U.S. patent application number 13/492237 was filed with the patent office on 2012-10-25 for substance binding human igg fc receptor iib (fcgammariib).
Invention is credited to Chiara Cabrele, Robert Huber, Uwe Jacob, Luis Moroder, Peter Sondermann, Kerstin Wendt.
Application Number | 20120269837 13/492237 |
Document ID | / |
Family ID | 34626375 |
Filed Date | 2012-10-25 |
United States Patent
Application |
20120269837 |
Kind Code |
A1 |
Huber; Robert ; et
al. |
October 25, 2012 |
SUBSTANCE BINDING HUMAN IgG Fc RECEPTOR IIb (FcgammaRIIb)
Abstract
This invention relates to novel immunogens carrying
conformationally discriminating epitopes (CDEs) and to immunization
methods for producing antibodies that specifically recognize
proteins with very closely related homologues. In particular, the
invention relates to antibodies which are specific for either
Fc.gamma.RIIb and Fc.gamma.RIIa.
Inventors: |
Huber; Robert; (Munchen,
DE) ; Sondermann; Peter; (Rudolfstetha, CH) ;
Jacob; Uwe; (Munchen, DE) ; Wendt; Kerstin;
(Brand - Erbisdort, DE) ; Cabrele; Chiara;
(Regensburg, DE) ; Moroder; Luis; (Martinsried,
DE) |
Family ID: |
34626375 |
Appl. No.: |
13/492237 |
Filed: |
June 8, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10580813 |
Feb 2, 2007 |
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PCT/EP2004/013450 |
Nov 26, 2004 |
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13492237 |
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Current U.S.
Class: |
424/185.1 ;
435/243; 435/320.1; 530/321; 530/387.9; 536/23.1; 536/23.53 |
Current CPC
Class: |
A61P 25/28 20180101;
A61P 35/00 20180101; A61P 35/02 20180101; A61P 19/00 20180101; A61P
43/00 20180101; A61P 19/02 20180101; A61K 2039/505 20130101; A61P
37/04 20180101; A61P 7/00 20180101; A61P 37/00 20180101; A61P 37/06
20180101; A61P 37/08 20180101; A61P 17/06 20180101; A61P 25/00
20180101; A61P 37/02 20180101; C07K 2317/34 20130101; C07K 16/283
20130101; C07K 14/70535 20130101; A61P 29/00 20180101 |
Class at
Publication: |
424/185.1 ;
530/321; 530/387.9; 536/23.1; 536/23.53; 435/320.1; 435/243 |
International
Class: |
C07K 7/64 20060101
C07K007/64; C07K 5/12 20060101 C07K005/12; C07K 16/28 20060101
C07K016/28; A61P 37/04 20060101 A61P037/04; C12N 15/11 20060101
C12N015/11; C12N 15/13 20060101 C12N015/13; C12N 15/63 20060101
C12N015/63; C12N 1/00 20060101 C12N001/00; C07K 2/00 20060101
C07K002/00; A61K 39/00 20060101 A61K039/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 26, 2003 |
EP |
03 027 000.3 |
Claims
1-53. (canceled)
54. An artificial peptide or polypeptide comprising a
conformationally discriminating epitope (CDE) in its native
conformation, wherein the CDE is structurally stabilized by
circularization.
55. The peptide or polypeptide claim 54, comprising artificial or
glycosylated amino acids.
56. The peptide or polypeptide claim 54, conjugated to a carrier
molecule.
57. The peptide or polypeptide of claim 54 comprising a CDE of an
Fc receptor.
58. The peptide or polypeptide of claim 57 comprising a CDE of
Fc.gamma.RIIb or Fc.gamma.RIIa, the CDE comprising at least one
residue which is unique to either Fc.gamma.RIIb or
Fc.gamma.RIIa.
59. The peptide or polypeptide of claim 58, wherein the CDE
comprises amino acids 27 to 30, or amino acids 127 to 135 or amino
acids 160 to 171 of Fc.gamma.RIIb of SEQ ID NO: 2 or the
corresponding amino acids of Fc.gamma.RIIa of SEQ ID NO: 1, or the
amino acid sequence of SEQ ID NO: 3.
60. The peptide or polypeptide of claim 57 conjugated to
Fc.gamma.RIIb or Fc.gamma.RIIa.
61. A method of producing a peptide carrying a conformationally
discriminating epitope (CDE) for the generation of antibodies
specifically recognizing a protein of interest carrying such an
epitope, comprising: (a) providing a protein of interest, (b)
identifying a CDE on said protein, (c) producing a peptide
comprising the sequence of the CDE, (d) structurally stabilizing
the peptide by circularization so that the CDE is present in its
native conformation.
62. The method of claim 61, wherein the circularization of the
peptide is achieved by generating cysteine bridges, or by bridging
amino acid side chains that form a pseudopeptide.
63. The method of claim 61, wherein the peptide is generated using
amino acids carrying glycosylation moieties which are present on
the protein of interest.
64. The method of claim 61, further comprising: (e) conjugating the
peptide to a carrier molecule selected from haptens, polypeptides,
peptides, and the protein of interest.
65. A peptide or polypeptide comprising a CDE, obtained by the
method of claim 61.
66. A method comprising generating immunomodulatory substances
specifically recognizing the CDE in its natural environment by
providing the peptide or polypeptide of claim 54 as an immunogen in
a environment suitable to generate the substance.
67. A method comprising immunizing an animal or a transgenic animal
expressing human Fc.gamma.RIIa by administering an effective amount
of the peptide or polypeptide of claim 59 to a subject in need
thereof.
68. A method comprising generating an antibody that can
specifically recognize alleles of the Fc.gamma.RIIa Arg/His
polymorphism at position 131 or the Fc.gamma.RIIa Val/Phe
polymorphism at position 155 by providing an effective amount of
the peptide or polypeptide of claim 59 in an environment effective
to a subject to generate the antibody.
69. A method of producing substances capable of discriminating
between an antigen of interest and closely related antigens,
comprising immunizing an animal with a peptide or polypeptide
according to claim 59 or with a correctly folded peptide derived
from Fc.gamma.RIIb or Fc.gamma.RIIa, or both, and isolating the
resulting antibodies.
70. A nucleic acid sequence encoding the peptide of claim 58 or an
antibody or fragment or derivative thereof that specifically binds
to human Fc.gamma.RIIb or Fc.gamma.RIIa in the natural environment
of the Fc receptor.
71. A nucleic acid vector comprising the nucleic acid sequence
according to claim 70.
72. A host cell transfected with a vector according to claim
71.
73. A diagnostic kit for the detection of autoimmune diseases or
cancer, comprising the antibody, fragment or derivative thereof
that specifically binds to human Fc.gamma.RIIb or Fc.gamma.RIIa in
the natural environment of the Fc receptor, or a recombinant
peptide or polypeptide comprising a conformationally discriminating
epitope (CDE) in its native conformation, wherein the CDE is
structurally stabilized by circulization.
74. The method of claim 69, further comprising generating
recombinant immunomodulatory substances with antibodies.
Description
[0001] The invention relates to novel immunogens carrying
conformationally discriminating epitopes (CDEs) and to immunization
methods for producing antibodies that specifically recognize
proteins with very closely related homologues. In particular, the
invention relates to antibodies which are specific for either
Fc.gamma.RIIb or Fc.gamma.RIIa and which are useful for the
diagnosis and treatment of autoimmune diseases, infections, tumors
and other conditions where the immune system is involved.
[0002] Fc receptors (FcRs) play a key role in defending the human
organism against infections. After pathogens have gained access to
the blood circulation they are opsonized by antibodies
(immunoglobulins, Igs). This leads to the formation of immune
complexes. The Fc portions of the antibodies can bind to Fc
receptors which are present on virtually all cells of the immune
system. Specific FcRs exist for all Ig classes. The Greek letter
indicates the Ig class to which it binds, i.e. Fc.gamma. receptors
recognize IgG etc.
[0003] It has been known for a number of years that the Fc
receptors for IgG (Fc.gamma.R) play an important role in triggering
effector responses (Metzger, 1992A). These include, depending on
the expressed Fc.gamma.R and cell type, endo- and phagocytosis
resulting in neutralization of the pathogens and antigen
presentation, antibody-dependent cell-mediated cytotoxicity (ADCC),
neutrophil activation, regulation of the antibody production or the
secretion of inflammatory mediators (Fridman et al., 1992; van de
Winkel and Capel, 1993; Ravetch and Bolland, 2001).
[0004] In contrast to the beneficial role FcRs play in the healthy
individual, they also transmit the stimulation of the immune system
in allergies (e.g. mediated by Fc.epsilon.RIa) or autoimmune
diseases. Moreover, some viruses employ Fc.gamma.Rs to get access
to cells like HIV (Homsy et al., 1989) and Dengue (Littaua et al.,
1990) or slow down the immune response by blocking Fc.gamma.Rs as
in the case of Ebola (Yang et al., 1998) and Measles (Ravanel et
al., 1997).
[0005] Fc receptors for IgG (Fc.gamma.R) are the most widespread of
the Fc receptor family and are expressed in a defined pattern on
all immunological active cells. Fc.gamma.RI is constitutively
expressed on monocytes and macrophages and can be induced on
neutrophils and eosinophils. The physiological role of Fc.gamma.RI
is still unknown as the expression on monocytes is not vital
(Ceuppens et al., 1988). The glycosylphosphatidylinositol-anchored
form (GPI) of Fc.gamma.RIII (Fc.gamma.RIIIb) is exclusively
expressed on granulocytes. Due to its missing cytoplasmic part, the
signal transduction into the cell occurs solely via other
transmembrane proteins like complement receptor type 3 (CR3) that
can at least associate with Fc.gamma.RIIIb (Zhou et al., 1993; Poo
et al., 1995). Fc.gamma.RIIIa is mainly expressed on monocytes and
macrophages but only in conjunction with an associated protein
called .gamma.-chain. Fc.gamma.RIIa is the receptor with the widest
distribution on immune competent cells and is mainly involved in
the endocytosis of immune complexes. Fc.gamma.RIIb is expressed on
B cells where it is the only IgG receptor, and on effector cells
such as macrophages, neutrophils and mast cells, but not on NK
cells and T cells.
[0006] Structurally, the extracellular part of the Fc.gamma.Rs
consists of three (Fc.gamma.RI, CD64) or two (Fc.epsilon.RI,
Fc.gamma.RII, CD32 and Fc.gamma.RIII, CD16) Ig-like domains (ca. 10
kDa/domain) and therefore belong to the immunoglobulin super
family. In addition to the extracellular domains, FcRs have a
transmembrane domain, and an intracellular domain with the
exception of the GPI-anchored Fc.gamma.RIIIb. The receptors are
homologous to each other, and the overall identity in so amino acid
sequence among the Fc.gamma.Rs and the Fc.epsilon.RIa exceeds 40%
in their extracellular regions. Fc.gamma.RIIa and Fc.gamma.RIIb
differ in their extracellular region by only 6% of the amino acid
residues. Nevertheless, both forms can be distinguished by their
binding characteristics to human and mouse IgG subclasses (van de
Winkel and Capel, 1993) and their differing affinity to human IgGs
(Sondermann et al., 1999A).
[0007] FcRs are highly glycosylated. The cDNA sequence of many Fc
receptors is known, and some soluble recombinant FcR have been
generated. Soluble recombinant Fc receptors which are characterised
by an absence of transmembrane domains, signal peptide and
glycosylation are disclosed in WO 00/32767.
[0008] Fc.gamma.Rs occur in various isoforms (Fc.gamma.RIa, b1, b2,
c; Fc.gamma.RIIa1-2, b1-3, c) and alleles (Fc.gamma.RIIa1-HR, -LR;
Fc.gamma.RIIIb-NA1, -NA2) (van de Winkel and Capel, 1993). In
contrast to the overall homologous extracellular parts, the
membrane spanning and the cytoplasmic domains of up to 8 kDa large
differ.
[0009] The Fc.gamma.Rs can be divided into two general classes
according to their function which may be an activating or an
inhibitory one. The activating receptors are associated with a
cytoplasmic 16 amino acid immunoreceptor tyrosine-based activation
motif (ITAM) having the consensus sequence
Y--X.sub.2-L/I--X.sub.8--Y--X.sub.2-L/I (Isakov, 1997). This motif
can be found, for example in Fc.gamma.RIIa. The other class of FcRs
are inhibitory receptors which have a cytoplasmic 6 amino acid
inhibitory motif (ITIM) having the consensus sequence
V/I--X--Y--X.sub.2--V/L (Isakov, 1997). An example of such an
inhibitory FcR is Fc.gamma.RIIb.
[0010] Activation and inhibition via the ITAM and ITIM motifs is
effected by tyrosine phosphorylation. Depending on the particular
cell type, activated by the Fc receptor, different tyrosine kinases
are involved in these signaling pathways (Amigorena et al., 1992).
Both activating and inhibiting Fc.gamma.Rs may be expressed on the
same cell which allows functioning of activation and inhibitory
receptors in concert for a fine tuning of the immune response.
[0011] Fc.gamma.RIIb has two inhibitory activities. One of these is
dependent on the ITIM motif and occurs when Fc.gamma.RIIb is
ligated to an ITAM-carrying receptor (e.g. Fc.gamma.RIIa) resulting
in the inhibition of ITAM-triggered calcium mobilization and
cellular proliferation. This means that calcium-dependent processes
such as degranulation, phagocytosis, ADCC, cytokine release and
pro-inflammatory activation, and also B cell proliferation are
blocked by Fc.gamma.RIIb. The second inhibitory activity of
Fc.gamma.RIIb involves homo-aggregation of the receptor
(Fc.gamma.RIIb clustering) which delivers a pro-apoptotic signal
into the cytoplasm. The pro-apoptotic signal has only been reported
in B cells and can be blocked by ligation of Fc.gamma.RIIb to the B
cell receptor (BCR). In vivo studies suggest that Fc.gamma.RIIb
plays a role in peripheral tolerance because Fc.gamma.RIIb-knockout
mice develop spontaneously autoimmune diseases. On the other hand,
Fc.gamma.RIIb has also been reported to down-regulate cytotoxicity
against tumors (Clynes et al., 2000). Mice deficient in
Fc.gamma.RIIb and treated with an anti-tumor antibody showed
enhanced ADCC resulting in a reduction of tumor metastasis, whereas
mice deficient in activating Fc receptors were unable to arrest
tumor growth, when treated with the same antibody.
[0012] The generation of antibodies by immunising animals with
proteins or peptides as immunogens is known in the art.
Conventional immunisation protocols use linear peptides as
immunogens which are derived from antigens of interest. The
disadvantage of such methods is that because the three-dimensional
structure of the epitopes is often completely lost, the resulting
antibodies are not very specific or they comprise a large fraction
of antibodies directed to epitopes other than the one of
interest.
[0013] During the last decade, immunization protocols using
Fc-receptor expressing cells or denatured Fc-receptors have only
resulted in antibodies that were able to specifically detect
denatured Fc-Receptors (Western Blot) or were not able to
discriminate between the related Fc.gamma.RIIa and Fc.gamma.RIIb on
cell lines (e.g. U-937, Raji) or blood cells. To date, there are no
antibodies or other binding substances which bind selectively and
specifically to Fc.gamma.RIIb in its native conformation and/or its
natural environment.
[0014] Conventional immunization protocols involving peptides or
recombinant proteins as antigens are not well suited to produce
specific antibodies against proteins for which homologues with very
high sequence identity exist. In general, antibodies are raised
using small linear peptides as immunogens. Such peptides do not
represent the native conformation of the epitope on the protein
from which they are derived. In addition, the large majority of the
antibodies produced by the immunized animal are directed against
epitopes of the carrier protein to which the antigen is conjugated
or against epitopes on the recombinant antigen that are common to
the homologues. In consequence, the produced antibodies are not
specific and/or fail to detect the antigen in its native
conformation. Furthermore, glycosylation sites might be located
within the epitopes of interest and mask these sites. Conventional
immunization protocols which use these epitopes without the
respective native glycosylation found in the target molecule result
in antibodies that fail to recognize the antigen in its native
conformation.
[0015] One object of the present invention is to provide
recombinant peptides or polypeptides which can be used as
immunogens to raise antibodies capable of discriminating between an
antigen of interest and closely related antigens, and a method of
generating such peptides and the corresponding antibodies and other
substances having immunological specificity.
[0016] It is a further object of the present invention to provide
substances which can selectively bind to and discriminate between
Fc receptor subtypes, thereby acting as an Fc receptor binding
substance useful for the treatment and diagnosis of immune
disorders, in particular autoimmune diseases, and as anti-tumor
agents which enhance the efficiency of such therapies by promoting
ADCC against tumor cells.
[0017] It is a further object of the present invention to provide
an immunization protocol that will allow the generation of such
Fc.gamma.RIIb-binding substances, in particular antibodies,
especially monoclonal antibodies with the above-mentioned
specificity.
[0018] The inventors of the present invention have found a novel
and inventive approach to developing substances, in particular
antibodies, that are capable of discriminating between very closely
related proteins and/or proteins and antigens with high
homology.
[0019] Surprisingly, it was found that it is possible to raise
specific antibodies against proteins of interest when so-called
conformationally discriminating epitopes (CDEs) are used as the
antigen to which the antibodies are raised.
[0020] The present invention therefore relates to an artificial
peptide or polypeptide comprising a conformationally discriminating
epitope (CDE) in its native conformation, wherein the CDE is
structurally stabilized by circularization.
[0021] For the purposes of the present invention, an artificial
peptide or polypeptide is one that is produced by any technical
process such as recombinant techniques or preferably by peptide
synthesis.
[0022] A CDE is an epitope in a protein which has a specific
conformation in the protein. Antibodies which are specific for such
an epitope can discriminate between a protein and very closely
related homologues. The CDE comprises at least one amino acid which
differs between the protein in which it is present and the
homologues of that protein (unique residue). The unique residues
need not be in close proximity in the linear sequence of the
protein in order to be part of the same epitope. The advantage of
the present invention is that the peptides of the invention do not
just have the linear sequence of those epitopes but mimic also
their structure. The CDE contains at least one of such unique
residues, preferably at least two, more preferably more than two of
such unique residues. The CDE represents the binding site for an
antibody.
[0023] The peptides of the present invention preferably have a
length of from 5, more preferably from about 8, more preferably
from about 10 to about 30, more preferably to about 20, more
preferably to about 18, more preferably to about 15 amino
acids.
[0024] Structural stabilization in this context means that the
peptide is stabilized so that the CDE is present in as close to its
native three-dimensional conformation in the original protein as
possible. Structural stabilization can be achieved by a number of
means. In particular, the peptide is circularized so that it forms
a stable three-dimensional structure such as a loop. Stabilizing
the peptide can be achieved by N- to C-terminal coupling, the
formation of cysteine bridges or by bridging amino acid side
chains. Pseudopeptides can be formed.
[0025] Preferably, the peptide or polypeptide of the present
invention also carries glycosylation moieties. The peptide is
preferably generated so that glycosylated amino acids are
incorporated at the same sites which are glycosylated in the native
protein from which the CDE is derived. Preferably, the glycosylated
amino acids are selected from N-acetyl-glucosamine, fucose, xylose,
mannose, and galactose conjugates but this list is not exhaustive.
If the discriminating epitope contains a N-glycosylation site, an
artificial conjugate of an asparagine residue with a
N-acetyl-glucosamine may be incorporated into the peptide, to
enhance the probability that the natively glycosylated substrate is
recognized by the resulting antibodies after successful
immunization. Accordingly for O-glycosylation sites, a serine or
threonine residue may be conjugated with a mannose, fucose, xylose,
galactose or N-Acetyl-galactosamine residue respectively.
[0026] The peptides and polypeptides of the invention may
additionally be coupled to a carrier molecule. Such carrier
molecules are preferably selected from haptens, peptides,
polypeptides and other immunogens. The peptides of the invention
may be grafted onto other peptides and proteins, even the same
protein or parts thereof from which the CDE was derived.
[0027] A preferred embodiment of the invention is a peptide
carrying a CDE from an Fc receptor. The inventors of the present
invention surprisingly found that there are specific epitopes on
the extracellular portion of Fc.gamma.RIIb which allow the
generation of antibodies which bind specifically to Fc.gamma.RIIb.
This is particularly useful because the family of Fc receptors
comprises unusually closely related homologues which are difficult
to distinguish using conventional antibodies. In particular, the
present invention makes it possible to generate substances which
bind to Fc.gamma.RIIb but not to Fc.gamma.RIIa and vice versa.
Similarly, the epitopes can be chosen so that Fc.gamma.RIIa is
specifically recognized.
[0028] In particular, the peptides or polypeptides according to the
present invention comprise an epitope comprising at least one,
preferably at least 2, preferably at least 3 of the following amino
acids of the amino acid sequence of human Fc.gamma.RIIb according
to FIG. 1 and SEQ ID NO: 2: Gln12, Arg27, Thr29, His 30, Val104,
Lys127, Ser132, Asn135, Tyr160, and Ala171, or the corresponding
amino acids of Fc.gamma.RIIa according to SEQ ID NO: 1. More
preferably, the epitopes useful for the purposes of the present
invention comprise amino acids 27 to 30, and/or 127 to 135, and/or
160 to 171 of the amino acid sequence of Fc.gamma.RIIb (FIG. 1,
SEQ. ID NO: 2), or the corresponding amino acids of Fc.gamma.RIIa
(FIG. 1). These peptides can represent Fc.gamma.RIIb-specific
conformationally discriminating epitopes (CDEs), when structurally
stabilized by circularization, in an adequate way as exemplified in
FIG. 2. Also, the corresponding epitopes of Fc.gamma.RIIa may be
used to produce antibodies that specifically bind only to
Fc.gamma.RIIa.
[0029] Especially peptides comprising the amino acid sequence
127-KKFSRSDPN-135 and flanking amino acids are preferred because
these peptides represent a Fc.gamma.RIIb-specific conformational
epitope within the binding region of the Fc-Receptor to the
Fc-fragment (WO 00/32767, Sondermann et al., 2000; Sondermann et
al., 2001). Furthermore peptides containing the amino acid sequence
28-RGTH-31 and flanking residues are preferred because they
represent a binding epitope apart from the binding region to the
Fc-fragment. Moreover, this epitope may be further adapted to the
native structure by circularisation and incorporation of a
glycosylated asparagine residue at position 135.
[0030] Thus, a preferred embodiment of the present invention is a
peptide or polypeptide carrying the CDE according SEQ ID NO: 3.
Preferably, the asparagine of position 135 (according to SEQ ID NO:
2) is glycosylated with N-acetyl-glucosamine. Preferably, the
peptide is as shown in FIG. 7, being circularized by linking the
first and last amino acids in the sequence as shown in FIG. 7.
[0031] These artificial peptides can then be used directly for the
immunization of animals or may be coupled to a carrier protein such
as haptens or peptides or polypeptides, or ideally to the target
protein itself. In a preferred embodiment, a CDE from Fc.gamma.RIIb
or Fc.gamma.RIIa or a peptide carrying such a CDE is conjugated to
Fc.gamma.RIIb or Fc.gamma.RIIa.
[0032] The peptides and polypeptides of the present invention are
preferably used as immunogens to immunize animals in order to
generate specific antibodies and, with the aid of the sequence of
those specific antibodies, further immunologically specific
substances. The CDEs and the peptides carrying them may be used for
the generation of immunomodulatory substances specifically
recognizing the CDE. This is particularly preferred when CDEs of Fc
receptors are chosen because they allow the generation of
antibodies specific for individual members of the family of
homologues. In particular, antibodies and other immunomodulatory
substance recognizing either Fc.gamma.RIIb or Fc.gamma.RIIa but not
both at the same time, can be generated. The present invention
allows the generation of antibodies which are capable not only of
specifically recognizing Fc.gamma.RIIb or Fc.gamma.RIIa and
discriminating between the two Fc receptors but also of doing so
when the Fc receptors are in their natural environment, for example
in cell culture or in vivo, e.g. in the blood stream.
[0033] By coupling of the CDE to the protein from which it was
derived, the background immune reaction against the carrier protein
is reduced. The artificial modification by the covalently coupled
CDE results in an increased immune response. This is especially
important if the immunized animal expresses similar proteins which
would be tolerated by its immune system. Thus, if the peptide of
the invention carries a CDE of Fc.gamma.RIIb or Fc.gamma.RIIa, it
is preferably coupled to the respective Fc receptor itself. This
increases the antigenicity. The coupling can occur by chemical
linkage or other suitable means.
[0034] This method preferably produces an immunogen with a high
density of CDEs thereby presenting them in a different thus
immunogenic environment. The initial immune response is directed
against the targeted region (CDE) which produces antibodies that
crossreact with the native structure to which they are coupled.
These crossreacting antibodies mature towards higher affinity also
recognizing the CDE in its natural environment.
[0035] The peptides of the invention and the CDEs may also be used
in screening of molecular libraries for binding molecules (e.g.
peptides, organic molecules, peptidometics etc.) or genetically
encoded libraries (e.g phage display of antibody variable domains
or other frameworks like lipocalines) to find specifically binding
substances to Fc.gamma.RIIb (or to Fc.gamma.RIIa). The peptides may
be used to screen libraries of molecules binding specifically to
either Fc.gamma.RIIa or Fc.gamma.RIIb on human cells.
[0036] Similar peptides according to the invention can be extracted
from the structure of other proteins, e.g. receptors, that are
related to each other but which have different functions (e.g.
human Fc.gamma.RIIa-specific antibodies can be developed that do
not recognize Fc.gamma.RIIb, which may be incorporated in Diabodies
or Triabodies, to promote ADCC which is mediated by Fc.gamma.RIIa
rather than by Fc.gamma.RIIIb) or which occur in different alleles
(e.g. Fc.gamma.RIIa Arg/His-polymorphism at position 131, or
Fc.gamma.RIIIa Phe/Val-polymorphism at position 155).
[0037] Another use of the novel peptides of the invention is a
direct use as inhibitors of promoters of immunological functions.
The peptides according to the invention may be used directly for
immunotherapies.
[0038] The above-described peptides of the invention can be
produced by a novel method, wherein the method comprises:
(a) providing a protein of interest, (b) identifying a CDE on that
protein, (c) producing a peptide comprising the sequence of the
CDE, (d) structurally stabilizing the peptide so that the CDE is
present in its native conformation.
[0039] The peptide is structurally stabilized by circularisation,
preferably by N- to C-terminal coupling, the formation of cysteine
bridges, and/or bridging amino acid side chains forming a
pseudopeptide. As stated above, the peptide is preferably generated
using amino acids carrying glycosylation moieties which are present
on the protein of interest. The method preferably comprises the
additional step of conjugating the peptide to a carrier molecule
which can be selected from any of the molecules mentioned above.
Another aspect of the present invention is a peptide or polypeptide
comprising a CDE, obtainable by the method of the invention.
[0040] In order to significantly raise the fraction of specifically
binding antibodies, the invention provides the following method for
generating specific binding substances capable of discriminating
between an antigen of interest and closely related antigens,
wherein the method comprises immunising an animal with a peptide or
polypeptide according to the present invention or/and with a
correctly folded portion of the antigen of interest, in particular
a peptide derived from an Fc receptor such as Fc.gamma.RIIb or
Fc.gamma.RIIa, and isolating the resulting antibodies, and
optionally using the antibodies to generate recombinant
immunomodulatory substances.
[0041] To produce antibodies that discriminate between an antigen A
and an antigen B with high sequence identity to A, the differing
amino acids are mapped to the structure of A or a respective
structure model of A. Differing amino acids that are separated by
several amino acids in the primary sequence may be in spatial
proximity. In case that these differing amino acids are accessible
from the solvent in the native structure these surface regions can
be regarded as conformationally discriminating epitopes (CDE). Such
epitopes can be artificially constructed by cyclic peptides or
peptide analogues and are especially useful for the generation of
antibodies that can discriminate between strongly related
antigens.
[0042] In a variation of this method transgenic animals are used
for immunization that are engineered to express the close
homologue(s) and are later immunized with the target antigen.
Animals that express the human Fc.gamma.RIIb are immunized with
human Fc.gamma.RIIa or vice versa.
[0043] In a particularly preferred aspect, the present invention
provides an Fc.gamma.RIIb-binding antibody or fragment or
derivative thereof, capable of specifically binding to
Fc.gamma.RIIb or to Fc.gamma.RIIa in the natural environment of the
Fc receptor. Such antibodies fragments or derivatives can
discriminate between the closely related homologues of
Fc.gamma.RIIb and Fc.gamma.RIIa in a natural environment, e.g in
cell culture or in vivo.
[0044] In a preferred embodiment, the Fc.gamma.RIIb- (or
Fc.gamma.RIIa-) binding antibody or fragment or derivative thereof
not only binds specifically to Fc.gamma.RIIb (or Fc.gamma.RIIa) but
also prevents the natural binding partners of Fc.gamma.RIIb (or
Fc.gamma.RIIa), i.e. IgG antibodies, from binding.
[0045] In another preferred embodiment of the present invention,
the specific anti-Fc.gamma.RIIb (or anti-Fc.gamma.RIIa) antibodies
are non-blocking and recognize an epitope distinct from the
Fc-receptor/Fc-fragment interaction site (e.g. an epitope of the
N-terminal domain around the amino acids 28-31). In contrast to
blocking antibodies these antibodies have the advantage that
binding of the receptor to immune complexes is not impaired. The
result is that the activation of the receptors by immune complexes
remains intact and additional receptors can be recruited to enhance
the activation.
[0046] It is thus possible to modulate the natural functions of
these Fc receptors independent of IgG binding. For example, the
antibody or fragment or derivative thereof may be chosen to be
capable of crosslinking the Fc receptor. That way, the receptor can
be activated. Preferably, the antibody or fragment or derivative
thereof of the invention does not interfere with immune complex
binding to Fc.gamma.RIIb or Fc.gamma.RIIa.
[0047] On the other hand, the antibody or fragment or derivative
thereof may be chosen so that it inhibits the physiological
function of human Fc.gamma.RIIa or Fc.gamma.RIIb.
[0048] The antibody or derivative or fragment of the invention
preferably binds with higher affinity to Fc.gamma.RIIb than to
Fc.gamma.RIIa. The antibody or fragment or derivative thereof binds
Fc.gamma.RIIb with at least 5 fold, preferably at least 10 fold,
preferably at least 100fold, more preferably at least 1,000fold,
more preferably at least 10,000fold, more preferably at least
100,000fold, more preferably at least 10.sup.6fold, more preferably
at least 10.sup.7fold, more preferably at least 10.sup.8fold, more
preferably at least 10.sup.9fold, more preferably 10.sup.10fold,
more preferably 10.sup.11fold, more preferably 10.sup.12fold higher
affinity than Fc.gamma.RIIa. Alternatively, the antibody or
fragment or derivative binds Fc.gamma.RIIa with at least 5fold,
preferably at least 10fold, preferably at least 100fold, more
preferably at least 1,000fold, more preferably at least 10,000fold,
more preferably at least 100,000fold, more preferably at least
10.sup.6fold, more preferably at least 10.sup.7fold, more
preferably at least 10.sup.8fold, more preferably at least
10.sup.9fold, more preferably 10.sup.10fold, more preferably
10.sup.11fold, more preferably 10.sup.12fold higher affinity than
Fc.gamma.RIIb. 5, 10, 100, 1000 or even more than 1,000,000 fold
tighter binding to the specific Fc-receptor is necessary to
overcome the much higher expression level of Fc.gamma.RIIa on
platelets over Fc.gamma.RIIb.
[0049] The antibody or fragment or derivative thereof can occur in
a monomeric or multimeric state.
[0050] The antibody or fragment or derivative thereof may be
capable of binding Fc receptor molecules with or without
cross-linking them on the cell surface. Preferably, the antibody or
fragment or derivative thereof is multimeric in order to cross-link
Fc.gamma.RIIa or Fc.gamma.RIIb. Alternatively, the antibody or
fragment or derivative thereof is monomeric and able to block IgG
binding to human Fc.gamma.RIIb, but preferably not able to
cross-link Fc.gamma.RIIb.
[0051] The antibody or fragment or derivative thereof of the
invention may also be modified in the Fc-fragment by the
modification of the glycosylation and/or mutagenesis to enhance the
binding towards subsets of the Fc-receptors.
[0052] The antibody or fragment or derivative thereof of the
invention is preferably able to bind to a CDE or/and peptide as
described above, in particular those comprising one or more of the
amino acids of human Fc.gamma.RIIb according to FIG. 1 and SEQ ID
NO: 2, selected from: Gln12, Arg27, Thr29, His30, Val104, Lys127,
Ser132, Asn135, Tyr160, and Ala171, or the corresponding amino
acids of Fc.gamma.RIIa according to SEQ ID NO: 1. More preferably,
the substance binds to an epitope comprising amino acids 27 to 30,
and/or 127 to 135, and/or 160 to 171 of the amino acid sequence of
Fc.gamma.RIIb (FIG. 1, SEQ ID NO: 2) or the corresponding epitopes
of Fc.gamma.RIIa.
[0053] In a similar way human Fc.gamma.RIIIa-specific antibodies
can be developed that do not recognize Fc.gamma.RIIIb, which may be
incorporated in Diabodies or Triabodies, to promote ADCC which is
mediated by Fc.gamma.RIIIa rather than by Fc.gamma.RIIIb.
[0054] The antibody or fragment or derivative thereof can be any
natural, artificial or recombinantly produced substance carrying a
region which can bind to the above-mentioned epitopes of
Fc.gamma.RIIb. Preferably, this region contains the complementarity
determining regions (CDRs) of the antibody which bind specifically
to Fc.gamma.RIIb. More preferably, the CDRs comprise the sequences
as depicted in FIGS. 5 and 6.
[0055] The described CDRs maybe the basis for variations to further
improve their specificity or designing new specific or
pan-antibodies (or binding molecules) for other selected
Fc-Receptors or receptor groups. Methods are known that include
random or site directed mutagenesis, screening for related
sequences and knowledge- or structure-based design.
[0056] Preferably, the antibody or fragment or derivative thereof
comprises one or both of the variable light and variable heavy
regions according to SEQ ID Nos: 5 and 7, and/or the variable light
and variable heavy regions according to SEQ ID Nos: 9 and 11. Most
preferably, the antibody is CE5 or GB3.
[0057] Monoclonal antibodies are preferred. Preferably, it is an
antibody or fragment or derivative thereof having an IgG, IgE, IgM
or IgA isotype. Preferably, the antibody is human or humanized, but
may also be of other origin, such as animal origin, in particular
of mouse or camel origin. The antibody may be in various forms,
such as a single chain antibody, bi- or tri-functional or
multi-functional antibody, Fab- of Fab.sub.2-fragment or as entire
antibody in which the Fc-fragment has a modified affinity towards
Fc receptors or complement. It may also be a Fab fragment, a
F(ab).sub.2 fragment, or a Fv fragment, or an scv fragment.
[0058] The antibody or fragment or derivative thereof may also be a
recombinantly produced polypeptide or polypeptide analogue which
has a specific binding region comprising the sequence of the CDRs
or a similar sequence related to more than 50%, preferably more
than 70%, preferably more than 90%, preferably more than 95% to the
provided sequences. These sequences may also be the starting point
for the design of inhibitors of Fc-receptors. Therefore, also
peptidomimetica are part of the invention that use or mimic
sequence motives of the provided CDRs.
[0059] In another preferred embodiment, the antibody or fragment or
derivative thereof is an anticaline or lipocaline-variant or
another antibody surrogate.
[0060] The obtained antibody or fragment or derivative thereof can
be coupled to an effector molecule such as an antigen of interest,
antibodies, antibody fragments, marker molecules, cytotoxic
substances, sterically bulky blocking substances and linker
molecules and linker substances.
[0061] Another aspect of the invention are nucleic acids, vectors
and host cells containing nucleic acids encoding the peptides
and/or the antibodies or fragments or derivatives thereof, of the
present invention as described above.
[0062] From the antibody or fragment or derivative thereof
according to the invention, a nucleic acid sequence encoding this
protein can be derived. Preferably, that sequence encodes the
variable regions, preferably the CDRs binding to the above
mentioned epitopes of Fc.gamma.RIIb. Most preferably, the nucleic
acid sequence encodes the CDRs according to one or more of the
sequences according to FIGS. 5 and 6. Preferably, the nucleic acid
encodes the sequence of monoclonal antibodies CE5 or GB3.
[0063] The nucleic acid sequence may be inserted into a vector for
the expression of the protein according to FIGS. 5 and 6, which
vector is also an aspect of the present invention. The vector
preferably comprises a promoter under the control of which the
above nucleic acid sequences are placed. The vector can be
prokaryotic or an eukaryotic expression vector, where the
recombinant nucleic acid is either expressed alone or in fusion to
other peptides or proteins or a vector suitable for
DNA-vaccination.
[0064] The invention also provides a host cell transfected with the
vector mentioned above. The host cell can be any cell, a
prokaryotic cell or a eukaryotic cell.
[0065] The present invention further provides a pharmaceutical
composition useful for the treatment of diseases associated with Fc
receptor mediated signaling, comprising an effective amount of the
antibody or fragment or derivative thereof according to the
invention, and pharmaceutically acceptable carrier substances.
[0066] The present invention further provides a diagnostic kit for
the detection of autoimmune diseases and/or cancer, comprising an
antibody or fragment or derivative thereof according to the
invention and/or the recombinant peptide or polypeptide according
to the invention which comprises or represents one of the epitopes
as described herein, and optionally marker reagents, carrier
reagents and/or suitable receptacles.
[0067] Immunization with unglycosylated correctly folded
Fc-receptors, e.g. derived from E. coli, and decorated with the
described epitopes surprisingly leads to antibodies that
specifically recognize natural Fc-receptors expressed on blood
cells and in cell culture (FIG. 3 and FIG. 4).
[0068] Another aspect of the present invention is a method of
producing antibodies characterized by the ability to specifically
bind to Fc.gamma.RIIb, wherein the method comprises: [0069] (a)
Providing the correctly folded Fc.gamma.RIIb molecule or portion
thereof as an immunogen, comprising at least a part of the
extracellular domain (conformational epitopes), their conjugation,
or conjugation with other carrier molecules (e.g. KLH, BSA). [0070]
(b) Immunizing a mammal with the immunogen of (a) and producing
antibodies according to known methods, [0071] (c) Isolating the
resulting antibodies or the cells producing these antibodies.
[0072] The antibodies are preferably monoclonal antibodies.
[0073] The CDRs may be grafted to other immunoglobulin classes
(e.g. IgM, IgE, IgG1-IgG4) or other scaffolds (e.g.
lipocaline-variants; camel antibodies), or mutated or derivatised
molecules (e.g. engineered antibodies containing a modified
Fc-fragment).
[0074] The above described method may be used to produce vehicles
for the immunization of animals and results in an anti-serum of
increased specificity towards Fc.gamma.RIIb, which after fusion of
isolated B-cells with myeloma cells results in hybridoma cells with
an increased fraction producing antibodies specific for
Fc.gamma.RIIb.
[0075] The antibody or fragment or derivative thereof according to
the present invention is useful for the production of a medicament
for the treatment and/or diagnosis of conditions involving the
immune system. Preferably, these conditions are autoimmune diseases
or cancer.
[0076] The diseases that can be treated with a medicament of the
invention include, but are not limited to rheumatoid arthritis,
psoriatic arthritis, ankylosing spondylitis, Rieter's syndrome,
psoriasis, multiple sclerosis, lupus erythematosus.
[0077] Autoimmune diseases which can be diagnosed or treated using
the substances of the present invention include, but are not
limited to systemic lupus erythematosus, rheumatoid arthritis,
Multiple Sclerosis, idiopathic thrombocytopenic purpura and
host-versus-graft disease.
[0078] Surprisingly, it has been found by the present inventors
that it is possible to enhance certain immunological processes by
using the Fc.gamma.RIIb-binding substances in vivo. In particular,
it is possible to use those substances of the invention to
specifically block the signaling of Fc.gamma.RIIb on cells and
thereby increasing the immune response of the individual. This may
be used to increase ADCC against tumor cells. In practice the
Fc.gamma.RIIb-binding substance is given as adjuvant with a
therapeutic antibody. The inhibitory signal transmitted by antigens
(e.g. tumor cells) opsonized with the therapeutic antibody to
activated macrophages or B-cells is blocked and the host immune
system will be more effective in combating the targeted antigen.
This can either be in a direct way by labeling tumor cells that
express Fc.gamma.RIIb (e.g. B cell lymphoma) or by using this
Fc.gamma.RIIb-binding substance as adjuvant in all approaches which
use known therapeutic antibodies and therefore depend on the ADCC
of the host.
[0079] The known therapeutic antibodies include but are not limited
to Herceptin.RTM., Rituxan.RTM., IC14, PANOREX.TM., IMC-225,
VITAXIN.TM., Campath 1H/LDP-03, LYMPHOCIDE.TM. and ZEVLIN.TM.. They
can also include antibodies binding to the following cancer
antigens: MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2,
N-acetylglucosaminyltransferase, p15, beta-catenin, MUM-1, CDK-4,
HER-2/neu, human papillomavirus E6, human papillomavirus E7 and
MUC-1.
[0080] In certain lymphomas B-cells or Mast-cells are transformed.
The antibody or fragment or derivative thereof is able to cross
link Fc.gamma.RIIb on the surface of these cells, which labels
these cells for elimination but additionally an inhibitory and pro
apoptotic signal is transmitted to these cells. This effect is an
improvement of previous therapeutic antibody approaches, which
completely depend on the ADCC of the host (e.g. Rituxan).
[0081] The same antibody that crosslinks or blocks Fc-receptors may
be used for the treatment of host-versus-graft disease or amyloid
associated diseases.
[0082] The same Fc.gamma.RIIb blocking and/or cross linking
constructs maybe used to inhibit mast cells for the treatment of
allergies.
[0083] The antibody or fragment or derivative thereof maybe coupled
to IgE (e.g. by transferring the CDRs shown in FIG. 5 or 6 to an
IgE molecule). In this case the IgE is bound by the Mast-cell
expressed Fc.epsilon.RI and the Fc.gamma.RIIb specific CDRs cross
link the ITAM of Fc.epsilon.RI with the ITIM of the Fc.gamma.RIIb.
Again an inhibitory and/or apoptotic signal is transmitted to
Mast-cells, which is useful in the therapy of allergies.
[0084] The antibody or fragment or derivative thereof (e.g
derivatives of the sequences depicted in FIGS. 5 and 6) maybe used
for the treatment of autoimmune diseases.
[0085] Such substances inhibit B-cells, dendritc cells and
activated ganulocytes (e.g Macrophages) which leads to a reduced
production of immune stimulatory mediators and to a reduction in
antibody production as well as antigen presentation (e.g. on
Dendritic Cells and Macrophages leading to a decrease in T-cell
recruitment). Taken together the feed back loop of antibody
production and restimulation of the immune system is inhibited.
[0086] Preferably the anti-Fc.gamma.RIIb or Fc.gamma.RIIa does not
interfere with Fc-fragment binding of the receptor. In this way the
normal function of the Fc-receptor is in contrast to blocking
antibodies maintained and enhances the activation of the cell by
the additional recruitment of further receptors.
[0087] On the other hand specific anti-Fc.gamma.RIIa antibodies or
fragments thereof maybe used in diabodies to direct an antigen
towards this receptor or fragments of these antibodies maybe used
to inhibit the uptake of immune complexes for example for the
treatment of ITP.
[0088] The CDRs can be used alone or in combination for the
production of specific inhibitors of the Fc.gamma.RIIa/IgG
interaction or the Fc.gamma.RIIb/IgG interaction. For the
generation of such inhibitors, derivatives or peptidomimetics as
well as non-natural amino acids may be used.
[0089] The inhibitors may in turn be used to generate crystal
structures or for structure based design or as subject for
evolutionary methods. A further use is the generation of modified
sequences from that depicted in FIG. 5 or 6 by evolutionary methods
(e.g. random or site directed mutagenesis or structure based
design).
[0090] In particular, the inhibitors of Fc receptors may be used to
reduce or enhance the specificity of the above for the selected
Fc-receptors. To this end, modifications can be carried out in the
CDRs of the specific antibodies, in particular of GB3 and CE5, in
order to enhance or lower their specificity to Fc.gamma.RIIb.
[0091] The peptides and polypeptides and substances of the
invention, in particular the antibody or fragment or derivative
thereof are useful for the production of a medicament for the
treatment and/or diagnosis of conditions involving the immune
system, in particular autoimmune diseases, preferably those
selected from Systemic Lupus Erythematosus, Rheumatoid Arthritis,
Immune Thrombocytopenic Purpura or Multiple Sclerosis. Further uses
of the peptides and antibodies or fragments or derivatives thereof
of the invention are in the diagnosis and/or treatment of cancer
and/or allergies. The mAbs CE5 or GB3 or derivatives or fragments
thereof are particularly useful for the treatment of autoimmune
diseases, Multiple sclerosis, Systemic Lupus Erythematosus,
Idiopathic Thrombocytopenic Purpura, Rheumatoid Arthritis, and
cancer, in particular lymphomas or leukemias.
[0092] The mAbs CE5 or GB3 or derivatives or fragments thereof can
also be used for the treatment of cancer in combination with other
therapeutics preferably biotherapeutics (e.g. antibodies).
[0093] The antibody or derivatives or fragments thereof generated
according to the present invention can be used for the treatment
and/or diagnosis of cancer, preferably in combination with other
therapeutics, preferably biotherapeutics (e.g. further antibodies).
The antibody or fragment or derivative thereof is then preferably
used as an adjuvant.
[0094] Further uses of the antibody or fragment or derivative
thereof of the invention include the use for the production of
pharmaceutical and/or diagnostic compositions for the treatment of
host-versus-graft disease, for the treatment of amyloid linked
diseases or to increase the effect of vaccination or for the
treatment of diseases associated activated dendritc cells and/or
macrophages.
[0095] It is also possible to use an antibody or fragment or
derivative thereof which comprises specific anti-Fc.gamma.RIIa
fragments in bi-specific antibodies to direct antigens towards
transport by thrombocytes and/or uptake by the liver and spleen
phagocytosis system. Preferably, the antibody or fragment or
derivative thereof is a specific anti-Fc.gamma.RIIa antibody or
fragment thereof for the treatment of ITP.
DESCRIPTION OF FIGURES AND SEQUENCE LISTING
[0096] FIG. 1:
[0097] Sequence alignment of the extracellular domains of the human
Fc.gamma.RIIb and Fc.gamma.RIIa. Differing amino acids are
boxed.
[0098] FIG. 2:
[0099] Structure of Fc.gamma.RIIb in ribbon representation. The
unique residues are shown in ball-and-stick and potential
glycosylation sites are indicated as larger spheres. Arrows point
to possible extractable substructures (epitopes 1 and 2) hat may be
artificially generated for the improvement of immunization
protocols towards specific Fc.gamma.RIIb-antisera and subsequently
for the production of isoform specific monoclonal antibodies.
[0100] FIG. 3:
[0101] Left diagram: Histogram of a FACS measurement of Raji cells
(Fc.gamma.RIIb-positive and Fc.gamma.RIIa-negative) using the
preimmune serum of the mouse (minus), the obtained antiserum after
the immunization procedure (antiserum) and the pan-Fc.gamma.RII-mAb
AT10 (Greenman et al., 1991). Right diagram: Fluorescence label on
U-937 cells (Fc.gamma.RIIa-positive and Fc.gamma.RIIb negative).
The antiserum reacts only marginally with the cells indicating the
presence of specific antibodies.
[0102] FIG. 4:
[0103] FACS analysis of human blood incubated either with normal
serum (negative control), antiserum of a mouse immunized with
Fc.gamma.RIIb-CDE[126-137], mAb AT10 or the specific monoclonal
antibody GB3 generated by using this invention. a): Dotblot
analysis of the blood sample in terms of cell size (FSC-H) and
granularity (SSC-H). The observed regions R1, R2 R3 contain
lymphocytes (B and T cells), monocytes and granulocytes
respectively. b) Fluorescence intensity of the cells found in
region R1 representing lymphocytes. The pan-Fc.gamma.RIIb mAb AT10,
the mAb GB3 and the antiserum stain the Fc.gamma.RIIb-positive
B-cells while the Fc.gamma.RII-negative T cells are not recognized.
c) Fluorescence intensity of the cells found in region R2
representing monocytes/macrophages. In contrast to the positive
controls mAb AT10 and the antiserum the mAb GB3 does not recognize
the Fc.gamma.RIIa-positive monocytes. d) Fluorescence intensity of
the cells found in region R3 representing granulocytes. In contrast
to the positive controls mAb AT10 and the antiserum the mAb GB3
does not recognize the Fc.gamma.RIIa-positive granulocytes.
[0104] FIG. 5:
[0105] The variable regions of the cloned antibody GB3. The boxed
regions represent the CDRs while the underlined termini may vary
due to cloning artifacts introduced by the primer. a) Variable
region of the light chain; b) Variable region of the heavy
chain.
[0106] FIG. 6:
[0107] The variable regions of the cloned antibody CE5. The boxed
regions represent the CDRs while the underlined termini may vary
due to cloning artifacts introduced by the primer. a) Variable
region of the light chain; b) Variable region of the heavy
chain.
[0108] FIG. 7:
[0109] The glycopeptide CDE[126-137] used for immunization and
generation of Fc.gamma.RIIb-specific antibodies.
[0110] FIG. 8:
[0111] Immunisation of SJL mice with a specific anti-mouse
Fc.gamma.RII antibody. SJLj were immunized with 200 .mu.g MOG an
day 0. Treatment with antiFc.gamma.RII antibody (dosis of 50
.mu.g/week) started at day 5. The clinical score was monitored
daily and is given as the average of the 8 mice per group.
TABLE-US-00001 SEQ ID NO: 1 amino acid sequence of Fc.gamma.RIIa
(as in FIG. 1) SEQ ID NO: 2 amino acid sequence of Fc.gamma.RIIb
(as in FIG. 1) SEQ ID NO: 3 sequence of the glycopeptide CDE
[126-137] SEQ ID NO: 4 nucleic acid sequence of the variable light
region of mAb GB3 SEQ ID NO: 5 corresponding amino acid sequence of
the variable light region of mAb GB3 SEQ ID NO: 6 nucleic acid
sequence of the variable heavy region of mAb GB3 SEQ ID NO: 7
corresponding amino acid sequence of the variable heavy region of
mAb GB3 SEQ ID NO: 8 nucleic acid sequence of the variable light
region of mAb CE5 SEQ ID NO: 9 corresponding amino acid sequence of
the variable light region of mAb CE5 SEQ ID NO: 10 nucleic acid
sequence of the variable heavy region of mAb CE5 SEQ INO: 11
corresponding amino acid sequence of the variable heavy region of
mAb CE5
EXAMPLES
Example 1
Synthesis of the
Cyclo-[N-.beta.-(2-acetylamino-deoxy-2-.beta.-glucopyranosyl)-Asn.sup.138-
, Gly.sup.141]-(129-141)-Fc.gamma.RIIb2, CDE[126-137]
[0112] Standard amino acid derivatives were from Alexis
(Laufelfingen, Switzerland), Fluorenylmethoxycarbonyl-derivative
(Fmoc) of
Asn(N-.beta.-3,4,6-tri-O-acetyl-2-acetylamino-deoxy-2-.beta.-glucopyranos-
yl)-OH from Merck-Novabiochem (Darmstadt, Germany), and the
preloaded chlorotrityl resin from Pepchem (Tubingen, Germany).
Reagents and solvents were of the highest quality commercially
available and were used without further purification. Analytical
reversed-phase HPLC was performed on Waters equipment (Eschbom,
Germany) with a Symmetry C.sub.18 column (5 .mu.m, 3.9.times.150
mm, Waters) by linear gradient elution: (1) 0-100% A in 15 min, or
(2) 0-30% A in 20 min, up to 50% A in 5 min and to 100% A in
further 5 min, (flow rate of 1.5 ml/min and UV detection at 210
nm). The binary elution system was (A) acetonitrile/2%
H.sub.3PO.sub.4 (90:10) and (B) acetonitrile/2% H.sub.3PO.sub.4
(5:95). Preparative reversed-phase HPLC was carried out on Abimed
equipment (Langenfeld, Germany) using Nucleosil C.sub.18 PPN (5
.mu.m, 100 .ANG., 10.times.250 mm, Macherey-Nagel, Duren, Germany)
and a gradient of 0.08% trifluoroacetic acid (TFA) in acetonitrile
(A) and 0.1% TFA in water (B) at a flow rate of 10 ml/min: 2% A for
7 min, up to 40% A in 50 min and to 70% A in further 5 min. ESI-MS
spectra were recorded on a Perkin-Elmer SCIEX API 165 triple
quadrupole spectrometer. LC-MS was carried out with a Nucleosil C18
column (5 .mu.m, 100 .ANG., 1.times.250 mm, Macherey-Nagel) using
linear gradients of 0.1% TFA in water and 0.08% TFA in acetonitrile
(flow rate: 30 .mu.l/min; detection at 210 nm).
a) Solid-Phase Peptide Synthesis.
[0113] The linear peptide precursor was synthesized manually on
Fmoc-Gly-chlorotrityl resin (232 mg, 0.13 mmol) following standard
procedures of Fmoc/tert-butyl (tBu) chemistry. The Fmoc group was
cleaved in each step with two successive treatments (3 and 20 min)
with 20% piperidine in N-methylpyrrolidone (NMP). For
Fmoc-Ser(tBu)-OH and Fmoc-Phe-OH double couplings (2.times.1 h)
with Fmoc-amino
acid/2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluoro-phosphate (HBTU)/N-hydroxybenzotriazole
(HOBt)/N,N-diisopropylethylamine (DIEA) (4:4:4:8 eq) in NMP were
applied, whereas the glycosylated Asn derivative was introduced by
single coupling using
Fmoc-aminoacid/(1H-benzotriazol-1-yloxy)-tripyrrolidinophosphonium
hexafluorophosphate (PyBOP)/HOBt/DIEA (2:2:2:5 eq) in NMP. The
reaction was complete after 5 h, as confirmed by the Kaiser test. A
capping step with acetic anhydride/DIEA (1:1, 3 eq) for 10 min was
performed prior to chain elongation. For acylation with the
remaining amino acid derivatives (Arg was introduced as
Arg-2,2,4,6,7-pentamethyl-dihydrobenzofurane-5-sulfonyl [Pbf]
derivative) again double couplings (2.times.1.5 h) were used with
Fmoc-amino acid/HBTU/HOBt/DIEA (6:6:6:12 eq) in NMP.
b) Cleavage of the Side-Chain-Protected Peptide.
[0114] The side-chain-protected linear peptide was cleaved from the
resin by treating the peptide-resin with 5 ml of 1% TFA in
dichloromethane (DCM) for 3 min. The filtrate was analyzed by thin
layer chromatography (TLC) (CH.sub.3Cl/MeOH/H.sub.2O, 8:3:1) prior
to addition of 1 ml of 1.0% pyridine in methanol. The TFA treatment
was repeated until the TLC control on the filtrate was negative
(overall four treatments). Finally, the resin was washed with DCM
and trifluoroethanol to improve the peptide recovery. The
peptide-containing filtrates and the final washes were combined and
concentrated to a small volume. The residue was diluted with MeOH,
and the product was precipitated with ice-cold water. The crude
product was collected by filtration (270 mg, 80% yield) and
characterized by analytical HPLC (gradient 1) and ESI-MS. A major
peak (t.sub.R 9.5 min; ESI-MS: m/z=2520 [M+H].sup.+; M.sub.r=2519.0
calcd for C.sub.120H.sub.188N.sub.20O.sub.36S) and a minor peak
(t.sub.R 9.3 min; ESI-MS: m/z=2478 [M-42+H].sup.+) at the ratio of
75:20 were found to correspond to the expected product and to a
side product, respectively. The mass difference was attributed to
the loss of one acetyl protecting group from
Asn(Ac.sub.3AcNH-.beta.-Glc).
c) Cyclization.
[0115] Backbone cyclization was accomplished at a peptide
concentration of 0.9 mM in N,N-dimethylformamide (DMF), in the
presence of PyBOP/HOBt/DIEA (1.5:1.5:3.5 eq). The base was added in
portions over 1 h. The conversion of the linear peptide to the
cyclic form was monitored by analytical HPLC, and was completed
after 2.5 h. The reaction mixture was taken to dryness, and the
residue was triturated and washed with ice-cold diethyl ether to
remove traces of DMF prior to the TFA cleavage.
d) Cleavage of the Side-Chain Protecting Groups.
[0116] The acid-labile side-chain protecting groups were removed by
dissolving the cyclic peptide in 10 ml the ice-cold
TFA/triisopropylsilane (TISH)/H.sub.2O (90:5:5). After 2 h shaking,
the TFA was removed under reduced pressure, the oily residue was
diluted with a small amount of MeOH and the crude product
precipitated with ice-cold diethyl ether. The precipitate was
collected by centrifugation, washed several times with ice-cold
ether and, finally, lyophilized from water. The crude glycopeptide
which in addition to the triacetylated form, according to LC-MS was
contaminated by the di- and mono-acetyl derivatives, was suspended
in MeOH and treated in portions with NaOMe over 30 min until an
apparent pH of >10 was reached. The reaction was monitored by
HPLC, and after 3.5 h it was quenched by addition of glacial acetic
acid until pH<5. The mixture was taken to dryness, and the solid
was suspended in MeOH and reprecipitated with ice-cold diethyl
ether. The precipitate was collected by filtration and lyophilized
from water. The crude product was purified by preparative HPLC and
the cyclic glycopeptide was isolated as lyophilized material;
yield: 20% yield (based on the starting resin loading of 0.13
mmol); HPLC: >95% (t.sub.R 7.37 min with gradient 2); ESI-MS:
m/z=1642.8 [M+H].sup.+; M=1641.8 Da calculated for
C.sub.71H.sub.108N.sub.120O.sub.25.
Coupling of the CDE[126-137] to Fc.gamma.RIIb Yielding
Fc.gamma.RIIb-CDE[126-137]
[0117] 100 .mu.l human soluble Fc.gamma.RIIb (10.6 mg/ml) were
added to 1490 .mu.l 50 mM borate pH10 and 410 .mu.l of the
glycopeptide CDE[126-137] (2 mg/ml) and stirred gently at room
temperature. 100 .mu.l of a 0.3% glutaraldehyde solution were
slowly added and the whole mixture stirred for another two hours at
RT before 100 .mu.l 1M glycine was added. The resulting
Fc.gamma.RIIb-CDE[126-137] was stirred for another 30 min and then
dialyzed against PBS and concentrated.
Example 2
Immunization with Fc.gamma.RIIb-CDE[126-137]
[0118] A female six weeks old C57B1/6 mouse was immunized
intraperitoneally every two weeks with an emulsion of 50 .mu.g
Fc.gamma.RIIb-CDE[126-137] in 100 .mu.l Complete Freunds Adjuvant
(CFA, Sigma/Deisenhofen, Germany) for three times. Three weeks
after the last immunization the mouse was boosted with 50 .mu.g of
the Fc.gamma.RIIb-CDE[126-137]: Three days later the spleen was
removed from the animal and the fusion of the extracted cells with
myeloma cells was performed according to Bazin, and Lemieux,
1989.
Example 3
Screening of the Hybridoma for
Fc.gamma.RIIb-CDE[126-137]-Specificity
[0119] Clones that were able to grow in the presence of
hypoxanthine, aminopterin, and thymidine were isolated and their
supernatant tested in ELISA assays where Fc.gamma.RIIb-CDE[126-137]
was precoated on microtitre plate with 120 ng sFc.gamma.RIIa/b per
well (in 100 .mu.l PBS, 20.degree. C., 12 h). The plate was washed
and incubated with PBS/T (PBS/0.2% Tween20, 30 min). 100 .mu.l of
the respective hybridoma were added to the well (100 .mu.l, 90
min). The plate was washed three times with blocking buffer before
100 .mu.l of a peroxidase labeled goat-anti mouse IgG+IgM antibody
(Dianova, Hamburg/Germany) diluted in PBS/T was added. After
incubating for 90 min and subsequent washing with PBS/T, 100 .mu.l
of substrate buffer (0.2 M citrate/phosphate buffer pH 5.2, 4 mg/ml
o-phenylenediamine, 0.024% (v/v) hydrogenperoxide) were applied to
the wells. The reaction was stopped by adding 50 .mu.l 8 N sulfuric
acid and the absorbance at 490 nm was measured in an ELISA
reader.
[0120] Clones that were positive in this assay were tested by flow
cytometry (FACS) using 10.sup.5 Raji cells per sample (ATCC CCL-86)
which strongly express human Fc.gamma.RIIb. After incubation with
100 .mu.l hybridoma supernatant for 30 min on ice the cells were
washed with 1 ml RPMI/10% FCS and precipitated by centrifugation
(400.times.g, 4.degree. C., 5 min). 100 .mu.l FITC labeled goat
anti human antibody (Dianova, Hamburg/Germany) were added. After
incubation for 30 min on ice the cells were washed (RPMI/10% FCS)
and subjected to flow cytometry (FACSort, Becton Dickinson,
Heidelberg/Germany). The median value of the fluorescence for 5,000
counted cells was determined for each sample. Hybridoma
supernatants that were positive in this assay were subjected in a
similar assay using U-937 cells (ATCC CRL-1593.2) which strongly
express Fc.gamma.RIIa to determine Fc.gamma.RIIb-specificity of the
hybridoma. As positive control for both cell lines the
pan-Fc.gamma.RII-mAb AT10 (Greenman et al., 1991) was used.
Example 4
Immunisation of SJL Mice with a Specific Anti-Mouse Fc.gamma.RII
Antibody
[0121] SJL-Mice were immunized with 200 .mu.g MOG to induce
Experimental Autoimmune Encephalomyelitis (EAE) an established
animal model of Multiple Sclerosis. Prophylactic as well as
therapeutic (data not shown) treatment of 8 mice per group with a
specific anti-mouse Fc.gamma.RII antibody (50 .mu.g/week)
significantly reduces the symptoms (clinical score) of the disease
(0=healthy, 1=light paralysis, 2=medium paralysis, 3=strong
paralysis, 4=complete paralysis, 5=death). The results are shown in
FIG. 8.
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Sequence CWU 1
1
111172PRThumanFc gamma RIIa 1Ala Pro Pro Lys Ala Val Leu Lys Leu
Glu Pro Pro Trp Ile Asn Val1 5 10 15Leu Gln Glu Asp Ser Val Thr Leu
Thr Cys Gln Gly Ala Arg Ser Pro 20 25 30Glu Ser Asp Ser Ile Gln Trp
Phe His Asn Gly Asn Leu Ile Pro Thr 35 40 45His Thr Gln Pro Ser Tyr
Arg Phe Lys Ala Asn Asn Asn Asp Ser Gly 50 55 60Glu Tyr Thr Cys Gln
Thr Gly Gln Thr Ser Leu Ser Asp Pro Val His65 70 75 80Leu Thr Val
Leu Ser Glu Trp Leu Val Leu Gln Thr Pro His Leu Glu 85 90 95Phe Gln
Glu Gly Glu Thr Ile Met Leu Arg Cys His Ser Trp Lys Asp 100 105
110Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly Lys Ser Gln Lys
115 120 125Phe Ser Arg Leu Asp Pro Thr Phe Ser Ile Pro Gln Ala Asn
His Ser 130 135 140His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly
Tyr Thr Leu Phe145 150 155 160Ser Ser Lys Pro Val Thr Ile Thr Val
Gln Val Pro 165 1702172PRThumanFc gamma RIIb 2Ala Pro Pro Lys Ala
Val Leu Lys Leu Glu Pro Gln Trp Ile Asn Val1 5 10 15Leu Gln Glu Asp
Ser Val Thr Leu Thr Cys Arg Gly Thr His Ser Pro 20 25 30Glu Ser Asp
Ser Ile Gln Trp Phe His Asn Gly Asn Leu Ile Pro Thr 35 40 45His Thr
Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn Asp Ser Gly 50 55 60Glu
Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp Pro Val His65 70 75
80Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln Thr Pro His Leu Glu
85 90 95Phe Gln Glu Gly Glu Thr Ile Val Leu Arg Cys His Ser Trp Lys
Asp 100 105 110Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly Lys
Ser Lys Lys 115 120 125Phe Ser Arg Ser Asp Pro Asn Phe Ser Ile Pro
Gln Ala Asn His Ser 130 135 140His Ser Gly Asp Tyr His Cys Thr Gly
Asn Ile Gly Tyr Thr Leu Tyr145 150 155 160Ser Ser Lys Pro Val Thr
Ile Thr Val Gln Ala Pro 165 170313PRThumanglycopeptide CDE
[126-137] 3Ser Lys Lys Phe Ser Arg Ser Asp Pro Asn Phe Ser Gly1 5
104312DNAUnknownCDS(1)..(312)variable light region of mAb GB3 4aga
att cag ctg acc cag tct cca tcc tcc tta tct gcc tct ctg gga 48Arg
Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly1 5 10
15gaa aga gtc agt ctc act tgt cgg gca agt cag gaa att agt ggt tac
96Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr
20 25 30tta agc tgg ctt cag cag aaa cca gat gga act att aaa cgc ctg
atc 144Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu
Ile 35 40 45tac gcc aca tcc gct tta gat tct ggt gtc cca aaa agg ttc
agt ggc 192Tyr Ala Thr Ser Ala Leu Asp Ser Gly Val Pro Lys Arg Phe
Ser Gly 50 55 60agt ggg tct ggg tca aat tat tct ctc acc atc agc agc
ctt gag tct 240Ser Gly Ser Gly Ser Asn Tyr Ser Leu Thr Ile Ser Ser
Leu Glu Ser65 70 75 80gaa gat ttt gca gac tat tac tgt cta caa tat
gct aat tat ccg tac 288Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr
Ala Asn Tyr Pro Tyr 85 90 95acg ttc gga ggg ggg acc aag ctg 312Thr
Phe Gly Gly Gly Thr Lys Leu 100 5104PRTUnknownSequence comprised by
an antibody 5Arg Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Leu Gly1 5 10 15Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu
Ile Ser Gly Tyr 20 25 30Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr
Ile Lys Arg Leu Ile 35 40 45Tyr Ala Thr Ser Ala Leu Asp Ser Gly Val
Pro Lys Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Ser Asn Tyr Ser Leu
Thr Ile Ser Ser Leu Glu Ser65 70 75 80Glu Asp Phe Ala Asp Tyr Tyr
Cys Leu Gln Tyr Ala Asn Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr
Lys Leu 1006312DNAUnknownCDS(1)..(312)variable heavy region of mAb
GB3 6gtg cag ctg cag cag tct gga cct gag ctg gtg aag cct ggg gct
tca 48Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
Ser1 5 10 15gtg aag att tcc tgc aag gct tct ggc tac acc ttc act gac
tac tat 96Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Tyr Tyr 20 25 30ata tac tgg gtg aaa cag tgg cct gga cag gga ctt gag
tgg att gga 144Ile Tyr Trp Val Lys Gln Trp Pro Gly Gln Gly Leu Glu
Trp Ile Gly 35 40 45tgg att ttt cct gga act ggt aat act tac tac aat
gaa aac ttc aag 192Trp Ile Phe Pro Gly Thr Gly Asn Thr Tyr Tyr Asn
Glu Asn Phe Lys 50 55 60gac aag gcc aca ctt act ata gat aga tcc tcc
agc aca gcc tac atg 240Asp Lys Ala Thr Leu Thr Ile Asp Arg Ser Ser
Ser Thr Ala Tyr Met65 70 75 80ttg ctc ggc agc ctg acc tct gag gac
tct gcg gtc tat ttc tgt tat 288Leu Leu Gly Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Phe Cys Tyr 85 90 95ggt ccg ttt gct tac tgg ggc caa
312Gly Pro Phe Ala Tyr Trp Gly Gln 1007104PRTUnknownSequence
comprised by an antibody 7Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
Val Lys Pro Gly Ala Ser1 5 10 15Val Lys Ile Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asp Tyr Tyr 20 25 30Ile Tyr Trp Val Lys Gln Trp Pro
Gly Gln Gly Leu Glu Trp Ile Gly 35 40 45Trp Ile Phe Pro Gly Thr Gly
Asn Thr Tyr Tyr Asn Glu Asn Phe Lys 50 55 60Asp Lys Ala Thr Leu Thr
Ile Asp Arg Ser Ser Ser Thr Ala Tyr Met65 70 75 80Leu Leu Gly Ser
Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Tyr 85 90 95Gly Pro Phe
Ala Tyr Trp Gly Gln 1008309DNAUnknownCDS(1)..(309)variable light
region of mAb CE5 8gag ctc acc cag tct cca gcc tcc ctt tct gcg tct
gtg gga gaa act 48Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser
Val Gly Glu Thr1 5 10 15gtc acc atc aca tgt cga gca agt ggg aat att
cac aat tat tta gca 96Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile
His Asn Tyr Leu Ala 20 25 30tgg tat cag cag aaa cag gga aaa tct cct
cag ctc ctg gtc tat tat 144Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro
Gln Leu Leu Val Tyr Tyr 35 40 45aca aca acc tta gca gat ggt gtg cca
tca agg ttc agt ggc agt gga 192Thr Thr Thr Leu Ala Asp Gly Val Pro
Ser Arg Phe Ser Gly Ser Gly 50 55 60tca gga aca caa tat tct ctc aag
atc aac agc ctg caa cct gaa gat 240Ser Gly Thr Gln Tyr Ser Leu Lys
Ile Asn Ser Leu Gln Pro Glu Asp65 70 75 80ttt ggg agt tat tac tgt
caa cat ttt tgg agt act cct cgg acg ttc 288Phe Gly Ser Tyr Tyr Cys
Gln His Phe Trp Ser Thr Pro Arg Thr Phe 85 90 95 ggt gga ggg acc
aag ctc gag 309Gly Gly Gly Thr Lys Leu Glu
1009103PRTUnknownSequence comprised by an antibody 9Glu Leu Thr Gln
Ser Pro Ala Ser Leu Ser Ala Ser Val Gly Glu Thr1 5 10 15Val Thr Ile
Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr Leu Ala 20 25 30Trp Tyr
Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val Tyr Tyr 35 40 45Thr
Thr Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 50 55
60Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro Glu Asp65
70 75 80Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Ser Thr Pro Arg Thr
Phe 85 90 95Gly Gly Gly Thr Lys Leu Glu
10010339DNAUnknownCDS(3)..(338)variable heavy region of mAb CE5
10tg cag gag tca gga cct ggc ctg gtg gcg ccc tca cag agc ctg tcc 47
Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser 1 5 10
15atc aca tgc acc gtc tca ggg ttc tca tta acc ggc tat ggt gta aac
95Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr Gly Val Asn
20 25 30tgg gtt cgc cag cct cca gga aag ggt ctg gag tgg ctg gga atg
att 143Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Met
Ile 35 40 45tgg ggt gat gga aac aca gac tat aat tca gct ctc aaa tcc
aga ctg 191Trp Gly Asp Gly Asn Thr Asp Tyr Asn Ser Ala Leu Lys Ser
Arg Leu 50 55 60agc atc agc aag gac aac tcc aag agc caa gtt ttc tta
aaa atg aac 239Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
Lys Met Asn 65 70 75agt ctg cac act gat gac aca gcc agg tac tac tgt
gcc aga gag aga 287Ser Leu His Thr Asp Asp Thr Ala Arg Tyr Tyr Cys
Ala Arg Glu Arg 80 85 90 95gat tat agg ctt gac tac tgg ggc caa ggg
acc acg gtc acc gtc tcc 335Asp Tyr Arg Leu Asp Tyr Trp Gly Gln Gly
Thr Thr Val Thr Val Ser 100 105 110tca g
339Ser11112PRTUnknownSequence comprised by an antibody 11Gln Glu
Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Ile1 5 10 15Thr
Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr Gly Val Asn Trp 20 25
30Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Met Ile Trp
35 40 45Gly Asp Gly Asn Thr Asp Tyr Asn Ser Ala Leu Lys Ser Arg Leu
Ser 50 55 60Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met
Asn Ser65 70 75 80Leu His Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala
Arg Glu Arg Asp 85 90 95Tyr Arg Leu Asp Tyr Trp Gly Gln Gly Thr Thr
Val Thr Val Ser Ser 100 105 110
* * * * *