U.S. patent application number 14/015952 was filed with the patent office on 2014-05-01 for bivalent, bispecific antibodies.
This patent application is currently assigned to Hoffman-La Roche Inc.. The applicant listed for this patent is Hoffman-La Roche Inc.. Invention is credited to Christian KLEIN, Wolfgang SCHAEFER.
Application Number | 20140120613 14/015952 |
Document ID | / |
Family ID | 39093026 |
Filed Date | 2014-05-01 |
United States Patent
Application |
20140120613 |
Kind Code |
A1 |
KLEIN; Christian ; et
al. |
May 1, 2014 |
BIVALENT, BISPECIFIC ANTIBODIES
Abstract
The present invention relates to a host cell for use in the
expression of a novel domain exchanged, bivalent, bispecific
antibody.
Inventors: |
KLEIN; Christian;
(Bonstetten, CH) ; SCHAEFER; Wolfgang; (Mannheim,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hoffman-La Roche Inc. |
Nutley |
NJ |
US |
|
|
Assignee: |
Hoffman-La Roche Inc.
Nutley
NJ
|
Family ID: |
39093026 |
Appl. No.: |
14/015952 |
Filed: |
August 30, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13362010 |
Jan 31, 2012 |
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14015952 |
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12332486 |
Dec 11, 2008 |
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13362010 |
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Current U.S.
Class: |
435/328 ;
435/252.33; 435/254.2 |
Current CPC
Class: |
C07K 16/468 20130101;
A61P 35/00 20180101; C07K 16/22 20130101; C07K 2317/21 20130101;
C07K 16/00 20130101; C07K 2319/00 20130101; C07K 2317/31 20130101;
C07K 16/2863 20130101 |
Class at
Publication: |
435/328 ;
435/252.33; 435/254.2 |
International
Class: |
C07K 16/00 20060101
C07K016/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 21, 2007 |
EP |
07024864.6 |
Claims
1. A host cell for use in expressing a domain exchanged, bivalent,
bispecific antibody, said host cell comprising: vectors comprising
nucleic acid molecules encoding the light chain and heavy chain of
an antibody specifically binding to a first antigen; vectors
comprising nucleic acid molecules encoding the light chain and
heavy chain of an antibody specifically binding to a second
antigen, wherein the variable domains VL and VH from the antibody
specifically binding to a second antigen are replaced by each
other.
2. A host cell according to claim 1 wherein said host cell
comprises: a) a vector comprising a nucleic acid molecule encoding
the light chain and a vector comprising a nucleic acid molecule
encoding the heavy chain, of an antibody specifically binding to a
first antigen b) a vector comprising a nucleic acid molecule
encoding the light chain and a vector comprising a nucleic acid
molecule encoding the heavy chain, of an antibody specifically
binding to a second antigen, wherein the variable domains VL and VH
are replaced by each other.
3. A host cell according to claim 1 wherein: in the final
bispecific antibody, the CH3 domain of one heavy chain meets at an
interface with the CH3 domain of the other heavy chain; and within
said interface, the CH3 domain of one heavy chain is altered so
that an amino acid residue is replaced with an amino acid residue
having a larger side chain volume thereby generating a
protuberance, and the CH3 domain of the other heavy chain is
altered so that an amino acid residue is replaced with an amino
acid residue having a smaller chain volume thereby generating a
cavity wherein said protuberance is positionable.
4. A host cell according to claim 3, wherein the amino acid residue
having a larger side chain volume is selected from the group
consisting of arginine (R), phenylalanine (F), tyrosine (Y),
tryptophan (W).
5. A host cell according to claim 3, wherein the amino acid residue
having a smaller side chain volume is selected from the group
consisting of alanine (A), serine (S), threonine (T), valine
(V).
6. A host cell according to claim 3, wherein both CH3 domains are
further altered by the introduction of cysteine (C) as amino acid
in the corresponding positions of each CH3 domain.
7. A host cell according to claim 1, wherein the CH3 domain on one
of the heavy chains is replaced by a constant heavy chain domain
CH1; and the CH3 domain on the other heavy chain is replaced by a
constant light chain domain CL.
Description
PRIORITY TO RELATED APPLICATION(S)
[0001] This application is a continuation of U.S. application Ser.
No. 13/362,010, filed Jan. 31, 2012, now abandoned; which is a
divisional of U.S. application Ser. No. 12/332,486, filed Dec. 11,
2008, now abandoned; which claims the benefit of European Patent
Application No. 07024864.6, filed Dec. 21, 2007. The entire
contents of the above-identified applications are hereby
incorporated by reference.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
146392019903 SubSeqList.txt, date recorded: Nov. 22, 2013, size:
35,263 bytes).
BACKGROUND OF THE INVENTION
[0003] Engineered proteins, such as bi- or multispecific antibodies
capable of binding two or more antigens are known in the art. Such
multispecific binding proteins can be generated using cell fusion,
chemical conjugation, or recombinant DNA techniques.
[0004] A wide variety of recombinant bispecific antibody formats
have been developed in the recent past, e.g. tetravalent bispecific
antibodies by fusion of, e.g. an IgG antibody format and single
chain domains (see e.g. Morrison, S. L., et al, Nature Biotech 15
(1997) 159-163; WO2001077342; and Coloma, M. J., Nature Biotech 25
(2007) 1233-1234).
[0005] Also several other new formats wherein the antibody core
structure (IgA, IgD, IgE, IgG or IgM) is no longer retained such as
dia-, tria- or tetrabodies, minibodies, several single chain
formats (scFv, Bis-scFv), which are capable of binding two or more
antigens, have been developed (Holliger P, et al, Nature Biotech 23
(2005) 1126-1136 2005; Fischer N., Leger O., Pathobiology 74 (2007)
3-14; Shen J, et al, Journal of Immunological Methods 318 (2007)
65-74; Wu, C. et al Nature Biotech 25 (2007) 1290-1297)
[0006] All such formats use linkers either to fuse the antibody
core (IgA, IgD, IgE, IgG or IgM) to a further binding protein (e.g.
scFv) or to fuse e.g. two Fab fragments or scFv. (Fischer N., Leger
O., Pathobiology 74 (2007) 3-14). While it is obvious that linkers
have advantages for the engineering of bispecific antibodies, they
may also cause problems in therapeutic settings. Indeed, these
foreign peptides might elicit an immune response against the linker
itself or the junction between the protein and the linker. Further
more, the flexible nature of these peptides makes them more prone
to proteolytic cleavage, potentially leading to poor antibody
stability, aggregation and increased immunogenicity. In addition
one may want to retain effector functions, such as e.g.
complement-dependent cytotoxicity (CDC) or antibody dependent
cellular cytotoxicity (ADCC), which are mediated through the
Fcpart, by maintaining a high degree of similarity to naturally
occurring antibodies.
[0007] Thus ideally, one should aim at developing bispecific
antibodies that are very similar in general structure to naturally
occurring antibodies (like IgA, IgD, IgE, IgG or IgM) with minimal
deviation from human sequences.
[0008] In one approach bispecific antibodies that are very similar
to natural antibodies have been produced using the quadroma
technology (see Milstein, C. and A. C. Cuello, Nature, 305 (1983)
537-40) based on the somatic fusion of two different hybridoma cell
lines expressing murine monoclonal antibodies with the desired
specificities of the bispecific antibody. Because of the random
pairing of two different antibody heavy and light chains within the
resulting hybrid-hybridoma (or quadroma) cell line, up to ten
different antibody species are generated of which only one is the
desired, functional bispecific antibody. Due to the presence of
mispaired byproducts, and significantly reduced production yields,
means sophisticated purification procedures are required (see e.g.
Morrison, S. L., Nature Biotech 25 (2007) 1233-1234). In general
the same problem of mispaired byproducts remains if recombinant
expression techniques are used.
[0009] An approach to circumvent the problem of mispaired
byproducts, which is known as `knobs-into-holes`, aims at forcing
the pairing of two different antibody heavy chains by introducing
mutations into the CH3 domains to modify the contact interface. On
one chain bulky amino acids were replaced by amino acids with short
side chains to create a `hole`. Conversely, amino acids with large
side chains were introduced into the other CH3 domain, to create a
`knob`. By coexpressing these two heavy chains (and two identical
light chains, which have to be appropriate for both heavy chains),
high yields of heterodimer formation (`knob-hole`) versus homodimer
formation (`hole-hole` or `knob-knob`) was observed (Ridgway J B,
Presta L G, Carter P; and WO1996027011). The percentage of
heterodimer could be further increased by remodeling the
interaction surfaces of the two CH3 domains using a phage display
approach and the introduction of a disulfide bridge to stabilize
the heterodimers (Merchant A. M, et al, Nature Biotech 16 (1998)
677-681; Atwell S, Ridgway J B, Wells J A, Carter P., J Mol Biol
270 (1997) 26-35). New approaches for the knobs-into-holes
technology are described in e.g. in EP 1870459A1. Although this
format appears very attractive, no data describing progression
towards the clinic are currently available. One important
constraint of this strategy is that the light chains of the two
parent antibodies have to be identical to prevent mispairing and
formation of inactive molecules. Thus this technique is not
appropriate for easily developing recombinant, bivalent, bispecific
antibodies against two antigens starting from two antibodies
against the first and the second antigen, as either the heavy
chains of these antibodies an/or the identical light chains have to
be optimized.
[0010] Xie, Z., et al, J Immunol Methods 286 (2005) 95-101 refers
to a new format of bispecific antibody using scFvs in combination
with knobs-into-holes technology for the FC part.
SUMMARY OF THE INVENTION
[0011] The invention relates to a host cell for use in expressing a
domain exchanged, bivalent, bispecific antibody, said host cell
comprising:
[0012] vectors comprising nucleic acid molecules encoding the light
chain and heavy chain of an antibody specifically binding to a
first antigen;
[0013] vectors comprising nucleic acid molecules encoding the light
chain and heavy chain of an antibody specifically binding to a
second antigen, wherein the variable domains VL and VH from the
antibody specifically binding to a second antigen are replaced by
each other.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The invention relates to a bivalent, bispecific antibody,
comprising:
a) the light chain and heavy chain of an antibody specifically
binding to a first antigen; and b) the light chain and heavy chain
of an antibody specifically binding to a second antigen, wherein
the variable domains VL and VH from the antibody specifically
binding to a second antigen are replaced by each other.
[0015] Therefore said bivalent, bispecific antibody, comprises:
a) a first light chain and a first heavy chain of an antibody
specifically binding to a first antigen; and b) a second light
chain and a second heavy chain of an antibody specifically binding
to a second antigen, wherein the variable domains VL and VH of the
second light chain and the second heavy chain are replaced by each
other.
[0016] Thus for said antibody specifically binding to a second
antigen the following applies:
within the light chain the variable light chain domain VL is
replaced by the variable heavy chain domain VH of said antibody;
and within the heavy chain the variable heavy chain domain VH is
replaced by the variable light chain domain VL of said
antibody.
[0017] The term "antibody" as used herein refers to whole,
monoclonal antibodies. Such whole antibodies consist of two pairs
of a "light chain" (LC) and a "heavy chain" (HC) (such light chain
(LC)/heavy chain pairs are abbreviated herein as LC/HC). The light
chains and heavy chains of such antibodies are polypeptides
consisting of several domains. In a whole antibody, each heavy
chain comprises a heavy chain variable region (abbreviated herein
as HCVR or VH) and a heavy chain constant region. The heavy chain
constant region comprises the heavy chain constant domains CH1, CH2
and CH3 (antibody classes IgA, IgD, and IgG) and optionally the
heavy chain constant domain CH4 (antibody classes IgE and IgM).
Each light chain comprises a light chain variable domain VL and a
light chain constant domain CL. The structure of one naturally
occurring whole antibody, the IgG antibody, is shown e.g. in FIG.
1. The variable domains VH and VL can be further subdivided into
regions of hypervariability, termed complementarity determining
regions (CDR), interspersed with regions that are more conserved,
termed framework regions (FR). Each VH and VL is composed of three
CDRs and four FRs, arranged from amino-terminus to carboxy-terminus
in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4
((Janeway C A, Jr et al (2001). Immunobiology., 5th ed., Garland
Publishing; and Woof J, Burton D Nat Rev Immunol 4 (2004) 89-99).
The two pairs of heavy chain and light chain (HC/LC) are capable of
specifically binding to same antigen. Thus said whole antibody is a
bivalent, monospecific antibody. Such "antibodies" include e.g.
mouse antibodies, human antibodies, chimeric antibodies, humanized
antibodies and genetically engineered antibodies (variant or mutant
antibodies) as long as their characteristic properties are
retained. Especially preferred are human or humanized antibodies,
especially as recombinant human or humanized antibodies.
[0018] There are five types of mammalian antibody heavy chains
denoted by the Greek letters: .alpha., .delta., .epsilon., .gamma.,
and .mu. (Janeway C A, Jr et al (2001). Immunobiology., 5th ed.,
Garland Publishing). The type of heavy chain present defines the
class of antibody; these chains are found in IgA, IgD, IgE, IgG,
and IgM antibodies, respectively (Rhoades R A, Pflanzer R G (2002).
Human Physiology, 4th ed., Thomson Learning). Distinct heavy chains
differ in size and composition; .alpha. and .gamma. contain
approximately 450 amino acids, while .mu. and .epsilon. have
approximately 550 amino acids.
[0019] Each heavy chain has two regions, the constant region and
the variable region. The constant region is identical in all
antibodies of the same isotype, but differs in antibodies of
different isotype. Heavy chains .gamma., .alpha. and .delta. have a
constant region composed of three constant domains CH1, CH2, and
CH3 (in a line), and a hinge region for added flexibility (Woof J,
Burton D Nat Rev Immunol 4 (2004) 89-99); heavy chains .mu. and
.epsilon. have a constant region composed of four constant domains
CH1, CH2, CH3, and CH4 (Janeway C A, Jr et al (2001).
Immunobiology., 5th ed., Garland Publishing). The variable region
of the heavy chain differs in antibodies produced by different B
cells, but is the same for all antibodies produced by a single B
cell or B cell clone. The variable region of each heavy chain is
approximately 110 amino acids long and is composed of a single
antibody domain.
[0020] In mammals there are only two types of light chain, which
are called lambda (.lamda.) and kappa (.kappa.). A light chain has
two successive domains: one constant domain CL and one variable
domain VL. The approximate length of a light chain is 211 to 217
amino acids. Preferably the light chain is a kappa (.kappa.) light
chain, and the constant domain CL is preferably derived from a
kappa (.kappa.) light chain (the constant domain C.kappa.)
[0021] The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of a single amino acid composition.
[0022] The "antibodies" according to the invention can be of any
class (e.g. IgA, IgD, IgE, IgG, and IgM, preferably IgG or IgE), or
subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, preferably
IgG1), whereby both antibodies, from which the bivalent bispecific
antibody according to the invention is derived, have an Fc part of
the same subclass (e.g. IgG1, IgG4 and the like, preferably IgG1),
preferably of the same allotype (e.g. Caucasian)
[0023] A "Fc part of an antibody" is a term well known to the
skilled artisan and defined on the basis of papain cleavage of
antibodies. The antibodies according to the invention contain as Fc
part, preferably a Fc part derived from human origin and preferably
all other parts of the human constant regions. The Fc part of an
antibody is directly involved in complement activation, C1q
binding, C3 activation and Fc receptor binding. While the influence
of an antibody on the complement system is dependent on certain
conditions, binding to C1q is caused by defined binding sites in
the Fc part. Such binding sites are known in the state of the art
and described e.g. by Lukas, T. J., et al., J. Immunol. 127 (1981)
2555-2560; Brunhouse, R., and Cebra, J. J., Mol. Immunol. 16 (1979)
907-917; Burton, D. R., et al., Nature 288 (1980) 338-344;
Thommesen, J. E., et al., Mol. Immunol. 37 (2000) 995-1004;
Idusogie, E. E., et al., J. Immunol. 164 (2000) 4178-4184; Hezareh,
M., et al., J. Virol. 75 (2001) 12161-12168; Morgan, A., et al.,
Immunology 86 (1995) 319-324; and EP 0 307 434. Such binding sites
are e.g. L234, L235, D270, N297, E318, K320, K322, P331 and P329
(numbering according to EU index of Kabat, see below). Antibodies
of subclass IgG1, IgG2 and IgG3 usually show complement activation,
C1q binding and C3 activation, whereas IgG4 do not activate the
complement system, do not bind C1q and do not activate C3.
Preferably the Fc part is a human Fc part.
[0024] The term "chimeric antibody" refers to an antibody
comprising a variable region, i.e., binding region, from one source
or species and at least a portion of a constant region derived from
a different source or species, usually prepared by recombinant DNA
techniques. Chimeric antibodies comprising a murine variable region
and a human constant region are preferred. Other preferred forms of
"chimeric antibodies" encompassed by the present invention are
those in which the constant region has been modified or changed
from that of the original antibody to generate the properties
according to the invention, especially in regard to C1q binding
and/or Fc receptor (FcR) binding. Such chimeric antibodies are also
referred to as "class-switched antibodies.". Chimeric antibodies
are the product of expressed immunoglobulin genes comprising DNA
segments encoding immunoglobulin variable regions and DNA segments
encoding immunoglobulin constant regions. Methods for producing
chimeric antibodies involve conventional recombinant DNA and gene
transfection techniques are well known in the art. See, e.g.,
Morrison, S. L., et al., Proc. Natl. Acad. Sci. USA 81 (1984)
6851-6855; U.S. Pat. Nos. 5,202,238 and 5,204,244.
[0025] The term "humanized antibody" refers to antibodies in which
the framework or "complementarity determining regions" (CDR) have
been modified to comprise the CDR of an immunoglobulin of different
specificity as compared to that of the parent immunoglobulin. In a
preferred embodiment, a murine CDR is grafted into the framework
region of a human antibody to prepare the "humanized antibody."
See, e.g., Riechmann, L., et al., Nature 332 (1988) 323-327; and
Neuberger, M. S., et al., Nature 314 (1985) 268-270. Particularly
preferred CDRs correspond to those representing sequences
recognizing the antigens noted above for chimeric antibodies. Other
forms of "humanized antibodies" encompassed by the present
invention are those in which the constant region has been
additionally modified or changed from that of the original antibody
to generate the properties according to the invention, especially
in regard to C1q binding and/or Fc receptor (FcR) binding.
[0026] The term "human antibody", as used herein, is intended to
include antibodies having variable and constant regions derived
from human germ line immunoglobulin sequences. Human antibodies are
well-known in the state of the art (van Dijk, M. A., and van de
Winkel, J. G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human
antibodies can also be produced in transgenic animals (e.g., mice)
that are capable, upon immunization, of producing a full repertoire
or a selection of human antibodies in the absence of endogenous
immunoglobulin production. Transfer of the human germ-line
immunoglobulin gene array in such germ-line mutant mice will result
in the production of human antibodies upon antigen challenge (see,
e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993)
2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258;
Bruggemann, M., et al., Year Immunol. 7 (1993) 33-40). Human
antibodies can also be produced in phage display libraries
(Hoogenboom, H. R., and Winter, G., J. Mol. Biol. 227 (1992)
381-388; Marks, J. D., et al., J. Mol. Biol. 222 (1991) 581-597).
The techniques of Cole et al. and Boerner et al. are also available
for the preparation of human monoclonal antibodies (Cole et al.,
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77
(1985); and Boerner, P., et al., J. Immunol. 147 (1991) 86-95). As
already mentioned for chimeric and humanized antibodies according
to the invention the term "human antibody" as used herein also
comprises such antibodies which are modified in the constant region
to generate the properties according to the invention, especially
in regard to C1q binding and/or FcR binding, e.g. by "class
switching" i.e. change or mutation of Fc parts (e.g. from IgG1 to
IgG4 and/or IgG1/IgG4 mutation.)
[0027] The term "recombinant human antibody", as used herein, is
intended to include all human antibodies that are prepared,
expressed, created or isolated by recombinant means, such as
antibodies isolated from a host cell such as a NS0 or CHO cell or
from an animal (e.g. a mouse) that is transgenic for human
immunoglobulin genes or antibodies expressed using a recombinant
expression vector transfected into a host cell. Such recombinant
human antibodies have variable and constant regions in a rearranged
form. The recombinant human antibodies according to the invention
have been subjected to in vivo somatic hypermutation. Thus, the
amino acid sequences of the VH and VL regions of the recombinant
antibodies are sequences that, while derived from and related to
human germ line VH and VL sequences, may not naturally exist within
the human antibody germ line repertoire in vivo.
[0028] The "variable domain" (variable domain of a light chain
(VL), variable region of a heavy chain (VH)) as used herein denotes
each of the pair of light and heavy chains which is involved
directly in binding the antibody to the antigen. The domains of
variable human light and heavy chains have the same general
structure and each domain comprises four framework (FR) regions
whose sequences are widely conserved, connected by three
"hypervariable regions" (or complementarity determining regions,
CDRs). The framework regions adopt a .beta.-sheet conformation and
the CDRs may form loops connecting the .beta.-sheet structure. The
CDRs in each chain are held in their three-dimensional structure by
the framework regions and form together with the CDRs from the
other chain the antigen binding site. The antibody heavy and light
chain CDR3 regions play a particularly important role in the
binding specificity/affinity of the antibodies according to the
invention and therefore provide a further object of the
invention.
[0029] The terms "hypervariable region" or "antigen-binding portion
of an antibody" when used herein refer to the amino acid residues
of an antibody which are responsible for antigen-binding. The
hypervariable region comprises amino acid residues from the
"complementarity determining regions" or "CDRs". "Framework" or
"FR" regions are those variable domain regions other than the
hypervariable region residues as herein defined. Therefore, the
light and heavy chains of an antibody comprise from N- to
C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
CDRs on each chain are separated by such framework amino acids.
Especially, CDR3 of the heavy chain is the region which contributes
most to antigen binding. CDR and FR regions are determined
according to the standard definition of Kabat et al., Sequences of
Proteins of Immunological Interest, 5th ed., Public Health Service,
National Institutes of Health, Bethesda, Md. (1991).
[0030] The "constant domains" of the heavy chain and of the light
chain are not involved directly in binding of an antibody to an
antigen, but exhibit various effector functions. Depending on the
amino acid sequence of the constant region of their heavy chains,
antibodies or immunoglobulins are divided into the classes:
[0031] The term "bivalent, bispecific antibody" as used herein
refers to an antibody as described above in which each of the two
pairs of heavy chain and light chain (HC/LC) is specifically
binding to a different antigen, i.e. the first heavy and the first
light chain (originating from an antibody against a first antigen)
are specifically binding together to a first antigen, and, the
second heavy and the second light chain (originating from an
antibody against a second antigen) are specifically binding
together to a second antigen (as depicted in FIG. 2); such
bivalent, bispecific antibodies are capable of specifically binding
to two different antigens at the same time, and not to more than
two antigens, in contrary to, on the one hand a monospecific
antibody capable of binding only to one antigen, and on the other
hand e.g. a tetravalent, tetraspecific antibody which can bind to
four antigen molecules at the same time.
[0032] According to the invention, the ratio of a desired bivalent,
bispecific antibody compared to undesired side products can be
improved by the replacement of certain domains in only one pair of
heavy chain and light chain (HC/LC). While the first of the two
HC/LC pairs originates from an antibody specifically binding to a
first antigen and is left essentially unchanged, the second of the
two HC/LC pairs originates from an antibody specifically binding to
a second antigen, and is altered by the following replacement:
[0033] light chain: replacement of the variable light chain domain
VL by the variable heavy chain domain VH of said antibody
specifically binding to a second antigen, and
[0034] heavy chain: replacement of the variable heavy chain domain
VH by the variable light chain domain VL of said antibody
specifically binding to a second antigen.
[0035] Thus the resulting bivalent, bispecific antibodies are
artificial antibodies which comprise
a) the light chain and heavy chain of an antibody specifically
binding to a first antigen; and b) the light chain and heavy chain
of an antibody specifically binding to a second antigen, wherein
said light chain (of an antibody specifically binding to a second
antigen) contains a variable domain VH instead of VL, and wherein
said heavy chain (of an antibody specifically binding to a second
antigen) contains a variable domain VL instead of VH.
[0036] In an additional aspect of the invention such improved ratio
of a desired bivalent, bispecific antibody compared to undesired
side products can be further improved by one of the following two
alternatives:
A) First Alternative (see FIG. 3):
[0037] The CH3 domains of said bivalent, bispecific antibody
according to the invention can be altered by the "knob-into-holes"
technology which described with in detail with several examples in
e.g. WO96/027011, Ridgway J B, et al, Protein Eng 9 (1996) 617-621;
and Merchant A. M., et al, Nat Biotechnol 16 (1998) 677-681. In
this method the interaction surfaces of the two CH3 domains are
altered to increase the heterodimerisation of both heavy chains
containing these two CH3 domains. Each of the two CH3 domains (of
the two heavy chains) can be the "knob", while the other is the
"hole". The introduction of a disulfide bridge stabilizes the
heterodimers (Merchant A. M, et al, Nature Biotech 16 (1998)
677-681; Atwell S, Ridgway J B, Wells J A, Carter P., J Mol Biol
270 (1997) 26-35) and increases the yield.
[0038] Therefore in preferred embodiment the CH3 domains of a
bivalent, bispecific antibody wherein the first CH3 domain and
second CH3 domain each meet at an interface which comprises an
original interface between the antibody CH3 domains are altered by
the "knob-into-holes" technology including further stabilization by
introduction of a disulfide bridge in the CH3 domains (described in
WO96/027011, Ridgway J B, et al, Protein Eng 9 (1996) 617-621;
Merchant A. M, et al, Nature Biotech 16 (1998) 677-681; and Atwell
S, Ridgway J B, Wells J A, Carter P., J Mol Biol 270 (1997) 26-35)
to promote the formation of the bivalent, bispecific antibody.
[0039] Thus in one aspect of the invention said bivalent,
bispecific antibody is characterized in that
the CH3 domain of one heavy chain and the CH3 domain of the other
heavy chain each meet at an interface which comprises an original
interface between the antibody CH3 domains; wherein said interface
is altered to promote the formation of the bivalent, bispecific
antibody, wherein the alteration is characterized in that: a) the
CH3 domain of one heavy chain is altered, so that within the
original interface the CH3 domain of one heavy chain that meets the
original interface of the CH3 domain of the other heavy chain
within the bivalent, bispecific antibody, an amino acid residue is
replaced with an amino acid residue having a larger side chain
volume, thereby generating a protuberance within the interface of
the CH3 domain of one heavy chain which is positionable in a cavity
within the interface of the CH3 domain of the other heavy chain and
b) the CH3 domain of the other heavy chain is altered, so that
within the original interface of the second CH3 domain that meets
the original interface of the first CH3 domain within the bivalent,
bispecific antibody an amino acid residue is replaced with an amino
acid residue having a smaller side chain volume, thereby generating
a cavity within the interface of the second CH3 domain within which
a protuberance within the interface of the first CH3 domain is
positionable.
[0040] Preferably said amino acid residue having a larger side
chain volume is selected from the group consisting of arginine (R),
phenylalanine (F), tyrosine (Y), tryptophan (W).
[0041] Preferably said amino acid residue having a smaller side
chain volume is selected from the group consisting of alanine (A),
serine (S), threonine (T), valine (V).
[0042] In one aspect of the invention both CH3 domains are further
altered the introduction of cysteine (C) as amino acid in the
corresponding positions of each CH3 domain such that a disulfide
bridge between both CH3 domains can be formed.
In another preferred embodiment of the invention both CH3 domains
are altered by the use of residues R409D; K370E (K409D) for knobs
residues and D399K; E357K for hole residues described eg. in EP
1870459A1; or
B) Second Alternative (see FIG. 4):
[0043] by the replacement of one constant heavy chain domain CH3 by
a constant heavy chain domain CH1; and the other constant heavy
chain domain CH3 is replaced by a constant light chain domain
CL.
[0044] The constant heavy chain domain CH1 by which the heavy chain
domain CH3 is replaced can be of any Ig class (e.g. IgA, IgD, IgE,
IgG, and IgM), or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and
IgA2).
[0045] The constant light chain domain CL by which the heavy chain
domain CH3 is replaced can be of the lambda (.lamda.) or kappa
(.kappa.) type, preferably the kappa (.kappa.) type.
[0046] Thus one preferred embodiment of the invention is a
bivalent, bispecific antibody, comprising:
a) the light chain and heavy chain of an antibody specifically
binding to a first antigen; and b) the light chain and heavy chain
of an antibody specifically binding to a second antigen, wherein
the variable domains VL and VH are replaced by each other, and
wherein optionally c) the CH3 domain of one heavy chain and the CH3
domain of the other heavy chain each meet at an interface which
comprises an original interface between the antibody CH3 domains;
wherein said interface is altered to promote the formation of the
bivalent, bispecific antibody, wherein the alteration is
characterized in that: ca) the CH3 domain of one heavy chain is
altered, so that within the original interface the CH3 domain of
one heavy chain that meets the original interface of the CH3 domain
of the other heavy chain within the bivalent, bispecific antibody,
an amino acid residue is replaced with an amino acid residue having
a larger side chain volume, thereby generating a protuberance
within the interface of the CH3 domain of one heavy chain which is
positionable in a cavity within the interface of the CH3 domain of
the other heavy chain and cb) the CH3 domain of the other heavy
chain is altered, so that within the original interface of the
second CH3 domain that meets the original interface of the first
CH3 domain within the bivalent, bispecific antibody an amino acid
residue is replaced with an amino acid residue having a smaller
side chain volume, thereby generating a cavity within the interface
of the second CH3 domain within which a protuberance within the
interface of the first CH3 domain is positionable; or d) one
constant heavy chain domain CH3 is replaced by a constant heavy
chain domain CH1; and the other constant heavy chain domain CH3 is
replaced by a constant light chain domain CL.
[0047] The terms "antigen" or "antigen molecule" as used herein are
used interchangeable and refer to all molecules that can be
specifically bound by an antibody. The bivalent, bispecific
antibody is specifically binding to a first antigen and a second
distinct antigen. The term "antigens" as used herein include e.g.
proteins, different epitopes on proteins (as different antigens
within the meaning of the invention), and polysaccharides. This
mainly includes parts (coats, capsules, cell walls, flagella,
fimbrae, and toxins) of bacteria, viruses, and other
microorganisms. Lipids and nucleic acids are antigenic only when
combined with proteins and polysaccharides. Non-microbial exogenous
(non-self) antigens can include pollen, egg white, and proteins
from transplanted tissues and organs or on the surface of
transfused blood cells. Preferably the antigen is selected from the
group consisting of cytokines, cell surface proteins, enzymes and
receptors cytokines, cell surface proteins, enzymes and
receptors.
[0048] Tumor antigens are those antigens that are presented by MHC
I or MHC II molecules on the surface of tumor cells. These antigens
can sometimes be presented by tumor cells and never by the normal
ones. In this case, they are called tumor-specific antigens (TSAs)
and typically result from a tumor specific mutation. More common
are antigens that are presented by tumor cells and normal cells,
and they are called tumor-associated antigens (TAAs). Cytotoxic T
lymphocytes that recognized these antigens may be able to destroy
the tumor cells before they proliferate or metastasize. Tumor
antigens can also be on the surface of the tumor in the form of,
for example, a mutated receptor, in which case they will be
recognized by B cells.
[0049] In one preferred embodiment at least one of the two
different antigens (first and second antigen), to which the
bivalent, bispecific antibody specifically binds to, is a tumor
antigen.
[0050] In another preferred embodiment both of the two different
antigens (first and second antigen), to which the bivalent,
bispecific antibody specifically binds to, are tumor antigens; in
this case the first and second antigen can also be two different
epitopes at the same tumor specific protein.
[0051] In another preferred embodiment one of the two different
antigens (first and second antigen), to which the bivalent,
bispecific antibody specifically binds to, is a tumor antigen and
the other is an effector cell antigen, as e.g. an T-Cell receptor,
CD3, CD16 and the like.
[0052] In another preferred embodiment one of the two different
antigens (first and second antigen), to which the bivalent,
bispecific antibody specifically binds to, is a tumor antigen and
the other is an anti-cancer substance such as a toxin or a kinase
inhibitor.
[0053] As used herein, "specifically binding" or "binds
specifically to" refers to an antibody specifically binding an
antigen. Preferably the binding affinity of the antibody
specifically binding this antigen is of KD-value of 10.sup.-9 mol/l
or lower (e.g. 10.sup.-10 mol/l), preferably with a KD-value of
10.sup.-10 mol/l or lower (e.g. 10.sup.-12 mol/l). The binding
affinity is determined with a standard binding assay, such as
surface plasmon resonance technique (Biacore.RTM.).
[0054] The term "epitope" includes any polypeptide determinant
capable of specific binding to an antibody. In certain embodiments,
epitope determinant include chemically active surface groupings of
molecules such as amino acids, sugar side chains, phosphoryl, or
sulfonyl, and, in certain embodiments, may have specific three
dimensional structural characteristics, and or specific charge
characteristics. An epitope is a region of an antigen that is bound
by an antibody. In certain embodiments, an antibody is said to
specifically bind an antigen when it preferentially recognizes its
target antigen in a complex mixture of proteins and/or
macromolecules.
[0055] An further embodiment of the invention is a method for the
preparation of a bivalent, bispecific antibody according to the
invention comprising
a) transforming a host cell with
[0056] vectors comprising nucleic acid molecules encoding the light
chain and heavy chain of an antibody specifically binding to a
first antigen
[0057] vectors comprising nucleic acid molecules encoding the light
chain and heavy chain of an antibody specifically binding to a
second antigen, wherein the variable domains VL and VH are replaced
by each other;
b) culturing the host cell under conditions that allow synthesis of
said antibody molecule; and c) recovering said antibody molecule
from said culture.
[0058] In general there are two vectors encoding the light chain
and heavy chain of said antibody specifically binding to a first
antigen, and further two vectors encoding the light chain and heavy
chain of said antibody specifically binding to a second antigen.
One of the two vectors is encoding the respective light chain and
the other of the two vectors is encoding the respective heavy
chain. However in an alternative method for the preparation of a
bivalent, bispecific antibody according to the invention, only one
first vector encoding the light chain and heavy chain of the
antibody specifically binding to a first antigen and only one
second vector encoding the light chain and heavy chain of the
antibody specifically binding to a second antigen can be used for
transforming the host cell.
[0059] The invention encompasses a method for the preparation of
the antibodies comprising culturing the corresponding host cells
under conditions that allow synthesis of said antibody molecules
and recovering said antibodies from said culture, e.g. by
expressing
[0060] a first nucleic acid sequence encoding the light chain of an
antibody specifically binding to a first antigen,
[0061] a second nucleic acid sequence encoding the heavy chain of
said antibody specifically binding to a first antigen,
[0062] a third nucleic acid sequence encoding the light chain of an
antibody specifically binding to a second antigen, wherein the
variable light chain domain VL is replaced by the variable heavy
chain domain VH, and
[0063] a fourth nucleic acid sequence encoding the heavy chain of
said antibody specifically binding to a second antigen, wherein
variable heavy chain domain VH by the variable light chain domain
VL.
[0064] A further embodiment of the invention is a host cell
comprising
[0065] vectors comprising nucleic acid molecules encoding the light
chain and heavy chain of an antibody specifically binding to a
first antigen
[0066] vectors comprising nucleic acid molecules encoding the light
chain and heavy chain of an antibody specifically binding to a
second antigen, wherein the variable domains VL and VH are replaced
by each other.
[0067] A further embodiment of the invention is a host cell
comprising
a) a vector comprising a nucleic acid molecule encoding the light
chain and a vector comprising a nucleic acid molecule encoding the
heavy chain, of an antibody specifically binding to a first antigen
b) a vector comprising a nucleic acid molecule encoding the light
chain and a vector comprising a nucleic acid molecule encoding the
heavy chain, of an antibody specifically binding to a second
antigen, wherein the variable domains VL and VH are replaced by
each other.
[0068] A further embodiment of the invention is a composition,
preferably a pharmaceutical or a diagnostic composition of the
bivalent, bispecific antibody according to the invention.
[0069] A further embodiment of the invention is a pharmaceutical
composition comprising a bivalent, bispecific antibody according to
the invention and at least one pharmaceutically acceptable
excipient.
[0070] A further embodiment of the invention is a method for the
treatment of a patient in need of therapy, characterized by
administering to the patient a therapeutically effective amount of
a bivalent, bispecific antibody according to the invention.
[0071] The term "nucleic acid or nucleic acid molecule", as used
herein, is intended to include DNA molecules and RNA molecules. A
nucleic acid molecule may be single-stranded or double-stranded,
but preferably is double-stranded DNA.
[0072] As used herein, the expressions "cell," "cell line," and
"cell culture" are used interchangeably and all such designations
include progeny. Thus, the words "transformants" and "transformed
cells" include the primary subject cell and cultures derived
therefrom without regard for the number of transfers. It is also
understood that all progeny may not be precisely identical in DNA
content, due to deliberate or inadvertent mutations. Variant
progeny that have the same function or biological activity as
screened for in the originally transformed cell are included. Where
distinct designations are intended, it will be clear from the
context.
[0073] The term "transformation" as used herein refers to process
of transfer of a vectors/nucleic acid into a host cell. If cells
without formidable cell wall barriers are used as host cells,
transfection is carried out e.g. by the calcium phosphate
precipitation method as described by Graham and Van der Eh,
Virology 52 (1978) 546ff. However, other methods for introducing
DNA into cells such as by nuclear injection or by protoplast fusion
may also be used. If prokaryotic cells or cells which contain
substantial cell wall constructions are used, e.g. one method of
transfection is calcium treatment using calcium chloride as
described by Cohen, F. N, et al, PNAS. 69 (1972) 7110ff.
[0074] Recombinant production of antibodies using transformation is
well-known in the state of the art and described, for example, in
the review articles of Makrides, S. C., Protein Expr. Purif. 17
(1999) 183-202; Geisse, S., et al., Protein Expr. Purif. 8 (1996)
271-282; Kaufman, R. J., Mol. Biotechnol. 16 (2000) 151-161;
Werner, R. G., et al., Arzneimittelforschung 48 (1998) 870-880 as
well as in U.S. Pat. No. 6,331,415 and U.S. Pat. No. 4,816,567.
[0075] As used herein, "expression" refers to the process by which
a nucleic acid is transcribed into mRNA and/or to the process by
which the transcribed mRNA (also referred to as transcript) is
subsequently being translated into peptides, polypeptides, or
proteins. The transcripts and the encoded polypeptides are
collectively referred to as gene product. If the polynucleotide is
derived from genomic DNA, expression in a eukaryotic cell may
include splicing of the mRNA.
[0076] A "vector" is a nucleic acid molecule, in particular
self-replicating, which transfers an inserted nucleic acid molecule
into and/or between host cells. The term includes vectors that
function primarily for insertion of DNA or RNA into a cell (e.g.,
chromosomal integration), replication of vectors that function
primarily for the replication of DNA or RNA, and expression vectors
that function for transcription and/or translation of the DNA or
RNA. Also included are vectors that provide more than one of the
functions as described.
[0077] An "expression vector" is a polynucleotide which, when
introduced into an appropriate host cell, can be transcribed and
translated into a polypeptide. An "expression system" usually
refers to a suitable host cell comprised of an expression vector
that can function to yield a desired expression product.
[0078] The bivalent, bispecific antibodies according to the
invention are preferably produced by recombinant means. Such
methods are widely known in the state of the art and comprise
protein expression in prokaryotic and eukaryotic cells with
subsequent isolation of the antibody polypeptide and usually
purification to a pharmaceutically acceptable purity. For the
protein expression, nucleic acids encoding light and heavy chains
or fragments thereof are inserted into expression vectors by
standard methods. Expression is performed in appropriate
prokaryotic or eukaryotic host cells like CHO cells, NS0 cells,
SP2/0 cells, HEK293 cells, COS cells, yeast, or E. coli cells, and
the antibody is recovered from the cells (supernatant or cells
after lysis). The bivalent, bispecific antibodies may be present in
whole cells, in a cell lysate, or in a partially purified or
substantially pure form. Purification is performed in order to
eliminate other cellular components or other contaminants, e.g.
other cellular nucleic acids or proteins, by standard techniques,
including alkaline/SDS treatment, column chromatography and others
well known in the art. See Ausubel, F., et al., ed., Current
Protocols in Molecular Biology, Greene Publishing and Wiley
Interscience, New York (1987).
[0079] Expression in NS0 cells is described by, e.g., Barnes, L.
M., et al., Cytotechnology 32 (2000) 109-123; and Barnes, L. M., et
al., Biotech. Bioeng. 73 (2001) 261-270. Transient expression is
described by, e.g., Durocher, Y., et al., Nucl. Acids. Res. 30
(2002) E9. Cloning of variable domains is described by Orlandi, R.,
et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837; Carter, P.,
et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and
Norderhaug, L., et al., J. Immunol. Methods 204 (1997) 77-87. A
preferred transient expression system (HEK 293) is described by
Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30 (1999)
71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996)
191-199.
[0080] The control sequences that are suitable for prokaryotes, for
example, include a promoter, optionally an operator sequence, and a
ribosome binding site. Eukaryotic cells are known to utilize
promoters, enhancers and polyadenylation signals.
[0081] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading frame. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0082] The bivalent, bispecific antibodies are suitably separated
from the culture medium by conventional immunoglobulin purification
procedures such as, for example, protein A-Sepharose,
hydroxylapatite chromatography, gel electrophoresis, dialysis, or
affinity chromatography. DNA or RNA encoding the monoclonal
antibodies is readily isolated and sequenced using conventional
procedures. The hybridoma cells can serve as a source of such DNA
and RNA. Once isolated, the DNA may be inserted into expression
vectors, which are then transfected into host cells such as HEK 293
cells, CHO cells, or myeloma cells that do not otherwise produce
immunoglobulin protein, to obtain the synthesis of recombinant
monoclonal antibodies in the host cells.
[0083] Amino acid sequence variants (or mutants) of the bivalent,
bispecific antibody are prepared by introducing appropriate
nucleotide changes into the antibody DNA, or by nucleotide
synthesis. Such modifications can be performed, however, only in a
very limited range, e.g. as described above. For example, the
modifications do not alter the above mentioned antibody
characteristics such as the IgG isotype and antigen binding, but
may improve the yield of the recombinant production, protein
stability or facilitate the purification.
[0084] The following examples, sequence listing and figures are
provided to aid the understanding of the present invention, the
true scope of which is set forth in the appended claims. It is
understood that modifications can be made in the procedures set
forth without departing from the spirit of the invention.
SEQUENCE LISTING
[0085] SEQ ID NO: 1 amino acid sequence of wild type <IGF-1R>
antibody heavy chain
[0086] SEQ ID NO: 2 amino acid sequence of wild type <IGF-1R>
antibody light chain
[0087] SEQ ID NO: 3 amino acid sequence of the heavy chain***
(HC***) of <IGF-1R> VL-VH exchange antibody, wherein the
heavy chain domain VH is replaced by the light chain domain
VL--variant A.
[0088] SEQ ID NO: 4 amino acid sequence of the light chain***
(LC***) of <IGF-1R> VL-VH exchange antibody, wherein the
light chain domain VL is replaced by the heavy chain domain
VH--variant A.
[0089] SEQ ID NO: 5 amino acid sequence of IGF-1R ectodomain
His-Streptavidin binding peptide-tag (IGF-1R-His-SBP ECD)
[0090] SEQ ID NO: 6 amino acid sequence of wild type Angiopoietin-2
<ANGPT2> antibody heavy chain
[0091] SEQ ID NO: 7 amino acid sequence of wild type Angiopoietin-2
<ANGPT2> antibody light chain
[0092] SEQ ID NO: 8 amino acid sequence of CH3 domain (Knobs) with
a T366W exchange for use in the knobs-into-holes technology
[0093] SEQ ID NO: 9 amino acid sequence CH3 domain (Hole) with a
T366S, L368A, Y407V exchange for use in the knobs-into-holes
technology
[0094] SEQ ID NO: 10 amino acid sequence of the heavy chain***
(HC***) of <IGF-1R> VL-VH exchange antibody, wherein the
heavy chain domain VH is replaced by the light chain domain
VL--variant B.
[0095] SEQ ID NO: 11 amino acid sequence of the light chain***
(LC***) of <IGF-1R> VL-VH exchange antibody, wherein the
light chain domain VL is replaced by the heavy chain domain
VH--variant B.
[0096] SEQ ID NO: 12 amino acid sequence of IGF-1R ectodomain
His-Streptavidin binding peptide-tag (IGF-1R-His-SBP ECD)
DESCRIPTION OF THE FIGURES
[0097] FIG. 1 Schematic figure of IgG, a naturally occurring whole
antibody specific for one antigen with two pairs of heavy and light
chain which comprise variable and constant domains in a typical
order.
[0098] FIG. 2 Schematic figure of a bivalent, bispecific antibody,
comprising: a) the light chain and heavy chain of an antibody
specifically binding to a first antigen; and b) the light chain and
heavy chain of an antibody specifically binding to a second
antigen, wherein the variable domains VL and VH are replaced by
each other.
[0099] FIG. 3 Schematic figure of a bivalent, bispecific antibody,
comprising: a) the light chain and heavy chain of an antibody
specifically binding to a first antigen; and b) the light chain and
heavy chain of an antibody specifically binding to a second
antigen, wherein the variable domains VL and VH are replaced by
each other, and wherein the CH3 domains of both heavy chains are
altered by the knobs-into-holes technology.
[0100] FIG. 4 Schematic figure of a bivalent, bispecific antibody,
comprising: a) the light chain and heavy chain of an antibody
specifically binding to a first antigen; and b) the light chain and
heavy chain of an antibody specifically binding to a second
antigen, wherein the variable domains VL and VH are replaced by
each other, and wherein one of the constant heavy chain domains CH3
of both heavy chains is replaced by a constant heavy chain domain
CH1; and the other constant heavy chain domain CH3 is replaced by a
constant light chain domain CL.
[0101] FIG. 5 Protein sequence scheme of the heavy
chain**<IGF-1R> HC** of the <IGF-1R> VL-VH exchange
antibody
[0102] FIG. 6 Protein sequence scheme of the light
chain**<IGF-1R> LC** of the <IGF-1R> VL-VH exchange
antibody (with a kappa constant light chain domain CL)
[0103] FIG. 7 Plasmid map of heavy chain***<IGF-1R> HC***
expression vector pUC-HC***-IGF-1R
[0104] FIG. 8 Plasmid map of light chain***<IGF-1R> LC***
expression vector pUC-LC***-IGF-1R
[0105] FIG. 9 Plasmid map of the 4700-Hyg-OriP expression
vector
[0106] FIG. 10 Assay principle of cellular FACS IGF-1R-ANGPT2
bridging assay on I24 IGF-1R expressing cells to detect the
presence of functional bispecific <ANGPT2-IGF-1R> VL-VH
exchange antibody
[0107] FIG. 11 Scheme IGF-1R ECD Biacore
[0108] FIG. 12 SDS-PAGE (A) and size exclusion chromatography (B)
of purified monospecific, bivalent <IGF-1R> VL-VH exchange
antibody (IgG1*) with HC* and LC* isolated from cell culture
supernatants after transient transfection of HEK293-F cells.
[0109] FIG. 13 Binding of monospecific <IGF-1R> VL-VH
exchange antibody (right panel) and wildtype <IGF-1R>
antibody (left panel) to the IGF-1R ECD in an ELISA-based binding
assay.
[0110] FIG. 14 SDS-PAGE of <ANGPT2-IGF-1R> VL-VH exchange
antibody mix purified from cell culture supernatants from
transiently transfected HEK293-F cells. SDS-PAGE run under reducing
conditions in the left panel and non-reducing conditions in the
right panel.
[0111] FIG. 15 Results for Samples A to F of cellular FACS
IGF-1R-ANGPT2 bridging assay on I24 IGF-1R expressing cells to
detect the presence of functional bispecific <ANGPT2-IGF-1R>
VL-VH exchange antibody in purified antibody mix.
Purified proteins Sample A to F, from top panel (Sample A) to
bottom panel (Sample F): A=I24 untreated B=I24+2 .mu.g/mL
hANGPT2+hIgG Isotype D=I24+2 .mu.g/mL hANGPT2+Mix from
co-expression of <IGF-1R> VL-VH exchange antibody and
<ANGPT2> wildtype antibody comprising bispecific
<ANGPT2-IGF-1R> VL-VH exchange antibody E=I24+2 .mu.g/mL
hANGPT2+<ANGPT2> wildtype antibody F=I24+2 .mu.g/mL
hANGPT2+<IGF-1R> wildtype antibody
EXAMPLES
Materials & General Methods
[0112] General information regarding the nucleotide sequences of
human immunoglobulins light and heavy chains is given in: Kabat, E.
A., et al., Sequences of Proteins of Immunological Interest, 5th
ed., Public Health Service, National Institutes of Health,
Bethesda, Md. (1991). Amino acids of antibody chains are numbered
and referred to according to EU numbering (Edelman, G. M., et al.,
Proc. Natl. Acad. Sci. USA 63 (1969) 78-85; Kabat, E. A., et al.,
Sequences of Proteins of Immunological Interest, 5th ed., Public
Health Service, National Institutes of Health, Bethesda, Md.,
(1991)).
Recombinant DNA Techniques
[0113] Standard methods were used to manipulate DNA as described in
Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The
molecular biological reagents were used according to the
manufacturer's instructions.
Gene Synthesis
[0114] Desired gene segments were prepared from oligonucleotides
made by chemical synthesis. The 600-1800 bp long gene segments,
which are flanked by singular restriction endonuclease cleavage
sites, were assembled by annealing and ligation of oligonucleotides
including PCR amplification and subsequently cloned via the
indicated restriction sites e.g. KpnI/SacI or AscI/PacI into a
pPCRScript (Stratagene) based pGA4 cloning vector. The DNA
sequences of the subcloned gene fragments were confirmed by DNA
sequencing. Gene synthesis fragments were ordered according to
given specifications at Geneart (Regensburg, Germany).
DNA Sequence Determination
[0115] DNA sequences were determined by double strand sequencing
performed at MediGenomix GmbH (Martinsried, Germany) or Sequiserve
GmbH (Vaterstetten, Germany).
DNA and Protein Sequence Analysis and Sequence Data Management
[0116] The GCG's (Genetics Computer Group, Madison, Wis.) software
package version 10.2 and Infomax's Vector NT1 Advance suite version
8.0 was used for sequence creation, mapping, analysis, annotation
and illustration.
Expression Vectors
[0117] For the expression of the described antibodies variants of
expression plasmids for transient expression (e.g. in HEK293 EBNA
or HEK293-F) cells based either on a cDNA organization with a
CMV-Intron A promoter or on a genomic organization with a CMV
promoter were applied.
[0118] Beside the antibody expression cassette the vectors
contained:
[0119] an origin of replication which allows replication of this
plasmid in E. coli, and
[0120] a .beta.-lactamase gene which confers ampicillin resistance
in E. coli.
[0121] The transcription unit of the antibody gene is composed of
the following elements:
[0122] unique restriction site(s) at the 5' end
[0123] the immediate early enhancer and promoter from the human
cytomegalovirus,
[0124] followed by the Intron A sequence in the case of the cDNA
organization,
[0125] a 5'-untranslated region of a human antibody gene,
[0126] a immunoglobulin heavy chain signal sequence,
[0127] the human antibody chain (wildtype or with domain exchange)
either as cDNA or as genomic organization with an the
immunoglobulin exon-intron organization
[0128] a 3' untranslated region with a polyadenylation signal
sequence, and
[0129] unique restriction site(s) at the 3' end.
[0130] The fusion genes comprising the described antibody chains as
described below were generated by PCR and/or gene synthesis and
assembled with known recombinant methods and techniques by
connection of the according nucleic acid segments e.g. using unique
restriction sites in the respective vectors. The subcloned nucleic
acid sequences were verified by DNA sequencing. For transient
transfections larger quantities of the plasmids were prepared by
plasmid preparation from transformed E. coli cultures (Nucleobond
AX, Macherey-Nagel).
Cell Culture Techniques
[0131] Standard cell culture techniques were used as described in
Current Protocols in Cell Biology (2000), Bonifacino, J. S., Dasso,
M., Harford, J. B., Lippincott-Schwartz, J. and Yamada, K. M.
(eds.), John Wiley & Sons, Inc.
[0132] Bispecific antibodies were expressed by transient
co-transfection of the respective expression plasmids in adherently
growing HEK293-EBNA or in HEK29-F cells growing in suspension as
described below.
Transient Transfections in HEK293-EBNA System
[0133] Bispecific antibodies were expressed by transient
co-transfection of the respective expression plasmids (e.g.
encoding the heavy and modified heavy chain, as well as the
corresponding light and modified light chain) in adherently growing
HEK293-EBNA cells (human embryonic kidney cell line 293 expressing
Epstein-Barr-Virus nuclear antigen; American type culture
collection deposit number ATCC # CRL-10852, Lot. 959 218)
cultivated in DMEM (Dulbecco's modified Eagle's medium, Gibco)
supplemented with 10% Ultra Low IgG FCS (fetal calf serum, Gibco),
2 mM L-Glutamine (Gibco), and 250 .mu.g/ml Geneticin (Gibco). For
transfection FuGENE.TM. 6 Transfection Reagent (Roche Molecular
Biochemicals) was used in a ratio of FuGENE.TM. reagent (.mu.l) to
DNA (.mu.g) of 4:1 (ranging from 3:1 to 6:1). Proteins were
expressed from the respective plasmids using a molar ratio of
(modified and wildtype) light chain and heavy chain encoding
plasmids of 1:1 (equimolar) ranging from 1:2 to 2:1, respectively.
Cells were feeded at day 3 with L-Glutamine ad 4 mM, Glucose
[Sigma] and NAA [Gibco]. Bispecific antibody containing cell
culture supernatants were harvested from day 5 to 11 after
transfection by centrifugation and stored at -20.degree. C. General
information regarding the recombinant expression of human
immunoglobulins in e.g. HEK293 cells is given in: Meissner, P. et
al., Biotechnol. Bioeng. 75 (2001) 197-203.
Transient Transfections in HEK293-F System
[0134] Bispecific antibodies were generated by transient
transfection of the respective plasmids (e.g. encoding the heavy
and modified heavy chain, as well as the corresponding light and
modified light chain) using the HEK293-F system (Invitrogen)
according to the manufacturer's instruction. Briefly, HEK293-F
cells (Invitrogen) growing in suspension either in a shake flask or
in a stirred fermenter in serumfree FreeStyle 293 expression medium
(Invitrogen) were transfected with a mix of the four expression
plasmids and 293fectin or fectin (Invitrogen). For 2 L shake flask
(Corning) HEK293-F cells were seeded at a density of 1.0E*6
cells/mL in 600 mL and incubated at 120 rpm, 8% CO2. The day after
the cells were transfected at a cell density of ca. 1.5E*6 cells/mL
with ca. 42 mL mix of A) 20 mL Opti-MEM (Invitrogen) with 600 .mu.g
total plasmid DNA (1 .mu.g/mL) encoding the heavy or modified heavy
chain, respectively and the corresponding light chain in an
equimolar ratio and B) 20 ml Opti-MEM+1.2 mL 293 fectin or fectin
(2 .mu.l/mL). According to the glucose consumption glucose solution
was added during the course of the fermentation. The supernatant
containing the secreted antibody was harvested after 5-10 days and
antibodies were either directly purified from the supernatant or
the supernatant was frozen and stored.
Protein Determination
[0135] The protein concentration of purified antibodies and
derivatives was determined by determining the optical density (OD)
at 280 nm, using the molar extinction coefficient calculated on the
basis of the amino acid sequence according to Pace et al., Protein
Science, 1995, 4, 2411-1423.
Antibody Concentration Determination in Supernatants
[0136] The concentration of antibodies and derivatives in cell
culture supernatants was estimated by immunoprecipitation with
Protein A Agarose-beads (Roche). 60 .mu.L Protein A Agarose beads
are washed three times in TBS-NP40 (50 mM Tris, pH 7.5, 150 mM
NaCl, 1% Nonidet-P40). Subsequently, 1-15 mL cell culture
supernatant were applied to the Protein A Agarose beads
pre-equilibrated in TBS-NP40. After incubation for at 1 h at room
temperature the beads were washed on an Ultrafree-MC-filter column
(Amicon] once with 0.5 mL TBS-NP40, twice with 0.5 mL 2.times.
phosphate buffered saline (2.times.PBS, Roche) and briefly four
times with 0.5 mL 100 mM Na-citrate pH 5.0. Bound antibody was
eluted by addition of 35 .mu.l NuPAGE.RTM. LDS Sample Buffer
(Invitrogen). Half of the sample was combined with NuPAGE.RTM.
Sample Reducing Agent or left unreduced, respectively, and heated
for 10 min at 70.degree. C. Consequently, 5-30 .mu.l were applied
to an 4-12% NuPAGE.RTM. Bis-Tris SDS-PAGE (Invitrogen) (with MOPS
buffer for non-reduced SDS-PAGE and MES buffer with NuPAGE.RTM.
Antioxidant running buffer additive (Invitrogen) for reduced
SDS-PAGE) and stained with Coomassie Blue.
[0137] The concentration of antibodies and derivatives in cell
culture supernatants was quantitatively measured by affinity HPLC
chromatography. Briefly, cell culture supernatants containing
antibodies and derivatives that bind to Protein A were applied to
an Applied Biosystems Poros A/20 column in 200 mM KH2PO4, 100 mM
sodium citrate, pH 7.4 and eluted from the matrix with 200 mM NaCl,
100 mM citric acid, pH 2.5 on an Agilent HPLC 1100 system. The
eluted protein was quantified by UV absorbance and integration of
peak areas. A purified standard IgG1 antibody served as a
standard.
[0138] Alternatively, the concentration of antibodies and
derivatives in cell culture supernatants was measured by
Sandwich-IgG-ELISA. Briefly, StreptaWell High Bind Strepatavidin
A-96 well microtiter plates (Roche) were coated with 100 .mu.L/well
biotinylated anti-human IgG capture molecule
F(ab')2<h-Fc.gamma.> BI (Dianova) at 0.1 .mu.g/mL for 1 h at
room temperature or alternatively over night at 4.degree. C. and
subsequently washed three times with 200 .mu.L/well PBS, 0.05%
Tween (PBST, Sigma). 100 .mu.L/well of a dilution series in PBS
(Sigma) of the respective antibody containing cell culture
supernatants was added to the wells and incubated for 1-2 h on a
microtiterplate shaker at room temperature. The wells were washed
three times with 200 .mu.L/well PBST and bound antibody was
detected with 100 .mu.l F(ab')2<hFc.gamma.>POD (Dianova) at
0.1 .mu.g/mL as detection antibody for 1-2 h on a microtiterplate
shaker at room temperature. Unbound detection antibody was washed
away three times with 200 .mu.L/well PBST and the bound detection
antibody was detected by addition of 100 .mu.L ABTS/well.
Determination of absorbance was performed on a Tecan Fluor
Spectrometer at a measurement wavelength of 405 nm (reference
wavelength 492 nm).
Protein Purification
[0139] Proteins were purified from filtered cell culture
supernatants referring to standard protocols. In brief, antibodies
were applied to a Protein A Sepharose column (GE healthcare) and
washed with PBS. Elution of antibodies was achieved at pH 2.8
followed by immediate neutralization of the sample. Aggregated
protein was separated from monomeric antibodies by size exclusion
chromatography (Superdex 200, GE Healthcare) in PBS or in 20 mM
Histidine, 150 mM NaCl pH 6.0. Monomeric antibody fractions were
pooled, concentrated if required using e.g. a MILLIPORE Amicon
Ultra (30 MWCO) centrifugal concentrator, frozen and stored at
-20.degree. C. or -80.degree. C. Part of the samples were provided
for subsequent protein analytics and analytical characterization
e.g. by SDS-PAGE, size exclusion chromatography or mass
spectrometry.
SDS-PAGE
[0140] The NuPAGE.RTM. Pre-Cast gel system (Invitrogen) was used
according to the manufacturer's instruction. In particular, 10% or
4-12% NuPAGE.RTM. Novex.RTM. Bis-TRIS Pre-Cast gels (pH 6.4) and a
NuPAGE.RTM. MES (reduced gels, with NuPAGE.RTM. Antioxidant running
buffer additive) or MOPS (non-reduced gels) running buffer was
used.
Analytical Size Exclusion Chromatography
[0141] Size exclusion chromatography for the determination of the
aggregation and oligomeric state of antibodies was performed by
HPLC chromatography. Briefly, Protein A purified antibodies were
applied to a Tosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM
KH2PO4/K2HPO4, pH 7.5 on an Agilent HPLC 1100 system or to a
Superdex 200 column (GE Healthcare) in 2.times.PBS on a Dionex
HPLC-System. The eluted protein was quantified by UV absorbance and
integration of peak areas. BioRad Gel Filtration Standard 151-1901
served as a standard.
Mass Spectrometry
[0142] The total deglycosylated mass of crossover antibodies was
determined and confirmed via electrospray ionization mass
spectrometry (ESI-MS). Briefly, 100 .mu.g purified antibodies were
deglycosylated with 50 mU N-Glycosidase F (PNGaseF, ProZyme) in 100
mM KH2PO4/K2HPO4, pH 7 at 37.degree. C. for 12-24 h at a protein
concentration of up to 2 mg/ml and subsequently desalted via HPLC
on a Sephadex G25 column (GE Healthcare). The mass of the
respective heavy and light chains was determined by ESI-MS after
deglycosylation and reduction. In brief, 50 .mu.g antibody in 115
.mu.l were incubated with 60 .mu.l 1M TCEP and 50 .mu.l 8 M
Guanidine-hydrochloride subsequently desalted. The total mass and
the mass of the reduced heavy and light chains was determined via
ESI-MS on a Q-Star Elite MS system equipped with a NanoMate
source.
IGF-1R ECD Binding ELISA
[0143] The binding properties of the generated antibodies were
evaluated in an ELISA assay with the IGF-1R extracellular domain
(ECD). For this sake the extracellular domain of IGF-1R (residues
1-462) comprising the natural leader sequence and the LI-cysteine
rich-12 domains of the human IGF-IR ectodomain of the alpha chain
(according to the McKern et al., 1997; Ward et al., 2001) fused to
an N-terminal His-Streptavidin binding peptide-tag (His-SBP) was
cloned into a pcDNA3 vector derivative and transiently expressed in
HEK293F cells. The protein sequence of the IGF-1R-His-SBP ECD is
given in SEQ ID NO: 12. StreptaWell High Bind Strepatavidin A-96
well microtiter plates (Roche) were coated with 100 .mu.L/well cell
culture supernatant containing soluble IGF-1R-ECD-SBP fusion
protein over night at 4.degree. C. and washed three times with 200
.mu.L/well PBS, 0.05% Tween (PBST, Sigma). Subsequently, 100
.mu.L/well of a dilution series of the respective antibody and as a
reference wildtype <IGF-1R> antibody in PBS (Sigma) including
1% BSA (fraction V, Roche) was added to the wells and incubated for
1-2 h on a microtiterplate shaker at room temperature. For the
dilution series the same amount of purified antibody were applied
to the wells. The wells were washed three times with 200 .mu.L/well
PBST and bound antibody was detected with 100 .mu.L/well
F(ab')2<hFc.gamma.>POD (Dianova) at 0.1 .mu.g/mL (1:8000) as
detection antibody for 1-2 h on a microtiterplate shaker at room
temperature. Unbound detection antibody was washed away three times
with 200 .mu.L/well PBST and the bound detection antibody was
detected by addition of 100 .mu.L ABTS/well. Determination of
absorbance was performed on a Tecan Fluor Spectrometer at a
measurement wavelength of 405 nm (reference wavelength 492 nm).
IGF-1R ECD Biacore
[0144] Binding of the generated antibodies to human IGF-1R ECD was
also investigated by surface plasmon resonance using a BIACORE T100
instrument (GE Healthcare Biosciences AB, Uppsala, Sweden).
Briefly, for affinity measurements Goat-Anti-Human IgG, JIR
109-005-098 antibodies were immobilized on a CMS chip via amine
coupling for presentation of the antibodies against human IGF-1R
ECD-Fc tagged. Binding was measured in HBS buffer (HBS-P (10 mM
HEPES, 150 mM NaCl, 0.005% Tween 20, ph 7.4), 25.degree. C. IGF-1R
ECD (R&D Systems or in house purified) was added in various
concentrations in solution. Association was measured by an IGF-1R
ECD injection of 80 seconds to 3 minutes; dissociation was measured
by washing the chip surface with HBS buffer for 3-10 minutes and a
KD value was estimated using a 1:1 Langmuir binding model. Due to
low loading density and capturing level of <IGF-1R>
antibodies monovalent IGF-1R ECD binding was obtained. Negative
control data (e.g. buffer curves) were subtracted from sample
curves for correction of system intrinsic baseline drift and for
noise signal reduction. Biacore T100 Evaluation Software version
1.1.1 was used for analysis of sensorgrams and for calculation of
affinity data. FIG. 11 shows a scheme of the Biacore assay.
Examples 1
Production, Expression, Purification and Characterization of
Monospecific, Bivalent <IGF-1R> Antibody, Wherein the
Variable Domains CL and CH1 are Replaced by Each Other (Abbreviated
Herein as <IGF-1R> VL-VH Exchange Antibody)
Example 1A
Making of the Expression Plasmids for the Monospecific, Bivalent
<IGF-1R> VL-VH Exchange Antibody
[0145] The sequences for the heavy and light chain variable domains
of the monospecific, bivalent <IGF-1R> VL-VH exchange
antibody including the respective leader sequences described in
this example are derived from a human <IGF-1R> antibody heavy
chain (SEQ ID NO: 1, plasmid 4843-pUC-HC-IGF-1R) and a light chain
(SEQ ID NO: 2, plasmid 4842-pUC-LC-IGF-1R) described in WO
2005/005635, and the heavy and light chain constant domains are
derived from a human antibody (C-kappa and IgG1).
[0146] The gene segments encoding the <IGF-1R> antibody
leader sequence, light chain variable domain (VL) and the human
heavy chain constant domain 1 (CH1) were joined and fused to the
5'-end of the Fc domains of the human .gamma.1-heavy chain constant
domains (Hinge-CH2-CH3). The DNA coding for the respective fusion
protein resulting from the exchange of the VH domain by the VL
domain (VH-VL exchange) was generated by gene synthesis and is
denoted <IGF-1R> HC*** (SEQ ID NO: 10) in the following.
Initially, the VL-CH1 domains were fused with a slightly different
sequence (SEQ ID NO: 3); due to the reduced expression yields of
this connection, SEQ10 that shows expression yields comparable to
wildtype antibodies, was chosen. The gene segments for the
<IGF-1R> antibody leader sequence, heavy chain variable
domain (VH) and the human light chain constant domain (CL) were
joined as independent chain. The DNA coding for the respective
fusion protein resulting from the exchange of the VL domain by the
VH domain (VL-VH exchange) was generated by gene synthesis and is
denoted <IGF-1R> LC*** (Heavy Chain***) (SEQ ID NO: 11) in
the following. Initially, the VH-CL domains were fused with a
slightly different sequence (SEQ ID NO: 4); due to the reduced
expression yields of this connection, SEQ ID NO: 11 that shows
expression yields comparable to wildtype antibodies was chosen.
[0147] FIG. 5 and FIG. 6 show a schematic view of the protein
sequence of the modified <IGF-1R> HC** heavy chain and the
modified <IGF-1R> LC** light chain.
[0148] In the following the respective expression vectors are
briefly described:
Vector pUC-HC**-IGF-1R Vector pUC-HC**-IGF-1R is an expression
plasmid e.g. for transient expression of a VL-VH exchange
<IGF-1R> heavy chain HC** (cDNA organized expression
cassette; with CMV-Intron A) in HEK293 (EBNA) cells or for stable
expression in CHO cells.
[0149] Beside the <IGF-1R> HC** Expression Cassette this
Vector Contains:
[0150] an origin of replication from the vector pUC18 which allows
replication of this plasmid in E. coli, and
[0151] a .beta.-lactamase gene which confers ampicillin resistance
in E. coli.
[0152] The transcription unit of the <IGF-1R> HC** gene is
composed of the following elements:
[0153] the AscI restriction site at the 5'-end
[0154] the immediate early enhancer and promoter from the human
cytomegalovirus,
[0155] followed by the Intron A sequence,
[0156] a 5'-untranslated region of a human antibody gene,
[0157] a immunoglobulin light chain signal sequence,
[0158] the human <IGF-1R> mature HC** chain encoding a fusion
of the human heavy chain variable domain (VH) and the human
kappa-light chain constant domain (CL) fused to the 5'-end of the
Fc domains of the human .gamma.1-heavy chain constant domains
(Hinge-CH2-CH3).
[0159] a 3' untranslated region with a polyadenylation signal
sequence, and
[0160] the restriction site SgrAI at the 3'-end.
[0161] The plasmid map of the heavy chain** VL-VH exchange
<IGF-1R> HC** expression vector pUC-HC**-IGF-1R is shown in
FIG. 7. The amino acid sequence of the <IGF-1R> HC**
(including signal sequence) is given in SEQ ID NO: 10.
Vector pUC-LC**-IGF-1R
[0162] Vector pUC-LC**-IGF-1R is an expression plasmid e.g. for
transient expression of a VL-VH exchange <IGF-1R> light chain
LC** (cDNA organized expression cassette; with CMV-Intron A) in
HEK293 (EBNA) cells or for stable expression in CHO cells.
[0163] Beside the <IGF-1R> LC** Expression Cassette this
Vector Contains:
[0164] an origin of replication from the vector pUC18 which allows
replication of this plasmid in E. coli, and
[0165] a .beta.-lactamase gene which confers ampicillin resistance
in E. coli.
[0166] The Transcription Unit of the <IGF-1R> LC** Gene is
Composed of the Following Elements:
[0167] the restriction site Sse8387I at the 5' end
[0168] the immediate early enhancer and promoter from the human
cytomegalovirus,
[0169] followed by the Intron A sequence,
[0170] a 5'-untranslated region of a human antibody gene,
[0171] a immunoglobulin heavy chain signal sequence,
[0172] the human <IGF-1R> antibody mature LC** chain encoding
a fusion of the human light chain variable domain (VL) and the
human .gamma.1-heavy chain constant domains (CH1).
[0173] a 3' untranslated region with a polyadenylation signal
sequence, and
[0174] the restriction sites SalI and FseI at the 3'-end.
[0175] The plasmid map of the light chain** VL-VH exchange
<IGF-1R> LC** expression vector pUC-LC**-IGF-1R is shown in
FIG. 8. The amino acid sequence of the <IGF-1R> LC**
(including signal sequence) is given in SEQ ID NO: 11.
[0176] Plasmids pUC-HC**-IGF-1R and pUC-LC**-IGF-1R can be used for
transient or stable co-transfections e.g. into HEK293, HEK293 EBNA
or CHO cells (2-vector system). For comparative reasons the
wildtype <IGF-1R> antibody was transiently expressed from
plasmids 4842-pUC-LC-IGF-1R (SEQ ID NO: 2) and 4843-pUC-HC-IGF-1R
(SEQ ID NO: 1) analogous to the ones described in this example.
[0177] In order to achieve higher expression levels in transient
expressions in HEK293 EBNA cells the <IGF-1R> HC** expression
cassette can be sub-cloned via AscI, SgrAI sites and the
<IGF-1R> LC** expression cassette can be sub-cloned via
Sse8387I and FseI sites into the 4700 pUC-Hyg_OriP expression
vector containing
[0178] an OriP element, and
[0179] a hygromycine resistance gene as a selectable marker.
[0180] Heavy and light chain transcription units can either be
sub-cloned into two independent 4700-pUC-Hyg-OriP vectors for
co-transfection (2-vector system) or they can be cloned into one
common 4700-pUC-Hyg-OriP vector (1-vector system) for subsequent
transient or stable transfections with the resulting vectors. FIG.
9 shows a plasmid map of the basic vector 4700-pUC-OriP.
Example 1B
Making of the Monospecific, Bivalent <IGF-1R> VL-VH Exchange
Antibody Expression Plasmids
[0181] The <IGF-1R> fusion genes (HC** and LC** fusion genes)
comprising the exchanged Fab sequences of the wildtype
<IGF-1R> antibody were assembled with known recombinant
methods and techniques by connection of the according nucleic acid
segments.
[0182] The nucleic acid sequences encoding the IGF-1R HC** and LC**
were each synthesized by chemical synthesis and subsequently cloned
into a pPCRScript (Stratagene) based pGA4 cloning vector at Geneart
(Regensburg, Germany). The expression cassette encoding the IGF-1R
HC** was ligated into the respective E. coli plasmid via PvuII and
BmgBI restriction sites resulting in the final vector
pUC-HC**-IGF-1R; the expression cassette encoding the respective
IGF-1R LC** was ligated into the respective E. coli plasmid via
PvuII and SalI restriction sites resulting in the final vector
pUC-LC**-IGF-1R. The subcloned nucleic acid sequences were verified
by DNA sequencing. For transient and stable transfections larger
quantities of the plasmids were prepared by plasmid preparation
from transformed E. coli cultures (Nucleobond AX,
Macherey-Nagel)
Example 1C
Transient Expression of Monospecific, Bivalent IGF-1R> VL-VH
Exchange Antibody, Purification and Confirmation of Identity by
Mass Spectrometry
[0183] Recombinant <IGF-1R> VL-VH exchange antibody was
expressed by transient co-transfection of plasmids pUC-HC**-IGF-1R
and pUC-LC**-IGF-1R in HEK293-F suspension cells as described
above.
[0184] The expressed and secreted monospecific, bivalent
<IGF-1R> VL-VH exchange antibody was purified from filtered
cell culture supernatants by Protein A affinity chromatography
according as described above. In brief, the <IGF-1R> VL-VH
exchange antibody containing cell culture supernatants from
transient transfections were clarified by centrifugation and
filtration and applied to a Protein A HiTrap MabSelect Xtra column
(GE Healthcare) equilibrated with PBS buffer (10 mM Na2HPO4, 1 mM
KH2PO4, 137 mM NaCl and 2.7 mM KCl, pH 7.4). Unbound proteins were
washed out with PBS equilibration buffer followed by 0.1 M sodium
citrate buffer, pH 5.5 and washed with PBS. Elution of antibody was
achieved with 100 mM sodium citrate, pH 2.8 followed by immediate
neutralization of the sample with 300 .mu.l 2 M Tris pH 9.0 per 2
ml fraction. Aggregated protein was separated from monomeric
antibodies by size exclusion chromatography on a HiLoad 26/60
Superdex 200 prep grade column (GE Healthcare) in 20 mM Histidine,
150 mM NaCl pH 6.0 and monomeric antibody fractions were
subsequently concentrated using a MILLIPORE Amicon Ultra-15
centrifugal concentrator. <IGF-1R> VL-VH exchange antibody
was frozen and stored at -20.degree. C. or -80.degree. C. The
integrity of the <IGF-1R> VL-VH exchange antibody was
analyzed by SDS-PAGE in the presence and absence of a reducing
agent and subsequent staining with Coomassie brilliant blue as
described above. Monomeric state of the <IGF-1R> VL-VH
exchange antibody was confirmed by analytical size exclusion
chromatography. (FIG. 12) Characterized samples were provided for
subsequent protein analytics and functional characterization. ESI
mass spectrometry confirmed the theoretical molecular mass of the
completely deglycosylated <IGF-1R> VL-VH exchange
antibody.
Example 1D
Analysis of the IGF-1R Binding Properties of Mono Specific,
Bivalent IGF-1R> VL-VH Exchange Antibody in an IGF-1R ECD
Binding ELISA and by Biacore
[0185] The binding properties of monospecific, bivalent
<IGF-1R> VL-VH exchange antibody were evaluated in an ELISA
assay with the IGF-1R extracellular domain (ECD) as descried above.
For this sake the extracellular domain of IGF-1R (residues 1-462)
comprising the natural leader sequence and the LI-cysteine rich-12
domains of the human IGF-IR ectodomain of the alpha chain
(according to the McKern et al., 1997; Ward et al., 2001) fused to
an N-terminal His-Streptavidin binding peptide-tag (His-SBP) was
cloned into a pcDNA3 vector derivative and transiently expressed in
HEK293F cells. The protein sequence of the IGF-1R-His-SBP ECD is
given in see above. The obtained titration curve showed that
<IGF-1R> VL-VH exchange antibody was functional and showed
comparable binding characteristics and kinetics as the wildtype
<IGF-1R> antibody within the error of the method and thus
appeared fully functional (FIG. 13).
[0186] These findings are being confirmed by Biacore with the
respective purified antibodies.
Example 1G
Analysis of the IGF-1R Binding Properties of Monospecific, Bivalent
IGF-1R> VL-VH Exchange Antibody by FACS with IGF-1R
Over-Expressing I24 Cells
[0187] In order to confirm the binding activity of <IGF-1R>
VL-VH exchange antibody to the IGF-1R over-expressed on the surface
of I24 cells (NIH3T3 cells expressing recombinant human IGF-1R,
Roche) is studied by FACS. Briefly, 5.times.10E5 I24 cells per FACS
tube are incubated with a dilution of purified <IGF-1R> VL-VH
exchange antibody and wildtype <IGF-1R> antibody as a
reference and incubated on ice for 1 h. Unbound antibody is washed
away with 4 ml ice cold PBS (Gibco)+2% FCS (Gibco). Subsequently,
cells are centrifuged (5 min at 400 g) and bound antibody is
detected with F(ab')2<hFc.gamma.>PE conjugate (Dianova) on
ice for 1 h protected from light. Unbound detection antibody is
washed away with 4 ml ice cold PBS+2% FCS. Subsequently, cells are
centrifuged (5 min 400 g), resuspended in 300-500 .mu.L PBS and
bound detection antibody is quantified on a FACSCalibur or FACS
Canto (BD (FL2 channel, 10,000 cells per acquisition). During the
experiment the respective isotype controls are included to exclude
any unspecific binding events. Binding of <IGF-1R> VL-VH
exchange antibody and wildtype <IGF-1R> reference antibody to
IGF-1R on I24 cells result in a comparable, concentration dependent
shift of mean fluorescence intensity.
Examples 2
Description of a Mono Specific, Bivalent <ANGPT2> Wildtype
Antibody
Example 2A
Making of the Expression Plasmids for the Monospecific, Bivalent
<ANGPT2> Wildtype Antibody
[0188] The sequences for the heavy and light chain variable domains
of a monospecific, bivalent ANGPT2 <ANGPT2> wildtype antibody
including the respective leader sequences described in this example
are derived from a human <ANGPT2> antibody heavy chain (SEQ
ID NO: 6) and a light chain (SEQ ID NO: 7) described in WO
2006/045049 and the heavy and light chain constant domains are
derived from a human antibody (C-kappa and IgG1).
[0189] The wildtype <ANGPT2> antibody was cloned into
plasmids SB04-pUC-HC-ANGPT2 (SEQ ID NO: 6) and SB06-pUC-LC-ANGPT2
(SEQ ID NO: 7) that are analogous to the vectors described in the
previous example 1A.
[0190] For comparative reasons and for co-expression experiments
(see example 3) the wildtype <ANGPT2> antibody was
transiently (co-) expressed from plasmids SB04-pUC-HC-ANGPT2 and
SB06-pUC-LC-ANGPT2.
Example 2B
Making of the Mono Specific, Bivalent <ANGPT2> Wildtype
Antibody Expression Plasmids
[0191] The nucleic acid sequences encoding the ANGPT2> HC and LC
were each synthesized by chemical synthesis and subsequently cloned
into a pPCRScript (Stratagene) based pGA4 cloning vector at Geneart
(Regensburg, Germany). The expression cassette encoding the
<ANGPT2> HC was cloned into the respective E. coli plasmid
resulting in the final vector SB04-pUC-HC-ANGPT2; the expression
cassette encoding the respective <ANGPT2> LC was cloned into
the respective E. coli plasmid resulting in the final vector
SB06-pUC-LC-ANGPT2. The subcloned nucleic acid sequences were
verified by DNA sequencing. For transient and stable transfections
larger quantities of the plasmids were prepared by plasmid
preparation from transformed E. coli cultures (Nucleobond AX,
Macherey-Nagel).
Examples 3
Expression of Bispecific, Bivalent <ANGPT2-IGF-1R> Antibody,
Wherein in the Heavy and Light Chain Specifically Binding to
IGF-1R, the Constant Domains VL and VH are Replaced by Each Other
(Abbreviated Herein as <ANGPT2-IGF-1R> VL-VH Exchange
Antibody)
Example 3A
Transient Co-Expression and Purification of <IGF-1R> VL-VH
Exchange Antibody and <ANGPT2> Wildtype Antibody in HEK293
EBNA Cells to Yield Bispecific <ANGPT2-IGF-1R> VL-VH Exchange
Antibody
[0192] In order to generate a functional bispecific antibody
recognizing IGF-1R via the <IGF-1R> VL-VH exchange antibody
Fab on one side and <ANGPT2> via the <ANGPT2> wildtype
Fab region on the other side the two expression plasmids coding for
the <IGF-1R> VL-VH exchange antibody (example 1A) were
co-expressed with two expression plasmids coding for the
<ANGPT2> wildtype antibody. (example 2A). Assuming a
statistical association of wildtype heavy chains HC and VL-VH
exchange heavy chains HC** this results in the generation of
bispecific and bivalent <IGF-1R-ANGPT2> VL-VH exchange
antibody. Under the assumption that both antibodies are equally
well expressed and without taking side products into account this
should result in a 1:2:1 ratio of the three main products A)
<IGF-1R> VL-VH exchange antibody, B) bispecific
<IGF-1R-ANGPT2> VL-VH exchange antibody, and C)
<ANGPT2> wildtype antibody. Several side products can be
expected. However, due to the exchange of only the VL-VH domains
the frequency of side products should be reduced compared to the
complete Fab crossover. Please note as the <ANGPT2> wildtype
antibody showed higher expression transient expression yields than
the <IGF-1R> wildtype and <IGF-1R> VL-VH exchange
antibodies the ratio of <ANGPT2> wildtype antibody plasmids
and <IGF-1R> VL-VH exchange antibody plasmids was shifted in
favour of the expression of <ANGPT2> wildtype antibody.
[0193] To generate the mix of the main products A) <IGF-1R>
VL-VH exchange antibody, B) bispecific <ANGPT2-IGF-1R> VL-VH
exchange antibody, and C) <ANGPT2> wildtype antibody the four
plasmids pUC-HC**-IGF-1R and pUC-LC**-IGF-1R and plasmids
SB04-pUC-HC-ANGPT2 and SB06-pUC-LC-ANGPT2 were transiently
co-transfected in suspension HEK293-F cells as described above The
harvested supernatant contained a mix of the main products A)
<IGF-1R> VL-VH exchange antibody, B) bispecific
<ANGPT2-IGF-1R> VL-VH exchange antibody, and C)
<ANGPT2> wildtype antibody and is denoted as "Bispecific
VL-VH exchange mix". Bispecific VL-VH exchange mix containing cell
culture supernatants, were harvested by centrifugation and
subsequently purified as described above.
[0194] The integrity of the antibody mix was analyzed by SDS-PAGE
in the presence and absence of a reducing agent and subsequent
staining with Coomassie brilliant blue and by size exclusion
chromatography as described. The SDS-PAGE showed that there were 2
different heavy and light chain presents in the preparation as
expected (reduced gel) (FIG. 14). Characterized samples were
provided for subsequent protein analytics and functional
characterization.
Example 3B
Detection of Functional Bispecific <ANGPT2-IGF-1R> VL-VH
Exchange Antibody in a Cellular FACS Bridging Assay on I24 IGF-1R
Expressing Cells
[0195] In order to confirm the presence of functional bispecific
<ANGPT2-IGF-1R> VL-VH exchange antibody in the purified
bispecific VL-VH exchange mix of the main products A)
<IGF-1R> VL-VH exchange antibody, B) bispecific
<ANGPT2-IGF-1R> VL-VH exchange antibody, and C)
<ANGPT2> wildtype antibody from the transient co-expression
described in example 3A, a cellular FACS IGF-1R-ANGPT2 bridging
assay on I24 cells (NIH3T3 cells expressing recombinant human
IGF-1R, Roche) was performed. The assay principle is depicted in
FIG. 10. A bispecific <ANGPT2-IGF-1R> VL-VH exchange antibody
that is present in the purified antibody mix is capable of binding
to IGF-1R in I24 cells and to ANGPT2 simultaneously; and thus will
bridge its two target antigens with the two opposed Fab
regions.
[0196] Briefly, 5.times.10E5 I24 cells per FACS tube were incubated
with total purified antibody mix and incubated on ice for 1 h
(titration 160 .mu.g/ml mix). The respective purified antibodies
wildtype <IGF-1R> and <ANGPT2> were applied to the I24
cells as controls. Unbound antibody was washed away with 4 ml ice
cold PBS (Gibco)+2% FCS (Gibco), cells were centrifuged (5 min at
400 g) and bound bispecific antibody was detected with 50 .mu.l 2
.mu.g/mL human ANGPT2 (R&D Systems) for 1 h on ice.
Subsequently, unbound ANGPT2 was washed away once or twice with 4
ml ice cold PBS (Gibco)+2% FCS (Gibco), cells were centrifuged (5
min at 400 g) and bound ANGPT2 was detected with 50 .mu.l 5
.mu.g/mL <ANGPT2>mIgG1-Biotin antibody (BAM0981, R&D
Systems) for 45 min on ice; alternatively, cells were incubated
with 50 .mu.l 5 .mu.g/mL mIgG1-Biotin-Isotype control (R&D
Systems). Unbound detection antibody was washed away with 4 ml ice
cold PBS (Gibco)+2% FCS (Gibco), cells were centrifuged (5 min at
400 g) and bound detection antibody was detected with 50 .mu.l
1:400 Streptavidin-PE conjugate (Invitrogen/Zymed) for 45 min on
ice protected from light. Unbound Streptavidin-PE conjugate was
washed away with 4 ml ice cold PBS+2% FCS. Subsequently, cells were
centrifuged (5 min 400 g), resuspended in 300-500 .mu.L PBS and
bound Streptavidin-PE conjugate was quantified on a FACSCalibur (BD
(FL2 channel, 10,000 cells per acquisition). During the experiment
the respective isotype controls were included to exclude any
unspecific binding events. In addition, purified monospecific,
bivalent IgG1 antibodies <IGF-1R> and <ANGPT2> were
included as controls.
[0197] The results in FIG. 15 show that the incubation with
purified antibody crossover mix (<ANGPT2-IGF-1R> VL-VH
exchange antibody) from the co-expression of a crossover antibody
(<IGF-1R> VL-VH exchange antibody) with a wildtype antibody
(<ANGPT2> wildtype antibody) resulted in a significant shift
in fluorescence indicating the presence of a functional bispecific
<ANGPT2-IGF-1R> VL-VH exchange antibody that was capable of
binding to IGF-1R in I24 cells and to ANGPT2 simultaneously; and
thus bridges its two target antigens with the two opposed Fab
regions. In contrast to this the respective <IGF-1R> and
<Ang-2> control antibodies did not result in shift in
fluorescence in the FACS bridging assay
[0198] Taken together these data show that by co-expressing the
respective wildtype and crossover plasmids functional bispecific
antibodies can be generated. The yields of correct bispecific
antibody can be increased by forcing the correct heterodimerization
of wildtype and modified crossover heavy chains e.g. using the
knobs-into-holes technology as well as disulfide stabilization (see
examples 4)
Example 4
Expression of Bivalent, Bispecific <ANGPT2-IGF-1R> VL-VH
Exchange Antibody with Modified CH3 Domains (Knobs-Into-Holes)
[0199] To further improve the yield of the bispecific
<ANGPT2-IGF-1R> VL-VH exchange antibody the knobs-into-holes
technology is applied to the co-expression of <IGF-1R> VL-VH
exchange and wildtype <ANGPT2> antibodies to obtain a
homogenous and functional bispecific antibody preparation. For this
purpose, the CH3 domain in the heavy chain* HC* of the
<IGF-1R> VL-VH exchange antibody is replaced by the CH3
domain (Knobs) of the SEQ ID NO: 8 with a T366W exchange and the
CH3 domain in the heavy chain of the wildtype <ANGPT2>
antibody is replaced by the CH3 domain (Hole) of the SEQ ID NO: 9
with a T366S, L368A, Y407V exchange or vice versa. In addition, a
disulfide can be included to increase the stability and yields as
well as additional residues forming ionic bridges and increasing
the heterodimerization yields (EP 1870459A1).
[0200] The transient co-expression, and the purification of the
resulting bivalent, bispecific <ANGPT2-IGF-1R> VL-VH exchange
antibody with modified CH3 domains (knobs-into-holes) is performed
as described in Example 3.
[0201] It should be noted that an optimization of
heterodimerization can be achieved e.g. by using different
knobs-in-holes technologies such as the introduction of an
additional disulfide bridge into the CH3 domain e.g. Y349C into the
"knobs chain" and D356C into the "hole chain" and/or combined with
the use of residues R409D; K370E (K409D) for knobs residues and
D399K; E357K for hole residues described by EP 1870459A1.
[0202] Analogously, further bivalent, bispecific VL-VH exchange
antibodies with modified CH3 domains (knobs-into-holes) directed
against ANGPT2 and another target antigen (using the above
described ANGPT2 heavy and light chain and the VL-VH exchange heavy
and light chain*** HC*** and LC*** of an antibody directed against
said other target, whereby both heavy chains are modified by
"knobs-in-holes"), or directed against IGF-1R and another target
(using the heavy and light chain of an antibody directed against
said other target and the above described IGF-1R VL-VH exchange
heavy and light chain*** HC*** and LC***, whereby both heavy chains
are modified by "knobs-in-holes") can be prepared.
Sequence CWU 1
1
121467PRTArtificial SequenceSynthetic polypeptide 1Met Glu Phe Gly
Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly1 5 10 15 Val Gln
Cys Gln Val Glu Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30
Pro Gly Arg Ser Gln Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35
40 45 Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu 50 55 60 Glu Trp Val Ala Ile Ile Trp Phe Asp Gly Ser Ser Thr
Tyr Tyr Ala65 70 75 80 Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Phe Cys Ala Arg Glu Leu
Gly Arg Arg Tyr Phe Asp Leu Trp Gly 115 120 125 Arg Gly Thr Leu Val
Ser Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala145 150 155 160
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165
170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys225 230 235 240 Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290
295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu385 390 395 400 Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410
415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 450 455 460 Pro Gly Lys465 2235PRTArtificial
SequenceSynthetic polypeptide 2Met Glu Ala Pro Ala Gln Leu Leu Phe
Leu Leu Leu Leu Trp Leu Pro1 5 10 15 Asp Thr Thr Gly Glu Ile Val
Leu Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser 35 40 45 Val Ser Ser
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 50 55 60 Arg
Leu Leu Ile Tyr Asp Ala Ser Lys Arg Ala Thr Gly Ile Pro Ala65 70 75
80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
85 90 95 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Arg Ser 100 105 110 Lys Trp Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys
Val Glu Ser Lys 115 120 125 Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu 130 135 140 Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe145 150 155 160 Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 165 170 175 Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 180 185 190 Thr
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 195 200
205 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
210 215 220 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys225 230 235
3459PRTArtificial SequenceSynthetic polypeptide 3Met Glu Ala Pro
Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro1 5 10 15 Asp Thr
Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30
Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser 35
40 45 Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro 50 55 60 Arg Leu Leu Ile Tyr Asp Ala Ser Lys Arg Ala Thr Gly
Ile Pro Ala65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser 85 90 95 Ser Leu Glu Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln Arg Ser 100 105 110 Lys Trp Pro Pro Trp Thr Phe
Gly Gln Gly Thr Lys Val Ser Val Ser 115 120 125 Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 130 135 140 Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp145 150 155 160
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 165
170 175 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr 180 185 190 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln 195 200 205 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp 210 215 220 Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr His Thr Cys Pro Pro225 230 235 240 Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 245 250 255 Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 260 265 270 Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 275 280 285
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 290
295 300 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val305 310 315 320 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser 325 330 335 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys 340 345 350 Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp 355 360 365 Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe 370 375 380 Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu385 390 395 400 Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 405 410
415 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
420 425 430 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr 435 440 445 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450
455 4243PRTArtificial SequenceSynthetic polypeptide 4Met Glu Phe
Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly1 5 10 15 Val
Gln Cys Gln Val Glu Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25
30 Pro Gly Arg Ser Gln Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
35 40 45 Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu 50 55 60 Glu Trp Val Ala Ile Ile Trp Phe Asp Gly Ser Ser
Thr Tyr Tyr Ala65 70 75 80 Asp Ser Val Arg Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Phe Cys Ala Arg Glu
Leu Gly Arg Arg Tyr Phe Asp Leu Trp Gly 115 120 125 Arg Gly Thr Leu
Val Glu Ser Lys Arg Thr Val Ala Ala Pro Ser Val 130 135 140 Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser145 150 155
160 Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
165 170 175 Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
Ser Val 180 185 190 Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser Ser Thr Leu 195 200 205 Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr Ala Cys Glu 210 215 220 Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser Phe Asn Arg225 230 235 240 Gly Glu
Cys5557PRTArtificial SequenceSynthetic polypeptide 5Met Lys Ser Gly
Ser Gly Gly Gly Ser Pro Thr Ser Leu Trp Gly Leu1 5 10 15 Leu Phe
Leu Ser Ala Ala Leu Ser Leu Trp Pro Thr Ser Gly Glu Ile 20 25 30
Cys Gly Pro Gly Ile Asp Ile Arg Asn Asp Tyr Gln Gln Leu Lys Arg 35
40 45 Leu Glu Asn Cys Thr Val Ile Glu Gly Tyr Leu His Ile Leu Leu
Ile 50 55 60 Ser Lys Ala Glu Asp Tyr Arg Ser Tyr Arg Phe Pro Lys
Leu Thr Val65 70 75 80 Ile Thr Glu Tyr Leu Leu Leu Phe Arg Val Ala
Gly Leu Glu Ser Leu 85 90 95 Gly Asp Leu Phe Pro Asn Leu Thr Val
Ile Arg Gly Trp Lys Leu Phe 100 105 110 Tyr Asn Tyr Ala Leu Val Ile
Phe Glu Met Thr Asn Leu Lys Asp Ile 115 120 125 Gly Leu Tyr Asn Leu
Arg Asn Ile Thr Arg Gly Ala Ile Arg Ile Glu 130 135 140 Lys Asn Ala
Asp Leu Cys Tyr Leu Ser Thr Val Asp Trp Ser Leu Ile145 150 155 160
Leu Asp Ala Val Ser Asn Asn Tyr Ile Val Gly Asn Lys Pro Pro Lys 165
170 175 Glu Cys Gly Asp Leu Cys Pro Gly Thr Met Glu Glu Lys Pro Met
Cys 180 185 190 Glu Lys Thr Thr Ile Asn Asn Glu Tyr Asn Tyr Arg Cys
Trp Thr Thr 195 200 205 Asn Arg Cys Gln Lys Met Cys Pro Ser Thr Cys
Gly Lys Arg Ala Cys 210 215 220 Thr Glu Asn Asn Glu Cys Cys His Pro
Glu Cys Leu Gly Ser Cys Ser225 230 235 240 Ala Pro Asp Asn Asp Thr
Ala Cys Val Ala Cys Arg His Tyr Tyr Tyr 245 250 255 Ala Gly Val Cys
Val Pro Ala Cys Pro Pro Asn Thr Tyr Arg Phe Glu 260 265 270 Gly Trp
Arg Cys Val Asp Arg Asp Phe Cys Ala Asn Ile Leu Ser Ala 275 280 285
Glu Ser Ser Asp Ser Glu Gly Phe Val Ile His Asp Gly Glu Cys Met 290
295 300 Gln Glu Cys Pro Ser Gly Phe Ile Arg Asn Gly Ser Gln Ser Met
Tyr305 310 315 320 Cys Ile Pro Cys Glu Gly Pro Cys Pro Lys Val Cys
Glu Glu Glu Lys 325 330 335 Lys Thr Lys Thr Ile Asp Ser Val Thr Ser
Ala Gln Met Leu Gln Gly 340 345 350 Cys Thr Ile Phe Lys Gly Asn Leu
Leu Ile Asn Ile Arg Arg Gly Asn 355 360 365 Asn Ile Ala Ser Glu Leu
Glu Asn Phe Met Gly Leu Ile Glu Val Val 370 375 380 Thr Gly Tyr Val
Lys Ile Arg His Ser His Ala Leu Val Ser Leu Ser385 390 395 400 Phe
Leu Lys Asn Leu Arg Leu Ile Leu Gly Glu Glu Gln Leu Glu Gly 405 410
415 Asn Tyr Ser Phe Tyr Val Leu Asp Asn Gln Asn Leu Gln Gln Leu Trp
420 425 430 Asp Trp Asp His Arg Asn Leu Thr Ile Lys Ala Gly Lys Met
Tyr Phe 435 440 445 Ala Phe Asn Pro Lys Leu Cys Val Ser Glu Ile Tyr
Arg Met Glu Glu 450 455 460 Val Thr Gly Thr Lys Gly Arg Gln Ser Lys
Gly Asp Ile Asn Thr Arg465 470 475 480 Asn Asn Gly Glu Arg Ala Ser
Cys Glu Ser Asp Val Ala Ala Ala Leu 485 490 495 Glu Val Leu Phe Gln
Gly Pro Gly Thr His His His His His His Ser 500 505 510 Gly Asp Glu
Lys Thr Thr Gly Trp Arg Gly Gly His Val Val Glu Gly 515 520 525 Leu
Ala Gly Glu Leu Glu Gln Leu Arg Ala Arg Leu Glu His His Pro 530 535
540 Gln Gly Gln Arg Glu Pro Ser Gly Gly Cys Lys Leu Gly545 550 555
6471PRTArtificial SequenceSynthetic polypeptide 6Met Glu Leu Gly
Leu Ser Trp Val Phe Leu Val Ala Ile Leu Glu Gly1 5 10 15 Val Gln
Cys Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln 20 25 30
Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35
40 45 Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu 50 55 60 Glu Trp Val Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile
Tyr Tyr Ala65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn 85 90 95 Ser Leu Tyr Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Asp Leu
Leu Asp Tyr Asp Ile Leu Thr Gly Tyr 115 120 125 Gly Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser145 150 155 160
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165
170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys 210 215 220 Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu225 230 235 240 Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290
295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr305 310
315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp385 390 395 400 Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435
440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys465 470
7219PRTArtificial SequenceSynthetic polypeptide 7Asp Ile Val Met
Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15 Glu Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30
Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Met Gln Gly 85 90 95 Thr His Trp Pro Pro Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165
170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 8107PRTArtificial SequenceSynthetic polypeptide 8Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu1 5 10 15
Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe 20
25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 100 105 9107PRTArtificial SequenceSynthetic
polypeptide 9Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys
Ala Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Val Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly65 70 75 80 Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 10440PRTArtificial
SequenceSynthetic polypeptide 10Glu Ile Val Leu Thr Gln Ser Pro Ala
Thr Leu Ser Leu Ser Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala
Ser Lys Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75
80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Lys Trp Pro Pro
85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ser Lys Ser Ser
Ala Ser 100 105 110 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr 115 120 125 Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro 130 135 140 Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val145 150 155 160 His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 165 170 175 Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 180 185 190 Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 195 200
205 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
210 215 220 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro225 230 235 240 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val 245 250 255 Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val 260 265 270 Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln 275 280 285 Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln 290 295 300 Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala305 310 315 320
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 325
330 335 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr 340 345 350 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser 355 360 365 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr 370 375 380 Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr385 390 395 400 Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 405 410 415 Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 420 425 430 Ser Leu
Ser Leu Ser Pro Gly Lys 435 440 11225PRTArtificial
SequenceSynthetic polypeptide 11Gln Val Glu Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Gln Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ile Ile
Trp Phe Asp Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Arg
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95 Ala Arg Glu Leu Gly Arg Arg Tyr Phe Asp Leu Trp Gly Arg
Gly Thr 100 105 110 Leu Val Ser Val Ser Ser Ala Ser Val Ala Ala Pro
Ser Val Phe Ile 115 120 125 Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
Gly Thr Ala Ser Val Val 130 135 140 Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu Ala Lys Val Gln Trp Lys145 150 155 160 Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu 165 170 175 Gln Asp Ser
Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu 180 185 190 Ser
Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr 195 200
205 His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
210 215 220 Cys225 12557PRTArtificial SequenceSynthetic polypeptide
12Met Lys Ser Gly Ser Gly Gly Gly Ser Pro Thr Ser Leu Trp Gly Leu1
5 10 15 Leu Phe Leu Ser Ala Ala Leu Ser Leu Trp Pro Thr Ser Gly Glu
Ile 20 25 30 Cys Gly Pro Gly Ile Asp Ile Arg Asn Asp Tyr Gln Gln
Leu Lys Arg 35 40 45 Leu Glu Asn Cys Thr Val Ile Glu Gly Tyr Leu
His Ile Leu Leu Ile 50 55 60 Ser Lys Ala Glu Asp Tyr Arg Ser Tyr
Arg Phe Pro Lys Leu Thr Val65 70 75 80 Ile Thr Glu Tyr Leu Leu Leu
Phe Arg Val Ala Gly Leu Glu Ser Leu 85 90 95 Gly Asp Leu Phe Pro
Asn Leu Thr Val Ile Arg Gly Trp Lys Leu Phe 100 105 110 Tyr Asn Tyr
Ala Leu Val Ile Phe Glu Met Thr Asn Leu Lys Asp Ile 115 120 125 Gly
Leu Tyr Asn Leu Arg Asn Ile Thr Arg Gly Ala Ile Arg Ile Glu 130 135
140 Lys Asn Ala Asp Leu Cys Tyr Leu Ser Thr Val Asp Trp Ser Leu
Ile145 150 155 160 Leu Asp Ala Val Ser Asn Asn Tyr Ile Val Gly Asn
Lys Pro Pro Lys 165 170 175 Glu Cys Gly Asp Leu Cys Pro Gly Thr Met
Glu Glu Lys Pro Met Cys 180 185 190 Glu Lys Thr Thr Ile Asn Asn Glu
Tyr Asn Tyr Arg Cys Trp Thr Thr 195 200 205 Asn Arg Cys Gln Lys Met
Cys Pro Ser Thr Cys Gly Lys Arg Ala Cys 210 215 220 Thr Glu Asn Asn
Glu Cys Cys His Pro Glu Cys Leu Gly Ser Cys Ser225 230 235 240 Ala
Pro Asp Asn Asp Thr Ala Cys Val Ala Cys Arg His Tyr Tyr Tyr 245 250
255 Ala Gly Val Cys Val Pro Ala Cys Pro Pro Asn Thr Tyr Arg Phe Glu
260 265 270 Gly Trp Arg Cys Val Asp Arg Asp Phe Cys Ala Asn Ile Leu
Ser Ala 275 280 285 Glu Ser Ser Asp Ser Glu Gly Phe Val Ile His Asp
Gly Glu Cys Met 290 295 300 Gln Glu Cys Pro Ser Gly Phe Ile Arg Asn
Gly Ser Gln Ser Met Tyr305 310 315 320 Cys Ile Pro Cys Glu Gly Pro
Cys Pro Lys Val Cys Glu Glu Glu Lys 325 330 335 Lys Thr Lys Thr Ile
Asp Ser Val Thr Ser Ala Gln Met Leu Gln Gly 340 345 350 Cys Thr Ile
Phe Lys Gly Asn Leu Leu Ile Asn Ile Arg Arg Gly Asn 355 360 365 Asn
Ile Ala Ser Glu Leu Glu Asn Phe Met Gly Leu Ile Glu Val Val 370 375
380 Thr Gly Tyr Val Lys Ile Arg His Ser His Ala Leu Val Ser Leu
Ser385 390 395 400 Phe Leu Lys Asn Leu Arg Leu Ile Leu Gly Glu Glu
Gln Leu Glu Gly 405 410 415 Asn Tyr Ser Phe Tyr Val Leu Asp Asn Gln
Asn Leu Gln Gln Leu Trp 420 425 430 Asp Trp Asp His Arg Asn Leu Thr
Ile Lys Ala Gly Lys Met Tyr Phe 435 440 445 Ala Phe Asn Pro Lys Leu
Cys Val Ser Glu Ile Tyr Arg Met Glu Glu 450 455 460 Val Thr Gly Thr
Lys Gly Arg Gln Ser Lys Gly Asp Ile Asn Thr Arg465 470 475 480 Asn
Asn Gly Glu Arg Ala Ser Cys Glu Ser Asp Val Ala Ala Ala Leu 485 490
495 Glu Val Leu Phe Gln Gly Pro Gly Thr His His His His His His Ser
500 505 510 Gly Asp Glu Lys Thr Thr Gly Trp Arg Gly Gly His Val Val
Glu Gly 515 520 525 Leu Ala Gly Glu Leu Glu Gln Leu Arg Ala Arg Leu
Glu His His Pro 530 535 540 Gln Gly Gln Arg Glu Pro Ser Gly Gly Cys
Lys Leu Gly545 550 555
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