherwise
stable isotope, for example as in boron neutron-capture therapy
(Guan et al., PNAS, 95: 13206-10, 1998).
[0254] In one embodiment, the therapeutic agent is selected from
MMAE, MMAF, and PEGS-Dol10.
[0255] Exemplary therapeutic effector moieties include the
structures:
##STR00051## ##STR00052##
In one embodiment, the effector moiety is selected from:
##STR00053## ##STR00054##
[0256] In certain embodiments, the effector moiety contains more
than one therapeutic agent. These multiple therapeutic agents can
be the same or different.
[0257] b) Diagnostic Effector Moieties
[0258] In certain embodiments, the binding polypeptides of the
current disclosure are conjugated to an effector moiety comprising
a diagnostic agent. In one embodiment, the diagnostic agent is a
detectable small molecule label e.g. biotin, fluorophores,
chromophores, spin resonance probes, imaging agents, or
radiolabels. Exemplary fluorophores include fluorescent dyes (e.g.
fluorescein, rhodamine, and the like) and other luminescent
molecules (e.g. luminal). A fluorophore may be
environmentally-sensitive such that its fluorescence changes if it
is located close to one or more residues in the modified binding
polypeptide that undergo structural changes upon binding a
substrate (e.g. dansyl probes). Exemplary radiolabels include small
molecules containing atoms with one or more low sensitivity nuclei
(.sup.13C, .sup.15N, .sup.2H, .sup.125I, .sup.124I, .sup.123I,
.sup.99Tc, .sup.43K, .sup.52Fe, .sup.64Cu, .sup.68Ga, .sup.111In
and the like). The radionuclide can be, for example, a gamma,
photon, or positron-emitting radionuclide with a half-life suitable
to permit activity or detection after the elapsed time between
administration and localization to the imaging site.
[0259] In one embodiment, the diagnostic agent is a polypeptide.
Exemplary diagnostic polypeptides include enzymes with fluorogenic
or chromogenic activity, e.g. the ability to cleave a substrate
which forms a fluorophore or chromophore as a product (i.e.
reporter proteins such as luciferase). Other diagnostic proteins
may have intrinsic fluorogenic or chromogenic activity (e.g.,
green, red, and yellow fluorescent bioluminescent aequorin proteins
from bioluminescent marine organisms) or they may comprise a
protein containing one or more low-energy radioactive nuclei
(.sup.13C, .sup.15N, .sup.2H, .sup.125I, .sup.124I, .sup.123I,
.sup.99Tc, .sup.43K, .sup.25Fe, .sup.64Cu, .sup.68Ga, .sup.111In
and the like).
[0260] With respect to the use of radiolabeled conjugates in
conjunction with the present disclosure, binding polypeptides of
the current disclosure may be directly labeled (such as through
iodination) or may be labeled indirectly through the use of a
chelating agent. As used herein, the phrases "indirect labeling"
and "indirect labeling approach" both mean that a chelating agent
is covalently attached to a binding polypeptide and at least one
radionuclide is associated with the chelating agent. Such chelating
agents are typically referred to as bifunctional chelating agents
as they bind both the polypeptide and the radioisotope. Exemplary
chelating agents comprise 1-isothiocycmatobenzyl-3-methyldiothelene
triaminepentaacetic acid ("MX-DTPA") and cyclohexyl
diethylenetriamine pentaacetic acid ("CHX-DTPA") derivatives. Other
chelating agents comprise P-DOTA and EDTA derivatives. Particularly
radionuclides for indirect labeling include .sup.111In and
.sup.90Y. Most imaging studies utilize 5 mCi .sup.111In-labeled
antibody, because this dose is both safe and has increased imaging
efficiency compared with lower doses, with optimal imaging
occurring at three to six days after antibody administration. See,
for example, Murray, (1985), J. Nuc. Med. 26: 3328 and Carraguillo
et al, (1985), J. Nuc. Med. 26: 67. The radionuclide for direct
labeling can be, for example, .sup.131I. Those skilled in the art
will appreciate that non-radioactive conjugates may also be
assembled depending on the selected agent to be conjugated.
[0261] In certain embodiments, the diagnostic effector moiety is a
FRET (Fluorescence Resonance Energy Transfer) probe. FRET has been
used for a variety of diagnostic applications including cancer
diagnostics. A FRET probe may include a cleavable linker (enzyme
sensitive or pH linker) connecting the donor and acceptor moieties
of the FRET probe, wherein cleavage results in enhanced
fluorescence (including near Infrared) (see, e.g., A. Cobos-Correa
et. al. Membrane-bound FRET probe visualizes MMP12 activity in
pulmonary inflammation, Nature Chemical Biology (2009), 5(9),
628-63; S. Gehrig et. al. Spatially Resolved Monitoring of
Neutrophil Elastase Activity with Ratiometric Fluorescent Reporters
(2012) Angew. Chem. Int. Ed., 51, 6258-6261).
[0262] In one embodiment, the effector moiety is selected from:
##STR00055## ##STR00056##
[0263] c) Functionalized Effector Moieties
[0264] In certain embodiments, effector moieties may be
functionalized to contain additional groups in addition to the
effector moiety itself. For example, the effector moiety may
contain cleavable linkers which release the effector moiety from
the binding polypeptide under particular conditions. In exemplary
embodiments, the effector moiety may include a linker that is
cleavable by cellular enzymes and/or is pH sensitive. Additionally
or alternatively, the effector moiety may contain a disulfide bond
that cleaved by intracellular glutathione upon uptake into the
cell. Exemplary disulfide and pH sensitive linkers are provided
below:
##STR00057##
[0265] In yet other embodiments, the effector moiety may include
hydrophilic and biocompatible moieties such as poly(glycine),
poly(oxazoline), or PEG moieties. Exemplary structures ("Y") are
provided below:
##STR00058##
[0266] In certain embodiments, the effector moiety contains an
aminooxy group which facilitates conjugation to a binding
polypeptide via a stable oxime linkage.
[0267] In other embodiments, the effector moiety contains a
hydrazide and/or N-alkylated hydrazine group to facilitate
conjugation to a binding polypeptide via a stable hydrazone
linkage. Exemplary effector moieties containing aminooxy groups are
set forth in Table 14 herein.
TABLE-US-00002 TABLE 14 Exemplary hydrazine and/or hydrazide
effector moieties ##STR00059## ##STR00060## ##STR00061##
##STR00062##
[0268] d) Targeting Moieties
[0269] In certain embodiments, effector moieties comprise targeting
moieties that specifically bind to one or more target molecules.
Any type of targeting moiety can be employed including, without
limitation, proteins, nucleic acids, lipids, carbohydrates (e.g.,
glycans), and combinations thereof (e.g., glycoproteins,
glycopeptides, and glycolipids). In certain embodiments, the
targeting moiety is a carbohydrate or glycopeptide. In one
embodiment, the targeting moiety is a trivalent glycopeptide (e.g.
a trivalent GalNAc glycan containing glycopeptide or a trivalent
galactose containing glycopeptide). In a specific embodiment, the
trivalent galactose containing polypeptide is lactose3-Cys3Gly4. In
certain embodiments, the targeting moiety is a glycan. Targeting
moieties can be naturally or non-naturally occurring molecules.
Targeting moieties suitable for conjugation may include those
containing aminooxy linkers (see, e.g., FIGS. 45 and 46).
[0270] The targeting moieties described in the present invention
may bind to any type of cell, including animal (e.g., mammalian),
plant, or insect cells either in vitro or in vivo, without
limitation. The cells may be of endodermal, mesodermal, or
ectodermal origins, and may include any cell type. In certain
embodiments, the targeting moiety binds to a cell, e.g., a
mammalian cell, a facilitates delivery of a binding polypeptide to
the targeted cell, e.g., to improve cell-targeting and/or uptake.
Exemplary target cells include, without limitation, immune cells
(e.g., lymphocytes such as B cells, T cells, natural killer (NK)
cells, basophils, macrophages, or dendritic cells), liver cells
(e.g., hepatocytes or non-parenchymal cells such as liver
sinusoidal endothelial cells, Kupffer cells, or hepatic stellate
cells), tumor cells (e.g., any malignant or benign cell including
hepatoma cells, lung cancer cells, sarcoma cells, leukemia cells,
or lymphoma cells), vascular cells (e.g., aortic endothelial cells
or pulmonary artery endothelial cells), epithelial cells (e.g.,
simple squamous epithelial cells, simple columnar epithelial cells,
pseudostratified columnar epithelial cells, or stratified squamous
epithelial cells), or mesenchymal cells (e.g., cells of the
lymphatic and circulatory systems, bone, and cartilage cells).
[0271] In one embodiment, the binding polypeptide is internalized
by the cell. In another embodiment, the amount of the binding
polypeptide internalized by the cell is greater than the amount of
a reference binding polypeptide lacking a targeting moiety
internalized by the cell.
[0272] In one embodiment, the targeting moiety binds to a receptor
on the target cell. For example, the targeting moiety may comprise
a mannose 6 phosphate moiety that binds to a mannose 6 phosphate
receptor on the cell. In other exemplary embodiments, the targeting
moiety binds to a Siglec on a target cell. Exemplary Siglecs
include sialoadhesin (Siglec-1), CD22 (Siglec-2), CD33 (Siglec-3),
MAG (Siglec-4), Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9,
Siglec-10, Siglec-11, Siglec-12, Siglec-14, or Siglec-15. In yet
other embodiments, the targeting moiety comprises an .alpha.2,3-,
.alpha.2,6-, or .alpha.2,8-linked sialic acid residue. In a further
embodiment, the targeting moiety comprises an
.alpha.2,3-siallylactose moiety or an .alpha.2,6-siallylactose
moiety. Other exemplary receptors include lectin receptors,
including but not limited to C-type lectin receptors, galectins,
and L-type lectin receptors. Exemplary lectin receptors include:
TDEC-205, macrophage mannose receptor (MMR), Dectin-1, Dectin-2,
macrophage-inducible C-type lectin (Mincle), dendritic
cell-specific ICAM3-grabbing nonintegrin (DC-SIGN, CD209), DC NK
lectin group receptor-1 (DNGR-1), Langerin (CD207), CD169, a
lectican, an asialoglycoprotein receptor, DCIR, MGL, a DC receptor,
a collectin, a selectin, an NK-cell receptor, a multi-CTLD
endocytic receptor, a Reg group (type VII) lectin, chondrolectin,
tetranectin, polycystin, attractin (ATRN), eosinophil major basic
protein (EMBP), DGCR2, Thrombomodulin, Bimlec, SEEC, and
CBCP/Frem1/QBRICK.
[0273] The binding polypeptides of the present invention may be
used to remove toxic compounds and harmful substances into liver in
multiple diseases by targeting carbohydrate receptors (e.g.,
mannose 6-phosphate receptor, mannose receptor, and
asialoglycoprotein receptor). Please see: Ganesan, L. P. et al:
Rapid and Efficient Clearance of Blood-borne Virus by Liver
Sinusoidal Endothelium. PLoS Pathogens 2011, 9: 1; and Monnier, V.
M. et al: Glucosepane: a poorly understood advanced glycation end
product of growing importance for diabetes and its complications.
Clin Chem Lab Med 2014; 52: 21.
[0274] The binding polypeptides of the present invention may also
be used to target tumor cells through targeting different cell
receptors including, but not limited to: carbohydrate receptors,
Asialoglycoprotein receptor, and Siglecs. Please see: Chen, W. C.
et al: In vivo targeting of B-cell lymphoma with glycan ligands of
CD22. Blood 2010, 115: 4778; Chen, W. C. et al: Targeting B
lymphoma with nanoparticles bearing glycan ligands of CD22. Leuk
Lymphoma 2012, 53: 208; Hatakeyama, S. et al: Targeted drug
delivery to tumor vasculature by a carbohydrate mimetic peptide.
PNAS, 2011, 108: 19587; Hong, F. et al: .beta.-Glucan Functions as
an Adjuvant for Monoclonal Antibody Immunotherapy by Recruiting
Tumoricidal Granulocytes as Killer Cells. Cancer Res. 2003, 23:
9023; Kawasakia, N. et al: Targeted delivery of lipid antigen to
macrophages via the CD169/sialoadhesin endocytic pathway induces
robust invariant natural killer T cell activation. PNAS 2013, 110:
7826; and Medina, S. H. et al: N-acetylgalactosamine-functionalized
dendrimers as hepatic cancer cell-targeted carriers. Biomaterials
2011, 32: 4118.
[0275] The binding peptides of the present invention may also be
used to regulate immune response through various receptors
including, but not limited to, carbohydrate receptors, DC-SIGN, or
Siglecs. Please see: Anthony, R. M. et al: Recapitulation of IVIG
Anti-Inflammatory Activity with a Recombinant IgG Fc. Science 2008,
320: 373; Anthony, R. M. et al: Identification of a receptor
required for the anti-inflammatory activity of IVIG. PNAS 2008,
105: 19571; Kaneko, Y. et al: Anti-Inflammatory Activity of
Immunoglobulin G Resulting from Fc Sialylation. Science 2006, 313:
670; and Manner, J. et al: Exogenous and endogenous glycolipid
antigens activate NKT cells during microbial infections. Nature
2005, 434: 525.
[0276] In one embodiment, the targeting moiety is a glycopeptide.
In a further embodiment, the targeting moiety is a
tri-galactosylated glycopeptide, e.g.,
lactose.sub.3-Cys.sub.3Gly.sub.4 (shown in Formula V, below):
##STR00063##
[0277] e) PEG Moieties
[0278] In other aspects, the effector moiety is a moiety comprising
poly(ethylene glycol) (PEG, PEO, or POE). PEG is an oligomer or
polymer of ethylene oxide and has the chemical structure
H--(O--CH.sub.2--CH.sub.2)n-OH wherein the element in parentheses
is repeated. PEGylation (or pegylation) is a process in which PEG
polymer chains are attached to another molecule (e.g., a binding
polypeptide), which is then described as PEGylated (or pegylated).
PEGylation can serve to reduce immunogenicity and antigenicity as
well as to increase the hydrodynamic size (size in solution) of the
molecule it is attached to, reducing renal clearance and prolonging
circulation time. PEGylation can also make molecules more water
soluble. In one embodiment of the present invention, the PEG moiety
may comprise mono-PEG, bi-PEG, or tri-PEG. In another embodiment,
the PEG moiety comprises 3 to 3.5 PEG.
VII. Conjugation of Effector Moieties to Binding Polypeptides
[0279] In certain embodiments, effector moieties are conjugated
(either directly or through a linker moiety) to an oxidized glycan
(e.g., an oxidized N-linked glycan) of an altered binding
polypeptide, (e.g., an engineered glycan at N114 of an antibody CH1
domain or a native glycan at N297 of an antibody F domain). The
term "oxidized glycan" means that an alcohol substituent on the
glycan has been oxidized, providing a carbonyl substituent. The
carbonyl substituent can react with suitable nitrogen nucleophile
to form a carbon-nitrogen double bond. For example, reaction of the
carbonyl group with an aminooxy group or hydrazine group would form
an oxime or hydrazine, respectively. In one embodiment, the
carbonyl substituent is an aldehyde. Suitable oxidized glycans
include oxidized galactose and oxidized sialic acid.
[0280] In one embodiment, the modified polypeptide of Formula (II)
may be of Formula (II):
Ab(Gal-C(O)H).sub.x(Gal-Sia-C(O)H).sub.y Formula (II),
wherein
[0281] A) Ab is an antibody or other binding polypeptide as defined
herein; [0282] B) Gal is a component derived from galactose; [0283]
C) Sia is a component derived from sialic acid; [0284] D) x is 0 to
5; and [0285] E) y is 0 to 5, [0286] wherein at least one of x and
y is not 0.
[0287] Any art recognized chemistry can be employed to conjugate an
effector moiety (e.g., an effector moiety comprising a linker
moiety) to a glycan (see e.g., Hermanson, G. T., Bioconjugate
Techniques. Academic Press (1996), which is incorporated herein ion
its entirety). In certain embodiments, a saccharide residue (e.g.,
a sialic acid or galactose residue) of the glycan is first oxidized
(e.g., using sodium periodate treatment of sialic acid or galactose
oxidase treatment of galactose) to generate a reactive aldehyde
group. This aldehyde group is reacted with effector moiety an
aminooxy group or hydrazine group to form an oxime or hydrazone
linker, respectively. Exemplary methods employing this general
reaction scheme are set forth in Examples 10 to 15.
[0288] In certain embodiments, the native or engineered glycans of
a binding polypeptide are first pre-treated with
glycosyltransferase enzymes in vitro to provide a terminal
saccharide residue that is suitably reactive. For example,
sialylation may be achieved first using a combination of
galactosyltransferase (Gal T) and sialyltransferase (Sial T). In
certain embodiments, biantennary glycans that lack galactose (G0F
or G0) or that contain only one galactose (G1F or G1) can be
converted to higher-order galactosylated or sialylated structures
suitable for conjugation (G1F, G1, G2F, G2, G1S1F, G1S1, G2S1F,
G2S1, G2S2F, or G2S2).
[0289] An exemplary conjugation scheme for producing sialylated
glycoconjugates is shown in FIG. 30C. An exemplary conjugation
scheme for producing sialylated glycoconjugates is shown in FIG.
30B. Sialic acid residues are introduced enzymatically and site
specifically into the glycan of an antibody (e.g., a native glycan
at Asn-297) using a combination of galactosyltransferase (Gal T)
and sialyltransferase (Sial T). Introduced sialic acid residues are
subsequently oxidized with a low concentration of sodium periodate
to yield reactive sialic acid aldehydes suitably reactive with
linkers (e.g., aminooxy linkers) to generate antibody-effector
moiety conjugates (e.g., oxime-linked antibody-effector moiety
conjugates). By controlling the number of glycan and the number of
sialic residues with in vitro remodeling, the skilled artisan may
have precise control over the drug-antibody ratio (DAR) of the
antibody-effector moiety conjugates. For example, if .about.1
sialic acid is added onto a single biantennary glycan (A1F) in each
of heavy chain, an antibody or binding polypeptide with a DAR of 2
can be homogeneously obtained.
Oxidation and Oxidation Agents
[0290] Oxidation can have adverse effects on the integrity of an
antibody, both through the oxidation of monosaccharides and through
the oxidation of amino acids. The oxidation of methionine residues,
including Met-252 and Met-428 (located in Fc CH.sub.3 region,
proximal to FcRn binding site) is known to affect FcRn binding,
which is critical for prolonging antibody serum half-life (Wang,
W., et al. (2011). Impact of methionine oxidation in human IgG1 Fc
on serum half-life of monoclonal antibodies. Mol Immunol 48,
860-6). Accordingly, attempts have previously been made to reduce
the amount of oxidizing agents (e.g. periodate oxidase or galactose
oxidase) used to treat binding proteins comprising glycans in order
to create oxidized groups for conjugation to effector moieties.
[0291] The method of the present invention uses CMP-sialic acid
derivatives comprising reactive moieties (including, but not
limited to, an aldehyde moiety, an alkyne, an aminooxy moiety, an
azide, a hydrazine, a keto moiety, or a thiol), which may be
reacted with a binding polypeptide in order to form a sialic acid
derivative-conjugated binding protein. These sialic acid
derivative-conjugated binding proteins can then be coupled to
different effector moieties without treatment with an oxidizing
agent.
Imine Chemistry
[0292] In some embodiments, the CMP-sialic acid derivative
comprises a reactive moiety including an aldehyde, a keto, a
hydrazine, or a hydrazone moiety. In some embodiments, the reactive
moiety is a terminal reactive moiety including, but not limited to,
a terminal aldehyde or a terminal keto moiety. In example
embodiments, the CMP-sialic acid derivative has one of the
following structural formulas:
##STR00064##
wherein R.sub.2 includes, but is not limited to, CH.sub.3,
CH.sub.2CH.sub.2(C.dbd.O)CH.sub.3, CH.sub.2OH, OH or H. In some
embodiments, the CMP-sialic acid derivative comprising a terminal
aldehyde moiety includes, but is not limited to, the following
structural formulas:
##STR00065##
[0293] In some embodiments, the sialic acid derivative-conjugated
binding polypeptide comprises a reactive moiety including an
aldehyde, a keto, a hydrazine, or a hydrazone moiety. In example
embodiments, the sialic acid derivative-conjugated binding
polypeptide may be represented by the following:
##STR00066##
wherein X represents the remainder of the sialic acid
derivative-conjugated binding polypeptide (i.e., other than the
reactive moiety).
[0294] In some embodiments, the effector or targeting moiety
comprises a reactive moiety including an aldehyde, a keto, a
hydrazine, or a hydrazone moiety. In example embodiments, the
effector or targeting moiety may be represented by the
following:
##STR00067##
wherein X represents the remainder of the effector or targeting
moiety (i.e., other than the reactive moiety).
[0295] In some embodiments, a targeting or effector moiety
conjugated binding polypeptide comprises an imine. In example
embodiments, a type of imine includes, but is not limited to, an
aldimine, a hydroxylamine, a hydrazone, a ketamine, or an oxime.
For example, see FIGS. 3A-3C for imine formation. In example
embodiments, the imine of a targeting or effector moiety conjugated
binding polypeptide is formed by reacting a sialic acid
derivative-conjugated binding polypeptide comprising an aldehyde or
a keto moiety with an effector or targeting moiety comprising an
aminooxy moiety or is bound to a moiety comprising an aminooxy
derivative or a hydrazine moiety. In example embodiments, the imine
of the targeting or effector moiety conjugated binding polypeptide
is formed by reacting a sialic acid derivative-conjugated binding
polypeptide comprising an aminooxy moiety or is bound to a moiety
comprising an aminooxy derivative or a hydrazine moiety with an
effector or targeting moiety comprising an aldehyde or a keto
moiety.
Click Chemistry
[0296] In some embodiments, the CMP-sialic acid derivative
comprises a terminal azide moiety. For example, the CMP-sialic acid
derivative may be a CMP-sialic acid C5 azide derivative or a sialic
acid C5 azide. In example embodiments, the CMP-sialic acid
derivative has the following structural formula:
##STR00068##
[0297] wherein R.sub.1 is a reactive moiety including, but not
limited to, NH(C.dbd.O)CH.sub.3, NH(C.dbd.O)C.sub.4H.sub.7O,
NH(C.dbd.O)CH.sub.2OH, NH(C.dbd.O)CH.sub.2N.sub.3, NH(C.dbd.O)SH,
OH or N.sub.3. In some embodiments, the CMP-sialic acid derivative
has a structural formula selected from the following:
##STR00069##
[0298] In some embodiments, the CMP-sialic acid derivative
comprises a moiety comprising an alkyne or is bound to a moiety
comprising an alkyne. In some embodiments, the CMP-sialic acid
derivative comprises or is bound to a cyclooctyne including, but
not limited to, an azadibenzocyclooctyne (DBCO, ADIBO, DIBAC)
moiety, a monofluorinated cyclooctyne, or a difluorinated
cyclooctyne.
[0299] In some embodiments, the sialic acid derivative-conjugated
binding polypeptide comprises a terminal azide moiety. For example,
the sialic acid derivative-conjugated binding polypeptide may be a
sialic acid C5 azide derivative-conjugated binding polypeptide. In
one exemplary embodiment, the sialic acid derivative-conjugated
binding polypeptide has the structural formulas represented in FIG.
95. In another example embodiment, the sialic acid
derivative-conjugated binding polypeptide has one of the following
structural formulas
##STR00070##
wherein X represents the remainder of the sialic
acid-derivative-conjugated binding polypeptide (i.e., other than
the terminal azide moiety).
[0300] In some embodiments, the sialic acid derivative-conjugated
binding polypeptide comprises a moiety comprising an alkyne or is
bound to a moiety comprising an alkyne. In some embodiments, the
sialic acid derivative-conjugated binding polypeptide comprises or
is bound to a cyclooctyne including, but not limited to, an
azadibenzocyclooctyne (e.g., DBCO, ADIBO, DIBAC) moiety, a
monoflorimated cyclooctyne, or a difluorinated cyclooctyne.
[0301] In some embodiments, the effector or targeting moiety
comprises an alkyne or is bound to a moiety comprising an alkyne.
In some embodiments, the effector or targeting moiety comprises or
is bound to a cyclooctyne including, but not limited to, an
azadibenzocyclooctyne (e.g., DBCO, ADIBO, DIBAC) moiety, a
monoflorimated cyclooctyne, or a difluorinated cyclooctyne. In
example embodiments, the effector or targeting moiety is bound to a
moiety comprising an alkyne and can be represented by the following
structural formula:
##STR00071##
In some embodiments, the effector or targeting moiety comprises a
terminal azide moiety.
[0302] In some embodiments, a targeting or effector moiety
conjugated binding polypeptide comprises a triazole ring. In
example embodiments, the triazole ring of a targeting or effector
moiety conjugated binding polypeptide is formed by reacting a
sialic acid derivative-conjugated binding polypeptide comprising a
terminal azide moiety with an effector or targeting moiety
comprising an alkyne or bound to a moiety comprising an alkyne
using click chemistry. In example embodiments, the triazole ring of
the targeting or effector moiety conjugated binding polypeptide is
formed by reacting a sialic acid derivative-conjugated binding
polypeptide comprising an alkyne or bound to a moiety comprising an
alkyne with an effector or targeting moiety comprising a terminal
azide moiety using click chemistry. In some embodiments, the click
chemistry reaction to form the targeting or effector moiety
conjugated binding polypeptide occurs at ambient temperatures. In
some embodiments, the click chemistry reaction to form a targeting
or effector moiety conjugated binding polypeptide occurs in the
presence of a metal catalyst, for example, a copper(I)-catalyzed
azide-alkyne cycloaddition. In some embodiments, the click
chemistry reaction to form a targeting or effector moiety
conjugated binding polypeptide is performed in the absence of
copper.
[0303] In some embodiments, the mechanism of a click chemistry
reaction to form a targeting or effector moiety conjugated binding
polypeptide includes, but is not limited to, a copper(I)-catalyzed
[3+2] azide-alkyne cycloaddition, a strain-promoted [3+2]
azide-alkyne cycloaddition, a [3+2] Huisgen cycloaddition between
an azide moiety and an activated alkyne, a [3+2] cycloaddition
between an azide moiety and an electron-deficient alkyne, a [3+2]
cycloaddition between an azide and an aryne, a Diels-Alder
retro-[4+2] cycloaddition between a tetrazine and an alkene, or a
radical addition between a thiol and an alkene.
Thioether Chemistry
[0304] In some embodiments, the CMP-sialic acid derivative or
sialic acid derivative comprises a reactive moiety including a
thiol or a maleimide moiety. In some embodiments, the CMP-sialic
acid derivative or sialic acid derivative comprises a terminal
thiol. In example embodiments, the CMP-sialic acid derivative
comprising a terminal thiol includes, but is not limited to a
structural formula of:
##STR00072##
[0305] In some embodiments, the sialic acid derivative-conjugated
binding polypeptide comprises a reactive moiety including a thiol
or a maleimide moiety. In some embodiments, the sialic acid
derivative-conjugated binding polypeptide comprises a terminal
thiol moiety. In example embodiments, the sialic acid
derivative-conjugated binding polypeptide may be represented by,
but is not limited to, the following:
##STR00073##
wherein X is the remainder of the sialic acid derivative-conjugated
binding polypeptide (i.e., other than the thiol or maleimide
moiety).
[0306] In some embodiments, the effector or targeting moiety
comprises a reactive moiety including a thiol or a maleimide
moiety. In some embodiments, the effector or targeting moiety
comprises a terminal thiol moiety. In some embodiments, the
effector or targeting moiety comprises a terminal maleimide moiety.
For example, the effector or targeting moiety comprising a
maleimide moiety includes, but is not limited to,
bis-mannose-6-phosphate hexamannose maleimide, or lactose
maleimide.
[0307] In example embodiments, the effector or targeting moiety may
be represented by, but is not limited to, the following:
##STR00074##
wherein X represents the remainder of the effector or targeting
moiety.
[0308] In some embodiments, a targeting or effector moiety
conjugated binding polypeptide comprises a thioether. In example
embodiments, the thioether of a targeting or effector moiety
conjugated binding polypeptide is formed by reacting a sialic acid
derivative-conjugated binding polypeptide comprising a thiol moiety
with an effector or targeting moiety comprising a maleimide moiety.
In example embodiments, the imine of the targeting or effector
moiety conjugated binding polypeptide is formed by reacting a
sialic acid derivative-conjugated binding polypeptide comprising a
maleimide moiety with an effector or targeting moiety comprising a
thiol moiety.
VIII. Modified Binding Polypeptides
[0309] In certain embodiments, the invention provides modified
polypeptides which are the product of the conjugating effector
moieties are conjugated (either directly or through a linker
moiety) to an oxidized glycan (e.g., an oxidized N-linked glycan)
of an altered binding polypeptide (e.g., an engineered glycan at
N114 of an antibody CH1 domain or a native glycan at N297 of an
antibody F domain).
[0310] In one embodiment, the binding polypeptide can be of Formula
(III):
Ab(Gal-C(H).dbd.N-Q-CON-X).sub.x(Gal-Sia-C(H).dbd.N-Q-CON-X).sub.y
Formula (III),
wherein:
[0311] A) Ab is an antibody as defined herein;
[0312] B) Q is NH or O;
[0313] C) CON is a connector moiety as defined herein; and
[0314] D) X is a targeting moiety as defined herein;
[0315] E) Gal is a component derived from galactose;
[0316] F) Sia is a component derived from sialic acid;
[0317] G) x is 0 to 5; and
[0318] H) y is 0 to 5, [0319] wherein at least one of x and y is
not 0.
[0320] In one embodiment, the binding polypeptide can be of Formula
(III) can be of Formula (Ma):
Ab(Gal-C(H).dbd.N-Q-CH.sub.2--C(O)--Z--X).sub.x(Gal-Sia-C(H).dbd.N-Q-CH.-
sub.2--C(O)--Z--X).sub.y Formula (IIIa),
wherein:
[0321] A) Ab is an antibody;
[0322] B) Q is NH or O;
[0323] C) Z is
Cys-(MC).sub.a-(VC).sub.b-(PABC).sub.c-(C.sub.16H.sub.32O.sub.8
C.sub.2H.sub.4).sub.f--, wherein [0324] i. Cys is a component
derived cysteinamide; [0325] ii. MC is a component derived from
maleimide; [0326] iii. VC is a component derived from valine
coupled with citruline; [0327] iv. PABC is a component derived from
4-aminobenzyl carbamate; [0328] v. X is an effector moiety (e.g., a
targeting moiety as defined herein); [0329] vi. a is 0 or 1; [0330]
vii. b is 0 or 1; [0331] viii. c is 0 or 1; and [0332] ix. f is 0
or 1;
[0333] D) X is a therapeutic agent as defined herein;
[0334] E) Gal is a component derived from galactose;
[0335] F) Sia is a component derived from sialic acid;
[0336] G) x is 0 to 5; and
[0337] H) y is 0 to 5, [0338] wherein at least one of x and y is
not 0.
[0339] It is to be understood that the Formula (III) is not
intended to imply that the antibody, the Gal substituent, and the
Gal-Sia substituent are connected in a chain-like manner. Rather,
when such substituents are present, the antibody is connected
directly connected to each substituent. For example, a binding
polypeptide of Formula (III) in which x is 1 and y is 2 could have
the arrangement shown below:
##STR00075##
[0340] The CON substituent in Formula (III) and components therein
are as described with regard to Formula (I) for effector
moieties.
[0341] In one embodiment, Q is NH. In another embodiment, Q is
O.
[0342] In one embodiment, x is 0.
[0343] The antibody Ab of Formula (III) may be any suitable
antibody as described herein.
[0344] In one embodiment, there is provided a method for preparing
the binding polypeptide of Formula (III), the method comprising
reacting an effector moiety of Formula (I):
NH.sub.2-Q-CON-X Formula (I),
wherein:
[0345] A) Q is NH or O;
[0346] B) CON is a connector moiety; and
[0347] C) X is an effector moiety (e.g., a targeting moiety as
defined herein),
with a modified antibody of Formula (II)
Ab(OXG).sub.r Formula (II)
wherein
[0348] A) OXG is an oxidized glycan; and
[0349] B) r is selected from 0 to 4;
[0350] In one embodiment, there is provided a method for preparing
the binding polypeptide of Formula (III), the method comprising
reacting an effector moiety of Formula (I):
NH.sub.2-Q-CON-X Formula (I),
wherein:
[0351] A) Q is NH or O;
[0352] B) CON is a connector moiety; and
[0353] C) X is an effector moiety (e.g., a targeting moiety as
defined herein),
with a modified antibody of Formula (IIa)
Ab(Gal-C(O)H).sub.x(Gal-Sia-C(O)H).sub.y Formula (IIa),
wherein
[0354] A) Ab is an antibody as described herein;
[0355] B) Gal is a component derived from galactose;
[0356] C) Sia is a component derived from sialic acid;
[0357] D) x is 0 to 5; and
[0358] E) y is 0 to 5, [0359] wherein at least one of x and y is
not 0.
IX. Methods of Treatment with Modified Antibodies
[0360] In one aspect, the invention provides methods of treating or
diagnosing a patient in need thereof comprising administering an
effective amount a binding polypeptide disclosed herein. In some
embodiments, the present invention includes kits and methods for
the diagnosis and/or treatment of disorders, e.g., neoplastic
disorders in a mammalian subject in need of such treatment. In some
embodiments, the subject is a human.
[0361] The binding polypeptides of the current disclosure are
useful in a number of different applications. For example, in one
embodiment, the subject binding polypeptide s are useful for
reducing or eliminating cells bearing an epitope recognized by the
binding domain of the binding polypeptide. In another embodiment,
the subject binding polypeptides are effective in reducing the
concentration of or eliminating soluble antigen in the circulation.
In one embodiment, the binding polypeptides may reduce tumor size,
inhibit tumor growth and/or prolong the survival time of
tumor-bearing animals. Accordingly, this disclosure also relates to
a method of treating tumors in a human or other animal by
administering to such human or animal an effective, non-toxic
amount of modified antibody. One skilled in the art would be able,
by routine experimentation, to determine what an effective,
non-toxic amount of modified binding polypeptide would be for the
purpose of treating malignancies. For example, a therapeutically
active amount of a modified antibody or fragments thereof may vary
according to factors such as the disease stage (e.g., stage I
versus stage IV), age, sex, medical complications (e.g.,
immunosuppressed conditions or diseases) and weight of the subject,
and the ability of the modified antibody to elicit a desired
response in the subject.
[0362] The dosage regimen may be adjusted to provide the optimum
therapeutic response. For example, several divided doses may be
administered daily, or the dose may be proportionally reduced as
indicated by the exigencies of the therapeutic situation.
[0363] In general, the compositions provided in the current
disclosure may be used to prophylactically or therapeutically treat
any neoplasm comprising an antigenic marker that allows for the
targeting of the cancerous cells by the modified antibody.
X. Methods of Administering Modified Antibodies or Fragments
Thereof
[0364] Methods of preparing and administering binding polypeptides
of the current disclosure to a subject are well known to or are
readily determined by those skilled in the art. The route of
administration of the binding polypeptides of the current
disclosure may be oral, parenteral, by inhalation or topical. The
term parenteral as used herein includes intravenous, intraarterial,
intraperitoneal, intramuscular, subcutaneous, rectal or vaginal
administration. While all these forms of administration are clearly
contemplated as being within the scope of the current disclosure, a
form for administration would be a solution for injection, in
particular for intravenous or intraarterial injection or drip.
Usually, a suitable pharmaceutical composition for injection may
comprise a buffer (e.g. acetate, phosphate or citrate buffer), a
surfactant (e.g. polysorbate), optionally a stabilizer agent (e.g.
human albumin), etc. However, in other methods compatible with the
teachings herein, the modified antibodies can be delivered directly
to the site of the adverse cellular population thereby increasing
the exposure of the diseased tissue to the therapeutic agent.
[0365] In one embodiment, the binding polypeptide that is
administered is a binding polypeptide of Formula (III):
Ab(Gal-C(H).dbd.N-Q-CON-X).sub.x(Gal-Sia-C(H).dbd.N-Q-CON-X).sub.y
Formula (III),
wherein:
[0366] A) Ab is an antibody as defined herein;
[0367] B) Q is NH or O;
[0368] C) CON is a connector moiety as defined herein; and
[0369] D) X is an effector moiety (e.g., a targeting moiety as
defined herein);
[0370] E) Gal is a component derived from galactose;
[0371] F) Sia is a component derived from sialic acid;
[0372] G) x is 0 to 5; and
[0373] H) y is 0 to 5, [0374] wherein at least one of x and y is
not 0.
[0375] 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. In the compositions and methods of the
current disclosure, pharmaceutically acceptable carriers include,
but are not limited to, 0.01-0.1 M or 0.05M phosphate buffer, or
0.8% saline. Other common parenteral vehicles include sodium
phosphate solutions, 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, antioxidants, chelating agents, and inert
gases and the like. More particularly, pharmaceutical compositions
suitable for injectable use include sterile aqueous solutions
(where water soluble) or dispersions and sterile powders for the
extemporaneous preparation of sterile injectable solutions or
dispersions. In such cases, the composition must be sterile and
should be fluid to the extent that easy syringability exists. It
should be stable under the conditions of manufacture and storage,
and should also be preserved against the contaminating action of
microorganisms, such as bacteria and fungi. The carrier can be a
solvent or dispersion medium containing, for example, water,
ethanol, polyol (e.g., glycerol, propylene glycol, and liquid
polyethylene glycol, and the like), and suitable mixtures thereof.
The proper fluidity can be maintained, for example, by the use of a
coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of
surfactants.
[0376] Prevention of the action of microorganisms can be achieved
by various antibacterial and antifungal agents, for example,
parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the
like. Isotonic agents, for example, sugars, polyalcohols, such as
mannitol, sorbitol, or sodium chloride may also be included in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0377] In any case, sterile injectable solutions can be prepared by
incorporating an active compound (e.g., a modified binding
polypeptide by itself or in combination with other active agents)
in the required amount in an appropriate solvent with one or a
combination of ingredients enumerated herein, as required, followed
by filtered sterilization. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle, which
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions,
methods of preparation typically include vacuum drying and
freeze-drying, which yield a powder of an active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof. The preparations for injections
are processed, filled into containers such as ampoules, bags,
bottles, syringes or vials, and sealed under aseptic conditions
according to methods known in the art. Further, the preparations
may be packaged and sold in the form of a kit such as those
described in co-pending U.S. Ser. No. 09/259,337 and U.S. Ser. No.
09/259,338 each of which is incorporated herein by reference. Such
articles of manufacture can include labels or package inserts
indicating that the associated compositions are useful for treating
a subject suffering from, or predisposed to autoimmune or
neoplastic disorders.
[0378] Effective doses of the compositions of the present
disclosure, for the treatment of the above described conditions
vary depending upon many different factors, including means of
administration, target site, physiological state of the patient,
whether the patient is human or an animal, other medications
administered, and whether treatment is prophylactic or therapeutic.
Usually, the patient is a human but non-human mammals including
transgenic mammals can also be treated. Treatment dosages may be
titrated using routine methods known to those of skill in the art
to optimize safety and efficacy.
[0379] For passive immunization with a binding polypeptide, the
dosage can range, e.g., from about 0.0001 to 100 mg/kg, and more
usually 0.01 to 5 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg,
0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), of the host body weight. For
example dosages can be 1 mg/kg body weight or 10 mg/kg body weight
or within the range of 1-10 mg/kg, e.g., at least 1 mg/kg. Doses
intermediate in the above ranges are also intended to be within the
scope of the current disclosure. Subjects can be administered such
doses daily, on alternative days, weekly or according to any other
schedule determined by empirical analysis. An exemplary treatment
entails administration in multiple dosages over a prolonged period,
for example, of at least six months. Additional exemplary treatment
regimens entail administration once per every two weeks or once a
month or once every 3 to 6 months. Exemplary dosage schedules
include 1-10 mg/kg or 15 mg/kg on consecutive days, 30 mg/kg on
alternate days or 60 mg/kg weekly. In some methods, two or more
monoclonal antibodies with different binding specificities are
administered simultaneously, in which case the dosage of each
antibody administered falls within the ranges indicated.
[0380] Binding polypeptides of the current disclosure can be
administered on multiple occasions. Intervals between single
dosages can be weekly, monthly or yearly. Intervals can also be
irregular as indicated by measuring blood levels of modified
binding polypeptide or antigen in the patient. In some methods,
dosage is adjusted to achieve a plasma modified binding polypeptide
concentration of 1-1000 .mu.g/ml and in some methods 25-300
.mu.g/ml. Alternatively, binding polypeptides can be administered
as a sustained release formulation, in which case less frequent
administration is required. For antibodies, dosage and frequency
vary depending on the half-life of the antibody in the patient. In
general, humanized antibodies show the longest half-life, followed
by chimeric antibodies and nonhuman antibodies.
[0381] The dosage and frequency of administration can vary
depending on whether the treatment is prophylactic or therapeutic.
In prophylactic applications, compositions containing the present
antibodies or a cocktail thereof are administered to a patient not
already in the disease state to enhance the patient's resistance.
Such an amount is defined to be a "prophylactic effective dose." In
this use, the precise amounts again depend upon the patient's state
of health and general immunity, but generally range from 0.1 to 25
mg per dose, especially 0.5 to 2.5 mg per dose. A relatively low
dosage is administered at relatively infrequent intervals over a
long period of time. Some patients continue to receive treatment
for the rest of their lives. In therapeutic applications, a
relatively high dosage (e.g., from about 1 to 400 mg/kg of antibody
per dose, with dosages of from 5 to 25 mg being more commonly used
for radioimmunoconjugates and higher doses for cytotoxin-drug
modified antibodies) at relatively short intervals is sometimes
required until progression of the disease is reduced or terminated,
or until the patient shows partial or complete amelioration of
disease symptoms. Thereafter, the patient can be administered a
prophylactic regime.
[0382] Binding polypeptides of the current disclosure can
optionally be administered in combination with other agents that
are effective in treating the disorder or condition in need of
treatment (e.g., prophylactic or therapeutic). Effective single
treatment dosages (i.e., therapeutically effective amounts) of
.sup.90Y-labeled modified antibodies of the current disclosure
range from between about 5 and about 75 mCi, such as between about
10 and about 40 mCi. Effective single treatment non-marrow ablative
dosages of .sup.131I-modified antibodies range from between about 5
and about 70 mCi, such as between about 5 and about 40 mCi.
Effective single treatment ablative dosages (i.e., may require
autologous bone marrow transplantation) of .sup.131I-labeled
antibodies range from between about 30 and about 600 mCi, such as
between about 50 and less than about 500 mCi. In conjunction with a
chimeric antibody, owing to the longer circulating half-life
vis-a-vis murine antibodies, an effective single treatment
non-marrow ablative dosages of iodine-131 labeled chimeric
antibodies range from between about 5 and about 40 mCi, e.g., less
than about 30 mCi. Imaging criteria for, e.g., the .sup.111In
label, are typically less than about 5 mCi.
[0383] While the binding polypeptides may be administered as
described immediately above, it must be emphasized that in other
embodiments binding polypeptides may be administered to otherwise
healthy patients as a first line therapy. In such embodiments the
binding polypeptides may be administered to patients having normal
or average red marrow reserves and/or to patients that have not,
and are not, undergoing one or more other therapies. As used
herein, the administration of modified antibodies or fragments
thereof in conjunction or combination with an adjunct therapy means
the sequential, simultaneous, coextensive, concurrent, concomitant,
or contemporaneous administration or application of the therapy and
the disclosed antibodies. Those skilled in the art will appreciate
that the administration or application of the various components of
the combined therapeutic regimen may be timed to enhance the
overall effectiveness of the treatment. For example,
chemotherapeutic agents could be administered in standard, well
known courses of treatment followed within a few weeks by
radioimmunoconjugates of the present disclosure. Conversely,
cytotoxin associated binding polypeptides could be administered
intravenously followed by tumor localized external beam radiation.
In yet other embodiments, the modified binding polypeptide may be
administered concurrently with one or more selected
chemotherapeutic agents in a single office visit. A skilled artisan
(e.g. an experienced oncologist) would be readily be able to
discern effective combined therapeutic regimens without undue
experimentation based on the selected adjunct therapy and the
teachings of the instant specification.
[0384] In this regard it will be appreciated that the combination
of the binding polypeptides and the chemotherapeutic agent may be
administered in any order and within any time frame that provides a
therapeutic benefit to the patient. That is, the chemotherapeutic
agent and binding polypeptides may be administered in any order or
concurrently. In selected embodiments the binding polypeptides of
the present disclosure will be administered to patients that have
previously undergone chemotherapy. In yet other embodiments, the
binding polypeptides and the chemotherapeutic treatment will be
administered substantially simultaneously or concurrently. For
example, the patient may be given the binding polypeptides while
undergoing a course of chemotherapy. In some embodiments the
modified antibody will be administered within one year of any
chemotherapeutic agent or treatment. In other embodiments the
binding polypeptides will be administered within 10, 8, 6, 4, or 2
months of any chemotherapeutic agent or treatment. In still other
embodiments the binding polypeptide will be administered within 4,
3, 2, or 1 week(s) of any chemotherapeutic agent or treatment. In
yet other embodiments the binding polypeptides will be administered
within 5, 4, 3, 2, or 1 day(s) of the selected chemotherapeutic
agent or treatment. It will further be appreciated that the two
agents or treatments may be administered to the patient within a
matter of hours or minutes (i.e. substantially simultaneously).
[0385] It will further be appreciated that the binding polypeptides
of the current disclosure may be used in conjunction or combination
with any chemotherapeutic agent or agents (e.g. to provide a
combined therapeutic regimen) that eliminates, reduces, inhibits or
controls the growth of neoplastic cells in vivo. Exemplary
chemotherapeutic agents that are compatible with the current
disclosure include alkylating agents, vinca alkaloids (e.g.,
vincristine and vinblastine), procarbazine, methotrexate and
prednisone. The four-drug combination MOPP (mechlethamine (nitrogen
mustard), vincristine (Oncovin), procarbazine and prednisone) is
very effective in treating various types of lymphoma, and can be
used in certain embodiments. In MOPP-resistant patients, ABVD
(e.g., adriamycin, bleomycin, vinblastine and dacarbazine), ChIVPP
(CH1orambucil, vinblastine, procarbazine and prednisone), CABS
(lomustine, doxorubicin, bleomycin and streptozotocin), MOPP plus
ABVD, MOPP plus ABV (doxorubicin, bleomycin and vinblastine) or
BCVPP (carmustine, cyclophosphamide, vinblastine, procarbazine and
prednisone) combinations can also be used. Arnold S. Freedman and
Lee M. Nadler, Malignant Lymphomas, in HARRISON'S PRINCIPLES OF
INTERNAL MEDICINE 1774-1788 (Kurt J. Isselbacher et al, eds., 13th
ed. 1994) and V. T. DeVita et al, (1997) and the references cited
therein for standard dosing and scheduling. These therapies can be
used unchanged, or altered as needed for a particular patient, in
combination with one or more binding polypeptides of the current
disclosure as described herein.
[0386] Additional regimens that are useful in the context of the
present disclosure include use of single alkylating agents such as
cyclophosphamide or chlorambucil, or combinations such as CVP
(cyclophosphamide, vincristine and prednisone), CHOP (CVP and
doxorubicin), C-MOPP (cyclophosphamide, vincristine, prednisone and
procarbazine), CAP-BOP (CHOP plus procarbazine and bleomycin),
m-BACOD (CHOP plus methotrexate, bleomycin and leucovorin),
ProMACE-MOPP (prednisone, methotrexate, doxorubicin,
cyclophosphamide, etoposide and leucovorin plus standard MOPP),
ProMACE-CytaBOM (prednisone, doxorubicin, cyclophosphamide,
etoposide, cytarabine, bleomycin, vincristine, methotrexate and
leucovorin) and MACOP-B (methotrexate, doxorubicin,
cyclophosphamide, vincristine, fixed dose prednisone, bleomycin and
leucovorin). Those skilled in the art will readily be able to
determine standard dosages and scheduling for each of these
regimens. CHOP has also been combined with bleomycin, methotrexate,
procarbazine, nitrogen mustard, cytosine arabinoside and etoposide.
Other compatible chemotherapeutic agents include, but are not
limited to, 2-Chlorodeoxyadenosine (2-CDA), 2'-deoxycoformycin and
fludarabine.
[0387] For patients with intermediate- and high-grade NHL, who fail
to achieve remission or relapse, salvage therapy is used. Salvage
therapies employ drugs such as cytosine arabinoside, carboplatin,
cisplatin, etoposide and ifosfamide given alone or in combination.
In relapsed or aggressive forms of certain neoplastic disorders the
following protocols are often used: IMVP-16 (ifosfamide,
methotrexate and etoposide), MIME (methyl-gag, ifosfamide,
methotrexate and etoposide), DHAP (dexamethasone, high dose
cytarabine and cisplatin), ESHAP (etoposide, methylpredisolone, HD
cytarabine, cisplatin), CEPP(B) (cyclophosphamide, etoposide,
procarbazine, prednisone and bleomycin) and CAMP (lomustine,
mitoxantrone, cytarabine and prednisone) each with well-known
dosing rates and schedules.
[0388] The amount of chemotherapeutic agent to be used in
combination with the modified antibodies of the current disclosure
may vary by subject or may be administered according to what is
known in the art. See for example, Bruce A Chabner et al,
Antineoplastic Agents, in GOODMAN & GILMAN'S THE
PHARMACOLOGICAL BASIS OF THERAPEUTICS 1233-1287 (Joel G. Hardman et
al, eds., 9th ed. 1996).
[0389] As previously discussed, the binding polypeptides of the
present disclosure, immunoreactive fragments or recombinants
thereof may be administered in a pharmaceutically effective amount
for the in vivo treatment of mammalian disorders. In this regard,
it will be appreciated that the disclosed binding polypeptides will
be formulated to facilitate administration and promote stability of
the active agent.
[0390] Pharmaceutical compositions in accordance with the present
disclosure typically include a pharmaceutically acceptable,
non-toxic, sterile carrier such as physiological saline, nontoxic
buffers, preservatives and the like. For the purposes of the
instant application, a pharmaceutically effective amount of the
modified binding polypeptide, immunoreactive fragment or
recombinant thereof, conjugated or unconjugated to a therapeutic
agent, shall be held to mean an amount sufficient to achieve
effective binding to an antigen and to achieve a benefit, e.g., to
ameliorate symptoms of a disease or disorder or to detect a
substance or a cell. In the case of tumor cells, the modified
binding polypeptide will typically be capable of interacting with
selected immunoreactive antigens on neoplastic or immunoreactive
cells and provide for an increase in the death of those cells. Of
course, the pharmaceutical compositions of the present disclosure
may be administered in single or multiple doses to provide for a
pharmaceutically effective amount of the modified binding
polypeptide.
[0391] In keeping with the scope of the present disclosure, the
binding polypeptides of the disclosure may be administered to a
human or other animal in accordance with the aforementioned methods
of treatment in an amount sufficient to produce a therapeutic or
prophylactic effect. The binding polypeptides of the disclosure can
be administered to such human or other animal in a conventional
dosage form prepared by combining the antibody of the disclosure
with a conventional pharmaceutically acceptable carrier or diluent
according to known techniques. It will be recognized by one of
skill in the art that the form and character of the
pharmaceutically acceptable carrier or diluent is dictated by the
amount of active ingredient with which it is to be combined, the
route of administration and other well-known variables. Those
skilled in the art will further appreciate that a cocktail
comprising one or more species of binding polypeptides described in
the current disclosure may prove to be particularly effective.
XI. Expression of Binding Polypeptides
[0392] In one aspect, the invention provides polynucleotides
encoding the binding polypeptides disclosed herein. A method of
making a binding polypeptide comprising expressing these
polynucleotides are also provided.
[0393] Polynucleotides encoding the binding polypeptides disclosed
herein are typically inserted in an expression vector for
introduction into host cells that may be used to produce the
desired quantity of the claimed antibodies, or fragments thereof.
Accordingly, in certain aspects, the invention provides expression
vectors comprising polynucleotides disclosed herein and host cells
comprising these vectors and polynucleotides.
[0394] The term "vector" or "expression vector" is used herein for
the purposes of the specification and claims, to mean vectors used
in accordance with the present invention as a vehicle for
introducing into and expressing a desired gene in a cell. As known
to those skilled in the art, such vectors may easily be selected
from the group consisting of plasmids, phages, viruses and
retroviruses. In general, vectors compatible with the instant
invention will comprise a selection marker, appropriate restriction
sites to facilitate cloning of the desired gene and the ability to
enter and/or replicate in eukaryotic or prokaryotic cells.
[0395] Numerous expression vector systems may be employed for the
purposes of this invention. For example, one class of vector
utilizes DNA elements which are derived from animal viruses such as
bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus,
baculovirus, retroviruses (RSV, MMTV or MOMLV), or SV40 virus.
Others involve the use of polycistronic systems with internal
ribosome binding sites. Additionally, cells which have integrated
the DNA into their chromosomes may be selected by introducing one
or more markers which allow selection of transfected host cells.
The marker may provide for prototrophy to an auxotrophic host,
biocide resistance (e.g., antibiotics) or resistance to heavy
metals such as copper. The selectable marker gene can either be
directly linked to the DNA sequences to be expressed, or introduced
into the same cell by cotransformation. Additional elements may
also be needed for optimal synthesis of mRNA. These elements may
include signal sequences, splice signals, as well as
transcriptional promoters, enhancers, and termination signals. In
some embodiments the cloned variable region genes are inserted into
an expression vector along with the heavy and light chain constant
region genes (e.g., human constant region genes) syntheticized as
discussed above.
[0396] In other embodiments, the binding polypeptides may be
expressed using polycistronic constructs. In such expression
systems, multiple gene products of interest such as heavy and light
chains of antibodies may be produced from a single polycistronic
construct. These systems advantageously use an internal ribosome
entry site (IRES) to provide relatively high levels of polypeptides
in eukaryotic host cells. Compatible IRES sequences are disclosed
in U.S. Pat. No. 6,193,980 which is incorporated by reference
herein. Those skilled in the art will appreciate that such
expression systems may be used to effectively produce the full
range of polypeptides disclosed in the instant application.
[0397] More generally, once a vector or DNA sequence encoding an
antibody, or fragment thereof, has been prepared, the expression
vector may be introduced into an appropriate host cell. That is,
the host cells may be transformed. Introduction of the plasmid into
the host cell can be accomplished by various techniques well known
to those of skill in the art. These include, but are not limited
to, transfection (including electrophoresis and electroporation),
protoplast fusion, calcium phosphate precipitation, cell fusion
with enveloped DNA, microinjection, and infection with intact
virus. See, Ridgway, A. A. G. "Mammalian Expression Vectors"
Chapter 24.2, pp. 470-472 Vectors, Rodriguez and Denhardt, Eds.
(Butterworths, Boston, Mass. 1988). Plasmid introduction into the
host can be by electroporation. The transformed cells are grown
under conditions appropriate to the production of the light chains
and heavy chains, and assayed for heavy and/or light chain protein
synthesis. Exemplary assay techniques include enzyme-linked
immunosorbent assay (ELISA), radioimmunoassay (RIA), or
flourescence-activated cell sorter analysis (FACS),
immunohistochemistry and the like.
[0398] As used herein, the term "transformation" shall be used in a
broad sense to refer to the introduction of DNA into a recipient
host cell that changes the genotype and consequently results in a
change in the recipient cell.
[0399] Along those same lines, "host cells" refers to cells that
have been transformed with vectors constructed using recombinant
DNA techniques and encoding at least one heterologous gene. In
descriptions of processes for isolation of polypeptides from
recombinant hosts, the terms "cell" and "cell culture" are used
interchangeably to denote the source of antibody unless it is
clearly specified otherwise. In other words, recovery of
polypeptide from the "cells" may mean either from spun down whole
cells, or from the cell culture containing both the medium and the
suspended cells.
[0400] In one embodiment, the host cell line used for antibody
expression is of mammalian origin; those skilled in the art can
determine particular host cell lines which are best suited for the
desired gene product to be expressed therein. Exemplary host cell
lines include, but are not limited to, DG44 and DUXB11 (Chinese
Hamster Ovary lines, DHFR minus), HELA (human cervical carcinoma),
CVI (monkey kidney line), COS (a derivative of CVI with SV40 T
antigen), R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse
fibroblast), HAK (hamster kidney line), SP2/O (mouse myeloma),
BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocyte), 293
(human kidney). In one embodiment, the cell line provides for
altered glycosylation, e.g., afucosylation, of the
antibodyexpressed therefrom (e.g., PER.C6.RTM. (Crucell) or
FUT8-knock-out CHO cell lines (Potelligent.RTM. Cells) (Biowa,
Princeton, N.J.)). In one embodiment NSO cells may be used. CHO
cells are particularly useful. Host cell lines are typically
available from commercial services, the American Tissue Culture
Collection or from published literature.
[0401] In vitro production allows scale-up to give large amounts of
the desired polypeptides. Techniques for mammalian cell cultivation
under tissue culture conditions are known in the art and include
homogeneous suspension culture, e.g. in an airlift reactor or in a
continuous stirrer reactor, or immobilized or entrapped cell
culture, e.g. in hollow fibers, microcapsules, on agarose
microbeads or ceramic cartridges. If necessary and/or desired, the
solutions of polypeptides can be purified by the customary
chromatography methods, for example gel filtration, ion-exchange
chromatography, chromatography over DEAE-cellulose and/or (immuno-)
affinity chromatography.
[0402] Genes encoding the binding polypeptides featured in the
invention can also be expressed non-mammalian cells such as
bacteria or yeast or plant cells. In this regard it will be
appreciated that various unicellular non-mammalian microorganisms
such as bacteria can also be transformed; i.e. those capable of
being grown in cultures or fermentation. Bacteria, which are
susceptible to transformation, include members of the
enterobacteriaceae, such as strains of Escherichia coli or
Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus;
Streptococcus, and Haemophilus influenzae. It will further be
appreciated that, when expressed in bacteria, the polypeptides can
become part of inclusion bodies. The polypeptides must be isolated,
purified and then assembled into functional molecules.
[0403] In addition to prokaryotes, eukaryotic microbes may also be
used. Saccharomyces cerevisiae, or common baker's yeast, is the
most commonly used among eukaryotic microorganisms although a
number of other strains are commonly available. For expression in
Saccharomyces, the plasmid YRp7, for example, (Stinchcomb et al.,
Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979);
Tschemper et al., Gene, 10:157 (1980)) is commonly used. This
plasmid already contains the TRP1 gene which provides a selection
marker for a mutant strain of yeast lacking the ability to grow in
tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics,
85:12 (1977)). The presence of the trpl lesion as a characteristic
of the yeast host cell genome then provides an effective
environment for detecting transformation by growth in the absence
of tryptophan.
EXAMPLES
[0404] The present invention is further illustrated by the
following examples which should not be construed as further
limiting. The contents of Sequence Listing, figures and all
references, patents, and published patent applications cited
throughout this application are expressly incorporated herein by
reference.
Example 1. Chemoenzyme Synthesis of CMP-Sialic Acid or CMP-Sialic
Acid Derivatives at C5
[0405] N-acetyl mannosamine or a derivative thereof can be treated
with sialic acid aldolase to form sialic acid or sialic acid
derivatives. Subsequent treatment of the sialic acid or sialic acid
derivative with CTP in the presence of CMP-sialic acid synthetase
would result in the creation of CMP-sialic acid or a CMP-sialic
acid derivative (FIG. 1).
[0406] CMP-sialic acid derivatives that could be created through
the chemoenzyme synthesis outlined in FIG. 1 include, but are not
limited to, the C5 CMP-sialic acid derivatives of FIG. 2. FIG. 2
also shows CMP-sialic acid derivatives at C7 and C8. The CMP-sialic
acid derivatives can be used as substrates to transfer the sialic
acid derivatives to antibodies through in vitro sialylation for
subsequent conjugation.
Example 2. Different Chemistries for Conjugation Through Sialic
Acid Derivatives
[0407] FIGS. 3A-E are depictions of different chemical reactions of
the instant invention, wherein the circles in combination with the
reactive moieties to which they are bonded represent sialic acid
derivative-conjugated binding polypeptides. The stars represent
targeting or effector moieties. FIG. 3A is a schematic showing the
reacting of a sialic acid derivative-conjugated binding polypeptide
comprising a terminal aldehyde with an aminooxy effector moiety,
e.g., a drug or a glycan, to form an imine. FIG. 3B is a schematic
showing the reacting of a sialic acid derivative-conjugated binding
polypeptide comprising a terminal keto group with an aminooxy
effector moiety, e.g., a drug or a glycan, to form an imine. FIG.
3C is a schematic showing the reacting of a sialic acid
derivative-conjugated binding polypeptide comprising a terminal
aldehyde or keto with an effector moiety comprising a hydrazine to
form a hydrazone, which is a type of imine. FIG. 3D is a schematic
showing the reacting of a sialic acid derivative-conjugated binding
polypeptide comprising a terminal azide with an effector moiety,
e.g., a drug or a glycan, comprising an alkyne or bound to a moiety
comprising an alkyne (here, DBCO) to form a triazole. FIG. 3E is a
schematic showing the reacting of a sialic acid
derivative-conjugated binding polypeptide comprising a terminal
thiol with an effector moiety, e.g., a drug or a glycan, comprising
a maleimide to form a thioester bond.
[0408] FIG. 4 depicts an effector moiety conjugated binding
polypeptide according to the methods of the instant invention. The
effector moiety conjugated binding polypeptide can be formed by (a)
reacting a sialic acid derivative with a glycan of a binding
polypeptide to form a sialic acid derivative-conjugated binding
polypeptide; and (b) reacting the sialic acid derivative-conjugated
binding polypeptide with an effector moiety to form the effector
moiety conjugated binding polypeptide, wherein an imine bond is
formed, and wherein neither the binding polypeptide nor the sialic
acid derivative-conjugated binding polypeptide are treated with an
oxidizing agent. FIG. 4 depicts the formation of an oxime, a type
of imine.
[0409] FIG. 5 depicts an effector moiety conjugated binding
polypeptide according to the methods of the instant invention. The
effector moiety conjugated binding polypeptide can be formed by (a)
reacting a sialic acid derivative comprising a terminal reactive
moiety at the C5 position with a glycan of a binding polypeptide to
form a sialic acid derivative-conjugated binding polypeptide; and
(b) reacting the sialic acid derivative-conjugated binding
polypeptide with an effector moiety to form the effector moiety
conjugated binding polypeptide using click chemistry. FIG. 5
depicts the formation of a triazole ring.
Example 3. Design, Preparation, and Characterization of 2C3
Anti-CD-52 Hyperglycosylation Antibody Mutants
[0410] Multiple hyperglycosylation mutations were designed in the
heavy chain of the anti-CD-52 antibody, 2C3, for the purpose of
adding a bulky group to an interaction interface (e.g., the FcRn
binding site to modulate antibody pharmacokinetics), for modulating
antibody effector function by changing its interaction with
Fc.gamma.Rs, or to introduce a novel cross-linking site subsequence
chemical modification for effector moiety conjugation, including
but not limited to, drugs, toxins, cytotoxic agents, and
radionucleotides. The hyperglycosylated 2C3 mutants are set forth
in Table 3.
TABLE-US-00003 TABLE 3 Hyperglycosylated 2C3 anti-CD-52 mutants
Mutation Desired Benefit Applications A114N Glycosylation at 1)
Control Asn-Ser-Thr 2) Effector moiety conjugation Y436T
Glycosylation at Asn434 1) Transplant and other Inhibition of FcRn
indications which need binding short half-life Y436S Glycosylation
at Asn434 1) Transplant and other Inhibition of FcRn indications
which need binding short half-life S440N Glycosylation at 1)
Control Asn-Leu-Ser 2) Effector moiety conjugation S442N
Glycosylation at 1) Control Asn-Leu-Ser 2) Effector moiety
conjugation Add NGT Glycosylation 1) Control to C-terminal 2)
Effector moiety conjugation S298N/ Glycosylation at Asn298 1)
Reduce effector function Y300S Reduced effector function 2)
Effector moiety conjugation
3A. Creation of 2C3 Anti-CD-52 Antibody Hyperglycosylation
Mutants
[0411] The A114N mutation, designated based upon the Kabat
numbering system, was introduced into the CH1 domain of 2C3 by
mutagenic PCR. To create the full-length antibody, the VH domain
plus the mutated A114N residue was inserted by ligation independent
cloning (LIC) into the pENTR-LIC-IgG1 vector encoding antibody CH
domains 1-3. All other mutations were introduced on pENTR-LIC-IgG1
by site-directed mutagenesis with a QuikChange site-directed
mutagenesis kit (Agilent Technologies, Inc., Santa Clara, Calif.,
USA). The WT 2C3 VH was cloned into mutated vectors by LIC.
Full-length mutants were cloned into the pCEP4(-E+I)Dest expression
vector by Gateway cloning. Fc mutations were designated based on
the EU numbering system. Mutations were confirmed by DNA
sequencing. Amino acid sequences of the WT 2C3 heavy and light
chains and the mutated 2C3 heavy chains are set forth in Table 4.
Mutated amino acids are highlighted in gray and the consensus
glycosylation target sites created by the mutation are
underlined.
TABLE-US-00004 TABLE 4 Amino acid sequences of 2C3 anti-CD-52
antibodies SEQ ID NO Name Amino Acid Sequence 1 Anti-CD-52
DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSNGKTY WT
LNWLLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSG light chain
TDFTLKISRVEAEDVGVYYCVQGTHLHTFGQGTRL
EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP
REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC 2 Anti-CD-52
VQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMN WT
WVRQAPGKGLEWVGQIRLKSNNYATHYAESVKGR heavy chain
FTISRDDSKNSLYLQMNSLKTEDTAVYYCTPVDFW
GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG
CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK
VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 3
Anti-CD-52 EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMN A114N
WVRQAPGKGLEWVGQIRLKSNNYATHYAESVKGR heavy chain
FTISRDDSKNSLYLQMNSLKTEDTAVYYCTPVDFW
GQGTTVTVSSNSTKGPSVFPLAPSSKSTSGGTAALG
CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK
VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 4
Anti-CD-52 EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMN Y436S heavy
WVRQAPGKGLEWVGQIRLKSNNYATHYAESVKGR chain
FTISRDDSKNSLYLQMNSLKTEDTAVYYCTPVDFW
GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG
CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK
VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHSTQKSLSLSPGK 5
Anti-CD-52 EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMN S440N heavy
WVRQAPGKGLEWVGQIRLKSNNYATHYAESVKGR chain
FTISRDDSKNSLYLQMNSLKTEDTAVYYCTPVDFW
GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG
CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK
VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKNLSLSPGK 6
Anti-CD-52 EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMN S442N heavy
WVRQAPGKGLEWVGQIRLKSNNYATHYAESVKGR chain
FTISRDDSKNSLYLQMNSLKTEDTAVYYCTPVDFW
GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG
CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK
VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLNLSPGK 7
Anti-CD-52 EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMN NGT
WVRQAPGKGLEWVGQIRLKSNNYATHYAESVKGR heavy chain
FTISRDDSKNSLYLQMNSLKTEDTAVYYCTPVDFW
GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG
CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK
VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGKNGT 8
Anti-CD-52 EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMN S298N/
WVRQAPGKGLEWVGQIRLKSNNYATHYAESVKGR Y300S
FTISRDDSKNSLYLQMNSLKTEDTAVYYCTPVDFW heavy chain
GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG
CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK
VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
NAKTKPREEQYNNTSRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK
[0412] The mutants and WT control were transfected into HEK293-EBNA
cells in a 6-well plate format. As shown in FIGS. 14A and B, the
expression level was found to be .about.0.1 .mu.g/ml, as analyzed
by SDS-PAGE and Western blot. Expression of mutants in conditioned
media was also measured by protein A capture on Biacore.
Concentration was determined using the dissociation response 6
minutes after injection into immobilized Protein A. CHO-produced WT
2C3 serially diluted in media from 90 .mu.g/mL to 1.5 ng/mL was
used as a standard curve. Concentrations were calculated down to
-0.2 .mu.g/mL by a calibration curve using a 4-parameter fit. As
shown in FIG. 14B, relative expressions levels were low and
generally corresponded with the Western blot results.
3B. Verification of Hyperglycosylation
[0413] To determine whether additional glycosylation sites were
introduced by mutation, 2C3 mutant and wild type proteins were
treated with the universal deglycosylating enzyme PNGase F and
protein samples were analyzed by SDS-PAGE and Western blot. As
shown in FIG. 15, only the A114N mutant had an increased apparent
molecular weight, indicating the presence of an additional N-linked
carbohydrate.
[0414] Small scale antibody preparations were produced to purify
the 2C3 mutants for further verification of glycosylation site
introduction. As shown in FIG. 16, it was confirmed by SDS-PAGE
that only the A114N mutant had additional glycosylation sites
introduced.
3C. Binding Properties of 2C3 Anti-CD-52 Mutants
[0415] Biacore was used to compare the binding properties of the
purified proteins. Mouse and SEC-purified human FcRn-HPC4 were
immobilized on a CMS chip via amine coupling. Each antibody was
diluted to 200, 50, and 10 nM and injected over the immobilized Fc
receptors. Campath, CHO-produced WT 2C3, and DEPC-treated Campath
were included as positive and negative controls. As shown in FIG.
18, the Y436S mutant displayed about a 2-fold decrease in binding
to human FcRn. Interestingly, binding of this mutant to mouse FcRn
was not affected. None of the other 2C3 mutations had any
considerable effect on human or mouse FcRn binding.
[0416] Biacore was used to compare the antigen binding properties
of the purified proteins using the CD-52 peptide 741 Biacore
binding assay. CD-52 peptide 741 and control peptide 777 were
immobilized to a CMS chip. Antibodies were serially diluted 2-fold
from 60 to 0.2 nM in HBS-EP and injected in duplicate for 3 min
followed by a 5 min dissociation in buffer at .alpha.50 .mu.L/min
flow-rate. GLD52 lot 17200-084 was included as a control. The
surface was regenerated with 1 pulse of 40 mM HCl. A 1:1 binding
model was used to fit the 7.5 to 0.2 nM curves. As shown in FIG.
21, the A114N mutant had a slightly lower CD-52 binding affinity
while the NGT mutant had a slightly higher affinity than the rest
of the mutants in this assay. The CD-52 peptide 741 Biacore binding
assay was repeated with protein purified from larger scale prep. As
shown in FIG. 22, the A114N mutant exhibited CD-52 peptide binding
that was comparable to WT 2C3.
3D. Charge Characterization of the A114N Mutant
[0417] Isoelectric focusing (IEF) was performed to characterize the
charge of the 2C3 mutants. Purified protein was run on immobilized
pH gradient (pH3-10) acrylamide (IPG) gels. As shown in FIG. 23A,
A114N was found to have more negative charges, likely due to sialic
acid residues. Intact MS data confirmed the complex structure with
sialic acids on the A114N mutant. In contrast, the WT 2C3 was shown
to have G0F and G1F as the dominant glycosylation species (FIGS.
23C and 23D, respectively).
Example 4. Preparation of Hyperglycosylation Mutants in Several
Antibody Backbones
[0418] In addition to the 2C3 anti-CD-52 antibody, the A114N
mutation was engineered in several other antibody backbones to
confirm that the unique hyperglycosylation site could be introduced
into unrelated heavy chain variable domain sequences. The
hyperglycosylated anti-TEM1, anti-FAP, and anti-Her2 mutants are
set forth in Table 5.
TABLE-US-00005 TABLE 5 A114N and/or S298N mutants designed in
several unrelated antibody backbones Mutation Antibody Desired
benefits Applications A114N anti-TEM1 Additional glycosylation 1)
Control anti-FAP site at the elbow hinge of 2) Aminooxy toxin
anti-Her2 heavy chain for site-specific conjugation via
carbohydrate-mediated exposed sialic acid conjugation or galactose
group (SAM or GAM) S298N/ anti-Her2 Switch the glycosylation 1)
Aminooxy toxin T299A/ from Asn297 to an conjugation via Y3005
engineered Asn298. exposed sialic acid (NNAS Expect solvent exposed
or galactose group ("NNAS" and complex carbohydrates (SAM or GAM)
disclosed at S298N, offering 2) Reduced effector as SEQ conjugation
site and means function ID NO: to remove effector function 40))
A114N/ anti-Her2 Potential for increased 1) Control NNAS
conjugation yield with two 2) Aminooxy toxin ("NNAS" conjugation
sites conjugation via as SEQ exposed sialic acid ID NO: or
galactose group 40) (SAM or GAM)
4A. Creation of Anti-TEM1 and Anti-FAP Antibody Hyperglycosylation
Mutants
[0419] The A114N mutation, designated based upon the Kabat
numbering system, was introduced into the CH1 domain of anti-TEM1
and anti-FAP by mutagenic PCR. To create the full-length antibody,
the mutated VH plus residue 114 was inserted by ligation
independent cloning (LIC) into the pENTR-LIC-IgG1 vector encoding
antibody CH domains 1-3. Full-length mutants were then cloned into
the pCEP4(-E+I)Dest expression vector by Gateway cloning. Mutations
were confirmed by DNA sequencing. Amino acid sequences of the
anti-TEM1 wild type and mutated heavy and light chains are set
forth in Table 6. Mutated amino acids are highlighted in gray and
the consensus glycosylation target sites created by the mutation
are underlined.
TABLE-US-00006 TABLE 6 Amino acid sequences of anti-TEM1 and
anti-FAP antibodies SEQ ID NO Name Amino Acid Sequence 9 Anti-TEM1
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWY WT light
QQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTL chain
TISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRT (clone #187)
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 10 Anti-TEM1
QVQLQESAPGLVKPSETLSLTCTVSGGSIRSYYWSW WT heavy
IRQPPGKGLEYIGYIYYTGSAIYNPSLQSRVTISVDTS chain
KNQFSLKLNSVTAADTAVYYCAREGVRGASGYYY (clone #187)
YGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS
NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK
11 Anti-TEM1 QVQLQESAPGLVKPSETLSLTCTVSGGSIRSYYWSW A114N
IRQPPGKGLEYIGYIYYTGSAIYNPSLQSRVTISVDTS
KNQFSLKLNSVTAADTAVYYCAREGVRGASGYYY
YGMDVWGQGTTVTVSSNSTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS
NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYTQKSLSLSPGK*
[0420] The mutants and wild type control were transfected into
HEK293-EBNA cells in a triple flask format and purified on HiTrap
protein A columns (GE Healthcare Biosciences, Pittsburgh, Pa.,
USA). As analyzed by A280 on a NanoDrop spectrophotometer, the
expression of anti-FAP A114N and anti-FAP A1 14C was about 3
.mu.g/ml and about 1 .mu.g/ml, respectively. The expression of
anti-TEM1 A114N was about 0.04n/ml.
4B. Verification of Hyperglycosylation
[0421] To confirm that the additional glycosylation site was
introduced into the A114N mutants, purified protein from the A114N
mutants was analyzed on reducing SDS-PAGE along with wild-type
control protein. One additional glycosylation site would add
2000-3000 Daltons to the molecular weight of the heavy chain. As
shown in FIG. 25, SDS-PAGE indicated that the anti-FAP and
anti-TEM1 A114N mutant heavy chain bands had increased apparent
molecular weight, consistent with successful introduction of an
additional glycosylation site to both antibodies.
4C. Creation of Anti-Her2 Antibody Hyperglycosylation Mutants
[0422] The Her-2 A114N, Her-2 A114N/NNAS ("NNAS" disclosed as SEQ
ID NO: 40), and WT Her-2 antibodies were created by ligation
independent cloning. The VH domain of Herceptin was synthesized and
PCR-amplified with two LIC-compatible sets of primers, either WT or
bearing the A114N mutation. To obtain a full-length antibody,
amplified VH inserts (WT or A114N) were cloned into two pENTR
vectors encoding CH 1-3 domains, pENTR-LIC-IgG1 WT and
pENTR-LIC-IgG1 NNAS ("NNAS" disclosed as SEQ ID NO: 40), resulting
in three full-length mutants (A114N, NNAS ("NNAS" disclosed as SEQ
ID NO: 40), A114N/NNAS ("NNAS" disclosed as SEQ ID NO: 40)) and WT
control as entry clones on pENTR. These mutants were cloned into
the pCEP4(-E+I)Dest expression vector, by Gateway cloning.
Mutations were confirmed by DNA sequencing. Amino acid sequences of
the anti-Her-2 wild type and mutated heavy and light chains are set
forth in Table 7. Mutated amino acids are highlighted in gray and
the consensus glycosylation target sites created by the mutation
are underlined.
TABLE-US-00007 TABLE 7 Amino acid sequences of anti-Her-2
antibodies SEQ ID NO Name Amino Acid Sequence 12 Anti-Her-2
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW WT
YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFT light chain
LTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRT
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 13 Anti-Her-2
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHW WT
VRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS heavy chain
ADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY
AMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT
KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT
LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK 14
Anti-Her-2 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHW A114N
VRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS heavy chain
ADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY
AMDYWGQGTLVTVSSNSTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT
KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT
LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK 15
Anti-Her2 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHW NAS
VRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS ("NNAS"
ADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY disclosed as
AMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG SEQ ID NO: 40)
GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA heavy chain
VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT
KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNNASRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT
LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK 16
Anti-Her2 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHW A114N /
VRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS NAS
ADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY ("NNAS"
AMDYWGQGTLVTVSSNSTKGPSVFPLAPSSKSTSG disclosed as
GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA SEQ ID NO: 40)
VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT heavy chain
KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNNASRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT
LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK
4D. Expression of the A114N Anti-Her2 Antibody Hyperglycosylation
Mutant
[0423] The A114N anti-Her2 and wild type constructs were
transfected with Lipofectamine-2000 (2.5:1 ratio of reagent to DNA)
and XtremeGene HP (3:1 ratio of reagent to DNA) into HEK293-EBNA
cells in 12 triple flasks. Octet measurement of aliquots from day 3
conditioned media (CM) showed that protein expression was
consistent across 6 flasks for both Lipofectamine-2000 and
XtremeGene HP. As shown in Table 8, the overall transfection
efficiency was about 30% higher with XtremeGene HP. Conditioned
media collected on day 3 was pooled together for both transfection
conditions and purified by protein A column. Octet measurement
showed 1.8 ug/ml antibody in the serum-containing mock media versus
0 ug/ml in no serum mock media.
TABLE-US-00008 TABLE 8 A114N anti-Her2 hyperglycosylation mutant
expression Lipofectamine- XtremeGene 2000 HP Purified protein
Concentration 1.72 3.18 from protein A (mg/ml) column Volume (ml)
3.5 3.5 Total protein (mg) 6.02 11.13 Buffer-exchanged
Concentration 15.59 16.86 protein (mg/ml) Volume (ml) 0.2 0.36
Total protein (mg) 3.1 6.07 % Recovery 51.8 54.5
[0424] Conditioned media from Day 6 was collected and purified
separately for each transfection condition. Both eluates were
buffer-exchanged separately into PBS, pH 7.2, and concentrated
.about.15-fold using Amicon-4 (50 kD cut-off) columns. Day 6 CM
showed higher expression level compared to Day 3 CM. As shown in
Table 8, a total of 3 mg of Herceptin A114N 15.59 mg/ml (from
Lipofectamine transfection) and 6 mg of Herceptin A114N 16.86 mg/ml
(from XtremeGene HP transfection) was produced from day 6
conditioned media for additional downstream applications, such as
antibody-drug conjugation.
4E. SDS-PAGE and HIC analysis of the A114N anti-Her2 mutant
[0425] Prior to conjugation, purified A114N Herceptin was
characterized by SDS-PAGE and HIC (hydrophobic interaction
chromatography). As shown in FIG. 26, the quality of purified A114N
Herceptin was determined to be suitable for further downstream
applications.
4F. Conjugation to engineered glycosylation
[0426] It was demonstrated that: a) a glycosylation site was
introduced at Kabat position 114 site on anti-TEM1; b) the A114N
mutant had hyperglycosylation on the heavy chain by reducing
SDS-PAGE; and c) the A114N hyperglycosylated mutant had complex
carbohydrate structure by intact LC/MS, including terminal sialic
acids and galactose, which are ideal for SAM and GAM conjugation.
To confirm that the engineered glycosylation site was suitable for
conjugation, anti-TEM1 A114N was conjugated with a 5 kDa PEG via
aminooxy chemistry. As shown in FIG. 27, PEG was successfully
conjugated to anti-TEM1 A114N through an aminooxy linkage. This
mutant was also successfully prepared on the anti-FAP and
anti-CD-52 2C3 backbones (not shown). These data demonstrate that
the glycosylation site at N114 is useful for conjugation of
effector moieties.
Example 5: Generation of S298N/Y300S Fe Mutants
[0427] Engineered Fc variants was designed and generated in which a
new glycosylation site was introduced at EU position Ser 298, next
to the naturally-occurring Asn297 site. The glycosylation at Asn297
was either maintained or ablated by mutation. Mutations and desired
glycosylation results are set forth in Table 9.
TABLE-US-00009 TABLE 9 Glycosylation states of various antibody
variants # Mutation Desired Glycosylation State Applications A
N297Q No glycosylation (agly) Agly Control B T299A No glycosylation
(agly) Agly Control, unknown effector function C N297Q/S298N/ No
glycosylation at 297 but Reduced effector Y300S (NSY) engineered
glycosylation site function; Conjugation at 298 via exposed sialic
acid or galactose groups. D S298N/T299A/ No glycosylation at 297
but Reduced effector Y300S (STY) engineered glycosylation site
function; Conjugation at 298 via exposed sialic acid or galactose
groups. E S298N/Y300S Two potential glycosylation Reduced effector
(SY) sites at 297 & 298; Alterations function; Conjugation in
glycosylation pattern. via exposed sialic acid or galactose groups.
F Wild-type 297 control
5A. Creation of H66 .alpha..beta.-TCR Antibody Altered
Glycosylation Variants
[0428] Mutations were made on the heavy chain of .alpha..beta.
T-cell receptor antibody clone #66 by Quikchange using a
pENTR_LIC_IgG1 template. The VH domain of HEBE1 .DELTA.ab IgG1 #66
was amplified with LIC primers before being cloned into mutated or
wild type pENTR_LIC_IgG1 by LIC to create full-length mutant or
wild-type antibodies. The subcloning was verified with DraIII/XhoI
double digest, producing an approximately 1250 bp-sized insert in
the successful clones. Those full-length mutants were then cloned
into an expression vector, pCEP4(-E+I)Dest, via Gateway cloning.
The mutations were confirmed by DNA sequencing. Amino acid
sequences of the WT H66 anti-.alpha..beta.TCR heavy and light
chains and the mutated H66 heavy chains are set forth in Table 10.
Mutated amino acids are highlighted in gray and the consensus
glycosylation target sites created by the mutation are
underlined.
TABLE-US-00010 TABLE 10 Amino acid sequences of H66 anti-aPTCR
antibodies SEQ ID NO Name Amino AcidSequence 23
Anti-.alpha..beta.TCR clone EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWYQQ
H66 light chain KPGQAPRRLIYDTSKLASGVPARFSGSGSGTSYTLTIS
SLEPEDFAVYYCQQWSSNPLTFGGGTKVEIKRTVAAP
SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC*
24 Anti-.alpha..beta.TCR clone EVQLLQSGGGLVQPGGSLRLSCAASGYKFTSYVMHW
H66 heavy chain VRQAPGKGLEWVGYINPYNDVTKYNEKFKGRFTLSR
DNSKNTLYLQMNSLRAEDTAVYYCARGSYYDYDGF
VYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA
LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK* 25
Anti-.alpha..beta.TCR clone EVQLLQSGGGLVQPGGSLRLSCAASGYKFTSYVMHW
H66 S298N/Y300S VRQAPGKGLEWVGYINPYNDVTKYNEKFKGRFTLSR heavy chain
DNSKNTLYLQMNSLRAEDTAVYYCARGSYYDYDGF
VYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA
LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYNNTSRVVSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK* 26
Anti-.alpha..beta.TCR clone EVQLLQSGGGLVQPGGSLRLSCAASGYKFTSYVMHW
H66 S298N/ VRQAPGKGLEWVGYINPYNDVTKYNEKFKGRFTLSR T299A/Y300S
DNSKNTLYLQMNSLRAEDTAVYYCARGSYYDYDGF heavy chain
VYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA
LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYNNASRVVSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK* 27
Anti-.alpha..beta.TCR clone EVQLLQSGGGLVQPGGSLRLSCAASGYKFTSYVMHW
H66 N297Q/ VRQAPGKGLEWVGYINPYNDVTKYNEKFKGRFTLSR S298N/Y300S
DNSKNTLYLQMNSLRAEDTAVYYCARGSYYDYDGF heavy chain
VYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA
LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYQNTSRVVSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK*
[0429] The mutant, wild-type, and two aglycosylated control (HEBE1
Agly IgG4 and HEBE1 .DELTA.ab IgG1 in pCEP4) constructs were
transfected into HEK293-EBNA cells in triple-flasks for expression.
Proteins were purified from 160 ml of conditioned media (CM) with 1
ml HiTrap protein A columns (GE) using a multi-channel peristaltic
pump. Five micrograms of each resulting supernatant were analyzed
on 4-20% Tris-Glycine reducing and non-reducing SDS-PAGE gels (see
FIG. 7). The heavy chains of the aglycosylated mutants (N297Q,
T299A, and Agly controls), have migrated further (arrowhead),
consistent with the loss of the glycans in these antibodies. The
heavy chains of the engineered glycosylated antibodies (NSY, STY,
SY, .DELTA.ab, and wt control, arrows), however, migrate similarly
to the wild-type control. This result is consistent with the
existence of an engineered glycosylation site at EU position 298.
SEC-HPLC analysis indicated that all mutants are expressed as
monomers.
5B. Glycosylation Analysis by LC-MS
[0430] The engineered H66 IgG1 Fc variants were partially reduced
with 20 mM DTT at 37.degree. C. for 30 min. The samples were then
analyzed by capillary LC/MS on an Agilent 1100 capillary HPLC
system coupled with a QSTAR qq TOF hybrid system (Applied
Biosystems). A Bayesian protein reconstruction with baseline
correction and computer modeling in Analyst QS 1.1 (Applied
Bisoystem) was used for data analysis. In the S298N/T299A/Y300S H66
antibody mutant, one glycosylation site was observed at amino acid
298 with bi-antennary and tri-antennary complex-type glycans
detected as the major species alongside G0F, GIF and G2F (see FIG.
39). This altered glycosylation profile is consistent which shifted
glycosylation at N.sub.298 instead of the wild-type glycosylation
site at N297.
5C. Binding Properties of .alpha..beta.TCR Antibody Mutants to
Human Fc.gamma.RIIIa and Fc.gamma.RI Using Biacore
[0431] Biacore was used to assess binding to recombinant human
Fc.gamma.RIIIa (V158 & F158) and Fc.gamma.RI. All four
flowcells of a CMS chip were immobilized with anti-HPC4 antibody
via the standard amine coupling procedure provided by Biacore. The
anti-HPC4 antibody was diluted to 50 .mu.g/mL in 10 mM sodium
acetate pH 5.0 for the coupling reaction and injected for 25 min at
5 .mu.L/min. Approximately 12,000 RU of antibody was immobilized to
the chip surface. Recombinant human Fc.gamma.RIIIa-V158 and
Fc.gamma.RIIIa-F158 were diluted to 0.6 .mu.g/mL in binding buffer
(HBS-P with 1 mM CaCl.sub.2) and injected to flowcells 2 and 4,
respectively, for 3 min at 54/min to capture 300-400 RU receptor on
the anti-HPC4 chip. In order to distinguish between the low
binders, three times more rhFc.gamma.RIIIa was captured on the
anti-HPC4 surface than usually used in this assay. Flowcells 1 and
3 were used as reference controls. Each antibody was diluted to 200
nM in binding buffer and injected over all four flowcells for 4
min, followed by 5 min dissociation in buffer. The surfaces were
regenerated with 10 mM EDTA in HBS-EP buffer for 3 min at 204/min.
The results of these experiments are shown in FIG. 8.
[0432] Biacore was also used to compare the Fc.gamma.RI binding.
Anti-tetra His antibody was buffer exchanged into 10 mM sodium
acetate pH 4.0 using a Zeba Desalting column and diluted to 25
.mu.g/mL in the acetate buffer for amine coupling. Two flowcells of
a CMS chip were immobilized with 9000 RU of the anti-Tetra-His
antibody after 20 min injection at 54/min. As in the previous
experiment, ten times more Fc.gamma.RI was captured to the
anti-tetra-His surface in order to compare samples with weak
binding. Recombinant human Fc.gamma.RI was diluted 10 .mu.g/mL in
HBS-EP binding buffer and injected to flowcell 2 for 1 min at
54/min to capture 1000 RU receptor to the anti-tetra-His chip. A
single concentration of antibody, 100 nM, was injected for 3 min at
304/min over the captured receptor and control surface.
Subsequently, dissociation was monitored for three minutes. The
surface was then regenerated with two 30 second injections of 10 mM
glycine pH 2.5 at 204/min. The results of these experiments are
shown in FIG. 9.
[0433] These results demonstrate a striking decrease in binding of
the glycoengineered mutants to Fc.gamma.RIIIa or Fc.gamma.RI. H66
S298N/T299A/Y300S in particular has almost completely abolished
binding to both receptors. This mutant was chosen for more detailed
analysis.
5D. Stability Characterization Using Circular Dichroism (CD)
[0434] The stability of the S298N/T299A/Y300S antibody mutant was
monitored by a Far-UV CD thermo melting experiment in which the CD
signal at 216 nm and 222 nm was monitored as increasing temperature
lead to the unfolding of the antibody (denaturation).
[0435] Temperature was controlled by a thermoelectric peltier
(Jasco model AWC100) and was increased at a rate of FC/min from
25-89.degree. C. The CD spectra were collected on a Jasco 815
spectrophotometer at a protein concentration of approximately 0.5
mg/mL in PBS buffer in a quartz cuvette (Hellma, Inc) with a path
length of 10 mm. The scanning speed was 50 nm/min and a data pitch
of 0.5 nm. A bandwidth of 2.5 nm was used with a sensitivity
setting of medium. The CD signal and HT voltage were collected from
210-260 nm with data intervals of 0.5 nm and at temperature
intervals of 1.degree. C. and four replicate scans were performed
for each sample. The results demonstrate that both delta AB H66 and
the S298N/T299A/Y300S H66 mutant exhibit similar thermal behaviors
and have approximately the same onset temperature for degradation
(around 63.degree. C.) (FIG. 40), further suggesting that they have
comparable stability.
Example 6: Functional Analysis of Fc-Engineered Mutants
[0436] Fc-engineered mutants were assessed through a PBMC
proliferation assay and a cytokine release assay. In the PBMC
proliferation assay, human PBMC were cultured with increasing
concentrations of therapeutic antibody for 72 hours,
.sup.3H-thymidine was added and cells were harvested 18 hours
later. For the T cell depletion/Cytokine Release assay, human PBMC
were cultured with increasing concentrations of therapeutic
antibody and were analyzed daily for cell counts and viability
(Vi-Cell, Beckman Coulter) out to day 7. Cell supernatants were
also harvested, stored at -20.degree. C. and analyzed on an 8-plex
cytokine panel (Bio-Rad).
[0437] Normal donor PBMC were thawed and treated under the
following conditions (all in media containing complement):
Untreated; BMA031, moIgG2b 10 ug/ml; OKT3, moIgG2a 10 ug/ml; H66,
huIgG1 deltaAB 10 ug/ml, 1 ug/ml and 0.1 ug/ml; H66, huIgG1
S298N/T299A/Y300S 10 ug/ml, 1 ug/ml and 0.1 ug/ml.
[0438] Cytokines were harvested at day 2 (D2) and day 4 (D4) for
Bioplex Analysis (IL2, IL4, IL6, IL8, IL10, GM-CSF, IFNg, TNFa).
Cells were stained at D4 for CD4, CD8, CD25 and abTCR
expression.
[0439] The results, shown in FIGS. 10-13, demonstrate that H66
S298N/T299A/Y300S behaved similarly to the H66 deltaAB in all the
cell based assays performed, showing minimal T-cell activation by
CD25 expression, binding to abTCR (with slightly different kinetics
to deltaAB), and minimal cytokine release at both D2 and D4 time
points. The S298N/T299A/Y300S mutant thus eliminated effector
function as effectively as the deltaAB mutation.
Example 7: Preparation and Characterization of an Engineered Fc
Variant in the Anti-CD52 Antibody Backbone
[0440] In addition to the H66 anti-.alpha..beta.TCR antibody, the
S298N/Y300S mutation was also engineered in an anti-CD52 antibody
backbone (clone 2C3). This mutant was then examined in order to
determine whether the observed effector function modulation seen in
the S298N/Y300S H66 anti-.alpha.TCR antibody was consistent in
another antibody backbone.
7A. Creation of 2C3 Anti-CD52 Antibody Altered Glycosylation
Variants
[0441] First, S298N/Y300S 2C3 variant DNA was prepared by quick
change mutagenesis using pENTR_LIC_IgG1, and WT 2C3 VH was cloned
into the mutated vector by LIC. Full-length mutants were cloned
into the pCEP4 (-E+I)Dest expression vector using Gateway
technology. Mutations were subsequently confirmed by DNA sequencing
and the sequences are set forth in Table 11. The mutants were then
transfected into HEK293-EBNA cells in a 6-well plate format and the
protein was purified from conditioned media. Anti-CD52 2C3
wild-type antibody was produced in parallel as a control. The
expression level was found to be 0.1 .mu.g/mL using SD-PAGE and
Western blot analyses (FIG. 15A). Expression of mutants in neat
conditioned media was also measured by protein A capture on
Biacore. Concentration was determined using the dissociation
response after a six-minute injection to immobilized protein A.
CHO-produced WT 2C3 serially diluted in media from 90 .mu.g/mL to
1.5 ng/mL was used as a standard curve. Concentrations were
calculated within approximately 0.2 .mu.g/mL by a calibration curve
using a 4-parameter fit. Relative expression levels were low and
generally agree with the Western blot data (FIG. 15B).
TABLE-US-00011 TABLE 11 Anti-CD52 clone 2C3 antibody sequences SEQ
ID NO Name Amino AcidSequence 28 Anti-CD-52
DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSNGKTYLNWL 2C3 WT
LQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISR light chain
VEAEDVGVYYCVQGTHLHTFGQGTRLEIKRTVAAPSVFIF
PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG
NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC* 29
Anti-CD-52 EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMNWVR 2C3 WT
QAPGKGLEWVGQIRLKSNNYATHYAESVKGRFTISRDDS heavy chain
KNSLYLQMNSLKTEDTAVYYCTPVDFWGQGTTVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK* 30 Anti-CD-52
EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMNWVR 2C3
QAPGKGLEWVGQIRLKSNNYATHYAESVKGRFTISRDDS S298N/Y300S
KNSLYLQMNSLKTEDTAVYYCTPVDFWGQGTTVTVSSAS heavy chain
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNNTSRVVSVLTVLHQDWLNGKE
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK*
7B. Glycosylation Analysis Using PNGaseF
[0442] To evaluate the additional glycosylation sites introduced by
the mutation, the enriched S298N/Y300S mutant was de-glycosylated
with PNGase F. It did not demonstrate any apparent change in
molecular weight, which indicates that no additional carbohydrate
was present (FIG. 15). Small scale preparations were performed in
order to purify these mutants for further characterization and the
results reconfirmed that there was not an additional carbohydrate
present on the S298N/Y300S mutant (FIG. 16).
7C. Binding Properties of 2C3 Anti-CD52 Antibody Mutants to Human
Fc.gamma.RIIIa Using Biacore
[0443] Biacore was also used to characterize the antigen-binding,
Fc.gamma.RIII, and binding properties of the purified antibodies
(see FIGS. 17A-C, 18, and 19A and B). The S298N/Y300S 2C3 variant
bound to the CD52 peptide tightly and the binding sensorgram was
undistinguishable from the wild-type control, demonstrating that
this mutation does not affect its antigen binding (FIG. 17A).
[0444] To assay for Fc effector function, Fc.gamma.RIII receptor
(Val158) was used in binding studies. The mutant and wild-type
control antibody were diluted to 200 nM and injected to HPC4-tag
captured Fc.gamma.RIIIa. Fc.gamma.RIII binding was almost
undetectable for the S298N/Y300S mutant, which indicated a loss of
effector function by this variant (FIG. 17B and FIG. 19A). To
further assay for Fc effector function, the Fc.gamma.RIII receptor
(Phe158) was also used in binding studies. The mutant and wild-type
control antibodies were diluted to 200 nM and injected to HPC4-tag
captured Fc.gamma.RIIIa. Fc.gamma.RIII binding was almost
undetectable for the S298N/Y300S mutant, which indicates a loss of
effector function with the Phe158 variant (FIG. 19B). Finally,
Biacore was used to compare the FcRn binding properties of the
purified proteins. Mouse and SEC-purified human FcRn-HPC4 were
immobilized to a CMS chip via amine coupling. Each antibody was
diluted to 200, 50, and 10 nM and injected over the receptors.
Campath, CHO-produced WT 2C3, and DEPC-treated Campath were
included as positive and negative controls. These data show that
the mutant binds to both human and murine FcRn receptor with the
same affinity as the wild-type antibody control and that it likely
has no alterations in its circulation half-life or other
pharmacokinetic properties. (see FIG. 17C, FIG. 18). Accordingly,
the S298N/Y300S mutation is applicable to antibodies in general, to
reduce or eliminate undesired Fc effector function, for example
through engagement of human Fc.gamma. receptors.
Example 8: Circulating Immune Complex Detection in the S298N/Y300S
Mutant
[0445] Circulating immune complex detection was also investigated
using a C1q binding assay for the S298N/Y300S mutant and WT
control. High binding Costar 96-well plates were coated overnight
at 4.degree. C. with 100 .mu.l of 2-fold serially diluted 2C3 Abs
at concentrations ranging from 10-0.001 .mu.g/ml in coating buffer
(0.1M NaCHO.sub.3 pH 9.2). ELISA analysis showed that C1q binding
is reduced for the S298N/Y300S mutant compared to WT (FIG. 20A).
The binding of anti-Fab Ab to the coated 2C3 Abs confirmed
equivalent coating of the wells (FIG. 20B).
Example 9: Separation and Analysis of S298N/Y300S Mutant Using
Isoelectric Focusing
[0446] A pH 3-10 Isoelectric Focusing (IEF) gel was run to
characterize the S298N/Y300S mutants. S298/Y300S was found to have
more negative charges, and therefore, likely more sialic acid
molecules (FIG. 23A). Both the S298N/Y300S mutant and WT 2C3 were
shown by intact MS to have G0F and G1F as the dominant
glycosylation species (FIGS. 23B and D, respectively).
Example 10: Antigen Binding Affinity of S298N/Y300S
[0447] Biacore was used to compare the antigen binding affinity of
WT anti-CD52 2C3 Ab and the S298N/Y300S mutant that had been
prepared and purified from both smaller (FIG. 21) and larger (FIG.
22) scale expressions. CMS chips immobilized with CD52 peptide 741
and control peptide 777 were obtained. Antibodies were serially
diluted 2-fold from 60 to 0.2 nM in HBS-EP and were then injected
over the chip surface for 3 min followed by a 5 min dissociation in
buffer at a flow rate of 50 .mu.l/min. The surface was then
regenerated with a pulse of 40 mM HCl. These analyses were
performed in duplicate and demonstrate that the S298N/Y300S mutant
and WT 2C3 antibodies show comparable CD52 peptide binding.
[0448] A media screening platform was designed to test functional
binding properties prior to purification in order to screen
antibodies created during small scale transfections. These tests
were performed using Octet (FIG. 24A) to determine concentration
and used Protein A biosensors and a GLD52 standard curve. Samples
were diluted to 7.5 and 2 nM in HBS-Ep for a CD52 binding
comparison using Biacore (FIG. 24B). The results of the peptide
binding assay showed that both the S298N/Y300S mutant and the WT
2C3 antibodies have comparable CD52 peptide binding. Furthermore,
these analyses demonstrate that Octet and Biacore work well to
predict antigen binding by antibodies from small scale
transfections.
Example 11: Preparation of S298N/Y300S, S298N/T299A/Y300S, and
N297Q/S298N/Y300S Altered Glycosylation Mutants in Additional
Antibody Backbones
[0449] In addition to the anti-.alpha..beta.-TCR antibody and 2C3
anti-CD-52 antibody, the 5298/Y300S, S298N/T299A/Y300S, and
N297Q/S298N/Y300S mutations were engineered in other antibody
backbones to confirm that the additional tandem glycosylation site
could be introduced into unrelated heavy chain variable domain
sequences. The alternatively glycosylated anti-CD-52 12G6 and
anti-Her2 mutants are set forth in Tables 12 and 13.
TABLE-US-00012 TABLE 12 Anti-CD52 clone 12G6 antibody sequences SEQ
ID NO Name Amino AcidSequence 31 Anti-CD-52
DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSNGKTYLNWV 12G6 WT
LQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRV light chain
EAEDVGVYYCVQGSHFHTFGQGTKLEIKRTVAAPSVFIFPP
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS
QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 32
Anti-CD-52 EVQLVESGGGLVQPGGSLRLSCAASGFPFSNYWMNWVRQ 12G6 WT
APGKGLEWVGQIRLKSNNYATHYAESVKGRFTISRDDSKN heavy chain
SLYLQMNSLKTEDTAVYYCTPIDYWGQGTTVTVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK* 33 Anti-CD-52
EVQLVESGGGLVQPGGSLRLSCAASGFPFSNYWMNWVRQ 12G6
APGKGLEWVGQIRLKSNNYATHYAESVKGRFTISRDDSKN S298N/Y300S
SLYLQMNSLKTEDTAVYYCTPIDYWGQGTTVTVSSASTKG heavy chain
PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYNNTSRVVSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK* 34 Anti-CD-52
EVQLVESGGGLVQPGGSLRLSCAASGFPFSNYWMNWVRQ 12G6S298N/
APGKGLEWVGQIRLKSNNYATHYAESVKGRFTISRDDSKN T299A/Y300S
SLYLQMNSLKTEDTAVYYCTPIDYWGQGTTVTVSSASTKG heavy chain
PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYNNASRVVSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK* 35 Anti-CD-52
EVQLVESGGGLVQPGGSLRLSCAASGFPFSNYWMNWVRQ 12G6 N297Q/
APGKGLEWVGQIRLKSNNYATHYAESVKGRFTISRDDSKN S298N/Y300S
SLYLQMNSLKTEDTAVYYCTPIDYWGQGTTVTVSSASTKG heavy chain
PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYQNTSRVVSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK*
TABLE-US-00013 TABLE 13 Anti-Her2 antibody sequences SEQ ID NO Name
Amino AcidSequence 36 Anti-Her2
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKP WT
GKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDF light chain
ATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQL
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC* 37 Anti-Her2
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQA WT
PGKGLEWVARTYPTNGYTRYADSVKGRFTISADTSKNTAY heavy chain
LQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTV
SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK* 38 Anti-Her2
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQA S298N/T299A/
PGKGLEWVARTYPTNGYTRYADSVKGRFTISADTSKNTAY Y300S
LQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTV heavy chain
SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNNASRVVSVLTVLHQDWLNGKE
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK*
Example 12. Generation of Altered Antibodies Containing Reactive
Glycan Moieties
[0450] In order to generate antibodies containing glycan moieties
capable of reacting with derivatized effector moieties, an anti-HER
antibody was first glycosylated in vitro using glycosyltransferase
and relevant sugar nucleotide donors. For example, to introduce the
sialic acid residues, donor antibodies were first galactosylated
with .beta.-galactosyltransferase, followed with sialylation with
.alpha.2,6-sialyltransferase according to the methods of Kaneko et
al. (Kaneko, Y., Nimmerjahn, F., and Ravetch, J. V. (2006)
Anti-inflammatory activity of immunoglobulin G resulting from Fc
sialylation. Science 313, 670-3). The reaction was performed in a
one-pot synthesis step using .beta.-galactosyltransferase (50
mU/mg, Sigma) and .alpha.2,6-sialyltranafrease (5 ug/mg, R&D
system) with donor sugar nucleotide substrates, UDP-galactose (10
mM) and CMP-sialic acid (10 mM) in 50 mM MES buffer (pH 6.5)
containing 5 mM MnCl2. The reaction mixture containing 5 mg/ml
anti-HER2 antibody was incubated for 48 hours at 37.degree. C. The
sialylation was verified using MALDI-TOF MS analysis of
permethylated glycans released from the antibody with PNGase F,
sialic acid content analysis using Dionex HPLC and lectin blotting
with SNA, a lectin specific for .alpha.2,6-sialic acid.
[0451] MALDI-TOF analysis of glycans released by PNGase F treatment
of the sialylated anti-HER2 antibody indicated that native glycans
had been completely remodeled with a mainly monosialylated
biantennary structure, A1F (FIG. 32A) together with small amount of
disialylated species. Treatment of the antibody with higher amounts
of .alpha.2,6-sialyltransferase produced more homogenous
populations of the A1F glycoform, suggesting that either the enzyme
activity or glycan localization may prevent full sialylation.
Sialic acid content was determined to be .about.2 mol per mol of
antibody, which is consistent with A1F glycan as the major
glycoform species (FIG. 32B). Lectin blotting with a SAN lectin,
Sambucus nigra agglutinin specific for .alpha.2,6-linked sialic
acid, confirmed that the sialic acid was present in an
.alpha.2,6-linkage configuration (FIG. 32C).
[0452] In conclusion, although the native protein glycans are
somewhat heterogeneous, remodeling through galactosyl and
sialyltransferases yields a nearly homogeneous antibody with
monosialylated but fully galactosylated biantennary glycans (A1F).
The introduction of only .about.1 sialic acid on the two galactose
acceptors on each branched glycan may be due to limited
accessibility of one of the galactoses from glycans which are often
buried in the antibody or non-covalent interactions of the glycans
with the protein surface.
Example 13. Alternate Method: Oxidation of Altered Antibodies
Containing Reactive Glycan Moieties
[0453] Oxidation of sialylated anti-HER2 antibody with various
concentrations of periodate (0.25 to 2 mM) was investigated. The
sialylated antibody was first buffer-exchanged into 25 mM Tris-HCl
(pH 7.5) containing 5 mM EDTA followed by buffer exchange with PBS
buffer. The buffered antibody mixture was then applied to protein A
Sepharose column pre-equilibrated with PBS buffer. After the column
was washed with 15 column volumes of PBS, 15 column volumes of PBS
containing 5 mM EDTA, and 30 column volumes of PBS, it was then
eluted with 25 mM citrate phosphate buffer (pH 2.9). The eluates
were immediately neutralized with dibasic phosphate buffer and the
antibody concentrated using Amicon ultra from Millipore. Following
purification, the sialylated anti-HER2 antibody then was oxidized
with sodium periodate (Sigma) in 100 mM sodium acetate buffer (pH
5.6) on ice in the dark for 30 minutes, and the reaction quenched
with 3% glycerol on ice for 15 minutes. The product was desalted
and exchanged into 100 mM sodium acetate (pH 5.6) by 5 rounds of
ultrafiltration over 50 kDa Amicons. FIG. 33A shows sialic acid
content analysis of sialylated antibody titrated with various
amounts of periodate. Complete oxidation of the sialic acid
residues was achieved at a periodate concentration above 0.5 mM. A
periodate concentration as low as 0.5 mM was enough to fully
oxidize the introduced sialic acid. Accordingly, a 1 mM
concentration of periodate was chosen for oxidation of sialylated
antibody for drug conjugation.
[0454] Oxidation can have adverse effects on the integrity of an
antibody. For example, the oxidation of methionine residues,
including Met-252 and Met-428, located in Fc CH.sub.3 region, close
to FcRn binding site is known to affect FcRn binding which is
critical for prolonging antibody serum half-life (Wang, W., et al.
(2011) Impact of methionine oxidation in human IgG1 Fc on serum
half-life of monoclonal antibodies. Mol Immunol 48, 860-6).
Accordingly, to examine the potential side effects of periodate
oxidation on methionine residues (e.g., Met-252) critical for FcRn
interaction, the oxidation state of the sialylated antibody was
determined by LC/MS analysis of a trypsin peptide digest. This
analysis revealed .about.30% oxidation of Met-252 and <10%
oxidation of Met-428 after treatment of the sialylated trastuzumab
with 1 mM periodate. To determine the impact of this degree of
methionine oxidation on FcRn binding, the FcRn binding kinetics for
each antibody was evaluated using surface plasmon resonance
(BIACORE). This analysis revealed that oxidation state correlated
with a minor loss in FcRn binding (12% and 26% reduction to mouse
and human FcRn, see FIGS. 33B and 33C respectively). Notably, a
.about.25% reduction in the Ka for human FcRn has been reported to
have no effect on the serum half-life in a human FcRn transgenic
mouse, since a single intact FcRn site on each antibody is
sufficient to provide functionality and the PK advantage (Wang et
al., Id).
[0455] In summary, these data indicate that the introduction of
periodate-sensitive sialic acid residues by sialyltransferase
treatment permits the use of much lower concentrations of
periodate, resulting in lowered side effects on antibody-FcRn
interactions and antibody integrity as assessed by aggregation
(.ltoreq.1%).
[0456] The galactose in a hyperglycosylated antibody mutant can
also be oxidized specifically using galactose oxidase to generate
an aldehyde group for conjugation. To confirm this approach, an
A114N anti-TEM1 antibody was concentrated to 13-20 mg/ml and then
treated with 20 mU/mg sialidase in PBS for 6 hours at 37.degree. C.
The desialated product was then oxidized with galactose oxidase
("GAO"), first with 5 ug GAO/mg protein overnight at 37.degree. C.
followed by addition of 2 ug GAO/mg protein and incubation for an
additional 5 hours. Sodium acetate was added to adjust the pH to
5.6 (0.1 v/v, pH5.6), and DMSO was added to achieve a final
reaction concentration of 16%, were added prior to conjugation. The
hyperglycosylation mutant A114N anti-HER antibody (15 mg/ml) was
similarly desialylated with sialidase (20 mU/mg) and oxidized with
5 ug GAO per mg protein in a single reaction overnight at
37.degree. C.
Example 14. Synthesis of Reactive Effector Moieties
[0457] In order to facilitate conjugation with the
aldehyde-derivatized antibody glycoforms, candidate drug effector
moieties (e.g., Momomethyl Auristatin E (MMAE) and Dolastatin 10
(Dol10)) were derivatized with aminooxy-cystamide to contain
functional groups (e.g., aminooxy-cys) specifically reactive with
the aldehyde.
[0458] Briefly, to generate aminooxy-cystamide as a starting
material, S-Trityl-L-cysteinamide (362 mg, 1 mmol) was added to a 3
mL of a DMF solution of t-BOC-aminooxyacetic acid
N-hydroxysuccinimide ester (289 mg, 1 mmol). The reaction was
complete after 3 h as evident from HPLC analysis. The reaction
mixture was subsequently diluted with 30 ml of dichloromethane and
was washed with 0.1 M sodium bicarbonate solution (2.times.20 mL),
water (2.times.20 mL), and brine (2.times.20 mL). The solution was
dried over anhydrous sodium sulfate, filtered and concentrated to
dryness. To this dried residue was added 3 mL of TFA followed by
150 .mu.L of triethylsilane. The resulting solution was
precipitated from t-butyl methyl ether and the process repeated
three times. After filtration, the residue was dried under reduced
pressure yielding 205 mg of an off white solid (67% yield). The
compound was used for next step without further purification.
[0459] To generate aminooxy-derivatized MMAE
(Aminooxy-Cys-MC-VC-PABC-MMAE), 30.1 mg of aminooxy-cystamide
(0.098 mmol, 2 eq.) was combined with 64.6 mg of MC-VC-PABC-MMAE
(0.049 mmol), and 100 .mu.L of triethylamine in 3 mL of DMF. The
resulting reaction mixture was stirred at room temperature for 15
minutes, by which time reaction was complete according to HPLC
analysis. The compound was purified by preparative HPLC yielding 45
mg (62%) of the desired product as an off-white solid.
Reversed-phase HPLC analysis suggested the purity of the compound
to be >96%. ESI calcd for C73H116N14O18S (MH)+1509.8501; found,
m/z 1509.8469.
[0460] To generate aminooxy-derivatized Dol10
(Aminooxy-Cys-MC-VC-PABC-PEG8-Dol10), 7.4 mg (0.024 mmol, 3 eq.) of
aminooxy-cystamide, 12 mg (0.008 mmol) of MC-VC-PABC-PEG8-Dol10 and
30 .mu.L triethylamine were combined in in 3 mL of DMF. The
reaction was complete within 15 minutes according to HPLC analysis.
Preparative HPLC purification resulted in 6.2 mg (46%) of the
desired product as an off-white solid. Reversed-phase HPLC analysis
suggests the purity of the compound to be >96%. ESI calcd for
C80H124N16O19S2 (MH)+1678.0664; found, m/z 1678.0613.
Example 15. Sialic Acid-Mediated (SAM) Conjugation of Reactive
Effector Moieties
[0461] Following desalting, drug-linkers of Example 13 were
combined with the oxidized, sialylated antibodies of Example 12
with 75% DMSO (0.167 v/v) at a concentration of 25 mM to achieve a
24:1 molar ratio of drug-linker to antibody and a final antibody
concentration at 5 mg/ml. The mixture was incubated overnight at
room temperature. The unincorporated drug-linkers and any free
drugs were scavenged using BioBeads. The product was
buffer-exchanged into Histidine-Tween buffer using PD-10 columns
and sterile filtered. The endotoxin levels were determined and less
than 0.1 EU/mg ADC was achieved for in vivo study.
[0462] FIG. 34A-C shows a hydrophobic interaction chromatograph
(HIC) of different sialylated antibodies (anti FAP B11 and G11 and
the anti-HER2 antibody of Example 13) glycoconjugated to AO-MMAE.
Sialylated HER2 antibody was also conjugated with the drug-linker,
AO-Cys-MC-VC-PABC-PEG8-Dol10 (FIG. 34D). This analysis reveals that
there are mainly one or two drug conjugates per antibody with a
drug-to-antibody ratio (DAR) ranging from 1.3-1.9. The increased
retention time of the Dol10 glycoconjugate (FIG. 34D) as compared
to the MMAE glycoconjugate (FIG. 34C) is likely due to the greater
hydrophobicity of Dol10.
[0463] LC-MS analysis was also conducted with an anti-HER antibody
conjugated with two different drug-linkers (AO-MMAE or
AO-PEG8-Dol10) at 30 mg scale. This analysis showed similar DAR
values of 1.7 and 1.5 following conjugation, which is comparable to
HIC analysis. Size-exclusion chromatograpy (SEC) showed very low
levels (1%) of aggregates in these conjugates.
Example 16 Galactose-Mediated (GAM) Conjugation of Reactive
Effector Moieties
[0464] The galactose aldehyde generated with galactose oxidase on
the A114N antiTEM1 hyperglycosylation mutant antibody as described
in Example 13 was conjugated with 24 molar excess of
aminooxy-MC-VC-PABC-MMAE drug-linker over antibody by overnight
incubation at 25.degree. C., yielding a ADC conjugate with a DAR of
1.72.
[0465] To the galactose oxidase-treated anti-HER antibody prepared
as described in Example 13, one tenth reaction volume of 1M sodium
acetate, pH5.6, was added to adjust the pH to 5.6 and DMSO was
added to make the final concentration of 14% before adding 24eq.
aminooxy MC-VC-PABC-MMAE drug linker. The reactions were incubated
for overnight at room temperature. Free drug and drug-linker were
scavenged with Biobeads and the product buffer exchanged by SEC
(65% yield). The product conjugate was analyzed by HIC. As shown in
FIG. 35, AO-MMAE had been conjugated to -60% of the molecules.
Example 17. In Vitro ADC Cell Proliferation Assays
[0466] The in vitro activity of the anti-HER and anti-FAP
glycoconjugate molecules were also compared with corresponding
thiol conjugates containing the same drug moiety linked via thiol
linkages to hinge region cysteines of the same donor antibody. The
thiol conjugates contained approximately twice the number of drugs
per antibody (DAR) than the glycoconjugates. Thiol-based
conjugation was performed as described by Stefano et al (Methods in
Molecular Biology 2013, in press). Her2+SK-BR-3 and Her2-
MDA-MB-231 cell lines were then employed to evaluate the relative
efficacy of each ADC. The results of this analysis are presented in
Table 15 below
TABLE-US-00014 TABLE 15 EC.sub.50 comparison of glycoconjugates and
thiol conjugates DAR EC.sub.50 (ng/ml) Anti-HER-MC-VC-PABC-MMAE
3.8* 2.3 (Thiol MMAE) Anti-HER-AO-Cys-MC-VC-PABC-MMAE 1.7* 4.7
(Glyco MMAE) Anti-HER-MC-VC-PABC-PEG8-Dol10 3.9* 0.45 (Thiol Dol10)
Anti-HER-AO-Cys-MC-VC-PABC-PEG8-Dol10 1.5* 0.97 (Glyco Dol10) Anti
FAP B11-MC-VC-PABC-MMAE 3.3** 382.4 (Thiol MMAE), CHO + FAP Anti
FAP B11-AO-Cys-MC-VC-PABC-MMAE 1.5** 682.4 (Glyco MMAE), CHO + FAP
Note: *DAR determined by LC-MS; **DAR determined by HIC
[0467] FIG. 36A-D shows a comparison of in vitro potency of
anti-HER glycoconjugate and its counterpart thiol conjugate. Cell
viability was determined following 72 hr exposure of the conjugates
to Her2 antigen expressing (SK-BR-3) cells (FIGS. 36A and C) or
non-expressing (MDA-MB-231) cells (FIGS. 36B and D). The ADCs
contained either MMAE or PEG8-Dol10 linked to the glycans ("glyco")
or by conventional chemistry to hinge region cysteines ("thiol").
As shown in FIGS. 36A and C, .about.2-fold lower EC.sub.50 was
observed for the thiol conjugates compared to the glycoconjugates,
which is consistent with 2-fold higher DAR in the former than the
latter. No toxicity was observed with the Her2- cell line with any
antibody up to 100 ug/ml.
[0468] Similar trends were also observed in the cell proliferation
for ADC prepared with antibodies against a tumor antigen (FAP)
which is highly expressed by reactive stromal fibroblasts in
epithelial cancers including colon, pancreatic and breast cancer
(Teicher, B. A. (2009) Antibody-drug conjugate targets. Curr Cancer
Drug Targets 9, 982-1004). These conjugates were again prepared by
conjugating either aminooxy MMAE drug-linker or maleimido MMAE
drug-linker to glycans or a thiol group. Cell proliferation assays
of these conjugates showed that EC.sub.50 of the thiol conjugate
had .about.100-fold higher potency on the CHO cells transfected
with human FAP than the same cells lacking FAP expression as
depicted in FIG. 37, which shows a comparison of in vitro potency
of anti FAP B11 glycoconjugate and thiol conjugate. Cell viability
was determined following exposure of the conjugates to CHO cells
transfected with or without FAP antigen. The ADCs contained MMAE
linked to the glycans ("glyco") or by conventional chemistry to
hinge region cysteines ("thiol"). Note that the .about.2-fold lower
EC50 for the thiol compared to the glycoconjugates is consistent
with the relative amounts of drug delivered per antibody assuming
similar efficiencies for target binding and internalization in
antigen expressing CHO cells. In parallel, a glycoconjugate of anti
FAP (B11) ADC with a DAR of 1.5 as described previously was assayed
and showed an .about.2-fold higher EC.sub.50 than comparator thiol
conjugate (DAR 3.3).
[0469] As shown in FIG. 41, similar trends were observed in the
cell proliferation assay for ADC prepared with the anti-HER
antibody bearing the A114N hyperglycosylation mutation and AO-MMAE
as described in Example 16, when assayed on SK-BR-3 expressing
cells or MDA-MB-231 cells. The A114N glycoconjugate clearly shows
enhanced cell toxicity against the Her2 expressing cell line over
the non-expressing line. The relative toxicity compared to the
SialT glycoconjugate prepared with the same antibody is consistent
with the lower drug loading of this preparation.
[0470] A cell proliferation assay was also performed for ADC
prepared with the anti-TEM1 antibody bearing the A114N
hyperglycosylation mutation and AO-MMAE prepared as described in
Example 16. Higher toxicity was observed with the TEM1-expressing
cells lines SJSA-1 and A673 compared to the non-expressing
MDA-MB-231 line. The level of toxicity compared with a conventional
thiol conjugate with the same antibody was in keeping with the drug
loading (DAR) of this preparation.
TABLE-US-00015 A673- A673- MDA- SJSA-1 RPMI DMEM-RPMI MB-231 IC50
IC50 IC50 IC50 antiTEM1 3 .mu.g/ml 3.2 .mu.g/ml 2.2 .mu.g/ml 40
.mu.g/ml A114N-AO-MC-VC- PABC-MMAE antiTEM1-MC-VC- 4 .mu.g/ml 1
.mu.g/ml 0.9 .mu.g/ml 20 .mu.g/ml PABC-MMAE
[0471] In summary, the site-specific conjugation of the drugs
through the glycans with cleavable linkers produces ADCs with
toxicities and in vitro efficacy that are equivalent to
conventional thiol-based conjugates, as demonstrated using
different antibodies and different drug-linkers. Moreover, below 2
mM periodate, the level of drug conjugation correlates with the
reduction of sialic acid. Increasing periodate concentration above
2 mM produces little benefit, as expected from the complete
conversion of sialic acid to the oxidized form. However, under all
conditions, the number of drugs per antibody was slightly lower
than the sialic acid content, indicating that some of the oxidized
sialic acids may similarly not be available for coupling, either
because of being buried or otherwise due to steric hindrance
arising from the bulk of the drug-linker.
Example 18. In Vivo Characterization of Antibody Drug
Conjugates
[0472] Efficacy of anti-HER glycoconjugates were also evaluated in
a Her2+ tumor cell xenograft mode and compared with thiol conjugate
comparators having .about.2-fold higher DAR. Beige/SCID mice were
implanted with SK-OV-3 Her2+ tumor cells which were allowed to
establish tumors of .about.150 mm.sup.3 prior to initiation of
treatment. ADCs at 3 or 10 mg/kg doses were injected through tail
vein on days 38, 45, 52 and 59. There were .about.10 mice per
group. The tumor volume of mice in different group was measured and
their survival was recorded. The survival curve was plotted based
on Kaplan-Meier method.
[0473] FIG. 38A-D shows a comparison of in vivo efficacy of the
anti-HER glycoconjugates and thiol conjugates in a Her2+ tumor cell
xenograft model. Beige/SCID mice implanted with SK-OV-3 Her2+ tumor
cells were dosed with MMAE (FIGS. 38A and B) and PEG8-Dol10 (FIGS.
38C and D) containing glycoconjugates or a thiol conjugate
comparators with .about.2-fold higher DAR. The tumor growth
kinetics of the MMAE conjugates is shown in FIG. 38A. In this case,
the glycoconjugate showed a significantly higher efficacy than the
naked antibody alone (black) but less than a thiol conjugate
comparator having a .about.2-fold higher DAR (green). The MMAE
glycoconjugate showed significant tumor regression and a .about.20
day delay in tumor growth (FIG. 38A) and .about.2-fold increase in
survival time from first dose (FIG. 38B). The thiol MMAE conjugate
showed near-complete tumor suppression at the same dose of ADC (10
mg/kg).
[0474] The in vivo efficacy of a PEG8-Dol10 glycoconjugate ("Glyco
Dol10`) and a thiol conjugate comparator with .about.2-fold higher
DAR ("Thiol Dol10") was also determined in the same Her2+ tumor
cell xenograft model. Both conjugates showed lower efficacy than
MMAE conjugates as described previously. However, the
aminooxy-PEG8-Dol10 glycoconjugate ("Glyco Dol10") at 10 mg/kg
showed a 15-day delay in tumor growth (FIG. 38C) and .about.20 day
(1.7-fold) increase in survival time following first administration
(FIG. 38D). The thiol conjugate was more efficacious at the same
dose, showing a 2-fold increase in survival. At a lower dose (3
mg/kg), the thiol conjugate showed a lesser efficacy than the
glycoconjugate at 10 mg/kg. This dose corresponds to 80 umol
PEGS-Dol10 drug per kg dose, compared to 110 umol PEGS-Dol10 drug
per kg dose for the glycoconjugate.
[0475] These data demonstrate that site-specific conjugation of
drugs onto sialic acid of antibody glycans yields molecules with
comparable potency as ADCs generated via thiol-based chemistry. The
somewhat lower in vivo efficacy likely stems from the fewer number
of drugs which are carried by each antibody into the tumor cells by
the internalization of each antibody-bound antigen. Although we
have not compared these glycoconjugates with thiol conjugates of
the same DAR, the efficacy observed at different doses of the two
ADCs representing comparable levels of administered drug shows that
the glycoconjugates have comparable intrinsic efficacy as their
thiol counterparts, indicating no deleterious effect of conjugation
at this site. Moreover, a 10 mg/kg dose of the Dol10 glycoconjugate
which introduced only 28% more drug provided a 2-fold increase in
survival over the thiol conjugate (at 3 mg/kg), suggesting these
conjugates may even provide superior efficacies at the same DAR.
Given the apparent limitation in sialic acid incorporation at
native glycans, higher drug loading could be achieved by a number
of different strategies including the use of branched drug linkers
or the introduction of additional glycosylation sites and using the
same method.
Example 19. Conjugation of Targeting Moieties
[0476] FIG. 42 demonstrates the overall scheme for the conjugation
of targeting moieties to existing carbohydrates or engineered
glycosylation sites. This conjugation can be performed through the
attachment of neoglycans, glycopeptides, or other targeting
moieties to oxidized sialylated antibodies (FIGS. 43 and 44).
Moieties suitable for conjugation may include those containing
aminooxy linkers (FIGS. 45 and 46).
Example 20. Conjugation Through Sialic Acid in Native Fc
Glycans
[0477] Mannose-6-P hexamannose aminooxy was conjugated to either a
polyclonal antibody or monoclonal antibody specifically targeting a
Man-6-P receptor. The SDS-PAGE and Maldi-TOF analyses of the
conjugation of the anti-Man-6-P-receptor rabbit polyclonal antibody
with Man-6-P hexamannose aminooxy is shown in FIG. 47. FIG. 48
depicts the results of surface plasmon resonance experiments used
to assess the binding of control and Man-6-P hexamannose conjugated
anti-Man-6-P-receptor rabbit polyclonal IgG antibodies to M6P
receptor. In vitro analyses of this conjugated antibody
demonstrates increased uptake into both HepG2 (Homo sapiens liver
hepatocellular carcinoma) and RAW (Mus musculus murine leukemia)
cell lines (FIG. 49). Cultures were stained with anti-rabbit-Alexa
488 antibody counterstained with DAPI.
[0478] Antibodies conjugated with M6P or lactose aminooxy moieties
were further tested through SDS-PAGE and lectin blotting and
compared with unconjugated antibodies (FIG. 50). MALDI-TOF intact
protein analyses of the control and conjugated antibodies
demonstrate that the conjugates have approximately two glycan
moieties per antibody, while control antibodies have none (FIG.
51).
Example 21. Conjugation Through Sialic Acid to Hinge Cysteine
Residues in Antibody
[0479] Mannose-6-P hexamannose maleimide was conjugated to either a
polyclonal antibody or monoclonal antibody specifically targeting a
Man-6-P receptor.
[0480] The conjugation of a polyclonal antibody with Man-6-P
hexamannose maleimide through hinge cysteines was examined through
SDS-PAGE, lectin blotting, and M6P quantitation (to determine the
number of glycans conjugated per antibody) (FIG. 52). Conjugation
of a polyclonal antibody with lactose maleimide was also examined
through the use of SDS-PAGE and galactose quantitation of the
control antibody, conjugated antibody, and filtrate are shown in
FIG. 53. Little increased aggregation was observed in hinge
cysteine-conjugated polyclonal antibodies by size exclusion
chromatography (SEC) (FIG. 55).
[0481] The conjugation of a monoclonal antibody with Man-6-P
hexamannose maleimide through hinge cysteines was also examined
through SDS-PAGE and glycan quantitation (to determine the number
of glycans conjugated per antibody) (FIG. 54). Little increased
aggregation was observed in hinge cysteine-conjugated polyclonal
antibodies by size exclusion chromatography (SEC) (FIG. 56).
[0482] The conjugation of bisM6P hexasaccharide to polyclonal and
monoclonal antibodies through native Fc glycans or hinge disulfides
was also examined through native PAGE (FIG. 60).
Example 22. Preparation of Sialylated Monoclonal Antibody and
Conjugation to a Trigalactosylated Glycopeptide or Glycopeptide
[0483] A mouse monoclonal antibody mutant with an STY mutation
(NNAS ("NNAS" disclosed as SEQ ID NO: 40)) was modified with
sialidase and galactosyltransferase for making mainly native
trigalactosylated glycans (2 glycans per antibody). The same mutant
was also sialylated with sialyltransferase and conjugated with a
glycopeptide using SAM approach. The sialic acid content of the
enzyme modified antibodies was examined (FIG. 57). Further,
MALDI-TOF analysis of the glycans released from control and
desialylated/galactosylated (FIG. 58) NNAS ("NNAS" disclosed as SEQ
ID NO: 40) as well as the glycans released from control and
sialylated (FIG. 59) NNAS ("NNAS" disclosed as SEQ ID NO: 40) were
examined. SDS-PAGE (4-12% NuPAGE) and lectin blotting of enzyme
modified and conjugated NNAS ("NNAS" disclosed as SEQ ID NO: 40)
are shown in FIG. 61. Terminal galactose quantitation was also
measured for the control NNAS ("NNAS" disclosed as SEQ ID NO: 40)
antibody, desialylated/galactosylated NNAS ("NNAS" disclosed as SEQ
ID NO: 40) antibody, and conjugated NNAS ("NNAS" disclosed as SEQ
ID NO: 40) antibody (FIG. 62).
Example 23. Preparation of .alpha.2,3 Sialylated Lactose Maleimide
Using a Chemoenzyme Approach and Subsequent Conjugation to
Non-Immune Rabbit IgG Through Hinge Disulfides
[0484] As carbohydrate-binding proteins (including Siglec proteins)
prefer multivalent binding for strong interaction, the
monosialylated glycans on a given antibody may not provide enough
sialic acid density for other Siglec proteins. Therefore, a hinge
disulfide conjugation approach for introducing multiple copies of
sialylated glycans was investigated. To produce sialylated glycans
for conjugation, lactose maleimide (5 mg) was sialylated in vitro
with .alpha.2,3 sialyltransferase from Photobacterium damsela in
Tris buffer (pH 7.5), for 2 hrs at 37.degree. C. A control glycan
was incubated without sialyltransferase and compared with the
original glycans. MALDI-TOF analysis showed that the incubation of
lactose maleimide without enzyme in Tris buffer (pH 7.5) for 2 hrs
at 37.degree. C. did not change the expected molecular weight of
the molecule, suggesting that the examined condition did not result
in maleimide hydrolysis. The MALDI-TOF and Dionex HPLC analysis of
glycans modified with .alpha.2,3 sialyltransferase indicate the
presence of sialyllactose, although not as major peak (data not
shown). Therefore, the sialyllactose maleimide was additionally
purified using QAE-sepharose columns and each fraction was
subsequently analyzed using MALDI-TOF and Dionex HPLC. These
analyses indicated that sialyllactose maleimide existed as major
species in the 20 mM NaCl eluate from QAE column (FIG. 63). The
amount of sialylated glycans purified was estimated using sialic
acid quantitation analysis of the samples, indicating a recovery of
.about.1.8 mg sialyllactose maleimide.
[0485] Subsequent conjugation of a rabbit polyclonal antibody with
this sialyllactose maleimide was tested using thiol chemistry. A
rabbit IgG antibody (1 mg) was reduced with TCEP at a 4 molar
excess (over the antibody) for 2 hrs at 37.degree. C. before being
conjugated to 24 molar excess of sialyllactose for 1 hr at room
temperature. The conjugate was then buffer-exchanged into PBS for
analysis on SDS-PAGE (FIG. 64A). Sialic acid quantitation was also
performed using Dionex HPLC (FIG. 64B). Aliquots of control and
thiol conjugate were treated with or without sialidase (1 U per mg)
overnight at 37.degree. C. before supernatants were recovered
through filtration (10 kDa MWCO). The sialic acid content of the
supernatants was measured and compared to samples treated without
sialidase. There are approximately 4 .alpha.2,3 sialyllactose
moieties coupled per antibody.
Example 24. Preparation of .alpha.2,6 Sialyllactose Maleimide by
Silylating Lactose Maleimide and Conjugation to Hinge Disulfides of
a Rabbit Polyclonal Antibody Through .alpha.2,3- or
.alpha.2,6-Linkages Resulting in High Sialylation
[0486] The conjugation of multiple copies of either .alpha.2,3- or
.alpha.2,6-sialylated glycans to the hinge disulfides of a rabbit
polyclonal antibody was investigated. Since the .alpha.2,3
sialyllactose maleimide was successfully produced using a
chemoenzyme approach (see above, Example 23), a similar method was
used to produce .alpha.2,6 sialyllactose maleimide (minor
modifications of the protocol included the use of a different
sialyltransferase). To produce .alpha.2,6 sialylated glycan for
conjugation, lactose maleimide (.about.5 mg) was sialylated in
vitro with 0.5 U of a bacterial .alpha.2,6 sialyltransferase from
Photobacterium damsela in Tris buffer (pH 8) for 1 hr at 37.degree.
C. After enzymatic reaction, the product was applied to a
QAE-sepharose column. The column was washed with 10 fractions of 1
ml 2 mM Tris (pH 8), 5 fractions of 1 ml of Tris buffer containing
20 mM NaCl, and 5 fractions of 1 ml Tris buffer containing 70 mM
NaCl. The aliquots from each fraction were analyzed using Dionex
HPLC alongside lactose and .alpha.2,6 sialyllactose standards. The
oligosaccharide profiles of the standards and one of the eluted
fractions are shown in FIG. 65 (A-D). The fractions containing
.alpha.2,6 sialyllactose maleimide were also analyzed and confirmed
by MALDI-TOF. The glycan in one of the fractions can be seen in
FIG. 66.
[0487] The amount of .alpha.2,6 sialyllactose maleimide purified
was then estimated using sialic acid quantitation analysis which
indicated a recovery of .about.1.5 mg sialyllactose maleimide. Once
the glycan was prepared, the conjugation of antibody with either
.alpha.2,6 sialyllactose maleimide or .alpha.2,3 sialyllactose
maleimide was tested using thiol chemistry. A rabbit polyclonal IgG
antibody (1 mg) was buffer-exchanged and reduced with TCEP at a 4
molar excess (over antibody) for 2 hrs at 37.degree. C. The reduced
antibody was then split in half: one portion was conjugated to 24
molar excess of .alpha.2,6 sialyllactose maleimide, and the other
to .alpha.2,3 sialyllactose maleimide for 1 hr at room temperature.
The two conjugates were then buffer-exchanged into PBS before
SDS-PAGE analysis (FIG. 67, A) and sialic acid quantitation using
Dionex HPLC (FIG. 67, B). Sialic acid quantitation was used to
estimate the number of glycan conjugated. Aliquots of control
antibody and thiol-conjugated antibody were treated with or without
sialidase (1 U per mg) overnight at 37.degree. C. before
supernatants were recovered through filtration (10 kDa MWCO). The
sialic acid content of the supernatants was measured and compared
to samples treated without sialidase (control). The analysis
demonstrated that approximately 7 glycans (either .alpha.2,3- or
.alpha.2,6-sialyllactose glycans) were conjugated to the polyclonal
antibody by this method.
Example 25. PEGylation of NNAS ("NNAS" Disclosed as SEQ ID NO: 40)
Using GAM Chemistry
[0488] A mouse NNAS ("NNAS" disclosed as SEQ ID NO: 40)
(S298N/T299A/Y300S) mutant monoclonal antibody was galactosylated
and disialylated, generating a Gal NNAS ("NNAS" disclosed as SEQ ID
NO: 40) monoclonal antibody without any protease degradation. This
antibody was modified with galactose oxidase (GAO) to generate
galactose aldehyde. The galactose aldehyde was then conjugated with
2 or 5 kDa of aminooxy polyethylene glycol (PEG). FIG. 68 depicts
the characterization of control and enzyme modified
(disalylated/galactosylated) NNAS ("NNAS" disclosed as SEQ ID NO:
40) mutant antibodies using SDS-PAGE and lectin blotting. FIG. 69
depicts the characterization through reducing SDS-PAGE of the
PEGylation of a control antibody and Gal NNAS ("NNAS" disclosed as
SEQ ID NO: 40) with various amounts of galactose oxidase. These
results demonstrate that Gal NNAS ("NNAS" disclosed as SEQ ID NO:
40) can be PEGylated efficiently with significant amounts of mono-,
bi-, and tri-PEG conjugated per heavy chain. FIG. 71 depicts the
characterization through reducing SDS-PAGE of the PEGylation of a
control antibody and Gal NNAS ("NNAS" disclosed as SEQ ID NO: 40)
with various molar excess of PEG over antibody. Protein Simple
scans characterizing the PEGylation the antibodies demonstrate that
approximately 1.5-1.7 PEG moieties are conjugated per heavy chain
(or about 3-3.4 PEG per antibody) (FIGS. 70 and 72).
Example 26. PEGylation of NNAS ("NNAS" Disclosed as SEQ ID NO: 40)
Using GAM Chemistry
[0489] An NNAS ("NNAS" disclosed as SEQ ID NO: 40) antibody was
galactosylated with 50 mU/mg galactosyltransferase and subsequently
desialylated with 1 U/mg sialidase in 50 mM MES buffer (pH 6.5).
Desialylated fetuin and NNAS ("NNAS" disclosed as SEQ ID NO: 40) as
well as galactosylated NNAS ("NNAS" disclosed as SEQ ID NO: 40)
were then treated with galactose oxidase (57 mU/mg)/catalase in the
presence or absence of 0.5 mM copper acetate before conjugation
with 25 molar excess of 5 kDa aminooxy PEG (FIG. 74, A). In another
experiment, galactosylated NNAS ("NNAS" disclosed as SEQ ID NO: 40)
was treated with galactose oxidase (57 mU/mg)/catalase in the
presence of 0, 0.02, 0.1 and 0.5 mM copper acetate before
conjugation with 25 molar excess of 5 kDa aminooxy PEG (FIG. 74,
B). Antibody oxidized with galactose oxidase in the presence of
copper acetate showed a higher degree of PEGylation than the same
antibody reacted with galactose oxidase in the absence of copper
acetate. Significantly higher levels of PEGylation were observed
when the conjugation was performed in a reaction containing copper
sulfate in concentrations above 0.1 mM.
Example 27. Modification of Wild-Type and Mutant Herceptin Using
Sialidase/Galactosyltransferase
[0490] Wild-type and mutant (A114N, NNAS ("NNAS" disclosed as SEQ
ID NO: 40), and A114N/NNAS) Herceptin antibodies were
enzymaticically modified with 50 mU/mg galactosyltransferase and
subsequently desialylated with 1 U/mg sialidase in 50 mM MES buffer
(pH 6.5). The modified antibodies were analyzed using SDS-PAGE
(reducing and nonreducing), lectin blotting with ECL (a plant
lectin specific for terminal galactose), and terminal galactose
quantitation using Dionex HPLC analysis of released galactose by
galactosidase (FIG. 75). Enzyme modified antibodies containing
approximately three to nine terminal galactose were obtained with
the NNAS ("NNAS" disclosed as SEQ ID NO: 40) and NNAS ("NNAS"
disclosed as SEQ ID NO: 40)/A114N double mutants demonstrating a
higher level of terminal galactose than the wild-type and A114N
mutant.
Example 28. PEGylation of Wild-Type and Mutant Antibodies Using the
SAM Conjugation Method
[0491] Wild-type and (A114N, NNAS ("NNAS" disclosed as SEQ ID NO:
40), and A114N/NNAS ("NNAS" disclosed as SEQ ID NO: 40)) Herceptin
antibodies were PEGylated using sialic acid-mediated (SAM)
conjugation. The antibodies were subsequently oxidized with 2 mM
periodate. After buffer exchange, the oxidized antibodies were
PEGylated with 25 molar excess of 5 kDa aminooxy PEG. The sialic
acid content of the wild-type and mutant antibodies was measured
using Dionex HPLC (FIG. 76). The PEGylated antibodies were then
analyzed using reducing and non-reducing SDS-PAGE (FIG. 77).
Further, the PEGylation (PAR, number of PEG per antibody) was
estimated by analyzing the scanned gels using ProteinSimple (FIG.
78). The NNAS ("NNAS" disclosed as SEQ ID NO: 40), A114N, and
A114N/NNAS ("NNAS" disclosed as SEQ ID NO: 40) mutants all showed
higher PAR (2.7-4.6) than wild-type Herceptin antibodies (1.4).
Example 29. Investigation of Uptake of Glycoengineered Antibodies
with Galactose Containing Glycan Ligands
[0492] A polyclonal antibody was either enzymatically modified with
galactosyltransferase (Gal Transferase), conjugated to lactose
aminooxy (Gal-Glc to 297: conjugated to sialic acid in glycans from
Asn-297 of sialylated antibody), or conjugated to lactose maleimide
(Gal-Glc to Hinge: conjugated to cysteines in hinge disulfides).
The control, modified, or conjugated antibodies were then incubated
with HepG2 cells (a hepatocyte cell line expressing ASGPR) for 1-2
hrs at 37.degree. C. before the uptaken antibodies were measured
using Immunofluorescence staining (FIG. 79). The results showed
increased HepG2 cell uptake of enzymatic modified or lactose
conjugated antibodies.
Example 30. Conjugation of a Trivalent GalNAc Glycan to
Herceptin
[0493] Herceptin (anti-Her2) was sialylated and conjugated with a
trivalent GalNAc glycan (FIG. 80) for targeting ASGPR using the SAM
approach. Subsequently, surface plasmon resonance experiments
(Biacore) were used to assesses the binding of these trivalent
GalNAc glycan-conjugated antibodies to ASGPR receptor subunit
H.sub.1 (FIG. 81).
Example 31. Conjugation of Trivalent GalNAc and Trivalent Galactose
to a Recombinant Lysosomal Enzyme
[0494] A recombinant lysosomal enzyme was conjugated with either
trivalent GalNAc glycan or trivalent galactose containing
glycopeptides (FIG. 82) for targeting ASGPR using the SAM
conjugation method. Subsequently, surface plasmon resonance
experiments (Biacore) were used to assesses the binding of these
trivalent GalNAc glycan-conjugated and trivalent
galactose-conjugated enzymes to ASGPR receptor subunit H.sub.1
(FIG. 83). The results showed strong ASGPR binding of trivalent
GalNAc glycan conjugated recombinant lysosomal enzyme.
Example 32. Use of Mannosamine Derivatives, Including ManLev, for
In Vitro Antibody Sialylation and Conjugation
[0495] Mannosamine derivatives, including ManLev, ManNAz, and
ManAz, were used to prepare sialic acid derivatives and then
CMP-sialic acid derivatives for antibody sialylation followed by
site-specific conjugation. The CMP-sialic acid derivatives prepared
were characterized using HPAEC-PAD and used for in vitro antibody
sialylation. Finally, the sialylated antibodies were PEGylated
without periodate oxidation using the SAM approach.
[0496] Sialic acid (0.2 .mu.mol) was titrated with various amounts
of CMP-sialic acid synthetase (N. mentingitidis) at 37.degree. C.
The generation of CMP-sialic acid was monitored using HPAEC-PAD as
compared to the retention time of CMP-sialic acid standard. The
CMP-sialic acid synthesized versus the amounts of enzyme used was
plotted and demonstrates that generation of CMP-sialic acid is
saturated by CMP-sialic acid synthetase at 5 mU per 0.2 .mu.mol
(FIG. 84).
[0497] ManNAc or ManLev (0.2 .mu.mol) was titrated with various
amounts of sialic acid aldolase (E. coli K-12) at 37.degree. C. The
generation of sialic acid (from ManNAc) or sialic acid derivative
(from ManLev) was monitored using HPAEC-PAD as compared to the
retention time of sialic acid standard. The synthesized sialic acid
or sialic acid derivative vs the amounts of the enzyme used are
shown in FIG. 85 (MacNAc) and FIG. 86 (ManLev).
[0498] In order to characterize sialic acid derivatives using
HPAEC-PAD, the CMP-sialic acid (from ManNAc) or CMP-sialic acid
derivative (from ManLev) was first digested with sialidase at
37.degree. C. The released sialic acid or sialic acid derivative
was monitored using HPAEC-PAD as compared to the retention time of
sialic acid standard and the identity of sialic acid was also
confirmed by disappearance of the sialic acid peak after periodate
treatment (FIG. 87). Sialic acid derivative (from ManLev) was
eluted later than sialic acid.
[0499] CMP-sialic acid (from ManNAc) and CMP-sialic acid
derivatives (from ManLev, ManNAz, ManAz) were also analyzed
directly on HPAEC-PAD without sialidase pretreatment. The
generation of CMP-sialic acid was compared to the retention time of
CMP-sialic acid standard. The CMP-sialic acid derivatives, produced
from ManLev, ManNAz, and ManAz, showed different retention time
compared to CMP-sialic acid standard (FIGS. 88 and 89).
[0500] Further, Herceptin was sialylated in vitro using .alpha.2,6
sialyltransferase and CMP-sialic acid derivatives. FIG. 90 is a
schematic representation demonstrating the sialylation of Herceptin
using a sialic acid derivative prepared from ManLev. The
sialylation was analyzed using LC-MS of CH.sub.2CH.sub.3 fragments
released by IdeS protease. FIG. 91 demonstrates the sialylation of
Herceptin with the sialic acid derivative prepared from ManLev
(with correct mass).
[0501] Finally, Herceptin sialylated with sialic acid derivatives
prepared from ManLev and ManNAz was PEGylated. FIG. 92 is a
schematic representation demonstrating the PEGylation of Herceptin
sialylated with a sialic acid derivative prepared from ManLev.
First, the Herceptin was sialylated in vitro using .alpha.2,6
sialyltransferase and CMP-sialic acid derivatives prepared from
ManLev. Subsequently, the sialylated antibodies were mixed with 5
kDa aminooxy PEG. The sialylated and PEGylated antibodies were then
analyzed using SDS-PAGE under reducing and non-reducing conditions.
An SDS-PAGE analysis of sialylated Herceptin PEGylated with a
sialic acid derivative prepared from ManLev is seen in FIG. 93.
FIG. 94 is a schematic representation demonstrating the sialylation
of antibody with a sialic acid derivative prepared from ManNAz. An
SDS-PAGE analysis of PEGylated Herceptin pre-sialylated with a
sialic acid derivative prepared from ManNAz in shown FIG. 95.
Sequence CWU 1
1
401218PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu
Ser Val Thr Pro Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser
Gln Ser Leu Leu Tyr Ser 20 25 30Asn Gly Lys Thr Tyr Leu Asn Trp Leu
Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Arg Leu Ile Tyr Leu Val
Ser Lys Leu Asp Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala
Glu Asp Val Gly Val Tyr Tyr Cys Val Gln Gly 85 90 95Thr His Leu His
Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg 100 105 110Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120
125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 2152443PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 2Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser1 5 10 15Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Trp 20 25 30Met Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 35 40 45Gln Ile Arg
Leu Lys Ser Asn Asn Tyr Ala Thr His Tyr Ala Glu Ser 50 55 60Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser Leu65 70 75
80Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr
85 90 95Cys Thr Pro Val Asp Phe Trp Gly Gln Gly Thr Thr Val Thr Val
Ser 100 105 110Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser 115 120 125Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp 130 135 140Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr145 150 155 160Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 180 185 190Thr Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 195 200
205Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
210 215 220Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro225 230 235 240Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr 245 250 255Cys Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn 260 265 270Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 290 295 300Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser305 310 315
320Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
325 330 335Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp 340 345 350Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe 355 360 365Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu 370 375 380Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe385 390 395 400Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 405 410 415Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 4403444PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
3Glu 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 Thr Phe Asn Thr
Tyr 20 25 30Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Gly Gln Ile Arg Leu Lys Ser Asn Asn Tyr Ala Thr His
Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys Asn Ser65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr
Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Pro Val Asp Phe Trp Gly
Gln Gly Thr Thr Val Thr Val 100 105 110Ser Ser Asn Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser 115 120 125Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 130 135 140Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu145 150 155
160Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
165 170 175Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr 180 185 190Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val 195 200 205Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr His Thr Cys Pro 210 215 220Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe225 230 235 240Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 260 265 270Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280
285Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
290 295 300Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val305 310 315 320Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala 325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg 340 345 350Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser385 390 395
400Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
405 410 415Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His 420 425 430Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 4404444PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 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 Thr Phe Asn Thr Tyr 20 25 30Trp Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Gln Ile Arg Leu Lys Ser Asn
Asn Tyr Ala Thr His Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asp Ser Lys Asn Ser65 70 75 80Leu Tyr Leu Gln Met
Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Pro
Val Asp Phe Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 115 120
125Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
130 135 140Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu145 150 155 160Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu 165 170 175Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr 180 185 190Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val 195 200 205Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 210 215 220Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe225 230 235
240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe 260 265 270Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr 290 295 300Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val305 310 315 320Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 340 345 350Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360
365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser385 390 395 400Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln 405 410 415Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His 420 425 430Ser Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 435 4405444PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 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 Thr Phe Asn Thr Tyr 20 25 30Trp Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Gln Ile
Arg Leu Lys Ser Asn Asn Tyr Ala Thr His Tyr Ala Glu 50 55 60Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser65 70 75
80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95Tyr Cys Thr Pro Val Asp Phe Trp Gly Gln Gly Thr Thr Val Thr
Val 100 105 110Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser 115 120 125Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys 130 135 140Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu145 150 155 160Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 165 170 175Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 180 185 190Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 195 200
205Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
210 215 220Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe 260 265 270Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315
320Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 340 345 350Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430Tyr
Thr Gln Lys Asn Leu Ser Leu Ser Pro Gly Lys 435
4406444PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 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 Thr Phe Asn Thr Tyr 20 25 30Trp Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Gln Ile Arg Leu Lys Ser Asn
Asn Tyr Ala Thr His Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asp Ser Lys Asn Ser65 70 75 80Leu Tyr Leu Gln Met
Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Pro
Val Asp Phe Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 115 120
125Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
130 135 140Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu145 150 155 160Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu 165 170 175Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr 180 185 190Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val 195 200 205Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 210 215 220Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe225 230 235
240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe 260 265 270Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr 290 295 300Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val305 310 315 320Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 340 345 350Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360
365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser385 390 395 400Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln 405
410 415Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His 420 425 430Tyr Thr Gln Lys Ser Leu Asn Leu Ser Pro Gly Lys 435
4407447PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 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 Thr Phe Asn Thr Tyr 20 25 30Trp Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Gln Ile Arg Leu Lys Ser Asn
Asn Tyr Ala Thr His Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asp Ser Lys Asn Ser65 70 75 80Leu Tyr Leu Gln Met
Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Pro
Val Asp Phe Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 115 120
125Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
130 135 140Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu145 150 155 160Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu 165 170 175Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr 180 185 190Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val 195 200 205Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 210 215 220Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe225 230 235
240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe 260 265 270Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr 290 295 300Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val305 310 315 320Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 340 345 350Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360
365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser385 390 395 400Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln 405 410 415Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His 420 425 430Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys Asn Gly Thr 435 440 4458444PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
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 Thr Phe Asn Thr
Tyr 20 25 30Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Gly Gln Ile Arg Leu Lys Ser Asn Asn Tyr Ala Thr His
Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys Asn Ser65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr
Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Pro Val Asp Phe Trp Gly
Gln Gly Thr Thr Val Thr Val 100 105 110Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser 115 120 125Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 130 135 140Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu145 150 155
160Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
165 170 175Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr 180 185 190Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val 195 200 205Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr His Thr Cys Pro 210 215 220Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe225 230 235 240Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 260 265 270Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280
285Arg Glu Glu Gln Tyr Asn Asn Thr Ser Arg Val Val Ser Val Leu Thr
290 295 300Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val305 310 315 320Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala 325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg 340 345 350Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser385 390 395
400Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
405 410 415Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His 420 425 430Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 4409215PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 9Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu
Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser
Gln Ser Val Ser Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala
Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95Trp Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120
125Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
130 135 140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
Asn Ser145 150 155 160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu 165 170 175Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val 180 185 190Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205Ser Phe Asn Arg Gly
Glu Cys 210 21510454PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 10Gln Val Gln Leu Gln Glu Ser Ala
Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr
Val Ser Gly Gly Ser Ile Arg Ser Tyr 20 25 30Tyr Trp Ser Trp Ile Arg
Gln Pro Pro Gly Lys Gly Leu Glu Tyr Ile 35 40 45Gly Tyr Ile Tyr Tyr
Thr Gly Ser Ala Ile Tyr Asn Pro Ser Leu Gln 50 55 60Ser Arg Val Thr
Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu
Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg
Glu Gly Val Arg Gly Ala Ser Gly Tyr Tyr Tyr Tyr Gly Met Asp 100 105
110Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly 130 135 140Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro145 150 155 160Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr 165 170 175Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 210 215 220Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu225 230
235 240Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp 245 250 255Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp 260 265 270Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly 275 280 285Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn 290 295 300Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp305 310 315 320Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345
350Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
355 360 365Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile 370 375 380Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr385 390 395 400Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys 405 410 415Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445Ser Leu Ser
Pro Gly Lys 45011454PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 11Gln Val Gln Leu Gln Glu Ser Ala
Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr
Val Ser Gly Gly Ser Ile Arg Ser Tyr 20 25 30Tyr Trp Ser Trp Ile Arg
Gln Pro Pro Gly Lys Gly Leu Glu Tyr Ile 35 40 45Gly Tyr Ile Tyr Tyr
Thr Gly Ser Ala Ile Tyr Asn Pro Ser Leu Gln 50 55 60Ser Arg Val Thr
Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu
Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg
Glu Gly Val Arg Gly Ala Ser Gly Tyr Tyr Tyr Tyr Gly Met Asp 100 105
110Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Asn Ser Thr Lys
115 120 125Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly 130 135 140Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro145 150 155 160Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr 165 170 175Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 210 215 220Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu225 230
235 240Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp 245 250 255Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp 260 265 270Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly 275 280 285Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn 290 295 300Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp305 310 315 320Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345
350Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
355 360 365Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile 370 375 380Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr385 390 395 400Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys 405 410 415Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445Ser Leu Ser
Pro Gly Lys 45012214PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 12Asp 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 21013450PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 13Glu 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 Asp Glu Leu 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
445Gly Lys 45014450PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 14Glu 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 Asn 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 Asp Glu Leu 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 445Gly Lys
45015450PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 15Glu 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 Asn Ala Ser 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 Asp Glu Leu 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 445Gly Lys
45016450PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 16Glu 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 Asn 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 Asn Ala Ser 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 Asp Glu Leu 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 445Gly Lys
450174PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 17Gly Gly Gly Gly1185PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 18Gly
Gly Gly Gly Gly1 5196PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 19Gly Gly Gly Gly Gly Gly1
5207PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 20Gly Gly Gly Gly Gly Gly Gly1 5218PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 21Gly
Gly Gly Gly Gly Gly Gly Gly1 5225PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 22Gly Gly Gly Gly Ser1
523213PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 23Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Ser Ala Thr
Ser Ser Val Ser Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Arg Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly
Val Pro Ala Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Ser Tyr Thr
Leu Thr Ile Ser Ser Leu Glu Pro Glu65 70 75 80Asp Phe Ala Val Tyr
Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120
125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln Glu145 150 155 160Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr Ala 180 185 190Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205Asn Arg Gly Glu Cys
21024450PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 24Glu Val Gln Leu Leu Gln Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Tyr Lys Phe Thr Ser Tyr 20 25 30Val Met His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp
Val Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys Gly Arg Phe Thr Leu 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 Gly Ser
Tyr Tyr Asp Tyr Asp Gly Phe Val 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 Asp Glu Leu 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 445Gly Lys 45025450PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 25Glu Val Gln Leu Leu Gln
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Tyr Lys Phe Thr Ser Tyr 20 25 30Val Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Tyr Ile
Asn Pro Tyr Asn Asp Val Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys Gly
Arg Phe Thr Leu 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 Gly Ser Tyr Tyr Asp Tyr Asp Gly Phe Val 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 Asn Thr Ser 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 Asp Glu Leu 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
445Gly Lys 45026450PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 26Glu Val Gln Leu Leu Gln Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Tyr Lys Phe Thr Ser Tyr 20 25 30Val Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Tyr Ile Asn Pro
Tyr Asn Asp Val Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys Gly Arg Phe
Thr Leu 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 Gly Ser Tyr Tyr Asp Tyr Asp Gly Phe Val 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 Asn Ala Ser 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 Asp Glu Leu 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 445Gly Lys
45027450PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 27Glu Val Gln Leu Leu Gln Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Tyr Lys Phe Thr Ser Tyr 20 25 30Val Met His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp
Val Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys Gly Arg Phe Thr Leu 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 Gly Ser
Tyr Tyr Asp Tyr Asp Gly Phe Val 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 Gln Asn Thr Ser 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 Asp Glu Leu 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 445Gly Lys
45028218PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 28Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu
Ser Val Thr Pro Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser
Gln Ser Leu Leu Tyr Ser 20 25 30Asn Gly Lys Thr Tyr Leu Asn Trp Leu
Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Arg Leu Ile Tyr Leu Val
Ser Lys Leu Asp Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala
Glu Asp Val Gly Val Tyr Tyr Cys Val Gln Gly 85 90 95Thr His Leu His
Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg 100 105 110Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120
125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 21529444PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 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 Thr Phe Asn Thr Tyr 20 25 30Trp Met Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Gln
Ile Arg Leu Lys Ser Asn Asn Tyr Ala Thr His Tyr Ala Glu 50 55 60Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser65 70 75
80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95Tyr Cys Thr Pro Val Asp Phe Trp Gly Gln Gly Thr Thr Val Thr
Val 100 105 110Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser 115 120 125Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys 130 135 140Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu145 150 155 160Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 165 170 175Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 180 185 190Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 195 200
205Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
210 215 220Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe 260 265 270Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315
320Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 340 345 350Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435
44030444PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 30Glu 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 Thr Phe Asn Thr Tyr 20 25 30Trp Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Gln Ile Arg Leu Lys Ser Asn
Asn Tyr Ala Thr His Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asp Ser Lys Asn Ser65 70 75 80Leu Tyr Leu Gln Met
Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Pro
Val Asp Phe Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 115 120
125Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
130 135 140Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu145 150 155 160Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu 165 170 175Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr 180 185 190Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val 195 200 205Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 210 215 220Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe225 230 235
240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe 260 265 270Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Tyr Asn Asn Thr Ser Arg
Val Val Ser Val Leu Thr 290 295 300Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val305 310 315 320Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 340 345 350Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360
365Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375
380Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser385 390 395 400Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln 405 410 415Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His 420 425 430Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 435 44031218PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 31Asp Ile Val Met Thr Gln
Thr Pro Leu Ser Leu Ser Val Thr Pro Gly1 5 10 15Gln Pro Ala Ser Ile
Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30Asn Gly Lys Thr
Tyr Leu Asn Trp Val Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Arg
Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75
80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Val Gln Gly
85 90 95Ser His Phe His Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
Arg 100 105 110Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln 115 120 125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr 130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200
205Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
21532444PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 32Glu 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 Pro Phe Ser Asn Tyr 20 25 30Trp Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Gln Ile Arg Leu Lys Ser Asn
Asn Tyr Ala Thr His Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asp Ser Lys Asn Ser65 70 75 80Leu Tyr Leu Gln Met
Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Pro
Ile Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 115 120
125Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
130 135 140Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu145 150 155 160Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu 165 170 175Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr 180 185 190Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val 195 200 205Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 210 215 220Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe225 230 235
240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe 260 265 270Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr 290 295 300Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val305 310 315 320Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 340 345 350Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360
365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser385 390 395 400Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln 405 410 415Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His 420 425 430Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 435 44033444PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 33Glu 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 Pro Phe Ser Asn Tyr 20 25 30Trp Met Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Gln
Ile Arg Leu Lys Ser Asn Asn Tyr Ala Thr His Tyr Ala Glu 50 55 60Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser65 70 75
80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95Tyr Cys Thr Pro Ile Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr
Val 100 105 110Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser 115 120 125Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys 130 135 140Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu145 150 155 160Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 165 170 175Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 180 185 190Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 195 200
205Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
210 215 220Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe 260 265 270Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Tyr
Asn Asn Thr Ser Arg Val Val Ser Val Leu Thr 290 295 300Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315
320Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 340 345 350Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435
44034444PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 34Glu 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 Pro Phe Ser Asn Tyr 20 25 30Trp Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Gln Ile Arg Leu Lys Ser Asn
Asn Tyr Ala Thr His Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asp Ser Lys Asn Ser65 70 75 80Leu Tyr Leu Gln Met
Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Pro
Ile Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 115 120
125Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
130 135 140Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu145 150 155 160Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu 165 170 175Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr 180 185 190Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val 195 200 205Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 210 215 220Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe225 230 235
240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe 260 265 270Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Tyr Asn Asn Ala Ser Arg
Val Val Ser Val Leu Thr 290 295 300Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val305 310 315 320Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 340 345 350Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360
365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser385 390 395 400Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln 405 410 415Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His 420 425 430Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 435 44035444PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 35Glu 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 Pro Phe Ser Asn Tyr 20 25 30Trp Met Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Gln
Ile Arg Leu Lys Ser Asn Asn Tyr Ala Thr His Tyr Ala Glu 50 55 60Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser65 70 75
80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95Tyr Cys Thr Pro Ile Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr
Val 100 105 110Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser 115 120 125Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys 130 135 140Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu145 150 155 160Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 165 170 175Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 180 185 190Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 195 200
205Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
210 215 220Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe 260 265 270Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg Glu Glu Gln Tyr
Gln Asn Thr Ser Arg Val Val Ser Val Leu Thr 290 295 300Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305 310 315
320Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 340 345 350Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser385 390 395 400Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435
44036214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 36Asp 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 21037450PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 37Glu 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 Asp Glu Leu 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
445Gly Lys 45038450PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 38Glu 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 Asn Ala Ser 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 Asp Glu Leu 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 445Gly Lys
4503910PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 39Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5
10404PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 40Asn Asn Ala Ser1
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