U.S. patent application number 11/594126 was filed with the patent office on 2007-05-17 for novel compounds.
This patent application is currently assigned to ACTIVE BIOTECH AB. Invention is credited to Thomas Brodin, Pia J. Karlstrom, Philip P. Kearney, Bo H. K. Nilson, Lennart G. Ohlsson, Jesper M. Tordsson.
Application Number | 20070112179 11/594126 |
Document ID | / |
Family ID | 20417520 |
Filed Date | 2007-05-17 |
United States Patent
Application |
20070112179 |
Kind Code |
A1 |
Brodin; Thomas ; et
al. |
May 17, 2007 |
Novel compounds
Abstract
An antibody, or a derivate or a fragment thereof, having a
binding structure for a target structure is described. The antibody
is displayed in, and on the cell surface of, human gastrointestinal
epithelial tumour cells and in a subpopulation of normal human
gastrointestinal epithelial cells. Said binding structure comprises
the complementarity determining region (CDR) sequences in the light
chain comprising essentially the amino acids number 23-33 (CDR1),
49-55 (CDR2), 88-98 (CDR3) of the amino acid sequence shown in SEQ
ID NO:2, and the CDR sequences in the heavy chain comprising
essentially the amino acids number 158-162 (CDR1), 177-193 (CDR2,
226-238 (CDR3) of the amino acid sequence shown in SEQ ID NO:2, or
other binding structures with similar unique binding properties.
There is also described a target structure displayed in, or on the
surface of tumour cells, vaccine compositions, pharmaceutical
compositions as well as methods related to human malignant
diseases.
Inventors: |
Brodin; Thomas; (Raa,
SE) ; Karlstrom; Pia J.; (Lund, SE) ; Ohlsson;
Lennart G.; (Lund, SE) ; Tordsson; Jesper M.;
(Lund, SE) ; Kearney; Philip P.; (Lund, SE)
; Nilson; Bo H. K.; (Lund, SE) |
Correspondence
Address: |
BUCHANAN, INGERSOLL & ROONEY PC
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Assignee: |
ACTIVE BIOTECH AB
Lund
SE
|
Family ID: |
20417520 |
Appl. No.: |
11/594126 |
Filed: |
November 8, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10088639 |
Mar 20, 2002 |
7189816 |
|
|
PCT/SE00/02082 |
Oct 26, 2000 |
|
|
|
11594126 |
Nov 8, 2006 |
|
|
|
Current U.S.
Class: |
530/388.8 ;
424/155.1; 530/391.1 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 35/04 20180101; C07K 16/3046 20130101; C07K 2317/622 20130101;
C07K 16/2839 20130101; C07K 16/303 20130101; Y02P 20/582 20151101;
C07K 2319/00 20130101; C07K 2317/21 20130101; A61K 2039/505
20130101; G01N 33/57446 20130101; C07K 2317/732 20130101; A61P
37/02 20180101 |
Class at
Publication: |
530/388.8 ;
530/391.1; 424/155.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/46 20060101 C07K016/46; C07K 16/30 20060101
C07K016/30 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 28, 1999 |
SE |
9903895-2 |
Claims
1-16. (canceled)
17. A target structure displayed in, or on the surface of, tumour
cells, said target structure a) having the ability of being
specifically blocked by and to specifically block a binding
structure, wherein the binding structure is an antibody or antigen
binding fragment of an antibody or a fragment thereof, and other
binding structures with similar binding properties, b) being
displayed in, and on the surface of, human gastrointestinal
epithelial cells, c) having substantial homology with .alpha.6
and/or .beta.4 integrin chains or variants thereof, representing a
shared or unique epitope, d) being highly expressed on the surface
of tumour cells, and e) being a target for cytotoxic effector
mechanism; wherein the antibody is an antibody, or a fragment
thereof, having a binding structure for a target structure
displayed in, and on the cell surface of, human gastrointestinal
epithelial tumour cells, said binding structure comprising the
complementarity determining region (CDR) sequences in the light
chain comprising essentially the amino acids number 23-33 (CDR1),
49-55 (CDR2), 88-98 (CDR3) of the amino acid sequence shown in SEQ
ID NO:2, and the CDR sequences in the heavy chain comprising
essentially the amino acids number 158-162 (CDR1), 177-193 (CDR2),
226-238 (CDR3) of the amino acid sequence shown in SEQ ID NO:2.
18. A target structure according to claim 17, wherein the antibody
or antigen binding fragment is labeled and the binding thereof is
inhibited by an unlabeled form of said binding structure and not by
other binding structures, and not inhibiting the binding of other
binding structures having other binding specificities.
19. A target structure according to claim 17, wherein the antibody
or antigen binding fragment comprises one or more of the
complementarity determining region (CDR) sequences comprising
essentially the amino acids number 23-33,
49-55,88-98,158-162,177-193,226-238 of the amino acid sequence
shown in SEQ ID NO : 2, or other binding structures with similar
unique binding properties.
20. A target structure according to claim 17, wherein the antibody
or antigen binding fragment is an antibody.
21. (canceled)
22. A target structure according to claim 17, which is expressed
homogenously in human colonic epithelial cells and less in
pancreatic duct and bile duct cells.
23. A target structure according to claim 17, the expression of
which is correlated to gastrointestinal epithelial
differentiation.
24. A target structure according to claim 17, which comprises
essentially the amino acid sequence of a6 integrin shown in SEQ ID
NO: 3 and/or of .beta.4 integrin shown in SEQ ID NO: 4, and/or one
or more fragments, and/or variants or splice variants, and or
subunits, thereof.
25. A target structure according to claim 24, which comprises homo-
or hetero-monomers or homo- or hetero-multimers of said .alpha.6p4
integrin and/or of said one or more fragments and/or variants
and/or subunits thereof.
26. A target structure according to claim 24, which has an apparent
molecular weight in its non-reduced form of from 90 to 140 kDa,
most preferred from 80 to 160 kDa.
27. A target structure according to claim 24, which comprises a
peptide or polypeptide(s) comprising essentially any one of the
amino acid sequences shown in SEQ ID NOs: 5-51, or comprises a
molecule complexed to said polypeptide(s).
28. A target structure according to claim 24 recognised,
exclusively or not, in its non-reduced form by the antibody or
antigen binding fragment comprised by an antibody or a fragment
thereof, having a binding structure for a target structure
displayed in, and on the cell surface of, human gastrointestinal
epithelial tumour cells, said binding structure comprising the
complementarity determining region (CDR) sequences in the light
chain comprising essentially the amino acids number 23-33 (CDR1),
49-55 (CDR2), 88-98 (CDR3) of the amino acid sequence shown in SEQ
ID NO:2, and the CDR sequences in the heavy chain comprising
essentially the amino acids number 158-162 (CDR1), 177-193 (CDR2),
226-238 (CDR3) of the amino acid sequence shown in SEQ ID NO:2.
29-34. (canceled)
35. A pharmaceutical composition comprising as an active principle
a target structure as defined in claim 17.
36-52. (canceled)
53. A method for therapy of human malignant disease, whereby an
antibody or a fragment thereof is administered to a human subject,
whereby said antibody or fragment thereof has been changed by being
genetically linked to molecules giving the combined molecule
changed pharmaco-kinetic properties; wherein the antibody is an
antibody or a fragment thereof, having a binding structure for a
target structure displayed in and on the cell surface of, human
gastrointestinal epithelial tumor cells, said binding structure
comprising the complementary determining region (CDR) sequences in
the light chain comprising essentially the amino acids number 23-33
(CDR1), 49-55 (CDR2), 88-98 (CDR3) of the amino acid sequence shown
in SEQ ID NO: 2, and the CDR sequence in the heavy chain comprising
essentially the amino acids number 158-162 (CDR1), 177-193 (CDR2),
226-238 (CDR3) of the amino acid sequence shown in SEQ ID NO:
2.
54. (canceled)
Description
[0001] The present invention is related to an antibody, or a
derivate, or a fragment thereof, having a binding structure for a
target structure displayed in, and on the cell surface of, human
gastrointestinal epithelial tumour cells and in a subpopulation of
normal human gastrointestinal epithelial cells; and to a target
structure displayed in, or on the surface of tumour cells; vaccine
compositions; pharmaceutical compositions; as well as methods
related to human malignant diseases.
BACKGROUND OF THE INVENTION
[0002] Surgery is the primary treatment of colorectal cancer
leading to five-year survival rates of 90 to 40 percent depending
on the state of tumour progression from Dukes Stage A to C.
Conventional adjuvant therapy that includes radiation therapy and
chemotherapy has been able to reduce the death rates further by
approximately 30 percent (1). Despite these achievements cancer of
the colon and rectum is one of the major causes of death in human
cancer. Immunological therapy has been extensively attempted.
However, colon cancer has generally been resistant to immunotherapy
and is considered to be of low immunogenicity. Patients with colon
cancer neither respond to IL-2 treatment or adoptive transfer of in
vitro cultured tumour infiltrating lymphocytes otherwise active in
patients with immunogenic malignancies such as melanoma. Most
encouraging however, Riethmuller et al. reported a 32 percent
decreased seven-year death rate for Dukes Stage C colorectal cancer
treated after primary tumour resection with a naked murine mAb
directed to a tumour and normal epithelial associated antigen
(Ep-CAM) (2), indicating that other immunotherapeutic modalities
could be effective.
[0003] A significant improvement of adjuvant immunotherapy and of
the treatment of more advanced stages of cancer should require a
more potent effector mechanism than provided by a naked mAb. In
principle, an increased potency should require an increased tumour
selectivity of the targeting antibody.
[0004] The limited number of colon cancer associated antigens
defined today have been discovered using hybridoma produced murine
mAbs resulting from xenogenic immunisations with human tumours
(3).
[0005] The use of large phage display libraries for the
identification of novel tumour-associated antigens can be expected
to significantly speed up the process of finding target molecules
useful for tumour immunotherapy and diagnosis. Such identification
of target molecules could be accomplished by the selection and
screening of antibody phage libraries on cultured tumour cells and
tissue sections to generate specific reagents defining in vitro and
in vivo expressed antigens (4). The phage display technology has
been established as an efficient tool to generate monoclonal
antibody reagents to various purified antigens, and the
construction and successful selection outcome from immune, naive
and synthetic antibody phage libraries have been described in
several studies (5).
[0006] Non-immune libraries are favourable with respect to their
general applicability, making unique libraries for every single
target unnecessary. On the other hand, sufficiently large and high
quality non-immune libraries are difficult to construct and a
target discovery process using these libraries should require
efficient subtractive selection methods when based on complex
antigens.
[0007] A phage library of a more moderate size has now been
constructed from a near human primate immunised with complex human
antigens. This represents an approach that takes advantage of an in
vivo pre-selected repertoire. Such libraries should be enriched for
specificities to tumour specific epitopes in a reduced background
reactivity to xenogeneic antigens (6). Furthermore, as compared to
the mouse, primate antibodies demonstrating close sequence homology
with human antibodies should not be immunogenic in man (7).
[0008] Novel primate antibodies from a phage library that define
selectively expressed colon cancer associated antigens have now
been identified. The therapeutic potential, demonstrated by T cell
mediated killing of cultured colon cancer cells coated with two of
these antibodies fused to engineered superantigens, is comparable
with superantigens fused to murine Fab fragment specific for colon
cancer associated antigens such as EP-CAM, for which there has
previously been established the therapeutic capacity in
experimental systems (8).
[0009] There is also provided a method for efficient positive and
subtractive cell selection of phage antibodies that should
facilitate future identification of novel phenotype specific
antigens including tumour associated antigens using antibodies from
large phage libraries.
BRIEF SUMMARY OF THE INVENTION
[0010] The present invention is related in a first aspect to an
antibody, or a derivative or a fragment thereof, having a binding
structure for a target structure displayed in, and on the cell
surface of, human gastrointestinal epithelial tumour cells and in a
subpopulation of normal human gastrointestinal epithelial cells,
said binding structure comprising the complementarity determining
region (CDR) sequences in the light chain comprising essentially
the amino acids number 23-33 (CDR1), 49-55 (CDR2), 88-98 (CDR3) of
the amino acid sequence shown in SEQ ID NO:2, and the CDR sequences
in the heavy chain comprising essentially the amino acids number
158-162 (CDR1), 177-193 (CDR2), 226-238 (CDR3) of the amino acid
sequence shown in NO: 2, or other binding structures with similar
unique binding properties.
[0011] In one embodiment the antibody is phage selected. In another
embodiment the sequences are of Macaca fascicularis origin. A
further embodiment of the invention is a derivative of said
antibody, which derivative is of human origin. The sequences
preferably have an identity of at least 84% to corresponding
sequences of human origin. Preferably, the antibody has low
immunogenicity or non-immunogenicity in humans.
[0012] In a further embodiment, the antibody has been derivatised
by genetically linking to other polypeptides, and/or by chemical
conjugation to organic or non-organic chemical molecules, and/or by
di-, oligo- or multimerisation.
[0013] In still a further embodiment, said antibody is genetically
linked or chemically conjugated to cytotoxic polypeptides or to
cytotoxic organic or non-organic chemical molecules.
[0014] In a further embodiment, said antibody is genetically linked
or chemically conjugated to biologically active molecules.
[0015] In still a further embodiment, said antibody is genetically
linked or chemically conjugated to immune activating molecules.
[0016] In another embodiment, said antibody has been changed to
increase or decrease the avidity and/or affinity thereof.
[0017] In still another embodiment, said antibody has been changed
to increase the production yield thereof.
[0018] In a further embodiement, said antibody has been changed to
influence the pharmacokinetic properties thereof.
[0019] In still a further embodiment, said antibody has been
changed to give new pharmacokinetic properties thereto.
[0020] In a further embodiment, said antibody is labeled and the
binding thereof is inhibited by an unlabeled form of said antibody
and not by other binding structures, and not inhibiting the binding
of other binding structures having other specificities.
[0021] A further embodiment is an antibody, the binding structure
of which recognizes a non-reduced form of .alpha.6.beta.4
integrin.
[0022] In another aspect the invention relates to a target
structure displayed in, or on the surface of, tumour cells, said
target structure
[0023] a) having the ability of being specifically blocked by and
to specifically block the binding structure of an antibody as
defined in any one of claims 1-14, and other binding structures
with similar binding specificities,
[0024] b) being displayed in, and on the surface of, human
gastrointestinal epithelial cells,
[0025] c) having substantial homology with .alpha.6 and/or .beta.4
integrin chains or variants thereof, representing a shared or
unique epitope,
[0026] d) being highly expressed on the surface of tumour cells,
and
[0027] e) being a target for cytotoxic effector mechanisms.
[0028] By substantial homology in this context is meant homology in
those parts of the target structure which are relevant for the
binding of the antibody.
[0029] In one embodiment of said target structure, the binding
structure is labeled and the binding thereof is inhibited by an
unlabeled form of said binding structure and not by other binding
structures, and not inhibiting the binding of other binding
structures having other binding specificies.
[0030] In a further embodiment of said target structure said
binding structure comprises one or more of the complementarity
determining region (CDR) sequences comprising essentially the amino
acids number 23-33, 49-55, 88-98, 158-162, 177-193, 226-238 of the
amino acid sequence shown in SEQ ID NO:2, or other binding
structures with similar unique binding properties.
[0031] In still a further embodiment of said target structure said
binding structure is an antibody, which antibody in a further
embodiment comprises the variable region of a light chain
comprising essentially the amino acids number 1-109 of the amino
acid sequence shown in SEQ ID NO:2, and the variable region of a
heavy chain comprising essentially the amino acids number 128-249
of the amino acid sequence shown in SEQ ID NO: 2.
[0032] Said target structure is in a further embodiment expressed
homogenously in human colonic epithelial cells and less in
pancreatic duct and bile duct cells.
[0033] In still a further embodiment, the expression of said target
structure is correlated to gastrointestinal epithelial
differentiation.
[0034] In another embodiment, said target structure comprises the
amino acid sequence of .alpha.6.beta.4 integrin, of which the
.alpha.6 part is shown in SEQ ID NO: 3 and the .beta.4 part is
shown in SEQ ID NO: 4. Another embodiment of the target structure
comprises homo- or heteromonomers or homo- or heteromultimers of
said .alpha.6.beta.4 integrin and/or of said one or more fragments
and/or variants and/or subunits thereof. Preferably, said target
structure has an apparent molecular weight in its non-reduced form
of from 90 to 140 kDa, most preferred fro 80 to 160 kDa.
[0035] In still further embodiments the target structure compirses
a peptide or polypeptide(s) comprising essentially any one of the
amino acid sequences shown in SEQ ID NOs: 5-51, or comprises a
molecule complexed to said polypeptide(s).
[0036] In the case of a target structure comprising amino acid
sequences from the .alpha.6.beta.4 integrin, said target structure
may in a further embodiment be recognised, exclusively or not, in
its non-reduced form by the binding structure comprised by the
antibody as defined above.
[0037] The invention relates in a further aspect to a substance
which binds to the target structure as defined above, which
substance is an organic chemnical molecule or a peptide. In one
embodiment, said substance is an anti-idiotype of said target
structure. Said anti-idiotype may be specifically blocked by and
specifically block a binding structure having similar binding
specificity for said target structure.
[0038] In a still further aspect, the invention relates to a
substance that blocks the function of the target structure as
defined above, which substance is an organic molecule or a
peptide.
[0039] In another aspect, the invention relates to a binding
structure which recognises a target structure as defined above and
which is of an organic chemical nature.
[0040] In a further aspect, the invention relates to a
pharmaceutical compositin comprising as an active principle an
antibody as defined above, or a target structure as defined above,
or a substance as defined above.
[0041] In still a further aspect, the invention is related to a
vaccine composition comprising as an active principle abn antibody
as defined above, or a target structure as defined above, or a
substance as defined above.
[0042] In a further aspect, the invention is related to a method of
therapy for treating conditions based on an anti-angiotenic
mechanism, whereby an antibody as defined above, or a target
structure as defined above, or a substance as defined above, is
administered to a human subject.
[0043] In another aspect, the invention is related to a method of
treating human metastatic diseases, wherein an antibody as defined
above is administered to a human subject.
[0044] In a further aspect the invention is related to a method of
in vitro histopathological diagnosis and prognosis of human
malignant disease, whereby a sample is contacted with an antibody
as defined above and an indicator.
[0045] Embodiments of said method comprise tumour typing, tumour
screening, tumour diagnosis and prognosis, and monitoring
premalignant conditions.
[0046] In still a further aspect, the invention is related to a
method for in vitro diagnosis and prognosis of human malignant
disease, whereby concentrations in bodily fluids of an antigen
comprising a target structure, as defined above, or an
anti-idiotype of said target structure, as defined above, is
assayed.
[0047] A further aspect of the invention is related to a method for
in vitro diagnosis and prognosis of human malignant disease,
whereby concentrations in bodily fluids of an antibody as defined
above is assayed.
[0048] A still further aspect of the invention is related to a
method for in vitro diagnosis and prognosis of human malignant
disease, whereby concentrations in bodily fluids of a complex of a)
an antigen comprising a target structure, as defined above, or an
anti-idiotype of said target structure, as defined above, and b) an
antibody, as defined above, is assayed.
[0049] In a still further aspect, the invention is related to a
method for in vivo diagnosis and prognosis of human malignant
disease, whereby the localisation of an antibody, as defined above,
to tumour deposits in a human subject is determined. Said antibody
is preferably administered to the subject before the determination.
In one embodiment said antibody is accumulated in tumour deposits.
In a further embodiment, said method is quantitative.
[0050] Another aspect of the invention is related to a method for
therapy of human malignant disease, whereby an antibody, as defined
above, is administered to a human subject. In one embodiment of
this method said antibody has been changed by being genetically
linked to molecules giving the combined molecule changed
pharmacokinetic properties. In another embodiment said antibody has
been changed by being derivatised.
DETAILED DESCRIPTION OF THE INVENTION
[0051] The identification of novel tumour associated antigens
(TAAs) is pivotal for the progression in the fields of tumour
immunotherapy and diagnosis. In relation to the present invention,
there was first developed, based on flow cytometric evaluation and
use of a mini-library composed of specific antibody clones linked
to different antibiotic resistance markers, methods for positive
and subtractive selection of phage antibodies employing intact
cells as the antigen source. An scFv phage library
(2.7.times.10.sup.7) was constructed from a primate (Macaca
fascicularis) immunised with pooled human colon carcinomas. This
library was selected for three rounds by binding to Colo205 colon
adenocarcinoma cells, and proteolytic elution followed by phage
amplification.
[0052] Several antibodies reactive with colon carcinomas and with
restricted reactivity with a few epithelial normal tissues were
identified by immunohistochemistry. One clone, A3 scFv, recognised
an epitope that was homogeneously expressed in 11/11 of colon and
4/4 pancreatic carcinomas studied and normal tissue expression
restricted to subtypes of epithelia in the gastrointestinal tract.
The A3 scFv had an apparent overall affinity about 100-fold higher
than an A3 Fab, indicating binding of scFv homodimers. The cell
surface density of the A3 epitope, calculated on the basis of Fab
binding, was exceptionally high, approaching 3 million per
cell.
[0053] Efficient T cell mediated killing of colon cancer cells
coated with A3 scFv fused to the low MHC class II binding
superantigen mutant SEA(D227A) is also demonstrated. The identified
A3 molecule thus represents a TAA with properties that suggests its
use for immuno-therapy of colon and pancreatic cancer.
Discussion
[0054] In relation to the present invention, efficient protocols
for phage selection to be used for the identification of cell
phenotype specific antibody fragments from large phage libraries
was developed. The target specificities for the applications as
exemplified were for colon tumour associated antigens.
[0055] First the frequency of pIII-scFv fusion protein surface
display in the phage population using the herein presented phagemid
construct for phage propagation was analysed. A higher level of
C215 scFv display was achieved as compared to previous reports.
This should favour subtractive selection efficiency, but also
increases the probability of avidity selection of low affinity
antibodies from libraries.
[0056] Specificity of C215 scFv phage binding to colon
adenocarcinoma Colo205 cells was clearly demonstrated. Bound phage
could be efficiently eluted by use of the protease Genenase that
specifically cleaves a target sequence between the phage protein
III and the scFv antibody leaving the cells intact after elution.
This non-chemical elution method should equally efficiently elute
phage antibodies irrespectively of their binding affinity and only
phage bound by scFv interactions, adding to the specificity of the
process.
[0057] The enrichment achieved after three selection rounds on
Colo205 cells (500 000.times.) using this selection protocol was
similar to that reported by other investigators for selections on
complex antigens.
[0058] After verifying the performance of the various
methodological steps the combined technology was applied to library
selections using Colo205 cells.
[0059] The library was constructed from a near human species
immunised with human tumours. The antibody pool generated this way
would potentially include affinity matured antibodies to tumour
specific antigens in a limited background of xeno reactivities to
widespread normal human tissue antigens (6). The antibodies
identified recognised tumour and tissue differentiation antigens
with restricted normal tissue distribution. All of the selected
antibodies identified as colon cancer tissue reactive in the
primary screening also reacted with viable Colo205 cells in flow
cytometry. This restriction to cell surface specificities should
reflect the selection process and not the composition of the
library, since a suspension of a mixture of tumour tissue
components was used for the immunisation.
[0060] In a similar previous study extra- and intracellular
specificities were identified in an anti-melanoma library produced
the same way and selected using tissue sections as the antigen
source (4). Tissue sections of resected human colorectal tumours
and normal colon (mounted in the same well) were used for the
primary screening using immunohistochemistry to assure the clinical
relevance of the selected specificities, to increase the efficiency
and to obtain more qualitative information as compared to flow
cytometric screening.
[0061] The selected antibodies could be classified into four
antibody specificity groups, distinguished by their reactivity
patterns to epithelia in different organs (see Example 1, Table 1).
Among these specificity groups, A3 scFv identified the most tumour
selective antigen. This A3 TAA was highly, homogeneously and
frequently expressed in samples of primary and metastatic colon
cancer and of pancreatic cancer. Furthermore, its cell surface
expression level as determined with the A3 Fab fusion protein (3
millions epitopes/cell) was exceptionally high and permissive for
cell surface mediated cytotoxic effects.
[0062] Few, if any, of the frequently expressed human tumour
antigens defined are tumour specific, but are commonly related to
tissue differentiation such as A3 and the Ep-CAM. However,
upregulated expression of these antigens in tumours should provide
a basis for a therapeutically active dose window. The availability
from the circulation of normal tissue compartments expressing the
antigen may also be more restricted due to limited capillary
permeability and their site of expression in the body (e.g. the
exposure of the apical side of gut epithelial cells to circulating
antibodies should be very limited).
[0063] The clinical experience with the pan-epithelial Ep-CAM
reactive 17-1A mAb supports the feasibility to identify an
effective non-toxic antibody dose. The restricted expression in
epithelia of all of the selected scFv clones in this work, indicate
that these clones in principal could be evaluated as candidates for
immuno-therapeutic applications analogously to the 17-1A, e.g. as
full-length mAbs. However, a particular advantage for the A3 TAA as
compared to the Ep-CAM is the lack of expression in most normal
epithelia such as of the lung and kidney, although the expression
in the colon is similar.
[0064] The tissue distribution to subtypes of normal epithelia is
supported by the selective expression in subtypes of carcinomas
originating from the gastro-intestinal tract (see Example 2, Table
2).
[0065] Several of the previously well-known colon cancer associated
antigens (CEA, CA50, CA19-9, CA242, Tag-72) (3) are expressed
equally or more restrictedly in normal tissues as compared to the
A3 epitope. However, in contrast to the A3 and the C215 Ep-CAM they
are more heterogeneously expressed in tumours.
[0066] Use of antibodies to the Ep-CAM has demonstrated good
clinical results including a survival advantage for colorectal
cancer patients in an adjuvant setting (2). With the objective to
induce tumour responses even in more advanced stage patients, the
introduction of potent effector molecules in conjunction with this
antibody will challenge the "normal tissue resistance" seen in the
treatment with the naked 17-1A mAb. Preclinically, this could be
studied in model systems using toxin-conjugated antibodies specific
to the murine version of this antigen or animals transgenic for
human colon cancer associated antigens.
[0067] Previously, antibody immunotoxins have been successfully
used to cure mice in models with metastatically growing tumours
expressing xeno (human) tumour antigens not expressed in mouse
tissues (10). However, the TAAs used are truly tumour specific and
the models do not reflect the potential for normal tissue targeted
toxicity.
[0068] In previous studies we have reported the potential of
superantigens as immunostimulatory toxins for tumour immunotherapy
(8). Antibody mediated targeting of superantigens attracted large
numbers of cytotoxic and cytokine-producing T cells to the tumour
site. The superantigen SEA(D227A), mutated for low MHC class II
binding affinity, was genetically linked to tumour targeting
antibodies. This "tumour-selective" agent was applied to recruit T
cells independent of MHC expression in the tumour, thus
short-cutting the problems of MHC down regulation and polymorphism
that represent significant obstacles for other active
immunotherapeutic approaches.
[0069] The mini-library of the established "tumour-selective", 1F
scFv phage, the "broadly-reactive" C215 phage and the non-specific
D1.3 phage antibody clones was an essential tool for the
development of protocols for efficient subtractive cell selection.
A requirement for this selection principle is that the negative
selection is followed by positive selection before phage rescue and
amplification, due to the high frequency of non-displaying phage
particles. Alternatively, non-displaying phage can be made
non-infective by selective proteolysis (G. Winter, pers. comm.).
Such a technique may allow the generation of "inert libraries",
i.e. libraries that have been extensively negatively preselected
(e.g. towards a cell in a resting state or a transfectable parental
cell).
[0070] In conclusion, the "non-wanted" model phage specificity
could selectively be subtracted from the phage population by a
factor of approx. 100 for each selection round. Future subtractive
selections using the developed protocol in combination with the use
of large non-immune phage libraries for identification of
differentially expressed cell surface antigens will demonstrate
whether such an approach prove to be superior to the strategy we
used in this study, i.e. positive selection using an in vivo
pre-selected immune repertoire, including restrictions and biases
such as immunodominance (4). The low affinity and high epitope
density demonstrated for the A3 Fab binding to tumour cells as
compared to the A3 scFv fusion protein suggests formation of scFv
multimers that interact with epitopes that cluster on cell
surfaces. Higher affinity monovalent variants of A3 Fab or
alternatively, stable divalent constructs such as full-length A3 Fv
grafted mAbs compatible with the putative low immunogenicity of A3
should be developed. Such constructs would be suitable for
targeting of appropriate effector molecules to this highly
expressed gastro-intestinal tumour associated antigen.
[0071] The invention is further illustrated in the following
nonlimiting experimental part of the description.
EXMPERIMENTAL PART
Materials and Methods
Animals
[0072] Cynomolgus Macaque (Macaca fascicularis) monkeys were kept
and immunised at the Swedish Institute for Infectious Decease
Control (SIIDC), Stockholm. Water and food were always available ad
libitum. Four monkeys were immunised subcutaneously with 2 ml of a
crude suspension of colon cancer tissues in 10% normal cynomolgus
serum in PBS. Booster doses were given day 21, 35, and 49. Antibody
responses were demonstrated in two monkeys where the antigen had
been admixed with alum adjuvant. All animals were kept according to
Swedish legislation and the experiments were approved by the local
ethical committees.
Tissues and Cells
[0073] Human tumours and normal tissue samples were obtained from
Lund University Hospital and Malm{umlaut over (o )} General
Hospital, Sweden. The human colorectal cell line Colo205, the human
B cell lymphoma cell line Raji and the murine B16 melanoma cell
line were from the American Tissue Culture Collection (ATCC,
Rockville, Md.). The mouse melanoma B16-C215.sup.+ cells
transfected with the expression vector pKGE839 containing the
Ep-CAM-1 gene (C215) has been described previously (9).
[0074] The human cells were cultured in RPMI 1640 medium (Gibco,
Middlesex, UK) supplemented with 10% heat inactivated foetal bovine
serum (Gibco) and 0.1 mg/ml gentamycin sulphate (Biological
Industries, Kibbutz Beit Haemek, Israel). The mouse cells were
cultured in medium additionally supplemented with 1 mM glutamine
(Hyclone, Cramlington, UK), 5.times.10.sup.-5 M
.beta.-mercaptoethanol (ICN, Costa Mesa, Calif.), 0.2% NaHCO.sub.3
(Seromed Biochrome, Berlin, Germany), 1.times.10.sup.-2 M HEPES
(HyClone, Utah) and 1.times.10.sup.-3 M sodium pyrovate (HyClone).
The cells were repeatedly tested for Mycoplasma contamination with
Gene-Probe Mycoplasma T. C. test (San Diego, Calif.).
Phagemid Vector and Phage Library Construction
[0075] Total spleen RNA was extracted from one of the responding
monkeys using an RNA isolation kit from Promega (Mannheim, Germany)
and cDNA was amplified using an RNA PCR kit from PE Biosystems
(Stockholm, Sweden). The primers for cDNA synthesis of lambda light
chain and heavy chain genes and for the assembly of these genes to
scFv genes have been reported previously (4). The scFv cDNA was
ligated into a phagemid vector (4) in fusion with the residues
249-406 of the M13 gene III. The scFv-gIII gene was expressed from
a phoA promoter and the resulting protein was directed by the E.
coli heat stable toxin II signal peptide.
[0076] Repeated electroporations of 7 .mu.g library vector with
scFv gene inserts resulted in a total of 2.7.times.10.sup.7 primary
transformed E. coli TG-1 growing as colonies on minimal agar
plates. The colonies were scraped from the plates and grown in 2xYT
at 150 rpm and 37.degree. C. for 1 h. The culture was superinfected
with M13K07 helper phage (Promega) in 50 times excess. Ampicillin
to a concentration of 100 mg/l was added and the culture grown for
a further hour. After addition of kanamycin to a concentration of
70 mg/l, the culture was grown for 15 h at 30.degree. C. and 250
rpm. The phage particles were harvested from the culture
supernatant using two repeated PEG/NaCl precipitations. The
precipitated phage was resolved in PBS 1% BSA.
Western Blot Analysis
[0077] A two-fold dilution series of scFv-C215 phage particles
(from an undiluted stock of PEG-precipitated/concentrated phage)
was applied to separation on a reducing 12% polyacrylamide gel with
1% SDS and 2% .beta.-mercaptoethanol. The proteins were transferred
to a nitrocellulose membrane (Bio-Rad, Hercules, Calif.) by
electrophoresis. The membrane was blocked with 5% low-fat milk
(Semper AB, Stockholm, Sweden) and then incubated with a rabbit
antiserum against a protein III derived peptide sequence,
AEGDDPAKAAFNSLQASATEC, conjugated to keyhole limpet hemocyanin.
Secondary horse radish peroxidase (HRP) conjugated goat-anti-rabbit
antibodies (Bio-Rad) were incubated for 30 min. Between all steps
the membrane was washed 3 times during 5 min in PBS/0.5% Tween 20.
The membrane was incubated in substrate (Amersham Pharmacia
Biotech, Little Chalfon Buckinghamshire, UK) for one min. A light
sensitive film (ECL hyperfilm, Amersham) was exposed to the
membrane and developed for 0.5-5 min.
[0078] Similarly, to analyse the integrity of purified Fab (A3,
including cynomolgus CH1 and Clambda domains), scFv- and Fab
(including murine CH1 and Ckappa)-SEA(D227A) fusion proteins
(produced as described previously (9)), 12% SDS-PAGEs were
performed. The membranes with transferred proteins were incubated
with purified polyclonal rabbit anti-SEA antibodies followed by the
reagent steps described above.
Model and Library Phage Selection on Cells
[0079] Phage suspensions of the lambda light chain library (or of
model phage), 10.sup.12 in 100 .mu.l PBS/1% BSA, were incubated
with 3 million Colo205 cells for 1 h on ice. The cells were washed
3 times including a 10-min incubation using 2 ml PBS/1% BSA for
each wash. The phage were eluted by adding 50 .mu.l of 33 .mu.g/ml
Genenase to the cell pellet and incubated for 15 min. Genenase,
which is a subtilisin BPN' mutant, S24C/H64A/E156S/G169A/Y217L, was
kindly provided by Dr. Poul Carter (San Francisco, Calif.). After
centrifugation the supernatant was transferred to a new tube and
250 .mu.l 1% BSA in PBS was added. To rescue and amplify the
selected library (and the model phage particles in the multi-pass
experiment), the eluted phage particles were allowed to infect 1
ml, E. coli DH5.alpha.F' (OD.sub.600 nm=1.0). The infected
bacterial culture was diluted 100 times with 2xYT supplemented with
the proper antibiotic and cultured until an OD>1.0 (up to two
days).
[0080] Finally, to produce soluble scFv the amber suppressor strain
HB2151 of E. coli was infected with the selected library from the
second and third round. After growth on agar plates containing
ampicillin, single colonies were cultured in 96 Micro well plates
in 2xYT medium supplemented with ampicillin at 30.degree. C. for 17
h. After centrifugation, removal of the supernatant to which an
equal volume of PBS/1% BSA was added, individual scFvs were
analysed for immunoreactivity to sections of human tumours and
normal tissues. Briefly, the C-terminal tag, ATPAKSE, was detected
using a rabbit antiserum followed by biotinylated goat anti-rabbit
antibodies (DAKO A/S, Copenhagen, Denmark) and StreptABComplex HRP
(DAKO A/S) (see "Immunohistochemistry").
Immunohistochemistry
[0081] Frozen cryosections (8 .mu.m) were air-dried on slides,
fixed in acetone at -20.degree. C. for 10 min and rehydrated in 20%
foetal bovine serum in PBS (FBS). Endogenous biotin was blocked
with avidin (diluted 1/6) for 15 min and then with biotin (diluted
1/6) for 15 min (Vector Laboratories, Burlingame, Calif.). Affinity
purified and biotinylated rabbit anti-SEA antibodies, 5 .mu.g/ml,
were incubated for 30 min followed by StreptABComplex HRP (DAKO
A/S, Copenhagen, Denmark), 1/110 diluted in 50 mM Tris pH 7.6 for
30 min. Between all steps the sections were washed 3 times in TBS.
The staining reaction was developed for 8 min in 0.5 mg/ml
3,3'-diaminobenzidine tetrahydrochloride (Sigma) dissolved in Tris
pH 7.6 with 0.01 percent H.sub.2O.sub.2. After 10 min
counterstaining in 0.5% methyl green, the slides were rinsed for 10
min in tap water and gradually dehydrated in 70-99% ethanol and
xylene before mounting in DPX medium (Sigma).
Flow Cytometry
[0082] The Colo205 colon cancer cells were dissociated with 0.02%
w/v EDTA and washed with PBS. To follow the development of an
antibody response in the monkeys the cells were incubated
consecutively with diluted serum, for 1 h at 4.degree. C.,
biotinylated rabbit anti-human IgG antibodies (Southern
Biotechnology Ass. Inc., Alabama, USA) for 30 min, and finally with
avidin-PE (Becton Dickinson, Mountain View, Calif.) for 30 min.
[0083] The binding of model phage to the cells was analysed using
rabbit-anti-M13 antibodies (produced by immunisation of rabbits
with M13 particles) and FITC conjugated donkey anti-rabbit
antibodies (Amersham Pharmacia Biotech). The binding of antibodies
fused to SEA(D227A) was detected using biotinylated rabbit anti-SEA
antibodies and avidin-PE. All reagents were diluted in PBS/1% BSA.
The cells were washed twice with PBS/1% BSA after incubations with
reagents and three times including 10 min incubations after binding
of phage particles.
[0084] Flow cytometric analysis was performed using a FACSort flow
cytometer (Becton Dickinson).
Affinity Determination on Cultured Cells
[0085] A3 scFv-SEA(D227A), A3 Fab-SEA(D227A) and 1F scFv SEA(D227A)
fusion proteins, 80 .mu.g of each protein, were labelled with
iodine as described by Bolton and Hunter to a specific activity of
10-15 .mu.Ci/.mu.g. Colo205 cells and Raji cells, 30 000/sample
were incubated with the iodinated fusion protein at 100 .mu.l/tube
in a two-fold dilution series in 1% BSA for 1 h and then washed
three times in PBS before measuring bound activity. The
concentration of added and bound fusion protein was used for
Scatchard analysis. The background binding to the Raji cells was
subtracted to calculate the specific binding to the Colo205
cells.
Cytotoxicity Assay
[0086] The T cell dependent cytotoxicity of the super-antigen
fusion protein (superantigen antibody dependent cellular
cytotoxicity, SADCC) was measured in a standard 4 h
chromium-release assay employing .sup.51Cr-labelled Colo205 cells
as target cells and human T cells as effector cells (9). The
percent specific lysis was calculated as: 100 .times. cpm .times.
.times. experimental .times. .times. release - cpm .times. .times.
background .times. .times. release cpm .times. .times. total
.times. .times. release - cpm .times. .times. background .times.
.times. release ##EQU1##
EXAMPLE 1
Generation of Tumour Binding Monoclonal Cynomolgus Antibodies
[0087] Cynomolgus monkeys, Macaca fascicularis (four individuals)
were repeatedly immunised with a suspension of human colon
carcinomas four times every other week. The gradual development of
an antibody response in the monkeys was followed by flow cytometric
staining of cultured colorectal cells, Colo205, using dilution
series of the preimmune and immune sera. An IgG antibody response
was elicited only when alum precipitated tumour tissue suspensions
were used (two individuals).
[0088] The monkey with the highest binding level of immune to
preimmune serum antibodies was used for the construction of a large
combinatorial scFv phage library of approximately
2.7.times.10.sup.7 (estimated from the number of primary
transformants). The primate phage library was selected using
Colo205 cells. The total phage yield (eluted/added number of phage
counted as colony forming units, CFU) from three consecutive
selection rounds increased gradually from 1.9.times.10.sup.-7,
1.4.times.10.sup.-5, to 1.2.times.10.sup.-3. Five percent (12/246)
of the monoclonal soluble scFv:s produced from the phage library
after the third round of selection were demonstrated to bind to
sections of a human colorectal cancer tissue and to intact Colo205
cells by flow cytometry. All of the selected antibodies
demonstrated individually unique nucleic acid sequences according
to Hinf I restriction patterns analysed by 1% agarose gel
electrophoresis.
[0089] The antibody genes were amplified by polymerase chain
reaction using 5 .mu.l of bacterial cultures and primers
complementary to regions 5' and 3' to the scFv gene in the phagemid
vector (regions in the phoA promoter and in the M13 gene III).
The Selected scFv Demonstrate Individually Unique Reactivity with
Epithelia in Normal Tissues
[0090] The colorectal cancer reactive scFv's were classified into
specificity groups based on their immunohistochemical reactivity
pattern with normal tissues (Table 1). The antibodies studied in
detail were A3 scFv (and A3 scFv-SEA(D227A)), A10 scFv, 3D scFv and
1D scFv. The representative antibodies could be distinguished from
each other by their fine specificity to epithelia in different
organs and by their binding to leukocytes. The 1D scFv strongly
reacted with gut epithelia and was the only antibody that reacted
with cells of polymorph nuclear granulocyte morphology. The 1D scFv
also differed from the other antibodies by staining the luminal
surface of kidney tubuli and collecting ducts whereas the A10 scFv
reacted homogeneously (non-polarly) with these epithelial cells and
3D scFv and A3 scFv were negative. 1D, A10 and 3D, but not A3 scFv
also reacted with macrophage-like cells in the lung.
[0091] A fifth group of antibodies, not extensively evaluated and
thus not included in Table 1, reacted with colon epithelia,
leukocytes and Kuppfer cells in the liver. The A3 scFv stands out
as demonstrating the most restricted reactivity with the panel of
normal tissues used. The most prominent normal tissue reactivity of
the A3 was staining of normal colon epithelium. Weak staining were
also detected in small ducts of the pancreas and bile ducts of the
liver and of substructures in small bowel epithelia. The surface
epithelium of one of the two stomach samples was strongly stained
by the A3 antibody.
[0092] The reactivity pattern of the A3 scFv was confirmed using
the fusion protein A3 scFv-SEA(D227A). This format permitted the
use of polyclonal rabbit anti-SEA antibodies for
immunohistochemical detection, which is a more sensitive detection
system demonstrating lower background and tissue crossreactivity as
compared to the use of secondary antibodies to the peptide tag,
ATPAKSE, at the C-terminus of the scFvs. TABLE-US-00001 TABLE 1
Immunohistochemical reactivity to normal human tissues of soluble
scFv fragments from the selected colorectal cancer phage library
scFv clone designation Tissue/sub-structure n* A3** A10 3 D 1 D
Esophagus /epithelial tissue 1 0 ND ND ND /non-epithelial tissue 0
ND ND ND Colon /epithelium 5 ++ + + ++ /non-epithelial tissue 0 0 0
granulocytes ++ Small bowel /villi epithelium 2 (+) heterogenously
+ + heterogenously (+) /basal glandulae + + + ++ /non-epithelial
tissue 0 0 0 0 Ventricle /surface epithelium 2 0, ++ 0 0, + ++
/glandular epithelium 0 +, ++ 0 ++ /non-epithelial tissue 0 0 0 0
Pancreas /acini 1 0 (+) + ++ /small ducts (+) (+) + ++ /large ducts
0 (+) + ++ /non-epithelial tissue 0 0 0 0 /endocrine 0 0 0 0 Liver
/hepatocytes 2 0 ND ND ND /Kuppfer cells 0 ND ND ND /bile ducts (+)
ND ND ND Kidney /proximal tubuli 1 0 + 0 luminal surface ++ /distal
tubuli 0 + 0 luminal surface ++ /collecting ducts 0 + 0 luminal
surface ++ /glomeruli 0 0 0 0 /non-epithelial tissue 0 0 0 0
Bladder /epithelial tissue 1 0 ND ND ND /non-epithelial tissue 0 ND
ND ND Prostate /epithelial tissue 1 0 ++ + and secreted material ++
/non-epithelial tissue 0 0 0 0 Lung /bronchial epithelium 1 0 (+)
(+) 0 /alveolar epithelium 0 (+) (+) 0 /non-epithelial tissue 0
macrophages + macrophages + granulocytes ++, macrophages + CNS
/gray matter 1 0 ND ND ND /white matter 0 ND ND ND Skeletal muscle
1 0 ND ND ND Notes to Table 1 0 = negative, (+) = weak, + =
moderate, ++ = strong, ND = not determined *Number of tissue
samples examined **The reactivity of A3 scFv has been confirmed
with the A3 scFv SEA(D227A) fusion protein
EXAMPLE 2
The A3 Tumour-Associated Antigen is Homogeneously and Frequently
Expressed in Colorectal and Pancreatic Tumours
[0093] The A3 scFv-SEA(D227A) fusion protein was used for
immunohistochemical staining of various tumours of epithelial
origin (Table 2 and FIG. 1). The fusion protein homogeneously and
strongly stained 11/11 samples of primary colon cancer tissues and
4/4 metastatic colon cancer samples resected from the ovary, a
lymph node and the liver. Pancreatic cancer tumours, 4/4 samples,
were equally strongly positive. In contrast, tissue samples of
gastric, prostate, breast and non-small cell lung carcinomas were
negative. TABLE-US-00002 TABLE 2 Tumor tissue reactivity of A3 scFv
SEA(D227A) Tumor tissue n Reactivity Colon cancer, 11 All tumor
cells are strongly and primary tumors homogenously stained Colon
cancer 4 As above metastasis in lymph node, liver and ovary
Pancreas cancer 4 As above Ventricle cancer 2 Negative Prostate
cancer 2 Negative Breast cancer 2 Negative Lung carcinoma 2
Negative (non-small cell) Malignant 2 Negative melanoma
EXAMPLE 3
The A3 TAA is Highly Expressed on the Surface of Colon Cancer
Cells
[0094] The results from several Scatchard plots for affinity
determination, based on the binding of the fusion proteins A3
scFv-SEA(D227A), A3 Fab and 1F scFv-SEA(D227A) (1F was classified
to the A3 specificity group) to Colo205 cells, are summarised in
Table 3. Specific binding was calculated by subtraction of
non-specific binding to human B cell lymphoma Raji cells, a cell
line not expressing the A3 and 1F TAAs, from the binding to Colo205
cells. Linear regression was used to calculate the slope and the
intercept of the extrapolated line in the Scatchard plot. The A3
scFv-SEA(D227A) fusion protein saturated approximately 10-fold less
binding sites per cell as compared to the A3 Fab (approx. 3 million
sites per cell) fusion protein, indicating that divalent
(multivalent) binding was involved for the scFv. This is supported
by the more than 100-fold higher overall affinity (3.6-5.5 nM) for
the A3 scFv fusion protein as compared to the A3 Fab (580-780
nM).
[0095] A single experiment performed using the 1F scFv-SEA(D227A)
fusion protein, demonstrated similar binding affinity and
saturation of binding sites as the A3 scFv-SEA(D227A) fusion
protein. TABLE-US-00003 TABLE 3 Scatchard analysis of iodinated
fusion proteins binding to Colo205 cells Fusion protein n* Kd (nM)
million sites/cell A3 Fab-SEA(D227A) 2 580-780 3.0-3.9 A3
scFv-SEA(D227A) 3 3.6-5.5 0.11-0.39 1F scFv-SEA(D227A) 1 4.2 0.18
*Number of experiments performed
EXAMPLE 4
A3 and 1F scFv-SEA(D227A) Mediate T Cell Lysis of Colo205 Cells
[0096] The capacity of the two fusion proteins A3 and 1F
scFv-SEA(D227A) to mediate superantigen antibody dependent cellular
cytotoxicity (SADCC) towards Colo205 cells was investigated and
compared with the positive control C215 Fab-SEA(D227A) and negative
control D1.3 scFv-SEA(D227A) fusion proteins. The A3
scFv-SEA(D227A) fusion protein titration approached a plateau for
maximal lysis which was similar, approx. 50 percent in this 4 h
assay, to that demonstrated for the C215 Fab-SEA(D227A) fusion
protein, although at a ten-fold higher concentration (FIG. 2). The
1F scFv-SEA(D227A) mediated a similar level of cytotoxicity at a
slightly higher concentration as compared to the A3
scFv-SEA(D227A).
[0097] The negative control D1.3 scFv SEA(D227A) fusion protein did
not mediate any cytotoxicity.
EXAMPLE 5
Purification of a Tumour Associated Antigen that is Recognised by
the Colon Cancer Reactive Antibody A3.
[0098] A tumour extract was made out of xenografted tumour cell
line Colo205. The extract was applied to a pre-column coupled with
C215Fab-SEAm9, and a column coupled with A3scFv-SEAm9. The columns
were in series, during the application of sample but separated
prior to elution under alkaline conditions.
[0099] A single peak was detected during elution by UV spectroscopy
(FIG. 3). This eluted fraction from the latter A3-column was
collected, neutralised, concentrated, and then analysed by SDS-PAGE
under non-reducing conditions (FIG. 4). Two bands visible by silver
staining (labelled I and II in FIG. 4) of apparent molecular weight
of approximately 90-140 kDa were cut out and examined by standard
peptid mapping methodologies. These two bands corresponds to bands
detected by A3 in Western Blot, see example 8. From band I 47
separate tryptic peptide masses were obtained (see SEQ ID NO: 3,
Table 4, and FIG. 5 for the sequnces and corresponing mass weights)
which completely matched to different tryptic peptide masses as
determined by MALDI-TOF) of the human .alpha.6 integrin or .beta.4
integrin (see SEQ ID NOs: 5-51 and 3-4, respectively, and FIGS. 3A
and B, respectively, where in FIG. 3A the underlinings correspond
to the peptides appearing in FIG. 3B/SEQ ID NOs: 5-51). From band
II 22 separate tryptic peptide masses were obtained which
completely matched to different tryptic peptide masses of .beta.4
integrin (data not shown). The data show that the .alpha.6.beta.4
integrin heterodimer is specifically isolated with the A3-affinity
column. TABLE-US-00004 TABLE 4 Peptides/polypeptides derived from
human .alpha.6.beta.4 integrin and masses thereof Sequence Measured
Calculated No. Sequence Mass Mass 5 LLLVGAPR 838.568 838.551 6
ANRTGGLYSCDITARGPCTR 2226.131 2226.050 7 VVTCAHRYEK 1262.637
1262.631 8 RQHVNTK 882.524 882.490 9 CYVLSQNLR 1152.618 1152.583 10
FGSCQQGVAATFTK 1501.706 1501.710 11 DFHYIVFGAPGTYNWK 1914.881
1914.917 12 DEITFVSGAPR 1191.625 1191.600 13 ANHSGAVVLLK 1108.600
1108.647 14 DGWQDIVIGAPQYFDR 1879.865 1879.897 15 DGEVGGAVYVYMNQQGR
1842.811 1842.844 16 WNNVKPIR 1026.608 1026.584 17
NIGDINQDGYPDIAVGAPYDDL 2520.213 2520.189 GK 18 GISPYFGYSIAGNMDLDR
1975.913 1975.922 19 NSYPDVAVGSLSDSVTIFR 2026.992 2027.008 20
SRPVINIQK 1054.644 1054.637 21 LRPIPITASVEIQEPSSR 1993.066 1993.108
22 VNSLPEVLPILNSDEPK 1863.920 1864.006 23 TAHIDVHFLK 1180.665
1180.647 24 FSYLPIQK 995.601 995.556 25 DIALEITVTNSPSNPR 1726.866
1726.897 26 SEDEVGSLIEYEFR 1672.764 1672.770 27 VESKGLEKVTCEPQK
1731.866 1731.895 28 REITEKQIDDNRK 1644.792 1644.866 29 FSLFAER
869.476 869.452 30 YQTLNCSVNVNCVNIR 1954.003 1953.927 31 LNYLDILMR
1150.644 1150.629 32 AFIDVTAAAENIR 1390.739 1390.733 33 LPNAGTQVR
955.523 955.532 34 VSVPQTDMRPEK 1386.727 1386.705 35 EPWPNSDPPFSFK
1547.730 1547.717 36 NVISLTEDVDEFR 1536.744 1536.754 37
TQDYPSVPTLVR 1375.718 1375.722 38 RGEVGIYQVQLR 1417.801 1417.791 39
ALEHVDGTHVCQLPEDQK 2075.965 2075.981 40 GNIHLKPSFSDGLK 1512.749
1512.817 41 MDAGIICDVCTCELQK 1928.901 1928.822 42 YEGQFCEYDNFQCPR
2012.795 2012.790 43 SCVQCQAWGTGEKKGR 1879.865 1879.890 44
DEDDDCTYSYTMEGDGAPGPNS 3103.229 3103.278 TVLVHK 45 QEVEENLNEVYR
1521.779 1521.718 46 VAPGYYTLTADQDAR 1640.779 1640.791 47
VPLFIRPEDDDEK 1572.778 1572.790 48 DVVSFEQPEFSVSR 1625.758 1625.781
49 LLELQEVDSLLR 1427.760 1427.810 50 VCAYGAQGEGPYSSLVSCR 2060.883
2060.916 51 VLVDNPKNR 1054.644 1054.600
Materials and Methods. Solubilisation of Tumour Tissue
[0100] Human colon cancer tissue expressing the A3 antigen was
provided by hospitals in Sweden and stored frozen at -70.degree. C.
in the tissue bank at ABR. Frozen colon cancer tissues were sliced
with a scalpel and transferred into a tube containing cold isotonic
sucrose buffer (0.25M sucrose, 10 mM KCl, 1.5M MgCl.sub.2, 50 mM
Tris-HCl pH 7.4 at 25.degree. C.) containing 1% (v/v) Nonidet P-40
(NP-40) and protease inhibitors (Completet.TM. Protease Inhibitor
Cocktail Tablet, Boehringer Mannheim). Tissue was homogenised with
an Ultra-Turrax homogeniser and were left to solubilise at
0.degree. C. The solubilised preparation was centrifuged at 11,000
rpm (Hettich centrifuge Universal 30 RF rotor), to remove cell
debris. The supernatant was further centrifuged at 108,000 g at
4.degree. C. (Beckman Ultracentrifuge Ti-60 rotor), and finally
filtered through a 0.2 .mu.m Minisart plus filter (Sartoriuis AG
Gottingen Germany).
Affinity Purification of Tissue Antigens
[0101] A3scFv-SEAm9 was coupled to a NHS-activated HiTrap.RTM.
column (Pharmacia Biotech Uppsala Sweden), according to the
manufacturer's recommendations. The control and pre-column were
coupled with C215Fab-SEAm9, and the control, pre-column and column
were set up in series. All columns were washed with pre-wash buffer
(20 mM Tris HCl pH7.5 at 4.degree. C. containing 0.2% NP 40). The
extract was loaded onto the column at 0.1 ml/min, and the flow
through was recirculated. The columns were then washed with start
buffer. Bound antigen was eluted in a pH gradient of diethylamine
starting at pH 7.5 up to 11.0. 2.5 ml of eluant was collected and
concentrated down to 75 .mu.l. The purification was performed at
4.degree. C. using an AKTA FPLC system (Amersham Pharmacia Biotech
Uppsala Sweden). Eluted protein was analysed by SDS PAGE and silver
staining. Individual bands were excised, digested with trypsin and
the masses of the peptide were determined using a MALDI-TOF
instrument by Protana A/S (Odense, Denmark). The peptide masses
were then compared in a computer search with all tryptic peptide
masses for each protein in the SWISSPROT database, a service
provided by Protana A/S (Odense Denmark).
EXAMPLE 6
A3scFv-SEAm9 Detects a Novel .alpha.6.beta.4 Integrin Epitope
[0102] Commercial antibodies to human .alpha.6 integrin and .beta.4
integrin were compared to A3 on normal and malignant colon
sections. The reactivity, shown in FIG. 6, demonstrates that A3 is
restricted to the colon epithelium (FIG. 6[i]), and malignant cell
in the tumour (FIG. 6[ii]). Commercial antibody NKI-GoH3 to
.alpha.6 integrin, also reacted with normal colon (FIG. 6[iii]) and
colon cancer (FIG. 6[iv]). Reaction was seen in epithelial cells of
colon and malignant cells (arrows) but also in blood vessels (BV),
some stromal components (s) and in muscularis mucosae (mm). The
reaction observed with commercial ASC-3 anti-.beta.4 integrin
antibody was similar to that noted with anti-.alpha.6 antibody but
weaker, in both normal colon (v) and colon cancer (vi).
Materials and Methods
Antibody
[0103] A3 scFv was selected from the M fascicularis library. The VH
and VL genes from this were released by restriction enzyme
digestion and fused to the Staphylococcal Enterotoxin AE chimeric
mutant (D227A) to generate the A3scFv-SEAm9. This demonstrated very
low levels of non-specific binding and allowed sensitive detection
by secondary antibodies. ASC-3 anti-human-.beta.4 integrin antibody
and NKI-GoH3 anti-human-.alpha.6 integrin antibody were from Becton
Dickinson (Copenhagen, Denmark)
Immunohistochemistry
[0104] Tumour and normal tissue samples were obtained from the
Department of Surgery Lund Hospital. These were rate-frozen in
iso-pentane, which had been pre-cooled in liquid nitrogen. Samples
were stored at -70.degree. C. until sectioned. After cryosectioning
the sections were air dried over night, fixed in cold acetone and
blocked with avidin/biotin (Vector Burlingame Calif.). Primary
antibody was then added to the section for one hour.
[0105] The secondary antibodies were incubated for 30 minutes
followed by streptavidin-biotin/HRP (Dakopatts Copenhagen Denmark)
for a further 30 minutes. Extensive washing was perfromed between
all these steps with 50 mM Tris pH 7.6, 0.15M NaCl.
Diaminobenzidine (DAB) was used as chromogen and the sections were
counterstained in 0.5% methyl green. Controls included a non-tissue
reactive Fab and SEA-D227A or no primary antibody. All antibodies
were used at a final concentration of 5 .mu.g/ml. Results were
expressed as negative, weak, moderate or strong staining.
EXAMPLE 7
The A3 Tumour Associated Antigen Reacted with .alpha.6 and .beta.4
Integrin Antibodies in a Capture ELISA
[0106] Crude tumor extract or A3 antigen purified by A3-affinity
chromatography (see example 5) was analysed by a capture ELISA.
Commercial antibodiy ASC-3 specific for beta 4 integrin were used
as capture antibody, to which different dilutions of crude tumor
extract was applied. This was then chased with A3scFv-SEAm9. Bound
A3scFv-SEAm9 was then detected with anti-SEA-HRP (FIG. 7A). In FIG.
7B the commercial anti-.alpha.6 integrin antibody NKI-GoH3 was used
to capture different dilutions of the concentrated A3-affinity
purified eluate. In a similar way as in FIG. 7A the captured
proteins were chased with A3scFv-SEAm9 and detected with
anti-SEA-HRP. In both experiments a concentration dependent signal
was detected. These results confirm the specificity of A3 to
.alpha.6.beta.4 intergin heterodimer, which was also shown to be
specifically isolated from the A3-affinity column in example 5.
Material and Methods
[0107] Commercial antibodies NKI-GoH3 or ASC-3 (Becton Dickinson
Copenhagen Denmark) 100 .mu.l, were used to coat the well of an
E.I.A./R.I.A.-plate (Costar) in 0.05 M NaHCO3, pH 9.6. The reaction
was allowed to continue overnight at 4.degree. C., after which the
plates were washed 4 times in DPBS+0.05% Tween 20. Wells were then
blocked with 200 .mu.l 3% non-fat milk powder in DPBS+0.05% Tween
20, for 1-2 h at room temperature (RT) with shaking. Wells were
again washed as above and 100 .mu.l antigen extract diluted in 3%
non-fat milk powder in DPBS+0.05% Tween 20, was applied for 2 h at
RT with shaking. Wells were again washed (4.times.DPBS+0.05% Tween
20) after which 100 .mu.l of the primary antibody diluted in 3%
non-fat milk powder in DPBS+0.05% Tween 20 was incuabted for 2 h at
RT with shaking. Wells were washed again as above and 100 .mu.l of
the secondary antibody diluted in 3% non-fat milkpowder in
DPBS+0.05% Tween 20 was added to each well for 1 h at RT with
shaking. Wells were again washed as above and colour developed by
the addition of 100 .mu.l peroxidase substrate (Sigma Fast OPD
Peroxidase Substrate Tablet Set P-9187). The reaction was allowed
to continue for 30 min at RT, in the dark and shaking before the
reaction was stopped by the addition of 50 .mu.l 3 M
H.sub.2SO.sub.4. The absorbance was read at 490 nm.
EXAMPLE 8
Western Blot Analysis of the A3 Tumour Antigen
[0108] A3-affinity purified tumour antigen extracts were separated
by SDS-PAGE and transferred to membranes for Western blot analysis.
Extracts were applied directly or heated to 100.degree. C. for 5
minutes or heated to 100.degree. C. for 5 minutes but in the
presence of mercaptoethanol (BME) (FIG. 8). The membranes were then
probed with A3scFv-SEAm9 and anti-SEA-HRP or anti-human-.alpha.6
integrin or anti-human-.beta.4 integrin antibodies. The
anti-.beta.4 integrin antibody did not react with any protein on
the membrane (FIG. 8[ii]). The anti-human-.alpha.6 integrin reacted
with a major specie with apparent molecular weight between 90-140
kDa in the A3-affinity purified tumour antigen extract (FIG.
8[iii]). The same species was also detected by A3scFv-SEAm9, which
also was detected after heating but was much weaker under reduced
conditions (with BME present) (FIG. 8[i]). The major band detected
in the 90-140 kDa interval corresponds to the bands in example 5,
that were analysed by peptide mapping and were found to contain
.alpha.6 integrin and .beta.4 integrin.
Materials and Methods
[0109] ASC-3 anti-human-.beta.4 integrin antibody and NKI-GoH3
anti-human-.alpha.6 integrin antibody were from Becton Dickinson
(Copenhagen, Denmark). Samples were resolved by SDS-PAGE in 0.25M
tris-glycine pH 8.9 and 0.1% SDS at 100V through the upper gel,
then 170V through the resolving gel. Molecular weight standards
(Biorad broad Range, Biorad) were included on all gels. Resolved
samples were transferred to nitrocellulose (Biorad) in transfer
buffer (10 mM Tris base, 2M glycine, 40% (v/v) methanol) at 100V
for 1 hour. Membranes were blocked with 5% (w/v) BSA/TBS for at
least 2 hours at 4.degree. C., then incubated with the appropriate
antibody diluted in 5% BSA/TBS/0.2% azide. This reaction was
allowed to proceed for at least 2 hours at RT, after which the
membrane was washed extensively in TBST-T. Bound antibody was
detected by incubation of membranes for 1 hour with HRP conjugated
antibody diluted in TSB-T containing 5% milk powder. Membranes were
then incubated with enhanced chemiluminescence (ECL) detection
reagents (Renaissance.RTM. NEN.TM. Life Science Products, Boston
Mass.) for 1 minute and exposed to film for up to 1 hour.
REFERENCES
[0110] 1. DeCosse J J, Tsioulias G J, Jacobson J S. Colorectal
cancer: detection, treatment, and rehabilitation. CA Cancer J Clin
1994; 44: 27-42.
[0111] 2. Riethmuller G, et al. Monoclonal antibody therapy for
resected Dukes' C colorectal cancer: seven-year outcome of a
multicenter randomized trial. J Clin Oncol 1998; 16: 1788-1794.
[0112] 3. Kuhn J A, Thomas G. Monoclonal antibodies and colorectal
carcinoma: a clinical review of diagnostic applications. Cancer
Invest 1994; 12: 314-323.
[0113] 4. Tordsson J, et al. Efficient selection of scFv antibody
phage by adsorption to in situ expressed antigens in tissue
sections. J Immunol Methods 1997; 210: 11-23.
[0114] 5. Aujame L, Geoffroy F, Sodoyer R. High affinity human
antibodies by phage display. Hum Antibodies 1997; 8: 155-168.
[0115] 6. Clark R K, Trainer D L, Bailey D S, Greig R G
Immunohistochemical analysis of antiserum from rhesus monkeys
immunized with human colon carcinoma. Cancer Res 1989; 49:
3656-3661.
[0116] 7. Lewis A P, et al. Cloning and sequence analysis of kappa
and gamma cynomolgus monkey immunoglobulin cDNAs. Dev Comp Immunol
1993; 17: 549-560.
[0117] 8. Brodin T N, et al. Man-made superantigens:
Tumor-selective agents for T-cell-based therapy. Adv Drug Deliv Rev
1998; 31: 131-142.
[0118] 9. Dohlsten M, et al. Monoclonal antibody-superantigen
fusion proteins: tumor-specific agents for T-cell-based tumor
therapy. Proc Natl Acad Sci USA 1994; 91: 8945-8949.
[0119] 10. Liu C, et al. Eradication of large colon tumor
xenografts by targeted delivery of maytansinoids. Proc Natl Acad
Sci USA 1996; 93: 8618-8623.
LEGENDS TO FIGURES
FIG. 1 The A3 Tumour-Associated Antigen is Homogeneously Expressed
in Primary and Metastatic Tumours
[0120] Immunohistochemical staining of frozen and acetone fixed
sections of human tumour tissues using A3 scFv-SEA(D227A) and C215
Fab-SEA(D227A) at 70 nM. The A3 scFv fusion protein reacted
strongly and homogeneously with both primary colon and pancreatic
carcinoma resected from tumour patients. A representative staining
of a primary colon cancer is shown for C215 Fab-SEA(D227A) in (A)
and for A3 scFv-SEA(D227A) in (B). Staining by A3 scFv-SEA(D227A)
of a colon cancer liver metastasis is shown in (C) and of a primary
pancreatic cancer in (D).
FIG. 2 A3 scFv-SEA(D227A) Coated Colo205 Tumour Cells are
Efficiently Killed by T Cells.
[0121] Superantigen antibody dependent cellular cytotoxicity
(SADCC) towards Colo205 cells mediated by A3 scFv-SEA(D227A)
reached the same maximal cytotoxicity as the anti-Ep-CAM fusion
protein C215 Fab-SEA(D227A), although at a ten-fold higher
concentration. The absence of cytotoxicity mediated by the D1.3
scFv-SEA(D227A) demonstrates the need of a tumour targeting
antibody moiety in the fusion protein.
FIG. 3
[0122] Immunoaffinity chromatography of tumor extract on a
A3scFv-SEAm9 coupled column. Protein bound to A3 coupled columns
was washed extensively then eluted as described in Materials and
Methods in Example 5. The eluted fractions were examined by UV
spectroscopy (arrow) and a single peak identified. The sample was
eluted with a pH gradient as indicated by an x.
FIG. 4
[0123] A3 antigen preparation was separated on a non-reduced SDS
PAGE and silver-stained. Previous Western analysis had defined a
molecular weight range in which the A3 antigen was believed to
reside. The bands evident within this region (Labelled I and II)
were excised for peptide mapping analysis
FIGS. 5A and 5B
[0124] Epithelial integrin .alpha.6.beta.4: complete primary
structure of .alpha.6 and variant forms of .beta.4 (precursor)
(Tamura et al J Cell Biol 111:1593-1604 (1990)). The matched
peptides shown in SEQ ID NOs: 5-51 are underlined in the sequences
of human .alpha.6 (FIG. 5A) integrin and .beta.4 (precursor) (FIG.
5B) integrin as published.
FIG. 6
[0125] Immunohistochemistry of normal and malignant colon using
A3scFv and commercial anti-human .alpha.6 and .beta.4 integrin
monoclonal antibodies.
FIGS. 7A and 7B
[0126] Capture ELISA. In FIG. 7A monoclonal antibody ASC-3 specific
for .beta.4 integrin was used as capture antibody, to which
different dilutions of crude tumor extract was applied. In FIG. 7B
the anti-.alpha.6 integrin monoclonal anti-body NKI-GoH3 was used
to capture different dilutions of the concentrated A3-affinity
purified eluate. In both FIGS. 7A and 7B the captured integrin
antigen was then successfully detected with A3scFv-SEAm9.
FIGS. 8A and 8B
[0127] Western blot analysis of the eluate from the A3-affinity
column. The primary antibodies used are (i) and (ii) A3scFv-SEAm9,
(iii) ASC-3 anti-human-.beta.4 integrin antibody and (iv) NKI-GoH3
anti-human-.alpha.6 integrin anti-body. Lane A--the eluate was
applied directly, lane B--the eluate was heated to 100.degree. C.
for 5 minutes, and lane C--the eluate was heated to 100.degree. C.
for 5 minutes but in the presence of mercaptoethanol. Positions of
molecular weight standards are indicated.
Sequence CWU 1
1
51 1 747 DNA Macaca fascicularis CDS (1)..(747) Coding sequence VL
(1-109) - modified Huston linker (110-127) - VH (128-249) 1 tct tct
gag ctg act cag ggc cct gca ttg tct gtg gcc ttg gga cat 48 Ser Ser
Glu Leu Thr Gln Gly Pro Ala Leu Ser Val Ala Leu Gly His 1 5 10 15
aca gtc agg atg acc tgc caa gga gac agc ctc aaa acc tat tat gca 96
Thr Val Arg Met Thr Cys Gln Gly Asp Ser Leu Lys Thr Tyr Tyr Ala 20
25 30 agc tgg tac cag cag aag cca ggc cag gtc cct gtg ctg gtc atc
tat 144 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Val Pro Val Leu Val Ile
Tyr 35 40 45 ggt aac aac tac cgg ccc tca ggg atc cca ggc cga ttc
tct ggc tcc 192 Gly Asn Asn Tyr Arg Pro Ser Gly Ile Pro Gly Arg Phe
Ser Gly Ser 50 55 60 tgg tca gga aac aca gct tcc ttg acc atc act
gcg gct cag gtg gaa 240 Trp Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr
Ala Ala Gln Val Glu 65 70 75 80 gat gag gct gac tat tac tgt aac tcc
tgg gac agc agc ggt acc cat 288 Asp Glu Ala Asp Tyr Tyr Cys Asn Ser
Trp Asp Ser Ser Gly Thr His 85 90 95 ccg gta ttc ggc gga ggg acc
cgg gtg acc gtc cta ggt caa gcc aac 336 Pro Val Phe Gly Gly Gly Thr
Arg Val Thr Val Leu Gly Gln Ala Asn 100 105 110 ggt gaa ggc ggc tct
ggt ggc ggg gga tcc gga ggc ggc ggt tct gag 384 Gly Glu Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 115 120 125 gtg cag ttg
gtg gag tct ggg gga ggc ttg gta aag cct ggg ggg tcc 432 Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser 130 135 140 ctg
aga ctc tct tgt gta gcc tct ggg tcc atc ttc agt agc tct gtt 480 Leu
Arg Leu Ser Cys Val Ala Ser Gly Ser Ile Phe Ser Ser Ser Val 145 150
155 160 atg cac tgg gtc cgc cag gct cca gga aag ggt ctg gag tgg gtc
tca 528 Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
Ser 165 170 175 gtt att agt gaa aat ggg cgt acc att aac tac gca gac
tct gtg aag 576 Val Ile Ser Glu Asn Gly Arg Thr Ile Asn Tyr Ala Asp
Ser Val Lys 180 185 190 ggc cga ttc acc atc tcc aga gac aac gcc aag
aac tca ctg ttt ctg 624 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Phe Leu 195 200 205 cag atg aac agc ctg aca ggc gag gac
acg gcc gtc tat tac tgt agt 672 Gln Met Asn Ser Leu Thr Gly Glu Asp
Thr Ala Val Tyr Tyr Cys Ser 210 215 220 aga gag ggg gga cct gga aca
acg tcc aac cgg ctc gat gcc tgg ggc 720 Arg Glu Gly Gly Pro Gly Thr
Thr Ser Asn Arg Leu Asp Ala Trp Gly 225 230 235 240 ccg gga gtc ctg
gtc acc gtt tcc tca 747 Pro Gly Val Leu Val Thr Val Ser Ser 245 2
249 PRT Macaca fascicularis Coding sequence VL (1-109) - modified
Huston linker (110-127) - VH (128-249) 2 Ser Ser Glu Leu Thr Gln
Gly Pro Ala Leu Ser Val Ala Leu Gly His 1 5 10 15 Thr Val Arg Met
Thr Cys Gln Gly Asp Ser Leu Lys Thr Tyr Tyr Ala 20 25 30 Ser Trp
Tyr Gln Gln Lys Pro Gly Gln Val Pro Val Leu Val Ile Tyr 35 40 45
Gly Asn Asn Tyr Arg Pro Ser Gly Ile Pro Gly Arg Phe Ser Gly Ser 50
55 60 Trp Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Ala Ala Gln Val
Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Trp Asp Ser Ser
Gly Thr His 85 90 95 Pro Val Phe Gly Gly Gly Thr Arg Val Thr Val
Leu Gly Gln Ala Asn 100 105 110 Gly Glu Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Glu 115 120 125 Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Lys Pro Gly Gly Ser 130 135 140 Leu Arg Leu Ser Cys
Val Ala Ser Gly Ser Ile Phe Ser Ser Ser Val 145 150 155 160 Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 165 170 175
Val Ile Ser Glu Asn Gly Arg Thr Ile Asn Tyr Ala Asp Ser Val Lys 180
185 190 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe
Leu 195 200 205 Gln Met Asn Ser Leu Thr Gly Glu Asp Thr Ala Val Tyr
Tyr Cys Ser 210 215 220 Arg Glu Gly Gly Pro Gly Thr Thr Ser Asn Arg
Leu Asp Ala Trp Gly 225 230 235 240 Pro Gly Val Leu Val Thr Val Ser
Ser 245 3 1073 PRT Human TA6-Human integrin alpha-6A 3 Met Ala Ala
Ala Gly Gln Leu Cys Leu Leu Tyr Leu Ser Ala Gly Leu 1 5 10 15 Leu
Ser Arg Leu Gly Ala Ala Phe Asn Leu Asp Thr Arg Glu Asp Asn 20 25
30 Val Ile Arg Lys Tyr Gly Asp Pro Gly Ser Leu Phe Gly Phe Ser Leu
35 40 45 Ala Met His Trp Gln Leu Gln Pro Glu Asp Lys Arg Leu Leu
Leu Val 50 55 60 Gly Ala Pro Arg Gly Glu Ala Leu Pro Leu Gln Arg
Ala Asn Arg Thr 65 70 75 80 Gly Gly Leu Tyr Ser Cys Asp Ile Thr Ala
Arg Gly Pro Cys Thr Arg 85 90 95 Ile Glu Phe Asp Asn Asp Ala Asp
Pro Thr Ser Glu Ser Lys Glu Asp 100 105 110 Gln Trp Met Gly Val Thr
Val Gln Ser Gln Gly Pro Gly Gly Lys Val 115 120 125 Val Thr Cys Ala
His Arg Tyr Glu Lys Arg Gln His Val Asn Thr Lys 130 135 140 Gln Glu
Ser Arg Asp Ile Phe Gly Arg Cys Tyr Val Leu Ser Gln Asn 145 150 155
160 Leu Arg Ile Glu Asp Asp Met Asp Gly Gly Asp Trp Ser Phe Cys Asp
165 170 175 Gly Arg Leu Arg Gly His Glu Lys Phe Gly Ser Cys Gln Gln
Gly Val 180 185 190 Ala Ala Thr Phe Thr Lys Asp Phe His Tyr Ile Val
Phe Gly Ala Pro 195 200 205 Gly Thr Tyr Asn Trp Lys Gly Ile Val Arg
Val Glu Gln Lys Asn Asn 210 215 220 Thr Phe Phe Asp Met Asn Ile Phe
Glu Asp Gly Pro Tyr Glu Val Gly 225 230 235 240 Gly Glu Thr Glu His
Asp Glu Ser Leu Val Pro Val Pro Ala Asn Ser 245 250 255 Tyr Leu Gly
Phe Ser Leu Asp Ser Gly Lys Gly Ile Val Ser Lys Asp 260 265 270 Glu
Ile Thr Phe Val Ser Gly Ala Pro Arg Ala Asn His Ser Gly Ala 275 280
285 Val Val Leu Leu Lys Arg Asp Met Lys Ser Ala His Leu Leu Pro Glu
290 295 300 His Ile Phe Asp Gly Glu Gly Leu Ala Ser Ser Phe Gly Tyr
Asp Val 305 310 315 320 Ala Val Val Asp Leu Asn Lys Asp Gly Trp Gln
Asp Ile Val Ile Gly 325 330 335 Ala Pro Gln Tyr Phe Asp Arg Asp Gly
Glu Val Gly Gly Ala Val Tyr 340 345 350 Val Tyr Met Asn Gln Gln Gly
Arg Trp Asn Asn Val Lys Pro Ile Arg 355 360 365 Leu Asn Gly Thr Lys
Asp Ser Met Phe Gly Ile Ala Val Lys Asn Ile 370 375 380 Gly Asp Ile
Asn Gln Asp Gly Tyr Pro Asp Ile Ala Val Gly Ala Pro 385 390 395 400
Tyr Asp Asp Leu Gly Lys Val Phe Ile Tyr His Gly Ser Ala Asn Gly 405
410 415 Ile Asn Thr Lys Pro Thr Gln Val Leu Lys Gly Ile Ser Pro Tyr
Phe 420 425 430 Gly Tyr Ser Ile Ala Gly Asn Met Asp Leu Asp Arg Asn
Ser Tyr Pro 435 440 445 Asp Val Ala Val Gly Ser Leu Ser Asp Ser Val
Thr Ile Phe Arg Ser 450 455 460 Arg Pro Val Ile Asn Ile Gln Lys Thr
Ile Thr Val Thr Pro Asn Arg 465 470 475 480 Ile Asp Leu Arg Gln Lys
Thr Ala Cys Gly Ala Pro Ser Gly Ile Cys 485 490 495 Leu Gln Val Lys
Ser Cys Phe Glu Tyr Thr Ala Asn Pro Ala Gly Tyr 500 505 510 Asn Pro
Ser Ile Ser Ile Val Gly Thr Leu Glu Ala Glu Lys Glu Arg 515 520 525
Arg Lys Ser Gly Leu Ser Ser Arg Val Gln Phe Arg Asn Gln Gly Ser 530
535 540 Glu Pro Lys Tyr Thr Gln Glu Leu Thr Leu Lys Arg Gln Lys Gln
Lys 545 550 555 560 Val Cys Met Glu Glu Thr Leu Trp Leu Gln Asp Asn
Ile Arg Asp Lys 565 570 575 Leu Arg Pro Ile Pro Ile Thr Ala Ser Val
Glu Ile Gln Glu Pro Ser 580 585 590 Ser Arg Arg Arg Val Asn Ser Leu
Pro Glu Val Leu Pro Ile Leu Asn 595 600 605 Ser Asp Glu Pro Lys Thr
Ala His Ile Asp Val His Phe Leu Lys Glu 610 615 620 Gly Cys Gly Asp
Asp Asn Val Cys Asn Ser Asn Leu Lys Leu Glu Tyr 625 630 635 640 Lys
Phe Cys Thr Arg Glu Gly Asn Gln Asp Lys Phe Ser Tyr Leu Pro 645 650
655 Ile Gln Lys Gly Val Pro Glu Leu Val Leu Lys Asp Gln Lys Asp Ile
660 665 670 Ala Leu Glu Ile Thr Val Thr Asn Ser Pro Ser Asn Pro Arg
Asn Pro 675 680 685 Thr Lys Asp Gly Asp Asp Ala His Glu Ala Lys Leu
Ile Ala Thr Phe 690 695 700 Pro Asp Thr Leu Thr Tyr Ser Ala Tyr Arg
Glu Leu Arg Ala Phe Pro 705 710 715 720 Glu Lys Gln Leu Ser Cys Val
Ala Asn Gln Asn Gly Ser Gln Ala Asp 725 730 735 Cys Glu Leu Gly Asn
Pro Phe Lys Arg Asn Ser Asn Val Thr Phe Tyr 740 745 750 Leu Val Leu
Ser Thr Thr Glu Val Thr Phe Asp Thr Pro Asp Leu Asp 755 760 765 Ile
Asn Leu Lys Leu Glu Thr Thr Ser Asn Gln Asp Asn Leu Ala Pro 770 775
780 Ile Thr Ala Lys Ala Lys Val Val Ile Glu Leu Leu Leu Ser Val Ser
785 790 795 800 Gly Val Ala Lys Pro Ser Gln Val Tyr Phe Gly Gly Thr
Val Val Gly 805 810 815 Glu Gln Ala Met Lys Ser Glu Asp Glu Val Gly
Ser Leu Ile Glu Tyr 820 825 830 Glu Phe Arg Val Ile Asn Leu Gly Lys
Pro Leu Thr Asn Leu Gly Thr 835 840 845 Ala Thr Leu Asn Ile Gln Trp
Pro Lys Glu Ile Ser Asn Gly Lys Trp 850 855 860 Leu Leu Tyr Leu Val
Lys Val Glu Ser Lys Gly Leu Glu Lys Val Thr 865 870 875 880 Cys Glu
Pro Gln Lys Glu Ile Asn Ser Leu Asn Leu Thr Glu Ser His 885 890 895
Asn Ser Arg Lys Lys Arg Glu Ile Thr Glu Lys Gln Ile Asp Asp Asn 900
905 910 Arg Lys Phe Ser Leu Phe Ala Glu Arg Lys Tyr Gln Thr Leu Asn
Cys 915 920 925 Ser Val Asn Val Asn Cys Val Asn Ile Arg Cys Pro Leu
Arg Gly Leu 930 935 940 Asp Ser Lys Ala Ser Leu Ile Leu Arg Ser Arg
Leu Trp Asn Ser Thr 945 950 955 960 Phe Leu Glu Glu Tyr Ser Lys Leu
Asn Tyr Leu Asp Ile Leu Met Arg 965 970 975 Ala Phe Ile Asp Val Thr
Ala Ala Ala Glu Asn Ile Arg Leu Pro Asn 980 985 990 Ala Gly Thr Gln
Val Arg Val Thr Val Phe Pro Ser Lys Thr Val Ala 995 1000 1005 Gln
Tyr Ser Gly Val Pro Trp Trp Ile Ile Leu Val Ala Ile Leu Ala 1010
1015 1020 Gly Ile Leu Met Leu Ala Leu Leu Val Phe Ile Leu Trp Lys
Cys Gly 1025 1030 1035 1040 Phe Phe Lys Arg Asn Lys Lys Asp His Tyr
Asp Ala Thr Tyr His Lys 1045 1050 1055 Ala Glu Ile His Ala Gln Pro
Ser Asp Lys Glu Arg Leu Thr Ser Asp 1060 1065 1070 Ala 4 1875 PRT
Human Integrin beta-4 precursor 4 Met Ala Gly Pro Arg Pro Ser Pro
Trp Ala Arg Leu Leu Leu Ala Ala 1 5 10 15 Leu Ile Ser Val Ser Leu
Ser Gly Thr Leu Ala Asn Arg Cys Lys Lys 20 25 30 Ala Pro Val Lys
Ser Cys Thr Glu Cys Val Arg Val Asp Lys Asp Cys 35 40 45 Ala Tyr
Cys Thr Asp Glu Met Phe Arg Asp Arg Arg Cys Asn Thr Gln 50 55 60
Ala Glu Leu Leu Ala Ala Gly Cys Gln Arg Glu Ser Ile Val Val Met 65
70 75 80 Glu Ser Ser Phe Gln Ile Thr Glu Glu Thr Gln Ile Asp Thr
Thr Leu 85 90 95 Arg Arg Ser Gln Met Ser Pro Gln Gly Leu Arg Val
Arg Leu Arg Pro 100 105 110 Gly Glu Glu Arg His Phe Glu Leu Glu Val
Phe Glu Pro Leu Glu Ser 115 120 125 Pro Val Asp Leu Tyr Ile Leu Met
Asp Phe Ser Asn Ser Met Ser Asp 130 135 140 Asp Leu Asp Asn Leu Lys
Lys Met Gly Gln Asn Leu Ala Arg Val Leu 145 150 155 160 Ser Gln Leu
Thr Ser Asp Tyr Thr Ile Gly Phe Gly Lys Phe Val Asp 165 170 175 Lys
Val Ser Val Pro Gln Thr Asp Met Arg Pro Glu Lys Leu Lys Glu 180 185
190 Pro Trp Pro Asn Ser Asp Pro Pro Phe Ser Phe Lys Asn Val Ile Ser
195 200 205 Leu Thr Glu Asp Val Asp Glu Phe Arg Asn Lys Leu Gln Gly
Glu Arg 210 215 220 Ile Ser Gly Asn Leu Asp Ala Pro Glu Gly Gly Phe
Asp Ala Ile Leu 225 230 235 240 Gln Thr Ala Val Cys Thr Arg Asp Ile
Gly Trp Arg Pro Asp Ser Thr 245 250 255 His Leu Leu Val Phe Ser Thr
Glu Ser Ala Phe His Tyr Glu Ala Asp 260 265 270 Gly Ala Asn Val Leu
Ala Gly Ile Met Ser Arg Asn Asp Glu Arg Cys 275 280 285 His Leu Asp
Thr Thr Gly Thr Tyr Thr Gln Tyr Arg Thr Gln Asp Tyr 290 295 300 Pro
Ser Val Pro Thr Leu Val Arg Leu Leu Ala Lys His Asn Ile Ile 305 310
315 320 Pro Ile Phe Ala Val Thr Asn Tyr Ser Tyr Ser Tyr Tyr Glu Lys
Leu 325 330 335 His Thr Tyr Phe Pro Val Ser Ser Leu Gly Val Leu Gln
Glu Asp Ser 340 345 350 Ser Asn Ile Val Glu Leu Leu Glu Glu Ala Phe
Asn Arg Ile Arg Ser 355 360 365 Asn Leu Asp Ile Arg Ala Leu Asp Ser
Pro Arg Gly Leu Arg Thr Glu 370 375 380 Val Thr Ser Lys Met Phe Gln
Lys Thr Arg Thr Gly Ser Phe His Ile 385 390 395 400 Arg Arg Gly Glu
Val Gly Ile Tyr Gln Val Gln Leu Arg Ala Leu Glu 405 410 415 His Val
Asp Gly Thr His Val Cys Gln Leu Pro Glu Asp Gln Lys Gly 420 425 430
Asn Ile His Leu Lys Pro Ser Phe Ser Asp Gly Leu Lys Met Asp Ala 435
440 445 Gly Ile Ile Cys Asp Val Cys Thr Cys Glu Leu Gln Lys Glu Val
Arg 450 455 460 Ser Ala Arg Cys Ser Phe Asn Gly Asp Phe Val Cys Gly
Gln Cys Val 465 470 475 480 Cys Ser Glu Gly Trp Ser Gly Gln Thr Cys
Asn Cys Ser Thr Gly Ser 485 490 495 Leu Ser Asp Ile Gln Pro Cys Leu
Arg Glu Gly Glu Asp Lys Pro Cys 500 505 510 Ser Gly Arg Gly Glu Cys
Gln Cys Gly His Cys Val Cys Tyr Gly Glu 515 520 525 Gly Arg Tyr Glu
Gly Gln Phe Cys Glu Tyr Asp Asn Phe Gln Cys Pro 530 535 540 Arg Thr
Ser Gly Phe Leu Cys Asn Asp Arg Gly Arg Cys Ser Met Gly 545 550 555
560 Gln Cys Val Cys Glu Pro Gly Trp Thr Gly Pro Ser Cys Asp Cys Pro
565 570 575 Leu Ser Asn Ala Thr Cys Ile Asp Ser Asn Gly Gly Ile Cys
Asn Gly 580 585 590 Arg Gly His Cys Glu Cys Gly Arg Cys His Cys His
Gln Gln Ser Leu 595 600 605 Tyr Thr Asp Thr Ile Cys Glu Ile Asn Tyr
Ser Ala Ile His Pro Gly 610 615 620 Leu Cys Glu Asp Leu Arg Ser Cys
Val Gln Cys Gln Ala Trp Gly Thr 625 630 635 640 Gly Glu Lys Lys Gly
Arg Thr Cys Glu Glu Cys Asn Phe Lys Val Lys 645 650 655 Met Val Asp
Glu Leu Lys Arg Ala Glu Glu Val Val Val Arg Cys Ser 660 665 670 Phe
Arg Asp Glu Asp Asp Asp Cys Thr Tyr Ser
Tyr Thr Met Glu Gly 675 680 685 Asp Gly Ala Pro Gly Pro Asn Ser Thr
Val Leu Val His Lys Lys Lys 690 695 700 Asp Cys Pro Pro Gly Ser Phe
Trp Trp Leu Ile Pro Leu Leu Leu Leu 705 710 715 720 Leu Leu Pro Leu
Leu Ala Leu Leu Leu Leu Leu Cys Trp Lys Tyr Cys 725 730 735 Ala Cys
Cys Lys Ala Cys Leu Ala Leu Leu Pro Cys Cys Asn Arg Gly 740 745 750
His Met Val Gly Phe Lys Glu Asp His Tyr Met Leu Arg Glu Asn Leu 755
760 765 Met Ala Ser Asp His Leu Asp Thr Pro Met Leu Arg Ser Gly Asn
Leu 770 775 780 Lys Gly Arg Asp Val Val Arg Trp Lys Val Thr Asn Asn
Met Gln Arg 785 790 795 800 Pro Gly Phe Ala Thr His Ala Ala Ser Ile
Asn Pro Thr Glu Leu Val 805 810 815 Pro Tyr Gly Leu Ser Leu Arg Leu
Ala Arg Leu Cys Thr Glu Asn Leu 820 825 830 Leu Lys Pro Asp Thr Arg
Glu Cys Ala Gln Leu Arg Gln Glu Val Glu 835 840 845 Glu Asn Leu Asn
Glu Val Tyr Arg Gln Ile Ser Gly Val His Lys Leu 850 855 860 Gln Gln
Thr Lys Phe Arg Gln Gln Pro Asn Ala Gly Lys Lys Gln Asp 865 870 875
880 His Thr Ile Val Asp Thr Val Leu Met Ala Pro Arg Ser Ala Lys Pro
885 890 895 Ala Leu Leu Lys Leu Thr Glu Lys Gln Val Glu Gln Arg Ala
Phe His 900 905 910 Asp Leu Lys Val Ala Pro Gly Tyr Tyr Thr Leu Thr
Ala Asp Gln Asp 915 920 925 Ala Arg Gly Met Val Glu Phe Gln Glu Gly
Val Glu Leu Val Asp Val 930 935 940 Arg Val Pro Leu Phe Ile Arg Pro
Glu Asp Asp Asp Glu Lys Gln Leu 945 950 955 960 Leu Val Glu Ala Ile
Asp Val Pro Ala Gly Thr Ala Thr Leu Gly Arg 965 970 975 Arg Leu Val
Asn Ile Thr Ile Ile Lys Glu Gln Ala Arg Asp Val Val 980 985 990 Ser
Phe Glu Gln Pro Glu Phe Ser Val Ser Arg Gly Asp Gln Val Ala 995
1000 1005 Arg Ile Pro Val Ile Arg Arg Val Leu Asp Gly Gly Lys Ser
Gln Val 1010 1015 1020 Ser Tyr Arg Thr Gln Asp Gly Thr Ala Gln Gly
Asn Arg Asp Tyr Ile 1025 1030 1035 1040 Pro Val Glu Gly Glu Leu Leu
Phe Gln Pro Gly Glu Ala Trp Lys Glu 1045 1050 1055 Leu Gln Val Lys
Leu Leu Glu Leu Gln Glu Val Asp Ser Leu Leu Arg 1060 1065 1070 Gly
Arg Gln Val Arg Arg Phe His Val Gln Leu Ser Asn Pro Lys Phe 1075
1080 1085 Gly Ala His Leu Gly Gln Pro His Ser Thr Thr Ile Ile Ile
Arg Asp 1090 1095 1100 Pro Asp Glu Leu Asp Arg Ser Phe Thr Ser Gln
Met Leu Ser Ser Gln 1105 1110 1115 1120 Pro Pro Pro His Gly Asp Leu
Gly Ala Pro Gln Asn Pro Asn Ala Lys 1125 1130 1135 Ala Ala Gly Ser
Arg Lys Ile His Phe Asn Trp Leu Pro Pro Ser Gly 1140 1145 1150 Lys
Pro Met Gly Tyr Arg Val Lys Tyr Trp Ile Gln Gly Asp Ser Glu 1155
1160 1165 Ser Glu Ala His Leu Leu Asp Ser Lys Val Pro Ser Val Glu
Leu Thr 1170 1175 1180 Asn Leu Tyr Pro Tyr Cys Asp Tyr Glu Met Lys
Val Cys Ala Tyr Gly 1185 1190 1195 1200 Ala Gln Gly Glu Gly Pro Tyr
Ser Ser Leu Val Ser Cys Arg Thr His 1205 1210 1215 Gln Glu Val Pro
Ser Glu Pro Gly Arg Leu Ala Phe Asn Val Val Ser 1220 1225 1230 Ser
Thr Val Thr Gln Leu Ser Trp Ala Glu Pro Ala Glu Thr Asn Gly 1235
1240 1245 Glu Ile Thr Ala Tyr Glu Val Cys Tyr Gly Leu Val Asn Asp
Asp Asn 1250 1255 1260 Arg Pro Ile Gly Pro Met Lys Lys Val Leu Val
Asp Asn Pro Lys Asn 1265 1270 1275 1280 Arg Met Leu Leu Ile Glu Asn
Leu Arg Glu Ser Gln Pro Tyr Arg Tyr 1285 1290 1295 Thr Val Lys Ala
Arg Asn Gly Ala Gly Trp Gly Pro Glu Arg Glu Ala 1300 1305 1310 Ile
Ile Asn Leu Ala Thr Gln Pro Lys Arg Pro Met Ser Ile Pro Ile 1315
1320 1325 Ile Pro Asp Ile Pro Ile Val Asp Ala Gln Ser Gly Glu Asp
Tyr Asp 1330 1335 1340 Ser Phe Leu Met Tyr Ser Asp Asp Val Leu Arg
Ser Pro Ser Gly Ser 1345 1350 1355 1360 Gln Arg Pro Ser Val Ser Asp
Asp Thr Gly Cys Gly Trp Lys Phe Glu 1365 1370 1375 Pro Leu Leu Gly
Glu Glu Leu Asp Leu Arg Arg Val Thr Trp Arg Leu 1380 1385 1390 Pro
Pro Glu Leu Ile Pro Arg Leu Ser Ala Ser Ser Gly Arg Ser Ser 1395
1400 1405 Asp Ala Glu Ala Pro Thr Ala Pro Arg Thr Thr Ala Ala Arg
Ala Gly 1410 1415 1420 Arg Ala Ala Ala Val Pro Arg Ser Ala Thr Pro
Gly Pro Pro Gly Glu 1425 1430 1435 1440 His Leu Val Asn Gly Arg Met
Asp Phe Ala Phe Pro Gly Ser Thr Asn 1445 1450 1455 Ser Leu His Arg
Met Thr Thr Thr Ser Ala Ala Ala Tyr Gly Thr His 1460 1465 1470 Leu
Ser Pro His Val Pro His Arg Val Leu Ser Thr Ser Ser Thr Leu 1475
1480 1485 Thr Arg Asp Tyr Asn Ser Leu Thr Arg Ser Glu His Ser His
Ser Thr 1490 1495 1500 Thr Leu Pro Arg Asp Tyr Ser Thr Leu Thr Ser
Val Ser Ser His Gly 1505 1510 1515 1520 Leu Pro Pro Ile Trp Glu His
Gly Arg Ser Arg Leu Pro Leu Ser Trp 1525 1530 1535 Ala Leu Gly Ser
Arg Ser Arg Ala Gln Met Lys Gly Phe Pro Pro Ser 1540 1545 1550 Arg
Gly Pro Arg Asp Ser Ile Ile Leu Ala Gly Arg Pro Ala Ala Pro 1555
1560 1565 Ser Trp Gly Pro Asp Ser Arg Leu Thr Ala Gly Val Pro Asp
Thr Pro 1570 1575 1580 Thr Arg Leu Val Phe Ser Ala Leu Gly Pro Thr
Ser Leu Arg Val Ser 1585 1590 1595 1600 Trp Gln Glu Pro Arg Cys Glu
Arg Pro Leu Gln Gly Tyr Ser Val Glu 1605 1610 1615 Tyr Gln Leu Leu
Asn Gly Gly Glu Leu His Arg Leu Asn Ile Pro Asn 1620 1625 1630 Pro
Ala Gln Thr Ser Val Val Val Glu Asp Leu Leu Pro Asn His Ser 1635
1640 1645 Tyr Val Phe Arg Val Arg Ala Gln Ser Gln Glu Gly Trp Gly
Arg Glu 1650 1655 1660 Arg Glu Gly Val Ile Thr Ile Glu Ser Gln Val
His Pro Gln Ser Pro 1665 1670 1675 1680 Leu Cys Pro Leu Pro Gly Ser
Ala Phe Thr Leu Ser Thr Pro Ser Ala 1685 1690 1695 Pro Gly Pro Leu
Val Phe Thr Ala Leu Ser Pro Asp Ser Leu Gln Leu 1700 1705 1710 Ser
Trp Glu Arg Pro Arg Arg Pro Asn Gly Asp Ile Val Gly Tyr Leu 1715
1720 1725 Val Thr Cys Glu Met Ala Gln Gly Gly Gly Pro Ala Thr Ala
Phe Arg 1730 1735 1740 Val Asp Gly Asp Ser Pro Glu Ser Arg Leu Thr
Val Pro Gly Leu Ser 1745 1750 1755 1760 Glu Asn Val Pro Tyr Lys Phe
Lys Val Gln Ala Arg Thr Thr Glu Gly 1765 1770 1775 Phe Gly Pro Glu
Arg Glu Gly Ile Ile Thr Ile Glu Ser Gln Asp Gly 1780 1785 1790 Gly
Pro Phe Pro Gln Leu Gly Ser Arg Ala Gly Leu Phe Gln His Pro 1795
1800 1805 Leu Gln Ser Glu Tyr Ser Ser Ile Thr Thr Thr His Thr Ser
Ala Thr 1810 1815 1820 Glu Pro Phe Leu Val Asp Gly Pro Thr Leu Gly
Ala Gln His Leu Glu 1825 1830 1835 1840 Ala Gly Gly Ser Leu Thr Arg
His Val Thr Gln Glu Phe Val Ser Arg 1845 1850 1855 Thr Leu Thr Thr
Ser Gly Thr Leu Ser Thr His Met Asp Gln Gln Phe 1860 1865 1870 Phe
Gln Thr 1875 5 8 PRT Human Amino acids 61-68 of SEQ ID NO 3 5 Leu
Leu Leu Val Gly Ala Pro Arg 1 5 6 20 PRT Human Amino acids 77-96 of
SEQ ID NO 3 6 Ala Asn Arg Thr Gly Gly Leu Tyr Ser Cys Asp Ile Thr
Ala Arg Gly 1 5 10 15 Pro Cys Thr Arg 20 7 10 PRT Human Amino acids
127-137 of SEQ ID NO 3 7 Val Val Thr Cys Ala His Arg Tyr Glu Lys 1
5 10 8 7 PRT Human Amino acids 138-144 of SEQ ID NO 3 8 Arg Gln His
Val Asn Thr Lys 1 5 9 9 PRT Human Amino acids 154-162 of SEQ ID NO
3 9 Cys Tyr Val Leu Ser Gln Asn Leu Arg 1 5 10 14 PRT Human Amino
acids 185-198 of SEQ ID NO 3 10 Phe Gly Ser Cys Gln Gln Gly Val Ala
Ala Thr Phe Thr Lys 1 5 10 11 16 PRT Human Amino acids 198-214 of
SEQ ID NO 3 11 Asp Phe His Tyr Ile Val Phe Gly Ala Pro Gly Thr Tyr
Asn Trp Lys 1 5 10 15 12 11 PRT Human Amino acids 272-282 of SEQ ID
NO 3 12 Asp Glu Ile Thr Phe Val Ser Gly Ala Pro Arg 1 5 10 13 11
PRT Human Amino acids 283-293 of SEQ ID NO 3 13 Ala Asn His Ser Gly
Ala Val Val Leu Leu Lys 1 5 10 14 16 PRT Human Amino acids 328-343
of SEQ ID NO 3 14 Asp Gly Trp Gln Asp Ile Val Ile Gly Ala Pro Gln
Tyr Phe Asp Arg 1 5 10 15 15 17 PRT Human Amino acids 344-360 of
SEQ ID NO 3 15 Asp Gly Glu Val Gly Gly Ala Val Tyr Val Tyr Met Asn
Gln Gln Gly 1 5 10 15 Arg 16 8 PRT Human Amino acids 361-368 of SEQ
ID NO 3 16 Trp Asn Asn Val Lys Pro Ile Arg 1 5 17 24 PRT Human
Amino acids 383-406 of SEQ ID NO 3 17 Asn Ile Gly Asp Ile Asn Gln
Asp Gly Tyr Pro Asp Ile Ala Val Gly 1 5 10 15 Ala Pro Tyr Asp Asp
Leu Gly Lys 20 18 18 PRT Human Amino acids 427-444 of SEQ ID NO 3
18 Gly Ile Ser Pro Tyr Phe Gly Tyr Ser Ile Ala Gly Asn Met Asp Leu
1 5 10 15 Asp Arg 19 19 PRT Human Amino acids 445-463 of SEQ ID NO
3 19 Asn Ser Tyr Pro Asp Val Ala Val Gly Ser Leu Ser Asp Ser Val
Thr 1 5 10 15 Ile Phe Arg 20 9 PRT Human Amino acids 464-472 of SEQ
ID NO 3 20 Ser Arg Pro Val Ile Asn Ile Gln Lys 1 5 21 18 PRT Human
Amino acids 577-594 of SEQ ID NO 3 21 Leu Arg Pro Ile Pro Ile Thr
Ala Ser Val Glu Ile Gln Glu Pro Ser 1 5 10 15 Ser Arg 22 17 PRT
Human Amino acids 597-613 of SEQ ID NO 3 22 Val Asn Ser Leu Pro Glu
Val Leu Pro Ile Leu Asn Ser Asp Glu Pro 1 5 10 15 Lys 23 10 PRT
Human Amino acids 614-623 of SEQ ID NO 3 23 Thr Ala His Ile Asp Val
His Phe Leu Lys 1 5 10 24 8 PRT Human Amino acids 652-659 of SEQ ID
NO 3 24 Phe Ser Tyr Leu Pro Ile Gln Lys 1 5 25 16 PRT Human Amino
acids 671-686 of SEQ ID NO 3 25 Asp Ile Ala Leu Glu Ile Thr Val Thr
Asn Ser Pro Ser Asn Pro Arg 1 5 10 15 26 14 PRT Human Amino acids
822-835 of SEQ ID NO 3 26 Ser Glu Asp Glu Val Gly Ser Leu Ile Glu
Tyr Glu Phe Arg 1 5 10 27 15 PRT Human Amino acids 871-885 of SEQ
ID NO 3 27 Val Glu Ser Lys Gly Leu Glu Lys Val Thr Cys Glu Pro Gln
Lys 1 5 10 15 28 13 PRT Human Amino acids 902-914 of SEQ ID NO 3 28
Arg Glu Ile Thr Glu Lys Gln Ile Asp Asp Asn Arg Lys 1 5 10 29 7 PRT
Human Amino acids 915-921 of SEQ ID NO 3 29 Phe Ser Leu Phe Ala Glu
Arg 1 5 30 16 PRT Human Amino acids 923-938 of SEQ ID NO 3 30 Tyr
Gln Thr Leu Asn Cys Ser Val Asn Val Asn Cys Val Asn Ile Arg 1 5 10
15 31 9 PRT Human Amino acids 968-976 of SEQ ID NO 3 31 Leu Asn Tyr
Leu Asp Ile Leu Met Arg 1 5 32 13 PRT Human Amino acids 977-989 of
SEQ ID NO 3 32 Ala Phe Ile Asp Val Thr Ala Ala Ala Glu Asn Ile Arg
1 5 10 33 9 PRT Human Amino acids 990-998 of SEQ ID NO 3 33 Leu Pro
Asn Ala Gly Thr Gln Val Arg 1 5 34 12 PRT Human Amino acids 178-189
of SEQ ID NO 4 34 Val Ser Val Pro Gln Thr Asp Met Arg Pro Glu Lys 1
5 10 35 13 PRT Human Amino acids 192-204 of SEQ ID NO 4 35 Glu Pro
Trp Pro Asn Ser Asp Pro Pro Phe Ser Phe Lys 1 5 10 36 13 PRT Human
Amino acids 205-217 of SEQ ID NO 4 36 Asn Val Ile Ser Leu Thr Glu
Asp Val Asp Glu Phe Arg 1 5 10 37 12 PRT Human Amino acids 301-312
of SEQ ID NO 4 37 Thr Gln Asp Tyr Pro Ser Val Pro Thr Leu Val Arg 1
5 10 38 12 PRT Human Amino acids 402-413 of SEQ ID NO 4 38 Arg Gly
Glu Val Gly Ile Tyr Gln Val Gln Leu Arg 1 5 10 39 18 PRT Human
Amino acids 414-431 of SEQ ID NO 4 39 Ala Leu Glu His Val Asp Gly
Thr His Val Cys Gln Leu Pro Glu 1 5 10 15 Asp Gln Lys 40 14 PRT
Human Amino acids 432-445 of SEQ ID NO 4 40 Gly Asn Ile His Leu Lys
Pro Ser Phe Ser Asp Gly Leu Lys 1 5 10 41 16 PRT Human Amino acids
446-461 of SEQ ID NO 4 41 Met Asp Ala Gly Ile Ile Cys Asp Val Cys
Thr Cys Glu Leu Gln Lys 1 5 10 15 42 15 PRT Human Amino acids
531-545 of SEQ ID NO 4 42 Tyr Glu Gly Gln Phe Cys Glu Tyr Asp Asn
Phe Gln Cys Pro Arg 1 5 10 15 43 16 PRT Human Amino acids 631-646
of SEQ ID NO 4 43 Ser Cys Val Gln Cys Gln Ala Trp Gly Thr Gly Glu
Lys Lys Gly Arg 1 5 10 15 44 28 PRT Human Amino acids 675-702 of
SEQ ID NO 4 44 Asp Glu Asp Asp Asp Cys Thr Tyr Ser Tyr Thr Met Glu
Gly Asp Gly 1 5 10 15 Ala Pro Gly Pro Asn Ser Thr Val Leu Val His
Lys 20 25 45 12 PRT Human Amino acids 845-856 of SEQ ID NO 4 45 Gln
Glu Val Glu Glu Asn Leu Asn Glu Val Tyr Arg 1 5 10 46 16 PRT Human
Amino acids 916-930 of SEQ ID NO 4 46 Val Ala Pro Gly Tyr Tyr Thr
Leu Thr Ala Asp Gln Asp Asp Ala Arg 1 5 10 15 47 13 PRT Human Amino
acids 946-958 of SEQ ID NO 4 47 Val Pro Leu Phe Ile Arg Pro Glu Asp
Asp Asp Glu Lys 1 5 10 48 14 PRT Human Amino acids 990-1003 of SEQ
ID NO 4 48 Asp Val Val Ser Phe Glu Gln Pro Glu Phe Ser Val Ser Arg
1 5 10 49 12 PRT Human Amino acids 1061-1072 of SEQ ID NO 4 49 Leu
Leu Glu Leu Gln Glu Val Asp Ser Leu Leu Arg 1 5 10 50 18 PRT Human
Amino acids 1196-1214 of SEQ ID NO 4 50 Val Cys Ala Tyr Ala Gln Gly
Glu Gly Pro Tyr Ser Ser Leu Val Ser 1 5 10 15 Cys Arg 51 9 PRT
Human Amino acids 1273-1281 of SEQ ID NO 4 51 Val Leu Val Asp Asn
Pro Lys Asn Arg 1 5
* * * * *